Search

Organotellurium compounds have been reported as an immune-modulator sensitizing chemotherapeutics. Herein, we report the design and synthesis of a series of novel tellurodibenzoic acids as mimics of diphenylarsenic acid (DPAA) and potential selective KGA inhibitors. Representative compound 3B exhibited potent inhibition of KGA and glutamine-dependent HCT-116 cells. Stability experiments indicated that 3B has excellent stability under acidic (HCOOH), basic (NH 3 ·H 2 O) and oxidative (H 2 O 2) conditions, but reacts with β-ME, DTT and lysine which suggested that compound 3B may interact with cysteine or lysine residues. Moreover, molecular docking disclosed that compound 3B binds to the allosteric site of the GAC tetramer containing Arg317-Lys320-Leu321-Phe322-Tyr394-Glu325, which helped to rationalize the SAR and further design and optimization. Taken together, compound 3B could be used as a starting point for the development of new KGA inhibitors. [ABSTRACT FROM AUTHOR]

There is currently considerable interest in SHP2 as a potential target for treatment of cancer. Mutation in SHP2, particularly the E76A mutation, has been found to seriously confer the phosphatase high activity. Recently, two compounds, 1 and 23, have been reported as potent allosteric inhibitors of both SHP2 wild type (SHP2WT) and the E76A mutant (SHP2E76A), with higher activity than other inhibitors. However, the structural and dynamic implications of their inhibitory mechanisms are yet unexplored which deserve further attention. Herein, the MD simulation applies to gain insight into the atomistic nature of each binding mode of inhibitors 1 and 23 in both SHP2WT and SHP2E76A. The comparative analysis reveals inhibitor 1 can freeze SHP2WT and SHP2E76A in their auto-inhibited conformation better than 23, in agreement with experimental data. GLU250 in both SHP2WT and SHP2E76A and ARG111 and ARG229 in SHP2E76A play a crucial role in the higher activity of 1 compared to 23. The mutation E76A increases the binding affinity of 1 and 23 compared to the wild type, implying that the two inhibitors have been well adopted by the E76A mutant. The findings here can substantially shed light on new strategies for developing novel classes of SHP2 inhibitors with increased potency. [ABSTRACT FROM AUTHOR]

The constitutive BCR–ABL1 active protein fusion has been identified as the main cause of chronic myeloid leukemia. The emergence of T334I and D381N point mutations in BCR–ABL1 confer drug resistance. Recent experimental studies show a synergistic effect in suppressing this resistance when Nilotinib and Asciminib are co-administered to target both the catalytic and allosteric binding site of BCR–ABL1 oncoprotein, respectively. However, the structural mechanism by which this synergistic effect occurs has not been clearly elucidated. To obtain insight into the observed synergistic effect, molecular dynamics simulations have been employed to investigate the inhibitory mechanism as well as the structural dynamics that characterize this effect. Structural dynamic analyses indicate that the synergistic binding effect results in a more compact and stable protein conformation. In addition, binding free energy calculation suggests a dominant energy effect of nilotinib during co-administration. van der Waals energy interactions were observed to be the main energy component driving this synergistic effect. Furthermore, per-residue energy decomposition analysis identified Glu481, Ser453, Ala452, Tyr454, Phe401, Asp400, Met337, Phe336, Ile334, And Val275 as key residues that contribute largely to the synergistic effect. The findings highlighted in this study provide a molecular understanding of the dynamics and mechanisms that mediate the synergistic inhibition in BCR–ABL1 protein in chronic myeloid leukemia treatment. [ABSTRACT FROM AUTHOR]

Abstract Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents. Our recent work has revealed the crucial role of one of the two structural domains (i.e. Bateman domain) in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs. Thus, we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH. These inhibitors were shown to counteract the activation by the natural positive effector, MgATP, and to block the enzyme in its apo conformation (low affinity for IMP). Our structural studies demonstrate the versatility of the Bateman domain to accommodate totally unrelated chemical scaffolds and pave the way for the development of allosteric inhibitors, an avenue little explored until now. Graphical abstract Image 1 Highlights • Four chemical series identified as novel allosteric IMPDH inhibitors by HTS. • Original binding mode in the Bateman domain solved by X-ray structure. • First in-class inhibitors trap IMPDH in its apo conformation. [ABSTRACT FROM AUTHOR]

Although a wealth of persuasive evidence was provided for the involvement of dual specificity phosphatase 1 (DUSP1) in cancers, depression, and hepatitis C virus replication, small‐molecule inhibitors have been reported rarely. Because most competitive phosphatase inhibitors are disadvantageous in terms of pharmacological properties due to the possession of highly polar groups, we aim in this work to identify the allosteric DUSP1 inhibitors through the structure‐based virtual screening. Using the modified protein‐ligand binding energy function involving an accurate molecular dehydration term, we identify six allosteric DUSP1 inhibitors that impair the enzymatic activity at low‐micromolar level. Consistent with the kinetic analysis on the inhibitory mechanism, docking simulation results indicate that the newly discovered inhibitors would bind in the peripheral binding pocket of DUSP1 in preference to the active site. Besides the micromolar‐level biochemical potency, the allosteric DUSP1 inhibitors found in this work may be meritorious over the competitive ones because of the lack of the reactive moiety to mimic the substrate phosphate group. Hence, they are anticipated to serve as a good starting point for the development of new therapeutics against cancers and neurological diseases. We have identified the allosteric inhibitors of dual specificity phosphatase 1 through the structure‐based virtual screening and in vitro enzyme assay. [ABSTRACT FROM AUTHOR]

Abstract Members of the RAS proto-oncogene superfamily are indispensable molecular switches that play critical roles in cell proliferation, differentiation, and cell survival. Recent studies have attempted to prevent the interaction of RAS/GTP with RAS guanine nucleotide exchange factors (GEFs), impair RAS-effector interactions, and suppress RAS localization to prevent oncogenic signalling. The present study aimed to investigate the effect of the natural triterpenoic acid inhibitor glycyrrhetinic acid, which is isolated from the roots of Glycyrrhiza plant species, on RAS stability. We found that glycyrrhetinic acid may bind to the P-loop of RAS and alter its stability. Based on our biochemical tests and structural analysis results, glycyrrhetinic acid induced a conformational change in RAS. Meanwhile, glycyrrhetinic acid abolishes the function of RAS by interfering with the effector protein RAF kinase activation and RAS/MAPK signalling. Graphical abstract Glycyrrhetinic acid binds to the conserved P-loop region of RAS protein, induces a conformational change, and alters its stability. Meanwhile, glycyrrhetinic acid abolishes the function of RAS by interfering with the effector protein RAF kinase activation and RAS/MAPK signalling. fx1 [ABSTRACT FROM AUTHOR]

Recently, allosteric regulations of HIV‐1 protease (PR) are suggested as a promising approach to relieve drug resistance of mutations toward inhibitors targeting the active site of PR. Replica‐exchange molecular dynamics (REMD) simulations and normal mode analysis (NMA) are integrated to enhance conformational sampling of PR. Molecular mechanics generalized Born surface area (MM‐GBSA) method was applied to calculate binding free energies of three inhibitors APV, DRV, and NIT to the wild‐type (WT) and multidrug resistance (MDR) PRs. The results suggest that binding free energies of APV and DRV are decreased in the MDR PR relative to the WT PR, suggesting drug resistance of mutations on these two inhibitors. However, the binding ability of the allosteric inhibitor NIT is not impaired in the MDR PR. In addition, internal dynamics analysis based on REMD simulations proves that mutations hardly produce obvious effect on the conformation of the MDR PR in comparison to the WT PR. Scanning of hydrophobic contacts and hydrogen bond contacts of inhibitors with residues of PRs on the concatenated trajectories of REMD demonstrates that mutations change the symmetric interaction networks of APV and DRV with PR, but do not generate obvious influence on the asymmetric interaction network of NIT with PR. In summary, allosteric inhibitor NIT can adapt the MDR PR better than those inhibitors toward the active site of PR, thus allosteric inhibitors of PR may be a possible channel to overcome drug resistance of PR. [ABSTRACT FROM AUTHOR]

[Display omitted] Methionine adenosyltransferase 2A (MAT2A) has been indicated as a drug target for oncology indications. Clinical trials with MAT2A inhibitors are currently on-going. Here, a structure-based virtual screening campaign was performed on the commercially available chemical space which yielded two novel MAT2A-inhibitor chemical series. The binding modes of the compounds were confirmed with X-ray crystallography. Both series have acceptable physicochemical properties and show nanomolar activity in the biochemical MAT2A inhibition assay and single-digit micromolar activity in the proliferation assay (MTAP -/- cell line). The identified compounds and the relating structural data could be helpful in related drug discovery projects. [ABSTRACT FROM AUTHOR]

[Display omitted] Extracellular signal-regulated kinase 2 (ERK2), a mitogen-activated protein kinase (MAPK), plays an essential role in physiological cellular processes and is a drug target for treating cancers and type 2 diabetes. A previous in silico screening study focusing on an allosteric site that plays a crucial role in substrate anchoring conferred an ERK2 inhibitor (compound 1). In this report, compound 1 was found to show high selectivity toward ERK2 compared with the nearest off-target p38α MAPK, and the crystal structure revealed that compound 1 binds to the allosteric site of ERK2. Fragment molecular orbital calculations based upon this crystal structure provided the structural basis to improve potency of compound 1 derivatives. Further computational studies uncovered that the low entropic cost of binding conferred the high selectivity of compound 1 toward ERK2 over p38α MAPK. These findings demonstrate the feasibility of developing potent and selective ERK2 inhibitors. [ABSTRACT FROM AUTHOR]

JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non- BCR-ABL1 myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations ( CALR and MPL) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targeting JAK2V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In contrast, the strong anti-inflammatory effects of the JAK inhibitors appear as a very promising therapeutic approach for many inflammatory and auto-immune diseases.

With the potential for coronaviruses to re-emerge and trigger future pandemics, the urgent development of antiviral inhibitors against SARS-CoV-2 is essential. The M pro enzyme is crucial for disease progression and the virus's life cycle. It possesses allosteric sites that can hinder its catalytic activity, with some of these sites located at or near the dimerization interface. Among them, sites #2 and #5 possess druggable pockets and are predicted to bind drug-like molecules. Consequently, a commercially available ligand library containing ~7 million ligands was used to target site #2 via structure-based virtual screening. After extensive filtering, docking, and post-docking analyses, 53 compounds were chosen for biological testing. An oxindole derivative was identified as a M pro non-competitive reversible inhibitor with a K i of 115 μM and an IC 50 of 101.9 μM. Throughout the 200 ns-long MD trajectories, our top hit has shown a very stable binding mode, forming several interactions with residues in sites #2 and #5. Moreover, derivatives of our top hit were acquired for biological testing to gain deeper insights into their structure-activity relationship. To sum up, drug-like allosteric inhibitors seem promising and can provide us with an additional weapon in our war against the recent pandemic, and possibly other coronaviruses-caused diseases., (© 2023 Wiley-VHCA AG, Zurich, Switzerland.)

Dengue fever is a neglected vector-borne disease and is more prevalent in Asia. Currently, no specific treatment is available. Given the time and cost of de novo drug discovery and development, an alternative option of drug repurposing is becoming an effective tool. We screened a library of 1127 pharmacologically active, metabolically stable, and structurally diverse small anticancer molecules to identify inhibitors of the dengue virus (DENV) NS2B/NS3 protease. Enzyme kinetics and inhibition data revealed four B-cell lymphoma 2 inhibitors, that is, ABT263, ABT737, AT101, and TW37, as potent inhibitors of DENV NS2B/NS3 protease, with IC 50 values of 0.86, 1.15, 0.81, and 0.89 µM, respectively. Mode of inhibition experiments and computational docking analyses indicated that ABT263 and ABT737 are competitive inhibitors, whereas AT101 and TW37 are noncompetitive inhibitors of the protease. With further evaluation, the identified inhibitors of the DENV NS2B/NS3 protease have the potential to be developed into specific anti-dengue therapeutics., (© 2023 Deutsche Pharmazeutische Gesellschaft.)

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the α C helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases., Competing Interests: TA, GD, SH, MN, CS, VH, AS, Jv, DF No competing interests declared, (© 2023, Aguirre et al.)

Abstract The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and replication in host cells and is a promising drug target. Through an enzymatic screen of the National Institute of Health clinical compound library, we report the discovery of zafirlukast, an FDA approved treatment for asthma, as an inhibitor for the WNV NS2B-NS3 protease. Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an IC 50 value of 32 μM. A structure activity relationship study of zafirlukast revealed the cyclopentyl carbamate and N -aryl sulfonamide as structural elements crucial for NS2B-NS3 protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an IC 50 of 22 μM. Experimental and computational docking analysis support the inhibition model of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the NS2B cofactor from binding, resulting in protease inhibition. Graphical abstract Image 1 Highlights • Zafirlukast was discovered as an inhibitor for the West Nile virus NS2B-NS3 protease. • Zafirlukast inhibits the NS2B-NS3 protease through a mixed mode inhibition mechanism. • An SAR study revealed the carbamate and aryl toluic acid sulfonamide vital for inhibition. • Substituting the cyclopentyl for a phenyl improved inhibitor activity. • Molecular modeling predicts zafirlukast disrupts NS2B binding to the NS3 protein. [ABSTRACT FROM AUTHOR]

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state. [ABSTRACT FROM AUTHOR]

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide “stapling” to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR. [ABSTRACT FROM AUTHOR]

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2. [ABSTRACT FROM AUTHOR]

This paper describes the identification of chlorhexidine, an agent commonly used in clinical as a novel potential allosteric inhibitor of PAK1. In cellular assays, chlorhexidine showed a good inhibitory profile, and its inhibitory profile was even better than IPA-3, a well-known allosteric inhibitor. In pharmacology experiments, chlorhexidine successfully inhibited the relief of PAK1 dimer and inhibited the activation of PAK1. Our findings offer an insight for the new drug development of PAK1 inhibitor. We also provide a possible explanation for the phenomenon that the application of the chlorhexidine in peritoneal lavage inhibited the development of tumor. [ABSTRACT FROM AUTHOR]

Antibodies are essential reagents that are increasingly used in diagnostics and therapy. Their specificity and capacity to recognize their native antigen are critical characteristics for their in vivo application. Follicle-stimulating hormone receptor is a GPCR protein regulating ovarian follicular maturation and spermatogenesis. Recently, its potentiality as a cancer biomarker has been demonstrated but no antibody suitable for in vivo tumor targeting and treatment has been characterized so far. In this paper we describe the first successful attempt to recover recombinant antibodies against the FSHR and that: i) are directly panned from a pre-immune library using whole cells expressing the target receptor at their surface; ii) show inhibitory activity towards the FSH-induced cAMP accumulation; iii) do not share the same epitope with the natural binder FSH; iv) can be produced inexpensively as mono- or bivalent functional molecules in the bacterial cytoplasm. We expect that the proposed biopanning strategy will be profitable to identify useful functional antibodies for further members of the GPCR class. [ABSTRACT FROM AUTHOR]

AKT1 is a family of serine/threonine kinases that play a key role in regulating cell growth, proliferation, metabolism, and survival. Two significant classes of AKT1 inhibitors (allosteric and ATP-competitive) are used in clinical development, and both of them could be effective in specific conditions. In this study, we investigated the effect of several different inhibitors on two conformations of the AKT1 by computational approach. We studied the effects of four inhibitors, including MK-2206, Miransertib, Herbacetin, and Shogaol, on the inactive conformation of AKT1 protein and the effects of four inhibitors, Capivasertib, AT7867, Quercetin, and Oridonin molecules on the active conformation of AKT1 protein. The results of simulations showed that each inhibitor creates a stable complex with AKT1 protein, although AKT1/Shogaol and AKT1/AT7867 complexes showed less stability than other complexes. Based on RMSF calculations, the fluctuation of residues in the mentioned complexes is higher than in other complexes. As compared to other complexes in either of its two conformations, MK-2206 has a stronger binding free energy affinity in the inactive conformation, −203.446 kJ/mol. MM-PBSA calculations showed that the van der Waals interactions contribute more than the electrostatic interactions to the binding energy of inhibitors to AKT1 protein. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania, and it currently affects 12 million people worldwide. The antileishmanial therapeutic arsenal remains very limited in number and efficacy, and there is no vaccine for this parasitic disease. One pathway that has been genetically validated as an antileishmanial drug target is the biosynthesis of uridine diphosphate-glucose (UDP-Glc), and its direct derivative UDP-galactose (UDP-Gal). De novo biosynthesis of these two nucleotide sugars is controlled by the specific UDP-glucose pyrophosphorylase (UGP). Leishmania parasites additionally express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. The inactivation of the two parasite pyrophosphorylases UGP and USP, results in parasite death. The present study reports on the identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening. Based on this screening, we selected a small set of commercially available compounds, and identified molecules that inhibit both Leishmania major USP and UGP, with a half-maximal inhibitory concentration in the 100 µM range. The inhibitors were predicted to bind at allosteric regulation sites, which were validated by mutagenesis studies. This study sets the stage for the development of potent USP inhibitors.

Vesicular storage of ATP is one of the processes initiating purinergic chemical transmission. Although an active transport mechanism was postulated to be involved in the processes, a transporter(s) responsible for the vesicular storage of ATP remained unidentified for some time. In 2008, SLC17A9, the last identified member of the solute carrier 17 type I inorganic phosphate transporter family, was found to encode the vesicular nucleotide transporter (VNUT) that is responsible for the vesicular storage of ATP. VNUT transports various nucleotides in a membrane potential-dependent fashion and is expressed in the various ATP-secreting cells. Mice with knockout of the VNUT gene lose vesicular storage and release of ATP from neurons and neuroendocrine cells, resulting in blockage of the initiation of purinergic chemical transmission. Thus, VNUT plays an essential role in the vesicular storage and release of ATP. The VNUT knockout mice exhibit resistance for neuropathic pain and a therapeutic effect against diabetes by way of increased insulin sensitivity. Thus, VNUT inhibitors and suppression of VNUT gene expression may be used for therapeutic purposes through suppression of purinergic chemical transmission. This review summarizes the studies to date on VNUT and discusses what we have learned about the relevance of vesicular ATP release as a potential drug target. [ABSTRACT FROM AUTHOR]

Despite a wealth of persuasive evidence for the involvement of human small C-terminal domain phosphatase 1 (Scp1) in the impairment of neuronal differentiation and in Huntington's disease, small-molecule inhibitors of Scp1 have been rarely reported so far. This study aims to the discovery of both competitive and allosteric Scp1 inhibitors through the two-track virtual screening procedure. By virtue of the improvement of the scoring function by implementing a new molecular solvation energy term and by reoptimizing the atomic charges for the active-site Mg ion cluster, we have been able to identify three allosteric and five competitive Scp1 inhibitors with low-micromolar inhibitory activity. Consistent with the results of kinetic studies on the inhibitory mechanisms, the allosteric inhibitors appear to be accommodated in the peripheral binding pocket through the hydrophobic interactions with the nonpolar residues whereas the competitive ones bind tightly in the active site with a direct coordination to the central Mg ion. Some structural modifications to improve the biochemical potency of the newly identified inhibitors are proposed based on the binding modes estimated with docking simulations. [ABSTRACT FROM AUTHOR]

The sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) signaling pathway is a crucial target for numerous human diseases from cancer to cardiovascular diseases. However, available SK1 inhibitors that target the active site suffer from poor potency, selectivity and pharmacokinetic properties. The selectivity issue of the kinases, which share a highly-conserved ATP-pocket, can be overcome by targeting the less-conserved allosteric sites. SK1 is known to function minimally as a dimer; however, the crystal structure of the SK1 dimer has not been determined. In this study, a template-based algorithm implemented in PRISM was used to predict the SK1 dimer structure and then the possible allosteric sites at the dimer interface were determined via SiteMap. These sites were used in a virtual screening campaign that includes an integrated workflow of structure-based pharmacophore modeling, virtual screening, molecular docking, re-screening of common scaffolds to propose a series of compounds with different scaffolds as potential allosteric SK1 inhibitors. Finally, the stability of the SK1-ligand complexes was analyzed by molecular dynamics simulations. As a final outcome, ligand 7 having a 4,9-dihydro-1H-purine scaffold and ligand 12 having a 2,3,4,9-tetrahydro-1H-β-carboline scaffold were found to be potential selective inhibitors for SK1. [ABSTRACT FROM AUTHOR]

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a. [ABSTRACT FROM AUTHOR]

Propofol is the most widely used i.v. general anesthetic to induce and maintain anesthesia. It is now recognized that this small molecule influences ligand-gated channels, including the GABAA receptor and others. Specific propofol binding sites have been mapped using photoaffinity ligands and mutagenesis; however, their precise target interaction profiles fail to provide complete mechanistic underpinnings for the anesthetic state. These results suggest that propofol and other common anesthetics, such as etomidate and ketamine, may target additional protein networks of the CNS to contribute to the desired and undesired anesthesia end points. Some evidence for anesthetic interactions with the cytoskeleton exists, but the molecular motors have received no attention as anesthetic targets. We have recently discovered that propofol inhibits conventional kinesin-1 KIF5B and kinesin-2 KIF3AB and KIF3AC, causing a significant reduction in the distances that these processive kinesins can travel. These microtubule-based motors are highly expressed in the CNS and the major anterograde transporters of cargos, such as mitochondria, synaptic vesicle precursors, neurotransmitter receptors, cell signaling and adhesion molecules, and ciliary intraflagellar transport particles. The single-molecule results presented show that the kinesin processive stepping distance decreases 40-60%with EC50 values <100 nM propofol without an effect on velocity. The lack of a velocity effect suggests that propofol is not binding at the ATP site or allosteric sites that modulate microtubule-activated ATP turnover. Rather, we propose that a transient propofol allosteric site forms when the motor head binds to the microtubule during stepping. [ABSTRACT FROM AUTHOR]

Purpose Early data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity. We investigated antitumor activity of alofanib (RPT835), a novel allosteric FGFR2 inhibitor, in ovarian cancer in vitro and in vivo. Methods Equal amounts of ovarian cancer cell (SKOV3) lysates were analyzed for FGFR1-3 protein expression using Wes. To assess the efficacy of alofanib on FGF-mediated cell proliferation, SKOV3 cells were incubated and were treated with serially diluted alofanib. Basic FGF was added at a concentration of 25 ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Immunocompromised mice were used for xenotransplantation of SKOV3 cancer cells. Seventy animals with measurable tumors were selected on day 10 and randomized into control groups (no treatment or chemotherapy alone (paclitaxel + carboplatin) and treatment groups (alofanib orally or intravenously (different dose levels) in combination with chemotherapy). Measurements of tumor volume (mm3) were performed by digital calipers every 3 days during 31 days after tumor inoculation. Number of tumor vessels and Ki-67 index were calculated. Results SKOV3 cells express FGFR1 and FGFR2 but not FGFR3. Basic FGF increased proliferation of the ovarian cancer cells in untreated control group ( P = 0.001). Alofanib inhibited growth of FGFR2-expressing SKOV3 cells with GI50 value of 0.37 μmol/L. Treatment with alofanib in combination with paclitaxel/carboplatin resulted in tumor growth delay phenotype in all treatment groups compared to control non-treatment groups. Compound exhibited a dose-dependent effect on tumor growth. Daily intravenous regimen of alofanib (total maximum dose per week was 350 mg/kg) demonstrated significant effect (inhibiting growth by 80 % and by 53 % in comparison with vehicle and chemotherapy group alone, respectively ( P < 0.001). Alofanib decreased number of vessels in tumor (−49 %; P < 0.0001) and number of Ki-67-positive SKOV3 cells (−42 %, P < 0.05 ). There were tumor necrosis and cell degeneration in alofanib group. Conclusions We suggest that FGFR2 inhibition has potent effects on ovarian cancer growth in preclinical studies. [ABSTRACT FROM AUTHOR]

Although numerous crystal structures for protein kinases have been reported, many include only the kinase domain but not the juxtamembrane (JM) region, a critical activity-controlling segment of receptor tyrosine kinases (RTKs). In this study, we determined the X-ray crystal structure of the tropomyosin receptor kinase (Trk) A selective inhibitor A1 complexed with the TrkA kinase domain and the JM region. This structure revealed that the unique inhibitor-binding pocket created by a novel JM configuration yields significant potency and high selectivity against TrkB and TrkC. Moreover, we validated the importance of the JM region for the potency of A1 using in vitro assays. The introduction of moieties that interact with the JM region will be one of the most effective strategies for producing highly selective RTK inhibitors. [ABSTRACT FROM AUTHOR]

Protein–protein interaction is a vital process which drives many important physiological processes in the cell and has also been implicated in several diseases. Though the protein–protein interaction network is quite complex but understanding its interacting partners using both in silico as well as molecular biology techniques can provide better insights for targeting such interactions. Targeting protein–protein interaction with small molecules is a challenging task because of druggability issues. Nevertheless, several studies on the kinetics as well as thermodynamic properties of protein–protein interactions have immensely contributed toward better understanding of the affinity of these complexes. But, more recent studies on hot spots and interface residues have opened up new avenues in the drug discovery process. This approach has been used in the design of hot spot based modulators targeting protein–protein interaction with the objective of normalizing such interactions. [ABSTRACT FROM AUTHOR]

Summary Metabolic reprogramming in cancer cells facilitates growth and proliferation. Increased activity of the serine biosynthetic pathway through the enzyme phosphoglycerate dehydrogenase (PHGDH) contributes to tumorigenesis. With a small substrate and a weak binding cofactor, (NAD + ), inhibitor development for PHGDH remains challenging. Instead of targeting the PHGDH active site, we computationally identified two potential allosteric sites and virtually screened compounds that can bind to these sites. With subsequent characterization, we successfully identified PHGDH non-NAD + -competing allosteric inhibitors that attenuate its enzyme activity, selectively inhibit de novo serine synthesis in cancer cells, and reduce tumor growth in vivo. Our study not only identifies novel allosteric inhibitors for PHGDH to probe its function and potential as a therapeutic target, but also provides a general strategy for the rational design of small-molecule modulators of metabolic enzyme function. [ABSTRACT FROM AUTHOR]

Currently, three HIV-1 integrase (IN) active site-directed inhibitors are in clinical use for the treatment of HIV infection. However, emergence of drug resistance mutations have limited the promise of a long-term cure. As an alternative, allosteric inhibition of IN activity has drawn great attention and several of such inhibitors are under early stage clinical development. Specifically, inhibitors of IN and the cellular cofactor LEDGF/p75 remarkably diminish proviral integration in cells and deliver a potent reduction in viral replicative capacity. Distinct from the extensively studied 2-(quinolin-3-yl) acetic acid or 1 H -indol-3-yl-2-hydroxy-4-oxobut-2-enoic acid chemotypes, this study discloses a new class of selective IN-LEDGF/p75 inhibitors without the carboxylic acid functionality. More significantly, 3-hydroxypicolinamides also show low micromolar inhibition against IN dimerization, providing novel dual IN inhibitors with in vitro therapeutically selective antiviral effect for further development. Finally, our shape-based ROCS pharmacophore model of the 3-hydroxypicolinamide class of compounds provides a new insight into the binding mode of these novel IN-LEDGF/p75 inhibitors. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase kinases 1/2 (MEK1/2) play critical roles in the canonical RAS/RAF/MEK/ERK pathway. Highly selective and potent non-ATP-competitive allosteric MEK1/2 inhibitors have been developed, and three of them were clinically approved for the treatment of BRAFV600 -mutant melanoma. However, the accompanying side effects of the systemically administered MEK1/2 drugs largely constrain their tolerable doses and efficacy. In this study, a series of mirdametinib-based optically activatable MEK1/2 inhibitors (opti-MEKi) were designed and synthesized. A structural-based design led to the discovery of photocaged compounds with dramatically diminished efficacy in vitro, whose activities can be spatiotemporally induced by short durations of irradiation of ultraviolet (365 nm) light. We demonstrated the robust photoactivation of MEK1/2 inhibition and antimelanoma activity in cultured human cells, as well as in a xenograft zebrafish model. Taken together, the modular approach presented herein provides a method for the optical control of MEK1/2 inhibitor activity, and these data support the further development of optically activatable agents for light-mediated antimelanoma phototherapy. [Display omitted] • Optically activatable MEK1/2 inhibitors were developed. • Upon UV irradiation, compound 14 inhibited proliferation of cancer cell lines. • Upon UV irradiation, compound 14 showed efficacy in a zebrafish xenograft model. • The first time to apply a photocaging concept to an allosteric kinase inhibitor. [ABSTRACT FROM AUTHOR]

Resistance to epidermal growth factor receptor (EGFR) inhibitors, from the first-generation erlotinib to the third generation osimertinib, is a clinical challenge in the treatment of patients with EGFR-mutant lung adenocarcinoma. Our previous work found that a novel allosteric inhibitor of phosphoglycerate mutase 1 (PGAM1), HKB99, restrains erlotinib resistance in lung adenocarcinoma cells. However, the role of HKB99 in osimertinib resistance and its underlying molecular mechanism remains to be elucidated. Herein, we found that IL-6/JAK2/STAT3 signaling pathway is aberrantly activated in both erlotinib and osimertinib resistant cells. Importantly, HKB99 significantly blocks the interaction of PGAM1 with JAK2 and STAT3 via the allosteric sites of PGAM1, which leads to inactivation of JAK2/STAT3 and thereby disrupts IL-6/JAK2/STAT3 signaling pathway. Consequently, HKB99 remarkably restores EGFR inhibitor sensitivity and exerts synergistic tumoricidal effect. Additionally, HKB99 alone or in combination with osimertinib down-regulated the level of p-STAT3 in xenograft tumor models. Collectively, this study identifies PGAM1 as a key regulator in IL-6/JAK2/STAT3 axis in the development of resistance to EGFR inhibitors, which could serve as a therapeutic target in lung adenocarcinoma with acquired resistance to EGFR inhibitors. [Display omitted] [ABSTRACT FROM AUTHOR]

Akt is a master regulator of pro-growth signaling in the cell. Akt is activated by phosphoinositides that disrupt the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains and then is phosphorylated at T308 and S473. Akt hyperactivation is oncogenic, which has spurred development of potent and selective inhibitors as therapeutics. Using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding Akt ATP-competitive and allosteric inhibitors. We compared inhibitors against three different states of Akt1. The allosteric inhibitor caused substantive conformational changes and restricts membrane binding. ATP-competitive inhibitors caused extensive allosteric conformational changes, altering the autoinhibitory interface and leading to increased membrane binding, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and conformational changes induced by Akt inhibitors and has important implications for the design of Akt targeted therapeutics. [Display omitted] • ATP-competitive inhibitors induce weakening of Akt1's autoinhibitory interface • ATP-competitive inhibitors lead to increased Akt1 membrane binding • Allosteric Akt inhibitors reorient the PH domain and decreases membrane binding Oncogenic mutations in the protein Akt are observed in cancer, leading to both allosteric and ATP-competitive inhibitor development. Here, Shaw et al., have shown how different classes of inhibitors alter the conformation of Akt1, and how this can be incorporated into future inhibitor design. [ABSTRACT FROM AUTHOR]

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.

HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein with the host factor LEDGF/p75. These small molecules act as molecular glues promoting hyper-multimerization of HIV-1 IN protein to severely perturb maturation of viral particles. Herein, we describe a new series of INLAIs based on a benzene scaffold that display antiviral activity in the single digit nanomolar range. Akin to other compounds of this class, the INLAIs predominantly inhibit the late stages of HIV-1 replication. A series of high-resolution crystal structures revealed how these small molecules engage the catalytic core and the C-terminal domains of HIV-1 IN. No antagonism was observed between our lead INLAI compound BDM-2 and a panel of 16 clinical antiretrovirals. Moreover, we show that compounds retained high antiviral activity against HIV-1 variants resistant to IN strand transfer inhibitors and other classes of antiretroviral drugs. The virologic profile of BDM-2 and the recently completed single ascending dose phase I trial (ClinicalTrials.gov identifier: NCT03634085) warrant further clinical investigation for use in combination with other antiretroviral drugs. Moreover, our results suggest routes for further improvement of this emerging drug class.