Goutam Chowdhury

The COVID‐19 pandemic caused by SARS‐CoV‐2 created a global health crisis, and the virus still circulates, mutates, and causes illness and death. Vaccination remains the primary defense, but effectiveness can be reduced in immunocompromised individuals and against new variants, and clinically approved antivirals are limited by side effects, drug interactions, and resistance. Herein, we propose a novel application of tanespimycin (17AAG), the chaperone heat shock protein (HSP)90 inhibitor, as an antiviral against the SARS‐CoV‐2 main protease (M ^pro ). M ^pro , due to its indispensable role in viral replication and absence of expression in the host, represents a key target for drug development. Through biochemical and cell‐based assays, structural analysis, and resistance‐mutant testing, we present data showing that 17AAG covalently binds to the active‐site cysteine of M ^pro , disrupts its secondary structure, and protects cells from M ^pro ‐induced toxicity more effectively than nirmatrelvir. We further showed that 17AAG retains its covalent binding and structure‐disrupting activity against the nirmatrelvir‐resistance M165I variant. Additionally, since HSP90 is important for viral protein stability, virion assembly, and modulation of host immune response, 17AAG is a promising, versatile drug candidate that could accelerate antiviral development for COVID‐19.

Atmospheric organosulfur compounds play critical roles in marine chemistry and climate processes. Methane sulfonamide (MSAM), a recently detected organosulfur species in the Arabian Sea, is estimated to have a a short atmospheric lifetime (∼2 days) that is inconsistent with laboratory-measured OH radical reaction rates, suggesting the existence of alternative removal pathways. The identification of all major sinks is crucial for accurately predicting atmospheric lifetimes, sulfur cycling, and related climate effects. Herein, we identified the reaction with Criegee intermediates (CIs), formed from alkene ozonolysis, as a previously unrecognized major sink for MSAM. Using Density Functional Theory and master-equation kinetic modeling studies, we show that CH₂OO and anti-CH₃CHOO react with MSAM at rates ∼10-fold higher than those of the hydroxyl radicals, corresponding to atmospheric lifetimes of ∼3.5 and ∼6.8 days at 298 K and 1 atm pressure. The reaction of MSAM with larger CIs, including anti-methacrolein oxide, is significantly dependent on the structure and temperature. Our estimates show that the lifetime of MSAM with respect to the reactions of anti-methacrolein oxide reduces from an upper bound of ∼19 days at 298 K to ∼1 day at 200 K, implying that the lifetime decreases under colder tropospheric conditions. The lifetimes of MSAM with respect to the reactions with most CIs considered here surpass those of OH reactions (∼80 days). CI-mediated reactions, thus, emerge as a key process controlling MSAM fate, with implications for marine aerosol composition and climate-relevant processes.

Lapatinib and neratinib are tyrosine kinase inhibitors (TKIs) approved for treating HER2-positive metastatic breast cancer, but their clinical use is limited by hepatotoxicity. Within hepatocytes, cytochrome P450 enzymes produce reactive metabolites of these drugs, causing DNA damage, mitochondrial dysfunction, and oxidative stress, which lead to hepatocyte injury. This cellular stress triggers hepatocyte senescence, a state of growth arrest accompanied by the secretion of pro-inflammatory and tissue-remodeling factors called the senescence-associated secretory phenotype (SASP). SASP significantly modifies the liver microenvironment, influences immune cell behavior, and worsens tissue damage. We show that lapatinib and neratinib induce senescence and SASP in the HepG2 liver cell line. Additionally, SASP from these senescent cells promotes M2-like polarization of RAW264.7 macrophages. Although M2 macrophages are associated with anti-inflammatory responses and tissue repair, their excessive or prolonged activity can lead to tissue damage and fibrosis. Our findings highlight a mechanism by which TKI-induced hepatocyte senescence and SASP modulate the immune response and may contribute to liver toxicity in breast cancer patients. This underscores the importance of targeting senescent cells or SASP factors to reduce liver toxicity and improve treatment outcomes, making the identification of effective senotherapeutics a vital research focus. There hasn't been any literature report demonstrating the effect of TKI-induced liver cell secretome on macrophage polarization.

Lapatinib and neratinib are tyrosine kinase inhibitors (TKIs) approved for treating HER2-positive metastatic breast cancer, but their clinical use is limited by hepatotoxicity. Within hepatocytes, cytochrome P450 enzymes produce reactive metabolites of these drugs, causing DNA damage, mitochondrial dysfunction, and oxidative stress, which lead to hepatocyte injury. This cellular stress triggers hepatocyte senescence, a state of growth arrest accompanied by the secretion of pro-inflammatory and tissue-remodeling factors called the senescence-associated secretory phenotype (SASP). SASP significantly modifies the liver microenvironment, influences immune cell behavior, and worsens tissue damage. We show that lapatinib and neratinib induce senescence and SASP in the HepG2 liver cell line. Additionally, SASP from these senescent cells promotes M2-like polarization of RAW264.7 macrophages. Although M2 macrophages are associated with anti-inflammatory responses and tissue repair, their excessive or prolonged activity can lead to tissue damage and fibrosis. Our findings highlight a mechanism by which TKI-induced hepatocyte senescence and SASP modulate the immune response and may contribute to liver toxicity in breast cancer patients. This underscores the importance of targeting senescent cells or SASP factors to reduce liver toxicity and improve treatment outcomes, making the identification of effective senotherapeutics a vital research focus. There hasn't been any literature report demonstrating the effect of TKI-induced liver cell secretome on macrophage polarization.

Therapy-induced senescence (TIS) is a reversible growth arrest induced by anticancer treatments, which may contribute to the development of long-term therapy resistance in tumor cells. Senotherapeutics, agents targeting senescent cells, are being tested in clinical trials to improve patient outcomes. Due to the transient nature of TIS, we hypothesized that senolytics would be most effective when administered at the appropriate time. We created a reliable TIS cell line model in triple-negative breast cancer (TNBC) using experimental drug YM155. We observed that a single dose of YM155 triggers a brief senescence, leading to a persistent drug-tolerant state that cannot be reversed by redosing. This reversibility is not limited to cancer cells. It extends to noncancerous human cells and live zebrafish larvae, suggesting a rapid adaptation mechanism against xenobiotics. We identified transforming growth factor-β (TGF-β), a cytokine linked to TNBC chemoresistance, as being expressed alongside the emergence of drug tolerance. We inhibited TGF-β signaling to eliminate the tolerant phenotype and promote the clearance of cancer cells by immune cells. However, this was most effective within a specific time window after TIS induction. We suggest that the timely use of senotherapeutics could improve the effectiveness of anticancer drugs in clinical settings.

Inflammation is an early immune response against invading pathogens and damaged tissue. Although beneficial, uncontrolled inflammation leads to various diseases including cancer in a chronic setting. Peroxynitrite (PN) is a major reactive nitrogen species generated during inflammation. It produces various DNA lesions including 8-nitro-guanine which spontaneously converts into abasic sites, resulting in DNA strand breakage, and is suspected to be mutagenic. Here, we report the discovery of a previously unrecognized function of the human repair protein O⁶-alkylguanine-DNA alkyltransferase (hAGT or MGMT). We showed that hAGT through its active site nucleophilic Cys145 thiolate spontaneously reacts with 8-nitro-guanine in DNA to form a stable DNA-protein cross-link (DPC). Interestingly, the process of DPC formation provided protection from PN-mediated genome instability, growth arrest, and apoptosis. The Cys145 mutant of hAGT failed to form a DPC and was unable to protect cells from inflammation-associated PN-mediated cytotoxicity. Gel-shift, dot blot, and UV-vis assays showed formation of a covalent linkage between PN-damaged DNA and hAGT through its active site Cys145. Finally, expression of hAGT was found to be significantly increased by induced macrophages and PN. The data presented here clearly demonstrated hAGT as a dual-function protein that along with DNA repair is capable of maintaining genomic integrity and providing protection from the toxicity caused by PN-mediated DNA damage. Although DPCs are known to be detrimental to the cell, recently, multiple pathways have been identified in normal cells for their repair.

Survivin, a cancer-cell-specific multifunctional protein, is regulated by many oncogenic signaling pathways and an effective therapeutic target. Although, several types of survivin-targeting agents have been developed over the past few decades, none of them received clinical approval. This could be because survivin expression is tightly controlled by the feedback interaction between different signaling molecules. Of the several signaling pathways that are known to regulate survivin expression, the phosphatidylinositol 3-kinase/AKT serine-threonine kinase/forkhead boxO (PI3K/AKT/FoxO) pathway is well-known for feedback loops constructed by cross-talk among different molecules. Using sepantronium bromide (YM155), the first of its class of survivin-suppressant, we uncovered the existence of an interesting cross-talk between Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) and FoxO transcription factors that also contributes to YM155 resistance in triple negative breast cancer (TNBC) cells. Pharmacological manipulation to interrupt this interaction not only helped restore/enhance the drug-sensitivity but also prompted effective immune clearance of cancer cells. Because the YM155-induced reactive oxygen species (ROS) initiates this feedback, we believe that it will be occurring for many ROS-producing chemotherapeutic agents. Our work provides a rational explanation for the poor efficacy of YM155 compared to standard chemotherapy in clinical trials. Finally, the triple drug combination approach used herein might help reintroducing YM155 into the clinical pipeline, and given the high survivin expression in TNBC cells in general, it could be effective in treating this subtype of breast cancer.

Inflammation is an early immune response against invading pathogens and damaged tissue. Although beneficial, uncontrolled inflammation leads to various diseases and may be fatal. Peroxynitrite (PN) is a major reactive nitrogen species (RNS) generated during inflammation. It produces various DNA lesions including labile 8-nitroguanine which spontaneously converts into abasic sites resulting in DNA strand breakage. Here, we report the discovery of a previously unrecognized function of the human repair protein O ⁶ -alkylguanine-DNA alkyltransferase (hAGT or MGMT). We showed that hAGT through its active site nucleophilic Cys145 thiolate can spontaneously react with 8-nitroguanine in DNA to form a stable DNA-protein crosslink (DPC). Interestingly, the process of DPC formation provides protection from PN-mediated genome instability. The Cys145-mutant of hAGT failed to form DPC and provide protection against inflammation-associated, PN-mediated cytotoxicity. Gel shift, dot blot and UV-Vis assays showed formation of a covalent linkage between PN-damaged DNA and hAGT through its active site Cys145. Finally, expression of hAGT was found to be significantly increased by induced macrophages and PN. The data presented here clearly demonstrated hAGT as a dual function protein that along with DNA repair is capable of maintaining genomic integrity and providing protection from the toxicity caused by PN-mediated DNA damage. Although DPCs may seem detrimental, there are multiple systems in place in normal cells for their repair.

The oxidation-reduction reactions of disulfides are important in both chemistry and biology. Dimethyl disulfide (DMDS), the smallest reduced sulfur species with a disulfide bond, is emitted in significant quantities from natural sources and contributes to the formation of aerosols and hazardous haze. Although atmospheric removal of DMDS via the reactions with OH or NO3 radicals and photolysis is known, the reactions of DMDS with other atmospheric oxidants are yet to be explored. Herein, using quantum chemical calculations, we explored the reactions of DMDS with CH2OO (formaldehyde oxide) and other methyl-substituted Criegee intermediates. The various reaction pathways evaluated were found to have positive energy barriers. However, in the presence of formic acid, a direct oxygen-transfer pathway leading to the corresponding sulfoxide (CH3SS(O)CH3) was found to proceed through a submerged transition state below the separated reactants. Calculations for the methyl-substituted Criegee intermediates, particularly for anti-CH3CHOO, show a significant increase in the rate of the direct oxygen-transfer reaction when catalyzed by formic acid. The presence of formic acid also alters the mechanism and reduces the enthalpic barrier of a second pathway, forming thioformaldehyde and hydroperoxide without any rate enhancement. Our data indicated that, although Criegee intermediates are unlikely to be an important atmospheric sink of DMDS under normal conditions, in regions rich in DMDS and formic acid, the formic acid-catalyzed Criegee intermediate-mediated oxidation of DMDS via the direct oxygen-transfer pathway could lead to organic sulfur compounds contributing to atmospheric aerosol.

The current COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) created a global health crisis. The ability of vaccines to protect immunocompromised individuals and from emerging new strains are major concerns. Hence antiviral drugs against SARS-CoV-2 are essential. The SARS-CoV-2 main protease M^pro is vital for replication and an important target for antivirals. Using CMap analysis and docking studies, withaferin A (wifA) and withanone (win), two natural products from the medicinal herb Withania somnifera (ashwagandha), were identified as promising candidates that can covalently inhibit the viral protease M^pro. Cell culture, enzymatic, LC-MS/MS, computational, and equilibrium dialysis based assays were performed. DFT calculations indicated that wifA and win can form stable adducts with thiols. The cytotoxicity of M^pro was significantly reduced by wifA and win. Both wifA and win were found to irreversibly inhibit 0.5 μM M^pro with IC₅₀ values of 0.54 and 1.8 μM, respectively. LC-MS/MS analysis revealed covalent adduct formation with wifA at cysteines 145 and 300 of M^pro. The natural products wifA and win can irreversibly inhibit the SARS-CoV-2 main protease M^pro. Based on the work presented here we propose that both wifA and win have the potential to be safely used as preventative and therapeutic interventions for COVID-19.