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Leucine rich repeat kinase 2 (LRRK2) is the most common genetic contributor to Parkinson's disease (PD), a complex neurodegenerative disorder affecting nearly 10 million people worldwide. Pathogenic mutations within LRRK2 often induce increased kinase activity, an effect that can be abolished with many small molecule inhibitors; however, these small molecule inhibitors are currently limited by their toxicities. Given the large and complex nature of LRRK2, more recent efforts have focused on protein–protein interactions (PPIs) involving LRRK2 and how they can contribute to PD. Here, we review recently resolved structures of LRRK2 and highlight unique interfaces driving both catalytic and non‐catalytic activities. Combining new structural information with established in vitro and in vivo data clarifies the role of PPIs in driving LRRK2‐mediated disease pathogenesis. Since constrained peptides and peptidomimetics have the potential to engage with elongated, hydrophobic interfaces that were previously considered "undruggable," they may provide a unique handle for LRRK2 targeting. Here, we discuss the use of constrained peptides and peptidomimetics to target LRRK2 as a strategy to downregulate its pathological activity. [ABSTRACT FROM AUTHOR]

Encoded by PTPN11 , the SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) is widely recognized as a carcinogenic phosphatase. As a promising anti-cancer drug target, SHP2 regulates many signaling pathways such as RAS-RAF-ERK, PI3K-AKT and JAK-STAT. Meanwhile, SHP2 plays a significant role in regulating immune cell function in the tumor microenvironment. Heretofore, five SHP2 allosteric inhibitors have been recruited in clinical studies for the treatment of cancer. Most recently, studies have proved the therapeutic potential of SHP2 inhibitor in overcoming drug resistance of kinase inhibitors and programmed cell death-1 (PD-1) blockade. Herein, we review the structure, function and small molecular inhibitors of SHP2, and highlight recent progress in overcoming drug resistance using SHP2 inhibitor. We hope this review would facilitate the future clinical development of SHP2 inhibitors. This review summarizes the structure, function and small molecular inhibitors of SHP2, and highlights recent progress in overcoming drug resistances combining SHP2 inhibitor with its related signaling pathway inhibition. [Display omitted] [ABSTRACT FROM AUTHOR]

NS4A is a non-structural multi-tasking small peptide that is essential for HCV maturation and replication. The central odd-numbered hydrophobic residues of NS4A (Val-23‘ to Leu-31‘)ii The prime symbol (`) is used to distinguish between NS4A residues and NS3 residues of (no prime). are essential for activating NS3 upon NS3/4A protease complex formation. This study aims to design new specific allosteric NS3/4A protease inhibitors by mutating Val-23‘, Ile-25‘, and Ile-29‘ into bulkier amino acids. Pep-15, a synthetic peptide, showed higher binding affinity towards HCV-NS3 subtype-4 than native NS4A. The Kd of Pep-15 (80.0 ± 8.0 nM) was twice as high as that of native NS4A (169 ± 37 nM). The mutant Pep-15 inhibited the catalytic activity of HCV-NS3 by forming an inactive complex. Molecular dynamics simulations suggested that a cascade of conformational changes occurred, especially in the catalytic triad arrangements, thereby inactivating NS3. A large shift in the position of Ser-139 was observed, leading to loss of critical hydrogen bonding with His-57. Even though this study is not a classic drug discovery study—nor do we propose Pep-15 as a drug candidate—it serves as a stepping stone towards developing a potent inhibitor of hitherto untargeted HCV subtypes.

Abstract Background Chronic myeloid leukaemia is in principle a treatable malignancy but drug resistance is lowering survival. Recent drug discoveries have opened up new options for drug combinations, which is a concept used in other areas for preventing drug resistance. Two of these are (I) Axitinib, which inhibits the T315I mutation of BCR-ABL1, a main source of drug resistance, and (II) Asciminib, which has been developed as an allosteric BCR-ABL1 inhibitor, targeting an entirely different binding site, and as such does not compete for binding with other drugs. These drugs offer new treatment options. Methods We measured the proliferation of KCL-22 cells exposed to imatinib–dasatinib, imatinib–asciminib and dasatinib–asciminib combinations and calculated combination index graphs for each case. Moreover, using the median–effect equation we calculated how much axitinib can reduce the growth advantage of T315I mutant clones in combination with available drugs. In addition, we calculated how much the total drug burden could be reduced by combinations using asciminib and other drugs, and evaluated which mutations such combinations might be sensitive to. Results Asciminib had synergistic interactions with imatinib or dasatinib in KCL-22 cells at high degrees of inhibition. Interestingly, some antagonism between asciminib and the other drugs was present at lower degrees on inhibition. Simulations revealed that asciminib may allow for dose reductions, and its complementary resistance profile could reduce the risk of mutation based resistance. Axitinib, however, had only a minor effect on T315I growth advantage. Conclusions Given how asciminib combinations were synergistic in vitro, our modelling suggests that drug combinations involving asciminib should allow for lower total drug doses, and may result in a reduced spectrum of observed resistance mutations. On the other hand, a combination involving axitinib was not shown to be useful in countering drug resistance.

Furin is a potential target protein associated with numerous diseases; especially closely related to tumors and multiple viral infections including SARS-CoV-2. Most of the existing efficient furin inhibitors adopt a substrate analogous structure, and other types of small molecule inhibitors need to be discovered urgently. In this study, a high-throughput screening combining virtual and physical screening of natural product libraries was performed, coupled with experimental validation and preliminary mechanistic assays at the molecular level, cellular level, and molecular simulation. A novel furin inhibitor, permethrin, which is a derivative from pyrethrin I generated by Pyrethrum cinerariifolium Trev. was identified, and this study confirmed that it binds to a novel allosteric pocket of furin through non-competitive inhibition. It exhibits a very favorable protease-selective inhibition and good cellular activity and specificity. In summary, permethrin shows a new parent nucleus with a new mode of inhibition. It could be used as a highly promising lead compound against furin for targeting related tumors and various resistant viral infections, including SARS-CoV-2.

The use of Fpocket and virtual screening techniques enabled us to identify potential allosteric druggable pockets within the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Of the compounds screened, compound 1 was identified as a promising inhibitor, lowering a SARS-CoV-2 RdRp activity to 57 % in an enzymatic assay at 10 μM concentration. The structure of compound 1 was subsequently optimized in order to preserve or enhance inhibitory activity. This involved the substitution of problematic ester and aromatic nitro groups with more inert functionalities. The N,N'-diphenylurea scaffold with two NH groups was identified as essential for the compound's activity but also exhibited high toxicity in Calu-3 cells. To address this issue, a scaffold hopping approach was employed to replace the urea core with potentially less toxic urea isosteres. This approach yielded several structural analogues with notable activity, specifically 2,2'-bisimidazol (in compound 55 with residual activity RA=42 %) and (1H-imidazol-2-yl)urea (in compounds 59 and 60, with RA=50 and 28 %, respectively). Despite these advances, toxicity remained a major concern. These compounds represent a promising starting point for further structure-activity relationship studies of allosteric inhibitors of SARS-CoV-2 RdRp, with the goal of reducing their cytotoxicity and improving aqueous solubility., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Immunogenic cell death (ICD) could activate anti-tumor immune responses, which is highly attractive for improving cancer treatment effectiveness. Here, this work reports a multifunctional arsenic(III) allosteric inhibitor Mech02, which induces excessive accumulation of 1 O 2 through sensitized biocatalytic reactions, leading to cell pyroptosis and amplified ICD effect. After Mech02 is converted to Mech03, it could actualize stronger binding effects on the allosteric pocket of pyruvate kinase M2, further interfering with the anaerobic glycolysis pathway of tumors. The enhanced DNA damage triggered by Mech02 and the pyroptosis of cancer stem cells provide assurance for complete tumor clearance. In vivo experiments prove nanomicelle Mech02-HA NPs is able to activate immune memory effects and raise the persistence of anti-tumor immunity. In summary, this study for the first time to introduce the arsenic(III) pharmacophore as an enhanced ICD effect initiator into nitrogen mustard, providing insights for the development of efficient multimodal tumor therapy agents., (© 2024 Wiley‐VCH GmbH.)

Currently, allosteric inhibitors have emerged as an effective strategy in the development of preservatives against the drug-resistant Botrytis cinerea ( B. cinerea ). However, their passively driven development efficiency has proven challenging to meet the practical demands. Here, leveraging the deep learning Neural Relational Inference (NRI) framework, we actively identified an allosteric inhibitor targeting B. cinerea Chitinase, namely, 2-acetonaphthone. 2-Acetonaphthone binds to the crucial domain of Chitinase, forming the strong interaction with the allosteric sites. Throughout the interaction process, 2-acetonaphthone diminished the overall connectivity of the protein, inducing conformational changes. These findings align with the results obtained from Chitinase activity experiments, revealing an IC 50 value of 67.6 μg/mL. Moreover, 2-acetonaphthone exhibited outstanding anti- B. cinerea activity by inhibiting Chitinase. In the gray mold infection model, 2-acetonaphthone significantly extended the preservation time of cherry tomatoes, positioning it as a promising preservative for fruit storage.

Coronavirus (CoV) infections have caused contagious and fatal respiratory diseases in humans worldwide. CoV 3-chymotrypsin-like proteases (3CLpro or Mpro) play an important role in viral maturation, and maintenance of their dimeric conformation is crucial for viral activity. Therefore, allosterically regulated dimerization of 3CLpro can be employed as a drug development target. Here, we investigated the allosteric regulatory mechanism of 3CLpro dimerization by using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) technology. We found that the FLAG tag directly coupled to the N-finger of 3CLpro significantly increased HDX kinetics at the dimer interface, and 3CLpro transformed from a dimer to a monomer. The 3CLpro mutants of SARS-CoV-2, which are monomeric, also exhibited increased deuterium exchange. Binding of the allosteric inhibitor Gastrodenol to most betacoronavirus 3CLpros led to increased allosteric deuterium exchange, resulting in the monomeric conformation of the CoV 3CLpro upon binding. Molecular dynamics (MD) simulation analysis further indicated the molecular mechanism of action of Gastrodenol on CoV 3CLpro: binding of Gastrodenol to SARS-CoV-2 3CLpro destroyed the hydrogen bond in the dimer interface. These results suggest that Gastrodenol may be a potential broad-spectrum anti-betacoronavirus drug., (© 2024 Wiley-VCH GmbH.)

Human epidermal growth factor receptor (EGFR) is an attractive anti‐non‐small cell lung cancer (anti‐NSCLC) druggable target, but many traditional adenosine triphosphate (ATP)‐competitive EGFR inhibitors have been clinically observed to eventually establish acquired resistance due to kinase mutations. Allosteric inhibitors represent a new direction of EGFR‐targeted therapy, which have been successfully developed since 2016 to overcome the widely occurring kinase drug‐resistant mutations T790M/C797S [Jia Y, et al. Nature 2016; 534: 129–132]. In this study, a systematic inhibitor response‐to‐mutation profile of 11 reported EGFR allosteric inhibitors against 17 clinically significant EGFR missense mutations in NSCLC was created by integrating computational analyses and kinase assays, from which we were able to identify a number of representative mutation/inhibitor pairs with sensitization and deactivation. It is unraveled that most mutations have only a moderate or modest effect on inhibitor binding, while few mutations located within or nearby the kinase's allosteric site can cause strong sensitization or deactivation to allosteric inhibitors; thus they can influence the inhibitor binding substantially by forming or breaking noncovalent interactions between them. In particular, the gatekeeper mutation T790M and constitutively activated mutation L858R were observed to effectively improve the inhibitory potency of allosteric inhibitors, whereas the gain‐of‐function mutation T854A was revealed to considerably reduce inhibitory activity by breaking a geometrically perfect hydrogen bond. In addition, we also found that the allosteric inhibitor‐addressed conformational change in EGFR kinase domain has only a marginal effect on ATP binding, suggesting that allosteric inhibitors do not use the substrate‐regulatory strategy to suppress kinase activity; instead, they just shift the EGFR conformational equilibrium from active to inactive states by inducing a displacement in kinase's αC‐helix. [ABSTRACT FROM AUTHOR]

Specifically targeting the With‐No‐Lysine (WNK1) kinase, which is implicated in hypertension, renders a significant challenge in discovering competitive inhibitors due to the highly conserved ATP‐binding pocket. However, an allosteric inhibitor may impart high specificity against the WNK kinase isoforms since it targets the less conserved site and can provide greater efficacy even under high physiological ATP concentration. In the current study, we have investigated the structural and energetic basis of the specificity of the allosteric inhibitor WNK476 against WNK kinase isoforms by combining molecular dynamics simulations and free energy calculations using molecular mechanics Poisson–Boltzmann surface area. Our study reveals that the conformational stabilization of αC‐helix near the allosteric binding site, including conformational changes in activation and glycine‐rich loop regions, favors the specificity of WNK476 toward WNK1. The MM/PBSA calculations suggest that the non‐polar contribution from hydrophobic residues and polar solvation energy influences WNK/WNK476 complexation. Despite more favorable electrostatic and van der Waals interactions in WNK2/WNK476, WNK476 is more potent against WNK1 due to the lower contribution of disfavoring components—polar solvation and entropy. Further, we have identified that the hydrophobic residues of DLG, αC‐helix, β4, and β5 regions, and H‐bond network near the β4 strand play a critical role in the specificity of WNK476 against WNK1. Finally, our study reveals that residues Leu272, Val281, Phe283, and Leu369 of WNK1 actively contribute to the overall hydrophobic interactions for WNK1/WNK476. Overall, our study might help in the rational design of novel allosteric inhibitors against hypertension. [ABSTRACT FROM AUTHOR]

The main protease enzyme (Mpro) of SARS-CoV-2 is one of the most promising targets for COVID-19 treatment. Accordingly, in this work, a structure-based virtual screening of 3.8 million ligand libraries was carried out. After rigorous filtering, docking, and post screening assessments, 78 compounds were selected for biological evaluation, 3 of which showed promising inhibition of the Mpro enzyme. The obtained hits (CB03, GR04, and GR20) had reasonable potencies with Ki values in the medium to high micromolar range. Interestingly, while our most potent hit, GR20, was suggested to act via a reversible covalent mechanism, GR04 was confirmed as a noncompetitive inhibitor that seems to be one of a kind when compared to the other allosteric inhibitors discovered so far. Moreover, all three compounds have small sizes (~300 Da) with interesting fittings in their relevant binding sites, and they possess lead-like characteristics that can introduce them as very attractive candidates for the future development of COVID-19 treatments.

The main proteinase (Mpro) of SARS-CoV-2 plays a critical role in cleaving viral polyproteins into functional proteins required for viral replication and assembly, making it a prime drug target for COVID-19. It is well known that noncompetitive inhibition offers potential therapeutic options for treating COVID-19, which can effectively reduce the likelihood of cross-reactivity with other proteins and increase the selectivity of the drug. Therefore, the discovery of allosteric sites of Mpro has both scientific and practical significance. In this study, we explored the binding characteristics and inhibiting process of Mpro activity by two recently reported allosteric inhibitors, pelitinib and AT7519 which were obtained by the X-ray screening experiments, to probe the allosteric mechanism via molecular dynamic (MD) simulations. We found that pelitinib and AT7519 can stably bind to Mpro far from the active site. The binding affinity is estimated to be −24.37 ± 4.14 and − 26.96 ± 4.05 kcal/mol for pelitinib and AT7519, respectively, which is considerably stable compared with orthosteric drugs. Furthermore, the strong binding caused clear changes in the catalytic site of Mpro, thus decreasing the substrate accessibility. The community network analysis also validated that pelitinib and AT7519 strengthened intra- and inter-domain communication of Mpro dimer, resulting in a rigid Mpro, which could negatively impact substrate binding. In summary, our findings provide the detailed working mechanism for the two experimentally observed allosteric sites of Mpro. These allosteric sites greatly enhance the 'druggability' of Mpro and represent attractive targets for the development of new Mpro inhibitors. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2) and p38α MAP kinase (p38α MAPK), regulate various cellular responses. ERK2 is a drug target for treating many diseases, such as cancer, whereas p38α has attracted much attention as a promising drug target for treating inflammatory disorders. ERK2 is a critical off-target for p38α MAPK and vice versa. In this study, an allosteric ERK2 inhibitor with a benzothiazole moiety (compound 1) displayed comparable inhibitory activity against p38α MAPK. Crystal structures of these MAPKs showed that compound 1 bound to the allosteric site of ERK2 and p38α MAPK in distinct manners. Compound 1 formed a covalent bond with Cys162 of p38α MAPK, whereas this covalent bond was absent in the ERK2 complex even though the corresponding cysteine is conserved in ERK2. Structural dissection combined with computational simulations indicated that an amino acid difference in the allosteric site is responsible for the distinct binding modes of compound 1 with ERK2 and p38α MAPK. These structural insights underline the feasibility of developing highly selective and potent ERK2 and p38α MAPK inhibitors. • Crystal structures of ERK2 and p38α MAPK complexed with an inhibitor were determined. • The structures showed that the inhibitor binds to these kinases in distinct manners. • Structural dissection and MD simulations elucidated the determinants for the distinct modes. • These advance the development of potent and selective inhibitors for both kinases. [ABSTRACT FROM AUTHOR]

[Display omitted] • Novel AurkA allosteric inhibitors of N -benzylbenzamide backbone were designed and synthesized. • The most potent analogue 6 h (AurkA IC 50 = 6.50 μM) was comparable to the activity of AurkinA, the most potent AurkA allosteric inhibitor. • 6 h dissipated the interaction between AurkA and its activator TPX2 by binding to the Y-pocket of AurkA. • 6 h suppressed the DNA replication during G/S transition, which is a non-catalytic function of AurkA. • Docking analysis of 6 h showed several crucial interactions with the residues in Y-pocket and was well-coherent with the structure–activity relationship. Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6 h) of N -benzylbenzamide backbone. Compound 6 h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6 h against AurkA (IC 50 = 6.50 μM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure–activity relationship. In addition, 6 h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors. [ABSTRACT FROM AUTHOR]

In over a century since its discovery, Alzheimer’s disease (AD) has continued to be a global health concern due to its incurable nature and overwhelming increase among older people. In this paper, we give an overview of the efforts of researchers towards identifying potent BACE1 exosite-binding antibodies and allosteric inhibitors. Herein, we apply computer-aided drug design (CADD) methods to unravel the interactions of some proposed psychotic and meroterpenoid BACE1 allosteric site inhibitors. This study is aimed at validating the allosteric potentials of these selected compounds targeted at BACE1 inhibition. Molecular docking, molecular dynamic (MD) simulations, and post-MD analyses are carried out on these selected compounds, which have been experimentally proven to exhibit allosteric inhibition on BACE1. The SwissDock software enabled us to identify more than five druggable pockets on the BACE1 structural surface using docking. Besides the active site region, a melatonin derivative (compound 1) previously proposed as a BACE1 allostery inhibitor showed appreciable stability at eight different subsites on BACE1. Refinement with molecular dynamic (MD) simulations shows that the identified non-catalytic sites are potential allostery sites for compound 1. The allostery and binding mechanism of the selected potent inhibitors show that the smaller the molecule, the easier the attachment to several enzyme regions. This finding hereby establishes that most of these selected compounds failed to exhibit strong allosteric binding with BACE1 except for compound 1. We hereby suggest that further studies and additional identification/validation of other BACE1 allosteric compounds be done. Furthermore, this additional allosteric site investigation will help in reducing the associated challenges with designing BACE1 inhibitors while exploring the opportunities in the design of allosteric BACE1 inhibitors.

Conopeptides are peptides in the venom of marine cone snails that are used for capturing prey or as a defense against predators. A new cysteine-poor conopeptide, Czon1107, has exhibited non-competitive inhibition with an undefined allosteric mechanism in the human (h) α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, the binding mode of Czon1107 to hα3β4 nAChR was investigated using molecular dynamics simulations coupled with mutagenesis studies of the peptide and electrophysiology studies on heterologous hα3β4 nAChRs. Overall, this study clarifies the structure–activity relationship of Czon1107 and hα3β4 nAChR and provides an important experimental and theoretical basis for the development of new peptide drugs.

Modern drug design aims to discover novel lead compounds with attractable chemical profiles to enable further exploration of the intersection of chemical space and biological space. Identification of small molecules with good ligand efficiency, high activity, and selectivity is crucial toward developing effective and safe drugs. However, the intersection is one of the most challenging tasks in the pharmaceutical industry, as chemical space is almost infinity and continuous, whereas the biological space is very limited and discrete. This bottleneck potentially limits the discovery of molecules with desirable properties for lead optimization. Herein, we present a new direction leveraging posttranslational modification (PTM) protein isoforms target space to inspire drug design termed as "Post‐translational Modification Inspired Drug Design (PTMI‐DD)." PTMI‐DD aims to extend the intersections of chemical space and biological space. We further rationalized and highlighted the importance of PTM protein isoforms and their roles in various diseases and biological functions. We then laid out a few directions to elaborate the PTMI‐DD in drug design including discovering covalent binding inhibitors mimicking PTMs, targeting PTM protein isoforms with distinctive binding sites from that of wild‐type counterpart, targeting protein‐protein interactions involving PTMs, and hijacking protein degeneration by ubiquitination for PTM protein isoforms. These directions will lead to a significant expansion of the biological space and/or increase the tractability of compounds, primarily due to precisely targeting PTM protein isoforms or complexes which are highly relevant to biological functions. Importantly, this new avenue will further enrich the personalized treatment opportunity through precision medicine targeting PTM isoforms. [ABSTRACT FROM AUTHOR]

The treatment of a variety of protozoal infections, in particular those causing disabling human diseases, is still hampered by a lack of drugs or increasing resistance to registered drugs. However, in recent years, remarkable progress has been achieved to combat neglected tropical diseases by sequencing the parasites’ genomes or the validation of new targets in the parasites by novel genetic manipulation techniques, leading to loss of function. The novel amino acid hypusine is a posttranslational modification (PTM) that occurs in eukaryotic initiation factor 5A (EIF5A) at a specific lysine residue. This modification occurs by two steps catalyzed by deoxyhypusine synthase (dhs) and deoxyhypusine hydroxylase (DOHH) enzymes. dhs from Plasmodium has been validated as a druggable target by small molecules and reverse genetics. Recently, the synthesis of a series of human dhs inhibitors led to 6-bromo-N-(1H-indol-4yl)-1-benzothiophene-2-carboxamide, a potent allosteric inhibitor with an IC50 value of 0.062 µM. We investigated this allosteric dhs inhibitor in Plasmodium. In vitro P. falciparum growth assays showed weak inhibition activity, with IC50 values of 46.1 µM for the Dd2 strain and 51.5 µM for the 3D7 strain, respectively. The antimalarial activity could not be attributed to the targeting of the Pfdhs gene, as shown by chemogenomic profiling with transgenically modified P. falciparum lines. Moreover, in dose-dependent enzymatic assays with purified recombinant P. falciparum dhs protein, only 45% inhibition was observed at an inhibitor dose of 0.4 µM. These data are in agreement with a homology-modeled Pfdhs, suggesting significant structural differences in the allosteric site between the human and parasite enzymes. Virtual screening of the allosteric database identified candidate ligand binding to novel binding pockets identified in P. falciparum dhs, which might foster the development of parasite-specific inhibitors.

N6A-methyladenosine (m6A) post-transcriptional modification, the most abundant internal RNA modification, is catalyzed by the METTL3-14 methyltransferase complex. Recently, attention has been drawn to the METTL3-14 complex regarding its significant roles in the pathogenesis of acute myeloid leukemia (AML), attracting the potential of novel therapeutic targets for the disease. Herein, we report the identification and characterization of eltrombopag as a selective allosteric inhibitor of the METTL3-14 complex. Eltrombopag exhibited selective inhibitory activity in the most active catalytic form of the METTL3-14 complex by direct binding, and the mechanism of inhibition was confirmed as a noncompetitive inhibition by interacting at a putative allosteric binding site in METTL3, which was predicted by cavity search and molecular docking studies. At a cellular level, eltrombopag displayed anti-proliferative effects in the relevant AML cell line, MOLM-13, in correlation with a reduction in m6A levels. Molecular mechanism studies of eltrombopag using m6A-seq analysis provided further evidence of its cellular function by determining the hypomethylation of leukemogenic genes in eltrombopag-treated MOLM-13 cells and the overlapping of the pattern with those of METTL3-knockdown MOLM-13 cells. In conclusion, eltrombopag was first disclosed as a functional METTL3-14 allosteric inhibitor in AML cells, which could be utilized for the further development of novel anti-AML therapy.

While Polo-like kinase 1 (PLK1) inhibitors have shown promise in clinical settings for treating triple-negative breast cancer tumors and other solid tumors, they are limited by their ability to bind non-selectively to the ATP kinase domain. Therefore, we sought to develop a PLK1 allosteric inhibitor targeting the PLK1 T-loop (a switch responsible for activation) and evaluate its effects in triple-negative breast cancer cells. A novel compound, RK-10, was developed based on an in silico model, and its effects on specificity, viability, migration, and cell cycle regulation in MCF-10A and MDA-MB 231 cells were evaluated. When MDA-MB 231 cells were treated with 0–50 µg/mL RK-10, phospho-PLK1 (Thr-210) was decreased in cells cultured adherently and cells cultured as mammospheres. RK-10 significantly inhibited viability after 24 h; however, by 48 h, 25–50 µM RK-10 caused >50% reduction. RK-10 attenuated wound healing by up to 99.7% and caused S and G2/M cell cycle arrest, which was associated with increased p21 expression. We developed a novel allosteric inhibitor which mediates anti-proliferative and anti-migratory properties through targeting phospho-PLK1 (Thr-210) in mammospheres and causing S phase and G2/M cell cycle arrest. Further development of PLK1 allosteric inhibitors may be a promising approach for TNBC treatment.

Src homology containing protein tyrosine phosphatase 2 (SHP2) represents a noteworthy target for various diseases, serving as a well-known oncogenic phosphatase in cancers. As a result of the low cell permeability and poor bioavailability, the traditional inhibitors targeting the protein tyrosine phosphate catalytic sites are generally suffered from unsatisfactory applied efficacy. Recently, a particularly large number of allosteric inhibitors with striking inhibitory potency on SHP2 have been identified. In particular, few clinical trials conducted have made significant progress on solid tumors by using SHP2 allosteric inhibitors. This review summarizes the development and structure–activity relationship studies of the small-molecule SHP2 inhibitors for tumor therapies, with the purpose of assisting the future development of SHP2 inhibitors with improved selectivity, higher oral bioavailability and better physicochemical properties. This review summarized the development and structure–activity relationship studies of the small-molecule SHP2 inhibitors, as well as the application of SHP2 inhibitors for tumor therapies. The discovery and development of each type inhibitors were discussed based on their chemotypes, activity, selectivity and cocrystal structures. Image 1 [ABSTRACT FROM AUTHOR]

At present, the resistance of New Delhi metallo-β-lactamase-1 (NDM-1) to carbapenems and cephalosporins, one of the mechanisms of bacterial resistance against β-lactam antibiotics, poses a threat to human health. In this work, based on the virtual ligand screen method, we found that carnosic acid1 (CA), a natural compound, exhibited a significant inhibitory effect against NDM-1 (IC50 = 27.07 μM). Although carnosic acid did not display direct antibacterial activity, the combination of carnosic acid and meropenem still showed bactericidal activity after the loss of bactericidal effect of meropenem. The experimental results showed that carnosic acid can enhance the antibacterial activity of meropenem against Escherichia coli ZC-YN3. To explore the inhibitory mechanism of carnosic acid against NDM-1, we performed the molecular dynamics simulation and binding energy calculation for the NDM-1-CA complex system. Notably, the 3D structure of the complex obtained from molecular modeling indicates that the binding region of carnosic acid with NDM-1 was not situated in the active region of protein. Due to binding to the allosteric pocket of carnosic acid, the active region conformation of NDM-1 was observed to have been altered. The distance from the active center of the NDM-1-CA complex was larger than that of the free protein, leading to loss of activity. Then, the mutation experiments showed that carnosic acid had lower inhibitory activity against mutated protein than wild-type proteins. Fluorescence experiments verified the results reported above. Thus, our data indicate that carnosic acid is a potential NDM-1 inhibitor and is a promising drug for the treatment of NDM-1 producing pathogens.

Molecular chaperones have recently emerged as fundamental regulators of salient biological routines, including metabolic adaptations to environmental changes. Yet, many of the molecular mechanisms at the basis of their functions are still unknown or at least uncertain. This is in part due to the lack of chemical tools that can interact with the chaperones to induce measurable functional perturbations. In this context, the use of small molecules as modulators of protein functions has proven relevant for the investigation of a number of biomolecular systems. Herein, we focus on the functions, interactions and signaling pathways of the HSP90 family of molecular chaperones as possible targets for the discovery of new molecular entities aimed at tuning their activity and interactions. HSP90 and its mitochondrial paralog, TRAP1, regulate the activity of crucial metabolic circuitries, making cells capable of efficiently using available energy sources, with relevant implications both in healthy conditions and in a variety of disease states and especially cancer. The design of small-molecules targeting the chaperone cycle of HSP90 and able to inhibit or stimulate the activity of the protein can provide opportunities to finely dissect their biochemical activities and to obtain lead compounds to develop novel, mechanism-based drugs.

Cystic fibrosis (CF) is caused by different mutations related to the cystic fibrosis transmembrane regulator protein (CFTR), with F508del being the most common. Pioneering the development of CFTR modulators, thanks to the development of effective correctors or potentiators, more recent studies deeply encouraged the administration of triple combination therapeutics. However, combinations of molecules interacting with other proteins involved in functionality of the CFTR channel recently arose as a promising approach to address a large rescue of F508del-CFTR. In this context, the design of compounds properly targeting the molecular chaperone Hsp70, such as the allosteric inhibitor MKT-077, proved to be effective for the development of indirect CFTR modulators, endowed with ability to amplify the accumulation of the rescued protein. Herein we performed structure-based studies of a number of allosteric HSP70 inhibitors, considering the recent X-ray crystallographic structure of the human enzyme. This allowed us to point out the main interaction supporting the binding mode of MKT-077, as well as of the related analogues. In particular, cation-π and π–π stacking with the conserve residue Tyr175 deeply stabilized inhibitor binding at the HSP70 cavity. Molecular docking studies had been followed by QSAR analysis and then by virtual screening of aminoaryl thiazoles (I–IIIa) as putative HSP70 inhibitors. Their effectiveness as CFTR modulators has been verified by biological assays, in combination with VX-809, whose positive results confirmed the reliability of the whole applied computational method. Along with this, the “in-silico” prediction of absorption, distribution, metabolism, and excretion (ADME) properties highlighted, once more, that AATs may represent a chemical class to be further investigated for the rational design of novel combination of compounds for CF treatment.

At present, the resistance of New Delhi metallo-β-lactamase-1 (NDM-1) to carbapenems and cephalosporins, one of the mechanisms of bacterial resistance against β-lactam antibiotics, poses a threat to human health. In this work, based on the virtual ligand screen method, we found that carnosic acid1 (CA), a natural compound, exhibited a significant inhibitory effect against NDM-1 (IC50 = 27.07 μM). Although carnosic acid did not display direct antibacterial activity, the combination of carnosic acid and meropenem still showed bactericidal activity after the loss of bactericidal effect of meropenem. The experimental results showed that carnosic acid can enhance the antibacterial activity of meropenem against Escherichia coli ZC-YN3. To explore the inhibitory mechanism of carnosic acid against NDM-1, we performed the molecular dynamics simulation and binding energy calculation for the NDM-1-CA complex system. Notably, the 3D structure of the complex obtained from molecular modeling indicates that the binding region of carnosic acid with NDM-1 was not situated in the active region of protein. Due to binding to the allosteric pocket of carnosic acid, the active region conformation of NDM-1 was observed to have been altered. The distance from the active center of the NDM-1-CA complex was larger than that of the free protein, leading to loss of activity. Then, the mutation experiments showed that carnosic acid had lower inhibitory activity against mutated protein than wild-type proteins. Fluorescence experiments verified the results reported above. Thus, our data indicate that carnosic acid is a potential NDM-1 inhibitor and is a promising drug for the treatment of NDM-1 producing pathogens. [ABSTRACT FROM AUTHOR]

Molecular chaperones have recently emerged as fundamental regulators of salient biological routines, including metabolic adaptations to environmental changes. Yet, many of the molecular mechanisms at the basis of their functions are still unknown or at least uncertain. This is in part due to the lack of chemical tools that can interact with the chaperones to induce measurable functional perturbations. In this context, the use of small molecules as modulators of protein functions has proven relevant for the investigation of a number of biomolecular systems. Herein, we focus on the functions, interactions and signaling pathways of the HSP90 family of molecular chaperones as possible targets for the discovery of new molecular entities aimed at tuning their activity and interactions. HSP90 and its mitochondrial paralog, TRAP1, regulate the activity of crucial metabolic circuitries, making cells capable of efficiently using available energy sources, with relevant implications both in healthy conditions and in a variety of disease states and especially cancer. The design of small-molecules targeting the chaperone cycle of HSP90 and able to inhibit or stimulate the activity of the protein can provide opportunities to finely dissect their biochemical activities and to obtain lead compounds to develop novel, mechanism-based drugs. [ABSTRACT FROM AUTHOR]

NS4A is a non-structural multi-tasking small peptide that is essential for HCV maturation and replication. The central odd-numbered hydrophobic residues of NS4A (Val-23' to Leu-31') i i The prime symbol (') is used to distinguish between NS4A residues and NS3 residues of (no prime). are essential for activating NS3 upon NS3/4A protease complex formation. This study aims to design new specific allosteric NS3/4A protease inhibitors by mutating Val-23', Ile-25', and Ile-29' into bulkier amino acids. Pep-15, a synthetic peptide, showed higher binding affinity towards HCV-NS3 subtype-4 than native NS4A. The K d of Pep-15 (80.0 ± 8.0 nM) was twice as high as that of native NS4A (169 ± 37 nM). The mutant Pep-15 inhibited the catalytic activity of HCV-NS3 by forming an inactive complex. Molecular dynamics simulations suggested that a cascade of conformational changes occurred, especially in the catalytic triad arrangements, thereby inactivating NS3. A large shift in the position of Ser-139 was observed, leading to loss of critical hydrogen bonding with His-57. Even though this study is not a classic drug discovery study—nor do we propose Pep-15 as a drug candidate—it serves as a stepping stone towards developing a potent inhibitor of hitherto untargeted HCV subtypes. [ABSTRACT FROM AUTHOR]

The existence of multiple serotypes renders vaccine development challenging for most viruses in the Enterovirus genus. An alternative and potentially more viable strategy for control of these viruses is to develop broad-spectrum antivirals by targeting highly conserved proteins that are indispensable for the virus life cycle, such as the 3C protease. Previously, two single-chain antibody fragments, YDF and GGVV, were reported to effectively inhibit human rhinovirus 14 proliferation. Here, we found that both single-chain antibody fragments target sites on the 3C protease that are distinct from its known drug site (peptidase active site) and possess different mechanisms of inhibition. YDF does not block the active site but instead noncompetitively inhibits 3C peptidase activity through an allosteric effect that is rarely seen for antibody protease inhibitors. Meanwhile, GGVV antagonizes the less-explored regulatory function of 3C in genome replication. The interaction between 3C and the viral genome 5' noncoding region has been reported to be important for enterovirus genome replication. Here, the interface between human rhinovirus 14 3C and its 5' noncoding region was probed by hydrogen-deuterium exchange coupled mass spectrometry and found to partially overlap with the interface between GGVV and 3C. Consistently, prebinding of GGVV completely abolishes interaction between human rhinovirus 14 3C and its 5' noncoding region. The epitopes of YDF and GGVV, therefore, represent two additional sites of therapeutic vulnerability in rhinovirus. Importantly, the GGVV epitope appears to be conserved across many enteroviruses, suggesting that it is a promising target for pan-enterovirus inhibitor screening and design. [ABSTRACT FROM AUTHOR]

The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation‐oriented strategies for therapeutic interference. Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient‐mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in tracking kinase activities. Chemical probes are needed to functionally annotate kinase functions, whose activities may not always depend on catalyzing phospho‐transfer. Such non‐catalytic kinase functions are related to transitions of full‐length kinase conformations. Recent findings underline that cell‐based reporter systems can be adapted to record conformation changes of kinases. Here, we discuss the possible applications of an extendable kinase conformation (KinCon) reporter toolbox for live‐cell recording of kinase states. KinCon is a genetically encoded bioluminescence‐based biosensor platform, which can be subjected for measurements of conformation dynamics of mutated kinases upon small molecule inhibitor exposure. We hypothesize that such biosensors can be utilized to delineate the molecular modus operandi for kinase and pseudokinase regulation. This should pave the path for full‐length kinase‐targeted drug discovery efforts aiming to identify single and combinatory kinase inhibitor therapies with increased specificity and efficacy. [ABSTRACT FROM AUTHOR]

Some canine cases of histiocytic sarcoma (HS) carry an activating mutation in the src homology two domain‐containing phosphatase 2 (SHP2) encoded by PTPN11. SHP099 is an allosteric inhibitor of SHP2 that stabilizes SHP2 in a folded, auto‐inhibited conformation. Here, we examined the expression and mutation status of SHP2 in five canine HS cell lines and evaluated the growth inhibitory properties of SHP099 against these cell lines. All five of the canine HS cell lines expressed SHP2, with three of the lines each harbouring a distinct mutation in PTPN11/SHP2 (p.Glu76Gln, p.Glu76Ala and p.Gly503Val). In silico analysis suggested that p.Glu76Gln and p.Glu76Ala, but not p.Gly503Val, promote shifting of the SHP2 conformation from folded to open‐active state. SHP099 potently suppressed the growth of two of the mutant cell lines (harbouring SHP2 p.Glu76Gln or p.Glu76Ala) but not that of the other three cell lines. In addition, SHP099 suppressed ERK activation in the cell line harbouring the SHP2 p.Glu76Ala mutation. The SHP2 p.Glu76Gln and p.Glu76Ala mutations are considered to be activating mutations, and the signal from SHP2 p.Glu76Ala is inferred to be transduced primarily via the ERK pathway. Moreover, SHP099‐sensitive HS cells, including those with SHP2 p.Glu76Gln or p.Glu76Ala mutations, may depend on these mutations for growth. Therefore, targeting cells harbouring SHP2 p.Glu76Gln and p.Glu76Ala with SHP099 may be an approach for the treatment of canine HS. [ABSTRACT FROM AUTHOR]

Small C‐terminal domain phosphatase 1(SCP1)'s biological function is significant in many cellular activities. Still, a recent study on neuroglioma cells emphasized the requirement of negative regulation of SCP1 in cancer invasion suppression. Due to the structural conservation of C‐terminal domain (CTD) phosphatases, we aimed to determine the peripherally targeting inhibitors, which reciprocally bind to an eccentric site on SCP1 using a multidisciplinary approach. From biochemical screening, we have identified two potential inhibitors, which showed twofold to threefold selectivity toward SCP1 compared to Dullard. Dullard was utilized as a negative control as it is a small CTD phosphatase that contains structural similarities to SCP1. Besides, from in silico approaches like protein–ligand docking and molecular dynamics analyses, we have successfully discovered two allosteric inhibitors of napthoquinone family compounds explicitly binding to the unique hydrophobic pocket of SCP1 from 1000 molecular dockings and 125 ns of dynamics simulation studies, respectively. [ABSTRACT FROM AUTHOR]

• Cloning, purification and biochemical characterization of CysE from Klebsiella pneumoniae • 3-D crystal structure of Kpn CysE complexed with L-cysteine at 2.8 Å • In silico identification and in vitro validation of inhibitory potential of quercetin • Identification of an allosteric site at the trimer-trimer interface in the hexameric CysE The emergence and spread of multidrug-resistant strains of Klebsiella pneumoniae is a major concern that necessitates the development of unique therapeutics. The essential requirement of serine acetyltransferase (SAT/CysE) for survival of several human pathogens makes it a very promising target for inhibitor designing and drug discovery. In this study, as an initial step to structure-based drug discovery, CysE from K. pneumonia was structurally and biochemically characterized. Subsequently, blind docking of selected natural products into the X-ray crystallography determined 3D structure of the target was carried out. Experimental validation of the inhibitory potential of the top-scorers established quercetin as an uncompetitive inhibitor of Kpn CysE. Molecular dynamics simulations carried out to elucidate the binding mode of quercetin reveal that this small molecule binds at the trimer-trimer interface of hexameric CysE, a site physically distinct from the active site of the enzyme. Detailed analysis of conformational differences incurred in Kpn CysE structure on binding to quercetin provides mechanistic understanding of allosteric modulation. Binding of quercetin to CysE leads to conformation changes in the active site loops and proximal loops that affect its internal dynamics and consequently its affinity for substrate/co-factor binding, justifying the reduced enzyme activity. [ABSTRACT FROM AUTHOR]

Background: Chronic myeloid leukaemia is in principle a treatable malignancy but drug resistance is lowering survival. Recent drug discoveries have opened up new options for drug combinations, which is a concept used in other areas for preventing drug resistance. Two of these are (I) Axitinib, which inhibits the T315I mutation of BCR-ABL1, a main source of drug resistance, and (II) Asciminib, which has been developed as an allosteric BCR-ABL1 inhibitor, targeting an entirely different binding site, and as such does not compete for binding with other drugs. These drugs offer new treatment options.Methods: We measured the proliferation of KCL-22 cells exposed to imatinib-dasatinib, imatinib-asciminib and dasatinib-asciminib combinations and calculated combination index graphs for each case. Moreover, using the median-effect equation we calculated how much axitinib can reduce the growth advantage of T315I mutant clones in combination with available drugs. In addition, we calculated how much the total drug burden could be reduced by combinations using asciminib and other drugs, and evaluated which mutations such combinations might be sensitive to.Results: Asciminib had synergistic interactions with imatinib or dasatinib in KCL-22 cells at high degrees of inhibition. Interestingly, some antagonism between asciminib and the other drugs was present at lower degrees on inhibition. Simulations revealed that asciminib may allow for dose reductions, and its complementary resistance profile could reduce the risk of mutation based resistance. Axitinib, however, had only a minor effect on T315I growth advantage.Conclusions: Given how asciminib combinations were synergistic in vitro, our modelling suggests that drug combinations involving asciminib should allow for lower total drug doses, and may result in a reduced spectrum of observed resistance mutations. On the other hand, a combination involving axitinib was not shown to be useful in countering drug resistance. [ABSTRACT FROM AUTHOR]

The anticoagulant activity of lignosulfonic acid sodium (LSAS), a non-saccharide heparin mimetic, was investigated in this study. LSAS is a relatively safe industrial byproduct with similar polyanionic characteristics to that of heparin. Human plasma clotting assays, fibrin polymerization testing, and enzyme inhibition assays were exploited to investigate the anticoagulant activity of LSAS. In normal human plasma, LSAS selectively doubled the activated partial thromboplastin time (APTT) at ~308 µg/mL. Equally, LSAS doubled APTT at ~275 µg/mL in antithrombin-deficient plasma. Yet, LSAS doubled APTT at a higher concentration of 429 µg/mL using factor XI-deficient plasma. LSAS did not affect FXIIIa-mediated fibrin polymerization at 1000 µg/mL. Enzyme assays revealed that LSAS inhibits factor XIa (FXIa) with an IC50 value of ~8 μg/mL. LSAS did not inhibit thrombin, factor IXa, factor Xa, factor XIIIa, chymotrypsin, or trypsin at the highest concentrations tested and demonstrated significant selectivity against factor XIIa and plasmin. In Michaelis–Menten kinetics, LSAS decreased the VMAX of FXIa hydrolysis of a tripeptide chromogenic substrate without significantly changing its KM indicating an allosteric inhibition mechanism. The inhibitor also disrupted the generation of FXIa–antithrombin complex, inhibited factor XIIa-mediated and thrombin-mediated activation of the zymogen factor XI to FXIa, and competed with heparin for binding to FXIa. Its action appears to be reversed by protamine sulfate. Structure–activity relationship studies demonstrated the advantageous selectivity and allosteric behavior of LSAS over the acetylated and desulfonated derivatives of LSAS. LSAS is a sulfonated heparin mimetic that demonstrates significant anticoagulant activity in human plasma. Overall, it appears that LSAS is a potent, selective, and allosteric inhibitor of FXIa with significant anticoagulant activity in human plasma. Altogether, this study introduces LSAS as a promising lead for further development as an anticoagulant.

The treatment of chronic pain is poorly managed by current analgesics, and there is a need for new classes of drugs. We recently developed a series of bioactive lipids that inhibit the human glycine transporter GlyT2 (SLC6A5) and provide analgesia in animal models of pain. Here, we have used functional analysis of mutant transporters combined with molecular dynamics simulations of lipid-transporter interactions to understand how these bioactive lipids interact with GlyT2. This study identifies a novel extracellular allosteric modulator site formed by a crevice between transmembrane domains 5, 7, and 8, and extracellular loop 4 of GlyT2. Knowledge of this site could be exploited further in the development of drugs to treat pain, and to identify other allosteric modulators of the SLC6 family of transporters.

ABSTRACT Indole propionic acid (IPA), produced by the gut microbiota, is active against Mycobacterium tuberculosis in vitro and in vivo. However, its mechanism of action is unknown. IPA is the deamination product of tryptophan (Trp) and thus a close structural analog of this essential aromatic amino acid. De novo Trp biosynthesis in M. tuberculosis is regulated through feedback inhibition: Trp acts as an allosteric inhibitor of anthranilate synthase TrpE, which catalyzes the first committed step in the Trp biosynthesis pathway. Hence, we hypothesized that IPA may mimic Trp as an allosteric inhibitor of TrpE and exert its antimicrobial effect by blocking synthesis of Trp at the TrpE catalytic step. To test our hypothesis, we carried out metabolic, chemical rescue, genetic, and biochemical analyses. Treatment of mycobacteria with IPA inhibited growth and reduced the intracellular level of Trp, an effect abrogated upon supplementation of Trp in the medium. Missense mutations at the allosteric Trp binding site of TrpE eliminated Trp inhibition and caused IPA resistance. In conclusion, we have shown that IPA blocks Trp biosynthesis in M. tuberculosis via inhibition of TrpE by mimicking the physiological allosteric inhibitor of this enzyme. IMPORTANCE New drugs against tuberculosis are urgently needed. The tryptophan (Trp) analog indole propionic acid (IPA) is the first antitubercular metabolite produced by human gut bacteria. Here, we show that this antibiotic blocks Trp synthesis, an in vivo essential biosynthetic pathway in M. tuberculosis. Intriguingly, IPA acts by decoupling a bacterial feedback regulatory mechanism: it mimics Trp as allosteric inhibitor of anthranilate synthase, thereby switching off Trp synthesis regardless of intracellular Trp levels. The identification of IPA’s target paves the way for the discovery of more potent TrpE ligands employing rational, target-based lead optimization.

The high expression or mutation of SHP2 can induce cancer, so targeting SHP2 has become a new strategy for cancer treatment. In this study, we used the previously reported SHP2 allosteric inhibitor IACS-13909 as a lead drug for structural derivation and modification, and synthesized three SHP2 inhibitors. Among them, 1 H -pyrazolo[3,4- b ]pyrazine derivative 4b was a highly selective SHP2 allosteric inhibitor, with an IC 50 value of 3.2 nM, and its inhibitory activity was 17.75 times than that of the positive control IACS-13909. The cell proliferation experiment detected that compound 4b would markedly inhibit the proliferation of various cancer cells. Interestingly, compound 4b was highly sensitive to KRAS G12C -mutant non-small cell lung cancer NCI-H358 cells, with an IC 50 value of 0.58 μM and its antiproliferative activity was 4.79 times than that of IACS-13909. Furthermore, the combination therapy of compound 4b and KRAS G12C inhibitor sotorasib would play a strong synergistic effect against NCI-H358 cells. The western blot experiment detected that compound 4b markedly downregulated the phosphorylation levels of ERK and AKT in NCI-H358 cells. Molecular docking study predicted that compound 4b bound to the allosteric site of SHP2 and formed H-bond interactions with key residues Thr108, Glu110, Arg111, and Phe113. In summary, this study aims to provide new ideas for the development of SHP2 allosteric inhibitors for the treatment of KRAS G12C mutant non-small cell lung cancer.Communicated by Ramaswamy H. Sarma.

Background: The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a worldwide public health crisis. At present, the development of effective drugs and/or related therapeutics is still the most urgent and important task for combating the virus. The viral entry and associated infectivity mainly rely on its envelope spike protein to recognize and bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2) through a conformational switch of the spike receptor binding domain (RBD) from inactive to active state. Thus, it is of great significance to design an allosteric inhibitor targeting spike to lock it in the inactive and ACE2-inaccessible state., Objective: This study aims to discover the potential broad-spectrum allosteric inhibitors capable of binding and stabilizing the diverse spike variants, including the wild type, Delta, and Omicron, in the inactive RBD down state., Methods: In this work, we first detected a potential allosteric pocket within the SARS-CoV-2 spike protein. Then, we performed large-scale structure-based virtual screening by targeting the putative allosteric pocket to identify allosteric inhibitors that could stabilize the spike inactive state. Molecular dynamics simulations were further carried out to evaluate the effects of compound binding on the stability of spike RBD., Results: Finally, we identified three potential allosteric inhibitors, CPD3, CPD5, and CPD6, against diverse SARS-CoV-2 variants, including Wild-type, Delta, and Omicron variants. Our simulation results showed that the three compounds could stably bind the predicted allosteric site and effectively stabilize the spike in the inactive state., Conclusion: The three compounds provide novel chemical structures for rational drug design targeting spike protein, which is expected to greatly assist in the development of new drugs against SARS-CoV-2., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: Mitochondrial LonP1 is an ATP-powered protease that also functions as an ATP-dependent chaperone. LonP1 plays a pivotal role in regulating mitochondrial proteostasis, metabolism and cell stress responses. Cancer cells exploit the functions of LonP1 to combat oncogenic stressors such as hypoxia, proteotoxicity, and oxidative stress, and to reprogram energy metabolism enabling cancer cell proliferation, chemoresistance, and metastasis., Areas Covered: LonP1 has emerged as a potential target for anti-cancer therapeutics. We review how cytoprotective functions of LonP1 can be leveraged by cancer cells to support oncogenic growth, proliferation, and survival. We also offer insights into small molecule inhibitors that target LonP1 by two distinct mechanisms: competitive inhibition of its protease activity and allosteric inhibition of its ATPase activity, both of which are crucial for its protease and chaperone functions., Expert Opinion: We highlight advantages of identifying specific, high-affinity allosteric inhibitors blocking the ATPase activity of LonP1. The future discovery of such inhibitors has potential application either alone or in conjunction with other anticancer agents, presenting an innovative approach and target for cancer therapeutics.

Evidence from clinical and experimental investigations reveals the role of AKT in oral cancer, which has led to the development of therapeutic and pharmacological medications for inhibiting AKT protein. Despite prodigious effort, researchers are searching for new allosteric inhibitors as orthosteric inhibitors are non-selective and exert off-target effects. In the current study, we proposed an integrated computational workflow for identifying allosteric AKT1 inhibitors as this isoform is highly correlated with poor prognosis and survival. To achieve this objective, 84 classification QSAR models with six different machine learning algorithms were developed. The models created with RDKit_RF and RDKit_kstar outperformed internal and test set validation with an ROC of 0.98. The outperformed models were then used to screen Chembl, which contains over a million compounds, for AKT1 inhibitors. The Tanimoto similarity search approach identified the compounds structurally resembling AKT allosteric inhibitors. The filtered compounds were further subjected to docking phases, molecular dynamic simulation and mmpbsa to verify the binding mode of selected ones. All these analyses suggested hit 5 (CHEMBL3948083) as the potential allosteric inhibitor of AKT1 as the stability parameters, favourable binding affinity (-107.78 ± 11.56 KJ/mol) and ligand interaction were better in comparison to other compounds and reference compound. The residual analysis demonstrated that allosteric and isoform-specific residues such as Trp80 and Val270 contributed the larger energy for ligand binding. The proposed integrated approach in this study might achieve a futuristic outcome when employed in a pharmaceutical scheme different from the conventional method.Communicated by Ramaswamy H. Sarma.

Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted MEK inhibition causes qualitative alterations of carcinogenic MAPK signals. Phosphorylation of the MEK1 activation loop at the positions S218 and S222 by RAF kinases triggers the conformational alignment of MEK’s catalytic pocket to enable ATP-binding and substrate phosphorylation. We have extended a kinase conformation (KinCon) biosensor platform to record MEK1 activity dynamics. In addition to MEK phosphorylation by BRAF, the integration of the phosphorylation-mimetic mutations S218D/S222D triggered opening of the kinase. Structural rearrangement may involve the flexibility of the N terminal MEK1 A-helix. Application of the allosterically acting MEK inhibitors (MEKi) trametinib, cobimentinib, refametinib, and selumetinib converted activated MEK1 KinCon reporters back into a more closed inactive conformation. We confirmed MEK1 KinCon activity dynamics upon drug engagement using the patient-derived melanoma cell line A2058, which harbors the V600E hotspot BRAF mutation. In order to confirm biosensor dynamics, we simulated structure dynamics of MEK1 kinase in the presence and absence of mutations and/or MEKi binding. We observed increased dynamics for the S218D/S222D double mutant particularly in the region of the distal A-helix and alpha-C helix. These data underline that MEK1 KinCon biosensors have the potential to be subjected to MEKi efficacy validations in an intact cell setting.

Hexylselen is a novel submicromolar dual KGA/GDH inhibitor, which demonstrates potent inhibition of cancer cells with minimal toxicity. To further investigation its mechanism of action, we designed and synthesized its biotinylated derivative 2 as a novel probe. From commercially available starting material, 2 was obtained in 6 steps with 13.4% overall yield. It is notable that this practical synthetic route give a template for the preparation of unsymmetrical di-benzo[ d ][1,2]selenazol-3(2 H)-ones. Based on probe 2 , we developed a novel biomolecular interaction assay for convenient and reliable test of KGA allosteric inhibitors and confirmed that hexylselen as an allosteric inhibitor of KGA sharing the same binding pocket with BPTES but not with Ebselen via competitive experiments. [ABSTRACT FROM AUTHOR]

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications. [ABSTRACT FROM AUTHOR]

RAS mutations account for >15% of all human tumors, and of these ~85% are due to mutations in a particular RAS gene: KRAS. Recent studies revealed that KRAS harbors four druggable allosteric sites. Here, we have (a) used molecular simulations to generate ensembles of wild type and four major oncogenic KRAS mutants (G12V, G12D, G13D, and Q61H); (b) characterized the druggability of each allosteric pocket in each protein; (c) conducted extensive ensemble‐based virtual screening using pocket‐tailored ligand libraries; (d) prioritized hits through hierarchical postdocking analysis; and (e) validated predicted hits with NMR. Of the 785 diverse potential hits identified by our in silico analysis, we tested 90 for their ability to bind KRAS using NMR and found that nine cause backbone amide chemical shift perturbations of residues near the functionally responsive switch loops, suggesting potential binding. We conducted detailed biophysical analyses on a novel indole‐based compound to demonstrate the potential of our workflow to yield lead compounds. We believe the detailed information documented in this work regarding the druggability profile of each allosteric site and the chemical fingerprints of compounds that target them will serve as vital resources for future structure‐based drug design efforts against KRAS, a high‐value target for cancer therapy. [ABSTRACT FROM AUTHOR]