Computational insights into diverse binding modes of the allosteric modulator and their regulation on dopamine D1 receptor.

Allosteric drugs hold the promise of addressing many challenges in the current drug development of GPCRs. However, the molecular mechanism underlying their allosteric modulations remain largely elusive. The dopamine D1 receptor (DRD1), a member of Class A GPCRs, is critical for treating psychiatric disorders, and LY3154207 serves as its promising positive allosteric modulator (PAM). In the work, we utilized extensive Gaussian-accelerated molecular dynamics simulations (a total of 41µs) for the first time probe the diverse binding modes of the allosteric modulator and their regulation effects, based on the DRD1 and LY3154207 as representative. Our simulations identify four binding modes of LY3154207 (one boat mode, two metastable vertical modes and a novel cleft-anchored mode), in which the boat mode is the most stable while there three modes are similar in the stability. However, it is interesting to observed that the most stable boat mode inversely exhibits the weakest positive allosteric effect on influencing the orthosteric ligand binding and maintaining the activity of the transducer binding site. It should result from its induced weaker correlation between the allosteric site and the orthosteric site, and between the orthosteric site and the transducer binding site than the other three binding modes, as well as its weakened interaction between a crucial activation-related residue (S2025.46) and the orthosteric ligand (dopamine). Overall, the work offers atomic-level information to advance our understanding of the complex allosteric regulation on GPCRs, which is beneficial to the allosteric modulator design and development.

Role of Conserved Tyrosine Lid Residues in the Activation of the M2 Muscarinic Acetylcholine Receptor.

The development of subtype selective small molecule drugs for the muscarinic acetylcholine receptor (mAChR) family has been challenging. The design of more selective ligands can be improved by understanding the structure and function of key amino acid residues that line ligand binding sites. Here we study the role of three conserved key tyrosine residues [Y1043.33, Y4036.51, and Y4267.39 (Ballesteros and Weinstein numbers in superscript)] at the human M2 mAChR, located at the interface between the orthosteric and allosteric binding sites of the receptor. We specifically focused on the role of the three tyrosine hydroxyl groups in the transition between the inactive and active conformations of the receptor by making phenylalanine point mutants. Single-point mutation at either of the three positions was sufficient to reduce the affinity of agonists by ∼100-fold for the M2 mAChR, whereas the affinity of antagonists remained largely unaffected. In contrast, neither of the mutations affected the efficacy of orthosteric agonists. When mutations were combined into double and triple M2 mAChR mutants, the affinity of antagonists was reduced by more than 100-fold compared with the wild-type M2 receptor. In contrast, the affinity of allosteric modulators, either negative or positive, was retained at all single and multiple mutations, but the degree of allosteric effect exerted on the endogenous ligand acetylcholine was affected at all mutants containing Y4267.39F. These findings will provide insights to consider when designing future mAChR ligands. SIGNIFICANCE STATEMENT: Structural studies demonstrated that three tyrosine residues between the orthosteric and allosteric sites of the M2 muscarinic acetylcholine receptor (mAChR) had different hydrogen bonding networks in the inactive and active conformations. The role of hydroxyl groups of the tyrosine residues on orthosteric and allosteric ligand pharmacology was unknown. We found that hydroxyl groups of the tyrosine residues differentially affected the molecular pharmacology of orthosteric and allosteric ligands. These results provide insights to consider when designing future mAChR ligands.

M1-selective muscarinic allosteric modulation enhances cognitive flexibility and effective salience in nonhuman primates.

Acetylcholine (ACh) in cortical neural circuits mediates how selective attention is sustained in the presence of distractors and how flexible cognition adjusts to changing task demands. The cognitive domains of attention and cognitive flexibility might be differentially supported by the M1 muscarinic acetylcholine receptor (mAChR) subtype. Understanding how M1 mAChR mechanisms support these cognitive subdomains is of highest importance for advancing novel drug treatments for conditions with altered attention and reduced cognitive control including Alzheimer's disease or schizophrenia. Here, we tested this question by assessing how the subtype-selective M1 mAChR positive allosteric modulator (PAM) VU0453595 affects visual search and flexible reward learning in nonhuman primates. We found that allosteric potentiation of M1 mAChRs enhanced flexible learning performance by improving extradimensional set shifting, reducing latent inhibition from previously experienced distractors and reducing response perseveration in the absence of adverse side effects. These procognitive effects occurred in the absence of apparent changes of attentional performance during visual search. In contrast, nonselective ACh modulation using the acetylcholinesterase inhibitor (AChEI) donepezil improved attention during visual search at doses that did not alter cognitive flexibility and that already triggered gastrointestinal cholinergic side effects. These findings illustrate that M1 mAChR positive allosteric modulation enhances cognitive flexibility without affecting attentional filtering of distraction, consistent with M1 activity boosting the effective salience of relevant over irrelevant objects specifically during learning. These results suggest that M1 PAMs are versatile compounds for enhancing cognitive flexibility in disorders spanning schizophrenia and Alzheimer's diseases.

Differential Activity of Orthosteric Agonists and Allosteric Modulators at Metabotropic Glutamate Receptor 7.

Metabotropic glutamate receptor 7 (mGlu7) is a G protein coupled receptor that has demonstrated promise as a therapeutic target across a number of neurologic and psychiatric diseases. Compounds that modulate the activity of mGlu7, such as positive and negative allosteric modulators, may represent new therapeutic strategies to modulate receptor activity. The endogenous neurotransmitter associated with the mGlu receptor family, glutamate, exhibits low efficacy and potency in activating mGlu7, and surrogate agonists, such as the compound L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4), are often used for receptor activation and compound profiling. To understand the implications of the use of such agonists in the development of positive allosteric modulators (PAMs), we performed a systematic evaluation of receptor activation using a system in which mutations can be made in either protomer of the mGlu7 dimer; we employed mutations that prevent interaction with the orthosteric site as well as the G-protein coupling site of the receptor. We then measured increases in calcium levels downstream of a promiscuous G protein to assess the effects of mutations in one of the two protomers in the presence of two different agonists and three positive allosteric modulators. Our results reveal that distinct PAMs, for example N-[3-Chloro-4-[(5-chloro-2-pyridinyl)oxy]phenyl]-2-pyridinecarboxamide (VU0422288) and 3-(2,3-Difluoro-4-methoxyphenyl)-2,5-dimethyl-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine (VU6005649), do exhibit different maximal levels of potentiation with L-AP4 versus glutamate, but there appear to be common stable receptor conformations that are shared among all of the compounds examined here. SIGNIFICANCE STATEMENT: This manuscript describes the systematic evaluation of the mGlu7 agonists glutamate and L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) in the presence and absence of three distinct potentiators examining possible mechanistic differences. These findings demonstrate that mGlu7 potentiators display subtle variances in response to glutamate versus L-AP4.

Mutual Cooperativity of Three Allosteric Sites on the Dopamine D1 Receptor.

An amine-containing molecule called Compound A has been reported by a group from Bristol-Myers Squibb to act as a positive allosteric modulator (PAM) at the dopamine D1 receptor. We synthesized the more active enantiomer of Compound A (BMS-A1) and compared it with the D1 PAMs DETQ and MLS6585, which are known to bind to intracellular loop 2 and the extracellular portion of transmembrane helix 7, respectively. Results from D1/D5 chimeras indicated that PAM activity of BMS-A1 tracked with the presence of D1 sequence in the N-terminal/extracellular region of the D1 receptor, a unique location compared with either of the other PAMs. In pairwise combinations, BMS-A1 potentiated the small allo-agonist activity of each of the other PAMs, while the triple PAM combination (in the absence of dopamine) produced a cAMP response about 64% of the maximum produced by dopamine. Each of the pairwise PAM combinations produced a much larger leftward shift of the dopamine EC50 than either single PAM alone. All three PAMs in combination produced a 1000-fold leftward shift of the dopamine curve. These results demonstrate the presence of three non-overlapping allosteric sites that cooperatively stabilize the same activated state of the human D1 receptor. SIGNIFICANCE STATEMENT: Deficiencies in dopamine D1 receptor activation are seen in Parkinson disease and other neuropsychiatric disorders. In this study, three positive allosteric modulators of the dopamine D1 receptor were found to bind to distinct and separate sites, interacting synergistically with each other and dopamine, with the triple combination causing a 1000-fold leftward shift of the response to dopamine. These results showcase multiple opportunities to modulate D1 tone and highlight new pharmacological approaches for allosteric modulation of G-protein-coupled receptors.

Commonly Used Therapeutics Associated with Changes in Arousal Inhibit GABAAR Activation.

GABAA receptor-positive modulators are well-known to induce sedation, sleep, and general anesthesia. Conversely, GABAA receptor negative allosteric modulators (GABAARNAMs) can increase arousal and induce seizures. Motivated by our studies with patients with hypersomnia, and our discovery that two GABAARNAMs can restore the Excitation/Inhibition (E/I) balance in vitro and arousal in vivo, we chose to screen 11 compounds that have been reported to modulate arousal, to see if they shared a GABA-related mechanism. We determined modulation with both conventional and microfluidic patch clamp methods. We found that receptor activation was variably modulated by all 11 compounds: Rifampicin (RIF), Metronidazole (MET), Minocycline (MIN), Erythromycin (ERY), Ofloxacin (OFX), Chloroquine (CQ), Hydroxychloroquine sulfate (HCQ), Flumazenil (FLZ), Pentylenetetrazol (PTZ), (-)-Epigallocatechin Gallate (EGCG), and clarithromycin (CLR). The computational modeling of modulator-receptor interactions predicted drug action at canonical binding sites and novel orphan sites on the receptor. Our findings suggest that multiple avenues of investigation are now open to investigate large and brain-penetrant molecules for the treatment of patients with diminished CNS E/I balance.

Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.

The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and ß-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance ß-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.

Novel neuroactive steroids as positive allosteric modulators of NMDA receptors: mechanism, site of action, and rescue pharmacology on GRIN variants associated with neurological conditions.

N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson's disease, Alzheimer's disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions.

Allosteric Modulators of Metabotropic Glutamate Receptors as Novel Therapeutics for Neuropsychiatric Disease.

Metabotropic glutamate (mGlu) receptors, a family of G-protein-coupled receptors, have been identified as novel therapeutic targets based on extensive research supporting their diverse contributions to cell signaling and physiology throughout the nervous system and important roles in regulating complex behaviors, such as cognition, reward, and movement. Thus, targeting mGlu receptors may be a promising strategy for the treatment of several brain disorders. Ongoing advances in the discovery of subtype-selective allosteric modulators for mGlu receptors has provided an unprecedented opportunity for highly specific modulation of signaling by individual mGlu receptor subtypes in the brain by targeting sites distinct from orthosteric or endogenous ligand binding sites on mGlu receptors. These pharmacological agents provide the unparalleled opportunity to selectively regulate neuronal excitability, synaptic transmission, and subsequent behavioral output pertinent to many brain disorders. Here, we review preclinical and clinical evidence supporting the utility of mGlu receptor allosteric modulators as novel therapeutic approaches to treat neuropsychiatric diseases, such as schizophrenia, substance use disorders, and stress-related disorders. SIGNIFICANCE STATEMENT: Allosteric modulation of metabotropic glutamate (mGlu) receptors represents a promising therapeutic strategy to normalize dysregulated cellular physiology associated with neuropsychiatric disease. This review summarizes preclinical and clinical studies using mGlu receptor allosteric modulators as experimental tools and potential therapeutic approaches for the treatment of neuropsychiatric diseases, including schizophrenia, stress, and substance use disorders.

High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP.

Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80-2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1-2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.

Positive Allosteric Modulation of α5-GABAA Receptors Reverses Stress-Induced Alterations in Dopamine System Function and Prepulse Inhibition of Startle.

BACKGROUND: Up to 64% of patients diagnosed with posttraumatic stress disorder (PTSD) experience psychosis, likely attributable to aberrant dopamine neuron activity. We have previously demonstrated that positive allosteric modulators of α5-GABAARs can selectively decrease hippocampal activity and reverse psychosis-like physiological and behavioral alterations in a rodent model used to study schizophrenia; however, whether this approach translates to a PTSD model remains to be elucidated. METHODS: We utilized a 2-day inescapable foot shock (IS) procedure to induce stress-related pathophysiology in male Sprague-Dawley rats. We evaluated the effects of intra-ventral hippocampus (vHipp) administration GL-II-73, an α5-GABAAR, or viral overexpression of the α5 subunit, using in vivo electrophysiology and behavioral measures in control and IS-treated rats. RESULTS: IS significantly increased ventral tegmental area dopamine neuron population activity, or the number of dopamine neurons firing spontaneously (n = 6; P = .016), consistent with observation in multiple rodent models used to study psychosis. IS also induced deficits in sensorimotor gating, as measured by reduced prepulse inhibition of startle (n = 12; P = .039). Interestingly, intra-vHipp administration of GL-II-73 completely reversed IS-induced increases in dopamine neuron population activity (n = 6; P = .024) and deficits in prepulse inhibition (n = 8; P = .025), whereas viral overexpression of the α5 subunit in the vHipp was not effective. CONCLUSIONS: Our results demonstrate that pharmacological intervention augmenting α5-GABAAR function, but not α5 overexpression in itself, can reverse stress-induced deficits related to PTSD in a rodent model, providing a potential site of therapeutic intervention to treat comorbid psychosis in PTSD.

The pocketome of G-protein-coupled receptors reveals previously untargeted allosteric sites.

G-protein-coupled receptors do not only feature the orthosteric pockets, where most endogenous agonists bind, but also a multitude of other allosteric pockets that have come into the focus as potential binding sites for synthetic modulators. Here, to better characterise such pockets, we investigate 557 GPCR structures by exhaustively docking small molecular probes in silico and converting the ensemble of binding locations to pocket-defining volumes. Our analysis confirms all previously identified pockets and reveals nine previously untargeted sites. In order to test for the feasibility of functional modulation of receptors through binding of a ligand to such sites, we mutate residues in two sites, in two model receptors, the muscarinic acetylcholine receptor M3 and ß2-adrenergic receptor. Moreover, we analyse the correlation of inter-residue contacts with the activation states of receptors and show that contact patterns closely correlating with activation indeed coincide with these sites.

Allosteric pluripotency: challenges and opportunities.

Allosteric pluripotency arises when the functional response of an allosteric receptor to an allosteric stimulus depends on additional allosteric modulators. Here, we discuss allosteric pluripotency as observed in the prototypical Protein Kinase A (PKA) as well as in other signaling systems, from typical multidomain signaling proteins to bacterial enzymes. We identify key drivers of pluripotent allostery and illustrate how hypothesizing allosteric pluripotency may solve apparent discrepancies currently present in the literature regarding the dual nature of known allosteric modulators. We also outline the implications of allosteric pluripotency for cellular signaling and allosteric drug design, and analyze the challenges and opportunities opened by the pluripotent nature of allostery.

Allosteric Modulation of Adenosine A2A Receptors as a New Therapeutic Avenue.

The therapeutic potential of targeting adenosine A2A receptors (A2ARs) is immense due to their broad expression in the body and central nervous system. The role of A2ARs in cardiovascular function, inflammation, sleep/wake behaviors, cognition, and other primary nervous system functions has been extensively studied. Numerous A2AR agonist and antagonist molecules are reported, many of which are currently in clinical trials or have already been approved for treatment. Allosteric modulators can selectively elicit a physiologic response only where and when the orthosteric ligand is released, which reduces the risk of an adverse effect resulting from A2AR activation. Thus, these allosteric modulators have a potential therapeutic advantage over classical agonist and antagonist molecules. This review focuses on the recent developments regarding allosteric A2AR modulation, which is a promising area for future pharmaceutical research because the list of existing allosteric A2AR modulators and their physiologic effects is still short.

Capturing intrinsic nanomechanics of allostery.

The Hsp70 chaperone exploits allosteric communication between its substrate binding domain and its nucleotide binding domain to regulate the loading and release of misfolded polypeptides in an ATP-hydrolysis-dependent manner. In this issue of Biophysical Journal, Singh, Rief, and Zoldák report an exquisitely detailed study of the nanomechanical aspects of the allosteric mechanism in DnaK, an Escherichia coli heat shock protein 70 chaperone.

Allosteric Modulator KM822 Attenuates Behavioral Actions of Amphetamine in Caenorhabditis elegans through Interactions with the Dopamine Transporter DAT-1.

Aberrant dopamine (DA) signaling is associated with several psychiatric disorders, such as autism, bipolar disorder, addiction, and Parkinson's disease, and several medications that target the DA transporter (DAT) can induce or treat these disorders. In addition, psychostimulants, such as cocaine and D-amphetamine (AMPH), rely on the competitive interactions with the transporter's substrate binding site to produce their rewarding effects. Agents that exhibit noncompetitive, allosteric modulation of DAT remain an important topic of investigation due to their potential therapeutic applications. We previously identified a novel allosteric modulator of human DAT, KM822, that can decrease the affinity of cocaine for DAT and attenuate cocaine-elicited behaviors; however, whether DAT is the sole mediator of KM822 actions in vivo is unproven given the large number of potential off-target sites. Here, we provide in silico and in vitro evidence that the allosteric site engaged by KM822 is conserved between human DAT and Caenorhabditis elegans DAT-1. KM822 binds to a similar pocket in DAT-1 as previously identified in human DAT. In functional dopamine uptake assays, KM822 affects the interaction between AMPH and DAT-1 by reducing the affinity of AMPH for DAT-1. Finally, through a combination of genetic and pharmacological in vivo approaches we provide evidence that KM822 diminishes the behavioral actions of AMPH on swimming-induced paralysis through a direct allosteric modulation of DAT-1. More broadly, our findings demonstrate allosteric modulation of DAT as a behavior modifying strategy and suggests that Caenorhabditis elegans can be operationalized to identify and investigate the interactions of DAT allosteric modulators. SIGNIFICANCE STATEMENT: We previously demonstrated that the dopamine transporter (DAT) allosteric modulator KM822 decreases cocaine affinity for human DAT. Here, using in silico and in vivo genetic approaches, we extend this finding to interactions with amphetamine, demonstrating evolutionary conservation of the DAT allosteric site. In Caenorhabditis elegans, we report that KM822 suppresses amphetamine behavioral effects via specific interactions with DAT-1. Our findings reveal Caenorhabditis elegans as a new tool to study allosteric modulation of DAT and its behavioral consequences.

Allosteric Modulation of the Sigma-1 Receptor Elicits Antipsychotic-like Effects.

Allosteric modulation represents an important approach in drug discovery because of its advantages in safety and selectivity. SOMCL-668 is the first selective and potent sigma-1 receptor allosteric modulator, discovered in our laboratory. The present work investigates the potential therapeutic effects of SOMCL-668 on phencyclidine (PCP)-induced schizophrenia-related behavior in mice and further elucidates underlying mechanisms for its antipsychotic-like effects. SOMCL-668 not only attenuated acute PCP-induced hyperactivity and PPI disruption, but also ameliorated social deficits and cognitive impairment induced by chronic PCP treatment. Pretreatment with the selective sigma-1 receptor antagonist BD1047 blocked the effects of SOMCL-668, indicating sigma-1 receptor-mediated responses. This was confirmed using sigma-1 receptor knockout mice, in which SOMCL-668 failed to ameliorate PPI disruption and hyperactivity induced by acute PCP and social deficits and cognitive impairment induced by chronic PCP treatment. Additionally, in vitro SOMCL-668 exerted positive modulation of sigma-1 receptor agonist-induced intrinsic plasticity in brain slices recorded by patch-clamp. Furthermore, in vivo lower dose of SOMCL-668 exerted positive modulation of improvement in social deficits and cognitive impairment induced by the selective sigma-1 agonist PRE084. Also, SOMCL-668 reversed chronic PCP-induced down-regulation in expression of frontal cortical p-AKT/AKT, p-CREB/CREB and BDNF in wide-type but not sigma-1 knockout mice. Moreover, administration of the PI3K/AKT inhibitor LY294002 abolished amelioration by SOMCL-668 of chronic PCP-induced schizophrenia-related behaviors by inhibition of BDNF expression. The present data provide initial, proof-of-concept evidence that allosteric modulation of the sigma-1 receptor may be a novel approach for the treatment of psychotic illness.

Structural Basis of the Negative Allosteric Modulation of 5-BDBD at Human P2X4 Receptors.

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site. SIGNIFICANCE STATEMENT: Activity of P2X4 receptor is associated with neuropathic pain, inflammation, and vasodilatation. Molecular information regarding small-molecule interaction with P2X4 is very limited. Here, this study provides a structural explanation for the action of the small-molecule antagonist 5-BDBD at the human P2X4 receptor.

Discovery of a Positive Allosteric Modulator of Cholecystokinin Action at CCK1R in Normal and Elevated Cholesterol.

Drugs useful in prevention/treatment of obesity could improve health. Cholecystokinin (CCK) is a key regulator of appetite, working through the type 1 CCK receptor (CCK1R); however, full agonists have not stimulated more weight loss than dieting. We proposed an alternate strategy to target this receptor, while reducing likelihood of side effects and/or toxicity. Positive allosteric modulators (PAMs) with minimal intrinsic agonist activity would enhance CCK action, while maintaining spatial and temporal characteristics of physiologic signaling. This could correct abnormal stimulus-activity coupling observed in a high-cholesterol environment observed in obesity. We utilized high-throughput screening to identify a molecule with this pharmacological profile and studied its basis of action. Compound 1 was a weak partial agonist, with PAM activity to enhance CCK action at CCK1R, but not CCK2R, maintained in both normal and high cholesterol. Compound 1 (10 µM) did not exhibit agonist activity or stimulate internalization of CCK1R. It enhanced CCK activity by slowing the off-rate of bound hormone, increasing its binding affinity. Computational docking of Compound 1 to CCK1R yielded plausible poses. A radioiodinatable photolabile analogue retained Compound 1 pharmacology and covalently labeled CCK1R Thr211, consistent with one proposed pose. Our study identifies a novel, selective, CCK1R PAM that binds to the receptor to enhance action of CCK-8 and CCK-58 in both normal and disease-mimicking high-cholesterol environments. This facilitates the development of compounds that target the physiologic spatial and temporal engagement of CCK1R by CCK that underpins its critical role in metabolic regulation.

The role of cholesterol recognition (CARC/CRAC) mirror codes in the allosterism of the human organic cation transporter 2 (OCT2, SLC22A2).

The human organic cation transporter 2 (OCT2) is a multispecific transporter with cholesterol-dependent allosteric features. The present work elucidates the role of evolutionarily conserved cholesterol recognition/interaction amino acid consensus sequences (CRAC and CARC) in the allosteric binding to 1-methyl-4-phenylpyridinium (MPP+) in human embryonic kidney 293 cells stably or transiently expressing OCT2. Molecular blind simulations docked two mirroring cholesterol molecules in the 5th putative transmembrane domain, where a CARC and a CRAC sequence lie. The impact of the conserved amino acids that may constitute the CARC/CRAC mirror code was studied by alanine-scanning mutagenesis. At a saturating extracellular concentration of substrate, at which the impact of cholesterol depletion is maximal, five mutants transported MPP+ at a significantly lower rate than the wild-type OCT2 (WT), resembling the behavior of the WT upon cholesterol depletion. MPP+ influx rate as a function of the extracellular concentration of substrate was measured for the mutants R234A, R235A, L252A and R263A. R234A kinetic behavior was similar to that of the WT, whereas R235A, L252A and R263A activity shifted from allosteric to one-binding site kinetics, very much like the WT upon cholesterol depletion. The impact of cholesterol on protein thermal stability was assessed for WT, R234A and R263A. While the thermal stability of WT and R234A was improved by the supplementation with cholesterol, R263A was not sensitive to the presence of cholesterol. To conclude, the disruption of the CARC/CRAC mirror code in the 5th putative transmembrane domain is sufficient to abolish the allosteric interaction between OCT2 and MPP+.