A pinboard by
Dharmaraja AT

I'm a Postdoc working in the area of chemical biology at the School of Medicine, CWRU, Ohio, USA

Apart from research life, I like listening tamil music, reading science histories, playing outdoor games like cricket, volley ball, table tennis and badminton.


Revisiting electrophilic molecules as cancer therapeutics is encouraged both in industry & Academia

Drug discovery primarily relied on non-covalent small molecule inhibitors of proteins due to their high selectivity and safe in vivo cytotoxicity profiles. Irreversible inhibitors associated with idiosyncratic toxicity related issues is the major concern and this could be overcome with moderate to low-reactivity electrophiles. Further, durable and potent target engagement by the covalent probes could offer benefits over reversible therapeutics. In this context, recently, many of the moderately reactive acrylamide-electrophile containing small molecules including Afatinib, Neratinib and Ibrutinib have been approved by FDA as cancer chemotherapeutics. These molecules engage cysteines adjacent to the active site of protein kinases in cancer cells. Recognition of irreversible inhibitors for the treatment of cancer have encouraged people in industry and academia to shift their focus on developing covalent probes based chemotherapeutics.

Multiple synthetic strategies have been developed to identify potent irreversible inhibitors of cellular proteins. Fragment-based library design, target-based drug synthesis, structure-guided drug design and combinatorial synthesis are some of the powerful synthetic approaches exploited in developing libraries of covalent chemical probes. In our group, we came up with a design of weakly reactive electrophilic warheads appended to diverse structural, stereochemical and complex scaffolds. The structural complexity is expected to provide strong affinity of protein by the scaffold and hence attenuate any non-selective reactivity effects. One of my projects utilized (S)-or (R)-2-chloropropionamide electrophile, a chiral, low-reactivity electrophile conjugated to diverse structural scaffolds and synthesized a library of small molecules. Activity-based protein profiling, mass-spectrometric chemical proteomics, and enzymatic analysis resulted in identification of highly selective, irreversible inhibitor of protein disulfide isomerase (PDI), an endoplasmic reticulum bound chaperone protein. Phenotypic screening results revealed a highly selective inhibition of multiple myeloma cells only by the PDI inhibitor. Advancement in proteomics have significantly helped in identifying many undraggable cellular proteins. Together, these versatile synthetic and advanced chemical proteomics approaches are highly useful for the identification of potential covalent small molecules based therapeutics.


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: 08 Aug '17

Target identification for small bioactive molecules: finding the needle in the haystack.

Abstract: Identification and confirmation of bioactive small-molecule targets is a crucial, often decisive step both in academic and pharmaceutical research. Through the development and availability of several new experimental techniques, target identification is, in principle, feasible, and the number of successful examples steadily grows. However, a generic methodology that can successfully be applied in the majority of the cases has not yet been established. Herein we summarize current methods for target identification of small molecules, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound. We describe the most frequently employed experimental approaches for target identification and provide several representative examples illustrating the state-of-the-art. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be most powerful and most frequently applied. To provide guidance for rapid entry into the field and based on our own experience we propose a typical workflow for target identification, which centers on the application of chemical proteomics as the key step to generate hypotheses for potential target proteins.

Pub.: 19 Feb '13, Pinned: 08 Aug '17

New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish.

Abstract: Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in the progression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requires attachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidated Shh has been challenging due to the limited array of tools available, and the roles of these posttranslational modifications are poorly understood. Herein, we describe the development and validation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable its visualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown to be an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shh from signalling cells, formation of multimeric transport complexes and signalling. We have used this probe to determine the size of transport complexes of lipidated Shh in culture medium and expression levels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitative chemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy and detection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probes provide a set of novel and well-validated tools that can be used to investigate the role of lipidation on activity of Shh, and potentially other members of the Hedgehog protein family.

Pub.: 13 Jan '15, Pinned: 08 Aug '17

Substrate Profiling and High Resolution Co-complex Crystal Structure of a Secreted C11 Protease Conserved Across Commensal Bacteria.

Abstract: Cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria, and this diverse family of proteins have significant biological roles in bacterial viability and environmental interactions. Members of the clostripain-like (C11) family of cysteine proteases from distal gut commensal strains have recently been shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. Development of substrates, inhibitors, and probes that target C11 proteases from enteric bacteria will help to establish the role of these proteins at the interface of the host and microbiome in health and disease. We employed mass spectrometry-based substrate profiling method to identify an optimal peptide substrate of PmC11, a C11 protease secreted by the commensal bacterium Parabacteroides merdae. Using this substrate sequence information, a panel of fluorogenic substrates were synthesized to calculate kcat and KM and to evaluate the importance of the P2 amino acid for substrate turnover. A potent and irreversible tetrapeptide inhibitor with an C-terminal acyloxymethyl ketone warhead, Ac-VLTK-AOMK, was then synthesized. We determined the crystal structure of PmC11 in complex with this inhibitor and uncovered key active-site interactions that govern PmC11 substrate recognition and specificity. This is the first C11 protease structure in complex with a substrate mimetic and is also the highest resolution crystal structure of a C11 protease to date at 1.12 Å resolution. Importantly, subjecting human epithelial cell lysates to PmC11 hydrolysis in combination with subtiligase-based N-terminal labeling and tandem mass spectrometry proteomics complemented the stringent substrate specificity observed in the in vitro substrate profiling experiment. The combination of chemical biological, biophysical, and biochemical techniques presented here to elucidate and characterize PmC11 substrate selectivity can be expanded to other proteases and the development of chemical tools to study these essential proteins in biologically relevant samples, such as the highly complex distal gut microbiome.

Pub.: 18 Apr '17, Pinned: 08 Aug '17

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: 08 Aug '17