HBK-15 bypasses BDNF via ERK1/2-biased 5-HT1A receptor signaling to deliver a rapid antidepressant-like effect

HBK-15 bypasses BDNF via ERK1/2-biased 5-HT1A receptor signaling to deliver a rapid antidepressant-like effect | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article HBK-15 bypasses BDNF via ERK1/2-biased 5-HT1A receptor signaling to deliver a rapid antidepressant-like effect Karolina Pytka, Kinga Sałaciak, Angelika Jagielska, Klaudia Lustyk, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7315313/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rapid-onset antidepressants hold transformative potential for treating affective and cognitive symptoms of depression, yet their mechanisms remain incompletely understood. Serotonin receptors orchestrate emotional and cognitive regulation, but current treatments poorly target their intracellular signaling. Here, we characterize HBK-15, a multi-target aminergic ligand, as a functionally selective compound that biases intracellular signaling at 5-HT 1A and 5-HT 7 receptors. HBK-15 acts as a partial agonist at the ERK1/2 arm of the 5-HT 1A receptor while blocking β-arrestin recruitment, cAMP inhibition, and calcium mobilization; at 5-HT 7 receptors, it preserves cAMP signaling but blocks β-arrestin recruitment. A single dose of HBK-15 reversed depressive- and cognitive-like deficits in two mouse strains subjected to chronic stress, engaging ERK1/2-linked kinases and plasticity-related signaling in the prefrontal cortex. Pharmacological blockade experiments showed that ERK1/2, but not PKA, signaling in the medial prefrontal cortex is required for HBK-15’s behavioral effects. Notably, HBK-15 retained antidepressant-like efficacy in mice carrying the human BDNF Val66Met polymorphism, a translational model characterized by impaired activity-dependent BDNF release, increased depression vulnerability, and reduced treatment responsiveness. The absence of cognitive rescue in this context uncovers a layered mechanism: ERK1/2 signaling is required for both behavioral domains, but BDNF-dependent pathways appear critical for cognitive restoration. These findings position HBK-15 as a mechanistically distinct compound with rapid behavioral efficacy, offering a prototype for signaling-driven strategies in next-generation antidepressant development. Health sciences/Diseases/Psychiatric disorders/Depression Biological sciences/Drug discovery Biological sciences/Neuroscience functional selectivity 5-HT1A receptor ERK1/2 signaling rapid-acting antidepressant procognitive chronic stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Major depressive disorder remains a leading cause of disability worldwide, with substantial limitations in current therapeutic approaches. Conventional antidepressants are associated with delayed onset, partial efficacy, and high non-response rates (reviewed in [ 1 , 2 ]). Although rapid-acting antidepressants such as ketamine offer new hope, concerns about psychotomimetic effects and abuse potential necessitate the search for mechanistically distinct alternatives that maintain rapid efficacy with improved safety and tolerability profiles. Biased agonism - the selective activation of specific intracellular pathways downstream of G protein-coupled receptors - offers a pharmacological strategy to enhance antidepressant efficacy and minimize side effects. In the serotonergic system, 5-HT 1A receptor agonists that preferentially activate ERK1/2 signaling produce rapid antidepressant-like effects in rodents, as shown for NLX-101 and NLX-204 in preclinical studies, including our own work [ 3 – 5 ]. Conversely, we have also demonstrated that compounds primarily stimulating β-arrestin recruitment through the 5-HT 1A receptor are associated with serotonin syndrome-like symptoms in rats, such as lower lip retraction, hypothermia, or a flat body posture [ 6 ]. Signaling selectivity at the 5-HT 1A receptor thus emerges as a key factor in uncoupling rapid antidepressant efficacy from unwanted serotonergic side effects. Multimodal compounds that simultaneously engage multiple neurotransmitter systems can, despite their complex pharmacology, exhibit functional selectivity at individual receptor subtypes. Such pathway-specific signaling offers the potential for precise modulation of intracellular cascades, with therapeutic benefits [ 7 ]. HBK-15, a compound developed and characterized in our laboratory, is a promising example. It produces rapid antidepressant-like, anxiolytic-, and procognitive effects in rodents and displays a multireceptor mechanism of action, involving several aminergic systems [ 8 – 10 ]. Among the serotonin receptors tested to date, HBK-15 shows its highest binding affinities at the 5-HT 1A and 5-HT 7 subtypes, which are likely key contributors to its pharmacological activity. This raises the question of whether HBK-15 engages these receptors in a functionally selective manner and whether such biased signaling underlies its rapid in vivo effects. Clarifying this point is critical, as kinases such as ERK1/2 - key intracellular mediators of plasticity and affective regulation (reviewed in [ 11 ] ) - can respond rapidly to receptor stimulation and integrate signaling within a time frame compatible with fast behavioral adaptation. Receptor-driven activation of plasticity-related kinases such as ERK1/2 may provide a mechanism for restoring behavioral function even in the presence of impaired neurotrophin signaling. This possibility is particularly relevant in the context of the BDNF Val66Met polymorphism, which disrupts activity-dependent BDNF release [ 12 ] and is associated with reduced responsiveness to conventional antidepressants [ 13 ]. Here, we investigated whether HBK-15 displays functional selectivity at serotonin 5-HT 1A and 5-HT 7 receptors and whether this property contributes to its rapid antidepressant-like and procognitive effects. To test this, we first extended radioligand binding studies to previously uncharacterized serotonin receptor subtypes, and then combined biosensor-based assays, behavioral models of chronic stress, pharmacological interventions, and genetically modified mice, focusing on plasticity-related kinases - particularly ERK1/2 - as candidate mediators of HBK-15’s rapid effects. Methods Animals 8–10 weeks old male CD-1 and BALB/c mice were sourced from either the Animal House at the Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, or the Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw. Male BDNF Val/Met mice were provided initially as a breeding pair by Alessandro Ieraci and subsequently bred by Janvier Labs, France [ 14 ]. Mice were housed under standard conditions (20–24°C, 45–65% humidity, 12:12 h light–dark cycle) with food and water available ad libitum, except during occasional eight-hour deprivation as part of the unpredictable chronic mild stress (UCMS) protocol. Animals were randomly assigned to groups using a computer-generated sequence, with all behavioral scoring and data analysis performed by researchers blinded to treatment. All experiments followed European (86/609/EEC; 2010/63/EU; 2012/707/EU) and Polish national regulations. The Local Ethics Committee for Experiments on Animals in Kraków approved the research protocols under the following reference numbers: 104/2016, 485/2021, 591/2022, 604/2022. Drugs HBK-15 (1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride) was resynthesized at the Jagiellonian University Medical College following previously described methods[ 15 ]. HBK-15, ketamine (Sigma, Germany), and WAY100135 (Sigma, Germany) were dissolved in saline and administered intraperitoneally ( ip ), with control groups receiving saline. U0126 or H-89 (STI, Poland) was prepared in 50% dimethyl sulfoxide (DMSO) and saline. Doses for all compounds were based on prior studies or literature [ 16 – 19 ]. Serotonin and NAN-190 (Sigma, Germany) were dissolved in DMSO (10 mM). The 5-CT (Tocris) and serodolin (synthesized as previously described [ 20 ]) were stored in DMSO (Sigma-Aldrich, Saint Louis, MO, USA) at -20°C and diluted in PBS before use. Coelenterazine H was purchased from INTERCHIM (Montluçon, France). Experimental procedures In vitro binding assays Binding studies were performed commercially in Eurofins Laboratories using testing procedures described elsewhere: 5-HT 1B [ 21 ], 5-HT 1D [ 22 ], 5-HT 2B [ 23 ], 5-HT 2C [ 24 ], 5-HT 4B [ 25 ], 5-HT 4E [ 26 ], and 5-HT 5A [ 27 ] receptors. The results are presented as the inhibition of control-specific binding in the presence of HBK-15. For inhibition values above 50%, the negative logarithm of the inhibition constant (pKi) was calculated. Functional assays 5-HT 1A receptor ERK phosphorylation CHO-5HT1A cells were tested for agonist-induced ERK phosphorylation using the SureFire ERK-Phosphorylation Alpha LISA kit (Perkin Elmer; Fig. 1 a). Thawed cells were cultured in medium (Advanced DMEM/F12 with 1% FBS dialyzed, 400µg/ml G-418, 4 mM L-Glutamine) and plated at 5x10 4 cells/well of 96-well plates, then incubated for 7 h (5% CO 2 , 37°C) and starved in DMEM/F12 medium with 0.1% BSA for 12 h. Serial dilutions of compounds were added and incubated for 15 min at 37°C. Following medium removal, 70 µl of "lysis buffer" was added, and the plate was shaken for 10 min before freezing at -80°C. The following day, thawed lysates (10 µl) were transferred to 384-well assay plates, and 10 µl of AlphaLISA reaction mix was added. After 2 h of incubation at 22°C, the plates were read using an EnVision plate reader (Perkin Elmer). E max values represent the ligand’s response expressed as a percentage of the maximal response to serotonin in agonist mode. β-Arrestin Recruitment : The functional assay used CHO-K1 cells expressing 5-HT 1A serotonin receptor, performed with the PathHunter Assay (DiscoverX; Fig. 1 c). Thawed cells (5x10 3 cells/well) were seeded in 96-well plates and incubated for 24 h at 37°C with 5% CO 2 . In antagonist mode, cells were preincubated with control antagonist and test compounds for 30 min, followed by the addition of serotonin (EC 80 concentration). In agonist mode, cells were stimulated with serotonin, tested compounds, and incubated for 90 min at 37°C with 5% CO 2 . Detection reagent was then added and incubated for 1 h at room temperature. The chemiluminescent signal was measured using a PolarStar Omega reader (BMG Labtech, Germany). I max values represent the ligand’s response as a percentage of the buffer (baseline) response in antagonist mode. 5-HT 2B receptor IP1 cellular functional assays The functional assay was performed commercially in Eurofins Laboratories using CHO-K1 cells stimulated for 30 min at 37°C with 5% CO 2 . In antagonist mode, serotonin (30 nM, EC 80 ) was added. After incubation, the HTRF signal was measured. E max or I max values were expressed as the ligand’s response percentage relative to serotonin's maximal response in agonist mode or SB 206553 in antagonist mode. 5-HT 7 receptor G𝛼s and β-Arrestin-2 recruitment assays HEK-293 cells were cultured in DMEM (Dulbecco Modified Eagle Medium) with 10% (vol/vol) FBS, 1 g/L glucose, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1 mM glutamine. Transient transfections were performed in 96-well plate using Lipofectamine 2000 (ThermoFisher, France) following the manufacturer’s protocol. For G𝛼s recruitment (Fig. 1 e), cells were co-transfected with 5-HT 7a receptor fused to Rluc (HA-5-HT7 Rluc) and acceptor NES-Venus-mGs (provided by Pr. N.A. Lambert, Auguysta University Augusta, USA)[ 28 , 29 ]. For β-arrestin-2 recruitment (Fig. 1 g), HEK293 cells were transiently co-transfected with 5-HT 7 R and Rluc-β-arrestin-2 YPET plasmids (provided by Dr. M.G. Scott, Cochin Institute, Paris, France). BRET measurements were taken 48 hours post-transfection after adding ligands at varying concentrations and 5 µM coelenterazine H. Signals were recorded for 25 min in a Mithras LB 940 Multireader (Berthold, Bad Widbad, Germany), with sequential integration of luminescence at 485 ± 10 nm (RLuc filter) and 530 ± 12 nm (YFP filter). Emission ratios (530 nm/485 nm) were calculated as induced-BRET changes, representing the difference between control (no ligands) and ligand-treated conditions. Results were expressed as the mean ± SEM from representative experiments, each replicated three times. Concentration-response and saturation curves were fitted using nonlinear regression and a hyperbolic one-binding site model, respectively. Behavioral assays Unpredictable chronic mild stress The unpredictable chronic mild stress model (UCMS) commenced with a one-week acclimatization for mice, after which they were grouped for testing [ 30 , 31 ]. While control mice were housed under standard conditions, except during housekeeping procedures and the sucrose preference test (social isolation), mice undergoing UCMS procedure experienced various unpredictable stimuli, including cold water swimming (13 ± 2°C for 5 min), warm water swimming (37 ± 2°C for 1 min), moist bedding (8 h), cage tilt (45° for 8 h), cage shaking (180 rpm, 10 min), tail pinch (1 cm from the tip of the tail for 1 min), food deprivation (6 or 12 h), water deprivation (12 h), overnight illumination (12 h), no bedding (8 h), exposure to rat odor (1 h), reversal of the day/night cycle, tube immobilization (5 min), or a no-stress condition (24 h) over a 4-week (CD-1 mice) or 6-week period (BALB/c, BDNF Val/Met mice). The protocols were adapted based on strain differences and followed the recommendations from the Local Ethics Committee. Forced swim test Mice were placed individually in glass cylinders filled with water (10 cm depth, 23–25°C) for 6 min [ 32 ]. Following a 2-minute habituation, immobility (passive floating with minimal head movements) was recorded for 4 min. A blinded observer analyzed videos using Eleven Maze software ( https://elevenmaze.com ). Sucrose preference test The sucrose preference test, adapted from Liu et al [ 33 ] involved providing mice with two bottles: one containing water and the other a 1% (w/v) sucrose solution during the unpredictable chronic mild stress procedure. Bottles with water and 1% sucrose were weighed before and after a 12-hour dark-phase exposure. Sucrose preference was calculated as: $$\:\varvec{s}\varvec{u}\varvec{c}\varvec{r}\varvec{o}\varvec{s}\varvec{e}\:\varvec{p}\varvec{r}\varvec{e}\varvec{f}\varvec{e}\varvec{r}\varvec{e}\varvec{n}\varvec{c}\varvec{e}=\:\frac{\varvec{s}\varvec{u}\varvec{c}\varvec{r}\varvec{o}\varvec{s}\varvec{e}\:\varvec{s}\varvec{o}\varvec{l}\varvec{u}\varvec{t}\varvec{i}\varvec{o}\varvec{n}\:\varvec{i}\varvec{n}\varvec{t}\varvec{a}\varvec{k}\varvec{e}\:\left[\varvec{g}\right]}{\varvec{s}\varvec{u}\varvec{c}\varvec{r}\varvec{o}\varvec{s}\varvec{e}\:\varvec{s}\varvec{o}\varvec{l}\varvec{u}\varvec{t}\varvec{i}\varvec{o}\varvec{n}\:\varvec{i}\varvec{n}\varvec{t}\varvec{a}\varvec{k}\varvec{e}\:\left[\varvec{g}\right]+\varvec{w}\varvec{a}\varvec{t}\varvec{e}\varvec{r}\:\varvec{i}\varvec{n}\varvec{t}\varvec{a}\varvec{k}\varvec{e}\:\left[\varvec{g}\right]}\varvec{*}100\varvec{\%}$$ Object recognition test The object recognition test was adapted from Leger et al [ 34 ]. In the familiarization session, mice were placed in an open field (35×35×35 cm) containing two identical objects and were allowed to explore until they reached a criterion of 20 s of total exploration, but no longer than 10 min. In the test phase, one object was replaced with a novel one; the positions of the objects were randomized. The mice were again allowed to explore until they reached the 20-second criterion of total exploration, but no longer than 10 min. The objects and the arena were cleaned between trials. The experiments were video recorded and scored using Eleven Maze software ( https://elevenmaze.com ) by a trained observer who was blind to the treatments. To assess the animals' performance in the object recognition test, the average novel object exploration times were compared to the chance level (10 s, equal exploration of the objects) according to the followed protocol [ 34 ]. Locomotor activity The locomotor activity test, as described previously [ 35 ], monitored the individual activity of mice using Opto M3 activity cages (Columbus Instruments, USA). After a 30-minute habituation period, beam crossings were recorded every minute for 6 min. The cages were disinfected with an odorless veterinary disinfectant after each mouse. Inhibition of ERK1/2 or PKA signaling pathways Surgery Animals were anesthetized with isoflurane (5% induction, 2.5% maintenance) and placed in a stereotaxic frame (ASI Instruments, Warren, MI, USA). Analgesia was achieved using subcutaneous lidocaine at the incision site, along with dorsal administration of carprofen (10 mg/kg). Bilateral guide cannulas (RWD-62052, USA) were implanted in the prelimbic cortex (AP + 1.93 mm; ML ± 0.4 mm; DV – 2.2 mm from the bregma; Fig. S1 a), according to the Franklin and Paxinos stereotaxis atlas[ 36 ]. Cannulas were secured with anchor screws, dental cement, dummy cannulas (protruding 0.5 mm, RWD-62123, USA) and caps (RWD-62523, USA). After surgery, mice were housed in a high-roofed cage with ad libitum access to water and food and monitored for recovery. Microinjection Following a one-week recovery, injection cannulas (RWD-62223, USA) were inserted into guide cannulas and connected via a polyethylene tubing to a microsyringe (NanoFil 10 µL, WPI, USA) driven by a microinfusion pump (UMP3 UltraMicroPump III with MICRO2T 2-Channel Controller, WPI, USA). U0126 (0.5 µg/0.5 µL per site), H-89 (2.6 ng/0.5 ul), or vehicle (50% DMSO in saline) was delivered at 250 nL/min over 2 min, with cannulas left in place for an additional 2 min to prevent backflow. Probe placement was confirmed post-mortem in coronal sections (Fig. S1 b). Ex vivo studies ELISA Following behavioral assessments, mice were euthanized, and their prefrontal cortices were dissected, frozen, and stored at -80°C. On the experimental day, tissues were thawed on ice, homogenized (1:10 w/v) in chilled PBS with protease and phosphatase inhibitors, and centrifuged at 2000 rpm for 20 min at 4°C, after which supernatants were collected for analysis. Protein concentrations of BDNF (201-02-0014), CREB (201-02-1579), pCREB (201-02-1586), ERK1/2 (201-02-3766), pERK1/2 (201-02-1572), PKA (201-02-1549), pPKA (201-02-1802), CaMKIV (201-02-1577), pCaMKIV (201-02-1801), RSK2 (201-02-1615), pRSK2 (-02-1597), CaMKII (201-02-1614), pCaMKII (201-02-1674), PKC (201-02-1613), and pPKC (201-02-0592) were determined using SunRed ELISA kits, following the manufacturer’s instructions. Tissue homogenates were normalized to wet weight prior to analysis. Samples were analyzed in duplicates, and mean concentrations calculated. Color intensity was measured at 450 nm using a plate reader (POLARstar Omega, BMG Labtech, Germany). Immunohistochemistry Tissue fixation Following behavioral testing, mice designated for immunofluorescence staining were deeply anesthetized with thiopental (75 mg/kg) and transcardially perfused with ~ 30 mL of ice-cold PBS, followed by 15 mL of 4% paraformaldehyde (PFA) in 0.1 M PBS. Extracted brains were post-fixed in 4% PFA overnight, then cryoprotected in graded sucrose solutions (10%, 20%, 30%) until fully submerged. Coronal sections (30 µm) were prepared using a Leica CM 1860 cryotome, mounted on SuperFrost slides (ThermoFisher, USA), and stored at − 20°C until staining. Immunofluorescent staining To visualize signaling proteins, slides with brain slices were immersed in 70% ethanol, air-dried, and washed twice for 10 min in PBS and once with 0.3% Triton X-100/PBS. Nonspecific binding was blocked with 10% goat serum in 0.3% Triton X-100/PBS for 1 h. Sections were incubated overnight at 4°C with anti-pERK1/2 (1:300, #4370, Cell Signaling) and anti-pPKA (1:150, STJ90386, St John’s) in 2% goat serum/0.03% Triton X-100/PBS. After washing (twice for 10 minutes in 0.3% Triton X-100/0.01 M PBS, then twice for 10 minutes in 2% goat serum with 0.3% Triton X-100/0.01 M PBS), slides were incubated with Alexa Fluor 488 secondary antibody (#A-21121, Thermo Fisher) for an hour in the absence of light. Afterward, the slides underwent five 10-minute washes in 0.01 M PBS. Finally, the slides were mounted with Vectashield Vibrance (with DAPI, Vectorlabs) and stored at 4°C. Images of the prelimbic cortex were captured using a Leica Stellaris 8 WLL, DLS confocal microscope (Leica, Germany), using the same microscope settings (i.a. laser power and frequency, detector power, pinhole), and processed with ImageJ2 version 2.14.0/1.54f [ 37 ]. To visualize regional distribution patterns, immunohistochemical staining for pERK1/2 and pPKA was performed on a subset of animals (separate from those used for ELISA). No quantitative analysis was performed on IHC images. Quantitative PCR analysis Total RNA was extracted from the medial prefrontal cortex using the Total RNA Mini kit (A&A Biotechnology, Poland). RNA concentration and purity were assessed using a NanoQuant Plate (Tecan, Switzerland), based on absorbance at 260 and 280 nm. Only samples with A260/280 ≥ 1.8 were included, and RNA concentrations were equalized prior to cDNA synthesis using the TranScriba kit with oligo(dT)18 as the primer (A&A Biotechnology). Gene expression was analyzed using TaqMan probes (ThermoFisher, USA): Mm04230607_s1 ( Bdnf ), Mm00442479_m1 ( Mapk1 ), Mm01278702_gH ( Mapk3 ), Mm00455829_m1 ( Rps6ka3 ), Mm00437967_m1 ( Camk2a ), Mm01135329_m1 ( Camk4 ), Mm01349190_m1 ( Crtc1 ), Mm00508404_m1 ( Riiad1 ) and normalized to the housekeeping reference gene: Mm99999915_g1 ( Gapdh ). A group of control animals was used as a reference. The level of housekeeping gene (GAPDH) was roughly equal in the samples and did not change significantly between experimental groups. RNA abundance was calculated by the standard ΔΔCt method. Statistical analysis Results are expressed as means ± SD or SEM for parametric data and medians with IQR for non-parametric data. Normality and homogeneity of variances were assessed using the Shapiro-Wilk and Brown-Forsythe tests. Group comparisons employed one-way ANOVA with Bonferroni post hoc for parametric data or Kruskal-Wallis with Dunn's post hoc for non-parametric data. Novel object exploration was analyzed with a one-sample t-test. Significance was set at p < 0.05. Analyses, including EC 50 , IC 50 , and Ki (via the Cheng-Prusoff formula), were conducted using GraphPad 9.5.0. Results HBK-15 displays pathway-selective signaling at serotonin 5-HT 1A and 5-HT 7 receptors To build on the existing binding profile of HBK-15, we extended its serotonergic receptor characterization beyond previously studied targets. Earlier studies demonstrated that HBK-15 exhibits high affinity for 5-HT 1A (pKi = 9.02), moderate affinity for 5-HT 2A (pKi = 7.21) and 5-HT 7 (pKi = 7.33) receptors (with pKi values reflecting the negative logarithm of Ki, where higher pKi indicates stronger binding), and negligible binding to 5-HT 3 and 5-HT 6 receptors [ 15 , 19 ]. In the current study, we focused on the 5-HT 1B , 5-HT 1D , 5-HT 2B , 5-HT 2C , 5-HT 4B , 5-HT 4E , and 5-HT 5A receptors to complete the pharmacological characterization across the major serotonin receptor subtypes. HBK-15 displayed high affinity for 5-HT 2B receptors (pKi = 7.58), while showing low or negligible binding to other receptors (Table 1 ). Table 1 In vitro binding assays for HBK-15. Molecular target Source % inhibition of control-specific binding 5-HT 1B human recombinant Chem-1 (RBL) cells 59.1 5-HT 1D rat recombinant CHO cells 59.2 5-HT 2B human recombinant CHO cells 94.2 5-HT 2C human recombinant HEK-293 cells 9.0 5-HT 4B human recombinant Chem-1 cells -7.7 5-HT 4E human recombinant CHO cells -0.4 5-HT 5A human recombinant HEK-293 cells 29.3 HBK-15 was tested at concentrations 10 − 6 M. The results are presented as % inhibition of control-specific binding in the presence of HBK-15. Results showing an activity > 60% were considered to represent significant effects of the test compound; results showing an inhibition between 25% and 60% indicate moderate to weak effect; results showing an inhibition < 25% are not considered significant and mostly attributable to the variability of the signal around the control level. Binding studies were performed commercially in Eurofins Laboratories (Poitiers, France). At the 5-HT 1A receptor, HBK-15 acted as a functionally selective ligand. While previous studies showed that HBK-15 antagonized serotonin-induced inhibition of cAMP synthesis [ 38 ] and blocked calcium mobilization [ 19 ] (pIC 50 = 6.47 and 7.72, respectively) with moderate potency and partial efficacy, here we extended its signaling profile to additional pathways. HBK-15 partially activated ERK1/2 phosphorylation (1.5-fold lower potency and 4-fold lower efficacy than serotonin, Fig. 1 b) and strongly inhibited β-arrestin recruitment (2-fold lower potency than NAN-190 with comparable maximal inhibition; Fig. 1 d). These findings indicate preferential engagement of selected downstream pathways while blocking others, consistent with functional selectivity. Given its high affinity for 5-HT 2B receptors - a target associated with cardiotoxicity due to agonist-induced valvulopathy - we specifically assessed whether HBK-15 exhibits any agonist activity at this receptor. In Gq-coupled assays, HBK-15 acted as an antagonist, inhibiting serotonin-induced calcium mobilization and inositol phosphate accumulation (pIC 50 = 6.74), with approximately 10-fold lower potency than the reference antagonist SB206553 (Table S1 ). As 5-HT 2B receptors play a limited role in central serotonergic neurotransmission, further pathway deconvolution was not pursued. These results suggest a favorable safety profile despite strong receptor binding affinity. At the 5-HT 7 receptor, we employed real-time BRET biosensors to characterize signaling bias. HBK-15 acted as a partial agonist in Gαs recruitment (pEC 50 = 7.3 ± 0.5, reaching 34% of the maximal effect relative to the full agonist; pEC 50 = 10.2 ± 0.1, Fig. 1 f), with no detectable β-arrestin engagement (Fig. 1 h). This contrasts with 5-CT and serodolin, a β-arrestin–biased ligand that acted as a Gs inverse agonist (pEC 50 = 8.1 ± 0.3) and strongly recruited β-arrestin (pEC 50 = 8.6 ± 0.6), supporting a Gαs-biased signaling profile for HBK-15 at this receptor. This finding diverges our previous results using a cAMP-based CRE-bla reporter assay in CHO-K1 cells, where HBK-15 behaved as an antagonist at the 5-HT 7 receptor [ 19 ]. This discrepancy likely reflects methodological differences between population-averaged cAMP accumulation assays and real-time, proximal G protein recruitment as measured by BRET. The partial Gαs engagement observed in the BRET assay may not have been sufficient to generate a detectable increase in cAMP in the previous system. These signaling characteristics provided the rationale for subsequent behavioral and molecular studies in animal models of depression. ERK1/2 and PKA activation by HBK-15 correlates with rapid antidepressant-like effects in vivo The functional selectivity of HBK-15 toward 5-HT 1A and 5-HT 7 receptor signaling pathways prompted us to investigate whether its preferential engagement of ERK1/2 phosphorylation in vitro translates into rapid antidepressant-like efficacy in vivo . To test this, we administered a single dose of HBK-15 (2.5 mg/kg) to CD-1 mice subjected to unpredictable chronic mild stress and compared its effects to ketamine (10 mg/kg), a fast-acting antidepressant, and fluoxetine (10 mg/kg), a conventional SSRI (Fig. 2 a). In the sucrose preference test, UCMS reduced sucrose intake by 42.9% relative to non-stressed controls (Fig. 2 b). Both HBK-15 and ketamine reversed this effect (+ 61.4% and + 52.0%, respectively), while fluoxetine was ineffective (Fig. 2 b). Similarly, in the forced swim test, UCMS increased immobility time by 21.2%, and both HBK-15 and ketamine significantly reduced this measure of behavioral despair (− 36.3% and − 31.9%, respectively; Fig. 2 c). None of the compounds altered locomotor activity (Fig. 2 d), excluding nonspecific behavioral stimulation. We next examined molecular correlates of these behavioral effects, focusing on intracellular kinases implicated in rapid serotonergic signaling. Previous studies on 5-HT 1A -biased agonists such as NLX-101 and NLX-204 have shown that their fast-onset behavioral effects coincide with ERK1/2 activation, specifically in the prefrontal cortex [ 3 – 5 ]. Therefore, we selected this region to define the kinase-level footprint of HBK-15. HBK-15 significantly increased phosphorylation of ERK1/2 (+ 28.6%; Fig. 2 f), PKA (+ 34.4%; Fig. 2 i), and CaMKIV (+ 16.4%; Fig. 2 l) in the prefrontal cortex, each of which was reduced by UCMS (− 21.9%, − 20.5%, and − 13.6%, respectively). We detected the expression of phosphorylated ERK1/2 and PKA in MAP2-positive neurons of the prefrontal cortex (Fig. 2 g,j, S2), consistent with HBK-15’s receptor-specific signaling profile observed in vitro . These results suggest that preferential activation of kinase pathways, particularly ERK1/2 and PKA, may contribute to the compound’s rapid antidepressant-like effects. While BDNF signaling is widely linked to antidepressant response, HBK-15 appears to act downstream, enhancing BDNF protein levels via intracellular kinase activation rather than by transcriptional upregulation of Bdnf . HBK-15 restored UCMS-induced reductions in BDNF protein levels (+ 167.3%), similar to ketamine (+ 186.9%), but without accompanying changes in Bdnf mRNA expression at the 24-hour timepoint (Fig. 2 o, S3f). Although transient transcriptional effects at earlier timepoints cannot be excluded, the data suggest that HBK-15 engages post-transcriptional or signaling-driven mechanisms downstream of kinase activation. Supporting this interpretation, HBK-15 also reversed stress-induced decreases in pCREB (+ 26.0%; Fig. 2 n), a transcription factor downstream of ERK1/2 and PKA, without altering total CREB expression (Fig. 2 m). To determine whether HBK-15-induced changes extended beyond phosphorylation, we measured total protein levels of key signaling components. UCMS markedly reduced the expression of CREB (− 42%; Fig. 2 m), ERK1/2 (− 38.1%; Fig. 2 e), and PKA (− 27.2%; Fig. 2 h), while CaMKIV levels remained unchanged (Fig. 2 k). HBK-15 did not normalize protein abundance, indicating that its molecular actions are driven by modulation of signaling activity rather than restoration of protein levels. These effects were not associated with transcriptional changes, as mRNA levels of these targets were unaltered at the same timepoint (Fig.S3a-f). Together, these findings support a model in which HBK-15 promotes rapid behavioral recovery through direct engagement of intracellular kinase signaling, with ERK1/2 and PKA as the most consistently regulated molecular targets. HBK-15 extends its rapid effects to cognitive function and recruits complementary neuroplasticity-related pathways To ensure the behavioral and molecular effects of HBK-15 were not strain-dependent, we replicated the UCMS protocol in BALB/c mice, a stress-sensitive inbred strain, and expanded the dose range (1.25, 2.5, and 5 mg/kg; Fig. 3 a). UCMS reduced sucrose preference by 24.9% compared to non-stressed controls (Fig. 3 b). HBK-15 reversed this effect at all doses tested (+ 13.8%, + 35.9%, and + 30.7%, respectively), similarly to ketamine (+ 23.8%), confirming its robust antianhedonic efficacy in this background (Fig. 3 b). Because depression is frequently accompanied by cognitive impairment and current antidepressants rarely address this dimension (reviewed in[ 39 ]), we evaluated whether HBK-15 could restore recognition memory in the object recognition test. Mice subjected to UCMS failed to explore the novel object above chance level, whereas HBK-15 significantly improved novel object exploration at all doses tested, mirroring the effects of ketamine (Fig. 3 c). These results indicate that HBK-15 produces rapid antidepressant-like and procognitive effects following a single administration, extending its efficacy profile beyond affective symptoms. To elucidate the molecular basis of these effects, we examined other kinases implicated in plasticity-related signaling. We focused particularly on RSK2, a well-characterized downstream target of ERK1/2, and evaluated PKC and CaMKII, which operate through independent calcium- and lipid-sensitive pathways. UCMS markedly reduced pRSK2 levels in the prefrontal cortex (− 40.7%), which were restored by both HBK-15 (+ 46.2%) and ketamine (+ 49.9%) (Fig. 3 e). Phosphorylation of CaMKII (Fig. 3 g) and PKC (Fig.S4c) was not significantly altered by UCMS; however, HBK-15 increased pPKC at the highest dose tested (Fig.S4c). The regulation of RSK2, together with previously observed activation of ERK1/2 and PKA (see Fig. 2 f,i) by HBK-15, suggests the engagement of a convergent kinase-signaling mechanism potentially underlying its behavioral efficacy. We next assessed whether these effects extended beyond phosphorylation. UCMS decreased total levels of RSK2 (− 63%; Fig. 3 d) and CaMKII (− 48.2%; Fig. 3 f), while PKC remained unaffected (Fig.S4d). HBK-15 restored RSK2 level (+ 106.3% at 5 mg/kg) and normalized CaMKII levels (+ 122.2% at 2.5 mg/kg), suggesting engagement of post-transcriptional or translational regulatory mechanisms. No significant changes in mRNA levels were detected for these targets (Fig.S4a-b), indicating that HBK-15 modulates kinase abundance at the protein level. Any upstream transcriptional contribution occurring before this timepoint cannot be ruled out, yet the observed changes are more consistent with translational or post-translational control. Finally, we assessed the temporal dynamics of these effects (Fig. 3 h). In the sucrose preference test, both HBK-15 and ketamine reversed UCMS-induced anhedonia at 24 h, but not at 72 h (Fig. 3 i). A similar time course was observed in the object recognition test: behavioral improvement was evident at 24 h but absent at 72 h (Fig. 3 j). These findings indicate that the behavioral effects of HBK-15 are rapid but transient, resembling the short-term profile of ketamine observed under the same experimental conditions. Although longer-lasting antidepressant effects of ketamine have been reported in other studies [ 40 , 41 ], such persistence appears to depend on experimental variables including species, strain, sex, dose, and stress paradigm [ 42 – 44 ]. The comparable temporal profile observed here suggests that HBK-15 may share core fast-acting mechanisms with ketamine, while its longer-term efficacy could emerge under different dosing regimens or behavioral contexts not explored in the present design. HBK-15 requires 5-HT 1A -ERK1/2 signaling to exert rapid antidepressant-like and procognitive effects Functional signaling assays demonstrated that HBK-15 partially activated ERK1/2 phosphorylation downstream of 5-HT 1A receptors and recruited Gαs at 5-HT 7 receptors, as detected by biosensor-based approaches. Consistent with these pathway-specific effects, HBK-15 increased phosphorylation of ERK1/2 (+ 28.6%) and PKA (+ 34.4%) in the prefrontal cortex of UCMS-exposed mice, supporting the selection of these kinases as candidate intracellular mediators of its behavioral efficacy. Given that HBK-15 displays its highest binding affinity for the 5-HT 1A receptor among its known targets (pKi = 9.02), and exhibits functional selectivity at this site, we next tested whether its behavioral effects are mediated by 5-HT 1A receptor activation. In signaling assays, HBK-15 acted as a biased ligand at the 5-HT 1A receptor: it partially activated ERK1/2 phosphorylation while antagonizing other receptor-coupled pathways, including cAMP production inhibition, β-arrestin recruitment, and calcium mobilization. To pharmacologically verify receptor involvement (Fig. 4 a), we used WAY-100635, a selective 5-HT 1A antagonist known to block all major downstream signaling pathways [ 45 ]. In vivo , pretreatment with WAY-100635 abolished the antidepressant-like effect of HBK-15 in the sucrose preference test, as sucrose intake dropped by 24.8% relative to HBK-15 alone (Fig. 4 b). In the object recognition test, mice pretreated with WAY-100635 failed to discriminate the novel object (Fig. 4 c), indicating that 5-HT 1A receptor activation is also necessary for the procognitive effects of HBK-15. To further delineate the intracellular pathways mediating HBK-15’s effects, we selectively inhibited either the ERK1/2 or PKA signaling in the prefrontal cortex (Fig. 4 d,g). Intra-prefrontal infusion of the MAPK/ERK kinase inhibitor U0126 completely abolished both the antidepressant-like and procognitive effects of HBK-15 (Fig. 4 e–f), supporting a central role for ERK1/2 signaling in mediating its behavioral efficacy. In contrast, inhibition of PKA signaling with H-89 had no effect on either behavioral endpoint (Fig. 4 h-i). These data indicate that ERK1/2 signaling is essential for both the affective and cognitive effects of HBK-15. These findings are consistent with the molecular signature of HBK-15, characterized by robust activation of ERK1/2 in the prefrontal cortex under chronic stress conditions. This in vivo effect reflects its in vitro signaling profile, where HBK-15 selectively promoted ERK1/2 phosphorylation downstream of 5-HT 1A receptors while blocking alternative pathways at the same site. Given the established role of ERK1/2 in regulating synaptic plasticity and fast behavioral adaptation [ 46 , 47 ], this kinase emerges as a central effector of HBK-15’s rapid action. In addition, HBK-15 increased phosphorylation of PKA in the prefrontal cortex, a kinase also implicated in stress resilience. This effect likely results from its dual receptor actions: partial Gαs recruitment at 5-HT 7 receptors, which enhances cAMP synthesis, and blockade of serotonin-mediated inhibition of cAMP production at 5-HT 1A receptors [ 48 ], preventing activation of canonical Gi/o signaling. While PKA may contribute to the compound’s effects, the convergence of these receptor-specific mechanisms on ERK1/2, together with the lack of behavioral response to PKA inhibition, highlights ERK1/2 as the principal intracellular mediator of HBK-15’s behavioral efficacy. HBK-15 retains affective, but not cognitive efficacy in BDNF Val/Met mice Given that HBK-15 increased pERK1/2, pPKA, and pCREB in the prefrontal cortex and restored BDNF protein levels after a single administration, we next examined whether BDNF signaling is required for its behavioral effects, focusing on the activity-dependent BDNF secretion (Fig. 5 a). To address this, we used BDNF Val66Met knock-in mice, which exhibit impaired stimulus-induced BDNF release but retain normal baseline expression levels [ 14 ], thereby avoiding developmental confounds of total BDNF knockout and modeling a clinically relevant human polymorphism[ 12 ]. In the UCMS model, sucrose preference was reduced by 20.4% relative to non-stressed controls (Fig. 5 b). HBK-15 reversed this effect at all tested doses (+ 19.2%, + 24.8%, and + 24.4% at 1.25, 2.5, and 5 mg/kg, respectively), whereas ketamine failed to exert an effect in this genotype (Fig. 5 b). This lack of efficacy is consistent with previous studies showing that the antidepressant-like effects of ketamine are abolished in BDNF Val/Met mice due to impaired activity-dependent BDNF release [ 49 ]. In contrast, HBK-15 retained its behavioral efficacy despite this deficit, pointing to an alternative mechanism, likely mediated by selective activation of intracellular kinase pathways such as ERK1/2. By contrast, recognition memory remained impaired in the object recognition test, and neither HBK-15 nor ketamine restored cognitive performance in BDNF Val/Met mice (Fig. 5 c). This pattern suggests that the procognitive effect of HBK-15 depend on intact BDNF signaling, while its antidepressant-like action is mediated via BDNF-independent, kinase-driven mechanisms. Discussion In this study, we identify HBK-15 as a functionally selective serotonergic compound that engages behaviorally relevant kinase pathways to produce rapid and simultaneous antidepressant-like and procognitive effects following a single administration in a chronic stress model. Through the use of pathway-specific biosensors, pharmacological tools, and stress-based behavioral assays, we demonstrate that HBK-15 preferentially activates ERK1/2 signaling via the 5-HT 1A receptor, while partially recruiting Gαs at 5-HT 7 receptors. This receptor-specific signaling translates into rapid in vivo behavioral efficacy, primarily driven by ERK1/2 activation and observed after a single dose, contrasting with the delayed onset typical of conventional monoaminergic antidepressants. These results directly address our initial hypothesis that HBK-15, despite its multimodal profile, acts through functionally selective engagement of intracellular effectors, particularly ERK1/2, to exert rapid behavioral effects (Fig. 6 ). ERK1/2 has emerged as a key intracellular effector linking serotonergic receptor activation to synaptic and behavioral plasticity, particularly in the context of rapid-acting antidepressant strategies [ 50 , 51 ]. Prior studies on 5-HT 1A -biased agonists such as NLX-101 and NLX-204 have demonstrated a rapid onset of behavioral effects, coinciding with enhanced ERK1/2 phosphorylation in the prefrontal cortex [ 3 – 5 ]. Consistent with this mechanism, HBK-15 selectively increased ERK1/2 phosphorylation both in vitro and in the prefrontal cortex of chronically stressed mice. Importantly, pharmacological inhibition of the ERK pathway abolished the antidepressant-like and procognitive effects of HBK-15, directly implicating ERK1/2 signaling in its behavioral efficacy. These effects occurred in the absence of transcriptional induction, consistent with a fast, signal-driven mechanism previously described for compounds such as ketamine and NLX-101. Unlike ERK1/2, PKA signaling was not necessary for either the affective or cognitive effects of HBK-15, indicating that HBK-15’s behavioral efficacy is selectively mediated through ERK1/2-dependent mechanisms. Although HBK-15 acts as a partial agonist at the ERK1/2 branch of 5-HT 1A signaling, our data indicate that full activation is not required for behavioral efficacy. Selective ERK1/2 recruitment, at moderate efficacy, appears sufficient when alternative receptor-mediated responses such as β-arrestin recruitment, cAMP inhibition, and calcium mobilization are suppressed. This suggests that biased, rather than strong, ERK1/2 activation may be optimal for therapeutic effects, aligning with current strategies to achieve targeted and safer 5-HT 1A receptor modulation. In addition to rapidly activating ERK1/2, HBK-15 increased phosphorylation of plasticity-related kinases, including RSK2 and CaMKIV, elevated total levels of CaMKII, and enhanced downstream effectors such as CREB and BDNF [ 9 , 18 ]. This pattern likely reflects a convergent intracellular response to receptor activation, in which ERK1/2 plays a central integrative role, while calcium-sensitive kinases may contribute modulatory input. However, given the preserved efficacy of HBK-15 in BDNF Val/Met mice, a model with impaired activity-dependent BDNF release [ 14 ], these transcriptional and calcium-dependent pathways are unlikely to be essential for its behavioral effects. Instead, the antidepressant-like effects of HBK-15 appear to rely primarily on ERK1/2-driven signaling, while its procognitive actions may additionally require intact BDNF-dependent mechanisms. Consistently, the preserved efficacy of HBK-15 in BDNF Val/Met mice further underscores its translational potential. The Val66Met polymorphism, present in approximately 25–30% of the population [ 52 ], has been associated with reduced synaptic plasticity, greater susceptibility to depression, and attenuated responses to classical antidepressants - including ketamine [ 49 , 53 , 54 ]. In this context, HBK-15’s ability to produce robust behavioral effects despite impaired BDNF release suggests that it may bypass traditional neurotrophic mechanisms. This property may prove particularly advantageous in genetically defined subpopulations with limited treatment options, supporting the relevance of HBK-15 as a mechanistically distinct candidate for precision psychiatry. Interestingly, BDNF Val/Met mice displayed intact object recognition memory at a 4-hour delay, consistent with reports that deficits typically emerge at longer intervals (≥ 24 h [ 55 ]). However, under chronic stress, this memory was disrupted and unresponsive to HBK-15, suggesting that its procognitive actions depend on intact BDNF signaling. In this context, the Val66Met variant may impair early-phase LTP or local protein synthesis in memory circuits, limiting HBK-15’s efficacy. Thus, while HBK-15 retains antidepressant-like effects in the BDNF-impaired brain, its cognitive benefits may be restricted to individuals with preserved neurotrophic capacity. Despite these promising outcomes, both the antidepressant-like and procognitive effects of HBK-15 were transient. This temporal profile mirrors that of ketamine under our experimental conditions. Although ketamine has been shown to induce behavioral improvements lasting from hours to weeks, depending on the model, dose, and context [ 56 – 58 ], in our paradigm, both compounds produced rapid but short-lived effects. This suggests that acute kinase activation may not be sufficient to sustain long-term behavioral benefits without repeated dosing or structural reinforcement. Notably, recent studies have shown that prolonging ERK1/2 signaling - e.g., through inhibition of the phosphatase DUSP6 - extends ketamine’s behavioral effects for up to several weeks [ 59 ]. This supports the idea that ERK1/2 activation is not only necessary for the onset of rapid antidepressant action but may also serve as a target for enhancing durability. Beyond efficacy, the signaling selectivity of HBK-15 may also inform its safety and tolerability profile. Preferential recruitment of β-arrestin at 5-HT 1A receptors has been associated with the so-called “serotonin syndrome” triad in rodents - lower-lip retraction, flat-body posture, and hypothermia - serving as a surrogate marker of excessive serotonergic activation [ 60 , 61 ]. In contrast, ERK-biased 5-HT 1A ligands, such as NLX-101, preserve antidepressant-like efficacy without eliciting these autonomic signs [ 6 ]. In humans, serotonergic toxicity manifests differently: even partial 5-HT 1A agonists like buspirone produce only transient drops in core temperature [ 62 ], and clinical signs include hyperthermia, nausea, and autonomic arousal [ 63 ]. Thus, while the rodent triad may offer only a heuristic indication of serotonergic overstimulation, the ERK-biased, β-arrestin-sparing signaling profile of HBK-15 is likely to enhance therapeutic precision, minimizing peripheral autonomic effects without compromising central antidepressant action. While our findings support ERK1/2-biased signaling as a key driver of HBK-15’s rapid behavioral effects, some limitations should be acknowledged. We identified kinase activation patterns and confirmed ERK1/2 involvement, but a full mapping of upstream–downstream signaling remains to be defined. Our behavioral studies focused on acute effects after a single dose; longer-term outcomes require investigation. Moreover, our experiments were conducted exclusively in male mice, and future studies should address potential sex differences in behavioral and molecular responses to HBK-15. Finally, although HBK-15 was effective in BDNF Val/Met mice, clinical validation in genetically defined populations will be crucial. These points highlight the need for further studies to assess the durability, generalizability, and translational scope of HBK-15’s therapeutic action. Taken together, these findings position HBK-15 as a mechanistically distinct example of functionally selective polypharmacology. Its “fine-tuned” ERK1/2 bias, together with the bypass of BDNF dependency in affective domains, highlights the translational relevance of HBK-15’s intracellular signaling profile. By shifting the focus from receptor binding to intracellular selectivity, HBK-15 exemplifies a paradigm in which biased signaling - rather than receptor agonism alone - emerges as a primary determinant of antidepressant efficacy. This framework may guide the rational design of next-generation therapies that act rapidly and effectively in biologically constrained populations. Declarations Competing interests The authors declare no competing interests. Funding This study was financially supported by the National Science Centre, Poland (grant number 2019/34/E/NZ7/00454 and 2017/01/X/NZ7/00818). CRediT author statement Conceptualization: KP (lead). Methodology: KS, MG, BM, SM, KP. Investigation: KS (lead), AJag, KL, MG, BM, EŻ (support), AJan (support), JD (support), AK (support), AS (support), WK (support), BP (support), JVG (support), BAFK, SM, KP. Formal Analysis: KS (lead), AJag, KL (support), MG, BM, JVG (support), SM, KP. Data Curation: KS, AJag, KL, MG, BM, AK, KP (lead). Writing – Original Draft: KS, AJag, KL, MG, BM, SM, KP (lead). Writing – Review & Editing: KS, AJag, KL, MG (support), BM (support), EŻ (support), AJan (support), JD (support), AK (support), AS (support), WK (support), BP (support), JVG (support), BAFK (support), SM (support), KP (lead). Supervision: KS (support), WK, BP, SM, KP (lead). Funding Acquisition: KP (lead). Acknowledgments We would like to thank Dr Małgorzata Więcek for the resynthesis of HBK-15 and Dr Alessandro Ieraci for providing a breeding couple of BDNF Val/Met mice. This research was carried out with the use of research infrastructure co-financed by the Smart Growth Operational Programme POIR 4.2 project no. 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Heliyon. 2024;10:e28918. Additional Declarations The authors have declared there is NO conflict of interest to disclose The authors declare no conflict of interests. Supplementary Files supplementalmaterials.docx Supplementary materials supplementalmaterialsline.docx Supplemental material FigureS1.pdf FigureS2.pdf FigureS4.pdf FigureS3.pdf Figure S2 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7315313","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":502797868,"identity":"52e13bc6-51be-4aa3-807e-88978a76d75e","order_by":0,"name":"Karolina 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Gómez-Lobo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"Valero","lastName":"Gómez-Lobo","suffix":""},{"id":502797882,"identity":"899a6357-6409-4189-8147-3e053598f677","order_by":14,"name":"Bila Abdoul Kabore","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bila","middleName":"Abdoul","lastName":"Kabore","suffix":""},{"id":502797883,"identity":"587c51e7-75d3-4031-bb2a-84ef0802653c","order_by":15,"name":"Severine Morisset-Lopez","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Severine","middleName":"","lastName":"Morisset-Lopez","suffix":""}],"badges":[],"createdAt":"2025-08-07 06:26:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7315313/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7315313/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90122393,"identity":"0b457814-bfd6-42dd-9594-096d0a6737f2","added_by":"auto","created_at":"2025-08-28 18:03:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":522770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctionally selective signaling of HBK-15 at 5-HT\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and 5-HT\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e receptors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,c,e,g: \u003c/strong\u003eSchematic illustrations of the signaling cascades tested at each receptor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb,d,f,h\u003c/strong\u003e: HBK-15 partially activated ERK1/2 phosphorylation (\u003cstrong\u003eb\u003c/strong\u003e) at 5-HT\u003csub\u003e1A\u003c/sub\u003e receptors without recruiting β-arrestin (\u003cstrong\u003ed\u003c/strong\u003e), while at 5-HT\u003csub\u003e7\u003c/sub\u003e receptors, it induced partial Gαₛ-mediated cAMP signaling (\u003cstrong\u003ef\u003c/strong\u003e) with no detectable β-arrestin recruitment (\u003cstrong\u003eh\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eData represent means ± SEM of three independent experiments (n=3; triplicates). Illustrations were created with BioRender.com.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/58942ae15de48101b30091ec.jpg"},{"id":90123119,"identity":"64bfefd1-7c8d-4bad-924e-bca06e230409","added_by":"auto","created_at":"2025-08-28 18:19:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":640476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHBK-15 rapidly induces antidepressant-like effects via biased 5-HT\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e–ERK1/2 signaling and plasticity-related kinase activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Experimental timeline: CD-1 mice were exposed to unpredictable chronic mild stress (UCMS) for 4 weeks with behavioral and molecular assessments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb–d\u003c/strong\u003e, Behavioral effects of treatment: single intraperitoneal (ip) injection of HBK-15, and ketamine, but not fluoxetine significantly increased sucrose consumption in the sucrose preference test (SPT; F\u003csub\u003e4,35 \u003c/sub\u003e=10.48, p\u0026lt;0.0001; \u003cstrong\u003eb\u003c/strong\u003e) and reduced immobility in the forced swim test (FST; F\u003csub\u003e4,35 \u003c/sub\u003e=13.81, p\u0026lt;0.0001; \u003cstrong\u003ec\u003c/strong\u003e), without affecting locomotor activity (LA, F\u003csub\u003e4,35 \u003c/sub\u003e= 0.3712, p=0.8276; \u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee–g\u003c/strong\u003e, HBK-15 and ketamine increased phosphorylated ERK1/2 levels in the prefrontal cortex (pERK1/2: F\u003csub\u003e3,28\u003c/sub\u003e=9.415, p=0.0002; \u003cstrong\u003ee\u003c/strong\u003e), while ERK1/2 was unchanged (F\u003csub\u003e3,16\u003c/sub\u003e=2.803, p=0.0733; \u003cstrong\u003ef\u003c/strong\u003e); representative confocal image of pERK1/2 staining in neuron (MAP2+ cell, arrow) is represented in \u003cstrong\u003eg\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh–j\u003c/strong\u003e, Level of phosphorylated PKA, but not PKA, was elevated following HBK-15 treatment (PKA: F\u003csub\u003e3,16\u003c/sub\u003e=8.195, p=0.0016;\u003cstrong\u003e h\u003c/strong\u003e, pPKA: F\u003csub\u003e3,28\u003c/sub\u003e=6.157, p=0.0024; \u003cstrong\u003ei\u003c/strong\u003e); representative confocal image of pPKA staining in neuron (MAP2+ cell, arrow) is shown in \u003cstrong\u003ej\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ek–o\u003c/strong\u003e, HBK-15 and ketamine upregulated key plasticity markers: pCaMKIV (F\u003csub\u003e3,27\u003c/sub\u003e=13.54, p\u0026lt;0.0001; \u003cstrong\u003el\u003c/strong\u003e), pCREB (F\u003csub\u003e3,20\u003c/sub\u003e=5.551, p=0.0061; \u003cstrong\u003en\u003c/strong\u003e), and BDNF (W\u003csub\u003e3,11.85\u003c/sub\u003e=23.01, p\u0026lt;0.0001; \u003cstrong\u003eo\u003c/strong\u003e), while effects on total CaMKIV (F\u003csub\u003e3,18\u003c/sub\u003e=7.277, p=0.0021; \u003cstrong\u003ek\u003c/strong\u003e), CREB (F\u003csub\u003e3,16\u003c/sub\u003e=2.942, p=0.0648; \u003cstrong\u003em\u003c/strong\u003e) were less pronounced.\u003c/p\u003e\n\u003cp\u003eThe data are presented as means ± SD. Statistical analysis: one-way ANOVA (Bonferroni \u003cem\u003epost hoc\u003c/em\u003e; \u003cstrong\u003eb,c,d,e,f,h,k,l,m,n\u003c/strong\u003e) and Welch’s ANOVA (Dunnett’s T3 \u003cem\u003epost hoc\u003c/em\u003e; \u003cstrong\u003eo\u003c/strong\u003e); *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, n=8-10 mice per group. Doses are expressed in milligrams per kilogram of body weight (mg/kg).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Experimental schemes were created with BioRender.com.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/3e9e310513e7a42e0b71c381.jpg"},{"id":90122400,"identity":"8f7168ec-ca9b-4423-99e7-42fdd8344e9c","added_by":"auto","created_at":"2025-08-28 18:03:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":441808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHBK-15 rapidly and persistently reverses affective and cognitive deficits by recruiting ERK1/2-linked neuroplasticity signaling pathways.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Experimental timeline: BALB/c mice were subjected to 6 weeks of unpredictable chronic mild stress (UCMS), followed by behavioral testing in the sucrose preference test (SPT) and object recognition test (ORT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb–c\u003c/strong\u003e: A single intraperitoneal (ip) injection of HBK-15 or ketamine significantly increased sucrose preference in the SPT (F\u003csub\u003e5,54\u003c/sub\u003e=15.85, p\u0026lt;0.0001, \u003cstrong\u003eb\u003c/strong\u003e) and enhanced novel object exploration time in the ORT (HBK-15: t\u003csub\u003e9\u003c/sub\u003e=2.904, p=0.0175 at 1.25 mg/kg; t\u003csub\u003e8\u003c/sub\u003e=2.347, p=0.0469 at 2.5 mg/kg; t\u003csub\u003e9\u003c/sub\u003e=2.624, p=0.0276 at 5 mg/kg; ketamine: t\u003csub\u003e7\u003c/sub\u003e=2.794, p=0.0267; \u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed–g\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eAcute treatment with HBK-15 or ketamine modulated prefrontal plasticity-related kinases, increasing RSK2 (F\u003csub\u003e5,24\u003c/sub\u003e=15.72, p\u0026lt;0.0001; \u003cstrong\u003ed\u003c/strong\u003e), pRSK2 (H\u003csub\u003e6,37\u003c/sub\u003e=21.08, p=0.0008; \u003cstrong\u003ee\u003c/strong\u003e), and CaMKII (F\u003csub\u003e5,24\u003c/sub\u003e=14.42, p\u0026lt;0.0001; \u003cstrong\u003ef\u003c/strong\u003e), while changes in pCaMKII did not reach significance (H\u003csub\u003e6,36\u003c/sub\u003e=10.31, p=0.067; \u003cstrong\u003eg\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh: \u003c/strong\u003eExperimental design: a separate cohort of BALB/c mice, exposed to 6 weeks of UCMS, received a single ip dose of HBK-15 or ketamine and were tested at 24 and 72 hours to assess the compounds’ effect duration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei–j\u003c/strong\u003e: HBK-15 and ketamine maintained behavioral effects up to 24 h: both compounds increased sucrose preference in the SPT at 24 h (F\u003csub\u003e5,45\u003c/sub\u003e=10.25, p\u0026lt;0.0001), but not 72 h (H\u003csub\u003e6,53\u003c/sub\u003e=17.42, p=0.0038; \u003cstrong\u003ei\u003c/strong\u003e), and improved novel object recognition in the ORT at 24 h (HBK-15: 1.25 mg/kg: t\u003csub\u003e7\u003c/sub\u003e=3.274, p=0.0136; 2.5 mg/kg: t\u003csub\u003e6\u003c/sub\u003e=3.708, p=0.01; ketamine: t\u003csub\u003e6\u003c/sub\u003e=12.06, p\u0026lt;0.0001), but not 72 h (\u003cstrong\u003ej\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe data are presented as means ± SD (\u003cstrong\u003eb,c,d,f,i\u003c/strong\u003e left graph,\u003cstrong\u003ej\u003c/strong\u003e) or medians with IQR (\u003cstrong\u003ee,g,i\u003c/strong\u003e right graph). Statistical analysis: one-way ANOVA (Bonferroni \u003cem\u003epost hoc\u003c/em\u003e; \u003cstrong\u003eb,d,f,i\u003c/strong\u003e left graph), Kruskal-Wallis (Dunn’s \u003cem\u003epost hoc\u003c/em\u003e; \u003cstrong\u003ee,g,i\u003c/strong\u003e right graph), one-sample t-test (chance level=10 s; \u003cstrong\u003ec,j\u003c/strong\u003e), *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, n=8-10 mice per group. Doses are expressed in milligrams per kilogram of body weight (mg/kg). Experimental schemes were created with BioRender.com.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/71299ea6799ecabc976e5451.jpg"},{"id":90123805,"identity":"8a4fc663-5327-4295-a5cc-13ccbe1847ea","added_by":"auto","created_at":"2025-08-28 18:27:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":503050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHBK-15 exerts behavioral effects via 5-HT\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e receptor-dependent ERK1/2 activation, independently of PKA signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Experimental timeline: mice were exposed to 6 weeks of unpredictable chronic mild stress (UCMS), followed by behavioral assessment using the sucrose preference test (SPT) and object recognition test (ORT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb–c\u003c/strong\u003e: A single intraperitoneal (ip) injection of HBK-15 significantly increased sucrose preference (F\u003csub\u003e4,39\u003c/sub\u003e=4.066, p=0.0075; \u003cstrong\u003eb\u003c/strong\u003e) and novel object exploration time (t\u003csub\u003e8\u003c/sub\u003e=2.307, p=0.0499; \u003cstrong\u003ec\u003c/strong\u003e), which was blocked by the 5-HT1A receptor antagonist WAY-100635 (SPT: \u003cstrong\u003eb\u003c/strong\u003e; ORT: t\u003csub\u003e8\u003c/sub\u003e=1.373, p=0.207; \u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e: Experimental timeline: after 6 weeks of UCMS and cannula implantation during the 5th week, mice received intra-prelimbic (iPrL) cortex microinjections of U0126 (ERK1/2 inhibitor) before behavioral testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee–f\u003c/strong\u003e: Co-administration of the ERK1/2 inhibitor U0126 into the iPrL cortex blocked the effects of HBK-15 on sucrose preference (F\u003csub\u003e4,35\u003c/sub\u003e=12.75, p\u0026lt;0.0001; \u003cstrong\u003ee\u003c/strong\u003e) and novel object recognition (HBK-15 2.5 mg/kg: t\u003csub\u003e7\u003c/sub\u003e=7.592, p\u0026lt;0.0001, HBK-15 2.5 mg/kg + U0126: t\u003csub\u003e7\u003c/sub\u003e=0.4423, p=0.6716; \u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e: Experimental timeline: mice were exposed to UCMS for 6 weeks, implanted with iPrL cannula in week 5, and tested following microinjection of PKA inhibitor, H-89.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh–i\u003c/strong\u003e: Intra-iPrL administration of the PKA inhibitor H-89 did not block the behavioral effects of HBK-15 in the SPT (F\u003csub\u003e4,35\u003c/sub\u003e=6.178, p=0.0007; \u003cstrong\u003eh\u003c/strong\u003e) or ORT (HBK-15 2.5 mg/kg: t\u003csub\u003e7\u003c/sub\u003e=2.494, p=0.0414, HBK-15 2.5 mg/kg + H-89: t\u003csub\u003e7\u003c/sub\u003e=3.642, p=0.0083; \u003cstrong\u003ei\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe data are presented as means ± SD. Statistical analysis: one-way ANOVA (Bonferroni \u003cem\u003epost hoc\u003c/em\u003e; \u003cstrong\u003eb,e,h\u003c/strong\u003e), one-sample t-test (chance level=10 s; \u003cstrong\u003ec,f,i\u003c/strong\u003e), *p\u0026lt;0.05, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, n=8-9 mice per group. Doses are expressed in milligrams per kilogram of body weight (mg/kg). Experimental schemes were created with BioRender.com.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/99f57a2de98d0e70081d0517.jpg"},{"id":90122394,"identity":"1dc3571e-cf92-4511-bde5-0c21ce345192","added_by":"auto","created_at":"2025-08-28 18:03:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":161335,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRapid antidepressant-like, but not procognitive, effect of HBK-15 does not require intact BDNF signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Experimental timeline: BDNF\u003csup\u003eVal/Met \u003c/sup\u003emice were subjected to 6 weeks of unpredictable chronic mild stress (UCMS), followed by behavioral assessment in the sucrose preference test (SPT) and object recognition test (ORT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e: Unlike ketamine, a single intraperitoneal (ip) injection of HBK-15 significantly improved sucrose preference in SPT (H\u003csub\u003e6,52\u003c/sub\u003e=16.02, p=0.0068).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e: No improvement in novel object recognition time was observed in ORT following HBK-15 treatment (1.25 mg/kg: t\u003csub\u003e7\u003c/sub\u003e=0.01038, p=0.992; 2.5 mg/kg: t\u003csub\u003e8\u003c/sub\u003e=0.2367, p=0.8188; 5 mg/kg: t\u003csub\u003e8\u003c/sub\u003e=0.9249, p=0.3821) or ketamine (t\u003csub\u003e7\u003c/sub\u003e=0.4979, p=0.9617).\u003c/p\u003e\n\u003cp\u003eThe data are presented as medians with IQR (\u003cstrong\u003eb\u003c/strong\u003e) or means ± SD (\u003cstrong\u003ec\u003c/strong\u003e). Statistical analysis: Kruskal-Wallis (Dunn’s \u003cem\u003epost hoc\u003c/em\u003e; \u003cstrong\u003eb\u003c/strong\u003e), one-sample t-test (chance level=10 s; \u003cstrong\u003ec\u003c/strong\u003e), *p\u0026lt;0.05, **p\u0026lt;0.01, n=8-9 mice per group. Doses are expressed in milligrams per kilogram of body weight (mg/kg). Experimental schemes were created with BioRender.com.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/6a1a02abd119667335303268.jpg"},{"id":90122408,"identity":"e6286781-61c9-4dbc-9f62-d88a9ba12be7","added_by":"auto","created_at":"2025-08-28 18:03:54","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":221000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHBK-15 selectively activates ERK1/2 via 5-HT\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e receptor to reverse stress-induced affective and cognitive deficits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIllustration was created with BioRender.com.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/5f59e611c6cea9084b25aff4.jpg"},{"id":97897814,"identity":"06f66984-d91a-4816-a28f-077fd678320b","added_by":"auto","created_at":"2025-12-10 15:38:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4003129,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/a01a7854-52a6-4efe-b58e-e3b3f18b1162.pdf"},{"id":90123121,"identity":"983d7fe8-ea60-4592-8cc1-884627a5ecbd","added_by":"auto","created_at":"2025-08-28 18:19:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4634642,"visible":true,"origin":"","legend":"Supplementary materials","description":"","filename":"supplementalmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/494b7a1e27e976f213b480c1.docx"},{"id":90122405,"identity":"2dc081f0-378c-4336-a8dc-5cbe61408c6b","added_by":"auto","created_at":"2025-08-28 18:03:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4634307,"visible":true,"origin":"","legend":"Supplemental material","description":"","filename":"supplementalmaterialsline.docx","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/a76fcbb04a322f92471b94f5.docx"},{"id":90122399,"identity":"e1b1658e-8429-4fab-8619-f072249f3aac","added_by":"auto","created_at":"2025-08-28 18:03:54","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":735758,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/1b0d5bd2b28b22e8bcd004d6.pdf"},{"id":90122401,"identity":"1e9d029d-5ec7-451e-844f-296600933ee2","added_by":"auto","created_at":"2025-08-28 18:03:54","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3295238,"visible":true,"origin":"","legend":" ","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/104fe508b715cf01ea68cee6.pdf"},{"id":90122921,"identity":"c344ae3f-9195-455b-824e-68869e1b930c","added_by":"auto","created_at":"2025-08-28 18:11:54","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":63844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/7af8d008c77cbf9d310cb302.pdf"},{"id":90122403,"identity":"be54d511-fd9e-42d4-89a9-60ee20983bdc","added_by":"auto","created_at":"2025-08-28 18:03:54","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":66484,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2\u003c/p\u003e","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7315313/v1/7a33a5702926f46b0aa515c0.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose\nThe authors declare no conflict of interests.","formattedTitle":"HBK-15 bypasses BDNF via ERK1/2-biased 5-HT1A receptor signaling to deliver a rapid antidepressant-like effect","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMajor depressive disorder remains a leading cause of disability worldwide, with substantial limitations in current therapeutic approaches. Conventional antidepressants are associated with delayed onset, partial efficacy, and high non-response rates (reviewed in [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]). Although rapid-acting antidepressants such as ketamine offer new hope, concerns about psychotomimetic effects and abuse potential necessitate the search for mechanistically distinct alternatives that maintain rapid efficacy with improved safety and tolerability profiles.\u003c/p\u003e\u003cp\u003eBiased agonism - the selective activation of specific intracellular pathways downstream of G protein-coupled receptors - offers a pharmacological strategy to enhance antidepressant efficacy and minimize side effects. In the serotonergic system, 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor agonists that preferentially activate ERK1/2 signaling produce rapid antidepressant-like effects in rodents, as shown for NLX-101 and NLX-204 in preclinical studies, including our own\u003c/p\u003e\u003cp\u003ework [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Conversely, we have also demonstrated that compounds primarily stimulating β-arrestin recruitment through the 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor are associated with serotonin syndrome-like symptoms in rats, such as lower lip retraction, hypothermia, or a flat body posture [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Signaling selectivity at the 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor thus emerges as a key factor in uncoupling rapid antidepressant efficacy from unwanted serotonergic side effects.\u003c/p\u003e\u003cp\u003eMultimodal compounds that simultaneously engage multiple neurotransmitter systems can, despite their complex pharmacology, exhibit functional selectivity at individual receptor subtypes. Such pathway-specific signaling offers the potential for precise modulation of intracellular cascades, with therapeutic benefits [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. HBK-15, a compound developed and characterized in our laboratory, is a promising example. It produces rapid antidepressant-like, anxiolytic-, and procognitive effects in rodents and displays a multireceptor mechanism of action, involving several aminergic systems [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among the serotonin receptors tested to date, HBK-15 shows its highest binding affinities at the 5-HT\u003csub\u003e1A\u003c/sub\u003e and 5-HT\u003csub\u003e7\u003c/sub\u003e subtypes, which are likely key contributors to its pharmacological activity. This raises the question of whether\u003c/p\u003e\u003cp\u003eHBK-15 engages these receptors in a functionally selective manner and whether such biased signaling underlies its rapid \u003cem\u003ein vivo\u003c/em\u003e effects.\u003c/p\u003e\u003cp\u003eClarifying this point is critical, as kinases such as ERK1/2 - key intracellular mediators of plasticity and affective regulation (reviewed in [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] ) - can respond rapidly to receptor stimulation and integrate signaling within a time frame compatible with fast behavioral adaptation. Receptor-driven activation of plasticity-related kinases such as ERK1/2 may provide a mechanism for restoring behavioral function even in the presence of impaired neurotrophin signaling. This possibility is particularly relevant in the context of the BDNF Val66Met polymorphism, which disrupts activity-dependent BDNF release [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and is associated with reduced responsiveness to conventional antidepressants [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHere, we investigated whether HBK-15 displays functional selectivity at serotonin 5-HT\u003csub\u003e1A\u003c/sub\u003e and 5-HT\u003csub\u003e7\u003c/sub\u003e receptors and whether this property contributes to its rapid antidepressant-like and procognitive effects. To test this, we first extended radioligand binding studies to previously uncharacterized serotonin receptor subtypes, and then combined biosensor-based assays, behavioral models of chronic stress, pharmacological interventions, and genetically modified mice, focusing on plasticity-related kinases - particularly ERK1/2 - as candidate mediators of HBK-15\u0026rsquo;s rapid effects.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e8\u0026ndash;10 weeks old male CD-1 and BALB/c mice were sourced from either the Animal House at the Faculty of Pharmacy, Jagiellonian University Medical College, Krak\u0026oacute;w, or the Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw. Male BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice were provided initially as a breeding pair by Alessandro Ieraci and subsequently bred by Janvier Labs, France [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mice were housed under standard conditions (20\u0026ndash;24\u0026deg;C, 45\u0026ndash;65% humidity, 12:12 h light\u0026ndash;dark cycle) with food and water available ad libitum, except during occasional eight-hour deprivation as part of the unpredictable chronic mild stress (UCMS) protocol. Animals were randomly assigned to groups using a computer-generated sequence, with all behavioral scoring and data analysis performed by researchers blinded to treatment. All experiments followed European (86/609/EEC; 2010/63/EU; 2012/707/EU) and Polish national regulations. The Local Ethics Committee for Experiments on Animals in Krak\u0026oacute;w approved the research protocols under the following reference numbers: 104/2016, 485/2021, 591/2022, 604/2022.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDrugs\u003c/h3\u003e\n\u003cp\u003eHBK-15 (1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride) was resynthesized at the Jagiellonian University Medical College following previously described methods[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. HBK-15, ketamine (Sigma, Germany), and WAY100135 (Sigma, Germany) were dissolved in saline and administered intraperitoneally (\u003cem\u003eip\u003c/em\u003e), with control groups receiving saline. U0126 or H-89 (STI, Poland) was prepared in 50% dimethyl sulfoxide (DMSO) and saline. Doses for all compounds were based on prior studies or literature [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSerotonin and NAN-190 (Sigma, Germany) were dissolved in DMSO (10 mM).\u003c/p\u003e\u003cp\u003eThe 5-CT (Tocris) and serodolin (synthesized as previously described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]) were stored in DMSO (Sigma-Aldrich, Saint Louis, MO, USA) at -20\u0026deg;C and diluted in PBS before use. Coelenterazine H was purchased from INTERCHIM (Montlu\u0026ccedil;on, France).\u003c/p\u003e\n\u003ch3\u003eExperimental procedures\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ebinding assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBinding studies were performed commercially in Eurofins Laboratories using testing procedures described elsewhere: 5-HT\u003csub\u003e1B\u003c/sub\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], 5-HT\u003csub\u003e1D\u003c/sub\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], 5-HT\u003csub\u003e2B\u003c/sub\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], 5-HT\u003csub\u003e2C\u003c/sub\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], 5-HT\u003csub\u003e4B\u003c/sub\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], 5-HT\u003csub\u003e4E\u003c/sub\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and 5-HT\u003csub\u003e5A\u003c/sub\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] receptors. The results are presented as the inhibition of control-specific binding in the presence of HBK-15. For inhibition values above 50%, the negative logarithm of the inhibition constant (pKi) was calculated.\u003c/p\u003e\n\u003ch3\u003eFunctional assays\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003e5-HT\u003c/b\u003e\u003csub\u003e\u003cb\u003e1A\u003c/b\u003e\u003c/sub\u003e \u003cb\u003ereceptor\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eERK phosphorylation\u003c/strong\u003e\u003cp\u003eCHO-5HT1A cells were tested for agonist-induced ERK phosphorylation using the SureFire ERK-Phosphorylation Alpha LISA kit (Perkin Elmer; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Thawed cells were cultured in medium (Advanced DMEM/F12 with 1% FBS dialyzed, 400\u0026micro;g/ml G-418, 4 mM L-Glutamine) and plated at 5x10\u003csup\u003e4\u003c/sup\u003e cells/well of 96-well plates, then incubated for 7 h (5% CO\u003csub\u003e2\u003c/sub\u003e, 37\u0026deg;C) and starved in DMEM/F12 medium with 0.1% BSA for 12 h. Serial dilutions of compounds were added and incubated for 15 min at 37\u0026deg;C. Following medium removal, 70 \u0026micro;l of \"lysis buffer\" was added, and the plate was shaken for 10 min before freezing at -80\u0026deg;C. The following day, thawed lysates (10 \u0026micro;l) were transferred to 384-well assay plates, and 10 \u0026micro;l of AlphaLISA reaction mix was added. After 2 h of incubation at 22\u0026deg;C, the plates were read using an EnVision plate reader (Perkin Elmer). E\u003csub\u003emax\u003c/sub\u003e values represent the ligand\u0026rsquo;s response expressed as a percentage of the maximal response to serotonin in agonist mode.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eβ-Arrestin Recruitment\u003c/em\u003e: The functional assay used CHO-K1 cells expressing 5-HT\u003csub\u003e1A\u003c/sub\u003e serotonin receptor, performed with the PathHunter Assay (DiscoverX; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Thawed cells (5x10\u003csup\u003e3\u003c/sup\u003ecells/well) were seeded in 96-well plates and incubated for 24 h at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. In antagonist mode, cells were preincubated with control antagonist and test compounds for 30 min, followed by the addition of serotonin (EC\u003csub\u003e80\u003c/sub\u003e concentration). In agonist mode, cells were stimulated with serotonin, tested compounds, and incubated for 90 min at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Detection reagent was then added and incubated for 1 h at room temperature. The chemiluminescent signal was measured using a PolarStar Omega reader (BMG Labtech, Germany). I\u003csub\u003emax\u003c/sub\u003e values represent the ligand\u0026rsquo;s response as a percentage of the buffer (baseline) response in antagonist mode.\u003c/p\u003e\u003cp\u003e\u003cb\u003e5-HT\u003c/b\u003e\u003csub\u003e\u003cb\u003e2B\u003c/b\u003e\u003c/sub\u003e \u003cb\u003ereceptor\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eIP1 cellular functional assays\u003c/strong\u003e\u003cp\u003eThe functional assay was performed commercially in Eurofins Laboratories using CHO-K1 cells stimulated for 30 min at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. In antagonist mode, serotonin (30 nM, EC\u003csub\u003e80\u003c/sub\u003e) was added. After incubation, the HTRF signal was measured. E\u003csub\u003emax\u003c/sub\u003e or I\u003csub\u003emax\u003c/sub\u003e values were expressed as the ligand\u0026rsquo;s response percentage relative to serotonin's maximal response in agonist mode or SB 206553 in antagonist mode.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e5-HT\u003c/b\u003e\u003csub\u003e\u003cb\u003e7\u003c/b\u003e\u003c/sub\u003e \u003cb\u003ereceptor\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eG\u0026#120572;s and β-Arrestin-2 recruitment assays\u003c/strong\u003e\u003cp\u003eHEK-293 cells were cultured in DMEM (Dulbecco Modified Eagle Medium) with 10% (vol/vol) FBS, 1 g/L glucose, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1 mM glutamine. Transient transfections were performed in 96-well plate using Lipofectamine 2000 (ThermoFisher, France) following the manufacturer\u0026rsquo;s protocol. For G\u0026#120572;s recruitment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), cells were co-transfected with 5-HT\u003csub\u003e7a\u003c/sub\u003e receptor fused to Rluc (HA-5-HT7 Rluc) and acceptor NES-Venus-mGs (provided by Pr. N.A. Lambert, Auguysta University Augusta, USA)[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For β-arrestin-2 recruitment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), HEK293 cells were transiently co-transfected with 5-HT\u003csub\u003e7\u003c/sub\u003eR and Rluc-β-arrestin-2 YPET plasmids (provided by Dr. M.G. Scott, Cochin Institute, Paris, France).\u003c/p\u003e\u003c/p\u003e\u003cp\u003eBRET measurements were taken 48 hours post-transfection after adding ligands at varying concentrations and 5 \u0026micro;M coelenterazine H. Signals were recorded for 25 min in a Mithras LB 940 Multireader (Berthold, Bad Widbad, Germany), with sequential integration of luminescence at 485\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nm (RLuc filter) and 530\u0026thinsp;\u0026plusmn;\u0026thinsp;12 nm (YFP filter). Emission ratios (530 nm/485 nm) were calculated as induced-BRET changes, representing the difference between control (no ligands) and ligand-treated conditions. Results were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from representative experiments, each replicated three times. Concentration-response and saturation curves were fitted using nonlinear regression and a hyperbolic one-binding site model, respectively.\u003c/p\u003e\n\u003ch3\u003eBehavioral assays\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eUnpredictable chronic mild stress\u003c/h2\u003e\u003cp\u003eThe unpredictable chronic mild stress model (UCMS) commenced with a one-week acclimatization for mice, after which they were grouped for testing [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. While control mice were housed under standard conditions, except during housekeeping procedures and the sucrose preference test (social isolation), mice undergoing UCMS procedure experienced various unpredictable stimuli, including cold water swimming (13\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 5 min), warm water swimming (37\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 1 min), moist bedding (8 h), cage tilt (45\u0026deg; for 8 h), cage shaking (180 rpm, 10 min), tail pinch (1 cm from the tip of the tail for 1 min), food deprivation (6 or 12 h), water deprivation (12 h), overnight illumination (12 h), no bedding (8 h), exposure to rat odor (1 h), reversal of the day/night cycle, tube immobilization (5 min), or a no-stress condition (24 h) over a 4-week (CD-1 mice) or 6-week period (BALB/c, BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice). The protocols were adapted based on strain differences and followed the recommendations from the Local Ethics Committee.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eForced swim test\u003c/h3\u003e\n\u003cp\u003eMice were placed individually in glass cylinders filled with water (10 cm depth, 23\u0026ndash;25\u0026deg;C) for 6 min [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Following a 2-minute habituation, immobility (passive floating with minimal head movements) was recorded for 4 min. A blinded observer analyzed videos using Eleven Maze software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://elevenmaze.com\u003c/span\u003e\u003cspan address=\"https://elevenmaze.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSucrose preference test\u003c/h3\u003e\n\u003cp\u003eThe sucrose preference test, adapted from Liu et al [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] involved providing mice with two bottles: one containing water and the other a 1% (w/v) sucrose solution during the unpredictable chronic mild stress procedure. Bottles with water and 1% sucrose were weighed before and after a 12-hour dark-phase exposure. Sucrose preference was calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{s}\\varvec{u}\\varvec{c}\\varvec{r}\\varvec{o}\\varvec{s}\\varvec{e}\\:\\varvec{p}\\varvec{r}\\varvec{e}\\varvec{f}\\varvec{e}\\varvec{r}\\varvec{e}\\varvec{n}\\varvec{c}\\varvec{e}=\\:\\frac{\\varvec{s}\\varvec{u}\\varvec{c}\\varvec{r}\\varvec{o}\\varvec{s}\\varvec{e}\\:\\varvec{s}\\varvec{o}\\varvec{l}\\varvec{u}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}\\:\\varvec{i}\\varvec{n}\\varvec{t}\\varvec{a}\\varvec{k}\\varvec{e}\\:\\left[\\varvec{g}\\right]}{\\varvec{s}\\varvec{u}\\varvec{c}\\varvec{r}\\varvec{o}\\varvec{s}\\varvec{e}\\:\\varvec{s}\\varvec{o}\\varvec{l}\\varvec{u}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}\\:\\varvec{i}\\varvec{n}\\varvec{t}\\varvec{a}\\varvec{k}\\varvec{e}\\:\\left[\\varvec{g}\\right]+\\varvec{w}\\varvec{a}\\varvec{t}\\varvec{e}\\varvec{r}\\:\\varvec{i}\\varvec{n}\\varvec{t}\\varvec{a}\\varvec{k}\\varvec{e}\\:\\left[\\varvec{g}\\right]}\\varvec{*}100\\varvec{\\%}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eObject recognition test\u003c/h2\u003e\u003cp\u003eThe object recognition test was adapted from Leger et al [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the familiarization session, mice were placed in an open field (35\u0026times;35\u0026times;35 cm) containing two identical objects and were allowed to explore until they reached a criterion of 20 s of total exploration, but no longer than 10 min. In the test phase, one object was replaced with a novel one; the positions of the objects were randomized. The mice were again allowed to explore until they reached the 20-second criterion of total exploration, but no longer than 10 min. The objects and the arena were cleaned between trials. The experiments were video recorded and scored using Eleven Maze software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://elevenmaze.com\u003c/span\u003e\u003cspan address=\"https://elevenmaze.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by a trained observer who was blind to the treatments. To assess the animals' performance in the object recognition test, the average novel object exploration times were compared to the chance level (10 s, equal exploration of the objects) according to the followed protocol [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eLocomotor activity\u003c/h2\u003e\u003cp\u003eThe locomotor activity test, as described previously [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], monitored the individual activity of mice using Opto M3 activity cages (Columbus Instruments, USA). After a 30-minute habituation period, beam crossings were recorded every minute for 6 min. The cages were disinfected with an odorless veterinary disinfectant after each mouse.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eInhibition of ERK1/2 or PKA signaling pathways\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eSurgery\u003c/strong\u003e\u003cp\u003eAnimals were anesthetized with isoflurane (5% induction, 2.5% maintenance) and placed in a stereotaxic frame (ASI Instruments, Warren, MI, USA). Analgesia was achieved using subcutaneous lidocaine at the incision site, along with dorsal administration of carprofen (10 mg/kg). Bilateral guide cannulas (RWD-62052, USA) were implanted in the prelimbic cortex (AP\u0026thinsp;+\u0026thinsp;1.93 mm; ML\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mm; DV \u0026ndash; 2.2 mm from the bregma; Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea), according to the Franklin and Paxinos stereotaxis atlas[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Cannulas were secured with anchor screws, dental cement, dummy cannulas (protruding 0.5 mm, RWD-62123, USA) and caps (RWD-62523, USA). After surgery, mice were housed in a high-roofed cage with \u003cem\u003ead libitum\u003c/em\u003e access to water and food and monitored for recovery.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMicroinjection\u003c/strong\u003e\u003cp\u003eFollowing a one-week recovery, injection cannulas (RWD-62223, USA) were inserted into guide cannulas and connected via a polyethylene tubing to a microsyringe (NanoFil 10 \u0026micro;L, WPI, USA) driven by a microinfusion pump (UMP3 UltraMicroPump III with MICRO2T 2-Channel Controller, WPI, USA). U0126 (0.5 \u0026micro;g/0.5 \u0026micro;L per site), H-89 (2.6 ng/0.5 ul), or vehicle (50% DMSO in saline) was delivered at 250 nL/min over 2 min, with cannulas left in place for an additional 2 min to prevent backflow. Probe placement was confirmed post-mortem in coronal sections (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003estudies\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eELISA\u003c/h2\u003e\u003cp\u003eFollowing behavioral assessments, mice were euthanized, and their prefrontal cortices were dissected, frozen, and stored at -80\u0026deg;C. On the experimental day, tissues were thawed on ice, homogenized (1:10 w/v) in chilled PBS with protease and phosphatase inhibitors, and centrifuged at 2000 rpm for 20 min at 4\u0026deg;C, after which supernatants were collected for analysis.\u003c/p\u003e\u003cp\u003eProtein concentrations of BDNF (201-02-0014), CREB (201-02-1579), pCREB (201-02-1586), ERK1/2 (201-02-3766), pERK1/2 (201-02-1572), PKA (201-02-1549), pPKA (201-02-1802), CaMKIV (201-02-1577), pCaMKIV (201-02-1801), RSK2 (201-02-1615), pRSK2 (-02-1597), CaMKII (201-02-1614), pCaMKII (201-02-1674), PKC (201-02-1613), and pPKC (201-02-0592) were determined using SunRed ELISA kits, following the manufacturer\u0026rsquo;s instructions. Tissue homogenates were normalized to wet weight prior to analysis. Samples were analyzed in duplicates, and mean concentrations calculated. Color intensity was measured at 450 nm using a plate reader (POLARstar Omega, BMG Labtech, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003eTissue fixation\u003c/h2\u003e\u003cp\u003eFollowing behavioral testing, mice designated for immunofluorescence staining were deeply anesthetized with thiopental (75 mg/kg) and transcardially perfused with ~\u0026thinsp;30 mL of ice-cold PBS, followed by 15 mL of 4% paraformaldehyde (PFA) in 0.1 M PBS. Extracted brains were post-fixed in 4% PFA overnight, then cryoprotected in graded sucrose solutions (10%, 20%, 30%) until fully submerged. Coronal sections (30 \u0026micro;m) were prepared using a Leica CM 1860 cryotome, mounted on SuperFrost slides (ThermoFisher, USA), and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until staining.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescent staining\u003c/h2\u003e\u003cp\u003eTo visualize signaling proteins, slides with brain slices were immersed in 70% ethanol, air-dried, and washed twice for 10 min in PBS and once with 0.3% Triton X-100/PBS. Nonspecific binding was blocked with 10% goat serum in 0.3% Triton X-100/PBS for 1 h. Sections were incubated overnight at 4\u0026deg;C with anti-pERK1/2 (1:300, #4370, Cell Signaling) and anti-pPKA (1:150, STJ90386, St John\u0026rsquo;s) in 2% goat serum/0.03% Triton X-100/PBS. After washing (twice for 10 minutes in 0.3% Triton X-100/0.01 M PBS, then twice for 10 minutes in 2% goat serum with 0.3% Triton X-100/0.01 M PBS), slides were incubated with Alexa Fluor 488 secondary antibody (#A-21121, Thermo Fisher) for an hour in the absence of light. Afterward, the slides underwent five 10-minute washes in 0.01 M PBS. Finally, the slides were mounted with Vectashield Vibrance (with DAPI, Vectorlabs) and stored at 4\u0026deg;C. Images of the prelimbic cortex were captured using a Leica Stellaris 8 WLL, DLS confocal microscope (Leica, Germany), using the same microscope settings (i.a. laser power and frequency, detector power, pinhole), and processed with ImageJ2 version 2.14.0/1.54f [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e To visualize regional distribution patterns, immunohistochemical staining for pERK1/2 and pPKA was performed on a subset of animals (separate from those used for ELISA). No quantitative analysis was performed on IHC images.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative PCR analysis\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from the medial prefrontal cortex using the Total RNA Mini kit (A\u0026amp;A Biotechnology, Poland). RNA concentration and purity were assessed using a NanoQuant Plate (Tecan, Switzerland), based on absorbance at 260 and 280 nm. Only samples with A260/280\u0026thinsp;\u0026ge;\u0026thinsp;1.8 were included, and RNA concentrations were equalized prior to cDNA synthesis using the TranScriba kit with oligo(dT)18 as the primer (A\u0026amp;A Biotechnology). Gene expression was analyzed using TaqMan probes (ThermoFisher, USA): Mm04230607_s1 (\u003cem\u003eBdnf\u003c/em\u003e), Mm00442479_m1 (\u003cem\u003eMapk1\u003c/em\u003e), Mm01278702_gH (\u003cem\u003eMapk3\u003c/em\u003e), Mm00455829_m1 (\u003cem\u003eRps6ka3\u003c/em\u003e), Mm00437967_m1 (\u003cem\u003eCamk2a\u003c/em\u003e), Mm01135329_m1 (\u003cem\u003eCamk4\u003c/em\u003e), Mm01349190_m1 (\u003cem\u003eCrtc1\u003c/em\u003e), Mm00508404_m1 (\u003cem\u003eRiiad1\u003c/em\u003e) and normalized to the housekeeping reference gene: Mm99999915_g1 (\u003cem\u003eGapdh\u003c/em\u003e). A group of control animals was used as a reference. The level of housekeeping gene (GAPDH) was roughly equal in the samples and did not change significantly between experimental groups. RNA abundance was calculated by the standard ΔΔCt method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eResults are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or SEM for parametric data and medians with IQR for non-parametric data. Normality and homogeneity of variances were assessed using the Shapiro-Wilk and Brown-Forsythe tests. Group comparisons employed one-way ANOVA with Bonferroni \u003cem\u003epost hoc\u003c/em\u003e for parametric data or Kruskal-Wallis with Dunn's \u003cem\u003epost hoc\u003c/em\u003e for non-parametric data. Novel object exploration was analyzed with a one-sample t-test. Significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Analyses, including EC\u003csub\u003e50\u003c/sub\u003e, IC\u003csub\u003e50\u003c/sub\u003e, and Ki (via the Cheng-Prusoff formula), were conducted using GraphPad 9.5.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eHBK-15 displays pathway-selective signaling at serotonin 5-HT\u003csub\u003e1A\u003c/sub\u003e and 5-HT\u003csub\u003e7\u003c/sub\u003e receptors\u003c/h2\u003e\u003cp\u003eTo build on the existing binding profile of HBK-15, we extended its serotonergic receptor characterization beyond previously studied targets. Earlier studies demonstrated that HBK-15 exhibits high affinity for 5-HT\u003csub\u003e1A\u003c/sub\u003e (pKi\u0026thinsp;=\u0026thinsp;9.02), moderate affinity for 5-HT\u003csub\u003e2A\u003c/sub\u003e (pKi\u0026thinsp;=\u0026thinsp;7.21) and 5-HT\u003csub\u003e7\u003c/sub\u003e (pKi\u0026thinsp;=\u0026thinsp;7.33) receptors (with pKi values reflecting the negative logarithm of Ki, where higher pKi indicates stronger binding), and negligible binding to 5-HT\u003csub\u003e3\u003c/sub\u003e and\u003c/p\u003e\u003cp\u003e5-HT\u003csub\u003e6\u003c/sub\u003e receptors [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the current study, we focused on the 5-HT\u003csub\u003e1B\u003c/sub\u003e, 5-HT\u003csub\u003e1D\u003c/sub\u003e, 5-HT\u003csub\u003e2B\u003c/sub\u003e, 5-HT\u003csub\u003e2C\u003c/sub\u003e, 5-HT\u003csub\u003e4B\u003c/sub\u003e, 5-HT\u003csub\u003e4E\u003c/sub\u003e, and 5-HT\u003csub\u003e5A\u003c/sub\u003e receptors to complete the pharmacological characterization across the major serotonin receptor subtypes. HBK-15 displayed high affinity for 5-HT\u003csub\u003e2B\u003c/sub\u003e receptors (pKi\u0026thinsp;=\u0026thinsp;7.58), while showing low or negligible binding to other receptors (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e binding assays for HBK-15.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMolecular target\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e% inhibition of control-specific binding\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-HT\u003csub\u003e1B\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehuman recombinant Chem-1 (RBL) cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-HT\u003csub\u003e1D\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003erat recombinant CHO cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-HT\u003csub\u003e2B\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehuman recombinant CHO cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e94.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-HT\u003csub\u003e2C\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehuman recombinant HEK-293 cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-HT\u003csub\u003e4B\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehuman recombinant Chem-1 cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-7.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-HT\u003csub\u003e4E\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehuman recombinant CHO cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-HT\u003csub\u003e5A\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehuman recombinant HEK-293 cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eHBK-15 was tested at concentrations 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e M. The results are presented as % inhibition of control-specific binding in the presence of HBK-15. Results showing an activity\u0026thinsp;\u0026gt;\u0026thinsp;60% were considered to represent significant effects of the test compound; results showing an inhibition between 25% and 60% indicate moderate to weak effect; results showing an inhibition\u0026thinsp;\u0026lt;\u0026thinsp;25% are not considered significant and mostly attributable to the variability of the signal around the control level. Binding studies were performed commercially in Eurofins Laboratories (Poitiers, France).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAt the 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor, HBK-15 acted as a functionally selective ligand. While previous studies showed that HBK-15 antagonized serotonin-induced inhibition of cAMP synthesis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and blocked calcium mobilization [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] (pIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.47 and 7.72, respectively) with moderate potency and partial efficacy, here we extended its signaling profile to additional pathways. HBK-15 partially activated ERK1/2 phosphorylation (1.5-fold lower potency and 4-fold lower efficacy than serotonin, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and strongly inhibited β-arrestin recruitment (2-fold lower potency than NAN-190 with comparable maximal inhibition; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). These findings indicate preferential engagement of selected downstream pathways while blocking others, consistent with functional selectivity.\u003c/p\u003e\u003cp\u003eGiven its high affinity for 5-HT\u003csub\u003e2B\u003c/sub\u003e receptors - a target associated with cardiotoxicity due to agonist-induced valvulopathy - we specifically assessed whether HBK-15 exhibits any agonist activity at this receptor. In Gq-coupled assays, HBK-15 acted as an antagonist, inhibiting serotonin-induced calcium mobilization and inositol phosphate accumulation (pIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.74), with approximately 10-fold lower potency than the reference antagonist SB206553 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As 5-HT\u003csub\u003e2B\u003c/sub\u003e receptors play a limited role in central serotonergic neurotransmission, further pathway deconvolution was not pursued. These results suggest a favorable safety profile despite strong receptor binding affinity.\u003c/p\u003e\u003cp\u003eAt the 5-HT\u003csub\u003e7\u003c/sub\u003e receptor, we employed real-time BRET biosensors to characterize signaling bias. HBK-15 acted as a partial agonist in Gαs recruitment (pEC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5, reaching 34% of the maximal effect relative to the full agonist; pEC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), with no detectable β-arrestin engagement (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). This contrasts with 5-CT and serodolin, a β-arrestin\u0026ndash;biased ligand that acted as a Gs inverse agonist (pEC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3) and strongly recruited β-arrestin (pEC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6), supporting a Gαs-biased signaling profile for HBK-15 at this receptor. This finding diverges our previous results using a cAMP-based CRE-bla reporter assay in CHO-K1 cells, where HBK-15 behaved as an antagonist at the 5-HT\u003csub\u003e7\u003c/sub\u003e receptor [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This discrepancy likely reflects methodological differences between population-averaged cAMP accumulation assays and real-time, proximal G protein recruitment as measured by BRET. The partial Gαs engagement observed in the BRET assay may not have been sufficient to generate a detectable increase in cAMP in the previous system.\u003c/p\u003e\u003cp\u003eThese signaling characteristics provided the rationale for subsequent behavioral and molecular studies in animal models of depression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eERK1/2 and PKA activation by HBK-15 correlates with rapid antidepressant-like effects\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe functional selectivity of HBK-15 toward 5-HT\u003csub\u003e1A\u003c/sub\u003e and 5-HT\u003csub\u003e7\u003c/sub\u003e receptor signaling pathways prompted us to investigate whether its preferential engagement of ERK1/2 phosphorylation \u003cem\u003ein vitro\u003c/em\u003e translates into rapid antidepressant-like efficacy \u003cem\u003ein vivo\u003c/em\u003e. To test this, we administered a single dose of HBK-15 (2.5 mg/kg) to CD-1 mice subjected to unpredictable chronic mild stress and compared its effects to ketamine (10 mg/kg), a fast-acting antidepressant, and fluoxetine (10 mg/kg), a conventional SSRI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the sucrose preference test, UCMS reduced sucrose intake by 42.9% relative to non-stressed controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Both HBK-15 and ketamine reversed this effect (+\u0026thinsp;61.4% and +\u0026thinsp;52.0%, respectively), while fluoxetine was ineffective (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Similarly, in the forced swim test, UCMS increased immobility time by 21.2%, and both HBK-15 and ketamine significantly reduced this measure of behavioral despair (\u0026minus;\u0026thinsp;36.3% and \u0026minus;\u0026thinsp;31.9%, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). None of the compounds altered locomotor activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), excluding nonspecific behavioral stimulation.\u003c/p\u003e\u003cp\u003eWe next examined molecular correlates of these behavioral effects, focusing on intracellular kinases implicated in rapid serotonergic signaling. Previous studies on 5-HT\u003csub\u003e1A\u003c/sub\u003e-biased agonists such as NLX-101 and NLX-204 have shown that their fast-onset behavioral effects coincide with ERK1/2 activation, specifically in the prefrontal cortex [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, we selected this region to define the kinase-level footprint of HBK-15. HBK-15 significantly increased phosphorylation of ERK1/2 (+\u0026thinsp;28.6%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), PKA (+\u0026thinsp;34.4%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei), and CaMKIV (+\u0026thinsp;16.4%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el) in the prefrontal cortex, each of which was reduced by UCMS (\u0026minus;\u0026thinsp;21.9%, \u0026minus;\u0026thinsp;20.5%, and \u0026minus;\u0026thinsp;13.6%, respectively). We detected the expression of phosphorylated ERK1/2 and PKA in MAP2-positive neurons of the prefrontal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg,j, S2), consistent with HBK-15\u0026rsquo;s receptor-specific signaling profile observed \u003cem\u003ein vitro\u003c/em\u003e. These results suggest that preferential activation of kinase pathways, particularly ERK1/2 and PKA, may contribute to the compound\u0026rsquo;s rapid antidepressant-like effects.\u003c/p\u003e\u003cp\u003eWhile BDNF signaling is widely linked to antidepressant response, HBK-15 appears to act downstream, enhancing BDNF protein levels via intracellular kinase activation rather than by transcriptional upregulation of \u003cem\u003eBdnf\u003c/em\u003e. HBK-15 restored UCMS-induced reductions in BDNF protein levels (+\u0026thinsp;167.3%), similar to ketamine (+\u0026thinsp;186.9%), but without accompanying changes in \u003cem\u003eBdnf\u003c/em\u003e mRNA expression at the 24-hour timepoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo, S3f). Although transient transcriptional effects at earlier timepoints cannot be excluded, the data suggest that HBK-15 engages post-transcriptional or signaling-driven mechanisms downstream of kinase activation. Supporting this interpretation, HBK-15 also reversed stress-induced decreases in pCREB (+\u0026thinsp;26.0%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en), a transcription factor downstream of ERK1/2 and PKA, without altering total CREB expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em).\u003c/p\u003e\u003cp\u003eTo determine whether HBK-15-induced changes extended beyond phosphorylation, we measured total protein levels of key signaling components. UCMS markedly reduced the expression of CREB (\u0026minus;\u0026thinsp;42%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em), ERK1/2 (\u0026minus;\u0026thinsp;38.1%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), and PKA (\u0026minus;\u0026thinsp;27.2%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh), while CaMKIV levels remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). HBK-15 did not normalize protein abundance, indicating that its molecular actions are driven by modulation of signaling activity rather than restoration of protein levels. These effects were not associated with transcriptional changes, as mRNA levels of these targets were unaltered at the same timepoint (Fig.S3a-f). Together, these findings support a model in which HBK-15 promotes rapid behavioral recovery through direct engagement of intracellular kinase signaling, with ERK1/2 and PKA as the most consistently regulated molecular targets.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eHBK-15 extends its rapid effects to cognitive function and recruits complementary neuroplasticity-related pathways\u003c/h2\u003e\u003cp\u003eTo ensure the behavioral and molecular effects of HBK-15 were not strain-dependent, we replicated the UCMS protocol in BALB/c mice, a stress-sensitive inbred strain, and expanded the dose range (1.25, 2.5, and 5 mg/kg; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). UCMS reduced sucrose preference by 24.9% compared to non-stressed controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). HBK-15 reversed this effect at all doses tested (+\u0026thinsp;13.8%, +\u0026thinsp;35.9%, and +\u0026thinsp;30.7%, respectively), similarly to ketamine (+\u0026thinsp;23.8%), confirming its robust antianhedonic efficacy in this background (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBecause depression is frequently accompanied by cognitive impairment and current antidepressants rarely address this dimension (reviewed in[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]), we evaluated whether HBK-15 could restore recognition memory in the object recognition test. Mice subjected to UCMS failed to explore the novel object above chance level, whereas HBK-15 significantly improved novel object exploration at all doses tested, mirroring the effects of ketamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These results indicate that HBK-15 produces rapid antidepressant-like and procognitive effects following a single administration, extending its efficacy profile beyond affective symptoms.\u003c/p\u003e\u003cp\u003eTo elucidate the molecular basis of these effects, we examined other kinases implicated in plasticity-related signaling. We focused particularly on RSK2, a well-characterized downstream target of ERK1/2, and evaluated PKC and CaMKII, which operate through independent calcium- and lipid-sensitive pathways. UCMS markedly reduced pRSK2 levels in the prefrontal cortex (\u0026minus;\u0026thinsp;40.7%), which were restored by both HBK-15 (+\u0026thinsp;46.2%) and ketamine (+\u0026thinsp;49.9%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Phosphorylation of CaMKII (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) and PKC (Fig.S4c) was not significantly altered by UCMS; however, HBK-15 increased pPKC at the highest dose tested (Fig.S4c). The regulation of RSK2, together with previously observed activation of ERK1/2 and PKA (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef,i) by HBK-15, suggests the engagement of a convergent kinase-signaling mechanism potentially underlying its behavioral efficacy.\u003c/p\u003e\u003cp\u003eWe next assessed whether these effects extended beyond phosphorylation. UCMS decreased total levels of RSK2 (\u0026minus;\u0026thinsp;63%; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) and CaMKII (\u0026minus;\u0026thinsp;48.2%; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), while PKC remained unaffected (Fig.S4d). HBK-15 restored RSK2 level (+\u0026thinsp;106.3% at 5 mg/kg) and normalized CaMKII levels (+\u0026thinsp;122.2% at 2.5 mg/kg), suggesting engagement of post-transcriptional or translational regulatory mechanisms. No significant changes in mRNA levels were detected for these targets (Fig.S4a-b), indicating that HBK-15 modulates kinase abundance at the protein level. Any upstream transcriptional contribution occurring before this timepoint cannot be ruled out, yet the observed changes are more consistent with translational or post-translational control.\u003c/p\u003e\u003cp\u003eFinally, we assessed the temporal dynamics of these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). In the sucrose preference test, both HBK-15 and ketamine reversed UCMS-induced anhedonia at 24 h, but not at 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). A similar time course was observed in the object recognition test: behavioral improvement was evident at 24 h but absent at 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). These findings indicate that the behavioral effects of HBK-15 are rapid but transient, resembling the short-term profile of ketamine observed under the same experimental conditions. Although longer-lasting antidepressant effects of ketamine have been reported in other studies [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], such persistence appears to depend on experimental variables including species, strain, sex, dose, and stress paradigm [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The comparable temporal profile observed here suggests that HBK-15 may share core fast-acting mechanisms with ketamine, while its longer-term efficacy could emerge under different dosing regimens or behavioral contexts not explored in the present design.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eHBK-15 requires 5-HT\u003csub\u003e1A\u003c/sub\u003e-ERK1/2 signaling to exert rapid antidepressant-like and procognitive effects\u003c/h2\u003e\u003cp\u003eFunctional signaling assays demonstrated that HBK-15 partially activated ERK1/2 phosphorylation downstream of 5-HT\u003csub\u003e1A\u003c/sub\u003e receptors and recruited Gαs at 5-HT\u003csub\u003e7\u003c/sub\u003e receptors, as detected by biosensor-based approaches. Consistent with these pathway-specific effects, HBK-15 increased phosphorylation of ERK1/2 (+\u0026thinsp;28.6%) and PKA (+\u0026thinsp;34.4%) in the prefrontal cortex of UCMS-exposed mice, supporting the selection of these kinases as candidate intracellular mediators of its behavioral efficacy.\u003c/p\u003e\u003cp\u003eGiven that HBK-15 displays its highest binding affinity for the 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor among its known targets (pKi\u0026thinsp;=\u0026thinsp;9.02), and exhibits functional selectivity at this site, we next tested whether its behavioral effects are mediated by 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor activation. In signaling assays, HBK-15 acted as a biased ligand at the 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor: it partially activated ERK1/2 phosphorylation while antagonizing other receptor-coupled pathways, including cAMP production inhibition, β-arrestin recruitment, and calcium mobilization. To pharmacologically verify receptor involvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), we used WAY-100635, a selective 5-HT\u003csub\u003e1A\u003c/sub\u003e antagonist known to block all major downstream signaling pathways [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. \u003cem\u003eIn vivo\u003c/em\u003e, pretreatment with WAY-100635 abolished the antidepressant-like effect of HBK-15 in the sucrose preference test, as sucrose intake dropped by 24.8% relative to HBK-15 alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In the object recognition test, mice pretreated with WAY-100635 failed to discriminate the novel object (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), indicating that 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor activation is also necessary for the procognitive effects of HBK-15.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further delineate the intracellular pathways mediating HBK-15\u0026rsquo;s effects, we selectively inhibited either the ERK1/2 or PKA signaling in the prefrontal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed,g). Intra-prefrontal infusion of the MAPK/ERK kinase inhibitor U0126 completely abolished both the antidepressant-like and procognitive effects of HBK-15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u0026ndash;f), supporting a central role for ERK1/2 signaling in mediating its behavioral efficacy. In contrast, inhibition of PKA signaling with H-89 had no effect on either behavioral endpoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh-i). These data indicate that ERK1/2 signaling is essential for both the affective and cognitive effects of HBK-15.\u003c/p\u003e\u003cp\u003eThese findings are consistent with the molecular signature of HBK-15, characterized by robust activation of ERK1/2 in the prefrontal cortex under chronic stress conditions. This \u003cem\u003ein vivo\u003c/em\u003e effect reflects its \u003cem\u003ein vitro\u003c/em\u003e signaling profile, where HBK-15 selectively promoted ERK1/2 phosphorylation downstream of 5-HT\u003csub\u003e1A\u003c/sub\u003e receptors while blocking alternative pathways at the same site. Given the established role of ERK1/2 in regulating synaptic plasticity and fast behavioral adaptation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], this kinase emerges as a central effector of HBK-15\u0026rsquo;s rapid action.\u003c/p\u003e\u003cp\u003eIn addition, HBK-15 increased phosphorylation of PKA in the prefrontal cortex, a kinase also implicated in stress resilience. This effect likely results from its dual receptor actions: partial Gαs recruitment at 5-HT\u003csub\u003e7\u003c/sub\u003e receptors, which enhances cAMP synthesis, and blockade of serotonin-mediated inhibition of cAMP production at 5-HT\u003csub\u003e1A\u003c/sub\u003e receptors [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], preventing activation of canonical Gi/o signaling. While PKA may contribute to the compound\u0026rsquo;s effects, the convergence of these receptor-specific mechanisms on ERK1/2, together with the lack of behavioral response to PKA inhibition, highlights ERK1/2 as the principal intracellular mediator of HBK-15\u0026rsquo;s behavioral efficacy.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eHBK-15 retains affective, but not cognitive efficacy in BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice\u003c/h2\u003e\u003cp\u003eGiven that HBK-15 increased pERK1/2, pPKA, and pCREB in the prefrontal cortex and restored BDNF protein levels after a single administration, we next examined whether BDNF signaling is required for its behavioral effects, focusing on the activity-dependent BDNF secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). To address this, we used BDNF Val66Met knock-in mice, which exhibit impaired stimulus-induced BDNF release but retain normal baseline expression levels [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], thereby avoiding developmental confounds of total BDNF knockout and modeling a clinically relevant human polymorphism[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the UCMS model, sucrose preference was reduced by 20.4% relative to non-stressed controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). HBK-15 reversed this effect at all tested doses (+\u0026thinsp;19.2%, +\u0026thinsp;24.8%, and +\u0026thinsp;24.4% at 1.25, 2.5, and 5 mg/kg, respectively), whereas ketamine failed to exert an effect in this genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This lack of efficacy is consistent with previous studies showing that the antidepressant-like effects of ketamine are abolished in BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice due to impaired activity-dependent BDNF release [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In contrast, HBK-15 retained its behavioral efficacy despite this deficit, pointing to an alternative mechanism, likely mediated by selective activation of intracellular kinase pathways such as ERK1/2.\u003c/p\u003e\u003cp\u003eBy contrast, recognition memory remained impaired in the object recognition test, and neither HBK-15 nor ketamine restored cognitive performance in BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). This pattern suggests that the procognitive effect of HBK-15 depend on intact BDNF signaling, while its antidepressant-like action is mediated via BDNF-independent, kinase-driven mechanisms.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identify HBK-15 as a functionally selective serotonergic compound that engages behaviorally relevant kinase pathways to produce rapid and simultaneous antidepressant-like and procognitive effects following a single administration in a chronic stress model. Through the use of pathway-specific biosensors, pharmacological tools, and stress-based behavioral assays, we demonstrate that HBK-15 preferentially activates ERK1/2 signaling via the 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor, while partially recruiting Gαs at 5-HT\u003csub\u003e7\u003c/sub\u003e receptors. This receptor-specific signaling translates into rapid \u003cem\u003ein vivo\u003c/em\u003e behavioral efficacy, primarily driven by ERK1/2 activation and observed after a single dose, contrasting with the delayed onset typical of conventional monoaminergic antidepressants. These results directly address our initial hypothesis that HBK-15, despite its multimodal profile, acts through functionally selective engagement of intracellular effectors, particularly ERK1/2, to exert rapid behavioral effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eERK1/2 has emerged as a key intracellular effector linking serotonergic receptor activation to synaptic and behavioral plasticity, particularly in the context of rapid-acting antidepressant strategies [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Prior studies on 5-HT\u003csub\u003e1A\u003c/sub\u003e-biased agonists such as NLX-101 and NLX-204 have demonstrated a rapid onset of behavioral effects, coinciding with enhanced ERK1/2 phosphorylation in the prefrontal cortex [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consistent with this mechanism, HBK-15 selectively increased ERK1/2 phosphorylation both \u003cem\u003ein vitro\u003c/em\u003e and in the prefrontal cortex of chronically stressed mice. Importantly, pharmacological inhibition of the ERK pathway abolished the antidepressant-like and procognitive effects of HBK-15, directly implicating ERK1/2 signaling in its behavioral efficacy. These effects occurred in the absence of transcriptional induction, consistent with a fast, signal-driven mechanism previously described for compounds such as ketamine and NLX-101. Unlike ERK1/2, PKA signaling was not necessary for either the affective or cognitive effects of HBK-15, indicating that HBK-15\u0026rsquo;s behavioral efficacy is selectively mediated through ERK1/2-dependent mechanisms.\u003c/p\u003e\u003cp\u003eAlthough HBK-15 acts as a partial agonist at the ERK1/2 branch of 5-HT\u003csub\u003e1A\u003c/sub\u003e signaling, our data indicate that full activation is not required for behavioral efficacy. Selective ERK1/2 recruitment, at moderate efficacy, appears sufficient when alternative receptor-mediated responses such as β-arrestin recruitment, cAMP inhibition, and calcium mobilization are suppressed. This suggests that biased, rather than strong, ERK1/2 activation may be optimal for therapeutic effects, aligning with current strategies to achieve targeted and safer 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor modulation.\u003c/p\u003e\u003cp\u003eIn addition to rapidly activating ERK1/2, HBK-15 increased phosphorylation of plasticity-related kinases, including RSK2 and CaMKIV, elevated total levels of CaMKII, and enhanced downstream effectors such as CREB and BDNF [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This pattern likely reflects a convergent intracellular response to receptor activation, in which ERK1/2 plays a central integrative role, while calcium-sensitive kinases may contribute modulatory input. However, given the preserved efficacy of HBK-15 in BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice, a model with impaired activity-dependent BDNF release [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], these transcriptional and calcium-dependent pathways are unlikely to be essential for its behavioral effects. Instead, the antidepressant-like effects of HBK-15 appear to rely primarily on ERK1/2-driven signaling, while its procognitive actions may additionally require intact BDNF-dependent mechanisms.\u003c/p\u003e\u003cp\u003eConsistently, the preserved efficacy of HBK-15 in BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice further underscores its translational potential. The Val66Met polymorphism, present in approximately 25\u0026ndash;30% of the population [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], has been associated with reduced synaptic plasticity, greater susceptibility to depression, and attenuated responses to classical antidepressants - including ketamine [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this context, HBK-15\u0026rsquo;s ability to produce robust behavioral effects despite impaired BDNF release suggests that it may bypass traditional neurotrophic mechanisms. This property may prove particularly advantageous in genetically defined subpopulations with limited treatment options, supporting the relevance of HBK-15 as a mechanistically distinct candidate for precision psychiatry.\u003c/p\u003e\u003cp\u003eInterestingly, BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice displayed intact object recognition memory at a 4-hour delay, consistent with reports that deficits typically emerge at longer intervals (\u0026ge;\u0026thinsp;24 h [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]). However, under chronic stress, this memory was disrupted and unresponsive to HBK-15, suggesting that its procognitive actions depend on intact BDNF signaling. In this context, the Val66Met variant may impair early-phase LTP or local protein synthesis in memory circuits, limiting HBK-15\u0026rsquo;s efficacy. Thus, while HBK-15 retains antidepressant-like effects in the BDNF-impaired brain, its cognitive benefits may be restricted to individuals with preserved neurotrophic capacity.\u003c/p\u003e\u003cp\u003eDespite these promising outcomes, both the antidepressant-like and procognitive effects of HBK-15 were transient. This temporal profile mirrors that of ketamine under our experimental conditions. Although ketamine has been shown to induce behavioral improvements lasting from hours to weeks, depending on the model, dose, and context [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], in our paradigm, both compounds produced rapid but short-lived effects. This suggests that acute kinase activation may not be sufficient to sustain long-term behavioral benefits without repeated dosing or structural reinforcement. Notably, recent studies have shown that prolonging ERK1/2 signaling - e.g., through inhibition of the phosphatase DUSP6 - extends ketamine\u0026rsquo;s behavioral effects for up to several weeks [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. This supports the idea that ERK1/2 activation is not only necessary for the onset of rapid antidepressant action but may also serve as a target for enhancing durability.\u003c/p\u003e\u003cp\u003eBeyond efficacy, the signaling selectivity of HBK-15 may also inform its safety and tolerability profile. Preferential recruitment of β-arrestin at 5-HT\u003csub\u003e1A\u003c/sub\u003e receptors has been associated with the so-called \u0026ldquo;serotonin syndrome\u0026rdquo; triad in rodents - lower-lip retraction, flat-body posture, and hypothermia - serving as a surrogate marker of excessive serotonergic activation [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In contrast, ERK-biased 5-HT\u003csub\u003e1A\u003c/sub\u003e ligands, such as NLX-101, preserve antidepressant-like efficacy without eliciting these autonomic signs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In humans, serotonergic toxicity manifests differently: even partial 5-HT\u003csub\u003e1A\u003c/sub\u003e agonists like buspirone produce only transient drops in core temperature [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], and clinical signs include hyperthermia, nausea, and autonomic arousal [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Thus, while the rodent triad may offer only a heuristic indication of serotonergic overstimulation, the ERK-biased, β-arrestin-sparing signaling profile of HBK-15 is likely to enhance therapeutic precision, minimizing peripheral autonomic effects without compromising central antidepressant action.\u003c/p\u003e\u003cp\u003eWhile our findings support ERK1/2-biased signaling as a key driver of HBK-15\u0026rsquo;s rapid behavioral effects, some limitations should be acknowledged. We identified kinase activation patterns and confirmed ERK1/2 involvement, but a full mapping of upstream\u0026ndash;downstream signaling remains to be defined. Our behavioral studies focused on acute effects after a single dose; longer-term outcomes require investigation. Moreover, our experiments were conducted exclusively in male mice, and future studies should address potential sex differences in behavioral and molecular responses to HBK-15. Finally, although HBK-15 was effective in BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice, clinical validation in genetically defined populations will be crucial. These points highlight the need for further studies to assess the durability, generalizability, and translational scope of HBK-15\u0026rsquo;s therapeutic action.\u003c/p\u003e\u003cp\u003eTaken together, these findings position HBK-15 as a mechanistically distinct example of functionally selective polypharmacology. Its \u0026ldquo;fine-tuned\u0026rdquo; ERK1/2 bias, together with the bypass of BDNF dependency in affective domains, highlights the translational relevance of HBK-15\u0026rsquo;s intracellular signaling profile. By shifting the focus from receptor binding to intracellular selectivity, HBK-15 exemplifies a paradigm in which biased signaling - rather than receptor agonism alone - emerges as a primary determinant of antidepressant efficacy. This framework may guide the rational design of next-generation therapies that act rapidly and effectively in biologically constrained populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was financially supported by the National Science Centre, Poland (grant number 2019/34/E/NZ7/00454 and 2017/01/X/NZ7/00818).\u003c/p\u003e\u003ch2\u003eCRediT author statement\u003c/h2\u003e\u003cp\u003eConceptualization: KP (lead). Methodology: KS, MG, BM, SM, KP. Investigation: KS (lead), AJag, KL, MG, BM, EŻ (support), AJan (support), JD (support), AK (support), AS (support), WK (support), BP (support), JVG (support), BAFK, SM, KP. Formal Analysis: KS (lead), AJag, KL (support), MG, BM, JVG (support), SM, KP. Data Curation: KS, AJag, KL, MG, BM, AK, KP (lead). Writing \u0026ndash; Original Draft: KS, AJag, KL, MG, BM, SM, KP (lead). Writing \u0026ndash; Review \u0026amp; Editing: KS, AJag, KL, MG (support), BM (support), EŻ (support), AJan (support), JD (support), AK (support), AS (support), WK (support), BP (support), JVG (support), BAFK (support), SM (support), KP (lead). Supervision: KS (support), WK, BP, SM, KP (lead). Funding Acquisition: KP (lead).\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe would like to thank Dr Małgorzata Więcek for the resynthesis of HBK-15 and\u003c/p\u003e\u003cp\u003eDr Alessandro Ieraci for providing a breeding couple of BDNF\u003csup\u003eVal/Met\u003c/sup\u003e mice.\u003c/p\u003e\u003cp\u003eThis research was carried out with the use of research infrastructure co-financed by the Smart Growth Operational Programme POIR 4.2 project no. POIR.04.02.00-00-D023/20.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCui L, Li S, Wang S, Wu X, Liu Y, Yu W, et al. Major depressive disorder: hypothesis, mechanism, prevention and treatment. Signal Transduct Target Ther. 2024;9:30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuman RS, Aghajanian GK, Sanacora G, Krystal JH. Synaptic plasticity and depression: new insights from stress and rapid-acting antidepressants. Nat Med. 2016;22:238\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGłuch-Lutwin M, Sałaciak K, Pytka K, Gawalska A, Jamrozik M, Śniecikowska J, et al. The 5-HT1A receptor biased agonist, NLX-204, shows rapid-acting antidepressant-like properties and neurochemical changes in two mouse models of depression. 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Heliyon. 2024;10:e28918.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"functional selectivity, 5-HT1A receptor, ERK1/2 signaling, rapid-acting antidepressant, procognitive, chronic stress","lastPublishedDoi":"10.21203/rs.3.rs-7315313/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7315313/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRapid-onset antidepressants hold transformative potential for treating affective and cognitive symptoms of depression, yet their mechanisms remain incompletely understood. Serotonin receptors orchestrate emotional and cognitive regulation, but current treatments poorly target their intracellular signaling. Here, we characterize HBK-15, a multi-target aminergic ligand, as a functionally selective compound that biases intracellular signaling at 5-HT\u003csub\u003e1A\u003c/sub\u003e and 5-HT\u003csub\u003e7\u003c/sub\u003e receptors. HBK-15 acts as a partial agonist at the ERK1/2 arm of the 5-HT\u003csub\u003e1A\u003c/sub\u003e receptor while blocking β-arrestin recruitment, cAMP inhibition, and calcium mobilization; at 5-HT\u003csub\u003e7\u003c/sub\u003e receptors, it preserves cAMP signaling but blocks β-arrestin recruitment. A single dose of HBK-15 reversed depressive- and cognitive-like deficits in two mouse strains subjected to chronic stress, engaging ERK1/2-linked kinases and plasticity-related signaling in the prefrontal cortex. Pharmacological blockade experiments showed that ERK1/2, but not PKA, signaling in the medial prefrontal cortex is required for HBK-15\u0026rsquo;s behavioral effects. Notably, HBK-15 retained antidepressant-like efficacy in mice carrying the human BDNF Val66Met polymorphism, a translational model characterized by impaired activity-dependent BDNF release, increased depression vulnerability, and reduced treatment responsiveness. The absence of cognitive rescue in this context uncovers a layered mechanism: ERK1/2 signaling is required for both behavioral domains, but BDNF-dependent pathways appear critical for cognitive restoration. These findings position HBK-15 as a mechanistically distinct compound with rapid behavioral efficacy, offering a prototype for signaling-driven strategies in next-generation antidepressant development.\u003c/p\u003e","manuscriptTitle":"HBK-15 bypasses BDNF via ERK1/2-biased 5-HT1A receptor signaling to deliver a rapid antidepressant-like effect","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-28 18:03:49","doi":"10.21203/rs.3.rs-7315313/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d1ca710-38d6-4a0a-acf4-984918aed18e","owner":[],"postedDate":"August 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53409776,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"},{"id":53409777,"name":"Biological sciences/Drug discovery"},{"id":53409778,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-12-09T10:46:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-28 18:03:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7315313","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7315313","identity":"rs-7315313","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}