Mechanisms of Allosteric Inhibition of Insulin-Regulated Aminopeptidase.

Inhibition of Insulin-Regulated Aminopeptidase is being actively explored for the treatment of several human diseases and several classes of inhibitors have been developed although no clinical applications have been reported yet. Here, we combine enzymological analysis with x-ray crystallography to investigate the mechanism employed by two of the most studied inhibitors of IRAP, an aryl sulfonamide and a 2-amino-4H-benzopyran named HFI-419. Although both compounds have been hypothesized to target the enzyme's active site by competitive mechanisms, we discovered that they instead target previously unidentified proximal allosteric sites and utilize non-competitive inhibition mechanisms. X-ray crystallographic analysis demonstrated that the aryl sulfonamide stabilizes the closed, more active, conformation of the enzyme whereas HFI-419 locks the enzyme in a semi-open, and likely less active, conformation. HFI-419 potency is substrate-dependent and fails to effectively block the degradation of the physiological substrate cyclic peptide oxytocin. Our findings demonstrate alternative mechanisms for inhibiting IRAP through allosteric sites and conformational restricting and suggest that the pharmacology of HFI-419 may be more complicated than initially considered. Such conformation-specific interactions between IRAP and small molecules can be exploited for the design of more effective second-generation allosteric inhibitors.

The energetic and allosteric landscape for KRAS inhibition.

Thousands of proteins have been validated genetically as therapeutic targets for human diseases1. However, very few have been successfully targeted, and many are considered 'undruggable'. This is particularly true for proteins that function via protein-protein interactions-direct inhibition of binding interfaces is difficult and requires the identification of allosteric sites. However, most proteins have no known allosteric sites, and a comprehensive allosteric map does not exist for any protein. Here we address this shortcoming by charting multiple global atlases of inhibitory allosteric communication in KRAS. We quantified the effects of more than 26,000 mutations on the folding of KRAS and its binding to six interaction partners. Genetic interactions in double mutants enabled us to perform biophysical measurements at scale, inferring more than 22,000 causal free energy changes. These energy landscapes quantify how mutations tune the binding specificity of a signalling protein and map the inhibitory allosteric sites for an important therapeutic target. Allosteric propagation is particularly effective across the central ß-sheet of KRAS, and multiple surface pockets are genetically validated as allosterically active, including a distal pocket in the C-terminal lobe of the protein. Allosteric mutations typically inhibit binding to all tested effectors, but they can also change the binding specificity, revealing the regulatory, evolutionary and therapeutic potential to tune pathway activation. Using the approach described here, it should be possible to rapidly and comprehensively identify allosteric target sites in many proteins.

The impact of cryo-EM on determining allosteric modulator-bound structures of G protein-coupled receptors.

G-protein coupled receptors (GPCRs) are important therapeutic targets for the treatment of human disease. Although GPCRs are highly successful drug targets, there are many challenges associated with the discovery and translation of small molecule ligands that target the endogenous ligand-binding site for GPCRs. Allosteric modulators are a class of ligands that target alternative binding sites known as allosteric sites and offer fresh opportunities for the development of new therapeutics. However, only a few allosteric modulators have been approved as drugs. Advances in GPCR structural biology enabled by the cryogenic electron microscopy (cryo-EM) revolution have provided new insights into the molecular mechanism and binding location of small molecule allosteric modulators. This review highlights the latest findings from allosteric modulator-bound structures of Class A, B, and C GPCRs with a focus on small molecule ligands. Emerging methods that will facilitate cryo-EM structures of more difficult ligand-bound GPCR complexes are also discussed. The results of these studies are anticipated to aid future structure-based drug discovery efforts across many different GPCRs.

Mechanistic insights on novel small molecule allosteric activators of cGMP-dependent protein kinase PKG1α.

cGMP-dependent protein kinase (PKG) represents a compelling drug target for treatment of cardiovascular diseases. PKG1 is the major effector of beneficial cGMP signaling which is involved in smooth muscle relaxation and vascular tone, inhibition of platelet aggregation and signaling that leads to cardioprotection. In this study, a novel piperidine series of activators previously identified from an ultrahigh-throughput screen were validated to directly bind partially activated PKG1α and subsequently enhance its kinase activity in a concentration-dependent manner. Compounds from initial optimization efforts showed an ability to activate PKG1α independent of the endogenous activator, cGMP. We demonstrate these small molecule activators mimic the effect of cGMP on the kinetic parameters of PKG1α by positively modulating the KM of the peptide substrate and negatively modulating the apparent KM for ATP with increase in catalytic efficiency, kcat. In addition, these compounds also allosterically modulate the binding affinity of cGMP for PKG1α by increasing the affinity of cGMP for the high-affinity binding site (CNB-A) and decreasing the affinity of cGMP for the low-affinity binding site (CNB-B). We show the mode of action of these activators involves binding to an allosteric site within the regulatory domain, near the CNB-B binding site. To the best of our knowledge, these are the first reported non-cGMP mimetic small molecules shown to directly activate PKG1α. Insights into the mechanism of action of these compounds will enable future development of cardioprotective compounds that function through novel modes of action for the treatment of cardiovascular diseases.

Druggable Allosteric Sites in ß-Propeller Lectins.

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a ß-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19 F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure-activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol-1 HA-1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens.

Xanthine Derivatives Reveal an Allosteric Binding Site in Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2).

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.

Discovery of Novel Allosteric Modulators Targeting an Extra-Helical Binding Site of GLP-1R Using Structure- and Ligand-Based Virtual Screening.

Allosteric modulators have emerged with many potential pharmacological advantages as they do not compete the binding of agonist or antagonist to the orthosteric sites but ultimately affect downstream signaling. To identify allosteric modulators targeting an extra-helical binding site of the glucagon-like peptide-1 receptor (GLP-1R) within the membrane environment, the following two computational approaches were applied: structure-based virtual screening with consideration of lipid contacts and ligand-based virtual screening with the maintenance of specific allosteric pocket residue interactions. Verified by radiolabeled ligand binding and cAMP accumulation experiments, two negative allosteric modulators and seven positive allosteric modulators were discovered using structure-based and ligand-based virtual screening methods, respectively. The computational approach presented here could possibly be used to discover allosteric modulators of other G protein-coupled receptors.

Potentiation of Muscarinic M3 Receptor Activation through a New Allosteric Site with a Novel Positive Allosteric Modulator ASP8302.

Muscarinic M3 (M3) receptors mediate a wide range of acetylcholine (ACh)-induced functions, including visceral smooth-muscle contraction and glandular secretion. Positive allosteric modulators (PAMs) can avoid various side effects of muscarinic agonists with their spatiotemporal receptor activation control and potentially better subtype selectivity. However, the mechanism of allosteric modulation of M3 receptors is not fully understood, presumably because of the lack of a potent and selective PAM. In this study, we investigated the pharmacological profile of ASP8302, a novel PAM of M3 receptors, and explored the principal site of amino-acid sequences in the human M3 receptor required for the potentiation of receptor activation. In cells expressing human M3 and M5 receptors, ASP8302 shifted the concentration-response curve (CRC) for carbachol to the lower concentrations with no significant effects on other subtypes. In a binding study with M3 receptor-expressing membrane, ASP8302 also shifted the CRC for ACh without affecting the binding of orthosteric agonists. Similar shifts in the CRC of contractions by multiple stimulants were also confirmed in isolated human bladder strips. Mutagenesis analysis indicated no interaction between ASP8302 and previously reported allosteric sites; however, it identified threonine 230 as the amino acid essential for the PAM effect of ASP8302. These results demonstrate that ASP8302 enhances the activation of human M3 receptors by interacting with a single amino acid distinct from the reported allosteric sites. Our findings suggest not only a novel allosteric site of M3 receptors but also the potential application of ASP8302 to diseases caused by insufficient M3 receptor activation. SIGNIFICANCE STATEMENT: The significance of this study is that the novel M3 receptor positive allosteric modulator ASP8302 enhances the activation of human M3 receptor by interacting with a residue distinct from the reported allosteric sites. The finding of Thr230 as a novel amino acid involved in the allosteric modulation of M3 receptors provides significant insight into further research of the mechanism of allosteric modulation of M3 and other muscarinic receptors.

Allosteric Modulation of GSK-3ß as a New Therapeutic Approach in Limb Girdle Muscular Dystrophy R1 Calpain 3-Related.

Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients' muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3ß (GSK-3ß), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.

Design of Negative and Positive Allosteric Modulators of the Cannabinoid CB2 Receptor Derived from the Natural Product Cannabidiol.

Cannabidiol (CBD), the second most abundant of the active compounds found in the Cannabis sativa plant, is of increasing interest because it is approved for human use and is neither euphorizing nor addictive. Here, we design and synthesize novel compounds taking into account that CBD is both a partial agonist, when it binds to the orthosteric site, and a negative allosteric modulator, when it binds to the allosteric site of the cannabinoid CB2 receptor. Molecular dynamic simulations and site-directed mutagenesis studies have identified the allosteric site near the receptor entrance. This knowledge has permitted to perform structure-guided design of negative and positive allosteric modulators of the CB2 receptor with potential therapeutic utility.

Affinity Mass Spectrometry-Based Fragment Screening Identified a New Negative Allosteric Modulator of the Adenosine A2A Receptor Targeting the Sodium Ion Pocket.

Allosteric ligands provide new opportunities to modulate G protein-coupled receptor (GPCR) function and present therapeutic benefits over orthosteric molecules. Negative allosteric modulators (NAMs) can inhibit the activation of a receptor and downstream signal transduction. Screening NAMs for a GPCR target is particularly challenging because of the difficulty in distinguishing NAMs from antagonists bound to the orthosteric site as they both show inhibitory effects in receptor signaling assays. Here we report an affinity mass spectrometry (MS)-based approach tailored to screening potential NAMs of a GPCR target especially from fragment libraries. Compared to regular surface plasmon resonance or NMR-based methods for fragment screening, our approach features a reduction of the protein and compound consumption by 2-4 orders of magnitude and an increase in the data acquisition speed by 2-3 orders of magnitude. Our affinity MS-based fragment screening led to the identification of a new NAM of the adenosine A2A receptor (A2AAR) bearing an unprecedented azetidine moiety predicted to occupy the allosteric sodium binding site. Molecular dynamics simulations, ligand structure-activity relationship (SAR) studies, and in-solution NMR analyses further revealed the unique binding mode and antagonistic property of this compound that differs considerably from HMA (5-(N,N-hexamethylene)amiloride), a well-characterized NAM of A2AAR. Taken together, our work would facilitate fragment-based screening of allosteric modulators, as well as guide the design of novel NAMs acting at the sodium ion pocket of class A GPCRs.

Structure-Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt.

The inhibition of the nuclear receptor retinoic-acid-receptor-related orphan receptor γt (RORγt) is a promising strategy in the treatment of autoimmune diseases. RORγt features an allosteric binding site within its ligand-binding domain that provides an opportunity to overcome drawbacks associated with orthosteric modulators. Recently, trisubstituted isoxazoles were identified as a novel class of allosteric RORγt inverse agonists. This chemotype offers new opportunities for optimization into selective and efficacious allosteric drug-like molecules. Here, we explore the structure-activity relationship profile of the isoxazole series utilizing a combination of structure-based design, X-ray crystallography, and biochemical assays. The initial lead isoxazole (FM26) was optimized, resulting in compounds with a ∼10-fold increase in potency (low nM), significant cellular activity, promising pharmacokinetic properties, and a good selectivity profile over the peroxisome-proliferated-activated receptor γ and the farnesoid X receptor. We envisage that this work will serve as a platform for the accelerated development of isoxazoles and other novel chemotypes for the effective allosteric targeting of RORγt.

Controlling the selectivity of aminergic GPCR ligands from the extracellular vestibule.

In addition to the orthosteric binding pocket (OBP) of GPCRs, recent structural studies have revealed that there are several allosteric sites available for pharmacological intervention. The secondary binding pocket (SBP) of aminergic GPCRs is located in the extracellular vestibule of these receptors, and it has been suggested to be a potential selectivity pocket for bitopic ligands. Here, we applied a virtual screening protocol based on fragment docking to the SBP of the orthosteric ligand-receptor complex. This strategy was employed for a number of aminergic receptors. First, we designed dopamine D3 preferring bitopic compounds from a D2 selective orthosteric ligand. Next, we designed 5-HT2B selective bitopic compounds starting from the 5-HT1B preferring ergoline core of LSD. Comparing the serotonergic profiles of the new derivatives to that of LSD, we found that these derivatives became significantly biased towards the desired 5-HT2B receptor target. Finally, addressing the known limitations of H1 antihistamines, our protocol was successfully used to eliminate the well-known side effects related to the muscarinic M1 activity of amitriptyline while preserving H1 potency in some of the designed bitopic compounds. These applications highlight the usefulness of our new virtual screening protocol and offer a powerful strategy towards bitopic GPCR ligands with designed receptor profiles.

Novel, natural allosteric inhibitors and enhancers of Candida rugosa lipase activity.

Candida rugosa lipase (CRL) is an enzyme commonly used in medicinal and biotechnological applications. Allosteric modulators of CRL could aid in modifying lipase-related diseases as well as improving biotechnological processes. Thus, a combinatorial approach of computational in-silico and high-throughput in-vitro screening was used to identify allosteric modulators of CRL. The screening of natural product libraries resulted in 132 compounds of which 53 were tested in-vitro. Subsequently, four inhibitors and three enhancers were identified of which rutin and cynaroside represented the strongest inhibitors of CRL activity (IC50: 227 ± 26 µM and 446 ± 15 µM, respectively) and NP-008496 the strongest enhancer (EC50: 425 ± 18 µM). All three compounds were predicted to bind the same allosteric site suggesting a common mechanism. Therefore, the present study demonstrated a reliable work-flow, identified an allosteric site of CRL and determined inhibitors and enhancers with numerous potential medical and biotechnological applications.

Cooperativity between the orthosteric and allosteric ligand binding sites of RORγt.

Cooperative ligand binding is an important phenomenon in biological systems where ligand binding influences the binding of another ligand at an alternative site of the protein via an intramolecular network of interactions. The underlying mechanisms behind cooperative binding remain poorly understood, primarily due to the lack of structural data of these ternary complexes. Using time-resolved fluorescence resonance energy transfer (TR-FRET) studies, we show that cooperative ligand binding occurs for RORγt, a nuclear receptor associated with the pathogenesis of autoimmune diseases. To provide the crucial structural insights, we solved 12 crystal structures of RORγt simultaneously bound to various orthosteric and allosteric ligands. The presence of the orthosteric ligand induces a clamping motion of the allosteric pocket via helices 4 to 5. Additional molecular dynamics simulations revealed the unusual mechanism behind this clamping motion, with Ala355 shifting between helix 4 and 5. The orthosteric RORγt agonists regulate the conformation of Ala355, thereby stabilizing the conformation of the allosteric pocket and cooperatively enhancing the affinity of the allosteric inverse agonists.

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

Modulating catalytic activity of human topoisomerase II α enzyme by fluorescent gold nanoclusters.

Precise monitoring of the enzyme activity by a suitable modulator is one of the very fundamental aspects of drug designing that provides the opportunity to overcome the challenges of several diseases. Herein, inhibition of human Topoisomerase IIα enzyme which serves as a potential target site for several anti-cancer drugs is demonstrated by using ultra-small size gold nanoclusters (Au NCs) with the dimension comparable with size of the active site of the enzyme. Molecular dynamics simulation results demonstrate that the Au NCs strongly interact with the human Topo IIα enzyme at its active site or allosteric site depending on forms of enzyme. Additionally, binding energy and interaction profile provides the molecular basis of understanding of interactions of ultra-small size Au NCs and human Topo IIα enzyme. Enthalpy change (ΔH) and binding constant (K) are measured based on a sequential binding model of the Au NCs with the enzyme as demonstrated by the ITC study. Moreover, the in-vitro inhibition study of the catalytic activity of the enzyme and gel electrophoresis indicates that the ultra-small size Au NCs may be used as a potent inhibitor of human Topo IIα enzyme.

Identification of benzimidazole containing 4H-chromen-4-one derivative as potential MAP kinase inhibitors by in-silico approaches.

MAP kinase is one of the important targets in the treatment of osteoarthritis, inflammation and cancer. Many p38 inhibitors with diverse chemical structures and modes of protein interaction have been designed on the basis of their ability to compete with ATP site or allosteric site for binding to MAP Kinase. This study involves the molecular docking of benzimidazoles containing 4H-chrome-4-one derivatives as potent inhibitors of the MAP kinase enzyme. The compounds were computationally designed and optimized with the molecular docking to investigate the interactions between the target compounds and the amino acid residues of the MAP Kinase. The inhibitory activities against human MAP kinase enzyme were investigated by molecular docking using the Autodock and discovery studio software. All the designed compounds were shown good binding energy when compared with the binging energies of standard drug Imatinib (anti-cancer). Among all the designed compounds, compound D1 and D6 have higher binding energy values when compared to standard drug. Here we also studied the molecular properties of designed compound using Molinspiration software. Further, we planned to synthesis these benzimidazole derivatives and screen for in-vitro and in-vivo of anti-cancer activity.

Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site.

TP53 is the most frequently mutated gene in cancer, yet these mutations remain therapeutically non-actionable. Major challenges in drugging p53 mutations include heterogeneous mechanisms of inactivation and the absence of broadly applicable allosteric sites. Here we report the identification of small molecules, including arsenic trioxide (ATO), an established agent in treating acute promyelocytic leukemia, as cysteine-reactive compounds that rescue structural p53 mutations. Crystal structures of arsenic-bound p53 mutants reveal a cryptic allosteric site involving three arsenic-coordinating cysteines within the DNA-binding domain, distal to the zinc-binding site. Arsenic binding stabilizes the DNA-binding loop-sheet-helix motif alongside the overall ß-sandwich fold, endowing p53 mutants with thermostability and transcriptional activity. In cellular and mouse xenograft models, ATO reactivates mutant p53 for tumor suppression. Investigation of the 25 most frequent p53 mutations informs patient stratification for clinical exploration. Our results provide a mechanistic basis for repurposing ATO to target p53 mutations for widely applicable yet personalized cancer therapies.

Activation of the IRE1 RNase through remodeling of the kinase front pocket by ATP-competitive ligands.

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.