Biology subjects

Rao, D. N.

Publications and source records attributed to Rao, D. N..

3 recordsLinked to original sources

bioRxiv2022

CRISPR correction of GBA mutation in hiPSCs restores normal function to Gaucher macrophages and increases their susceptibility to Mycobacterium tuberculosis

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the {beta}-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. To investigate whether correction of GBA mutations restores normal function to Gaucher macrophages, we performed CRISPR editing of homozygous L444P (1448T[->]C) GBA mutation in Type 2 GD (GBA-/-) hiPSCs, which yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs, were compared for GCase enzymatic activity, motility, and phagocytosis, all of which showed that GBA mutation correction restores normal macrophage functions. Furthermore, we investigated whether lysosomal disorders drive susceptibility to Mycobacterium tuberculosis, by infecting GBA-/-, GBA+/- and GBA+/+ macrophages with the virulent H37Rv lab strain. The results showed that impaired mobility and phagocytic activity of Gaucher macrophages, correlated with reduced levels of TB engulfment and TB multiplication, supporting the hypothesis that GD may be protective against tuberculosis.

genomics↗

bioRxiv2021

Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

biochemistry↗

bioRxiv2019

Structural insights into the molecular mechanisms of the Mycobacterium evolvability factor Mfd

Mfd is a highly conserved ATP dependent DNA translocase that mediates the role of Transcription-Coupled-DNA-Repair(TCR) in bacteria. The molecular mechanisms and conformational remodelling that occurs in Mfd upon ATP binding, hydrolysis, and DNA translocation are poorly defined. Here we report a series of crystal and electron microscopy(EM) structures of Mfd from Mycobacterium tuberculosis (MtbMfd) and Mycobacterium smegmatis Mfd, solved in both the apo and nucleotide-bound states. The structures reveal the mechanism of nucleotide-binding, which lead to the remodeling of the Walker A motif at the catalytic pocket, inducing a flip-flop action of the hinge and flexible linker regions. Specifically, nucleotide binding unlocks the Translocation in RecG motif of the D6-domain to induce a ratchet-like motion. Functional studies of MtbMfd-RNAP complexes show that MtbMfd rescues stalled Transcription Elongation Complexes. We also report negative-stain and cryo-EM single particle reconstructions of MtbMfd higher order oligomer, that reveal an unexpected dodecameric assembly state. Given that Mfd accelerates the evolution of antimicrobial resistance(AMR), our results establish a framework for the design of new \"anti-evolution\" therapeutics to counter AMR.

molecular biology↗