ACS Pharmacology & Translational Science

The PACAP receptor PAC1 is a Gs-coupled family B1 GPCR for which the highest-affinity endogenous peptide ligands are the pituitary adenylate cyclase-activating peptides PACAP38 and PACAP27, and whose most abundant endogenous ligand is PACAP38. PACAP action at PAC1 is implicated in neuropsychiatric disorders, atherosclerosis, pain chronification, and protection from neurodegeneration and ischemia. As PACAP also interacts with two related receptors, VPAC1 and VPAC2, highly selective ligands, both agonists and antagonists, for PAC1 have been sought. To date, the peptide PACAP(6-38) and polypeptide M65, which is related to maxadilan, a sandfly vasodilator peptide, have been identified as selective for PAC1. Several non-peptide small molecule compounds (SMOLs) have been reported to be specific antagonists at PAC1, albeit their specificities have not been rigorously documented. Here, we present a platform of cellular assays for the screening of biologically relevant antagonists at PAC1 and show that some currently proposed SMOL antagonists do not have activity in this cell reporter assay, while we confirm that PACAP(6-38) and M65 are competitive antagonists. We have used this assay system to explore other peptide antagonists at PAC1, guided by molecular dynamics analysis of the PACAP-PAC1 interaction based on cryo-EM structural models of PAC1 complexed with a number of biologically active ligands. The affinity-trap model for the PAC1-ligand interaction successfully predicts the engagement behavior of PACAP27 and PACAP38 peptide-based PAC1 inhibitors. In particular, C-terminal deletants of PACAP(6-38) that maintain equipotency to PACAP(6-38) allow the shorter sequence to function as a scaffold for further peptide-based antagonist exploration.

The inhibitory analysis of intracellular signaling pathways is widely employed to gain insight into molecular mechanisms underlying diverse physiological processes. Unfortunately, the essential drawback of this basically effective methodology is that many, if not all, inhibitors, antagonists, modulators, and blockers can affect cellular functions not only acting through specified cellular targets, but also causing off-target effects. In particular, the class I phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and its PI3K-inactive structural analog LY303511 have been shown to affect agonist-induced Ca2+ signaling in cells of various types independently of PI3K activity. Here we studied serotonin-induced Ca2+ signaling in HEK293 cells expressing the recombinant mouse 5-HT2C receptor and analyzed the effects of LY294002 and LY303511 on cell responsiveness. As shown with Ca2+ imaging, both LY294002 and LY303511 affected intracellular Ca2+ but via distinct mechanisms. LY294002 suppressed responsiveness of assayed cells to serotonin in a manner suggesting that this substance acted as a competitive antagonist of the 5-HT2C receptor. In turn, LY303511 itself triggered Ca2+ transients in 5-HT2C-positive cells, exhibiting traits of a 5-HT2C agonist. In support of these findings, molecular docking and molecular dynamics simulations validated the binding of both LY294002 and LY303511 to the 5-HT2C receptor and occupying its orthosteric site. Altogether, physiological findings and computational data suggested that the observed effects of these compounds were most likely mediated by extracellular mechanisms associated with the direct interaction of both with the 5-HT2C receptor. This expands the list of non-specified cellular targets of LY294002 and LY303511 with 5-HT2C subtype of serotonin receptors.

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, previously developed D1R selective agonists possess a catechol moiety which displays poor pharmacokinetic properties. The first selective non-catechol D1R agonists were recently discovered and unexpectedly many of these ligands showed G protein biased signaling. Here, we investigate both catechol and non-catechol D1R agonists to validate potential biased signaling and examine if this impacts agonist-induced D1R endocytosis. We determined that most, but not all, non-catechol agonists display G protein biased signaling at the D1R and have reduced or absent {beta}-arrestin recruitment. A notable exception was compound (Cmpd) 19, a non-catechol agonist with full efficacy at both D1R-G protein or D1R-{beta}-arrestin pathways. In addition, the catechol ligand A-77636 was a highly potent, super agonist for D1R-{beta}-arrestin activity. When examined for agonist-induced D1R endocytosis, balanced agonists SKF-81297 and Cmpd 19 induced robust D1R endocytosis while the G protein biased agonists did not. The {beta}-arrestin super agonist, A-77636, showed significantly increased D1R endocytosis. Moreover, {beta}-arrestin recruitment efficacy of tested agonists strongly correlated with total D1R endocytosis. Taken together, these results indicate the degree of D1R signaling functional selectivity profoundly impacts D1R endocytosis regardless of pharmacophore. The range of functional selectivity of these D1R agonists will provide valuable tools to further investigate D1R signaling, trafficking and therapeutic potential. Significance StatementThe D1R is a validated therapeutic target and the recently discovered non-catechol D1R agonists have translational potential. We have systematically characterized several structurally distinct D1R agonists including non-catechols with balanced or G protein biased activity. When examined for agonist-induced D1R endocytosis, balanced agonists induced robust D1R endocytosis while G protein biased agonists did not. These results indicate the degree of D1R signaling functional selectivity profoundly impacts receptor endocytosis. This work also independently validates agonist tools to further investigate D1R activation in basic and translational research.

Synthetic cannabinoid receptor agonists (SCRAs) represent one of the most rapidly growing classes of novel psychoactive substances (NPS) and exhibit higher efficacy and potency at CB1 and CB2 cannabinoid receptors (CB1 and CB2) compared to {Delta}9-tetrahydrocannabinol, contributing to distinct adverse effects absent in cannabis use. The indole-based SCRAs GBD-002 and GBD-003, incorporating 2,2-dimethylindane, differ by a single substituent position yet display markedly different receptor binding profiles. We evaluated both the GBD-002/GBD-003 pair and an analogous structural isomer pair using bioluminescence resonance energy transfer (BRET) assays to assess key CB1 and CB2 transducer pathways. Site-directed mutagenesis characterized contributions of non-conserved residues between CB1 and CB2 and aromatic residues on CB1 transmembrane helix 2 (TM2). This study provided insights into the molecular determinants of cannabinoid receptor selectivity and efficacy.

AbstractUncovering a drugs mechanism of action and possible adverse effects are critical components in drug discovery and development. Moreover, it provides evidence for why some drugs prove more effective than others, and how to design better drugs altogether. Here we demonstrate the utility of a high- throughput in vitro screening platform along with a comprehensive panel to aid in the characterization of fifteen BTK inhibitors that are either approved by the FDA or presently under clinical evaluation. To compare the potency of these drugs, we measured the binding affinity of each to wild-type BTK, as well as a clinically relevant resistance mutant of BTK (BTK C481S). In doing so, we discovered a considerable difference in the selectivity and potency of these BTK inhibitors to the wild-type and mutant proteins. Some of this potentially contributes to the adverse effects experienced by patients undergoing therapy using these drugs. Overall, non-covalent BTK inhibitors showed stronger potency for both the wild-type and mutant BTK when compared with that of covalent inhibitors, with the majority demonstrating a higher specificity and less off-target modulation. Additionally, we compared biological outcomes for four of these inhibitors in human cell-based models. As expected, we found different phenotypic profiles for each inhibitor. However, the two non-covalent inhibitors had fewer off-target biological effects when compared with the two covalent inhibitors. This and similar in-depth preclinical characterization of drug candidates can provide critical insights into the efficacy and mechanism of action of a compound that may affect its safety in a clinical setting. Table of Contents/Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/611550v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@744cf2org.highwire.dtl.DTLVardef@167cf2forg.highwire.dtl.DTLVardef@5326c4org.highwire.dtl.DTLVardef@1163b29_HPS_FORMAT_FIGEXP M_FIG C_FIG

Oral peptide therapeutics typically suffer from short half-lives as they are readily degraded by digestive enzymes. Systematic peptide engineering along with formulation optimization led to the development of a clinical candidate MEDI7219, an orally-bioavailable GLP-1 peptide, that is much more stable than wild type GLP-1 or semaglutide. In this study, we elucidated peptide biotransformation products using in vitro pancreatin assay that employed both collision-induced dissociation (CID) and electron-activated dissociation (EAD) LC-MS/MS methods. Using this approach, we have confidently identified a total of 13 metabolites. Relative quantification of these metabolites over time showed sequential cleavage pattern as peptides were further digested to smaller fragments. These 13 metabolites mapped to 8 cleavage sites on MEDI7219 structure. Most of these cleavage sites can be explained by the specificity of digestive enzymes, e.g. ,trypsin, pepsin and elastase. -methyl-L-phenylalanine appeared to be well protected from chymotrypsin and pepsin digestion since no cleavage peptides ending with -methyl-L-phenylalanine were observed. These study results expand upon previously published stability data and provide new insights on potential GLP1 proteolytic liabilities for future engineering. Furthermore, this study exemplifies the application of pancreatin in vitro system methodology as a valuable tool for understanding metabolism of oral peptide therapeutics in vitro. Additionally, orthogonal MS fragmentation modes offered improved confidence in identification for peptide unknown metabolites. Significance StatementIn vitro metabolite identification of oral GLP1 peptide that uncovers potential proteolytic hotspots that can inform future oral peptide engineering.

Background and PurposeCD28 is a pivotal costimulatory receptor that governs T cell activation through interaction with B7 ligands (CD80/CD86). While antibody-based inhibitors of CD28 signaling have advanced clinically, the development of small molecule modulators remains limited due to the receptors shallow, flexible surface. We sought to discover small-molecule modulators with favorable pharmacokinetic properties capable of disrupting CD28-B7 interactions in translational models of T cell activation. Experimental ApproachStructure-based virtual screening was conducted using pharmacophore filtering and consensus docking against the human CD28 ectodomain. Hit compounds were validated using temperature-related intensity change (TRIC) and microscale thermophoresis (MST). Functional antagonism was assessed through ELISA, NanoBit luciferase complementation, and a CD28 Blockade Bioassay. In vitro ADME and safety pharmacology profiling were performed, and immunosuppressive activity was evaluated in tumor-PBMC and mucosal-PBMC co-culture assays. Key ResultsLead compound 22VS bound CD28 in biophysical screening, targeting a lipophilic canyon anchored by K24, Q25, and P27. 22VS inhibited CD28-CD80/CD86 interactions in ELISA and cell-based assays with submicromolar potency. 22VS robustly suppressed T cell activation markers in both tumor- PBMC and human mucosal epithelial-PBMC co-culture models, phenocopying the anti-CD28 biologic FR104. It showed no cytotoxicity up to 300 {micro}M and exhibited high solubility, low clearance, strong membrane permeability, and minimal off-target effects in pharmacokinetic screens. Conclusion and ImplicationsThis study identifies a novel druggable site on CD28 and validates 22VS as a selective, non-toxic small molecule inhibitor with translational potential for immune modulation in autoimmunity, transplantation, and cancer. Bullet Point SummaryO_ST_ABSWhat is already knownC_ST_ABSO_LICD28 is a key T cell costimulatory receptor essential for immune activation. C_LIO_LISmall-molecule inhibitors of CD28 are largely unexplored compared to biologics. C_LI What this study addsO_LIIdentifies a novel druggable pocket on CD28 via structure-based virtual screening. C_LIO_LIDiscovers 22VS, a selective small molecule CD28 inhibitor with cellular activity. C_LIO_LIDemonstrates that 22VS suppresses T cell activation in tumor-PBMC and mucosal-PBMC co- culture assays, phenocopying a benchmark biologic (FR104). C_LIO_LIEstablishes 22VS as a drug-like compound with favorable in vitro pharmacokinetic properties, including metabolic stability, permeability, and low off-target toxicity. C_LI Clinical significanceO_LIHighlights the potential of 22VS as a lead for immunomodulatory therapeutic development. C_LIO_LISupports small-molecule targeting of CD28-B7 interactions in T cell-driven diseases. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/665260v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f1d2forg.highwire.dtl.DTLVardef@451c98org.highwire.dtl.DTLVardef@158446corg.highwire.dtl.DTLVardef@1f42ef1_HPS_FORMAT_FIGEXP M_FIG C_FIG

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of GZESTY components. GZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with GZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through the D1- (D1R and D5R) and D2-class (D2R, D3R and D4R) of seven transmembrane receptors, and activates both G-protein- and {beta}-arrestin-dependent signaling pathways, to mediate its physiological effects. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinsons disease, or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side-effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling towards the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side-effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity-relationship (SAR) studies have embarked on discovering {beta}-arrestin-biased dopamine agonists, focused on D2 partial agonists, non-catechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R {beta}-arrestin biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold. Previously described and novel analogs of A-86929 were synthesized and screened in vitro for structure-functional-selectivity relationships (SFSR) studies to identify chemical motifs responsible for {beta}-arrestin biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein biased but none of them were exclusively arrestin-biased. Additionally, various catechol aryl fragments were designed and synthesized. Other compounds surveyed included hydroxyl and chloro analogs of dopamine to test for hydrogen bonding and ionic interactions. Some of these small molecular probes displayed weak bias towards the {beta}-arrestin pathway. Continued in-depth SFSR studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate discovery of potent and efficacious arrestin-biased dopamine receptor ligands.

The dopamine D1 receptor (D1R) couples to Gs and Golf and plays a crucial role in regulating voluntary movement and other cognitive functions, making it a potential therapeutic target for several neurological and neuropsychiatric disorders, such as Parkinsons disease and schizophrenia. In the central nervous system, Gs is widely expressed in the cortex and Golf is predominantly found in the striatum. We used two different configurations of bioluminescence resonance energy transfer (BRET) assays and a fluorescence-based cyclic AMP (cAMP) production functional assay to test a series of tetracyclic catechol (dihydrexidine, methyl-dihydrexidine, doxanthrine) and non-catechol (tavapadon, PF-8294, PF-6142) D1R agonists for their activity at these G proteins. We discovered that these tetracyclic catechol compounds, PF-8294 and PF-6142 exerted full agonism when D1R coupled to Gs but partial agonism when D1R coupled to Golf. In contrast, tavapadon acted as a full agonist at Golf and a partial agonist at Gs. The effects of these compounds on the cortical and nigral electrophysiological events agree with their selectivity profiles. This suggests the possibility of achieving region-specific pharmacology and opens new directions for developing D1R drugs to treat relevant neurological and neuropsychiatric disorders.

Radioligands are well-established tools for measuring ligand binding affinities at receptors. Determining affinities of test ligands at many G protein-coupled receptors (GPCRs), including serotonin (5-HT) GPCRs, often involves incubating a radioligand, test ligands, and receptors expressed in cell membranes in Tris buffers, and commonly in a standard binding buffer (SBB) containing Tris HCl, MgCl2, and EDTA until ligand- receptor equilibrium binding is established. However, the composition of extracellular fluid (ECF), where ligands first encounter GPCRs in vivo, differs from that of SBB, which we hypothesized impacts ligand affinity. We conducted radioligand binding assays to compare the affinities of the agonist 5-carboxamidotryptamine (5-CT) and two antagonists/inverse agonists, lurasidone and SB-269970, at [3H]5-CT-labeled 5-HT7 GPCRs stably expressed in HEK293 cells using SBB or artificial brain ECF (abECF) as the medium at room or physiological temperatures (RT or 37{degrees}C). The rank order of ligand potencies, as well as 5-CTs affinity, was unaffected by the different experimental environments. [3H]5-CT 5-HT7R Bmax values increased in abECF and modestly at 37{degrees}C, without affecting Kd, suggesting an increase in active state conformations. In contrast to 5-CT, antagonist/inverse agonist affinities depended on both media and temperature. The affinities of lurasidone and SB-269970 at 5-HT7 receptors were substantially higher at 37{degrees}C than at RT. Also, incubation of lurasidone and SB-269970 in abECF resulted in significantly higher affinities compared to incubation in SBB (e.g., [~]10-fold higher for lurasidone), indicating that temperature and the buffer and ionic composition of abECF influence 5-HT7 antagonist/inverse agonist ligand binding. As a high concentration of NaCl in abECF is a remarkable difference from the composition of SBB, we probed the impact of removing NaCl from abECF; removal of NaCl had a minor affinity-enhancing effect on the antagonists, inferring that other ions, glucose, or sodium phosphates in abECF underlie significant changes in ligand-receptor binding interactions. Overall, the observations indicate that measuring 5-HT7 antagonist affinities at [3H]5-CT-labeled 5-HT7Rs with abECF at physiological temperature--modeling the in vivo brain environment where ligands and GPCRs interact--yields distinct affinity values that may be more physiologically accurate than values obtained from SBB. Moreover, several historical reports demonstrate that temperature, ions, and buffers have no consistent effect on the affinities of distinct ligands at various other GPCRs, and there is no consensus binding buffer used in the literature for any GPCR, which may contribute to the variability in ligand-GPCR affinities reported. These findings show that buffer and temperature impacted 5-HT7R ligand binding affinities and highlight the importance of considering such conditions when performing experiments.

G protein coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at this class of receptors offer further potential for exploitation. In particular GPCR intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor-effector interface (e.g. G protein) and so inhibit agonist responses non-competitively. This potentially offers a tailored mode of action and greater selectivity between conserved receptor subtypes compared to classical orthosteric ligands. However, while specific examples of the IAM class of ligands are well described (particularly for chemokine receptors), a more general methodology for assessing compound interactions at the GPCR IAM site is lacking. Here fluorescent labelled peptides based on the G peptide C terminus are developed as novel binding and activation biosensors for the GPCR IAM binding site. In TR-FRET binding studies, unlabelled peptides derived from the GS subunit C-terminus were first characterised for their ability to positively modulate agonist affinity at the {beta}2-adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labelled tracer was synthesized based on the 19 amino acid C terminal GS peptide (TMR-GS19cha18, where cha=cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-GS19cha18 was recruited to Gs coupled {beta}2-adrenoceptor and EP2 receptors in an agonist dependent manner (correlated with ligand efficacy), but not to the Gi coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-GS19cha18 enabled direct assessment of the affinity of unlabelled ligands for {beta}2-adrenoceptor IAM site. Thus the NanoBRET platform using fluorescent-labelled G protein peptide mimetics offers novel potential for medium-throughput affinity screens to identify new IAMs, applicable across GPCRs coupled to a G protein class. Using the same platform, Gs peptide biosensors also represent useful tools to probe orthosteric agonist efficacy and the dynamics of receptor activation.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as the -opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G protein biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at MOR using adenylate cyclase inhibition and {beta}-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and {beta}-arrestin2 recruitment pathways. Compared to the reference agonist DAMGO, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting {beta}-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and {beta}-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. Instead, our results show that, regardless of bias, opioids with sufficiently high intrinsic efficacy can be lethal, especially given the extremely high potency of many of these compounds that are now pervading the illicit drug market.

Non-selective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson disease and dystonia. Despite their efficacy in these and other central nervous system disorders, anti-muscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the anti-parkinsonian and anti-dystonic efficacy observed with the use of non-selective anti-muscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of non-selective anti-muscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here we utilize genetic mAChR knockout animals in combination with non-selective mAChR antagonists to confirm that the M4 receptor underlies the locomotor-stimulating and anti-parkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have anti-parkinsonian and anti-dystonic efficacy in pharmacological and genetic models of movement disorders.

HIV continues to impose a significant global burden despite success of effective antiretroviral therapy and pre-exposure prophylaxis (PrEP), due to stigma, access, and continuity of treatment that impact real-world effectiveness. Broadly neutralizing antibodies (bNAbs) are novel monoclonal antibody therapeutics being studied for long-acting ART and PrEP, and part of curative HIV regimens. Clinical translation for dose optimization remains a major challenge for the development of these therapeutics. Further, methods to characterize tissue levels of these agents are limited and often impractical due to the need for invasive tissue biopsies. Here we demonstrate the ability of serial whole body positron emission tomography (PET) imaging, following microdosing of the bNAb VRC01 in people without HIV, coupled with physiological-based pharmacokinetic (PBPK) modeling, to accurately predict therapeutic plasma, tissue exposure and prevention efficacy in two major VRC01 prevention trials. Based on our PBPK model, we determined a >51-fold anorectal tissue VRC01 level:inhibitory concentration (IC80) target would achieve 90% prevention efficacy compared to >200 based on plasma levels in the primary trial analysis. Thus, these PET-PBPK approaches are promising for noninvasive determination of bNAb penetration more closely linked with concentrations needed to prevent virus acquisition, and may be leveraged to improve efficient development of bNAbs.

Previous studies show ATP-sensitive potassium (KATP) channel openers can reduce hypersensitivity associated with chronic pain models in rodents, and reduce morphine tolerance. Many agonists of KATP channels are not soluble in physiologically relevant vehicles, requiring adaptation for clinical use. This study compared the antinociception activity of novel KATP channel targeting prodrugs, CKLP1, CKLP2, and CF3-CKLP. These prodrugs are activated by endogenous alkaline phosphatase enzymes present in the peripheral and central nervous systems. Analgesic capabilities of intrathecally injected prodrugs were tested in rodent models of spinal nerve ligation (SNL) and Complete Freunds Adjuvant (CFA) as models for neuropathic and inflammatory pain, respectively. CKLP1 and CKLP2 significantly increased mechanical paw withdrawal thresholds 1-2 hours after intrathecal administration in the SNL model, but all three prodrugs were able to attenuate hypersensitivity up to 7 days after CFA treatment. The reduction of opioid tolerance and opioid-induced hypersensitivity in mice treated chronically with morphine was significantly reduced in CKLP1 and CKLP2 treated animals. Prodrug cleavage was confirmed in mouse spinal cords using liquid chromatography. These studies may aid in the further development of KATP channel prodrugs for use in treatments of chronic pain, opioid tolerance, and withdrawal.

The endocannabinoid (eCB) system regulates several brain functions and is implicated in neurological disorders. The pharmacological blockade of cannabinoid receptors has a therapeutic potential for various cognitive deficits, but also produces severe psychiatric side effects. Hence, new cannabinoid compounds that potentiate therapeutic effects, while minimizing toxicity, are required. In this study, we synthesized and characterized a novel antagonist/inverse agonist of CB1 receptors. UVI3502 showed affinity for two [3H]CP55,940 binding sites (IC50Hi 0.47 {+/-} 1.94 nM and IC50Lo 1470 {+/-} 1.80 nM). Subsequent binding assays performed in CB1 and CB2 overexpressing membranes determined that the low affinity binding site corresponded to CB1, but the high-affinity binding site of UVI3502 did not correspond to CB2 and the possibility of it corresponding to GPR55 was analyzed. The affinity of UVI3502 for CB1 receptors was further confirmed with neuroanatomical specificity by autoradiography in key brain areas, in which functional [35S]GTP{gamma}S assays demonstrated that UVI3502 behaved as an antagonist/inverse agonist of CB1 receptors, blocking the stimulation evoked by potent cannabinoid receptor agonist CP55,940 and decreasing basal [35S]GTP{gamma}S binding. The in silico characterization of the binding to CB1 receptor through molecular docking and molecular dynamics suggests that this activity is explained by the planar and rigid structure of UVI3502, which is optimal for interactions with the inactive state of the receptor. These results indicate that UVI3502 is a novel antagonist/inverse agonist of CB1 receptors, making it a compelling candidate for pharmacologically blocking cannabinoid receptors in the central nervous system. Significance StatementUVI3502 is a novel antagonist/inverse agonist of CB1 receptors, with almost no affinity for CB2 receptors and an additional high-affinity binding site for a third, cannabinoid-like receptor, potentially GPR55. In relevant brain areas for learning and memory processes with a high expression of CB1, UVI3502 blocks the stimulation evoked by the cannabinoid receptor agonist CP55,940, rendering it as an interesting compound for the pharmacological blockade of cannabinoid receptors in the central nervous system.

Synthetic opioids such as fentanyl and related analogs have been widely used for pain management. However, their negative side effects, including respiratory depression and high potential for addiction, underscore the need for a deeper understanding of fentanyls interactions with proteins throughout the human body. Fentanyl analogs bind and activate opioid receptors in the central and peripheral nervous systems, triggering numerous downstream signaling pathways. Increasingly, fentanyl has been shown to interact with non-opioid receptors, and elucidation of these non-canonical fentanyl-protein interactions may provide insights into the mechanisms contributing to fentanyls adverse effects and illuminate novel countermeasure strategies. To identify proteins in mammalian tissues that may interact with fentanyl, we designed and synthesized three affinity-based probes (AfBPs) that include the fentanyl core and feature a diazirine photoaffinity group and alkyne handle for click chemistry at different positions. Molecular docking simulations predicted that these AfBPs bind the mu opioid receptor similarly to fentanyl. Affinity-based protein profiling using the FA-T1 probe in vitro in tissues from six animal species identified histamine N-methyltransferase (HNMT), endophilin-B1 (SH3GLB1), fructosamine-3-kinase (FN3K), cutA divalent cation tolerance analog (CUTA), and monoamine oxidase B (MAOB) among the top proteins that bind fentanyl in multiple species and tissue types. Molecular docking of fentanyl and remifentanil with these protein structures identified putative binding sites. The interaction of fentanyl with specific proteins was empirically assessed through protein structural analyses. These findings highlight potential fentanyl-protein interactions that may contribute to the acute and long-term impacts of fentanyl exposures.

S1P (sphingosine 1-phosphate) receptor modulator (SRM) drugs interfere with lymphocyte trafficking by downregulating lymphocyte S1P receptors. While the immunosuppressive activity of SRM drugs has proved useful in treating autoimmune diseases such as multiple sclerosis, that drug class is beset by on-target liabilities such as initial dose bradycardia. The S1P that binds to cell surface lymphocyte S1P receptors is provided by S1P transporters. Mice born deficient in one of these, spinster homolog 2 (Spns2), are lymphocytopenic and have low lymph S1P concentrations. Such observations suggest that inhibition of Spns2-mediated S1P transport might provide another therapeutically beneficial method to modulate immune cell positioning. We report here results using a novel S1P transport blocker (STB), SLF80821178, to investigate the consequences of S1P transport inhibition in rodents. We found that SLF80821178 is efficacious in a multiple sclerosis model but - unlike the SRM fingolimod - neither decreases heart rate nor compromises lung endothelial barrier function. Notably, although Spns2 null mice have a sensorineural hearing defect, mice treated chronically with SLF80821178 have normal hearing acuity. STBs such as SLF80821178 evoke a dose-dependent decrease in peripheral blood lymphocyte counts, which affords a reliable pharmacodynamic marker of target engagement. However, the maximal reduction in circulating lymphocyte counts in response to SLF80821178 is substantially less than the response to SRMs such as fingolimod (50% vs. 90%) due to a lesser effect on T lymphocyte sub-populations by SLF80821178. Finally, in contrast to results obtained with Spns2 deficient mice, lymph S1P concentrations were not significantly changed in response to administration of STBs at doses that evoke maximal lymphopenia, which indicates that current understanding of the mechanism of action of S1P transport inhibitors is incomplete.

Neurotrophins are critical regulators of neuronal development and have been implicated as therapeutic targets in a range of neurodegenerative and psychiatric disorders. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) signal through the receptor tyrosine kinase family of tropomyosin receptor kinase (Trk) receptors. These include TrkA responding to canonical ligand NGF, TrkB responding to BDNF or NT-4, and TrkC responding to NT-3. While TrkA and TrkB have been comparatively well studied, the fundamental pharmacological properties of TrkC remain largely unexplored. Here, we developed and utilised real-time bioluminescence- or fluorescence-based resonance energy transfer (BRET or FRET) biosensors to study the real-time spatial and temporal dynamics at 37{degrees}C to profile Trk receptor dimerisation, trafficking and nuclear ERK signalling in response to neurotrophin stimulation. TrkA and TrkB displayed consistent concentration-dependent dimerisation, trafficking, and signalling. TrkC, on the other hand, exhibited considerable dimerisation but reduced trafficking and ERK signalling relative to TrkA or TrkB. There was also evidence for comparable activation by both canonical and some non-canonical ligands across the Trk family in response to NGF, BDNF, NT-3, or NT-4 across signalling and trafficking assays. The divergence between robust receptor oligomerisation and minimal trafficking suggests TrkC is subject to unique molecular mechanisms distinct from TrkA or TrkB.