Dharmaraja Allimuthu

Our lab is interested in identifying high-quality covalent chemical probes as irreversible inhibitors of disease relevant cellular proteins, which couples the inter-disciplinary research expertise of chemistry and biology.
Rationally designing targeted covalent therapeutics as irreversible enzyme inhibitors has led to the approval of several anticancer therapeutics including afatinib, neratinib and ibrutinib. Many of these FDA approved therapeutics or small molecules that are in clinical trials are equipped with reactive electrophilic warheads. In these molecules, electrophiles possessing high to moderate reactivity have been associated with off-target activity as they are prone to irreversibly modify several proteins at the nucleophilic amino acid residues including cysteine and serine, which is considered a potential hurdle for successful translation/usage of covalent therapies in clinical applications.

We are interested in developing library of small molecules tagged to low reactivity electrophiles such as 2-chloropropinamides, sulfonyl fluorides and their modified warhead analogs which are expected to possess weak reactivity with protein nucleophiles. Our initial focus is to develop selective and irreversible inhibitors of redox active cellular proteins containing cysteine residues in its active sites, glutaredoxin (GRX1) and peroxyredoxin-1 (PRDX-1). Both GPRX1 and PRDX1 are highly expressed in many of the cancer cell lines including drug-resistant ones, disturbing the levels of these proteins in cells using small interference ribonucleic acid (siRNA) mediated knock down or CRISPR-cas9 mediated knock out have been shown to affecttheir survival. However, these disease causing proteins lack selective and potent functional inhibitors. Our focus is to synthesize a library of rationally designed targeted-covalent small moleculesbased on existing ligand scaffolds of GPRX-1 and PRDX-1, which are tagged with electrophilic warheads that are capable of reacting with active site cysteine residues. Then, we evaluate their inhibitory potency using in vitro biochemical assays.Subsequently, liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the proteins exposed to the inhibitors will establish the direct engagement of the covalent small molecule at the active site cysteines of the redox proteins. Evaluating the top hits in cells to identify their selectivity in cellular proteome will be the next step.Finally, the leading hits identified will be used in cell death-based phenotypic screening of PRDX1/GRX1 sensitive cancer cell lines/drug-resistant cancer phenotypes and their paired non-cancerous cells to find selective anticancer agents that mediate their effect through the inhibition of PRDX1/GRX1 redox active proteins.
Further, the synthesized library also will be used in screening against several cancer lines using both cell death-based phenotypic screening and activity-based protein profiling, followed by chemical proteomics to identify novel small molecule anticancer therapeutics and to elucidate their mechanism of action.