Postdoctoral Research Scholar, Case Western Reserve University


Low-reactivity electrophiles for the identification of covalent chemical probes

Traditionally used drugs including penicillin antibiotics and aspirin impart their biological functions through covalent modification of cellular proteins. However, due to poor selectivity and idiosyncratic off-targets of irreversible small molecules in general to cellular proteins, covalent small molecules are not favorites for in vivo applications. Recently, there are resurgent interests in developing covalent small molecules based drug discovery following the approval of acrylamide-containing electrophilic small molecules such as ibrutinib, afatinib, and neratinib by FDA for the treatment of cancer. We are interested in developing a library of small molecules with structurally diverse and complex scaffolds conjugated to various electrophiles and evaluate them in Click-activity based protein profiling (ABPP) assay to identify high-quality covalent chemical probes. When synthesized and screened a library of ~200 electrophilic small molecules, we have identified many molecules labeled cellular proteins that are relevant in cancer biology. One of the low-reactivity electrophiles, 2-chloropropionamide is less reactive than typical acrylamide electrophiles could be amenable for in vivo applications. One of the proteins being selectively targeted by a small molecule probe containing 2-chloropropionamide (CPA) electrophile is protein disulfide isomerase (PDI). This CPA probe was found to selectively label PDI protein and inhibit its function by modifying the catalytic cysteine residues of the protein PDI. We have derived a correlation of the functional of PDI by small molecules responsible for the killing of multiple myeloma cells. This novel, low-reactivity electrophile could be used to re-engineer many of the commercially available electrophilic small molecules to enhance their selectivity to proteins and facilitate in vivo applications


1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor

Abstract: 1,6-epi-Cyclophellitol cyclosulfate (“α-cyclosulfate”) is a conceptually new, potent and selective irreversible α-glucosidase inhibitor that acts through mimicry of the α-glucosidase Michaelis complex 4C1 chair conformation.The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.

Pub.: 13 Jul '17, Pinned: 28 Aug '17

Proteome-wide covalent ligand discovery in native biological systems.

Abstract: Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

Pub.: 17 Jun '16, Pinned: 28 Aug '17

1,3,5-Triazine as a modular scaffold for covalent inhibitors with streamlined target identification.

Abstract: Small-molecule inhibitors can accelerate the functional annotation and validate the therapeutic potential of proteins implicated in disease. Phenotypic screens provide an effective platform to identify such pharmacological agents but are often hindered by challenges associated with target identification. For many protein targets, these bottlenecks can be overcome by incorporating electrophiles into small molecules to covalently trap interactions in vivo and by employing bioorthogonal handles to enrich the protein targets directly from a complex proteome. Here we present the trifunctionalized 1,3,5-triazine as an ideal modular scaffold for generating libraries of irreversible inhibitors with diverse target specificities. A divergent synthetic scheme was developed to derivatize the triazine with an electrophile for covalent modification of target proteins, an alkyne as a click-chemistry handle for target identification, and a diversity element to direct the compounds toward distinct subsets of the proteome. We specifically targeted our initial library toward cysteine-mediated protein activities through incorporation of thiol-specific electrophiles. From this initial screen we identified two compounds, RB-2-cb and RB-11-ca, which are cell permeable and highly selective covalent modifiers for Cys239 of β-tubulin (TUBB) and Cys53 of protein disulfide isomerase (PDI) respectively. These compounds demonstrate in vitro and cellular potencies that are comparable to currently available modulators of tubulin polymerization and PDI activity. Our studies demonstrate the versatility of the triazine as a modular scaffold to generate potent and selective covalent modifiers of diverse protein families for chemical genetics applications.

Pub.: 06 Feb '13, Pinned: 28 Aug '17

Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles.

Abstract: Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or over-expressed in) disease cells. In this context, fragment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compounds for biological inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-molecule fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidation in cells. Such reactions are particularly relevant in diseases, like cancer, which are linked to excessive production of reactive oxygen species (ROS). Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that react selectively with cysteine sulfenic acid (Cys-SOH) formed in proteins via oxidation or hydrolysis reactions. Here, we report analysis of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were identified for >1280 S-sulfenylated cysteines present in 'druggable' proteins and orphan targets, revealing disparate reactivity profiles and target preferences. Among the unique ligand-protein interactions identified was that of a pyrrolidinedione nucleophile, PYD that reacted preferentially with protein tyrosine phosphatases (PTPs). Fragment-based covalent ligand discovery with C-nucleophiles affords an expansive snapshot of the ligandable 'redoxome' with significant implications for covalent inhibitor pharmacology and also affords new chemical tools to investigate redox-regulation of protein function.

Pub.: 31 Mar '17, Pinned: 28 Aug '17

High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38alpha.

Abstract: Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases between inactive and enzymatically competent conformations and vice versa. The development of novel kinase inhibitors is mainly fostered by high-throughput screening initiatives where the small molecule perturbation of the phosphorylation reaction is measured to identify inhibitors. Such setups require enzymatically active kinase preparations and present a risk of solely identifying classical ATP-competitive Type I inhibitors. Here we report the high-throughput screening of a library of approximately 35000 small organic molecules with an assay system that utilizes enzymatically inactive human p38alpha MAP kinase to detect stabilizers of the pharmacologically more desirable DFG-out conformation. We used protein X-ray crystallography to characterize the binding mode of hit compounds and reveal structural features which explain how these ligands stabilize and/or induce the DFG-out conformation. Lastly, we show that although some of the hit compounds were confirmed by protein X-ray crystallography, they were not detected in classic phosphorylation assays, thus validating the unique sensitivity of the assay system used in this study and highlighting the potential of screening with inactive kinase preparations.

Pub.: 03 Dec '09, Pinned: 28 Aug '17

Chemical probes unravel an antimicrobial defense response triggered by binding of the human opioid dynorphin to a bacterial sensor kinase.

Abstract: Host-microbe communication via small molecule signals is important for both symbiotic and pathogenic relationships, but is often poorly understood at the molecular level. Under conditions of host stress, levels of the human opioid peptide dynorphin are elevated, triggering virulence in the opportunistic pathogenic bacterium Pseudomonas aeruginosa via an unknown pathway. Here we apply a multilayered chemical biology strategy to unravel the mode of action of this putative interkingdom signal. We designed and applied dynorphin-inspired photoaffinity probes to reveal the protein targets of the peptide in live bacteria via chemical proteomics. ParS, a largely uncharacterized membrane sensor of a two-component system, was identified as the most promising hit. Subsequent full proteome studies revealed that dynorphin(1-13) induces an antimicrobial peptide-like response in Pseudomonas, with specific upregulation of membrane defence mechanisms. No such response was observed in a parS mutant, which was more susceptible to dynorphin-induced toxicity. Thus, P. aeruginosa exploits the ParS sensing machinery to defend itself against the host in response to dynorphin as a signal. This study highlights interkingdom communication as a potential essential strategy not only for induction of P. aeruginosa virulence but also for maintaining viability in the hostile environment of the host.

Pub.: 30 Mar '17, Pinned: 27 Jun '17