Browsing by Author "Meroueh, Samy O."

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    Analogues of Nitrofuran Antibiotics are Potent GroEL/ES Pro-drug Inhibitors with Efficacy against Enterococcus Faecium, Staphylococcus Aureus, and Escherichia Coli
    (2020-05) Howe, Christopher Ryan; Johnson, Steven M.; Hoang, Quyen Q.; Meroueh, Samy O.
    In 2019, the ESKAPE pathogens were highlighted by the World Health Organization as some of the most prominent threats to human health, capable of developing significant antibiotic resistance. These pathogens contribute to over 2 million annual infections and ~23,000 annual deaths in the U.S. In addition, various strains of E. coli have also shown a proclivity to develop resistance against common drug classes. The prevalence of these infections highlights an urgency to discover new antibiotics that target previously unexploited pathways essential to bacterial survival. The bacterial GroEL/ES chaperonin system is viewed as a viable antibiotic target. It has been proven essential to bacterial survival and homeostasis under all conditions. Previous studies identified hit GroEL/ES inhibitors with potential antibiotic activity. One promising hit (1) was shown to be a moderate but selective GroEL/ES inhibitor with antibacterial effects against Gram-negative pathogens (K. pneumoniae and A. baumannii). The structural similarity of 1 to known antibiotics – nitroxoline, nifuroxazide, and nitrofurantoin – prompted me to develop two series of hydroxyquinoline and nitrofuran-based analogs. I then assessed these compounds’ abilities to inhibit in vitro GroEL/ES activity, as well as to selectively target ESKAPE/E. coli bacteria over human cells. Initially, I found the nitrofuran analogs were stronger inhibitors of bacterial growth than the hydroxyquinolines, but were weaker at blocking GroEL/ES functions. However, considering nitrofuran-based antibiotics behave as pro-drugs, it was found that they became much more effective GroEL/ES inhibitors when E. coli NfsB nitroreductase was introduced into the GroEL/ES-dMDH refolding assay, metabolizing the nitro groups to their active species. Importantly, lead analogs that potently inhibited bacterial growth exhibited low cytotoxicity to human colon and intestine cells. Although I found E. coli were able to generate varying degrees of irreversible resistance to nifuroxazide, nitrofurantoin, and lead inhibitor 17, perhaps through mutations known to effect NfsA and NfsB nitroreductases, the resulting strains were not necessarily cross-resistant to the other inhibitors. Thus, combination therapy may help bypass these resistance mechanisms. In summary, this study identified key structure-activity relationships to selectively inhibit GroEL/ES and the growth of several bacterial species. Results from this study will aid future efforts to improve inhibitor potency.
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    Atractylenolide I enhances responsiveness to immune checkpoint blockade therapy by activating tumor antigen presentation
    (The American Society for Clinical Investigation, 2021-05-17) Xu, Hanchen; Van der Jeught, Kevin; Zhou, Zhuolong; Zhang, Lu; Yu, Tao; Sun, Yifan; Li, Yujing; Wan, Changlin; So, Ka Man; Liu, Degang; Frieden, Michael; Fang, Yuanzhang; Mosley, Amber L.; He, Xiaoming; Zhang, Xinna; Sandusky, George E.; Liu, Yunlong; Meroueh, Samy O.; Zhang, Chi; Wijeratne, Aruna B.; Huang, Cheng; Ji, Guang; Lu, Xiongbin; Medical and Molecular Genetics, School of Medicine
    One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell–mediated cytotoxicity, we identified atractylenolide I (ATT-I), which substantially promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non–ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced MHC-I–mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient–derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation and empowers T cell cytotoxicity, thus elevating the tumor response to immunotherapy.
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    BioDrugScreen: a computational drug design resource for ranking molecules docked to the human proteome
    (Oxford University Press, 2009-11-18) Li, Liwei; Bum-Erdene, Khuchtumur; Baenziger, Peter H.; Rosen, Joshua J.; Hemmert, Jamison R.; Nellis, Joy A.; Pierce, Marlon E.; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine
    BioDrugScreen is a resource for ranking molecules docked against a large number of targets in the human proteome. Nearly 1600 molecules from the freely available NCI diversity set were docked onto 1926 cavities identified on 1589 human targets resulting in >3 million receptor–ligand complexes requiring >200 000 cpu-hours on the TeraGrid. The targets in BioDrugScreen originated from Human Cancer Protein Interaction Network, which we have updated, as well as the Human Druggable Proteome, which we have created for the purpose of this effort. This makes the BioDrugScreen resource highly valuable in drug discovery. The receptor–ligand complexes within the database can be ranked using standard and well-established scoring functions like AutoDock, DockScore, ChemScore, X-Score, GoldScore, DFIRE and PMF. In addition, we have scored the complexes with more intensive GBSA and PBSA approaches requiring an additional 120 000 cpu-hours on the TeraGrid. We constructed a simple interface to enable users to view top-ranking molecules and access purchasing and other information for further experimental exploration.
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    Chemical Space Overlap with Critical Protein–Protein Interface Residues in Commercial and Specialized Small-Molecule Libraries
    (Wiley, 2018-12-20) Si, Yubing; Xu, David; Bum-Erdene, Khuchtumur; Ghozayel, Mona K.; Yang, Baocheng; Clemons, Paul A.; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine
    There is growing interest in the use of structure-based virtual screening to identify small molecules that inhibit challenging protein–protein interactions (PPIs). In this study, we investigated how effectively chemical library members docked at the PPI interface mimic the position of critical side-chain residues known as “hot spots”. Three compound collections were considered, a commercially available screening collection (ChemDiv), a collection of diversity-oriented synthesis (DOS) compounds that contains natural-product-like small molecules, and a library constructed using established reactions (the “screenable chemical universe based on intuitive data organization”, SCUBIDOO). Three different tight PPIs for which hot-spot residues have been identified were selected for analysis: uPAR·uPA, TEAD4·Yap1, and CaVα·CaVβ. Analysis of library physicochemical properties was followed by docking to the PPI receptors. A pharmacophore method was used to measure overlap between small-molecule substituents and hot-spot side chains. Fragment-like conformationally restricted small molecules showed better hot-spot overlap for interfaces with well-defined pockets such as uPAR·uPA, whereas better overlap was observed for more complex DOS compounds in interfaces lacking a well-defined binding site such as TEAD4·Yap1. Virtual screening of conformationally restricted compounds targeting uPAR·uPA and TEAD4·Yap1 followed by experimental validation reinforce these findings, as the best hits were fragment-like and had few rotatable bonds for the former, while no hits were identified for the latter. Overall, such studies provide a framework for understanding PPIs in the context of additional chemical matter and new PPI definitions.
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    Chloroacetamide fragment library screening identifies new scaffolds for covalent inhibition of the TEAD·YAP1 interaction
    (Royal Society of Chemistry, 2023-08-03) Bum-Erdene, Khuchtumur; Ghozayel, Mona K.; Zhang, Mark J.; Gonzalez-Gutierrez, Giovanni; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine
    Transcriptional enhanced associate domain (TEAD) binding to co-activator yes-associated protein (YAP1) leads to a transcription factor of the Hippo pathway. TEADs are regulated by S-palmitoylation of a conserved cysteine located in a deep well-defined hydrophobic pocket outside the TEAD·YAP1 interaction interface. Previously, we reported the discovery of a small molecule based on the structure of flufenamic acid that binds to the palmitate pocket, forms a covalent bond with the conserved cysteine, and inhibits TEAD4 binding to YAP1. Here, we screen a fragment library of chloroacetamide electrophiles to identify new scaffolds that bind to the palmitate pocket of TEADs and disrupt their interaction with YAP1. Time- and concentration-dependent studies with wild-type and mutant TEAD1-4 provided insight into their reaction rates and binding constants and established the compounds as covalent inhibitors of TEAD binding to YAP1. Binding pose hypotheses were generated by covalent docking revealing that the fragments and compounds engage lower, middle, and upper sub-sites of the palmitate pocket. Our fragments and compounds provide new scaffolds and starting points for the design of derivatives with improved inhibition potency of TEAD palmitoylation and binding to YAP1.
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    Covalent Fragment Screening Identifies Rgl2 RalGEF Cysteine for Targeted Covalent Inhibition of Ral GTPase Activation
    (Wiley, 2022) Bum-Erdene, Khuchtumur; Ghozayel, Mona K.; Xu, David; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine
    Ral GTPases belong to the RAS superfamily, and they are directly activated by K-RAS. The RalGEF pathway is one of the three major K-RAS signaling pathways. Ral GTPases do not possess a cysteine nucleophile to develop a covalent inhibitor following the strategy that led to a K-RAS G12C therapeutic agent. However, several cysteine amino acids exist on the surface of guanine exchange factors that activate Ral GTPases, such as Rgl2. Here, we screen a library of cysteine electrophile fragments to determine if covalent bond formation at one of the Rgl2 surface cysteines could inhibit Ral GTPase activation. We found several chloroacetamide and acrylamide fragments that inhibited Ral GTPase exchange by Rgl2. Site-directed mutagenesis showed that covalent bond formation at Cys-284, but not other cysteines, leads to inhibition of Ral activation by Rgl2. Follow-up time- and concentration-dependent studies of derivatives identified by substructure search of commercial libraries further confirmed Cys-284 as the reaction site and identified the indoline fragments as the most promising series for further development. Cys-284 is located outside of the Ral•Rgl2 interface on a loop that has several residues that come in direct contact with Ral GTPases. Our allosteric covalent fragment inhibitors provide a starting point for the development of small-molecule covalent inhibitors to probe Ral GTPases in animal models.
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    CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy
    (Sage, 2012-07-24) Piekarz, Andrew D.; Due, Michael R.; Khanna, May; Wang, Bo; Ripsch, Matthew S.; Wang, Ruizhong; Meroueh, Samy O.; Vasko, Michael R.; White, Fletcher A.; Khanna, Rajesh; Anesthesia, School of Medicine
    Background: The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822-829 (2011)]. Results and discussion: Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca²⁺ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca²⁺ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. Conclusions: Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target.
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    Crystal Packing Reveals a Potential Autoinhibited KRAS Dimer Interface and a Strategy for Small-Molecule Inhibition of RAS Signaling
    (American Chemical Society, 2023) Brenner, Robert J.; Landgraf, Alexander D.; Bum-Erdene, Khuchtumur; Gonzalez-Gutierrez, Giovanni; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine
    KRAS GTPases harbor oncogenic mutations in more than 25% of human tumors. KRAS is considered to be largely undruggable due to the lack of a suitable small-molecule binding site. Here, we report a unique crystal structure of His-tagged KRASG12D that reveals a remarkable conformational change. The Switch I loop of one His-KRASG12D structure extends into the Switch I/II pocket of another His-KRASG12D in an adjacent unit cell to create an elaborate interface that is reminiscent of high-affinity protein-protein complexes. We explore the contributions of amino acids at this interface using alanine-scanning studies with alchemical free energy perturbation calculations based on explicit-solvent molecular dynamics simulations. Several interface amino acids were found to be hot spots as they contributed more than 1.5 kcal/mol to the protein-protein interaction. Computational analysis of the complex revealed the presence of two large binding pockets that possess physicochemical features typically found in pockets considered druggable. Small-molecule binding to these pockets may stabilize this autoinhibited structure of KRAS if it exists in cells to provide a new strategy to inhibit RAS signaling.
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    Design and Synthesis of Fragment Derivatives with a Unique Inhibition Mechanism of the uPAR·uPA Interaction
    (American Chemical Society, 2020) Bum-Erdene, Khuchtumur; Liu, Degang; Xu, David; Ghozayel, Mona K.; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine;
    There is substantial interest in the development of small molecules that inhibit the tight and highly challenging protein-protein interaction between the glycophosphatidylinositol (GPI)-anchored cell surface receptor uPAR and the serine protease uPA. While preparing derivatives of a fragment-like compound that previously emerged from a computational screen, we identified compound 5 (IPR-3242), which inhibited binding of uPA to uPAR with submicromolar IC50s. The high inhibition potency prompted us to carry out studies to rule out potential aggregation, lack of stability, reactivity, and nonspecific inhibition. We designed and prepared 16 derivatives to further explore the role of each substituent. Interestingly, the compounds only partially inhibited binding of a fluorescently labeled α-helical peptide that binds to uPAR at the uPAR·uPA interface. Collectively, the results suggest that the compounds bind to uPAR outside of the uPAR·uPA interface, trapping the receptor into a conformation that is not able to bind to uPA. Additional studies will have to be carried out to determine whether this unique inhibition mechanism can occur at the cell surface.
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    Design, Synthesis, Biochemical Studies, Cellular Characterization, and Structure-Based Computational Studies of Small Molecules Targeting the Urokinase Receptor
    (Elsevier, 2012) Wang, Fang; Knabe, W. Eric; Li, Liwei; Jo, Inha; Mani, Timmy; Roehm, Hartmut; Oh, Kyungsoo; Li, Jing; Khanna, May; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine
    The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.