The current agonists and positive allosteric modulators ofα7 nAChR for CNS indications in clinical trials

The alpha-7 nicotinic acetylcholine receptor(α7 nAChR), consisting of homomeric α7 subunits, is a ligand-gated Ca^(2+)-permeable ion channel implicated in cognition and neuropsychiatric disorders. Enhancement of α7 nAChR function is considered to be a potential therapeutic strategy aiming at ameliorating cognitive deficits of neuropsychiatric disorders such as Alzheimer's disease(AD) and schizophrenia. Currently, a number of α7 nAChR modulators have been reported and several of them have advanced into clinical trials. In this brief review, we outline recent progress made in understanding the role of the α7 nAChR in multiple neuropsychiatric disorders and the pharmacological effects of α7 nAChR modulators used in clinical trials.

Sonogashira coupling reaction in the synthesis of novel positive allosteric modulators ofα7 nicotinic acetylcholine receptors

A series of 2-arylamino-1,3,5-triazine derivatives(4a–4g),which were designed and synthesized via Sonogashira coupling reaction,were evaluated using two-electrode voltage clamp(TEVC)recordings of humanα7 nAChR expressed in Xenopus ooctyes.Compound 4g as a positive allosteric modulator(PAM)showed better efficacy than lead compound 3(HZZ-A-11)with an EC50 value of 1.23±0.41μM.Further pharmacological evaluation of compound 4g might lead to the developmental potential for therapy of cognitive deficits commonly shared by neuropsychiatric disorders,such as schizophrenia and Alzheimer’s disease.

Computational Investigation on the Allosteric Modulation of Androgen Receptor

Androgens have similar structures with different biological activities. To identify molecular determinants responsible for the activity difference, we have docked six steroidal androgens to the binding site or the surface of androgen receptor by using molecular docking with computational investigation. The energy was calculated respectively based on the QM （quantum mechanics） and MM （molecular mechanics） methods. The result shows that the allosteric modulation of androgen receptor plays an important role in the binding process between androgens and receptor. The open state receptor is less stable than the close state one, but the latter is more favorable for binding with androgens. It is worthy of note that when the androgen receptors binding or without binding with androgen are in close state, they are difficult to return to their open state. This phenomenon is an exception of the well known two-state model theory in which the two states are reversible. Whether the internal of close state androgen receptor has a combination of androgen or not, the androgen receptor surface can be combined with another androgen, and their surface binding energies could be very close. The result is consistent with the experimental observations, but this phenomenon of continuous combination from open state is also an exception of the two-state model theory.

Allosteric modulation of G protein-coupled receptors as a novel therapeutic strategy in neuropathic pain

Neuropathic pain is a debilitating pathological condition that presents significant therapeutic challenges in clinical practice.Unfortunately,current pharmacological treatments for neuropathic pain lack clinical efficacy and often lead to harmful adverse reactions.As G protein-coupled receptors(GPCRs)are widely distributed throughout the body,including the pain transmission pathway and descending inhibition pathway,the development of novel neuropathic pain treatments based on GPCRs allosteric modulation theory is gaining momentum.Extensive research has shown that allosteric modulators targeting GPCRs on the pain pathway can effectively alleviate symptoms of neuropathic pain while reducing or eliminating adverse effects.This review aims to provide a comprehensive summary of the progress made in GPCRs allosteric modulators in the treatment of neuropathic pain,and discuss the potential benefits and adverse factors of this treatment.We will also concentrate on the development of biased agonists of GPCRs,and based on important examples of biased agonist development in recent years,we will describe universal strategies for designing structure-based biased agonists.It is foreseeable that,with the continuous improvement of GPCRs allosteric modulation and biased agonist theory,effective GPCRs allosteric drugs will eventually be available for the treatment of neuropathic pain with acceptable safety.

Therapeutic potential of α7 nicotinic receptor agonists to regulate neuroinflammation in neurodegenerative diseases

Neurodegenerative diseases, such as Alzheimer＇s, Parkinson＇s and Huntington＇s diseases, are all character- ized by a component of innate immunity called neuroinflammation. Neuronal loss and neuroinflammation are two phenomena closely linked. Hence, the neuroinflammation is a relevant target for the management of the neurodegenerative diseases given that, to date, there is no treatment to stop neuronal loss. Several studies have investigated the potential effects of activators of alpha 7 nicotinic acetylcholine receptors in animal models of neurodegenerative diseases. These receptors are widely distributed in the central nervous system. After activation, they seem to mediate the cholinergic anti-inflammatory pathway in the brain. This anti-inflammatory pathway, first described in periphery, regulates activation of microglial cells considered as the resident macrophage population of the central nervous system. In this article, we shortly review the agonists of the alpha 7 nicotinic acetylcholine receptors that have been evaluated in vivo and we focused on the selective positive allosteric modulators of these receptors. These compounds represent a key element to enhance receptor activity only in the presence of the endogenous agonist.

Biphasic modulation of chemerin peptide-induced calcium flux and ERK phosphorylation by amyloid beta peptide

OBJECTIVE The chemokine-like receptor 1(CMKLR1,Chem R23) is a functional receptor for chemerin,the chemerin-derived nonapeptide(C9),and the amyloid β peptide 1-42(Aβ_(42)).Because these peptides share little sequence homology,studies were conducted to investigate their pharmacological properties and regulation at CMKLR1.METHODS Cells expressing CMKLR1 were incubated with Aβ_(42) before stimulation with a strong agonist,the C9 peptide.Calcium mobilization,c AMP inhibition and MAP kinase activation were measured.Intramolecular FRET were determined using CMKLR1 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first intracellular loop(IL1).RESULTS Binding of both Aβ_(42) and the C9 peptide induced CMKLR1 internalization,but only the Aβ_(42)-induced receptor internalization involved clathrin-coated pits.Likewise,Aβ_(42) but not C9 stimulated β-arrestin 2 translocation to plasma membranes.A robust Ca^(2+)flux was observed following C9 stimulation,whereas Aβ_(42) was ineffective even at micromolar concentrations.Despite its low potency in calcium mobilization assay,Aβ_(42) was able to alter C9-induced Ca^(2+) flux in dose-dependent manner:a potentiation effect at 100 pmol·L^(-1) of Aβ_(42) was followed by a suppression at 10 nmol·L^(-1) and further potentiation at 1 μmol·L^(-1).This unusual and biphasic modulatory effect was also seen in the C9-induced ERK phosphorylation but the dose curve was opposite to that of Ca^(2+) flux and c AMP inhibition,suggesting a reciprocal regulatory mechanism.Intramolecular FRET assay confirmed that Aβ_(42) modulates CMKLR1 rather than its downstream signaling pathways.CONCLUSION These findings suggest Aβ_(42) as an allosteric modulator that can both positively and negatively regulate the activation state of CMKLR1 in a manner that differs from existing allosteric modulatory mechanisms.

Functionally diverse ligands modulate different activation states of the formyl peptide receptor 2,a G protein-coupled receptor

OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2(FPR2)mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner.METHODS Cells expressing FPR2 were incubated with weak agonists,Aβ42 and Ac2-26,before stimulation with a strong agonist,WKYMVm.Calcium mobilization,c AMP inhibition and MAP kinase activation were measured.Intramolecular FRET were determined using FPR2 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first or third intracellular loop(IL1 or IL3,respectively).RESULTS Aβ42 did not induce significant Ca^(2+) mobilization,but positively modulated WKYMVm-induced Ca^(2+) mobilization and c AMP reduction in a dose-variable manner within a narrow range of ligand concentrations.Treating FPR2-expressing cells with Ac2-26,a peptide with anti-inflammatory activity,negatively modulated WKYMVm-induced Ca^(2+) mobilization and c AMP reduction.Intramolecular FRET assay showed that stimulation of the receptor constructs with Aβ42 brought the C-terminal domain closer to IL1 but away from IL3.An opposite conformational change was induced by Ac2-26.The FPR2 conformation induced by Aβ42 corresponded to enhanced ERK phosphorylation and attenuated p38 MAPK phosphorylation,whereas Ac2-26 induced FPR2 conformational change corresponding to elevated p38 MAPK phosphorylation and reduced ERK phosphorylation.CONCLUSION Aβ42 and Ac2-26 induce different conformational changes in FPR2.These findings provide a structural basis for FPR2 mediation of inflammatory vs anti-inflammatory functions and identify a type of receptor modulation that differs from the classic positive and negative allosteric modulation.

SARS-CoV-2 spike variants differ in their allosteric responses to linoleic acid

The SARS-CoV-2 spike protein contains a functionally important fatty acid(FA)binding site,which is also found in some other coronaviruses,e.g.SARS-CoV and MERS-CoV.The occupancy of the FA site by linoleic acid(LA)reduces infectivity by‘locking’the spike in a less infectious conformation.Here,we use dynamical-nonequilibrium molecular dynamics(D-NEMD)simulations to compare the allosteric responses of spike variants to LA removal.D-NEMD simulations show that the FA site is coupled to other functional regions of the protein,e.g.the receptor-binding motif(RBM),N-terminal domain(NTD),furin cleavage site,and regions surrounding the fusion peptide.D-NEMD simulations also identify the allosteric networks connecting the FA site to these functional regions.The comparison between the wild-type spike and four variants(Alpha,Delta,Delta plus,and Omicron BA.1)shows that the variants differ significantly in their responses to LA removal.The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein,with the exception of the RBM and the S71–R78 region,which show a weaker link to the FA site.In contrast,Omicron is the most different variant,exhibiting significant differences in the RBM,NTD,V622–L629,and furin cleavage site.These differences in the allosteric modulation may be of functional relevance,potentially affecting transmissibility and virulence.Experimental comparison of the effects of LA on SARS-CoV-2 variants,including emerging variants,is warranted.

Muscarinic acetylcholine receptor modulators derived from natural toxins and diverse interaction modes

Muscarinic acetylcholine receptors （mAChRs） play crucial roles in various physiological functions and pathophysiological processes. Acetylcholine （ACh）, as a classical ligand and one of the pivotal neurotransmitters, serves as a prototype for the elucidation of molecular interaction and the development of mimicked and antagonized agents. With the advances in medicinal chemistry and structural biology, more and more mAChRs modulators derived from natural toxins have been identified. Based on the chemical structures and the receptor-ligand interaction modes, these mAChRs modulators can be divided into orthosteric modulators, allosteric modulators and other modulators. Moreover, allosteric modulators can be further divided into three groups： alcuronium-like modulators, staurosporine-like modulators, and phlegrnarine-like modulators. In this review, we focus on various mAChRs modulators derived from natural toxins on the basis of the receptor-ligand interaction modes. The under- standing of the affinity, the intrinsic efficacy, and the selectivity of mAChRs modulators may lead to the discovery of new drug leads for the treatment of diseases related to mAChRs.

The design, synthesis and α7 nicotinic acetylcholine receptors positive allosteric modulative evaluation of 3H-quinazolin-4-one derivatives

A series of new 6-substituted 3 H-quinazolin-4-ones（3 a-3 d） were designed, synthesized and evaluated as the type I positive allosteric modulators（PAMs） of human α7 n ACh R expressed in Xenopus ooctyes by two-electrode voltage clamp. However, no compound showed a better efficacious PAM than lead compound 2 in the presence of acetylcholine（100 μM）. The structure-activity relationship（SAR） analysis suggested that thiazolo[4,5-d]pyrimidin-7（6 H）-one was the key biological skeleton.

Identifying Qingkailing(清开灵)ingredients-dependent mesenchymal-epithelial transition factor-axiation“π”structuring module with angiogenesis and neurogenesis effects

OBJECTIVE:To explore the functional role of the drugdependent mesenchymal-epithelial transition(Met)-axiation"π"structural module of neurogenesis after processing by three components of Qingkailing injection in neurogenesis and angiogenesis in cerebral ischemia.METHODS:We used a Glutathione S-transferase(GST)-pull down assay,isothermal titration calorimetry assay,and other related methods to identify the relationships among Met,inositol polyphosphate phosphatase like 1(Inppl1),and death associated protein kinase 3(Dapk3)in this allosteric module.The biological effects of the modules of neurons generation composed of Met,Inppl1,and Dapk3 were measured through Western blot,apoptosis analysis,and double immunofluorescence labeling.RESULTS:The GST-pull down assay revealed that proline-serine-threonine rich domain of Met binds to the Src homology domain of Inppl1 to form a protein-protein complex;Dapk3 with a C-terminal domain interacts weakly with the protein kinase C domain of Met in the intracellular region.Thus,we obtained a“π”structuring module considered a neural regeneration module.The biological effects of angiogenesis and neurogenesis modules composed of Met,Inppl1,and Dapk3 were also verified.CONCLUSION:The study suggested that understanding the functional modules that contribute to pharmaceutics might provide novel signatures that can be used as endpoints to define disease processes under stroke or cerebral ischemia conditions.

Exploring structure-based drug discovery of GPCRs beyond the orthosteric binding

G-protein coupled receptors(GPCRs)are the largest family of druggable targets.In recent years,GPCR structural biology has made great advances,revealing the three-dimensional structures of many GPCRs and their interactions with ligands,proteins,and membrane components,which also have inspired a surge of structure-based drug discovery campaigns.This article provides a comprehensive summary of the currently available structural insights into the allosteric pockets of GPCRs and their regulatory mechanisms governing GPCR conformational changes.Furthermore,this article also presents several structure-inspired studies that utilize both orthosteric and allosteric modulation to discover small molecular modulators targeting GPCRs.The article emphasizes the promising potential of drug discovery targeting GPCR allosteric sites,while acknowledging the challenges arising from the limited structural information regarding the lipids and cholesterols in the membrane.Finally,the article discusses the future prospects of using large-scale or focused compound libraries to discover novel chemotypes,as well as the application of artificial intelligence(AI)in structure-based virtual screening(SBVS)against GPCRs.

Allosteric Modulation of Human Serum Albumin Induced by Peptide Ligand

Human serum albumin （HSA） is an abundant protein in plasma that can bind and transport many small molecules, and the corresponding affinity-controlled drug delivery shows great advantage in the biological system. Peptide SA06 is a reported ligand comprising 20 amino acids, and is known to non-covalently bind with HSA to extend the lifetime and improve the pharmacokinetic performance. The structural information of the HSA-peptide complex is keen for obtainingmolecular insight of the binding mechanism. We studied the secondary structural change and structure-affinity relations of Peptide SA06 with HSA by using circular dichroism （CD） spectroscopy in solution. Noticeable allosteric effect can be identified by compositional increase of a-helix structures when the peptide was co-incubated with HSA. Furthermore, the equilibrium dissociation constant of Peptide SA06 with HSA can be determined by CD-baged method. This work provides structural evidence on the allosteric interaction between peptide ligand and HSA, and sheds light on optimization of therapeutic properties in the affinity-controlled delivery systems.

Met-Controlled Allosteric Module of Neural Generation as A New Therapeutic Target in Rodent Brain Ischemia

Objective To investigate a Met-controlled allosteric module(AM)of neural generation as a potential therapeutic target for brain ischemia.Methods We selected Markov clustering algorithm(MCL)to mine functional modules in the related target networks.According to the topological similarity,one functional module was predicted in the modules of baicalin(BA),jasminoidin(JA),cholic acid(CA),compared with I/R model modules.This functional module included three genes:Inppl1,Met and Dapk3(IMD).By gene ontology enrichment analysis,biological process related to this functional module was obtained.This functional module participated in generation of neurons.Western blotting was applied to present the compound-dependent regulation of IMD.Co-immunoprecipitation was used to reveal the relationship among the three members.We used IF to determine the number of newborn neurons between compound treatment group and ischemia/reperfusion group.The expressions of vascular endothelial growth factor(VEGF)and matrix metalloproteinase 9(MMP-9)were supposed to show the changing circumstances for neural generation under cerebral ischemia.Results Significant reduction in infarction volume and pathological changes were shown in the compound treatment groups compared with the I/R model group(P<0.05).Three nodes in one novel module of IMD were found to exert diverse compound-dependent ischemic-specific excitatory regulatory activities.An anti-ischemic excitatory allosteric module(AME)of generation of neurons(AME-GN)was validated successfully in vivo.Newborn neurons increased in BJC treatment group(P<0.05).The expression of VEGF and MMP-9 decreased in the compound treatment groups compared with the I/R model group(P<0.05).Conclusions AME demonstrates effectiveness of our pioneering approach to the discovery of therapeutic target.The novel approach for AM discovery in an effort to identify therapeutic targets holds the promise of accelerating elucidation of underlying pharmacological mechanisms in cerebral ischemia.

Electrophysiological characterization of furo[3,2-b]pyridine derivatives as negative allosteric modulator of a7 nicotinic acetylcholine receptor

A series of lH-pyrrolo[3,2-b]pyridine (3a-3f) and fUroP,2-b]pyridine derivatives (4a-4g) were evaluated on human a7 nicotinic acetylcholine receptors (nAChRs) using two-electrode voltage clamp (TEVC) recording. A representative 2-(2-methoxyphenyl)- furo[3,2-b]pyridine 4f as negative allosteric modulator (NAM) selectively inhibited alpha7 nAChR over a3p4, a4p2 nAChRs and 5-HT3a receptor, with a potency of IC50 of 5.51 |1M and a maximum inhibition rate of 87.8%. The preliminary analysis of structure-activity relationship (SAR) suggested that compound 4f could serve as a basis for further discovery of potent and selective a7 nAChR NAMs.

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

As a general mechanism for governing the bioactivity of membrane receptors,allosteric modulation is critical in cell signaling and cell communication but remains difficult to measure in situ.Herein,we introduce a data mining-integrated tracking microscopy(DMITM)to investigate allosteric modulation of membrane receptors in the native state in live cells.Using Kmeans clustering-based hidden Markov modeling to uncover the ligand binding and unbinding events with diffusivity variations of ligand-conjugated nanoprobes as observations.

Modulating effects of RAMPs on signaling profiles of the glucagon receptor family

Receptor activity-modulating proteins(RAMPs)are accessory molecules that form complexes with specific G protein-coupled receptors(GPCRs)and modulate their functions.It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood.In this study,we used a bioluminescence resonance energy transfer(BRET)approach to comprehensively investigate such interactions.In conjunction with c AMP accumulation,Gaqactivation andβ-arrestin1/2 recruitment assays,we not only verified the GPCR-RAMP pairs previously reported,but also identified new patterns of GPCR-RAMP interaction.While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor(GCGR)and glucagon-like peptide-1 receptor(GLP-1 R),and RAMP2 mainly affectedβ-arrestin1/2 recruitment by GCGR,GLP-1 R and glucagon-like peptide-2 receptor,RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways.Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner.Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation.