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theses

Tuberculosis global epidemics is a serious threat to human population, the disease is estimated to be the second highest cause of death due to infection. In this study we present a lead hit-compound “AAP1” identified through high-throughput screening performed against Mycobacterium tuberculosis RNA polymerase (RNAP). AAP1 displays great potential as a mycobacterial RNAP inhibitor and mycobacterial growth inhibitor. AAP1 can be synthesized from commercially available precursors in single reaction step. AAP1 has a modular structure, facilitating the synthesis of novel AAP1 analogs and facilitating the interpretation of structure-activity relationships for novel AAP1 analogs. Currently, more than 400 AAP1 analogs have been synthesized and tested and based on improved characteristics, an AAP1 analog AAP12 is used in this study with AAP1 (AAPs). By isolating, sequencing, and characterizing spontaneous resistant mutants, we have shown that mycobacterial RNAP is the functional cellular target for the antibacterial activity of AAPs By isolating, sequencing, and characterizing resistant mutants, we have shown AAPs function through a site on RNAP that is different from, and does not overlap with the binding sites for rifamycins. The site is located at the base of RNAP " lobe" and N’- terminal of β’ bridge helix and corresponds, essentially exactly, to the site for CBR703, a compound that does not inhibit mycobacterial RNAP and does not inhibit mycobacterial growth. It appears that AAPs are mycobacterial-specific ligands of RNAP β lobe and that CBR703 is a Gram-negative-specific ligand of the RNAP β lobe. By observing the effect of AAPs on individual transcription step, we have shown that AAPs affect the nucleotide addition cycle of RNAP. Based on the fact that AAPs interfere with the nucleotide addition cycle of the RNAP without making any contact with the active center residues we propose that AAPs function allosterically by binding at the N-terminal sub-region of β’ bridge helix, and by interfering with its conformational dynamics In our current work, we are systematically preparing and evaluating novel AAP1 analogs. The objectives are to increase the antimycobacterial potency, to improve physical and pharmacological properties, and to identify compounds suitable for evaluation in an animal model of tuberculosis.

ETD_5293

Ph.D.

Includes bibliographical references

by Soma Mandal

doi:10.7282/T3Q52MQZ

ETD doctoral

The author owns the copyright to this work.