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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., Graphical abstract 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.Image 1

Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby, activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based design of allosteric inhibitors.

Graphical abstract, 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 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.

Significance The 3C protease has long been the focus of enterovirus antiviral development due to its conservation and critical role in the virus life cycle. Nevertheless, although antienterovirus drugs have a potentially enormous global market, the nucleophilic active site of 3C has frustrated drug discovery efforts. Here, we identified two disparate sites on 3C and unraveled the unconventional inhibitory mechanism of two antibodies that target these sites. We show that the regulatory role of 3C in enterovirus replication is another drug-targetable function besides its protease activity. These findings together with the antiviral effects of these antibodies suggest that these two sites on 3C may represent new chemotherapeutic intervention opportunities against enterovirus infections., 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.

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.

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.

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

International audience; Significant inhibition of Aurora B was achieved by the synthesis of simplified fragments of benzosceptrins and oroidin belonging to the marine pyrrole-2-aminoimidazoles metabolites isolated from sponges. Evaluation of kinase inhibition enabled the discovery of a synthetically accessible rigid acetylenic structural analogue EL-228 (1), whose structure could be optimized into the potent CJ2-150 (37). Here we present the synthesis of new inhibitors of Aurora B kinase, which is an important target for cancer therapy through mitosis regulation. The biologically oriented synthesis yielded several nanomolar inhibitors. The optimized compound CJ2-150 (37) showed a non-ATP competitive allosteric mode of action in a mixed-type inhibition for Aurora B kinase. Molecular docking identified a probable binding mode in the allosteric site "F" and highlighted the key interactions with the protein. We describe the improvement of the inhibitory potency and specificity of the novel scaffold as well as the characterization of the mechanism of action.; Une inhibition significative d'Aurora B a été obtenue par la synthèse de fragments simplifiés de benzosceptrines et d'oroïdine appartenant aux métabolites marins pyrrole-2-aminoimidazoles isolés à partir d'éponges. L'évaluation de l'inhibition de la kinase a permis la découverte d'un analogue structurel acétylénique rigide synthétiquement accessible EL-228 (1), dont la structure pourrait être optimisée dans le puissant CJ2-150 (37). Nous présentons ici la synthèse de nouveaux inhibiteurs de l'Aurora B kinase, qui est une cible importante pour le traitement du cancer par la régulation de la mitose. La synthèse biologiquement orientée a donné plusieurs inhibiteurs nanomolaires. Le composé optimisé CJ2-150 (37) a montré un mode d'action allostérique compétitif non ATP dans une inhibition de type mixte de la kinase Aurora B. L'arrimage moléculaire a identifié un mode de liaison probable dans le site allostérique «F» et a mis en évidence les interactions clés avec la protéine. Nous décrivons l'amélioration de la puissance inhibitrice et la spécificité du nouvel échafaudage ainsi que la caractérisation du mécanisme d'action.

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules&rsquo, mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.

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.

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., Graphical abstract 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

Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.

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.

The heat shock protein 70 (Hsp70) family of molecular chaperones are highly expressed in tumors. Inhibitors containing a pyridinium-modified benzothiazole, such as JG-98, bind to a conserved, allosteric site in Hsp70, showing promising anti-proliferative activity in cancer cells. When bound to Hsp70, the charged pyridinium makes favorable contacts; however, this moiety also increases the inhibitor’s fluorescence, giving rise to undesirable interference in biochemical and cell-based assays. Here, we explore whether the pyridinium can be replaced with a neutral pyridine. We report that pyridine-modified benzothiazoles, such as compound 17h (JG2-38), have reduced fluorescence, yet retain promising anti-proliferative activity (EC50 values ~0.1 to 0.07 µM) in breast and prostate cancer cell lines. These chemical probes are expected to be useful in exploring the roles of Hsp70s in tumorigenesis and cell survival.

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.

Indole propionic acid (IPA), produced by the gut microbiota, is active against Mycobacterium tuberculosisin 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.

International audience; 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.

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 &micro, M 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.

The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CLpro) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies.

The role of lipids in modulating membrane protein function is an emerging and rapidly growing area of research. The rational design of lipids that target membrane proteins for the treatment of pathological conditions is a novel extension in this field and provides a step forward in our understanding of membrane transporters. Bioactive lipids show considerable promise as analgesics for the treatment of chronic pain and bind to a high-affinity allosteric-binding site on the human glycine transporter 2 (GlyT2 or SLC6A5). Here, we use a combination of medicinal chemistry, electrophysiology, and computational modeling to develop a rational structure-activity relationship for lipid inhibitors and demonstrate the key role of the lipid tail interactions for GlyT2 inhibition. Specifically, we examine how lipid inhibitor head group stereochemistry, tail length, and double-bond position promote enhanced inhibition. Overall, the l-stereoisomer is generally a better inhibitor than the d-stereoisomer, longer tail length correlates with greater potency, and the position of the double bond influences the activity of the inhibitor. We propose that the binding of the lipid inhibitor deep into the allosteric-binding pocket is critical for inhibition. Furthermore, this provides insight into the mechanism of inhibition of GlyT2 and highlights how lipids can modulate the activity of membrane proteins by binding to cavities between helices. The principles identified in this work have broader implications for the development of a larger class of compounds that could target SLC6 transporters for disease treatment.

The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015&ndash, 2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (KD) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.

HIV-1 protease (HIV-PR) performs a vital step in the virus life cycle which makes it an excellent target for drug therapy. However, due to the error-prone of HIV reverse transcriptase, mutations in HIV-PR often occur, inducing drug-resistance to inhibitors. Some HIV-PR mutations can make the flaps of the enzyme more flexible thus increasing the flaps opening rate and inhibitor releasing. It has been shown that by targeting novel binding sites on HIV-PR with small molecules, it is possible to alter the equilibrium of flap conformational states. A previous fragment-based crystallographic screen have found two novel binding sites for small fragments in the inhibited, closed form of HIV-PR, termed flap and exo sites. While these experiments were performed in wild type HIV-PR, it still remains to be proven whether these small fragments can stabilize the closed conformation of flaps in resistant forms of the enzyme. Here we performed Molecular Dynamics simulations of wild type and mutant form of HIV-PR bound to inhibitor TL-3. Simulations show that on going from wild type to 6X mutant the equilibrium shifts from closed to semi-open conformation of flaps. However, when fragment Br6 is placed at flap site of mutant form, the enzyme is restored back to closed conformation. This finding supports the hypothesis that allosteric inhibitors, together with active site inhibitors could increase the number of point mutations necessary for appreciable clinical resistance to AIDS therapy. Fil: Luchi, Adriano Martín. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura. Departamento de Química. Laboratorio de Estructura Molecular y Propiedades; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina Fil: Angelina, Emilio Luis. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura. Departamento de Química. Laboratorio de Estructura Molecular y Propiedades; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina Fil: Bogado, María Lucrecia. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura. Departamento de Química. Laboratorio de Estructura Molecular y Propiedades; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina Fil: Forli, Stefano. Scripps Research Institute; Estados Unidos Fil: Olson, Arthur. Scripps Research Institute; Estados Unidos Fil: Peruchena, Nelida Maria. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura. Departamento de Química. Laboratorio de Estructura Molecular y Propiedades; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina

// Ju-Yi Hsieh 1, 2, * , Shao-Yu Li 1, * , Wen-Chen Tsai 1, 2 , Jyung-Hurng Liu 3, 4 , Chih-Li Lin 5 , Guang-Yaw Liu 2 , Hui-Chih Hung 1, 3, 4 1 Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan 2 Institute of Microbiology & Immunology, Chung Shan Medical University, and Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan 3 Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan 4 Agricultural Biotechnology Center (ABC), National Chung Hsing University, Taichung, Taiwan 5 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan * These authors have contributed equally to this work Correspondence to: Hui-Chih Hung, e-mail: hchung@dragon.nchu.edu.tw Guang-Yaw Liu, e-mail: liugy@csmu.edu.tw Keywords: allosteric inhibitor, selective inhibitor, non-competitive inhibition, mutagenesis analysis, cellular senescence Received: February 20, 2015 Accepted: May 06, 2015 Published: May 19, 2015 ABSTRACT Here, we found a natural compound, embonic acid (EA), that can specifically inhibit the enzymatic activity of mitochondrial NAD(P) + -dependent malic enzyme (m-NAD(P)-ME, ME2) either in vitro or in vivo . The in vitro IC 50 value of EA for m-NAD(P)-ME was 1.4 ± 0.4 μM. Mutagenesis and binding studies revealed that the putative binding site of EA on m-NAD(P)-ME is located at the fumarate binding site or at the dimer interface near the site. Inhibition studies reveal that EA displayed a non-competitive inhibition pattern, which demonstrated that the binding site of EA was distinct from the active site of the enzyme. Therefore, EA is thought to be an allosteric inhibitor of m-NAD(P)-ME. Both EA treatment and knockdown of m-NAD(P)-ME by shRNA inhibited the growth of H1299 cancer cells. The protein expression and mRNA synthesis of m-NAD(P)-ME in H1299 cells were not influenced by EA, suggesting that the EA-inhibited H1299 cell growth occurs through the suppression of in vivo m-NAD(P)-ME activity EA treatment further induced the cellular senescence of H1299 cells. However, down-regulation of the enzyme-induced cellular senescence was not through p53. Therefore, the EA-evoked senescence of H1299 cells may occur directly through the inhibition of ME2 or a p53-independent pathway.

There is a current and pressing need for improved cancer therapies. The use of small molecule kinase inhibitors and their application in combinatorial regimens represent an approach to personalized targeted cancer therapy. A number of AGC kinases, including atypical Protein Kinase C enzymes (PKCs), are validated drug targets for cancer treatment. Most drug development programs for protein kinases focus on the development of drugs that bind at the ATP-binding site. Alternatively, allosteric drugs have great potential for the development of future innovative drugs. However, the rational development of allosteric drugs poses important challenges because the compounds not only must bind to a given site but also must stabilize forms of the protein with a desired effect at a distant site. Here we describe the development of a new class of compounds targeting a regulatory site (PIF-pocket) present in the kinase domain and provide biochemical and crystallographic data showing that these compounds allosterically inhibit the activity of atypical PKCs. PS432, a representative compound, decreased the rate of proliferation of non-small cell lung cancer cells more potently than aurothiomalate, an atypical PKC inhibitor currently under evaluation in clinical trials, and significantly reduced tumor growth without side effects in a mouse xenograft model. The druglike chemical class provides ample possibilities for the synthesis of derivative compounds, with the potential to allosterically modulate the activity of atypical PKCs and other kinases. Fil: Arencibia, Jose M.. Universitätsklinikum Frankfurt; Alemania Fil: Fröhner, Wolfgang. Universitat Saarland; Alemania Fil: Krupa, Magdalena. Universitätsklinikum Frankfurt; Alemania Fil: Pastor Flores, Daniel. Universitätsklinikum Frankfurt; Alemania Fil: Merker, Piotr. Universitätsklinikum Frankfurt; Alemania Fil: Oellerich, Thomas. Goethe Universitat Frankfurt; Alemania Fil: Neimanis, Sonja. Universitätsklinikum Frankfurt; Alemania Fil: Schmithals, Christian. Universitätsklinikum Frankfurt; Alemania Fil: Köberle, Verena. Universitätsklinikum Frankfurt; Alemania Fil: Süß, Evelyn. Universitätsklinikum Frankfurt; Alemania Fil: Zeuzem, Stefan. Universitätsklinikum Frankfurt; Alemania Fil: Stark, Holger. Goethe Universitat Frankfurt; Alemania Fil: Piiper, Albrecht. Universitätsklinikum Frankfurt; Alemania Fil: Odadzic, Dalibor. Goethe Universitat Frankfurt; Alemania Fil: Schulze, Jörg O.. Universitätsklinikum Frankfurt; Alemania Fil: Biondi, Ricardo Miguel. Universitätsklinikum Frankfurt; Alemania. German Cancer Consortium; Alemania. German Cancer Research Center ; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts.

Most nucleoside kinases, besides the catalytic domain, feature an allosteric domain which modulates their activity. Generally, non-substrate analogs, interacting with allosteric sites, represent a major opportunity for developing more selective and safer therapeutics. We recently developed a series of non-nucleoside non-competitive inhibitors of human adenosine kinase (hAK), based on a pyrrolobenzoxa(thia)zepinone scaffold. Based on computational analysis, we hypothesized the existence of a novel allosteric site on hAK, topographically distinct from the catalytic site. In this study, we have adopted a multidisciplinary approach including molecular modeling, biochemical studies, and site-directed mutagenesis to validate our hypothesis. Based on a three-dimensional model of interaction between hAK and our molecules, we designed, cloned, and expressed specific, single and double point mutants of hAK (Q74A, Q78A, H107A, K341A, F338A, and Q74A-F338A). Kinetic characterization of recombinant enzymes indicated that these mutations did not affect enzyme functioning; conversely, mutated enzymes are endowed of reduced susceptibility to our non-nucleoside inhibitors, while maintaining comparable affinity for nucleoside inhibitors to the wild-type enzyme. This study represents the first characterization and validation of a novel allosteric site in hAK and may pave the way to the development of novel selective and potent non-nucleoside inhibitors of hAK endowed with therapeutic potential.

BACKGROUND: Maraviroc (MVC) is an allosteric CCR5 inhibitor used against HIV-1 infection. While MVC-resistant viruses have been identified in patients, it still remains incompletely known how they adjust their CD4 and CCR5 binding properties to resist MVC inhibition while preserving their replicative capacity. It is thought that they maintain high efficiency of receptor binding. To date however, information about the binding affinities to receptors for inhibitor-resistant HIV-1 remains limited. RESULTS: Here, we show by means of viral envelope (gp120) binding experiments and virus-cell fusion kinetics that a MVC-resistant virus (MVC-Res) that had emerged as a dominant viral quasispecies in a patient displays reduced affinities for CD4 and CCR5 either free or bound to MVC, as compared to its MVC-sensitive counterpart isolated before MVC therapy. An alanine insertion within the GPG motif (G310_P311insA) of the MVC-resistant gp120 V3 loop is responsible for the decreased CCR5 binding affinity, while impaired binding to CD4 is due to sequence changes outside V3. Molecular dynamics simulations of gp120 binding to CCR5 further emphasize that the Ala insertion alters the structure of the V3 tip and weakens interaction with CCR5 ECL2. Paradoxically, infection experiments on cells expressing high levels of CCR5 also showed that Ala allows MVC-Res to use CCR5 efficiently, thereby improving viral fusion and replication efficiencies. Actually, although we found that the V3 loop of MVC-Res is required for high levels of MVC resistance, other regions outside V3 are sufficient to confer a moderate level of resistance. These sequence changes outside V3, however, come with a replication cost, which is compensated for by the Ala insertion in V3. CONCLUSION: These results indicate that changes in the V3 loop of MVC-resistant viruses can augment the efficiency of CCR5-dependent steps of viral entry other than gp120 binding, thereby compensating for their decreased affinity for entry receptors and improving their fusion and replication efficiencies. This study thus sheds light on unsuspected mechanisms whereby MVC-resistant HIV-1 could emerge and grow in treated patients. This work was supported by Agence Nationale de la Recherche sur le SIDA (ANRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur, Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” (Grant ANR-10- LABEX-62-IBEID), Instituto de Salud Carlos III (Intrasalud PI12/0056), the Spanish Ministry of Health (EC11-175) and Red de investigación en SIDA RD12/0017/0015 as part of the Plan Nacional R + D + I and cofinanced by ISCIII- Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER). Sí

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.