Allosteric Modulators Research Articles

Discovery of 4-(5-Membered)Heteroarylether-6-methylpicolinamide Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5

Discovery of 4-(5-Membered)Heteroarylether-6-methylpicolinamide Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5

Mechanical stress and anionic lipids synergistically stabilize an atypical structure of the angiotensin II type 1 receptor (AT1).

Environmental factors, including mechanical stress and surrounding lipids, can influence the response of GPCRs, such as the mechanosensitive angiotensin II type 1 receptor (AT1). To investigate the impact of these factors on AT1 activation, we developed a steered molecular dynamics simulations protocol based on quaternion formalism. In this protocol, a pulling force was applied to the N-terminus of transmembrane helix 6 (TM6) to induce the TM6 opening characteristic of activation. Subsequently, the simulations were continued without constraints to allow the receptor to relax around the novel TM6 conformation under different conditions. We analyzed the responses of AT1 to membrane stretching, modeled by applying surface tension, in different bilayers. In phosphocholine bilayers without surface tension, we could observe a transient atypical structure of AT1, with an outward TM7 conformation, at the beginning of the activation process. This atypical structure then evolved toward a pre-active structure with outward TM6 and inward TM7. Strikingly, the presence of anionic phosphoglycerol lipids and application of surface tension synergistically favored the atypical structure, which led to an increase in the cross-section area of the receptor intracellular domain. Lipid internalization and H-bonds between lipid heads and the receptor C-terminus increased in phosphoglycerol vs phosphocholine bilayers, but did not depend on surface tension. The difference in the cross-section area of the atypical and pre-active conformations makes the conformational transition sensitive to lateral pressure, and favors the atypical conformation upon surface tension. Anionic lipids act as allosteric modulators of the conformational transition, by stabilizing the atypical conformation. These findings contribute to decipher the mechanisms underlying AT1 activation, highlighting the influence of environmental factors on GPCR responses. Moreover, our results reveal the existence of intermediary conformations that depend on receptor environment and could be targeted in drug design efforts.

In-Silico discovery of 17alpha-hydroxywithanolide-D as potential neuroprotective allosteric modulator of NMDA receptor targeting Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by cognitive decline, memory impairment, and behavioral alterations. The N-methyl-D-aspartate (NMDA) receptor has emerged as a promising target for AD pharmacotherapy due to its role in the disease's pathogenesis. This study leverages advanced computational methods to screen 80 active constituents of Withania somnifera (Ashwagandha), a traditional herb known for its neuroprotective effects, against the NMDA receptor, using FDA-approved Ifenprodil as a reference. Our blind virtual screening results demonstrated that all tested compounds could bind to various domains of the NMDA receptor, with binding energies ranging from - 4.1 to -11.9kcal/mol, compared to Ifenprodil's -7.8kcal/mol. Binding preference analysis revealed 7 compounds bound to the A-chain, 37 to the B-chain, 7 to the C-chain, and 29 to the D-chain of the receptor. Notable binding was observed predominantly at the Amino Terminal Domain (ATD) core site, some at the ATD-Ligand Binding Domain (LBD) interface, and a few at the Transmembrane Domain (TMD). Particularly, 17alpha-hydroxywithanolide D, with a binding energy of -11.9kcal/mol, emerged as a prime candidate for further investigation. Molecular dynamics simulations of this compound revealed key interactions, including direct hydrogen bonding with residues ASP165, ARG431, THR433, LYS466, and TYR476 on the D-chain, as well as additional hydrophobic and water-bridging interactions. These simulations highlighted the compound's influence on dynamic conformational states of the GluN1b-GluN2B receptor complex, modulating interactions between GluN1b Lys178 and GluN2B Asn184. Furthermore, the compound affected the distance between LBD heterodimers and the tension within the LBD-M30 linker, demonstrating its potential to modulate NMDA receptor activity. This comprehensive study not only underscores the therapeutic promise of Withania somnifera derivatives for AD but also provides a detailed molecular basis for their efficacy, offering valuable insights for targeted drug development and innovative therapeutic strategies against Alzheimer's disease.

Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis.

Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2h resulted in an increase in the frequency of Ca2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.

A cyclic peptide toolkit reveals mechanistic principles of peptidylarginine deiminase IV regulation.

Peptidylarginine deiminase IV (PADI4, PAD4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due toa lack of appropriate systems and tools. Here, we develop and apply a set of potent and selective PADI4 modulators. Using the mRNA-display-based RaPID system, we screen >1012 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_3, a cell-active inhibitor specific for the active conformation of PADI4; PADI4_7, an inert binder, which we functionalise for the isolation and study of cellular PADI4; and PADI4_11, a cell-active PADI4 activator. Structural studies with PADI4_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work contributes to our understanding of PADI4 regulation and provides a toolkit for the study and modulation of PADI4 across (patho)physiological contexts.

Enhancement of Glutamate Uptake as Novel Antiseizure Approach: Preclinical Proof of Concept.

Excitotoxicity is a common hallmark of epilepsy and other neurological diseases associated with elevated extracellular glutamate levels. Thus, here, we studied the protective effects of (R)-AS-1, a positive allosteric modulator (PAM) of glutamate uptake in epilepsy models. (R)-AS-1 was evaluated in a range of acute and chronic seizure models, while its adverse effect profile was assessed in a panel of standard tests in rodents. The effect of (R)-AS-1 on glutamate uptake was assessed in COS-7 cells expressing the transporter. WAY 213613, a selective competitive EAAT2 inhibitor, was used to probe the reversal of the enhanced glutamate uptake in the same transporter expression system. Confocal microscopy and Western blotting analyses were used to study a potential influence of (R)-AS-1 on GLT-1 expression in mice. (R)-AS-1 showed robust protection in a panel of animal models of seizures and epilepsy, including the maximal electroshock- and 6 Hz-induced seizures, corneal kindling, mesial temporal lobe epilepsy, lamotrigine-resistant amygdala kindling, as well as seizures induced by pilocarpine or Theiler's murine encephalomyelitis virus. Importantly, (R)-AS-1 displayed a favorable adverse effect profile in the rotarod, the minimal motor impairment, and the Irwin tests. (R)-AS-1 enhanced glutamate uptake in vitro and this effect was abolished by WAY 213613, while no influence on GLT-1 expression in vivo was observed after repeated treatment. Collectively, our results show that (R)-AS-1 has favorable tolerability and provides robust preclinical efficacy against seizures. Thus, allosteric enhancement of EAAT2 function could offer a novel therapeutic strategy for treatment of epilepsy and potentially other neurological disorders associated with glutamate excitotoxicity. ANN NEUROL 2024.

LPM682000012, a Synthetic Neuroactive Steroid That Ameliorates Epileptic Seizures by Downregulating the Serpina3n/NF-κB Signaling Pathway

Epilepsy is characterized by abnormal neuronal firing in the brain. Several therapeutic strategies exist for epilepsy; however, several patients remain poorly treated. Therefore, the development of effective treatments remains a high priority in the field. Neuroactive steroids can potentiate extra-synaptic and synaptic GABAA receptors, thereby providing therapeutic benefits relative to benzodiazepines. This research study investigated the therapeutic effectiveness and underlying mechanisms of LPM682000012, a new synthetic neuroactive steroid-positive allosteric modulator (PAM) of GABAA receptors employed for treating epilepsy. Acute and chronic rat epilepsy models were established to identify the anti-seizure potency of LPM682000012. The dose-dependent sedative effects of LPM682000012 and Ganaxolone in normal rats were evaluated, which revealed that they both dose-dependently alleviated acute epileptic seizure in the pentylenetetrazol (PTZ)-mediated seizure model. Furthermore, LPM682000012 indicated an enhanced safety profile than Ganaxolone. Moreover, LPM682000012 also indicated therapeutic effects in the kainic acid (KA)-induced chronic spontaneous seizure model. Morphologically, LPM682000012 decreased neuronal loss in the hippocampal CA1 and CA3 regions and increased dendritic spine density in the CA1 region. In addition, mechanical analyses, including transcriptomics, Western blot, and proteomics analyses, revealed that the Serpina3n/NF-κB signaling pathway was up-regulated in epileptic rat hippocampal tissue, and LPM682000012 treatment reversed these changes. In summary, this report demonstrated that the novel neurosteroid GABAA PAM LPM682000012 activated the synaptic and extra-synaptic GABAA receptors and alleviated KA-induced neuronal loss and synaptic remodeling, potentially by down-regulating the Serpina3n/NF-κB signaling pathways. The results provide evidence that LPM682000012 is a potential anti-seizure pharmacotherapy candidate for epilepsy and warrants further research.

Evaluation of allosteric NMDA receptor modulation by GluN2A-selective antagonists using pharmacological equilibrium modeling.

NMDA-type ionotropic glutamate receptors are critically involved in excitatory neurotransmission and their dysfunction is implicated in many brain disorders. Allosteric modulators with selectivity for specific NMDA receptor subtypes are therefore attractive as therapeutic agents, and sustained drug discovery efforts have resulted in a wide range of new allosteric modulators. However, evaluation of allosteric NMDA receptor modulators is limited by the lack of operational ligand-receptor models to describe modulator binding dissociation constants (KB) and effects on agonist binding affinity (α) and efficacy (β). Here, we describe a pharmacological equilibrium model that encapsulates activation and modulation of NMDA receptors, and we apply this model to afford deeper understanding of GluN2A-selective negative allosteric modulators (NAMs), TCN-201, MPX-004, and MPX-007. We exploit slow NAM unbinding to examine receptors at hemi-equilibrium when fully occupied by agonists and modulators to demonstrate that TCN-201 display weaker binding and negative modulation of glycine binding affinity (KB = 42 nM, α = 0.0032) compared to MPX-004 (KB = 9.3 nM, α = 0.0018) and MPX-007 (KB = 1.1 nM, α = 0.00053). MPX-004 increases agonist efficacy (β = 1.19), whereas TCN-201 (β = 0.76) and MPX-007 (β = 0.82) reduce agonist efficacy. These values describing allosteric modulation of diheteromeric GluN1/2A receptors with two modulator binding sites are unchanged in triheteromeric GluN1/2A/2B receptors with a single binding site. This evaluation of NMDA receptor modulation reveals differences between ligand analogs that shape their utility as pharmacological tool compounds and facilitates the design of new modulators with therapeutic potential. Significance Statement Detailed understanding of allosteric NMDA receptor modulation requires pharmacological methods to quantify modulator binding affinity and the strengths of modulation of agonist binding and efficacy. We describe a generic ligand-receptor model for allosteric NMDA receptor modulation and use this model for the characterization of GluN2A-selective NAMs. The model enables quantitative evaluation of a broad range of NMDA receptor modulators and provides opportunities to optimize these modulators by embellishing the interpretation of their structure-activity relationships.

Progress in GABAA receptor agonists for insomnia disorder

Insomnia is the most common sleep disorder in which an individual has trouble falling or staying asleep. Chronic sleep loss interferes with daily functioning and adversely affects health. The main clinical drugs for insomnia are the positive allosteric modulator of the GABA (gamma-aminobutyric acid) A receptors (GABAARs) at the benzodiazepine site with selectivity of the GABA-α1 receptor. They are divided into benzodiazepine drugs and non-benzodiazepine drugs. Most recently, the first partial positive allosteric modulator of GABAAR Dimdazenil was approved by National Medical Products Administration (NMPA) and launched in China. This review summarized the mechanism of actions of current clinical drugs for insomnia, and the clinical applications of these drugs, which may help to understand their involvement in insomnia, and to search for more selective and potent ligands to be used in the treatment of insomnia.

Quinoline- and pyrimidine-based allosteric modulators of the sarco/endoplasmic reticulum calcium ATPase.

Small-molecule allosteric activators of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA) hold promise as novel experimental tools to manipulate intracellular calcium concentrations and as therapeutic agents to treat medical conditions associated with elevated cytosolic calcium levels. Here, we synthesized and characterized 20 analogs of the known allosteric SERCA activator CDN1163 and tested their ability to stimulate SERCA activity. The structures of the compounds varied in the alkyl group of the parent scaffold's ether moiety as well as in the composition of the nitrogenous aromatic ring system. The most active compounds exhibited potencies in the sub-micromolar range while increasing enzyme activity by more than 25%. The observed structure-activity relationships indicated that bulky alkyl groups in the ether moiety along with a quinoline ring methyl substituent were beneficial for activity. Replacement of the quinoline by a pyrimidine ring system reduced activity. To conceive a potential mechanism of action, we generated a molecular model of the transition state of SERCA when undergoing the rate-limiting step of its catalytic cycle. Subsequent blind docking with CDN1163 identified a high-affinity binding site close to the enzyme's ATP binding pocket, suggesting that the activators may accelerate SERCA's catalytic cycle by aiding in ATP binding and positioning.

Role of cholesterol in modulating brain hyperexcitability.

Cholesterol is a critical molecule in the central nervous system, and imbalances in the synthesis and metabolism of brain cholesterol can result in a range of pathologies, including those related to hyperexcitability. The impact of cholesterol on disorders of epilepsy and developmental and epileptic encephalopathies is an area of growing interest. Cholesterol cannot cross the blood-brain barrier, and thus the brain synthesizes and metabolizes its own pool of cholesterol. The primary metabolic enzyme for brain cholesterol is cholesterol 24-hydroxylase (CH24H), which metabolizes cholesterol into 24S-hydroxycholesterol (24HC). Dysregulation of CH24H and 24HC can affect neuronal excitability through a range of mechanisms. 24HC is a positive allosteric modulator of N-methyl-D-aspartate (NMDA) receptors and can increase glutamate release via tumor necrosis factor-α-dependent pathways. Increasing cholesterol metabolism can lead to dysfunction of excitatory amino acid transporter 2 and impair glutamate reuptake. Finally, overstimulation of NMDA receptors can further activate metabolism of cholesterol, leading to a vicious cycle of overactivation. All of these mechanisms increase extracellular glutamate and can lead to hyperexcitability. For these reasons, the cholesterol pathway represents a new potential mechanistic target for antiseizure medications. CH24H inhibition has been shown to decrease seizure behavior and improve survival in multiple animal models of epilepsy and could be a promising new mechanism of action for the treatment of neuronal hyperexcitability and developmental and epileptic encephalopathies.

Detailed functional characterization of four nanobodies as positive allosteric modulators of the human Calcium-Sensing receptor

The calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis, and small-molecule and peptide positive allosteric modulators (PAMs) of CaSR, so-called calcimimetics, are used in the treatment of hyperparathyroidism and hypocalcemic disorders. In this study, four monovalent nanobodies − representing four distinct nanobody families with CaSR PAM activity − were subjected to elaborate pharmacological profiling at the receptor. While Nb5 displayed negligible PAM activity at CaSR in all assays, Nb4, Nb10 and Nb45 all potently potentiated Ca2+-evoked signalling through a myc epitope-tagged CaSR expressed in HEK293 or HEK293T cells in Gαq and Gαi1 protein activation assays and in a Ca2+/Fluo-4 assay. Nb4 and Nb10 also displayed comparable PAM properties at a stable CaSR-HEK293 cell line in a Ca2+/Fura-2 imaging assay, but surprisingly Nb45 was completely inactive at this cell line in both the Ca2+/Fura-2 and Ca2+/Fluo-4 assays. Investigations into this binary difference in Nb45 activity revealed that the nanobody only possesses modulatory activity at CaSRs tagged N-terminally with various epitopes (myc, HA, Flag-SNAP), whereas it is inactive at the untagged wild-type receptor. In conclusion, overall each of the nanobodies exhibit similar CaSR PAM properties in a range of assays, and thus none of them display pathway bias as modulators. However, of the four nanobodies Nb4 and Nb10 would be applicable as pharmacological tools for the wild-type CaSR, and the complete inactivity of Nb45 at the untagged CaSR serves as an reminder that epitope-tagging of a receptor, even if deemed functionally silent, can have profound implications for ligand discovery efforts.

Challenge for Deep Learning: Protein Structure Prediction of Ligand-Induced Conformational Changes at Allosteric and Orthosteric Sites.

In the realm of biomedical research, understanding the intricate structure of proteins is crucial, as these structures determine how proteins function within our bodies and interact with potential drugs. Traditionally, methods like X-ray crystallography and cryo-electron microscopy have been used to unravel these structures, but they are often challenging, time-consuming and costly. Recently, a breakthrough in computational biology has emerged with the development of deep learning algorithms capable of predicting protein structures based on their amino acid sequences (Jumper, J., et al. Nature 2021, 596, 583. Lane, T. J. Nature Methods 2023, 20, 170. Kryshtafovych, A., et al. Proteins: Structure, Function and Bioinformatics 2021, 89, 1607). This study focuses on predicting the dynamic changes that proteins undergo upon ligand binding, specifically when they bind to allosteric sites, i.e. a pocket different from the active site. Allosteric modulators are particularly important for drug discovery, as they open new avenues for designing drugs that can target proteins more effectively and with fewer side effects (Nussinov, R.; Tsai, C. J. Cell 2013, 153, 293). To study this, we curated a data set of 578 X-ray structures comprised of proteins displaying orthosteric and allosteric binding as well as a general framework to evaluate deep learning-based structure prediction methods. Our findings demonstrate the potential and current limitations of deep learning methods, such as AlphaFold2 (Jumper, J., et al. Nature 2021, 596, 583), NeuralPLexer (Qiao, Z., et al. Nat Mach Intell 2024, 6, 195), and RoseTTAFold All-Atom (Krishna, R., et al. Science 2024, 384, eadl2528) to predict not just static protein structures but also the dynamic conformational changes. Herein we show that predicting the allosteric induce-fit conformation still poses a challenge to deep learning methods as they more accurately predict the orthosteric bound conformation compared to the allosteric induce fit conformation. For AlphaFold2, we observed that conformational diversity, and sampling between the apo and holo state could be increased by modifying the MSA depth, but this did not enhance the ability to generate conformations close to the allosteric induced-fit conformation. To further support advancements in protein structure prediction field, the curated data set and evaluation framework are made publicly available.

A Perspective On A Promising Role For Caffeine In Modulating The Casr In Pancreatic Β-Cells

The extracellular calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that plays a crucial role in the parathyroid glands and kidneys in maintaining calcium (Ca2+) homeostasis. The extracellular CaSR is present in various cell types not implicated in controlling plasma Ca2+ levels. There is accumulating evidence that CaSR plays a significant role in regulating the function of β-cells in the pancreas. Divalent cations and insulin are released together during exocytosis, and their concentration in the restricted intercellular compartments of the pancreatic islet increases to activate CaSR in neighboring cells. Subsequently, the activated CaSR stimulates insulin vesicle release in the adjacent cells, and by repeating these steps, the signal is amplified in the whole islet. Recent work developed a photoaffinity-tagged glutathione derivative (DAZ-G) as a chemical tool. The study demonstrated that DAZ-G can identify and analyze CaSR ligands. The use of DAZ-G identified caffeine as a positive allosteric modulator of the CaSR, suggesting that the physiological effects of caffeine are partly attributed to its stimulation of the CaSR. Future work may utilize caffeine to activate the CaSR in pancreatic β-cells to improve their secretory function

Mechanism of negative μ-opioid receptor modulation by sodium ions.

Negative allosteric modulation of G-protein coupled receptors (GPCRs) by Na+ ions was first described in the 1970s for opioid receptors (ORs) and has subsequently been detected for most class A GPCRs. In high-resolution structures of inactive-state class A GPCRs, a Na+ ion binds to a conserved pocket near residue D2.50, whereas active-state structures of GPCRs are incompatible with Na+ binding. Correspondingly, Na+ diminishes agonist affinity, stabilizes the receptors in the inactive state, and reduces basal signaling. We applied a mutual-information based analysis to μs-timescale biomolecular simulations of the μ-opioid receptor (μ-OR). Our results reveal that Na+ binding is coupled to a water wire linking the Na+ binding site with the agonist binding pocket and to rearrangements in polar networks propagating conformational changes to the agonist and G-protein binding sites. These findings provide a new mechanistic link between the presence of the ion, altered agonist affinity, receptor deactivation, and lowered basal signaling levels.

Allosteric modulation of the Lon protease via ssDNA binding and local charge changes.

The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduce ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements, we find this change in activity correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.

Biased allosteric modulator of neurotensin receptor 1 reduces ethanol drinking and responses to ethanol administration in rodents

Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7th leading cause of premature death [1]. Despite this, there are currently only 3 FDA approved pharmacological approaches for the treatment of AUDs in the United States. The neurotensin (Nts) system has long been implicated in modulating behaviors associated with alcohol misuse. Recently, a novel compound, SBI-553, that biases the action of Nts receptor 1 (NTSR1) activation, has shown promise in preclinical models of psychostimulant use. Here we investigate the efficacy of this compound to alter ethanol-mediated behaviors in a comprehensive battery of experiments assessing ethanol consumption, behavioral responses to ethanol, physiological sensitivity to ethanol, and ethanol metabolism. Additionally, we investigated behavior in avoidance and cognitive assays to monitor potential side effects of SBI-553. We find that SBI-553 reduces ethanol consumption in mice without altering avoidance behavior or novel object recognition. We also observe sex-dependent differences in physiological responses to sequential ethanol injections in mice. In rats, we show that SBI-553 attenuates sensitivity to the interoceptive effects of ethanol (using a Pavlovian drug discrimination task). Our data suggest that targeting NTSR1 signaling may be promising to attenuate alcohol misuse, and adds to a body of literature that suggests NTSR1 may be a common downstream target involved in the psychoactive effects of multiple reinforcing substances.

Targeting adhesion G protein-coupled receptors. Current status and future perspectives.

G protein-coupled receptors (GPCRs) orchestrate many physiological functions and are a crucial target in drug discovery. Adhesion GPCRs (aGPCRs), the second largest family within this superfamily, are promising yet underexplored targets for treating various diseases, including obesity, psychiatric disorders, and cancer. However, the receptors' unique and complex structure and miscellaneous interactions complicate comprehensive pharmacological studies. Despite recent progress in determining structures and elucidation of the activation mechanism, the function of many receptors remains to be determined. This review consolidates current knowledge on aGPCR ligands, focusing on small molecule orthosteric ligands and allosteric modulators identified for the ADGRGs subfamily (subfamily VIII), (GPR56/ADGRG1, GPR64/ADGRG2, GPR97/ADGRG3, GPR114/ADGRG5, GPR126/ADGRG6, and GPR128/ADGRG7). We discuss challenges in hit identification, target validation, and drug discovery, highlighting molecular compositions and recent structural breakthroughs. ADGRG ligands can offer new insights into aGPCR modulation and have significant potential for novel therapeutic interventions targeting various diseases.

Gliflozins, sucrose and flavonoids are allosteric activators of lecithin-cholesterol acyltransferase

Lecithin-cholesterol acyltransferase (LCAT) serves as a pivotal enzyme in preserving cholesterol homeostasis via reverse cholesterol transport, a process closely associated with the onset of atherosclerosis. Impaired LCAT function can lead to severe LCAT deficiency disorders for which no pharmacological treatment exists. LCAT-based therapies, such as small molecule positive allosteric modulators (PAMs), against LCAT deficiencies and atherosclerosis hold promise, although their efficacy against atherosclerosis remains challenging. Herein we utilized a quantitative in silico metric to predict the activity of novel PAMs and tested their potencies with in vitro enzymatic assays. As predicted, sodium-glucose cotransporter 2 (SGLT2) inhibitors (gliflozins), sucrose and flavonoids activate LCAT. This has intriguing implications for the mechanism of action of gliflozins, which are commonly used in the treatment of type 2 diabetes, and for the endogenous activation of LCAT. Our results underscore the potential of molecular dynamics simulations in rational drug design.

Heterotropic Allosteric Modulation of CYP3A4 In Vitro by Progesterone: Evidence for Improvement in Prediction of Time Dependent Inhibition for Macrolides.

Predictions of drug-drug interactions resulting from time-dependent inhibition (TDI) of CYP3A4 have consistently overestimated or mis-predicted (i.e. false positives) the interaction that is observed in vivo. Recent findings demonstrated that the presence of the allosteric modulator progesterone (PGS) in the in vitro assay could alter the in vitro kinetics of CYP3A4 TDI with inhibitors that interact with the heme moiety, such as metabolic-intermediate complex (MIC) forming inhibitors. The impact of the presence of 100 µM PGS on the TDI of molecules in the class of macrolides typically associated with MIC formation was investigated. Presence of PGS resulted in varied responses across the inhibitors tested. The TDI signal was eliminated for five inhibitors, and unaltered in the case of one, fidaxomicin. The remaining molecules erythromycin, clarithromycin, and troleandomycin, were observed to have a decrease in both potency and maximum inactivation rate ranging from 1.7-fold to 6.7-fold. These changes in TDI kinetics led to a >90% decrease in inactivation efficiency. In order to determine in vitro conditions that could reproduce in vivo inhibition, varied concentrations of PGS were incubated with clarithromycin and erythromycin. Resulting in vitro TDI kinetics were incorporated into dynamic physiologically-based pharmacokinetic (PBPK) models to predict clinically observed interactions. The results suggested that a concentration of ~45 µM PGS would result in TDI kinetic values that could reproduce in vivo observations and could potentially improve predictions for CYP3A4 TDI. Significance Statement The impact of the allosteric heterotropic modulator progesterone on the CYP3A4 time-dependent inhibition kinetics was quantified for a set of metabolic-intermediate complex forming mechanism-based inhibitors. We identify the in vitro conditions that optimally predict time-dependent inhibition for in vivo drug-drug interactions through dynamic physiologically-based pharmacokinetic modeling. The optimized assay conditions improve in vitro to in vivo translation and prediction of time-dependent inhibition.