Harinath Chakrapani

Cholesteryl esters (CEs) are widely regarded as inert storage forms of cholesterol, yet their potential to directly engage cellular proteins remains largely unexplored. Here, we establish an integrated chemical proteomic platform to systematically define CE–protein interactions in mammalian cells. We developed and comparatively deployed three orthogonal photoaffinity labeling strategies: (i) metabolic assembly of a bifunctional CE probe through endogenous acyl-CoA:cholesterol acyltransferase activity, (ii) direct delivery of a structurally defined diazirine–alkyne CE analog, and (iii) fragment-assisted subtraction using sterol and fatty acyl control probes to resolve interactions dependent on the intact esterified scaffold. Each probe system was rigorously validated by lipidomics and UV-dependent crosslinking prior to quantitative proteomic analysis. Integration of these complementary modalities identified 495 CE-associated proteins spanning enzymes, transporters, scaffolding proteins, and canonical sterol-binding families. The limited overlap across strategies reveals that lipid–protein engagement is strongly conditioned by biosynthetic origin, probe topology, and intracellular routing. Functional and database annotations further demonstrate enrichment of druggable and disease-linked proteins, connecting CE interactions to metabolic, neurological, and cardiovascular pathways. Collectively, this work provides the first systems-level map of CE–protein interactions and establishes a generalizable, multimodal framework for chemically resolving lipid–protein interactomes.

Current tuberculosis (TB) treatment suffers from drawbacks such as long regimens, high pill burden and side effects leading to non-adherence and poor treatment outcomes. Dissolution-controlled drug depot formulation with high drug loading is a clinically successful drug delivery strategy. Such depots reduce the dosing frequency for treatments requiring daily administration, thereby improving treatment adherence and compliance. However, dissolution-controlled depots for first-line TB drugs have not been demonstrated due to their high solubility and high dose requirements. In this study, we overcame this challenge by developing injectable, extended-release, dissolution-controlled depots of nanocrystalline rifapentine (NCRPT), microcrystalline rifapentine (MCRPT) and amorphous rifapentine microparticles (ARPT) with more than 75% loading. Crystalline formulations resulted in much slower depot dissolution compared to amorphous formulations. A single intramuscular (IM) injection of NCRPT in mice resulted in therapeutic serum concentrations for over a week. We then demonstrated the efficacy of NCRPT in both pre-exposure prophylaxis and therapeutic models of mice TB. NCRPT administered at 60 mg/kg once every two weeks demonstrated excellent efficacy in a mouse model of TB infection. In each case, a ~ 4-log-fold reduction in lung bacterial load compared to untreated mice was observed. These results open new avenues for developing LAI formulations of TB drugs and could improve patient compliance and TB management.

Cellular hydropersulfides (RS‐SH) derived from hydrogen sulfide (H ₂ S) such as glutathione hydropersulfide (GS‐SH) are excellent hydrogen atom transfer agents, and quench radicals to protect cells from ferroptosis, a form of iron‐mediated cell death associated with an unchecked build‐up of lipid radicals. Here, using principles of enzyme‐inhibitor design, a series of new artificial substrates for the endogenous hydropersulfide‐generating enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST) was developed. We find that the lead molecules generated GS‐SH, catalyzed by 3‐MST, permeated cells to enhance endogenous hydropersulfides, protected cells from ferroptosis, and reduced systemic inflammation in an animal model. Together, this calibrated approach to promote cell's own radical trapping antioxidants using its biosynthetic machinery to prevent ferroptosis has tremendous implications in redox biology and therapeutics.

Cellular hydropersulfides (RS‐SH) derived from hydrogen sulfide (H ₂ S) such as glutathione hydropersulfide (GS‐SH) are excellent hydrogen atom transfer agents, and quench radicals to protect cells from ferroptosis, a form of iron‐mediated cell death associated with an unchecked build‐up of lipid radicals. Here, using principles of enzyme‐inhibitor design, a series of new artificial substrates for the endogenous hydropersulfide‐generating enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST) was developed. We find that the lead molecules generated GS‐SH, catalyzed by 3‐MST, permeated cells to enhance endogenous hydropersulfides, protected cells from ferroptosis, and reduced systemic inflammation in an animal model. Together, this calibrated approach to promote cell's own radical trapping antioxidants using its biosynthetic machinery to prevent ferroptosis has tremendous implications in redox biology and therapeutics.

Most front-line tuberculosis (TB) drugs are ineffective against hypoxic nonreplicating Mycobacterium tuberculosis (Mtb), largely due to poor permeability, leading to reduced drug accumulation and target engagement. To overcome this phenotypic antimicrobial resistance (AMR), we developed nitroheteroaryl prodrugs for Moxifloxacin (MXF), a front-line TB drug. These prodrugs are activated by bacterial nitroreductases (NTR), which are overexpressed in hypoxic Mtb. NTR-mediated electron transfer and protonation facilitate rapid cleavage of the protective group, releasing active MXF. The lead prodrug exhibited comparable efficacy to MXF in replicating Mtb and significantly enhanced lethality in nonreplicating Mtb. Drug accumulation studies confirmed a modest but significant increase in MXF levels in nonreplicating Mtb treated with the prodrug, suggesting improved permeability. A mathematical model integrating growth and drug-killing kinetics further elucidated how permeability differences impact lethality. Together, these findings highlight enzyme-activated prodrugs as a promising strategy to address phenotypic AMR in Mtb

Lysophosphatidylglycerol (Lyso-PG) is an important membrane lipid that has critical roles in immune regulation and tissue repair. However, precise mechanisms of how lyso-PG does this are yet to be completely characterized due to its limited commercial availability. Here, an efficient stereoselective synthesis of C18:1 lyso-PG is reported, and this new lipid probe should help interrogate cellular and other physiological functions of lyso-PG.

Signaling lipids are important hormone-like biomolecules that regulate many physiological processes in humans. However, a significant number of them still remain poorly characterized, despite their direct association to human diseases. While the signaling pathways regulated by the endocannabinoid 2-arachidonoyl-glycerol in mammals are extensively characterized, the same cannot be said about other members of the monoacylglycerol (MAG) family of signaling lipids. Here, we report the synthesis of a bifunctional MAG probe, containing a photoreactive group and an alkyne handle. Using established chemoproteomics approaches coupled to bioorthogonal chemistry, we profile this bifunctional MAG probe in mouse brain and mammalian cell proteomes, and identify hitherto unknown protein ligands for MAG lipids. Finally, we find that the neuronal calcium sensor Hippocalcin is indeed a putative MAG protein ligand, and show that MAG lipids may have a role to play in calcium sensing and downstream signaling in the mammalian brain.

A tool that generates sulfane sulfur species such as persulfides and polysulfides to promote endogenous antioxidant response is reported.

Mechanisms controlling innate immune responses and coagulation are interdependent, evolutionarily entangled and make a complex network to form immuno-thrombosis axis which is an integral part of host-defence response. During infections, immunothrombosis generates intravascular scaffold enabling recognition, trap and destruction of pathogens facilitating tissue integrity. However, the accompanying dysregulation fosters into pathologies associated with thrombosis and regulates severity, morbidity and mortality in infections. Several extrinsic and intrinsic factors such as (epi)genetic mechanisms, age, metabolism and lifestyle regulate immunothrombosis during infections. Mounting evidence demonstrates that homocysteine, a metabolic intermediate of methionine synthesis pathway activate cells participating in immuno-thrombosis such as neutrophils, platelets, monocytes and endothelial cells. Interestingly, multiple infections are significantly associated with perturbed homocysteine metabolism. In the present review, we describe mechanistic insights into how homocysteine drives immuno-thrombotic crosstalk that generate a vicious cycle of inflammation and coagulation that fuels organ failure during infections with an emphasis on sepsis, COVID-19, and other infectious diseases caused by parasites, viral, and bacterial pathogens. Subsequently, we discuss therapeutic strategies targeting homocysteine metabolism that may improve clinical outcomes in infections.

A new class of mercapto-NSAIDs that generate persulfide, H₂S, and the NSAID is reported.