Protein Secretion as a Novel Drug Target for Tuberculosis
Abstract
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major human pathogen causing significant human suffering worldwide. There is an urgent unmet need for new therapeutic agents and an increased understanding of the disease process. We propose to develop anti-tubercular agents that work in new ways by targeting protein secretion, a key virulence mechanism, and to understand how these compounds kill bacteria. We previously identified several novel anti-tubercular series using a high throughput screen directed at protein secretion in live cells. We selected a set of representative compounds for each of these five series and commenced testing compounds for activity against multiple strains of M. tuberculosis which under-express LepB. We generated recombinant strains of M. tuberculosis to monitor protein secretion using a reporter protein (alkaline phosphatase). We screened our over-expression library against key molecules to identify potential resistance mechanisms and determined activity of compounds on agar plates in preparation for isolating resistant mutants. We designed and synthesized a new set of analogs for the hydrazine series and tested these for both anti-tubercular activity and cytotoxicity. Several potent molecules were identified but these are also cytotoxic. We designed a second set of molecules aimed at reducing cytotoxicity while retaining potency. We designed new analogs for two additional series and prepared synthetic schemes. We initiated studies to understand the essentiality and function of LepB in M. tuberculosis. We constructed new plasmids to generate controllable knockdown strains of LepB by CRISPRi in M.tuberculosis. We generated recombinant strains of M. tuberculosis with mutant alleles of LepB, which demonstrate that 1 of 6 cysteine residues in the protein is essential.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 2021
- Accession Number
- AD1152637
Entities
People
- Tanya Parish
Organizations
- Seattle Children's Research Institute