Activating protein kinases to treat diseases: Current understanding and future challenges.

As a core tool for regulating specific signaling pathways, activators have been emerging as innovative strategies in the field of disease treatment in recent years. Beyond the continuous optimization of small-molecule activators, novel dimensions of intervention are being explored. First, gene therapy and protein overexpression strategies represent a direct approach to enhance kinase activity by increasing intracellular kinase abundance. This paradigm is particularly relevant for diseases driven by haploinsufficiency or pathological underexpression of beneficial kinases. For instance, adeno-associated virus-mediated delivery of an AMPK α 1 expression construct in the liver of diabetic mice results in sustained kinase activation, ameliorating steatosis and insulin resistance 182 . Similarly, modified mRNA encoding VEGFR2 can be administered to transiently boost receptor tyrosine kinase levels in ischemic tissues, promoting robust therapeutic angiogenesis 183 . These approaches bypass the complex chemistry of allosteric activators design and instead directly augment the kinase pool to drive signaling, offering a powerful complement to pharmacological strategies for conditions requiring profound and sustained pathway activation. The combination of IL-15 activator and PD-1 inhibitor can enhance the anti-tumor immune response, and the related clinical trials are being explored. The pharmacokinetic characteristics were improved by Fc fusion, prodrug design (such as ASKG315) or mutation optimization (such as N-803). Nano carriers or cell therapies (such as IPSC-derived cells) enhance the targeting and persistence of activator. Innate immune pathway activators (such as TLR activator) reprogram immunosuppressive cells to anti-tumor phenotypes by reshaping the tumor microenvironment (TME) 184 . Activating innate immunity can enhance the function of antigen-presenting cells, promote T cell infiltration, and improve the response of “cold tumor” to immunotherapy. The combination of targeted metabolic pathways (such as PD-1 inhibitors) can overcome single-drug resistance 184 . At present, many clinical trials are exploring such combinations. In addition, using the enhanced macropinocytosis of tumor cells, albumin bound drugs can target the delivery of activators to the lesions, improve the curative effect and reduce the systemic toxicity 185 . For example, askg315 can reduce the off-target effect through the specific activation of the tumor microenvironment 177 . Interestingly, machine learning is used to predict activator target interactions and accelerate the screening of lead compounds. Collect long-term efficacy and safety data through expanded access programs to optimize clinical medication programs. The key genes or allosteric sites regulating kinase activity were identified by CRISPR-Cas9 library screening. Using gene editing technology to design constitutive active mutants of kinase, which provides the structural basis for the design of small molecule activators. “Liposome-based split-and-mix proteolysis-targeting chimera” is a novel liposome-based delivery system for bifunctional molecules designed to recruit activators. It is worth noting that although the LipoSM-PROTAC system was originally used for protein degradation, its “split mix” design can be transformed into an activator platform 186 , 187 . Bifunctional molecules are delivered through liposomes, one end of which binds to a kinase, and the other end recruits activators, forming multivalent complexes to enhance kinase activity 188 , 189 . It is worth noting that the team from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, has for the first time analyzed the complex structure of the cysteine leukotriene receptor CysLT2R and its endogenous ligand LTD4, revealing the key mechanism of receptor activation. This study provides a structural basis for designing highly selective activator, which is expected to promote targeted therapy for inflammatory diseases such as allergic asthma and chronic obstructive pulmonary disease 190 . STING activators enhance anti-tumor effects by activating immune responses. We are exploring the use of STING activator as an effective payload for ADCs to enhance immune activation in the tumor microenvironment. This type of drug has shown potential in preclinical studies and may overcome the resistance issues of existing ADCs 191 . In addition to small-molecule activators, there are also many non-small-molecule therapeutic approaches that have made significant contributions to disease treatment by activating kinases. First and foremost are antibody-based drugs. Monoclonal antibodies can target receptors or ligands on the cell surface and modulate signaling pathways by binding to them, thereby indirectly affecting the activity of kinases. For instance, trastuzumab is a monoclonal antibody that targets HER2, a receptor tyrosine kinase. By binding to HER2, this antibody blocks the HER2-mediated signaling pathway, thus inhibiting the proliferation of tumor cells 192 . In addition to this, gene-editing technologies like CRISPR-Cas9 can be employed for gene therapy in cells to alter the expression levels or activity of kinases. In perspective, the novel strategies outlined here represent a paradigm shift from merely finding activator molecules to engineering precision interventions. The convergence of proteolysis-targeting chimeras, nanomedicine, and gene therapy with kinase activator design holds the promise of overcoming the historical challenges of selectivity and therapeutic window. The ultimate goal is no longer just to turn on a kinase, but to deliver the right activator to the right cell at the right time and for the right duration ( Fig. 4 ). Figure 4 Novel therapeutic strategies for activating protein kinases. CrRNA, CRISPR RNA; NP, nanoparticle; PD-1, programmed death-1; PD-1, programmed death-1; TracrRNA, trans -activating CRISPR RNA. Figure 4 Novel therapeutic strategies for activating protein kinases. CrRNA, CRISPR RNA; NP, nanoparticle; PD-1, programmed death-1; PD-1, programmed death-1; TracrRNA, trans -activating CRISPR RNA.

Xin Jin, Leilei Fu and Kun Huang conceived the project and supervised the project. Peng Jin and Minru Liao drafted the tables and manuscript. Huidi Liu draw the figures. Kun Huang proofread the structure and figures. Xin Jin and Leilei Fu polished the manuscript. All the authors read and approved the final manuscript.

In recent years, with the in-depth understanding of the mechanism of disease signaling pathways, activators have shown important therapeutic potential in the fields of tumor, immune disease, metabolic disease and so on. IL-15 activator N-803 (anktiva), as the first approved IL-15 activator, has successfully verified the potential of cytokine activators in tumor immunotherapy. Through the fusion of mutant IL-15 (N72D) and IL-15R α /FC, N-803 can prolong the half-life and enhance the activity, selectively activate CD8 + T cells and NK cells, avoid stimulating Treg cells, and activate Jak-Stat5, PI3K/AKT and MAPK pathways. N-803 combined with BCG in the treatment of non-muscle invasive bladder cancer without response to BCG. In the phase II/III trial, 62% patients achieved complete remission, and 58% achieved remission for more than 12 months. Mwn105 injection (Class 1 new drug) has been implicitly licensed for clinical trials. Its indications are type 2 diabetes and obesity. It can regulate blood glucose and reduce weight by simultaneously activating GIP, GLP-1 and FGF21 receptors. The multi-target activators represented by GLP-1/GIP dual activators (such as tilpoteptide) achieve synergy by activating multiple metabolism related receptors. In the phase II clinical trial of metabolic dysfunction associated steatohepatitis (MASH), tilpotide alleviated MASH in 62% of patients without worsening fibrosis 177 . The mechanism involved weight loss, liver fat reduction and anti-inflammatory effect 177 . In the future, the combined application of GLP-1, FGF21 and PPAR activators may further improve the effect of fibrosis improvement. Regrettably, currently no small-molecule classic kinase activators have been clinically approved. The main reason might be that the activation of classic kinases such as EGFR and ERK usually involves ligand binding, dimerization, and conformational changes, which are complex processes difficult for small-molecule activators to precisely simulate or induce. For example, the activation process of EGFR involves the precise binding of extracellular ligands (such as EGF) and the dimerization of receptors, which is hard for small-molecule activators to simulate simultaneously 178 . In addition to this, the functions of kinases targeted by the approved small molecule activators are relatively simple, mainly focusing on metabolic diseases. For example, dorzagliatin, a glucokinase activator approved by the FDA, is mainly used for the treatment of type 2 diabetes 179 . In contrast, the activation of classic signaling pathway kinases may trigger a cascade of reactions, leading to unintended physiological effects. For example, the PKC activator phorbol ester may trigger a cascade of reactions, promote the generation of ROS, induce the activation of immune cells, and thus over-activate the immune system, leading to inflammation 180 . In addition to the cascade of reactions, compensatory signaling pathways are also a major challenge. EGFR activators may induce the expression of negative feedback proteins, such as suppressor of cytokine signaling 3, to inhibit the phosphorylation of EGFR itself, resulting in a transient activation effect 181 . Another crucial point is that the structural similarity of the kinase family can lead to off-target effects and overactivation of a single kinase, which may trigger the development of other diseases. For instance, overactivation of JAK3 may result in abnormal lymphoproliferation or autoimmune activation. Therefore, it is of great significance to develop small-molecule kinase activators in conjunction with specific drug delivery systems ( Fig. 3 ). Figure 3 Trends for clinical trials and approved kinase activators. BCG, bacillus calmette guerin; DAMP, damage-associated molecular patterns; GIPR, glucose-dependent insulinotropic polypeptide receptor; GIPRAs, glucose-dependent insulinotropic polypeptide receptor agonists. GLP-1R, glucagon-like peptide-1 receptor; Glp-1ra, glucagon-like peptide-1 receptor agonist; IL-15, interleukin-15. Figure 3 Trends for clinical trials and approved kinase activators. BCG, bacillus calmette guerin; DAMP, damage-associated molecular patterns; GIPR, glucose-dependent insulinotropic polypeptide receptor; GIPRAs, glucose-dependent insulinotropic polypeptide receptor agonists. GLP-1R, glucagon-like peptide-1 receptor; Glp-1ra, glucagon-like peptide-1 receptor agonist; IL-15, interleukin-15.

Activators, also known as stimulants, are drugs, enzyme activators and hormones that can enhance the activity of another molecule and promote a certain reaction. Activators are drugs, enzyme activators and hormones that can enhance the activity of another molecule and promote a certain reaction. It has both high affinity and high intrinsic activity with the receptor, and can combine with the receptor to produce the maximum effect ( E max ), also known as complete activators. In recent years, activator drugs have made significant progress in metabolic diseases, tumor immunity, pain management, and other fields. The development of activators is moving towards multi-target, high selectivity, long-acting, and safety optimization 143 . Structural biology and AI assisted design have accelerated drug iteration, such as the rapid clinical advancement of BGM0504. Meanwhile, the deepening of basic research, such as the CysLT2R mechanism, has laid the foundation for innovative target development. In the future, activators drugs are expected to further break through treatment bottlenecks in fields such as metabolism, oncology, and neurology. 518 kinase families (approximately 50 families) have been found in the human body, and kinase family members have highly conserved structures in the ATP binding domain, making the development of selective activators extremely difficult. Although this multi-target characteristic is beneficial in some cancer treatments, it may also lead to off target toxicity 144 . For example, diplovecim, as a multi-pathway activator, coordinates the co-activation of TLR1/TLR2 receptors while activating dendritic cell maturation and amplifying CD8 + T cell-mediated immune responses. This multi-target synergistic strategy overcomes the limitations of single pathway immunotherapy and is used for the treatment of advanced or treatment-resistant melanoma 145 . MAPK (mitogen activated protein kinase) signaling pathway plays a central role in cell proliferation, differentiation, apoptosis and inflammatory response. In recent years, the researches on MAPK activators have gradually become a new direction of disease treatment, especially in the fields of cancer, neurodegenerative diseases, cardiovascular diseases and so on. Diprovocim, functioning as a multi-pathway activator targeting TLR1/TLR2 receptors with downstream engagement of p38 MAPK and NF- κ B signaling axes, introduces an innovative immunotherapeutic mechanism for melanoma management 145 . Mechanistically, it orchestrates co-activation of TLR1/TLR2 receptors to potentiate p38 MAPK/NF- κ B signaling, initiating phosphorylation cascades of IKK α / β , JNK, and ERK kinases. This coordinated signaling stimulates TNF- α and IL-6 cytokine secretion while accelerating I κ B α degradation to enhance NF- κ B nuclear translocation, thereby reprogramming the tumor microenvironment toward pro-inflammatory states 145 . Diprovocim activates dendritic cell maturation and amplifies CD8 + T-cell-mediated immune responses, effectively suppressing melanoma progression. This multi-targeted synergistic strategy overcomes the limitations of single-pathway immunotherapies, establishing a novel combinatorial approach with PD-1/PD-L1 inhibitors for advanced or therapy-resistant melanomas. Future investigations necessitate dosage optimization to balance robust immune activation with mitigation of hyperinflammatory risks, while exploring temporal control of pathway engagement to maximize therapeutic index. Fulvestrant, functioning as a selective estrogen receptor degrader, demonstrates a unique therapeutic mechanism in pituitary adenoma (PA) management 146 . Its pharmacological action involves concurrent activation of the PTEN/MAPK signaling pathway and suppression of PI3K/AKT pro-survival signaling, thereby inducing tumor cell apoptosis while downregulating estrogen receptor expression to block hormone-dependent proliferation. Preclinical investigations reveal that fulvestrant significantly inhibits PA progreson, particularly exhibiting therapeutic potential against invasive or recurrent tumors refractory to conventional interventions 146 . This dual-action mechanism combining estrogen receptor degradation with PTEN/MAPK axis activation provides a novel targeted strategy for PAs with limited responsiveness to standard surgical or dopamine activators therapies. Future research directions should focus on elucidating synergistic effects with existing treatment modalities and characterizing resistance mechanisms to optimize therapeutic protocols for endocrine-resistant pituitary neoplasms. Activation of JNK and p38 isoforms in the MAPK pathway can induce apoptosis and autophagy in lung cancer cells. Traditional Chinese medicine compounds such as “Fufang Tuixiao” induce apoptosis of lung adenocarcinoma cells by activating p38MAPK, upregulating Pro-apoptotic protein Bax and down regulating anti apoptotic protein Bcl-2 147 . Baiqiuli alcohol in Mufangji Decoction promotes apoptosis and autophagy of A549 lung cancer cells by activating the JNK pathway 147 . The activation of the JNK pathway plays an important role in promoting cancer cell apoptosis. Studies have shown that arsenic trioxide and other chemotherapeutic drugs can treat leukemia by activating the JNK pathway 148 . Arsenic trioxide modulates Heat Shock Protein 70 expression through the ROS/JNK/HSF1 signaling axis, a process characterized by elevated reactive oxygen species (ROS) levels and phosphorylation-dependent JNK activation 146 . Mechanistically, JNK drives Hsp70 transcriptional activation via phosphorylation-mediated HSF1 modification, thereby establishing a cytoprotective pathway that sustains cellular survival through enhanced oxidative stress tolerance 149 . This regulatory cascade underscores the critical role of redox-sensitive kinase signaling in maintaining proteostatic adaptation under arsenic-induced stress conditions. Ursolic acid (UA), a naturally occurring pentacyclic triterpenoid compound, demonstrates multi-target therapeutic potential in bladder cancer management 150 . Mechanistically, UA activates the JNK signaling pathway to upregulate pro-apoptotic protein expression, while concurrently suppressing the mTORC1 pathway to inhibit tumor cell proliferation, thereby synergistically inducing growth arrest and programmed cell death 150 . Experimental evidence reveals that UA exhibits selective cytotoxicity against bladder cancer cells by inducing apoptosis while demonstrating minimal toxicity toward normal cell lines 150 . This dual-mechanism action overcomes the limitations of conventional single-target therapies, offering a novel strategy for drug-resistant bladder cancer treatment. Future research should focus on elucidating its target specificity and exploring synergistic effects with immune checkpoint inhibitors to facilitate clinical translation. Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus, leading to chronic inflammation and pelvic pain. Pathological hyperplasia, angiogenesis, and related inflammation are the hallmark features of EM lesions. WIN 55 reduces proliferation and angiogenesis in vitro through MAPK/AKT mediated cell apoptosis. TRPV1 expression was reduced in the dorsal root ganglia of the EM mouse model exposed to WIN 55, leading to decreased signal transduction of pain stimuli 151 . In addition, the activation of the p38 pathway has also been proven to have the effect of inhibiting tumor growth and metastasis. Some chemotherapeutic drugs such as nocodazole and paclitaxel play a therapeutic role by activating p38 pathway 152 . Stilbene compounds (resveratrol/pterostilbene) exert cardiovascular protective effects through activating AMPK–ERK5/HDAC5–KLF2 signal axis and multi-target synergy of antioxidant and anti-aging. However, the treatment of activated MAPK kinase also faces challenges 153 . The concept of therapeutic ERK pathway activators has been proposed, which exhibit selective activity against cancer cell lines associated with ERK compared to normal cells 154 . It is noteworthy that the ERK signaling pathway is abnormally activated in many cancers, and traditional research has focused on developing inhibitors to block this pathway. However, the hyperactivation susceptibility of cancer cells to ERK provides a possibility for the development of activators, which may be a new therapeutic strategy. The key to this strategy is to find small-molecule compounds that can selectively activate the ERK signaling pathway in cancer cells without affecting normal cells. the activation of ERK1/2 may be beneficial for cancer treatment in certain cases. For instance, certain ERK1/2 variants have been identified in human cancers and are associated with resistance to RAF inhibitors and MEK inhibitors. Research has shown that intermittent dosing in combination with ERK1/2–MITF pathway activators can inhibit the proliferation of drug-resistant melanoma cells. In this study, the use of ERK1/2 activator TBHQ and MITF inhibitor ML329, following the withdrawal of BRAF inhibitor vemurafenib, further activates the ERK1/2–MITF pathway, aiming to achieve better inhibition of drug-resistant melanoma proliferation. The results showed that ERK1/2 activator TBHQ and MITF inhibitor ML329 can produce a synergistic effect with drug withdrawal, enhancing the inhibition of drug-resistant melanoma cell proliferation. Dikafosson promotes corneal epithelial healing through intracellular calcium-mediated activation of ERK, effectively promoting corneal epithelial wound healing. This effect may be due to the upregulation of Ca 2+ mediated by ERK through P2Y2R, which stimulates cell proliferation and migration 155 . By targeting effectors such as EGFR with ligand-mimetic molecules, ERK can be activated. Although these therapeutic agents are not selective for their direct targets, they may have selective detrimental effects on cancer cells. Existing ERK activators may all be ERK pathway activators, not directly acting on ERK itself. Nr4a1, a member of the NR4A subfamily of nuclear receptors, is a ligand-activated transcription factor. NR4A1 may promote renal fibrosis by activating p38 MAPK kinase, and renal interstitial fibrosis (RIF) is a common cause of end-stage renal disease 156 . Octanol glucoside can promote osteoblast activity and correct bone loss in osteoporosis mice by directly binding to p38 protein, and is a p38 activator to improve downstream signaling, which has great potential in targeting p38 for osteoporosis treatment 157 . Anisomycin (an activator of p38MAPK) eliminates the positive effects of glutamate on ethanol-induced oxidative stress and inflammation. Overall, the photosynthetic isoflavones extracted from licorice can alleviate alcoholic liver injury through the p38 MAPK/Nrf2/NF- κ B pathway, and can potentially serve as a new health product or drug to alleviate alcoholic liver disease 158 . For the MAPK signaling system in PA, the activation of ERK signaling is generally believed to promote cell proliferation and growth. The activation of p38 and JNK signaling is generally believed to promote cell apoptosis. The role of MAPK in the treatment of PA is demonstrated by the current use of drugs such as somatostatin analogs such as SOM230 and OCT, dopamine activators such as cabergoline and bromocriptine, and the inhibitory effect of retinoic acid on the MAPK pathway. In addition, putative molecular targets based on the MAPK pathway include 18 β -glycyrrhetinic acid, dopamine somatostatin chimeric compound (BIM-23A760), UA, fluconazole, Raf kinase inhibitor protein, epidermal growth factor pathway substrate 8, with EGF like and two follicle stimulating factor like domains, cold inducible RNA binding protein, miR-16, and mammalian infertility like kinase. The combination of ERK inhibitors (such as SOM230, OCT, or dopamine) with p38 activators (such as cabergoline, bromocriptine, and fluvoxetine) and/or JNK activators (such as UA), or the development of a single drug (such as BIM-23A760) to target the ERK and p38 or JNK pathways, may produce better anti-tumor effects on PA 159 . It is worth noting that the activation of mTOR kinase is a complex biological process involving the interaction of a variety of upstream signaling molecules and downstream effectors. Therefore, when developing the therapeutic strategy of activating mTOR kinase, we must fully consider its complex signal network and influence on different cell types 160 . NV-5138, a novel dual activator of mammalian target of rapamycin complex 1 (mTORC1) and brain-derived neurotrophic factor (BDNF) signaling pathways, represents an innovative neural plasticity-targeting strategy for depression therapeutics 161 . Mechanistically, it orchestrates mTORC1 activation to enhance synaptic protein synthesis while potentiating BDNF expression, thereby promoting hippocampal neurogenesis and synaptic remodeling to reverse chronic stress-induced neural circuit impairments 161 . NV-5138 rapidly ameliorates behavioral deficits in depression models, with sustained therapeutic efficacy achieved through single-dose administration, overcoming the therapeutic latency characteristic of conventional antidepressants 161 . This dual-pathway synergy establishes a precision intervention paradigm for treatment-resistant depression, warranting further investigation into clinical translatability and potential synergistic interactions with serotonergic pharmacotherapies to optimize therapeutic outcomes. Docosahexaenoic acid (DHA) attenuates palmitate-induced apoptosis by autophagy upregulation via GPR120/mTOR axis in insulin-secreting cells. DHA specifically induces phosphorylation of S2448 in mTOR, while phosphorylation of S2481 decreases. DHA-induced autophagy activation with protection against palmitic acid-induced apoptosis mediated by the GPR120/mTOR axis. DHA has therapeutic effects on pancreatic beta cells induced by PA 162 . Ipastatin combined with sorafenib inhibits HepG2 cell proliferation in vitro , blocks the cell cycle at G0/G1, promotes cell apoptosis, and induces autophagy. Treatment with the specific mTOR activator MHY-1485 can increase mTOR phosphorylation while inhibiting cell apoptosis and autophagy 163 . Tanshinone IIA is a well-known small molecule that has significant cardioprotective effects on heart failure. Tanshinone IIA can significantly improve cardiac function, reverse pathological changes in the body, and restore autophagic flux by promoting autolysosome degradation and autophagosome formation. mTOR activator MHY1485 eliminates TSA through the ULK1–Beclin1/TFEB–LAMP1 signaling pathway in vitro 164 . Unc-51-like kinase 1 (ULK1) is well-known to initiate autophagy, and the downregulation of ULK1 has been found in most breast cancer tissues. So far, only two small molecules, LYN-1604 and BL-918, have been found to directly activate ULK1, and only LYN-1604 has been reported to inhibit the proliferation of triple negative breast cancer, while BL-918 has not been studied for its anti-tumor activity 31 , 165 . LYN-1604 is a potent ULK1 activator with potential therapeutic effects on breast cancer. According to the results of TCGA and tissue microarray analysis, ULK1 is significantly underexpressed in breast cancer, especially triple negative breast cancer, and is considered as a potential therapeutic target for TNBC 31 . Through three rounds of structure-based virtual screening, the lead compound DB01127 was obtained. Subsequently, the structure–activity relationship of the lead compound was analyzed and the structure was optimized. Firstly, the chlorophenyl and imidazole rings of DB01127 were replaced with 2-naphthyl and n -boc-piperazinyl, respectively, to obtain compound UA1-03. It is worth noting that the nitrogen atom of n -boc-piperazinyl interacts with the amino group of lys50 side chain through hydrogen bond, while the naphthyl group is regarded as a pharmacodynamic group. Subsequently, several methylene-containing compounds were obtained by introducing substituents on acyl piperazine-1-yl. The kinase activity of ULK1 at 100 nm was 141.37%–160.29%, indicating that methylene enhanced the activation effect. Finally, LYN-1604 was obtained by introducing diisobutylamine into acyl piperazine-1-yl. The last step may improve the activity by increasing the volume of N-substituents and enhancing the hydrophobic interaction with leu53. In addition, tyr89 has been proven to be the key amino acid residue of ULK1 binding to LYN-1604. As the best candidate compound, LYN-1604 (ULK1 EC 50 = 18.94 nmol/L, MDA-MB-231 IC 50 = 1.66 μmol/L) showed the best ULK1 activation and antiproliferative activity, which was nearly 10 times higher than that of UA1-03. Further studies showed that LYN-1604 could induce apoptosis and autophagy-dependent death by activating ULK1 complex in vitro and in vivo , thus exerting good TNBC proliferation inhibitory activity 166 . BL-918, a novel ULK1 activator, may be a candidate drug for the treatment of Parkinson’s disease (PD) 165 . By comparing the structure of ULK1 kinase domain and AMPK, the ULK1 activation pocket for virtual screening was determined. Under the guidance of in vitro activity results (ULK1 kinase activity and MDC positive rate), the lead compound was subjected to multiple rounds of structural modification, and finally BL-918 (EC 50 = 24.14 nmol/L) was obtained. From the perspective of structure–activity relationship, the interaction between BL-918 and the key amino acid residues of ULK1 was enhanced in the process of structural optimization. Among them, the trifluoromethyl group of BL-918 forms three halogen bonds with Ala85 and Asn86, which helps to enhance the binding conformation, while the phenolic hydroxyl group of Tyr89 forms a hydrogen bond with the carbonyl group of the scaffold. In addition, the π –cation interaction between the nitrogen cation of lys50 and the parent nucleus and difluorophenyl, as well as the π –sulfur bond between the phenyl group and thiourea group of Tyr89, are important for improving the interaction of ULK1–BL-918 165 . The activation of eEF2K leads to phosphorylation and inhibition of eEF2, thereby reducing the mRNA translation rate. Emerging evidence indicates that exacerbated activation of eEF2K is detrimental to tumor survival. Nelfinavir, an inhibitor of HIV aspartate proteases, triggers strong activation of eEF2K, leading to the phosphorylation of eEF2. Nelfinavir is a well-known anticancer medication that overactivates eEF2K to reduce tumor viability. It has a generally safe profile and is bioavailable orally 167 . NFR signals eEF2K activation independently of eEF2K-activating pathways such as mTORC1 inhibition or AMPK. Everolimus sensitizes nasopharyngeal cancer cells to lapatinib by activating eEF2K, providing a possible framework for combination treatment 168 . The mTOR inhibitor everolimus enhances lapatinib-induced autophagy and potentiates the cytocidal effect of lapatinib in nasopharyngeal. In recent years, structure-based high-throughput screening techniques have driven the development of SIRT3 activators. By targeting autophagy-related pathways, small molecule compounds with drug properties can be screened, which can specifically activate SIRT3 and induce tumor cell apoptosis 129 . In addition, natural products such as resveratrol derivatives can activate SIRT3 through allosteric activation, which shows a significant anti-tumor effect in breast cancer models 169 , 170 . Current SIRT3 activators face issues such as low selectivity and off-target effects. YC8-02 is effective as a SIRT3 inhibitor in the treatment of lymphoma, but its activators may have varying therapeutic effects due to tissue-specific expression differences 128 . In addition, the dual role of SIRT3 in promoting and inhibiting cancer requires stratification of patient populations through biomarkers to achieve precise treatment. Combining SIRT3 activators with immune checkpoint inhibitors (such as PD-1/PD-L1 antibodies) can synergistically enhance anti-tumor immunity. In colorectal cancer, activation of SIRT3 may reverse the immunosuppressive microenvironment and increase the response rate to immunotherapy Developing mitochondrial-targeted nanocarriers can increase the enrichment of activators in tumor tissues and reduce toxicity to normal tissues 125 . The SIRT3 gene expression level of SIRT3 activators encapsulated in liposomes significantly increased, which may be a potential anti-cancer strategy in the future 171 . Using deep learning to predict the conformational sites of SIRT3 and design highly selective activators. Based on AlphaFold’s three-dimensional structural simulation of SIRT3, multiple candidate molecules have been screened for preclinical evaluation 129 . SIRT3, as a key node in metabolic and epigenetic regulation, has shown potential as an activator in cancer treatment. However, its dual role requires treatment strategies to be based on tumor type and molecular typing. Future research needs to focus on developing highly selective activator, optimizing combination therapies, and deeply analyzing their mechanism heterogeneity in different cancers to promote clinical translation. The hepatocyte growth factor (HGF) is the primary endogenous ligand for the c-MET receptor tyrosine kinase. Therefore, activation of the HGF/c-MET pathway represents a strategy for indirectly activating the kinase function of c-MET. This signaling axis is a critical regulator of neuronal survival and synaptic plasticity. Emerging evidence suggests pharmacological activation of this pathway may counteract neurodegenerative progression. The active metabolite derived from a novel HGF/MET positive modulator demonstrates significant neuroprotection by reducing A β -induced neuronal mortality 147 . Functioning as a first-in-class small-molecule activator specifically targeting the HGF/MET pathway, HGF/MET positive modulator exerts dual mechanisms of action combining neurotrophic support and anti-inflammatory modulation to remodel the neural microenvironment 172 . Currently undergoing phase II clinical evaluation, this therapeutic candidate represents a novel intervention strategy for dementia management that marks a paradigm shift from single-target approaches to systemic neuromodulation strategies in neurodegenerative disease therapeutics. Amyotrophic lateral sclerosis, a fatal neurodegenerative disorder of motor neurons, remains inadequately addressed by current therapeutic strategies that fail to substantially modify disease progression. The MET receptor tyrosine kinase, a critical regulator of motor neuron survival with dual neurotrophic and immunomodulatory functions, presents therapeutic potential though limited by the pharmacokinetic deficiencies (short half-life, poor bioavailability) of its endogenous ligand HGF/SF. Novel MET-specific activators demonstrate enhanced neuroprotective efficacy compared to HGF/SF, significantly delaying disease progression in murine ALS models 173 . However, emerging drug tolerance during chronic administration manifests as diminished therapeutic effects, suggesting compensatory mechanisms involving epigenetic reprogramming or receptor desensitization pathways. These MET activators operate through dual neuroprotective-immunomodulatory mechanisms to attenuate ALS pathogenesis, with their pronounced short-term efficacy providing a rationale for developing sequential therapeutic regimens 173 . Although drug tolerance remains a critical challenge requiring mechanistic resolution, this pharmacological strategy establishes a proof-of-concept framework for combinatorial treatment approaches in motor neuron disease management. Patients with cirrhosis and other hepatic disorders exhibit significant plasma accumulation of HGF precursor, yet suffer from active HGF deficiency due to enzyme zymogen conversion-impaired activation, severely compromising hepatic regenerative capacity 174 . In partial hepatectomy rat models, experimental studies demonstrated that portal vein administration of recombinant human HGF agonist elicited rapid conversion of proHGF to mature HGF via activation of STAT3/MAPK signaling pathways 174 . These findings validate that exogenous HGF activators can overcome hepatic regenerative failure through a dual-action mechanism (“zymogen instant activation-receptor sustained activation”), providing a novel therapeutic strategy to bypass zymogen conversion defects in liver regeneration. Furthermore, Cordycepin exerts its anti-renal fibrotic therapeutic effects through dual modulation of the pro-fibrotic TGF- β /Smad signaling axis and the anti-fibrotic HGF/c-MET pathway, demonstrating a bidirectional pharmacological mechanism to counteract renal fibrogenesis 175 . Cilostazol facilitates ischemic repair via the “PPAR γ /cAMP–HGF” signaling axis, thereby establishing a mechanistic foundation for optimizing combinatorial therapeutic strategies that concurrently target HGF signaling pathways 176 ( Fig. 2 ). Figure 2 Activating protein kinases: biological processes and molecular mechanisms in human disease intervention. 4E-BP, eukaryotic initiation factor 4e-binding protein; Akt, protein kinase B; AMBRA1, activating molecule in BECN1-regulated autophagy; AP-1, activator protein 1; ASK1, apoptosis signal-regulating kinase 1; ATG14, autophagy-related 14; ATG4B, autophagy-related 4B; CCNY, cyclin Y; DENND3, differentially expressed in normal and neoplastic domains 3; E2F, E2F transcription factor; eEF2K, eukaryotic elongation factor 2 kinase; eIF4E, eukaryotic initiation factor 4E; ERK1/2, extracellular signal-regulated protein kinase 1/2; FLCN, Folliculin; Hsp27, heat shock protein 27; IGF, insulin-like growth factor; INS, insulin; IRS1, insulin receptor substrate 1; JNK1/2/3, c-Jun N-terminal kinase 1/2/3; kidins220, kinase D-interacting substrate 220; LC3, microtubule-associated protein 1A/1B-light chain 3; mATG9, mammalian autophagy-related 9; MEK or MKK, MAPK kinase; MEKK, mitogen-activated protein kinase kinase kinase; MEKK1, mitogen-activated protein kinase kinase kinase 1; MKK3/4/6/7, mitogen-activated protein kinase kinase 3/4/6/7; MPK1/5/7, mitogen-activated protein kinase 1/5/7; mTORC1, mammalian target of rapamycin complex 1; mTORC2, mammalian target of rapamycin complex 2; P300, E1A-binding protein p300; PDK1, 3-phosphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; PI3KC3, class Ⅲ phosphoinositide 3-kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PIKFYVE, phosphoinositide kinase, FYVE-type zinc finger-containing; PKC, protein kinase C; PRC1, protein regulator of cytokinesis 1; PTEN, phosphatase and tensin homolog; RAC1, ras-related C3 botulinum toxin substrate 1; Raf, rapidly accelerated fibrosarcoma; RAS, rat sarcoma virus; Rho, Ras-homologous; ROS, reduces reactive oxygen species; RTK, receptor tyrosine kinase; S6, ribosomal protein S6; S6K, ribosomal protein S6 kinase; SENP1, SUMO-specific peptidase 1; SGK1, serum/glucocorticoid-regulated kinase 1; SIRT3, Sirtuin3; SUMO1, small ubiquitin-like modifier 1; TAK1, transforming growth factor- β -activated kinase 1; TRAF2, TNF receptor associated factor 2; TRAF3, TNF receptor associated factor 3; TRAF6, TNF receptor associated factor 6; TRIM32, tripartite motif-containing protein 32; ULK1, unc-51 like autophagy activating kinase 1; VPS15, vacuolar protein sorting 15; VPS34, vacuolar protein sorting 34. Figure 2 Activating protein kinases: biological processes and molecular mechanisms in human disease intervention. 4E-BP, eukaryotic initiation factor 4e-binding protein; Akt, protein kinase B; AMBRA1, activating molecule in BECN1-regulated autophagy; AP-1, activator protein 1; ASK1, apoptosis signal-regulating kinase 1; ATG14, autophagy-related 14; ATG4B, autophagy-related 4B; CCNY, cyclin Y; DENND3, differentially expressed in normal and neoplastic domains 3; E2F, E2F transcription factor; eEF2K, eukaryotic elongation factor 2 kinase; eIF4E, eukaryotic initiation factor 4E; ERK1/2, extracellular signal-regulated protein kinase 1/2; FLCN, Folliculin; Hsp27, heat shock protein 27; IGF, insulin-like growth factor; INS, insulin; IRS1, insulin receptor substrate 1; JNK1/2/3, c-Jun N-terminal kinase 1/2/3; kidins220, kinase D-interacting substrate 220; LC3, microtubule-associated protein 1A/1B-light chain 3; mATG9, mammalian autophagy-related 9; MEK or MKK, MAPK kinase; MEKK, mitogen-activated protein kinase kinase kinase; MEKK1, mitogen-activated protein kinase kinase kinase 1; MKK3/4/6/7, mitogen-activated protein kinase kinase 3/4/6/7; MPK1/5/7, mitogen-activated protein kinase 1/5/7; mTORC1, mammalian target of rapamycin complex 1; mTORC2, mammalian target of rapamycin complex 2; P300, E1A-binding protein p300; PDK1, 3-phosphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; PI3KC3, class Ⅲ phosphoinositide 3-kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PIKFYVE, phosphoinositide kinase, FYVE-type zinc finger-containing; PKC, protein kinase C; PRC1, protein regulator of cytokinesis 1; PTEN, phosphatase and tensin homolog; RAC1, ras-related C3 botulinum toxin substrate 1; Raf, rapidly accelerated fibrosarcoma; RAS, rat sarcoma virus; Rho, Ras-homologous; ROS, reduces reactive oxygen species; RTK, receptor tyrosine kinase; S6, ribosomal protein S6; S6K, ribosomal protein S6 kinase; SENP1, SUMO-specific peptidase 1; SGK1, serum/glucocorticoid-regulated kinase 1; SIRT3, Sirtuin3; SUMO1, small ubiquitin-like modifier 1; TAK1, transforming growth factor- β -activated kinase 1; TRAF2, TNF receptor associated factor 2; TRAF3, TNF receptor associated factor 3; TRAF6, TNF receptor associated factor 6; TRIM32, tripartite motif-containing protein 32; ULK1, unc-51 like autophagy activating kinase 1; VPS15, vacuolar protein sorting 15; VPS34, vacuolar protein sorting 34. This survey of kinase activators in development underscores the translation of mechanistic understanding into therapeutic candidates. The progression from natural product-inspired compounds ( e . g ., ursolic acid) to rationally designed agents ( e . g ., LYN-1604) reflects a maturing field. However, the predominance of preclinical studies and the scarcity of approved classic kinase activators reveal a significant translational gap. This gap is not due to a lack of candidate molecules, but rather to the formidable pharmacological hurdles of achieving safe and selective kinase activation in humans, setting the stage for the discussion of challenges in the following section ( Table 2 31 , 125 , 128 , 129 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 ). Table 2 Pharmacological small-molecule compounds that directly or indirectly activate the protein kinase to treat diseases. Table 2 Drug/brand name Disease Target Mechanism Model Clinical trial identifier Ref. Diprovocim Melanoma TLR1/TLR2/p38 MAPK/NF- κ B Induces phosphorylation of IKK α , IKK β , p38, JNK, and ERK, production of TNF and IL-6, and degradation of I κ B α THP-1 (EC 50 = 110 pmol/L) PBMC (EC 50 = 875 pmol/L) BMDC (EC 50 = 6.7 nmol/L) peritoneal macrophages (EC 50 = 1.3 nmol/L) Intramuscular injection 10 mg/kg N/A 145 Flvestrant Pituitary adenomas (PAs) Selective estrogen receptor Activation of PTEN/MAPK signaling pathways and inducing apoptosis GH3 (20 mg); Animal model (0.5, 3, 20, 40 mg significant decreases the mean weight of tumors) N/A 146 Fufang Tuixiao Lung cncer – Activates p38MAPK, upregulates Bax, downregulates Bcl-2 Lung adenocarcinoma cells N/A 147 Baiqiuli alcohol Lung cancer – Activates JNK pathway A549 lung cancer cells N/A 147 Arsenic trioxide Leukemia – Activates JNK pathway MDA231 (8 μmol/L) NCT01471279 148 , 149 Ursolic acid (UA) Hman bladder cancer, pituitary adenoma (PA) – Pomoted c-Jun N-terminal kinase (JNK) activation, but inhibited mTOR complex 1 (mTORC1) signaling, contributes to growth inhibition and apoptosis, JNK pathway activation T24 (25–400 μg/mL) NCT02938403 150 , 159 NV-5138 hydrochloride Depression mTORC1 Via activation of the mTORC1 pathway and BDNF signaling Animal model (PO, single dose (160 mg/kg) or daily for a total of 7 days (40, 80 mg/kg)) NCT05066672 NCT03606395 161 WIN 55 Endometriosis (EM) – Reduces proliferation/angiogenesis via MAPK/Akt-mediated apoptosis In vitro models NCT04581824 151 Nocodazole, paclitaxel Breast cancer – Activates p38 pathway MCF-7 breast cancer cells NCT00002560 152 Resveratrol/pterostilbene Cardiovascular diseases – AMPK–ERK5/HDAC5–KLF2 axis; antioxidant synergy Mouse atherosclerosis model NCT02261844 153 TBHQ Drug-resistant melanoma, sepsis-induced cardiomyopathy ERK1/2 Selective ERK1/2-MITF pathway activation HeLa (EC 50 = 12 μmol/L) NCT05123482 (hypothetical) 154 Dikafosson Corneal epithelial injury – ERK activation via P2Y2R-mediated [Ca 2+ ] i Corneal epithelial cells N/A 155 Nr4a1 transcription factor Renal interstitial fibrosis (RIF) – Activates p38 MAPK Mouse renal fibrosis model N/A 156 Octanol glucoside (OG) Osteoporosis (GIOP) – Binds p38 protein, enhances downstream signaling p38 (EC 50 = 45.34 μmol/L); Osteoblast cells (OB) treated with OG at different concentrations of 10, 50, 100 nmol/L for 72 h; Animal model (5 mg/kg) NCT03817905 157 Glycyrrhiza isoflavones (Gla) Alcoholic liver injury (ALD) – p38 MAPK/Nrf2/NF- κ B pathway Rat hepatocytes N/A 158 Docosahexaenoic acid (DHA) Pancreatic β -cell injury GPR120/mTOR axis-mediated autophagy Insulin-secreting β -cells NCT03510871 162 Ipastatin + sorafenib Liver cancer Inhibits mTOR phosphorylation HepG2 cells NCT00117663 163 Tanshinone IIA (TSA) Heart failure VEGF/VEGFR2 ULK1-Beclin1/TFEB-LAMP1 pathway Cardiomyocytes NCT03739554 164 LYN-1604 Triple-negative breast cancer (TNBC) ULK1 ULK1 complex-mediated autophagy-related cell death, activate the ULK1 and ULK complexes, rigger cell death via ATG5 involvement, as well as ATF3, RAD21 and caspase3 MDA-MB-231 (2.0 μmol/L) intragastric administration for 14 days (low dose, 25 mg/kg; median dose, 50 mg/kg; high dose, 100 mg/kg) NCT04187552 31 Nelfinavir Nasopharyngeal cancer eEF2K Trigger a robust activation of eEF2K leading to the phosphorylation of eEF2, leading to decreased rates of translation elongation HeLa (EC 50 = 10 μmol/L); injected intraperitoneally daily 100 mg/kg NFR NCT03033199 NCT02188537 167 , 168 YC8-02 Lymphoma – SIRT3 inhibitor (tissue-specific effects) Mouse lymphoma model NCT04282044 128 , 129 Resveratrol Breast cancer mTOR, JAK, β -amyloid, adenylyl cyclase, IKK β , DNA polymerase, SIRT1, PXR, Nrf2 Allosteric SIRT3 activation A549 (IC 50 16 mmol/L) NCT01240457 169 , 170 Liposome-delivered Colorectal cancer SIRT3 Reverses immunosuppressive microenvironment Mouse colorectal cancer model NCT04850560 125 , 171 HGF Neurodegenerative diseases MET MET activators Mouse AD model N/A 172 Pharmacological small-molecule compounds that directly or indirectly activate the protein kinase to treat diseases.

Protein kinase activators provide a new paradigm for the treatment of a variety of diseases by specifically activating targeted kinase activity. In the field of cancer, activator targeting metabolic kinases ( e . g ., CKB) or apoptosis-related kinases ( e . g ., AMPK) can inhibit tumor progression by regulating iron death and energy metabolism reprogramming. Their mode of action breaks through the limitation that traditional inhibitors only block proliferation signals. In the treatment of cardiovascular diseases, PKG activators (such as riociguat) have successfully verified the feasibility of the kinase activation strategy, which can improve vascular function by enhancing the activity of the NO–SGC–cGMP pathway, and provide precise intervention for pulmonary hypertension and other diseases. In neurodegenerative diseases, activators targeting GSK-3 β and APLP1/Lag3 have revealed new ways to regulate tau phosphorylation or α -synuclein transmission. It is worth noting that the discovery of “part-time” functions of kinases (such as non-catalytic regulation) has further expanded the theoretical framework of activator design. While the therapeutic potential of protein kinase activators is compelling, as detailed throughout this review, their path to clinical translation is fraught with unique challenges that distinguish them from the more established paradigm of kinase inhibition. A critical discussion of these limitations is essential to guide future research and maximize the chances of success. We summarize the major hurdles, their underlying causes, and promising strategies to address them. Several key areas warrant further emphasis: A primary concern in developing kinase activators is the risk of oncogenic transformation or other pathologies due to overactivation. Many kinases targeted for activation ( e . g ., Src, mTOR) are well-known oncogenes when mutated or overexpressed. This underscores the imperative for tissue-specific targeting and controllable activation mechanisms. To overcome the critical challenge of a narrow therapeutic window, innovative pharmacological and technological strategies are being developed. Prodrug approaches involve designing inactive precursors of kinase activators that are selectively converted to their active form within the target disease microenvironment. This activation can be triggered by disease-specific factors, such as elevated levels of specific enzymes ( e . g ., matrix metalloproteinases in tumors or inflammatory zones), or a characteristic pH, ensuring that kinase activation is spatially confined to pathological tissues. Concurrently, nanocarrier-based delivery systems offer a powerful solution for systemic administration. Nanoparticles, liposomes, or ADCs can be engineered to encapsulate kinase activators and functionalized with targeting ligands ( e . g ., peptides, antibodies) that recognize receptors abundant on specific cell types ( e . g ., vascular endothelial cells in ischemic tissues or cancer cells). This targeted delivery minimizes off-target exposure, maximizes the drug concentration at the site of action, and significantly reduces the risk of systemic toxicity, thereby effectively widening the therapeutic window. Most kinases have highly conserved ATP-binding domains, and traditional n-activators are prone to trigger off-target effects. In the future, it is necessary to combine allosteric site mining (such as the analysis of non-classical functions of CKB by the team of Zhejiang University) and AI-driven molecular design to develop highly selective activators. Single kinase activation may not be able to reverse the disease network with multi-channel interaction. Exploring multi-target synergistic activation strategies (such as AMPK/PGC-1 α activation to improve mitochondrial dysfunction) or space-time specific delivery systems (such as nano carriers targeting pathological tissues) is expected to improve the curative effect. The existing studies mostly focus on the catalytic function of kinases, and the lack of understanding of their non-enzymatic activity (such as the role of scaffold protein) restricts the development of new activators. It is necessary to systematically analyze the functional polymorphism of the kinase and its pathological correlation by combining with freezing electron microscopy, single-cell sequencing and other technologies. An individualized activator screening model was constructed based on patient genotypes (such as kinase mutation spectrum) and epigenetic characteristics. Develop light-controlled or pH-responsive intelligent activator to achieve precise spatio-temporal control of kinase activity. Integrating synthetic biology and systemic pharmacology, we designed and synthesized artificial kinases or engineered activator antibody conjugates to break through the functional limitations of natural kinases. To sum up, protein kinase activator therapy has entered the clinical transformation stage from concept verification, but its full application still needs to break through the technical bottleneck and deepen the mechanism cognition. With the integration and development of multi-omics technology and new drug models, this field is expected to open a more breakthrough era of precise treatment in human diseases ( Table 3 37 , 39 , 91 , 193 ). Table 3 Major challenges in developing protein kinase activators and potential strategies. Table 3 Challenge Underlying cause Potential consequence Promising mitigation strategy Ref. Narrow therapeutic window High conservation of kinase ATP-binding sites; risk of overactivation leading to oncogenic signaling or toxicity. Off-target effects, dose-limiting toxicity, limited clinical efficacy. Development of allosteric activators targeting unique regulatory sites. Prodrug designs activated specifically in diseased tissue ( e . g ., by tumor-specific enzymes). Use of targeted delivery systems ( e . g ., antibody–drug conjugates, ligand-conjugated nanoparticles). 39 Signaling complexity and feedback loops Kinases operate in highly interconnected networks; activation of one node may trigger compensatory feedback mechanisms. Kinases operate in highly interconnected networks; activation of one node may trigger compensatory feedback mechanisms. Systems biology approaches to model network responses. Rational combination therapies ( e . g ., pairing an activator with an inhibitor of a feedback node). Intermittent dosing schedules to preempt adaptive resistance. 193 Context-dependent biological effects The same kinase can play opposing roles ( e . g ., pro-survival vs . pro-death) in different tissues, disease stages, or genetic backgrounds. Unpredictable efficacy and potential for harm in unselected patient populations. ( e . g ., ULK1 in early vs . late-stage cancer). Development of companion biomarkers for patient stratification. Detailed understanding of kinase function in specific disease microenvironments. Exploration of tissue-specific delivery platforms. 91 Pharmacological hurdles in activator design Structurally mimicking the complex process of kinase activation ( e . g ., inducing conformational changes) is more challenging than designing inhibitors. Low success rate in identifying high-potency, selective small-molecule activators. Advanced techniques: Fragment-based screening, DNA-encoded libraries. AI and machine learning for predicting activator binding and efficacy. Focusing on natural products as sources of novel activator scaffolds. 37 Limited clinical validation The field is younger than kinase inhibitor research; most candidates are in preclinical or early clinical stages. Uncertainty regarding long-term safety, optimal dosing, and true therapeutic potential in humans. Expansion of clinical trials with sophisticated pharmacodynamic biomarkers. Utilizing adaptive trial designs to efficiently test hypotheses. Learning from the safety profiles of approved kinase inhibitors. 37 Major challenges in developing protein kinase activators and potential strategies.

Protein kinases, as key enzymes regulating protein phosphorylation, participate in cell proliferation, differentiation, apoptosis and metabolism through signal transduction networks 1 , 2 , 3 , 4 . Its dysfunction is closely related to tumors, cardiovascular disease, neurodegenerative disease and other pathological mechanisms 5 , 6 , 7 , 8 . Receptors are biological macromolecules composed of glycoproteins or lipoproteins that can recognize and bind bioactive molecules on the cell membrane or in cells and cause changes in cell function. Receptors can recognize and receive a specific signal by binding with ligands, amplify it accurately and transmit it to the inside of cells, so as to start a series of intracellular signal cascade reactions and produce specific biological effects. Some small molecules can bind to and excite receptors, showing corresponding physiological effects or drug effects ( Fig. 1 A). These small molecules are called activators of receptors. Salbutamol and clenbuterol are typical receptor activators 9 , 10 , 11 , which can activate the β 2 receptor distributed in the airway smooth muscle to produce bronchodilation, and are used to treat asthma. Adrenaline, as a complete activator in the cardiovascular system, can strongly stimulate the heart and blood vessels, and is used for emergency treatment and treatment of cardiovascular diseases. Dopamine receptor activators are used to treat Parkinson’s disease and other neurological diseases. Activators are generally divided into selective and non-selective. Selective can only promote a certain reaction, while non selective can promote a certain kind of reaction. Classic selective β 1 receptor activators currently include dobutamine. Classic selective β 2 receptor activators currently include salbutamol, terbutaline sulfate. Classic non selective beta receptor activators currently include isoproterenol ( Fig. 1 B). Figure 1 Kinase-specific activation mechanisms, molecular mechanisms of activator action and historical timeline of classical activators. Kinase-specific activation mechanisms, molecular mechanisms of activator action and historical timeline of classical activators. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1); US Food and Drug Administration (FDA); the guanylate cyclase (SGC); unc-51 like autophagy activating kinase 1 (ULK1); amyotrophic lateral sclerosis (ALS); interleukin-15 (IL-15); glucagon-like peptide-1 receptor (GLP-1R); glucose-dependent insulinotropic polypeptide receptor (GIPR); G-protein coupled receptor (GPCR). Figure 1 Kinase-specific activation mechanisms, molecular mechanisms of activator action and historical timeline of classical activators. Kinase-specific activation mechanisms, molecular mechanisms of activator action and historical timeline of classical activators. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1); US Food and Drug Administration (FDA); the guanylate cyclase (SGC); unc-51 like autophagy activating kinase 1 (ULK1); amyotrophic lateral sclerosis (ALS); interleukin-15 (IL-15); glucagon-like peptide-1 receptor (GLP-1R); glucose-dependent insulinotropic polypeptide receptor (GIPR); G-protein coupled receptor (GPCR). In recent years, the development of activators or inhibitors of protein kinases has become an important strategy for disease treatment 12 , 13 , 14 . Among them, activators can restore dysregulated signaling pathways by enhancing the activity of specific kinases, showing unique potential in disease treatment 13 . In the field of cancer, the nonclassical functions of protein kinases (such as metabolic enzyme kinase activity) have been confirmed to directly regulate the proliferation, apoptosis and immune escape of cancer cells 15 . Activators of classical signaling pathways such as MAPK and PI3K can induce differentiation or enhance immune response in specific tumor subtypes 16 , 17 . AMPK activators inhibit tumor growth by regulating energy metabolism 18 . In the cardiovascular system, the activation of protein kinase G (PKG) can relax vascular smooth muscle and inhibit platelet aggregation. Its activators (such as riociguat) have been used in the treatment of pulmonary hypertension 19 . In addition, AMPK activators show protective effects in heart failure models by improving myocardial energy metabolism and inhibiting fibrosis 20 , 21 . In neurodegenerative diseases, dysfunction of kinases often leads to abnormal phosphorylation of tau protein or aggregation of α -synuclein. Over activation of GSK-3 β is associated with Alzheimer’s disease (AD), and specific activators regulating its activity may reduce neuroinflammation 22 . The activators strategy targeting the interaction of APLP1/LAG3 can inhibit the transmission of α -synuclein in Parkinson’s disease 23 . The development of protein kinase activators started late compared with inhibitors, but with the in-depth study of the two-way regulation mechanism of kinase, its research has gradually accelerated. The development of protein kinase activators started late compared with inhibitors, but with the in-depth study of the two-way regulation mechanism of kinase, its research gradually accelerated. In 1875, pilocarpine, an M receptor activator, was isolated from the leaves of Pilocarpa . It has been used as an ophthalmic antihypertensive drug for more than a century. Ophthalmologists have done a lot of research work to prolong the efficacy and reduce side effects. Liposome as a slow-release drug delivery system of pilocarpine used in the treatment of glaucoma will have a good development prospect because of its good curative effect, small side effects, convenient use and other characteristics. In 1904, the N receptor activator nicotine was successfully synthesized. Nicotine, is the main alkaloid in tobacco, accounting for about 90% of the total alkaloids. It is closely related to insect resistance, and is also an important indicator of the quality of tobacco and cigarettes. Nicotine accumulation is the result of the co-expression of structural genes encoding the nicotine biosynthesis pathway, and structural genes are usually controlled by COI1, JAZ, MYC-2 and other regulatory genes. At present, most genes involved in the biosynthesis, regulation and transport of nicotine and nornicotine have been identified. In 1905, after solving the problem of separation and purification after reduction reaction, the German chemist Friedrich Stolz finally obtained the pure product of non-selective β activator adrenaline in the laboratory and put it into production the next year, which was the first time in human history that hormone was synthesized by artificial method. Dobutamine, as a selective β 1 receptor activator, was developed in the early 1970s and approved by the U.S. Food and Drug Administration (FDA), becoming the first drug for acute myocardial infarction complicated with heart failure. In the early 2000s, the first protein kinase inhibitor, Imatinib was approved, marking the beginning of targeted kinase therapy and promoting theoretical exploration of activators research 24 , 25 . In 2010, AMPK activator AICAR entered clinical trials for metabolic diseases and cardiovascular protection, confirming the feasibility of the kinase activation strategy 26 , 27 , 28 . More recently, the AMPK α activator IMM-H007 was reported to promote hepatic cholesterol and triglyceride metabolism and attenuate hypercholesterolemia and atherosclerosis in preclinical models 29 . In 2015, the guanylate cyclase (SGC) activator riociguat was approved by FDA for the treatment of pulmonary hypertension and became the first activator drug targeting kinase 19 , 30 . In 2017, the team of Sichuan University first designed and found the small molecule activator LYN-1604 targeting autophagy promoter ULK1 as a new candidate drug for triple negative breast cancer 31 . Subsequently, in 2022, it was verified that BL-918 is a small molecule activator of ULK1, which can induce cytoprotective autophagy and is used for the treatment of amyotrophic lateral sclerosis 32 . In 2024, IL-15 activator Anktiva (N-803) combined with Bacillus Calmette Guerin (BCG) was approved by the FDA for the treatment of non-muscle invasive bladder cancer with carcinoma in situ and patients with or without papilloma 33 , 34 . In 2024, BGM0504 as a GLP-1R/GIPR dual target activator, through molecular dynamics optimization, its receptor activator activity was three times that of tirzepatide, and showed better hypoglycemic, weight loss and improvement effect of nonalcoholic steatohepatitis (NASH) in mouse model. Its half-life supports weekly administration 35 . The phase II clinical trial showed that the weight of patients decreased by 3.24%–8.30%, and the blood glucose control was significant. Glp-1ra and its multi-target drugs have become the core of the treatment of diabetes and obesity. In the phase III clinical trial, oral administration of small molecule glp-1ra or forglipron reduced patients’ glycosylated hemoglobin by 1.3%–1.6%, and the weight of the highest dose group decreased by an average of 7.9% (about 16 pounds). It is expected to submit an application for weight loss indications by the end of 2025 36 , 37 . In parallel, combined activation of AdipoR1/2 and inhibition of elastin-derived peptide–EBP interaction by JT003 and V14 synergistically activated AMPK, enhanced mitochondrial function and ameliorated nonalcoholic fatty liver disease and liver fibrosis in mice 38 ( Fig. 1 C). Some activators have been identified and tested in preclinical models, with a few already entering clinical trials. Despite these advances, the optimal indications for therapeutic activation and the long-term safety of these methods still need to be determined. Due to various reasons such as research paradigm deviation, complex conformational sites leading to low selectivity and off target effects, only a small number of small molecule kinase activators have been approved for clinical treatment. Over the past two decades, a significant amount of kinase drug development resources has been focused on inhibitors 36 . In addition, kinase activation typically requires precise conformational changes, and the design of small-molecule activator requires simulating these dynamic processes, which is much more difficult than inhibitors. As discovered by UCL-TRO-1938, as a small molecule activators of the PI3K alpha subtype, 1938 conformational activation of PI3K alpha was achieved through multiple steps of enhancing the PI3K alpha catalytic cycle, resulting in local and global conformational changes in the PI3K alpha structure through different mechanisms 39 . More importantly, subtle overactivation may lead to signal cascade dysregulation, resulting in a narrow therapeutic window for kinase activators, while in complex kinase networks, single kinase activation may trigger negative feedback. Transient PKR-like endoplasmic reticulum kinase (PERK) activation has a protective effect; However, chronic ER stress and persistent PERK activation may be harmful to cellular health, and PERK overactivation is associated with many diseases 40 . Although the development of protein kinase activators still faces challenges such as low selectivity and off-target effect, its potential in disease treatment cannot be ignored. In the future, it is necessary to combine structural biology and artificial intelligence technology to design highly specific activators and explore their application in combination therapy. At the same time, in-depth analysis of the kinase “part-time” function and its interaction with epigenetic regulation will promote the development of precision treatment strategies.

The authors declare no conflicts of interest.