Design, Synthesis and Evaluation of Small Molecules for Profiling Thiol Proteome of Microbes

Abstract

Thiols are biologically important molecules and play a vital role in a number of cellular processes. Cysteine is the most important biological thiol, least abundant (3.3%), yet highly conserved on the proteome. Along with other cytosolic thiols, it helps in regulating the redox balance of the cell. Any perturbation to the levels of thiols inside cells can cause redox stress that can lead to lethality. Cysteine residues are also responsible for retaining the structural stability of the protein, which ensures the smooth functioning of a number of cellular processes. They are involved in different post-translational modifications such as S-phosphorylation, S-glutathionylation, disulfide formation etc. Additionally, cysteine serves as the reactive nucleophile in many enzymatic biochemical reactions. The nucleophilicity of the thiol is profoundly dependent upon the microenvironment around the cysteine residue in the native state of the protein, which eventually dictates the pKa of the thiol. Even in bacteria, thiols play important roles in maintaining redox homeostasis. For example, Bacillithiol in Staphylococcus aureus (S. aureus) and Glutathione in Escherichia coli (E. coli) essay the role of antioxidants. Cysteines are also key residues on bacterial proteins responsible for generating resistance and virulence in pathogenic bacteria. Due to all the aforementioned reasons, thiols and in particular cysteine, becomes an attractive biological target from a therapeutic and diagnostic point-of-view. Thus, in order to target cellular thiols, we designed and evaluated the ability of Indole-based naphthoquinone epoxides (INDQEs) to react with cellular thiols via the opening of the epoxide ring. A series of analogues were synthesized and their reactivity with thiol was evaluated. We could propose a sterics-based model that explains that the reactivity with thiol could be tuned by varying the groups neighbouring the epoxide. The epoxide-thiol reaction resulted in an irreversible covalent modification. When tested against a panel of pathogenic bacteria, these compounds were found to inhibit Gram-positive S. aureus at low concentrations. The key finding was a good correlation between reactivity with a thiol and the antibacterial potency. Additionally, the kinetics of the reaction with a thiol was a major driving force in bacterial inhibition. The lead compound identified, was found to inhibit clinically acquired extremely drug-resistant strains of S. aureus such as Methicillin-resistant S. aureus (MRSA) and Vancomycin-resistant S. aureus (VRSA) at potent Minimum Inhibitory Concentrations (MIC). Clinically, infections caused by these drug-resistant strains are extremely difficult to treat. Daptomycin is the antibiotic of last resort against VRSA infections. Thus, development of better antibacterial candidates with novel targets is of urgent need. In order to decipher the targets of the lead compound in S. aureus, INDQE alkyne probes were synthesized and Activity-Based Protein Profiling (ABPP) technique was employed to profile the thiol proteome of S. aureus to identify the target proteins. An active INDQE alkyne probe was identified with similar reactivity with thiol and inhibitory activity against S. aureus as that of the lead compound. A series of ABPP experiments were conducted using this probe to establish the selectivity of these compounds towards cysteine residues as well as the permeability specific to S. aureus only. Further, chemoproteomics experiments using this probe fished out certain transcriptional virulence factors as potential targets. The identified target proteins were obtained by routine techniques of cloning and purification and were further validated using ABPP techniques. Cysteine-point mutants were generated using site-directed mutagenesis and ABPP experiments corroborated cysteine as the target site for these compounds. Two Mar R (Multiple antibiotic resistance regulator) family of proteins were identified and validated from this study. These identified targets are novel and hence targeting these can help in generating improved and more potent antibacterial agents. This development may help in reducing the burden of antibacterial resistance in the near future.