Biology subjects

Shriwas, P.

Publications and source records attributed to Shriwas, P..

6 recordsLinked to original sources

bioRxiv2026

Human CYP1A2 metabolism of organophosphorus pesticides and nerve agent surrogates

CYP1A2 contributes 13-15% of hepatic CYP450 content and plays a critical role in the metabolism of approximately 10% of pharmaceutical drugs in the human body. CYP1A2 has been previously reported to bioactivate and detoxify both the thion (P=S) and oxon (P=O) forms of organophosphorus (OP) compounds. In this study, we determined the capacity of CYP1A2 to degrade OP compounds using LC-MS/MS, as well as the inhibition of its activity by OP compounds using the previously developed pFluor50 assay. CYP1A2 was selective in metabolizing thions over oxons, and several OP compounds inhibited its activity in a time- and dose-dependent manner. To further investigate the mechanism of CYP1A2-OP interactions, molecular docking studies were performed using a crystal structure of CYP1A2 (PDB ID: 2HI4). Linear correlation analysis between CYP1A2 inhibition at 10 M with 30 minute preincubation and the % OP compound remaining at 2 hours (R = -0.71, P<0.001) showed a moderate but significant correlation. Based on these findings, CYP1A2 was identified as a potential bioscavenger for OP compound metabolism, supporting the future integration of experimental and in silico data for developing artificial intelligence (AI) / machine learning (ML) models for prediction of CYP1A2-mediated metabolism of existing and emerging OP compounds, including those of toxicological and chemical warfare relevance.

pharmacology and toxicology↗

bioRxiv2026

Organophosphorus pesticide and nerve agent surrogate metabolism by human CYP3A4

Of the cytochrome P450 enzymes, CYP3A4 is the most abundant isoform in the human liver, and this enzyme plays a dominant role in the metabolism of a wide range of clinical drugs and xenobiotics. Previous studies have demonstrated that CYP3A4 participates in the oxidative metabolism of several organophosphorus (OP) pesticides involving both thion (P=S) and oxon (P=O) forms. In the present study, we evaluated the capacity of CYP3A4 to metabolize a structurally diverse set of OP compounds using LC-MS/MS methods and assessed their potential to inhibit CYP3A4 activity using previously developed pFlour50 fluorogenic assay. Our results demonstrate that CYP3A4 preferentially metabolizes thions, as compared to oxons, and several OP compounds were also found to inhibit CYP3A4 activity in a time-dependent manner. To gain further mechanistic structural insight into the CYP3A4-OP interactions, molecular docking studies were performed using a crystal structure of CYP3A4 (PDB ID: 3NXU). Linear correlation analysis between in silico parameters like molecular weight or binding energy correlated with experimental data including inhibition data for 10 or 30 minutes or the LC-MS/MS data showing the degradation at 1 or 2 hours showed moderate but significant correlation. Soman surrogate PiMP, and cyclosarin surrogate CMP, were both effectively metabolized by CYP3A4, while docking of these surrogates and authentic agents with CYP3A4 receptor revealed very similar binding poses and interactions. Collectively, these findings highlight the important role of CYP3A4 in OP metabolism and support the potential of integrating experimental and in silico data to predict CYP3A4-mediated metabolism of existing and emerging OP compounds, including those of toxicological and chemical warfare relevance.

pharmacology and toxicology↗

bioRxiv2026

Dual Pathways of Extracellular ATP Action in Cancer Cells: Purinergic Signaling Driven Senescence and Macropinocytic ATP Internalization

BackgroundOpportunistic nutrient uptake is a hallmark of cancer metabolism. Cancer cells upregulate macropinocytosis to acquire extracellular nutrients to support growth and stress adaptation. We previously showed that extracellular ATP (eATP) is internalized by macropinocytosis and promotes multiple cancer phenotypes. Here, we tested whether eATP uptake is prevalent across cancers and whether eATP also induces senescence through purinergic receptor (PR) signaling. MethodsIntracellular ATP (iATP) levels were measured following eATP exposure across multiple cancer cell lines. eATP internalization was visualized in vitro and in vivo using a non-hydrolyzable fluorescent ATP analog together with high-molecular-weight dextran as a macropinocytosis marker. Senescence was quantified using three SA-{beta}-galactosidase assays and flow cytometry. Pharmacologic inhibitors of macropinocytosis and purinergic receptors were used to define pathway dependence. Combination treatments with the glucose transporter inhibitor DRB18 and the senolytic navitoclax were evaluated for antiproliferative effects. ResultseATP produced dose- and time-dependent increases in iATP across diverse cancer cell types. Imaging demonstrated widespread macropinocytic internalization of ATP in vitro and in tumor xenografts. eATP induced senescence in NSCLC cells, confirmed by multiple {beta}-gal assays and flow cytometry. PR inhibition significantly reduced senescence, whereas macropinocytosis inhibition had minimal effect on senescence induction. ConclusionseATP acts through dual pathways in cancer cells: macropinocytic internalization that elevates iATP and PR signaling that drives senescence. Targeting metabolic uptake together with senolytic therapy may offer a novel anticancer strategy.

cancer biology↗

bioRxiv2025

Human CYP2C9 metabolism of organophosphorus pesticides and nerve agent surrogates

Of the Cytochrome P450 enzymes, the CYP2C9 variant is very important and is the cytochrome P450 (CYP) involved in the metabolism of several human drugs where it acts as a natural bioscavenger. Previously, CYP2C9 was demonstrated to remove sulfur from different organophosphorus (OP) pesticides, such as dimethoate, diazinon and parathion. In this study, we have tested the ability of CYP2C9 to degrade other OP compounds. We investigated the metabolism of OP compounds by CYP2C9 using LC-MS/MS approaches as well as the time-dependent inhibition of CYP2C9 by OP compounds using the previously developed pFluor50 fluorogenic assay. We found that CYP2C9 metabolizes thions (P=S) preferentially over oxons (P=O) and there were many OP compounds that inhibited CYP2C9 activity in a time-dependent manner. Additionally, we performed molecular docking based on the Protein Data Bank (PDB)crystal structure (1OG5) of the CYP2C9 receptor in order to gain atomistic insight from the in vitro experimental results. We observed a positive though moderate correlation between the calculated binding energy and the CYP2C9 metabolism of various OP compounds (R = 0.61) as well as the time-dependent changes in inhibition of OPs with binding energy (R = 0.59). Although the R values were modest,59). These in vitro data combined with analysis of additional OP derivatives could be used to develop artificial intelligence (AI)machine learning models to predict the metabolism of specific OP compounds by CYP2C9. This type of approach could be particularly relevant for the prediction of the metabolism of current and emerging chemical warfare agents.

pharmacology and toxicology↗

bioRxiv2024

A novel pan class-I glucose transporter inhibitor DRB18 exhibits synergistic effects in vitro and in vivo with paclitaxel against human non-small cell lung cancer

PurposeCancer cells depend on glucose for biomass synthesis, cell proliferation, and drug resistance. Glucose transporter (GLUT) transcripts as well as proteins are upregulated in human lungs and other cancers and are negatively correlated with patient survival, particularly GLUT1 and GLUT3. Thus, inhibiting GLUT function has been an attractive anticancer strategy. We previously characterized WZB117 and DRB18, first- and second-generation pan-class I GLUT inhibitors, respectively. DRB18 strongly inhibits glucose transport mediated by GLUT1-4 in non-small lung cancer (NSCLC) A549 cells in vitro and in vivo. Here, we report DRB18 as a more stable and potent anticancer compound, compared to WZB117. MethodsImmunohistochemistry analysis was performed in Lung adenocarcinoma (LUAD) tissue array to investigate GLUT1 and GLUT3 protein expression between normal, lower and higher stage LUAD patients. Bioinformatics analysis was performed to examine additive effect of GLUT3 to GLUT1 mediated prognosis in LUAD. Glucose uptake and resazurin dye-based proliferation assays were used to determine glucose uptake inhibitory and cell proliferation inhibitory against panel of human cancer cell lines A549, Panc1 and Hela. DRB18 potency was tested against the presence of extracellular nutrients glucose, glutamine and ATP. Synergism between DRB18 and clinically approved anticancer drugs was tested against cancer cells. DRB18 and advanced NSCLC drug Paclitaxel were tested for synergy in vitro and in vivo. ResultsGLUT1/3 combination exhibited higher hazard ratio than either GLUT1 and GLUT3 alone in many cancer types including LUAD. DRB18 reduced glucose uptake in NSCLC A549, pancreatic Panc1, and cervical Hela cancer cells with varied but strong anticancer potencies in the presence or absence of extracellular nutrients such as ATP and glucose. Combined with different clinical and pre-clinical anticancer compounds such as V9302, CB839, Sutent, Brigatinib, DRB18 significantly increased death of A549 and Panc1 cells. Noteworthy, DRB18 exhibited strong anticancer synergy with paclitaxel, an approved chemo drug for NSCLC, drastically reducing cancer cell proliferation in vitro and growth of A549 tumors grafted on the flank of nude mice without significant side effects, compared to single drug treatments. ConclusionsCollectively, our results demonstrate anticancer efficacy of pan class-I GLUT inhibitor DRB18 in combination with paclitaxel, providing a potentially more efficacious therapeutic strategy for treating advanced NSCLC and other cancers.

cancer biology↗

bioRxiv2023

Development and characterization of pFluor50, a fluorogenic-based kinetic assay system for high-throughput inhibition screening and characterization of time-dependent inhibition and inhibition type for six human CYPs

1Cytochrome P450 enzymes (CYPs) play an integral role in drug and xenobiotic metabolism in humans and thus understanding CYP inhibition or activation by new therapeutic candidates is an important step in the drug development process. Ideally, CYP inhibition/activation assays should be high-throughput, use commercially available components, allow for analysis of metabolism by the majority of human CYPs, and allow for kinetic analysis of inhibition type and time-dependent inhibition. Here, we developed pFluor50, a 384-well microtiter plate-based fluorogenic kinetic enzyme assay system using substrates metabolized by six human CYPs to generate fluorescent products and determined the Michaelis-Menten kinetics constant (KM) and product formation rate (Vmax) for each substrate-CYP pair. The substrate-CYP pairs were as follows: resorufin ethyl ether for CYP1A2 (KM= 0.8 M), CYP2C9 (KM= 0.6 M), and CYP2D6 (KM= 2.7 M); resorufin benzyl ether for CYP2B6 (KM= 46 M); 3-O-methyl fluorescein for CYP2C19 (KM= 3.0 M); and dibenzyl fluorescein for CYP3A4 (KM= 2.9 M). We then validated each assay using known inhibitors: -naphthoflavone for CYP1A2 (IC50= 13.5 nM); sertraline for CYP2B6 (IC50= 410 nM) and CYP2D6 (IC50= 2.4 M); sulfaphenazole for CYP2C9 (IC50= 1 M); ticlopidine for CYP2C19 (IC50= 1.2 M); and CYP3cide for CYP3A4 (IC50= 56 nM). pFluor50 was also used to elucidate inhibition type and time-dependent inhibition for some inhibitors demonstrating its utility for characterizing the observed inhibition, even mechanism-based inhibition. The pFluor50 assay system developed in this study using commercially available components should be very useful for high-throughput screening and further characterization of potential therapeutic candidates for inhibition/activation with the most prevalent human CYPs.

pharmacology and toxicology↗