Biology subjects

Sarathy, J.

Publications and source records attributed to Sarathy, J..

4 recordsLinked to original sources

bioRxiv2026

In vitro dormancy models improve ability to predict treatment response in severe marmoset tuberculosis lesions

Tuberculosis lesions are structurally and physiologically heterogeneous, creating microenvironments that restrict antibiotic penetration and alter bacterial susceptibility. This heterogeneity remains a major impediment to shortening tuberculosis treatment, as it is difficult to predict which regimens will sterilize bacteria in these hard-to-treat lesion niches. Many in vitro systems and commonly used animal models fail to recapitulate the necrotic, caseous lesion pathology associated with treatment failure and relapse. Here, we show that resource-efficient in vitro assays engineered to mimic key lesion microenvironments, including those with lipid-rich, caseum-like matrices with controlled oxygenation and pH, generate drug-response metrics that predict lesion-level treatment responses in a non-human primate model. We stratified individual marmoset lesions by baseline 2-deoxy-2-[18F] fluoro-D-glucose ([18F] FDG) positron emission tomography/computed tomography (PET/CT) features, then quantified drug potency and interactions across three lipid-induced dormancy (LIDs) conditions under both equipotent and lesion pharmacokinetic-informed dosing schemes. LIDs-based and pharmacokinetic-informed measurements aligned more closely with treatment responses in severe lesions than conventional in vitro data and recapitulated interaction patterns consistent with known regimen performance. Integrating imaging-derived and in vitro features in multivariate, sequential computational models improved predictive accuracy over baseline imaging alone and identified a subset of informative in vitro metrics. Together, these results establish a scalable framework linking lesion-mimicking assays to lesion-specific outcomes, enabling earlier, more cost-effective prioritization of pre-clinical regimens aimed at sterilizing hard-to-treat tuberculosis lesions and shortening therapy.

molecular biology↗

bioRxiv2024

The kinetics of bedaquiline diffusion in tuberculous cavities opens a window for emergence of resistance.

Cavitary tuberculosis (TB) is difficult to cure and a site of relapse. Bedaquiline has been a wonder drug in the treatment of multidrug resistant TB, but emergence of resistance threatens its sustained success. To investigate the role of drug distribution in resistance development, we designed a novel laser-capture microdissection scheme to spatially resolve the penetration of bedaquiline in the necrotic center (caseum) of cavities, a recalcitrant site of disease. Working with preclinical models that form large necrotic lesions, we profiled bedaquiline and two next generation diarylquinolines TBAJ-587 and TBAJ-876. Drug concentrations were measured in laser-captured areas of cavity caseum as a function of time and distance from blood supply. To simulate drug coverage in patient cavities, the data were modeled, and drug penetration parameter estimates were linked to clinical plasma pharmacokinetics for bedaquiline and the new diarylquinolines. Pharmacokinetic-pharmacodynamic (PK-PD) simulations revealed that bedaquiline reaches efficacious concentrations in outer and deep caseum after several weeks to months and lingers at subtherapeutic concentrations up to 3 years after therapy ends. TBAJ-587 and TBAJ-876, currently in clinical development, achieve bactericidal concentrations in caseum more rapidly and shorten the window of suboptimal concentrations post treatment compared to bedaquiline. Simulations of clinically plausible dosing schemes were conducted to guide the design of clinical trials for cavitary TB and help mitigate resistance development. In summary, the slow kinetics of diffusion of bedaquiline into and out of cavity caseum creates spatio-temporal windows of subtherapeutic concentrations. Site-of-disease simulations of TBAJ-587 and TBAJ-876 predict reduced opportunities for resistance development. SIGNIFICANCEClinical resistance to bedaquiline has emerged faster than anticipated. Understanding potential contributing factors could help curb further resistance development, not only for bedaquiline but also for the next generation diarylquinolines currently in phase 2, TBAJ-587 and TBAJ-876. Here we hypothesized and confirmed that the extended time to reach steady state and slow clearance of bedaquiline leads to extremely slow diffusion into and out of cavity caseum, a recalcitrant site of TB disease and relapse. Through modeling of experimental data in a preclinical model of cavitary TB and clinical simulations, we show that the next generation diarylquinolines may reduce spatio-temporal windows of resistance development compared to bedaquiline. Our results can inform dosing schemes of diarylquinoline-based therapies that limit resistance development.

pharmacology and toxicology↗

bioRxiv2022

Macrophage internalization creates a multidrug-tolerant fungal persister population, providing a permissive reservoir for the emergence of drug resistance

Candida glabrata is a major fungal pathogen notable for causing recalcitrant infections, rapid emergence of drug-resistant strains, and its ability to survive and proliferate within macrophages. Resembling bacterial persisters, a subset of genetically drug-susceptible C. glabrata cells can survive lethal exposure to the fungicidal echinocandin drugs. Herein, we show that macrophage internalization induces cidal drug tolerance in C. glabrata, expanding the persister reservoir from which echinocandin-resistant mutants emerge. We show that this drug tolerance is associated with non-proliferation and is triggered by macrophage-induced oxidative stress, and that deletion of genes involved in reactive oxygen species detoxification significantly increases the emergence of echinocandin-resistant mutants. Finally, we show that the fungicidal drug amphotericin B can kill intracellular C. glabrata echinocandin persisters, reducing emergence of resistance. Our study supports the hypothesis that intra-macrophage C. glabrata is a reservoir of recalcitrant/drug-resistant infections, and that drug alternating strategies can be developed to eliminate this reservoir.

microbiology↗

bioRxiv2019

Pyrazinamide triggers degradation of its target aspartate decarboxylase

The introduction of pyrazinamide (PZA) in the tuberculosis drug regimen shortened treatment from 12 to 6 months 1. PZA is a prodrug that is activated by a Mycobacterium tuberculosis (Mtb) amidase to release its bioactive component pyrazinoic acid (POA) 2. Aspartate decarboxylase PanD, a proenzyme activated by autocatalytic cleavage (Supplementary Fig. 1A, 3) and required for Coenzyme A (CoA) biosynthesis, emerged as a target of POA 4-7. In vitro and in vivo screening to isolate spontaneous POA-resistant Mtb mutants identified missense mutations in either panD or the unfoldase clpC1, encoding a component of the caseinolytic protease ClpC1-ClpP 4,6-9. Overexpression and binding studies of PanD or ClpC1 pointed to PanD as the direct target of POA whereas clpC1 mutations appeared to indirectly cause resistance 4,5,7,9,10. Indeed, supplementing growth media with CoA precursors downstream of the PanD catalyzed step conferred POA resistance 4,7,11. Metabolomic analyses and biophysical studies using recombinant proteins confirmed targeting of PanD by POA 5. However, the exact molecular mechanism of PanD inhibition by POA remained unknown. While most drugs act by inhibiting protein function upon target binding, we show here that POA is not a bona fide enzyme inhibitor. Rather, POA binding to PanD triggers degradation of the protein by ClpC1-ClpP. Thus, the old tuberculosis drug PZA promotes degradation of its target. While novel for an antibacterial, drug-induced target degradation has recently emerged as a strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA.

microbiology↗