Browsing by Author "Meroueh, Samy"
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Item BIOCHEMICAL CHARACTERIZATION OF SMALL MOLECULES TARGETING RAL GTPASE(Office of the Vice Chancellor for Research, 2012-04-13) Ishikawa, Megan; Khanna, May; Jo, Inha; Meroueh, SamyThe Ral subfamily of GTPases consists of highly similar RalA and RalB isoforms that participate in diverse cellular functions including endocytosis, exocytosis, actin cytoskeletal dynamics, and transcription. A large body of evidence has implicated Ral GTPases with tumor cell growth, migration, and angiogenesis in bladder, prostate, lung, and pancreatic cancer. The purpose of this project was to target the activity of Ral GTPases and their association with effector proteins through the identification of small molecule inhibitors that block this interaction. In order to accomplish this, both direct binding to RalB as well as disruption of protein-protein interaction were investigated. The top 200 compounds from a larger computational library of 500,000 compounds targeting the RalBP1 binding site on RalB were tested. Differen-tial scanning fluorimetry (DSF) was used to measure the degree of direct binding between compound and protein through thermal melting shift. To measure disruption between RalB and RalBP1 by small molecules, a novel enzyme-linked immunosorbent assay (ELISA) was developed. Identification of a few key compounds binding to RalB as well as optimization of an ELISA assay for RalBP1 was accomplished. Further direction of this project would be to utilize the ELISA assay to test inhibition of the protein-protein interac-tion between RalB and RalBP1 using the top compounds from the DSF trials.Item Characterization of a Putative Acid Phosphatase in Toxoplasma Gondii and Its Role in Parasite Propagation(2020-11) Blakely, William James; Arrizabalaga, Gustavo; Gilk, Stacey; Meroueh, Samy; Wek, RonaldThe parasite Toxoplasma gondii infects approximately one-third of people worldwide. Infection can lead to severe disease in those with a compromised immune system and primary infection during pregnancy can lead to severe birth defects or miscarriage. Treatment options are limited, have significant side effects, and are ineffective for all infection stages. Imperative to the discovery of novel therapeutic targets is a thorough understanding of how Toxoplasma propagates within a host. To replicate, the parasite must enter the cells of an infected organism where, during the invasion process, it surrounds itself with host cell membrane to form a parasitophorous vacuole (PV), within which it freely divides. To endure the intracellular environment of a host cell, Toxoplasma secretes a large repertoire of proteins beyond the PV to manipulate important host cellular functions. How these Toxoplasma proteins transit from parasites to host cell is not well understood. Protein translocation into the host cell is mediated by three proteins hypothesized to function as a putative translocon complex inside the PV, but whether other proteins are involved in the structure or regulation of this putative translocon remains unknown. The secreted protein GRA44, which contains a putative acid phosphatase domain, has been discovered to interact with members of this translocon and is required for downstream alteration of host cells. GRA44 was found to be post-translationally cleaved in a region homologous to sequences targeted by protozoan proteases of the secretory pathway with both major cleavage products secreted to the PV. Conditional knockdown of GRA44 resulted in loss of host cell cMyc upregulation, a phenotype also seen in translocon member disruption. Therefore, the putative acid phosphatase GRA44, in association with the translocon complex, is critical for host cell manipulation during infection, a process Toxoplasma relies upon for successful propagation as an intracellular pathogen.Item Chemical Proteomics Reveals Soluble Epoxide Hydrolase as a Therapeutic Target for Ocular Neovascularization(ACS, 2018) Sulaiman, Rania S.; Park, Bomina; Sardar Pasha, Sheik Pran Babu; Si, Yubing; Kharwadkar, Rakshin; Mitter, Sayak K.; Lee, Bit; Sun, Wei; Qi, Xiaoping; Boulton, Michael E.; Meroueh, Samy; Fei, Xiang; Seo, Seung-Yong; Corson, Timothy W.; Ophthalmology, School of MedicineThe standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound’s mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.Item A Computational Investigation of Small-Molecule Engagement of Hot Spots at Protein–Protein Interaction Interfaces(ACS, 2017-08) Xu, David; Bum-Erdene, Khuchtumur; Si, Yubing; Zhou, Donghui; Liu, Degang; Ghozayel, Mona; Meroueh, Samy; Biochemistry and Molecular Biology, School of MedicineThe binding affinity of a protein–protein interaction is concentrated at amino acids known as hot spots. It has been suggested that small molecules disrupt protein–protein interactions by either (i) engaging receptor protein hot spots or (ii) mimicking hot spots of the protein ligand. Yet, no systematic studies have been done to explore how effectively existing small-molecule protein–protein interaction inhibitors mimic or engage hot spots at protein interfaces. Here, we employ explicit-solvent molecular dynamics simulations and end-point MM-GBSA free energy calculations to explore this question. We select 36 compounds for which high-quality binding affinity and cocrystal structures are available. Five complexes that belong to three classes of protein–protein interactions (primary, secondary, and tertiary) were considered, namely, BRD4•H4, XIAP•Smac, MDM2•p53, Bcl-xL•Bak, and IL-2•IL-2Rα. Computational alanine scanning using MM-GBSA identified hot-spot residues at the interface of these protein interactions. Decomposition energies compared the interaction of small molecules with individual receptor hot spots to those of the native protein ligand. Pharmacophore analysis was used to investigate how effectively small molecules mimic the position of hot spots of the protein ligand. Finally, we study whether small molecules mimic the effects of the native protein ligand on the receptor dynamics. Our results show that, in general, existing small-molecule inhibitors of protein–protein interactions do not optimally mimic protein–ligand hot spots, nor do they effectively engage protein receptor hot spots. The more effective use of hot spots in future drug design efforts may result in smaller compounds with higher ligand efficiencies that may lead to greater success in clinical trials.Item Computational Methods to Identify and Target Druggable Binding Sites at Protein-Protein Interactions in the Human Proteome(2019-09) Xu, David; Wu, Huanmei; Meroueh, Samy; Liu, Xiaowen; Janga, Sarath Chandra; Liu, YunlongProtein-protein interactions are fundamental in cell signaling and cancer progression. An increasing prevalent idea in cancer therapy is the development of small molecules to disrupt protein-protein interactions. Small molecules impart their action by binding to pockets on the protein surface of their physiological target. At protein-protein interactions, these pockets are often too large and tight to be disrupted by conventional design techniques. Residues that contribute a disproportionate amount of energy at these interfaces are known as hot spots. The successful disruption of protein-protein interactions with small molecules is attributed to the ability of small molecules to mimic and engage these hot spots. Here, the role of hot spots is explored in existing inhibitors and compared with the native protein ligand to explore how hot spot residues can be leveraged in protein-protein interactions. Few studies have explored the use of interface residues for the identification of hit compounds from structure-based virtual screening. The tight uPAR•uPA interaction offers a platform to test methods that leverage hot spots on both the protein receptor and ligand. A method is described that enriches for small molecules that both engage hot spots on the protein receptor uPAR and mimic hot spots on its protein ligand uPA. In addition, differences in chemical diversity in mimicking ligand hot spots is explored. In addition to uPAR•uPA, there are additional opportunities at unperturbed protein-protein interactions implicated in cancer. Projects such as TCGA, which systematically catalog the hallmarks of cancer across multiple platforms, provide opportunities to identify novel protein-protein interactions that are paramount to cancer progression. To that end, a census of cancer-specific binding sites in the human proteome are identified to provide opportunities for drug discovery at the system level. Finally, tumor genomic, protein-protein interaction, and protein structural data is integrated to create chemogenomic libraries for phenotypic screening to uncover novel GBM targets and generate starting points for the development of GBM therapeutic agents.Item Design and Synthesis of Small-Molecule Protein-Protein Interaction Antagonists(2014) Han, Xu; Meroueh, Samy; Long, Eric C. (Eric Charles); McLeish, Michael J.Protein-protein interactions play a crucial role in a wide range of biological processes. Research on the design and synthesis of small molecules to modulate these proteinprotein interactions can lead to new targets and drugs to modulate their function. In Chapter one, we discuss the design and synthesis of small molecules to probe a proteinprotein interaction in a voltage-gated Ca2+ channel. Virtual screening identified a compound (BTT-3) that contained a 3,4-dihydro-3,4’-pyrazole core. This compound had modest biological activity when tested in a fluorescence polarization (FP) assay. The synthetic route to BTT-3 consisted of six steps. In addition, analogs of BTT-3 were made for a structure-activity study to establish the importance of a carboxylate moiety. We also synthesized a biotinylated benzophenone photo-affinity probe and linked it to BTT-3 to identify additional protein targets of the compound. In Chapter two, small-molecule antagonists targeting uPA-uPAR protein-protein interaction are presented. A total of 500 commercially-available compounds were previously identified by virtual screening and tested by a FP assay. Three classes of compounds were found with biological activity. The first class of compounds contains pyrrolidone core structures represented by IPR- 1110, the second class has a novel pyrrolo[3,4-c]pyrazole ring system, represented by xv IPR-1283 and the last series had compounds with a 1,2-disubstituted 1,2- dihydropyrrolo[3,4-b]indol-3(4H)-one core structure, represented by IPR-540. Each of these three compounds were synthesized and assessed by FP and ELISA assays. A binding mode of IPR-1110 with uPA was subsequently proposed. Based on this binding mode, another 61 IPR-1110 derivatives were synthesized by us to illustrate the SAR activity. Analogs of the other two series were also synthesized.Item Discovery of Novel Regulators of Aldehyde Dehydrogenase Isoenzymes(2011-05) Parajuli, Bibek; Kimble-Hill, Ann C.; Khanna, May; Ivanova, Yvelina; Meroueh, Samy; Hurley, Thomas DOver the past three years we have been involved in high-throughput screening in an effort to discover novel small molecular modulators of aldehyde dehydrogenase (ALDH) activity. In particular, we have been interested in both the activation and inhibition of the three commonly studied isoenzymes, ALDH1A1, ALDH2 and ALDH3A1, as their distinct, yet overlapping substrate specificities, present a particularly difficult challenge for inhibitor discovery and design. Activation of ALDH2 has been shown to benefit cardiovascular outcome following periods of ischemia and renewed interest in specific inhibition of ALDH2 has application for alcohol aversion therapy, and more recently, in cocaine addiction. In contrast, inhibition of either ALDH1A1 or ALDH3A1 has application in cancer treatments where the isoenzymes are commonly over-expressed and serve as markers for cancer stem cells. We are taking two distinct approaches for these screens: in vitro enzyme activity screens using chemical libraries and virtual computational screens using the structures of the target enzymes as filters for identifying potential inhibitors, followed by in vitro testing of their ability to inhibit their intended targets. We have identified selective inhibitors of each of these three isoenzymes with inhibition constants in the high nanomolar to low micromolar range from these screening procedures. Together, these inhibitors provide proof for concept that selective inhibition of these broad specificity general detoxication enzymes through small molecule discovery and design is possible.Item Exploring a structural protein-drug interactome for new therapeutics in lung cancer(Royal Society of Chemistry, 2014-03-04) Peng, Xiaodong; Wang, Fang; Li, Liwei; Bum-Erdene, Khuchtumur; Xu, David; Wang, Bo; Sinn, Tony; Pollok, Karen; Sandusky, George; Li, Lang; Turchi, John; Jalal, Shadia I.; Meroueh, Samy; Department of Biochemistry & Molecular Biology, IU School of MedicineThe pharmacology of drugs is often defined by more than one protein target. This property can be exploited to use approved drugs to uncover new targets and signaling pathways in cancer. Towards enabling a rational approach to uncover new targets, we expand a structural protein-ligand interactome () by scoring the interaction among 1000 FDA-approved drugs docked to 2500 pockets on protein structures of the human genome. This afforded a drug-target network whose properties compared favorably with previous networks constructed using experimental data. Among drugs with the highest degree and betweenness two are cancer drugs and one is currently used for treatment of lung cancer. Comparison of predicted cancer and non-cancer targets reveals that the most cancer-specific compounds were also the most selective compounds. Analysis of compound flexibility, hydrophobicity, and size showed that the most selective compounds were low molecular weight fragment-like heterocycles. We use a previously-developed screening approach using the cancer drug erlotinib as a template to screen other approved drugs that mimic its properties. Among the top 12 ranking candidates, four are cancer drugs, two of them kinase inhibitors (like erlotinib). Cellular studies using non-small cell lung cancer (NSCLC) cells revealed that several drugs inhibited lung cancer cell proliferation. We mined patient records at the Regenstrief Medical Record System to explore the possible association of exposure to three of these drugs with occurrence of lung cancer. Preliminary in vivo studies using the non-small cell lung cancer (NCLSC) xenograft model showed that losartan- and astemizole-treated mice had tumors that weighed 50 (p < 0.01) and 15 (p < 0.01) percent less than the treated controls. These results set the stage for further exploration of these drugs and to uncover new drugs for lung cancer therapy.Item Mimicking Intermolecular Interactions of Tight Protein–Protein Complexes for Small-Molecule Antagonists(Wiley, 2017-11) Xu, David; Bum-Erdene, Khuchtumur; Si, Yubing; Zhou, Donghui; Ghozayel, Mona; Meroueh, Samy; Biochemistry and Molecular Biology, School of MedicineTight protein–protein interactions (Kd<100 nm) that occur over a large binding interface (>1000 Å2) are highly challenging to disrupt with small molecules. Historically, the design of small molecules to inhibit protein–protein interactions has focused on mimicking the position of interface protein ligand side chains. Here, we explore mimicry of the pairwise intermolecular interactions of the native protein ligand with residues of the protein receptor to enrich commercial libraries for small-molecule inhibitors of tight protein–protein interactions. We use the high-affinity interaction (Kd=1 nm) between the urokinase receptor (uPAR) and its ligand urokinase (uPA) to test our methods. We introduce three methods for rank-ordering small molecules docked to uPAR: 1) a new fingerprint approach that represents uPA′s pairwise interaction energies with uPAR residues; 2) a pharmacophore approach to identify small molecules that mimic the position of uPA interface residues; and 3) a combined fingerprint and pharmacophore approach. Our work led to small molecules with novel chemotypes that inhibited a tight uPAR⋅uPA protein–protein interaction with single-digit micromolar IC50 values. We also report the extensive work that identified several of the hits as either lacking stability, thiol reactive, or redox active. This work suggests that mimicking the binding profile of the native ligand and the position of interface residues can be an effective strategy to enrich commercial libraries for small-molecule inhibitors of tight protein–protein interactions.Item Protein function prediction by integrating sequence, structure and binding affinity information(2014-02-03) Zhao, Huiying; Zhou, Yaoqi; Liu, Yunlong; Meroueh, Samy; Janga, Sarath ChandraProteins are nano-machines that work inside every living organism. Functional disruption of one or several proteins is the cause for many diseases. However, the functions for most proteins are yet to be annotated because inexpensive sequencing techniques dramatically speed up discovery of new protein sequences (265 million and counting) and experimental examinations of every protein in all its possible functional categories are simply impractical. Thus, it is necessary to develop computational function-prediction tools that complement and guide experimental studies. In this study, we developed a series of predictors for highly accurate prediction of proteins with DNA-binding, RNA-binding and carbohydrate-binding capability. These predictors are a template-based technique that combines sequence and structural information with predicted binding affinity. Both sequence and structure-based approaches were developed. Results indicate the importance of binding affinity prediction for improving sensitivity and precision of function prediction. Application of these methods to the human genome and structure genome targets demonstrated its usefulness in annotating proteins of unknown functions and discovering moon-lighting proteins with DNA,RNA, or carbohydrate binding function. In addition, we also investigated disruption of protein functions by naturally occurring genetic variations due to insertions and deletions (INDELS). We found that protein structures are the most critical features in recognising disease-causing non-frame shifting INDELs. The predictors for function predictions are available at http://sparks-lab.org/spot, and the predictor for classification of non-frame shifting INDELs is available at http://sparks-lab.org/ddig.