large-scale-sars-cov-2-testing-utilizing-saliva-transposition-sample

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. ...

Michigan State University

The  mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in  Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel  growth inhibitors for differential growth inhibition of wild-type  compared to the growth of a pool of 24 unique  mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both  and against intracellular   cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species  Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24  mutants defined two clades of inhibitors and two clades of  mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.

Kayla N. Conner

Biography

JoVE Journal Publications

Other Publications