Gaining Insights into Key Structural Hotspots within the Allosteric Binding Pockets of Protein Kinases.

Protein kinases are essential regulators of cell function and represent one of the largest and most diverse protein families. They are particularly influential in signal transduction and coordinating complex processes like the cell cycle. Out of the 518 human protein kinases identified, 478 are part of a single superfamily sharing catalytic domains that are related in sequence. The dysregulation of protein kinases due to certain mutations has been associated with various diseases, including cancer. Although most of the protein kinase inhibitors identified as type I or type II primarily target the ATP-binding pockets of kinases, the structural and sequential resemblances among these pockets pose a significant challenge for selective inhibition. Therefore, targeting allosteric pockets that are beside highly conserved ATP pockets has emerged as a promising strategy to prevail current limitations, such as poor selectivity and drug resistance. In this article, we compared the binding pockets of various protein kinases for which allosteric (type III) inhibitors have already been developed. Additionally, understanding the structure and shape of existing ligands could aid in identifying key interaction sites within the allosteric pockets of kinases. This comprehensive review aims to facilitate the design of more effective and selective allosteric inhibitors.

Synthesis, In Vivo Anticonvulsant Activity Evaluation and In Silico Studies of Some Quinazolin-4(3H)-One Derivatives.

Two series, "a" and "b", each consisting of nine chemical compounds, with 2,3-disubstituted quinazolin-4(3H)-one scaffold, were synthesized and evaluated for their anticonvulsant activity. They were investigated as dual potential positive allosteric modulators of the GABAA receptor at the benzodiazepine binding site and inhibitors of carbonic anhydrase II. Quinazolin-4(3H)-one derivatives were evaluated in vivo (D1-3 = 50, 100, 150 mg/kg, administered intraperitoneally) using the pentylenetetrazole (PTZ)-induced seizure model in mice, with phenobarbital and diazepam, as reference anticonvulsant agents. The in silico studies suggested the compounds act as anticonvulsants by binding on the allosteric site of GABAA receptor and not by inhibiting the carbonic anhydrase II, because the ligands-carbonic anhydrase II predicted complexes were unstable in the molecular dynamics simulations. The mechanism targeting GABAA receptor was confirmed through the in vivo flumazenil antagonism assay. The pentylenetetrazole experimental anticonvulsant model indicated that the tested compounds, 1a-9a and 1b-9b, present a potential anticonvulsant activity. The evaluation, considering the percentage of protection against PTZ, latency until the onset of the first seizure, and reduction in the number of seizures, revealed more favorable results for the "b" series, particularly for compound 8b.

Exploring Binding Pockets in the Conformational States of the SARS-CoV-2 Spike Trimers for the Screening of Allosteric Inhibitors Using Molecular Simulations and Ensemble-Based Ligand Docking.

Understanding mechanisms of allosteric regulation remains elusive for the SARS-CoV-2 spike protein, despite the increasing interest and effort in discovering allosteric inhibitors of the viral activity and interactions with the host receptor ACE2. The challenges of discovering allosteric modulators of the SARS-CoV-2 spike proteins are associated with the diversity of cryptic allosteric sites and complex molecular mechanisms that can be employed by allosteric ligands, including the alteration of the conformational equilibrium of spike protein and preferential stabilization of specific functional states. In the current study, we combine conformational dynamics analysis of distinct forms of the full-length spike protein trimers and machine-learning-based binding pocket detection with the ensemble-based ligand docking and binding free energy analysis to characterize the potential allosteric binding sites and determine structural and energetic determinants of allosteric inhibition for a series of experimentally validated allosteric molecules. The results demonstrate a good agreement between computational and experimental binding affinities, providing support to the predicted binding modes and suggesting key interactions formed by the allosteric ligands to elicit the experimentally observed inhibition. We establish structural and energetic determinants of allosteric binding for the experimentally known allosteric molecules, indicating a potential mechanism of allosteric modulation by targeting the hinges of the inter-protomer movements and blocking conformational changes between the closed and open spike trimer forms. The results of this study demonstrate that combining ensemble-based ligand docking with conformational states of spike protein and rigorous binding energy analysis enables robust characterization of the ligand binding modes, the identification of allosteric binding hotspots, and the prediction of binding affinities for validated allosteric modulators, which is consistent with the experimental data. This study suggested that the conformational adaptability of the protein allosteric sites and the diversity of ligand bound conformations are both in play to enable efficient targeting of allosteric binding sites and interfere with the conformational changes.

G protein-coupled receptors (GPCRs): advances in structures, mechanisms, and drug discovery.

G protein-coupled receptors (GPCRs), the largest family of human membrane proteins and an important class of drug targets, play a role in maintaining numerous physiological processes. Agonist or antagonist, orthosteric effects or allosteric effects, and biased signaling or balanced signaling, characterize the complexity of GPCR dynamic features. In this study, we first review the structural advancements, activation mechanisms, and functional diversity of GPCRs. We then focus on GPCR drug discovery by revealing the detailed drug-target interactions and the underlying mechanisms of orthosteric drugs approved by the US Food and Drug Administration in the past five years. Particularly, an up-to-date analysis is performed on available GPCR structures complexed with synthetic small-molecule allosteric modulators to elucidate key receptor-ligand interactions and allosteric mechanisms. Finally, we highlight how the widespread GPCR-druggable allosteric sites can guide structure- or mechanism-based drug design and propose prospects of designing bitopic ligands for the future therapeutic potential of targeting this receptor family.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

[Computer-aided prediction and molecular mechanism investigation of active components in compound Kushen injection inhibiting p21-activated kinase 1].

Targeting p21-activated kinase 1 (PAK1) is a novel strategy for pancreatic cancer treatment. Compound Kushen injection contains many anti-pancreatic cancer components, but the specific targets are unknown. In this study, 14α-hydroxymatrine, an active component of Kushen injection, was found to possess high binding free energy with the allosteric site of PAK1 by molecular docking based virtual screening. Molecular dynamics simulations suggested that 14α-hydroxymatrine caused the α1 and α2 helices of the allosteric site of PAK1 to extend outward to form a deep allosteric regulatory pocket. Meanwhile, 14α-hydroxymatrine induced the ß-folding region at the adenosine triphosphate (ATP)-binding pocket of PAK1 to close inward, resulting in the ATP-binding pocket in a "semi-closed" state which caused the inactivation of PAK1. After removal of 14α-hydroxymatrine, PAK1 showed a tendency to change from the inactive conformation to the active conformation. We supposed that 14α-hydroxymatrine of compound Kushen injection might be a reversible allosteric inhibitor of PAK1. This study used modern technologies and methods to study the active components of traditional Chinese medicine, which laid a foundation for the development and utilization of natural products and the search for new treatments for pancreatic cancer.

Identification of two novel chemical classes of Autotaxin (ATX) inhibitors using Enalos Asclepios KNIME nodes.

Autotaxin is a secreted lysophospholipase D which is a member of the ectonucleotide pyrophosphatase/phosphodiesterase family converting extracellular lysophosphatidylcholine and other non-choline lysophospholipids, such as lysophosphatidylethanolamine and lysophosphatidylserine, to the lipid mediator lysophosphatidic acid. Autotaxin is implicated in various fibroproliferative diseases including interstitial lung diseases, such as idiopathic pulmonary fibrosis and hepatic fibrosis, as well as in cancer. In this study, we present an effort of identifying ATX inhibitors that bind to allosteric ATX binding sites using the Enalos Asclepios KNIME Node. All the available PDB crystal structures of ATX were collected, prepared, and aligned. Visual examination of these structures led to the identification of four crystal structures of human ATX co-crystallized with four known inhibitors. These inhibitors bind to five binding sites with five different binding modes. These five binding sites were thereafter used to virtually screen a compound library of 14,000 compounds to identify molecules that bind to allosteric sites. Based on the binding mode and interactions, the docking score, and the frequency that a compound comes up as a top-ranked among the five binding sites, 24 compounds were selected for in vitro testing. Finally, two compounds emerged with inhibitory activity against ATX in the low micromolar range, while their mode of inhibition and binding pattern were also studied. The two derivatives identified herein can serve as "hits" towards developing novel classes of ATX allosteric inhibitors.

Synergizing structure and function: Cinnamoyl hydroxamic acids as potent urease inhibitors.

The current investigation encompasses the structural planning, synthesis, and evaluation of the urease inhibitory activity of a series of molecular hybrids of hydroxamic acids and Michael acceptors, delineated from the structure of cinnamic acids. The synthesized compounds exhibited potent urease inhibitory effects, with IC50 values ranging from 3.8 to 12.8 µM. Kinetic experiments unveiled that the majority of the synthesized hybrids display characteristics of mixed inhibitors. Generally, derivatives containing electron-withdrawing groups on the aromatic ring demonstrate heightened activity, indicating that the increased electrophilicity of the beta carbon in the Michael Acceptor moiety positively influences the antiureolytic properties of this compounds class. Biophysical and theoretical investigations further corroborated the findings obtained from kinetic assays. These studies suggest that the hydroxamic acid core interacts with the urease active site, while the Michael acceptor moiety binds to one or more allosteric sites adjacent to the active site.

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.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Probe Substrate Dependencies in CYP3A4 Allosteric Inhibition: A Novel Molecular Mechanism Involving F-F' Loop Perturbations.

The biochemical basis for substrate dependences in apparent inhibition constant values (Ki) remains unknown. Our study aims to elucidate plausible structural determinants underpinning these observations. In vitro steady-state inhibition assays conducted using human recombinant CYP3A4 enzyme and testosterone substrate revealed that fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and pemigatinib noncompetitively inhibited CYP3A4 with apparent Ki values of 10.2 ± 1.1 and 3.3 ± 0.9 µM, respectively. However, when rivaroxaban was adopted as the probe substrate, there were 2.0- and 3.2-fold decreases in its apparent Ki values. To glean mechanistic insights into this phenomenon, erdafitinib and pemigatinib were docked to allosteric sites in CYP3A4. Subsequently, molecular dynamics (MD) simulations of apo- and holo-CYP3A4 were conducted to investigate the structural changes induced. Comparative structural analyses of representative MD frames extracted by hierarchical clustering revealed that the allosteric inhibition of CYP3A4 by erdafitinib and pemigatinib did not substantially modulate its active site characteristics. In contrast, we discovered that allosteric binding of the FGFR inhibitors reduces the structural flexibility of the F-F' loop region, an important gating mechanism to regulate access of the substrate to the catalytic heme. We surmised that the increased rigidity of the F-F' loop engenders a more constrained entrance to the CYP3A4 active site, which in turn impedes access to the larger rivaroxaban molecule to a greater extent than testosterone and culminates in more potent inhibition of its CYP3A4-mediated metabolism. Our findings suggest a potential mechanism to rationalize probe substrate dependencies in Ki arising from the allosteric noncompetitive inhibition of CYP3A4.

A three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D.

Modulation of protein function through allosteric regulation is central in biology, but biomacromolecular systems involving multiple subunits and ligands may exhibit complex regulatory mechanisms at different levels, which remain poorly understood. Here, we discover an aldo-keto reductase termed AKRtyl and present its three-level regulatory mechanism. Specifically, by combining steady-state and transient kinetics, X-ray crystallography and molecular dynamics simulation, we demonstrate that AKRtyl exhibits a positive synergy mediated by an unusual Monod-Wyman-Changeux (MWC) paradigm of allosteric regulation at low concentrations of the cofactor NADPH, but an inhibitory effect at high concentrations is observed. While the substrate tylosin binds at a remote allosteric site with positive cooperativity. We further reveal that these regulatory mechanisms are conserved in AKR12D subfamily, and that substrate cooperativity is common in AKRs across three kingdoms of life. This work provides an intriguing example for understanding complex allosteric regulatory networks.

Machine learning approaches in predicting allosteric sites.

Allosteric regulation is a fundamental biological mechanism that can control critical cellular processes via allosteric modulator binding to protein distal functional sites. The advantages of allosteric modulators over orthosteric ones have sparked the development of numerous computational approaches, such as the identification of allosteric binding sites, to facilitate allosteric drug discovery. Building on the success of machine learning (ML) models for solving complex problems in biology and chemistry, several ML models for predicting allosteric sites have been developed. In this review, we provide an overview of these models and discuss future perspectives powered by the field of artificial intelligence such as protein language models.

Structure, cooperativity and inhibition of the inosine 5'-monophosphate-specific phosphatase from Saccharomyces cerevisiae.

The nucleoside inosine is a main intermediate of purine nucleotide catabolism in Saccharomyces cerevisiae and is produced via the dephosphorylation of inosine monophosphate (IMP) by IMP-specific 5'-nucleotidase 1 (ISN1), which is present in many eukaryotic organisms. Upon transition of yeast from oxidative to fermentative growth, ISN1 is important for intermediate inosine accumulation as purine storage, but details of ISN1 regulation are unknown. We characterized structural and kinetic behavior of ISN1 from S. cerevisiae (ScISN1) and showed that tetrameric ScISN1 is negatively regulated by inosine and adenosine triphosphate (ATP). Regulation involves an inosine-binding allosteric site along with IMP-induced local and global conformational changes in the monomer and a tetrameric re-arrangement, respectively. A proposed interaction network propagates local conformational changes in the active site to the intersubunit interface, modulating the allosteric features of ScISN1. Via ATP and inosine, ScISN1 activity is likely fine-tuned to regulate IMP and inosine homeostasis. These regulatory and catalytic features of ScISN1 contrast with those of the structurally homologous ISN1 from Plasmodium falciparum, indicating that ISN1 enzymes may serve different biological purposes in different organisms.

Discovery of novel MAT2A inhibitors by an allosteric site-compatible fragment growing approach.

The methionine adenosyltransferase MAT2A catalyzes the synthesis ofthe methyl donor S-adenosylmethionine (SAM) and thereby regulates critical aspects of metabolism and transcription. Aberrant MAT2A function can lead to metabolic and transcriptional reprogramming of cancer cells, and MAT2A has been shown to promote survival of MTAP-deficient tumors, a genetic alteration that occurs in âˆ¼ 13 % of all tumors. Thus, MAT2A holds great promise as a novel anticancer target. Here, we report a novel series of MAT2A inhibitors generated by a fragment growing approach from AZ-28, a low-molecular weight MAT2A inhibitor with promising pre-clinical properties. X-ray co-crystal structure revealed that compound 7 fully occupies the allosteric pocket of MAT2A as a single molecule mimicking MAT2B. By introducing additional backbone interactions and rigidifying the requisite linker extensions, we generated compound 8, which exhibited single digit nanomolar enzymatic and sub-micromolar cellular inhibitory potency for MAT2A.

Inhibition of polymorphic MexXY-OprM efflux system in Pseudomonas aeruginosa clinical isolates by Berberine derivatives.

The MexXY-OprM multidrug efflux pump (EP) in aminoglycosides resistant Pseudomonas aeruginosa is one of the major resistance mechanisms, which is often overexpressed in strains isolated from pulmonary chronic disease such as cystic fibrosis.[1-3] In this research, we focused on the design of potential efflux pumps inhibitors, targeting MexY, the inner membrane component, in an allosteric site. Berberine[4] has been considered as lead molecule since we previously demonstrated its effectiveness in targeting MexY in laboratory reference strains.[5,6] Since this protein is often present in polymorphic variants in clinical strains, we sequenced and modeled all the mutated forms and we synthesized and evaluated by computational techniques, some berberine derivatives carrying an aromatic functionalization in its 13-C ring position. These compounds were tested in vitro against clinical P. aeruginosa strains for antimicrobial and antibiofilm activity. In conclusion, the results demonstrated the importance of the aromatic moiety functionalization in exerting the EP inhibitory activity in synergy with the aminoglycoside tobramycin. More, we found that aminoacidic composition of MexY in different strains must be considered for predicting potential binding site and affects the different activity of berberine derivatives. Finally, the antibiofilm effect of these new EPIs is promising, particularly for o-CH3-berberine derivative.

Allo-targeting of the kinase domain: Insights from in silico studies and comparison with experiments.

The eukaryotic protein kinase domain has been a broadly explored target for drug discovery, despite limitations imposed by its high sequence conservation as a shared modular domain and the development of resistance to drugs. One way of addressing those limitations has been to target its potential allosteric sites, shortly called allo-targeting, in conjunction with, or separately from, its conserved catalytic/orthosteric site that has been widely exploited. Allosteric regulation has gained importance as an alternative to overcome the drawbacks associated with the indiscriminate effect of targeting the active site, and it turned out to be particularly useful for these highly promiscuous and broadly shared kinase domains. Yet, allo-targeting often faces challenges as the allosteric sites are not as clearly defined as its orthosteric sites, and the effect on the protein function may not be unambiguously assessed. A robust understanding of the consequence of site-specific allo-targeting on the conformational dynamics of the target protein is essential to design effective allo-targeting strategies. Recent years have seen important advances in in silico identification of druggable sites and distinguishing among them those sites expected to allosterically mediate conformational switches essential to signal transmission. The present opinion underscores the utility of such computational approaches applied to the kinase domain, with the help of comparison between computational predictions and experimental observations.

Degraders upgraded: the rise of PROTACs in hematological malignancies.

ABSTRACT: Targeted protein degradation (TPD) is a revolutionary approach to targeted therapy in hematological malignancies that potentially circumvents many constraints of existing small-molecule inhibitors. Heterobifunctional proteolysis-targeting chimeras (PROTACs) are the leading TPD drug class, with numerous agents now in clinical trials for a range of blood cancers. PROTACs harness the cell-intrinsic protein recycling infrastructure, the ubiquitin-proteasome system, to completely degrade target proteins. Distinct from targeted small-molecule inhibitor therapies, PROTACs can eliminate critical but conventionally "undruggable" targets, overcome resistance mechanisms to small-molecule therapies, and can improve tissue specificity and off-target toxicity. Orally bioavailable, PROTACs are not dependent on the occupancy-driven pharmacology inherent to inhibitory therapeutics, facilitating substoichiometric dosing that does not require an active or allosteric target binding site. Preliminary clinical data demonstrate promising therapeutic activity in heavily pretreated populations and novel technology platforms are poised to exploit a myriad of permutations of PROTAC molecular design to enhance efficacy and targeting specificity. As the field rapidly progresses and various non-PROTAC TPD drug candidates emerge, this review explores the scientific and preclinical foundations of PROTACs and presents them within common clinical contexts. Additionally, we examine the latest findings from ongoing active PROTAC clinical trials.

What is allosteric regulation? Exploring the exceptions that prove the rule!

"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation.

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.