{"paper_id":"29e09556-c6d8-4a5a-b7f6-e04225a28f6f","body_text":"Off-target interaction of the amyloid PET imaging tracer PiB with acetylcholinesterase \n \nAlberto Granzotto 1,2,*, Rosa Fullone 1,2, Ludovico Miccoli 1,2,3, Manuela Bomba 1,2, Claudia Di Marzio 1,2, \nStefano Delli Pizzi 2,4, Giuseppe Floresta 5,6, Stefano L. Sensi 1,2,3,4 \n \n1 Center for Advanced Studies and Technology – CAST , University G. d’ Annunzio of Chieti-Pescara, Chieti, \nItaly  \n2 Department of Neuroscience, Imaging, and Clinical Sciences, University G. d’ Annunzio of Chieti-Pescara, \nChieti, Italy \n3 Institute of Neurology, SS Annunziata University Hospital, University G. d’ Annunzio of Chieti -Pescara, \nChieti, Italy \n4 Institute for Advanced Biomedical Technologies – ITAB, University G. d’ Annunzio of Chieti-Pescara, Chieti, \nItaly \n5 Department of Drug and Health Sciences, University of Catania, Catania, Italy \n6 Psychopharmacology, Drug Misuse and Novel Psychoactive Substances Research Unit, School of Life and \nMedical Sciences, University of Hertfordshire, Hatfield, United Kingdom \n \nKeywords: in silico studies; computational analysis; Alzheimer’s disease; molecular dynamics; molecular \ndocking; amyloid, tau \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n* To whom correspondence should be addressed: \nDr. Alberto Granzotto \nCenter for Advanced Studies and Technology – CAST \nDepartment of Neuroscience, Imaging, and Clinical Sciences \nUniversity G. d’ Annunzio of Chieti-Pescara,  \nVia L. Polacchi, 11, 66100, Chieti (CH), Italy \nEmail: alberto.granzotto@unich.it \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\nAbstract \n \nPittsburgh compound B (PiB) is a widely used Positron Emission Tomography (PET) tracer for detecting \namyloid-β (Aβ) deposits in Alzheimer’s disease (AD). While PiB is assumed to bind selectively to Aβ, \nemerging evidence suggests off-target interactions that may complicate PET signal interpretation. Here, \nwe report that PiB can interact with acetylcholinesterase (AchE), a key enzyme in the cholinergic system. \nSimilarity screening identified the AchE ligand thioflavin T (ThT) as the top structural analog of PiB. \nDocking studies and molecular dynamics simulations showed that PiB stably binds the peripheral anionic \nsite (PAS) of AchE, with binding energies comparable to ThT and clinically relevant AchE inhibitors. In \nvitro fluorescence-based assays confirmed this interaction and suggest an involvement of the PAS. These \nfindings indicate a stable off-target interaction between PiB and AChE with implications for interpreting \nPiB-PET signals in AD, particularly in regions with altered AchE expression or under AchE inhibitor \ntherapy. \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\nIntroduction \n \nPositron emission tomography (PET)-based biomarkers are largely employed in research and \nclinical settings for disease diagnosis and monitoring, patient stratification, or as an efficacy outcome of \ninterventions [1]. In Alzheimer’s disease (AD), PET tracers have been developed to quantitatively detect \nchanges in the accumulation of key pathological markers, like cortical amyloid-β (Aβ) deposits, \nhyperphosphorylated tau (p-tau) protein buildup, and neurodegeneration [2,3]. Alterations in these \nbiomarkers mirror disease progression and are the “gold standard” for diagnosing AD and for the early \ndetection of people at-risk of developing the condition [4,5]. The shift from a clinical- to a biomarker-\nbased definition of AD is also at the basis of the “ATN research framework”, a biological definition of the \ndisease (i.e., 'A' – amyloid, ‘T′ – tau, and ‘N′ – neurodegeneration) aimed at offering a quantifiable and \nunbiased staging of AD [4]. The approach is relevant since the pathological alterations of AD can occur \nand are detectable long before the onset of cognitive and behavioral symptoms [6,7]. Early identification \nof individuals in the very early stages of the condition represents a transformative step for the effective \ndevelopment and targeted implementation of disease-modifying interventions. \nAlterations of Aβ levels are widely recognized as one of the earliest molecular changes that can \nforeshadow the onset of AD pathology, although the specific contribution of Aβ to disease pathogenesis \nis debated [8,9]. Quantitative assessment of Aβ is either performed in biological fluids like liquor and \nplasma, where decreases in Aβ abundance reflect the cerebral deposition of the peptide, or by PET-\nbased imaging, where specific radioligands are employed to detect the presence of fibrillar Aβ (fAβ) \naggregates in the brain. Several Aβ radiotracers have been developed since the early 2000s with \nPittsburgh compound B (11C-PiB), a thioflavin T (ThT) analog, being the first of this class of imaging \nagents. The short half-life of 11C-PiB led to the development of fluorine-18 derivatives more suitable for \nclinical applications, like 18F-flutemetamol or the trans-stilbene-based compounds 18F-florbetapir and 18F-\nflorbetaben. Nevertheless, 11C-PiB is still broadly adopted in clinical research settings. \n Although these radioligands are widely employed for the diagnosis of AD and for monitoring \ntarget engagement of Aβ-targeting interventions, doubts have been cast on their specificity and \nsensitivity [10–13]. Previous studies demonstrated that 2-aryl-6-hydroxybenzothiazole-based tracers can \neffectively bind to off-target molecules, like sulfotransferases, that likely contribute to PET signals \nunrelated to the overall Aβ load [10,12]. However, it is unclear whether this class of Aβ radioligands has \nadditional off-target effects. \nThis study aims to investigate PiB binding characteristics at the molecular level and, by \nemploying unbiased in silico screening, docking calculations, molecular dynamics (MD) simulations, and \nin vitro assay, we surveyed for potential novel binding partners unrelated to Aβ pathology. \n \n \nMaterials and methods \n \nReagents and chemicals \n PiB was purchased from TargetMol. Acetylcholinesterase from Electrophorus electricus (eeAchE), \nCatalase from bovine liver and all the other chemicals were from Sigma-Aldrich.  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\nLibrary screening \nSimilarity screening for the PiB amyloid PET tracer was performed with the SwissSimilarity 2021 \nWeb Tool (http://www.swisssimilarity.ch/ [14,15]) on August 3, 2024. The search was limited to ligands \npresent in the Protein Data Bank (LigandExpo; 19500 compounds) using a consensus 2D/3D screening \nusing a score based on both FP2 Tanimoto coefficient and Electroshape-5D Manhattan distance [14]. \nScreening scores and SMILES notations were downloaded for further analysis. \n \nMolecular modeling \nAll the molecules investigated in this study were downloaded as three-dimensional conformer \n.sdf files from PubChem [16]. The Energy Minimization Experiment function, using YASARA AutoSMILES \nfor automatic force field parameter assignment, was used to optimize the 3D structure before docking. \nDocking calculations were performed using VINA default docking parameters as implemented in \nthe YASARA suite, as previously described [17]. Briefly, the crystal structure of the target protein was \ndownloaded from the PDB (PDB ID: pdb_00004ey7), a cell encompassing all atoms extending 5 Å from \nthe surface of the structure of the ligand was generated, and the crystallized ligand was removed. Global \nligand docking was performed using VINA using the default parameters and further refined with VINA \nLocal Search [18]. \nThe molecular dynamics simulations of the acetylcholinesterase complexes were run with the \nsame YASARA suite [19] by employing the macro md_runfast. A cuboid periodic simulation cell extending \n20 Å from the protein surface was set and filled with water (density: 0.997 g/mL). The setup included an \noptimization of the hydrogen bonding network [20] to increase the solute stability, and a pKa prediction \nto fine-tune the protonation states of protein residues at pH 7.4 [21]. NaCl ions were added at a \nphysiological concentration of 0.9%. After steepest descent and simulated annealing minimizations to \nremove clashes, the simulation was run for 300 nanoseconds using the AMBER14 force field [22] for the \nsolute, GAFF2 [23] and AM1BCC [24] for ligands and TIP3P for water. The cutoff was 8 Å for Van der \nWaals forces [25], no cutoff was applied to electrostatic forces (using the Particle Mesh Ewald \nalgorithm) [26]. The equations of motions were integrated with a multiple timestep of 2.5 fs for bonded \ninteractions and 5.0 fs for non-bonded interactions at a temperature of 298K and a pressure of 1 atm \n(NPT ensemble) using algorithms described previously [27]. MD conformations were recorded every \n250 ps. The energies of binding and the MD trajectory have been calculated using the \nmd_analyzebindenergy macro implemented in the YASARA suite employing the MM/PBSA method as \npreviously described [28]. Ligand movement root mean square displacement (RMSD) was calculated \nwith the YASARA md_analyze function after superposing on the receptor. \n \nPiB spectra \n A 20 µM PiB (0.8 % DMSO final concentration) solution was prepared in a 100 mM potassium \nphosphate buffer solution (KPi, pH 7.4). Absorbance spectrum was measured by employing a \nPerkinElmer Lambda 35 spectrophotometer (Range: 200 – 900 nm; slit: 2nm; resolution: 1 nm; speed: \n240 nm/min). Fluorescence emission spectrum was measured with a BioTek Synergy H1 plate reader (Ex \nλ: 350 nm; Em range: 380 – 700 nm; resolution: 1 nm; gain: 60 a.u.). \n \nTurbidity assay \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\nThe turbidity assay was performed as previously described [29]. In brief, the absorbance of increasing \nconcentrations of PiB (1.56 µM to 1600 µM) was measured at 405 nm using a PerkinElmer SPECTRAmax \n190 microplate reader. Absorbance readings from the buffer alone (KPi containing 0.8% DMSO) served as \nreference. The purpose of the assay was to determine the highest PiB concentration that does not result \nin precipitation of the compound. \n \nFluorescence-based interaction assay \n A fluorescence-based binding assay was performed to assess the potential interaction between \nPiB and eeAChE. PiB (25 µM final concentration; dissolved in 100mM KPi, 0.1 % DMSO) was incubated in \nvitro either in the presence or absence of 2 µg of eeAChE in a total volume of 50 µL. Incubations were \ncarried out for 5 hours at 30 °C under gentle agitation (1100 rpm). Following incubation, each mixture \nwas filtered using Amicon Ultra-0.5 centrifugal filters with a 30 kDa molecular weight cutoff (Millipore), \ncentrifuged at 14,000 × g for 10 minutes at room temperature to separate unbound PiB from AChE-\nbound PiB. The retentate, containing eeAChE and any bound PiB, was recovered and transferred to a \nblack walled 96-well plate for fluorescence measurement. Fluorescence was measured using a BioTek \nSynergy H1 plate reader with excitation at 350 nm and emission at 440 nm. The retentate was \nsubsequently spotted onto a nitrocellulose membrane, stained with Ponceau S, and imaged to assess \nprotein recovery. \n \nPropidium iodide displacement assay \n To evaluate the interaction between PiB and the PAS of eeAChE, we employed a propidium \niodide (PI) displacement assay. A total of 25 U of eeAChE were incubated overnight with PI (1 µM), either \nalone or in the presence of PiB (20 µM, 0.8% DMSO). A parallel experiment using donepezil (20 µM, 0.8% \nDMSO) served as a positive control. PI fluorescence was measured using a BioTek Synergy H1 plate \nreader with excitation at 535 nm and emission at 630 nm. Background fluorescence from PI alone was \nsubtracted from all readings. Data were then normalized as Fx/Fvehicle, where Fx represents the PI \nfluorescence for each condition, and Fvehicle is the PI fluorescence in the presence of eeAChE and 0.8% \nDMSO. \n \nStatistical analysis \n Microsoft Excel (Microsoft) and OriginPro (OriginLab) were employed for statistical analysis and \ndata plotting. Data in Fig. 2 are represented as mean ± 1 standard error of the mean (s.e.m.); data points \nrepresent individual experiments. Exact P values are reported for each relevant comparison. The number \nof replicates and the statistical test used are provided in the figure legends. \n \n \nResults \n \nTo identify potential off-target partners of Aβ PET tracers, we performed an unbiased screening \nof biologically relevant molecules that show structural similarity with PiB by employing the \nSwissSimilarity 2021 Web Tool. Our analysis returned the score of 400 molecules (Fig. 1A and \nSupplementary Table 1) with ThT being the top-scoring molecule (score 0.996). More importantly, ThT \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\nwas identified as the molecule is a ligand for acetylcholinesterase (AchE) in the PDB (PDB ID: \npdb_00002j3q). To test the hypothesis that PiB interacts with AchE, we performed docking studies within \nthe AchE pocket using the crystal structure of the human AchE (PDB ID: pdb_00004ey7). AchE has two \nbinding sites: the catalytic site and the peripheral anionic site (PAS; [30,31]). We focused on the latter, \nlocated at the entrance of the catalytic gorge, since it mediates the interaction of AchE with ThT [32–34]. \nAnalysis of docking results shows that PiB has binding energy comparable with that of ThT (8.603 and \n8.771 kcal/mol, respectively; Fig. 1B). Binding energy of clinically approved AchE inhibitors (donepezil, \ngalantamine, and rivastigmine) was calculated for comparison (Fig. 1B). Fig. 1C and D show the 2D and \n3D poses and the interaction of PiB with the amino acid residues in the AchE PAS. PiB forms a π–π-sulfur \ninteraction with residue Phe297 along with several hydrophobic, alkyl, and Van der Waals interactions \nwith residues Trp86, His447, Tyr337, Phe338, Tyr72, Trp286, Phe295, Tyr341, and Tyr124 (Fig. 1C, D). \nWe further investigated the PiB-AchE complex by performing a 300 ns molecular dynamics (MD) \nsimulation. Analysis of the energy of binding shows that PiB maintains a high and stable binding energy \nthroughout the simulation (Fig. 1E). The stability of the PiB-AchE complex is also supported by the RMSD \nanalysis of the ligand movement after superimposing the molecule on the enzyme structure (Fig. 1F). \nAfter a stabilization phase, the ligand remains within the AchE PAS. The compound exhibits only a few \nmodest, sharp fluctuations that return to baseline levels during the simulation (Fig. 1F). We attribute the \nstability of the complex to the sulfur interaction, along with the dense network of hydrophobic \ninteractions that keep PiB within the AchE PAS.  \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\n \n \nFigure 1. Identification and in silico characterization of AchE as a potential target of PiB. (A) The plot \nillustrates the similarity score of each compound screened with the SwissSimilarity 2021 Web Tool. (B) \nThe histogram depicts the binding energy calculation of the listed ligands after docking on AchE. (C - D) \nTwo-dimensional (C) and three-dimensional (D) docking poses and interactions of PiB in the AchE PAS. \nThe dashed yellow line indicates π–π-sulfur interaction; dashed pink lines indicate π–π-alkyl interactions; \nmagenta lines indicate π–π and T-shaped interactions; green residues show van der Waals interactions. \n(E - F) Time course of energy of binding (E) and root mean squared displacement (RMSD; F) for the PiB-\nAchE complex over a 300 ns MD simulation. \n \nTo further evaluate PiB binding to the PAS of eeAChE, we performed PI displacement, an assay \nused for probing the interaction of candidate drugs with the PAS of AchE [35]. The binding of PI to the \nPAS increases dye fluorescence. Meanwhile its displacement by PAS-interacting compounds leads to \nsignal reduction [36]. In the presence of 20 µM PiB, PI fluorescence was reduced by approximately 15% \ncompared to control conditions (Fig. 2F). Although this difference did not reach statistical significance (P \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\n= 0.10), the data suggest a potential trend toward PI displacement. The reduction increased to ≈ 78% in \nthe presence of the high-affinity, PAS-binding AchE inhibitor donepezil (20 µM; Fig. 2F) [30,37]. \nTogether, these findings support the formation of a stable PiB–eeAchE complex in vitro and are \nconsistent with the possibility that PiB interacts with the PAS of the enzyme. \n \n \n \n \nFigure 2. Experimental validation of the formation of the PiB-eeAchE complex. (A) Absorption (dashed \nline) and emission (solid line) normalized spectra of PiB in KPi buffer (pH 7.4). (B) The bar graph depicts \nnormalized fluorescence of PiB following incubation of the compound with or without eeAchE (2 µg) and \nsize-exclusion filtration (PiB n = 6 and PiB + eeAchE n = 5 independent experiments). (C) Ponceau S \nstaining of the retentate was spotted onto a nitrocellulose membrane to assess protein recovery. (D) The \nbar graph depicts normalized fluorescence of PiB following incubation of the compound with or without \nCatalase (2 µg) and size-exclusion filtration (PiB n = 4 and PiB + Catalase n = 4 independent experiments). \n(E) Ponceau S staining of the retentate was spotted onto a nitrocellulose membrane to assess protein \nrecovery. (F) The bar graph depicts normalized fluorescence of PI following incubation with eeAchE in \nthe presence of vehicle (Control; 0.8% DMSO), PiB (20 µM, 0.8% DMSO), or Donepezil (20 µM, 0.8% \nDMSO). Note the ≈ 15% signal reduction in the presence of PiB. In B and D, the comparison of mean \nvalues was assessed by the Mann-Whitney U Test. In F, mean values were compared by one-way ANOVA \nfollowed by Tukey's post-hoc test. \n \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\nDiscussion \n  \nIn this study, we provide computational and experimental evidence that the amyloid PET tracer \nPiB can bind AchE, suggesting a previously unrecognized off-target interaction. Our findings extend prior \nobservations that ThT-based compounds may interact with non-amyloid targets and raise important \nquestions on the specificity of PiB and related PET tracers used in AD research and diagnostics [32]. \nOur in silico similarity screening identified ThT – a known AchE ligand – as the compound most \nstructurally related to PiB among biologically relevant molecules in the PDB database, pointing to a \npotential interaction between PiB and AchE. We further tested this hypothesis using molecular docking \nand MD simulations. Docking results showed that PiB binds the PAS of AchE with binding energy \ncomparable to ThT and within the range of clinically relevant AchE inhibitors. PiB establishes π–sulfur \nand hydrophobic interactions with residues located in the PAS and near the gorge of the active site, like \nPhe297, Trp286, Tyr337, and His447. These residues were found to be key for the interaction with AchE-\ntargeting drugs [30]. MD simulations further confirmed the persistence of these interactions, indicating a \nstable and energetically favorable complex. \nWe validated the computational predictions with a binding assay that exploits the intrinsic \nfluorescence properties of PiB, supporting the formation of a stable PiB–AchE complex in vitro and \nsuggesting that the interaction occurs at the PAS of the enzyme. It is important to notice that a more \nthorough investigation of the interaction between PiB and AchE was hampered by both the \nphysicochemical properties of PiB and the sensitivity of AchE to PiB-compatible solvents. We found that \nin conditions suitable for the AchE enzymatic assay, PiB began to precipitate at concentrations above 25 \nµM (Supplementary Fig. 1B). The use of alternative solvents or surfactants was unsuccessful \n(unpublished observations). Moreover, higher concentrations of DMSO were shown to substantially \nimpair AchE activity [38]. In a further attempt to directly examine PiB-PAS interaction, we also tested an \nin vitro competition assay in the presence of donepezil [30,37]. However, preliminary control \nexperiments showed a substantial spectral overlap between PiB and donepezil (Supplementary Fig. 1B), \nmaking fluorescence-based comparisons difficult to interpret and prone to bias. While these technical \nconstraints limit our ability to perform orthogonal or competitive binding assays, they do not undermine \nthe core observation that PiB interacts with eeAchE, as suggested in our fluorescence-based filtration \nassay and PI displacement. \nThe identification of AChE as a potential off-target of PiB has several implications. First, it \nimposes the need to carefully interpret PET signals in brain regions where AchE is abundantly expressed, \nparticularly in early-stage or atypical AD presentations, where amyloid deposition may not be the unique \ncontributor to the tracer uptake. Second, given that AchE expression and activity can change in the aging \nbrain and neurodegenerative conditions beyond AD [39,40]. The off-target binding of PiB to AchE could \ncontribute to false positives or elevated baseline signals in specific populations. Third, the PiB signal \ncould be influenced by the use of AchE inhibitors that act by binding the PAS of the enzyme, leading to \nfalse negative results. Moreover, the high lipophilicity of PiB could also explain the elevated retention of \nthe tracer in lipid-enriched white matter regions [41,42]. \nOur findings align with previous reports of off-target binding for other radiotracers used in AD, \nfor whom interactions with enzymes like monoamine oxidases and sulfotransferases have been reported \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\n[10,11,43]. In addition, our similarity virtual screening does not rule out the existence of additional PiB \nbinding partners. \nHowever, to our knowledge, this is the first study to indicate an interaction between PiB and \nAchE at both the computational and experimental levels. While the functional consequences of this \nbinding remain to be explored, such interactions may alter AchE activity or affect PiB signals in vivo. \nFurther studies using radiolabeled PiB and AchE inhibitors in vivo are warranted to confirm whether this \ninteraction occurs under pathophysiological conditions and contributes to PET signals. \nIn conclusion, these results underscore the importance of integrative approaches combining \ncomputational modeling with biochemical validation to uncover and assess the biological relevance of \nsuch interactions. \n  \n \nAcknowledgments \nA.G. is supported by the European Union - Next Generation EU, Mission 4 Component 1, CUP: \nD53D23019280001. \nAI-assisted technology (ChatGPT 4o) has been used in the writing process to improve the readability and \nlanguage of the manuscript. \n \n \nReferences \n \n[1]  Nasrallah I, Dubroff J (2013) An overview of PET neuroimaging. 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It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\nSupplementary Information \n \n \nSupplementary Figure 1. Limited PiB solubility and spectral properties of PiB and donepezil complicate \nfluorescence- and enzymatic-based assays. (A) The scatter plot depicts absorbance of PiB measured \nacross increasing concentrations (1.56 – 1600 µM) and reveals precipitation above 25 µM, limiting its use \nin assays requiring higher concentrations. (B) Normalized absorbance spectra of PiB (blue) and donepezil \n(orange) highlight substantial spectral overlap which complicates interpretation of fluorescence-based \ncompetition assays involving both compounds. Data are representative of at least two independent \nexperiments. a.u., arbitrary units.  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\n \nSupplementary table 1 \n \nLigand \nPDB ID \nSimilarity \nScore \nSMILES \nTFL 0.996 CN(C)C1=CC=C(C=C1)C1=[N+](C)C2=CC=C(O)C=C2S1 \nGK1 0.767 CC1=CC=C(O)C=C1NC1=CC=NC2=CC(=CC=C12)C1=CSC(C=O)=N1 \nEVW 0.715 NC1=NC2=CC=C(C=C2S1)C(=O)NC(C1=CC=CC=C1)C1=CC=CC=C1 \nUUL 0.672 OC1=CC=C(NC2=NC(=CS2)C2=CC=C(Cl)C=C2)C=C1 \nJMT 0.616 OC1=CC=C2NC=C(CN3CCN(CC3)C3=NC4=CC=CC=C4S3)C2=C1 \n09H 0.606 O=C(NC1=CC=CC=C1N1CCNCC1)C1=CSC(=N1)C1=CC=C2OCCC2=C1 \nJMW 0.594 OC1=CC=C2NC=C(CN3CCN(CC3)C3=NC4=CC(Cl)=CC=C4S3)C2=C1 \nEWK 0.585 NC1=NC2=CC=C(SCC3=CC=C(C=C3)C(=O)NCC3=CC=CC=C3)C=C2S1 \nEWT 0.574 NC1=NC2=CC=C(C=C2S1)C(=O)NCC1=CC(Cl)=C(Cl)C=C1 \n2WJ 0.565 CC(=O)NC1=NC2=CC=C(C=C2S1)C1=CC=CN=C1 \nAQE 0.563 C1CC[C@@]2(CCCN(C2)C2=C3C(NC=C3C3=NC=CS3)=NC=C2)NC1 \n94U 0.522 CCN(CC)C1=CC=C(NC(=O)C2=CC3=C(N2)N=CS3)C=C1 \nG4A 0.514 CN1\\\\C(OC2=CC=CC=C12)=C\\\\C1=[N+](CCCS(O)(=O)=O)C2=CC=CC=C2S1 \n3TI 0.51 OC1=CC=C(C=C1)\\\\N=C\\\\C1=C2C=CC=CC2=CC=C1O \nN0E 0.505 OC1=CC=C(NC(=O)CCC2=CC=CC=C2)C=C1 \n2JR 0.5 C1CCN(C1)C1(CCCCC1)C1=CN=C(S1)C1=CC=C2NC=CC2=C1 \nEV8 0.498 COC(=O)C1CCN(CC1)C(=O)C1=CC=C(CNC(=O)C2=CC=C3N=C(N)SC3=C2)C=C1 \nCP9 0.497 CC1=NC2=CN=CC=C2N1C1=CC=C(CN2C(=O)SC3=CC=CC=C23)C=C1 \nX1H 0.492 COC1=CC=C(C=C1)C(=O)C1=C(SC2=CC(O)=CC=C12)C1=CC=C(O)C=C1 \nB4K 0.484 CC(=O)NC1=C2C=CC(=NC2=NN1)C1=CC=C(O)C(O)=C1 \nP2X 0.48 CC(C)N1N=C(C2=CC3=CC(O)=CC=C3N2)C2=C(N)N=CN=C12 \nRU5 0.475 NC(=O)C1=CC=C2NC(=NC2=C1)C1=CC=C(OC2=CC=C(Cl)C=C2)C=C1 \n3F4 0.474 OC1=CC=C(C=C1)C1=NC(=O)C2=CC=CC=C2N1 \nMKY 0.472 CCOC(=O)CN1\\\\C(SC2=CC(O)=CC=C12)=N\\\\C(N)=N \nOFI 0.471 CCCC(=O)NC1=NNC2=CC(=CC=C12)C1=CC=C(O)C=C1 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\nNU3 0.466 NC(=O)C1=C2N=C(NC2=CC=C1)C1=CC=C(O)C=C1 \nLNJ 0.462 COC1=CC=C(CC2=CC=C(C=C2)C2=CSC(N)=N2)C=C1 \nC2J 0.442 OC1=CC=C(C=C1)C1=CC=C2C(NN=C2NC(=O)C2CC2)=C1 \n57X 0.44 CCCN1C2=NNC(C3=CN=C(S3)C3=CC=CN=C3)=C2C=CC1=O \n972 0.419 CC(C)COC1=CC=CC(C2=NC3=CC(C(N)=N)=C(Cl)C=C3N2)=C1O \nJV5 0.418 CC1=CC=CC=C1OCC(=O)NC1=CC2=NNN=C2C=C1 \n917 0.417 CC(=O)NC[C@H]1CN(C(=O)O1)C1=CC=C(C=C1)C1=CN=CS1 \n2RE 0.416 OC1=CC=C(C=C1)C1=NC(=C(N1)C1=CC=NC=C1)C1=CC=C(F)C=C1 \n0HD 0.416 O=C(NCCC1=CC=CC=C1)NC1=CC2=CNN=C2C=C1 \n656 0.413 CC(C)COC1=CC=CC(C2=NC3=CC(=CC=C3N2)C(N)=N)=C1O \n3U6 0.411 NC1=NN=C(S1)C1=CC=C2NC=C(C2=C1)C1=NC(NC2CCCC2)=CC=C1 \nD58 0.408 C[C@H]1NCCC[C@@H]1NC1=C2C=C(SC2=C(C=N1)C(N)=O)C1=CC=CC=C1 \nEK7 0.404 CN(C)C1=C2C(CCC3=C2N=C(NC2=CC=CC(O)=C2)N=C3)=C(S1)C#N \n3TX 0.4 OC1=CC=C(C=C1)N1C=C(N=N1)C1=NC2=CC=CC=C2C=C1 \n3J7 0.4 CC(C)(N)CNC1=C2C=CN=CC2=NC(=N1)C1=CC2=CNN=C2C=C1 \n41Z 0.394 CC1=NC2=C(C=CC=C2C(NCC2=C(C)C=CC=C2C)=C1)C(N)=O \n97K 0.393 O=C1N=C(NC2=CC=CC=C12)C1=CC2=CNN=C2C=C1 \nI0D 0.379 CN1CCC2=C(C1)C=CC=C2NC1=C(Cl)C(=O)N(C)N=C1 \n79X 0.379 COC1=CC2=C(C=C1OC)C1=CC3=CC(O)=CC=C3N1C2=O \n4GM 0.379 NC(=O)C1=CC=C(NCC2=CC=CC=C2O)N=C1 \nIOK 0.377 C[C@H](CCC1=CC=C(O)C=C1)NC(=O)CC1=C(NC2=CC=CC=C12)C1=CC=CC=C1 \n3T9 0.376 COC1=CC(=CC=C1O)C1=NC2=NNC(=C2C=C1)C1=CC=CC=C1 \n655 0.374 NC(=N)C1=CC=C2NC(=NC2=C1)C1=C(O)C(OC2CCCC2)=CC=C1 \nWTF 0.373 CCS(=O)(=O)C1=CC=CC=C1C(=O)N1CCN(C[C@@H]1C)C1=NC2=CC=C(F)C=C2S1 \nWAM 0.37 COC1=CC(\\\\C=C\\\\C2=[N+](C)C3=CC=CC=C3C(=C2)C(N)=O)=CC=C1O \nC70 0.368 NC(=O)C1=C2SC(=CC2=C(N[C@H]2CCCNC2)N=N1)C1=CC=C(Cl)C=C1 \nNUW 0.367 CNC(=O)C1=CC=C2N(CC3CCN(CC3)C(C)=O)C(=NC2=C1)C1=CC(C)=C(O)C(C)=C1 \n4KK 0.365 COC1=CC=CC(CC(=O)NC2=NC(=CS2)C2=CC=NC=C2)=C1 \n824 0.365 OC1=CC2=C(NC3=C2C2=C(C(=O)NC2=O)C(=C3)C2=CC=CC=C2)C=C1 \n950 0.364 CC(C)COC1=CC=CC(C2=NC3=CC(F)=C(C=C3N2)C(N)=N)=C1O \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\n879 0.364 NC1=NC(CN2C(=CC=C2C2=CC=CC=C2Cl)C2=CC=C(OC3=CN=CN=C3)C=C2)=CC=C1 \n133 0.364 CC(C)COC1=C(O)C(=CC=C1)C1=NC2=CC(F)=C(C=C2N1)C(N)=N \nG4E 0.362 CC1=CC=CC(NC2=NNC(=N2)C2=CC=C(OC3=CC=NC=C3)C=C2)=C1C \nCJH 0.361 CCOC1=CC=CC(=C1)C1=CC=C(NC(=O)C(C#N)C(C)=O)C=C1 \n6QJ 0.356 CC1=CC(=CC=C1O)C1=CC=CC(=N1)C(=O)C1=CC=C(F)C(O)=C1 \n9ET 0.354 CC(=O)OC[C@]1(C)OC2=C(C=C1)C1=C(C=C2C)C2=C(N1)C=C(O)C=C2 \nYVQ 0.353 N1C=C(C=N1)C1=CN2C(C=N1)=NC=C2C1=CNC2=CC=CC=C12 \nQ4A 0.352 COC1=CC2=NC(=NC(NC3CCN(CC4=CC=CC=C4)CC3)=C2C=C1OC)N1CCCN(C)CC1 \n0VN 0.35 CC(C)(C)C1=CC=C(NC2=NC3=CC(=CC=C3N2)C#N)C=C1 \nQP8 0.347 CC(C)(C)OC(=O)N1CCN(CC1)C1=CC(=NN=C1N)C1=CC=CC=C1O \n859 0.347 NC(=O)C1=CC=CC=C1NC1=CC=NC(NC2=CC=CC(O)=C2)=N1 \n6W3 0.346 CN1C(=CC2=C1C=CS2)C(=O)NC1=CC=CC=C1COC1=CC=C(OC2CCN(C)CC2)C=C1 \nC72 0.345 NC(=O)C1=C2SC(=CC2=C(N[C@H]2CCCNC2)N=C1)C1=CC=C(Cl)C=C1 \n28C 0.345 CC1=NN2C=NN=C2C(NCCC2=CC=C(O)C=C2)=C1 \nU81 0.343 BrC1=CC2=C(OCC[C@H]2NCCCNC2=CC(=O)C3=C(N2)C=CS3)C(Br)=C1 \nA3F 0.343 COC1=CC(=CC(OC)=C1OC)C1=CC(=CN=C1N)C1=CC=CC(O)=C1 \n6H2 0.343 OC1=CC=C(C=C1O)C1=CN2C=CC=CC2=N1 \n2YX 0.341 NC1=NC(=O)C2=CC3=C(NC(NCCC4=CC=C(C=C4)C#N)=N3)C=C2N1 \nZZF 0.34 CC1=CC=C(OC2=CC=NC(NC3=CC=C(C=C3)S(N)(=O)=O)=C2)C(C)=N1 \nMCV 0.338 COC1=CC=C(OC)C(CCC2=CSC3=NC(N)=NC(N)=C23)=C1 \n8UN 0.338 C[C@@H](C1CCCCC1)N1C2=CC=C(C=C2N=C1C1=CC2=C(OCO2)C=C1Br)C(=O)NC1=CC=C(C=C1)C#N \nPKJ 0.337 CC1=CC=C(C=C1)C1=CSC2=NN=C(SCC(=O)NC3=CC=C4OCOC4=C3)N12 \nO1Q 0.332 CC1=CC=CC(=C1)N1N=CC=C1C1=CC(Cl)=C2N=NN(C2=C1)C1=CC2=NNC=C2C=C1 \nD62 0.332 COC1=CC=C(C=C1OC)C1=NN(C2CCN(CC2)C2=C3C=CSC3=NC(N)=N2)C(=O)[C@@H]2CC=CC[C@H]12 \nCK6 0.332 CNC1=NC(C)=C(S1)C1=CC=NC(NC2=CC=C(O)C=C2)=N1 \n826 0.332 OC1=CC=C(CN2C3=C(CCN(C3)C(=O)C3=CC=C(O)C=C3)C3=CC=CC=C23)C=C1 \nKTQ 0.331 COC1=CC=C(CCNC2=C(N=C3C=CC=CN23)C2=CC=C(C=C2)[N+]([O-])=O)C=C1 \n8HZ 0.331 CC1=CC=C(NC2=C(N=C3N2C=CC=C3C)C2=CC=C(O)C=C2)C=C1 \n5ES 0.33 OC1=CC=C(C=C1)C(=CC1=CC(NC2=CC=C(F)C=C2)=CC=C1)C1=CC=C(O)C=C1 \n5C4 0.33 CC(=C(C1=CC=C(O)C=C1)C1=CC=C(O)C=C1)C1=CC=CC(NC2=CC=CC=C2)=C1 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 21, 2025. ; https://doi.org/10.1101/2025.05.16.654428doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}