Project I: Determination of DNA damage and DNA-protein crosslinks resulting from acute inflammation due to SARS-CoV-2 and its long-term consequences
(For references see the paper below and references therein)
Inflammation is a defensive immune response against infectious agents including bacteria, viruses and parasites. If unchecked, acute inflammation can turn against us resulting in life-threatening conditions. This is being observed with the critically ill SARS-CoV-2-infected patients in the current coronavirus pandemic. For severe cases, acute inflammation can lead to genome instability. Peroxynitrite (PN) is one of the major oxidants produced by the infiltrating immune cells responsible for inflammation-associated pathogenesis through macromolecular (protein and DNA) damages. 8-nitroguanine (8-nitro-G), one of the primary DNA lesions produced from PN, is recognized as a mutagenic inflammatory biomarker routinely found in inflammation-associated cancers. However, 8-nitro-G being a labile lesion results in abasic site formation that is efficiently repaired by endogenous enzyme. We have recently demonstrated that thiols including glutathione (GSH) can react with 8-nitro-G in DNA to form a stable GSH-DNA adduct. This process provides an immediate protection against the detrimental effect of PN on DNA. But, DNA-peptide (as in GSH) and DNA-protein adducts in general are extremely toxic for cells due to their bulky nature and tendency to hinder information transactions from DNA. If unrepaired, the most noticeable long-term consequence of such adducts is induction of somatic mutations and cancer. We recently reported the DNA-GSH adduct formed between GSH and 8-nitro-G is in fact mutagenic in bacteria. Taken together, we hypothesize that generation of toxic DNA-peptide and DNA-protein cross-link adducts at the sites of acute inflammation like that in severe covid-19 cases, have long-term consequences on genome stability and subsequent cancer predisposition.
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Ahmed N, Chakrabarty A, Guengerich FP and Chowdhury G*. Protective Role of Glutathione Against Peroxynitrite-Mediated DNA Damage During Acute Inflammation. Chem. Res. Toxicol. 2020,
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Project 2: Mechanism of DNA damage by the antitumor agent sepantronium bromide (YM155)
Sepantronium bromide (YM155) is a small molecule drug developed by Astellas Pharma that selectively inhibits the expression of survivin, presumably by suppressing its transcription. YM155 is currently in phase II clinical trials for the treatment of various cancers, including triple negative breast cancer (Int. J. Oncol. 2011, 39, 569−575). In addition to survivin suppression, there are contradictory reports that it causes DNA damage. Using low nanomolar levels of YM155, we found that it causes significant cytotoxicity and DNA damage, particularly in triple negative breast cancer cells. These results provide strong evidence that DNA damage does play a crucial role in the pharmacology and antitumor property of YM155. The efficiency of DNA damage by YM155 is significantly better than the prototypical quinone, menadione. Interestingly, the mechanism of DNA damage by YM155 was found to be distinct from that of typical quinones. We have shown that DNA damage by YM155 is reductively activated, oxygen independent and oxidative in nature, and presence of iron is absolutely necessary. The species responsible for DNA damage is not known and extensive DNA cleavage by YM155 in absence of oxygen is surprising. Thus, a detailed understanding of the mechanism of DNA damage by YM155, particularly the role of iron and the non-requirement of oxygen, is necessary.
We plan to extend our studies to other agents including lapatinib, thalidomide and pomalidomide. (all of which have alternative mode of action other than DNA damage).
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TH Wani, S Surendran, VS Mishra, J Chaturvedi, G Chowdhury, A Chakrabarty (2018) Adaptation to chronic exposure to sepantronium bromide (YM155), a prototypical survivin suppressant is due to persistent DNA damage-response in breast cancer cells. Oncotarget 9 (71), 33589.
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Wani, T. H.; Suendran, S.; Jana, A.; Chakrabarty, A. and Chowdhury, G*. (2018) The Quinone Based Antitumor Agent Sepantronium Bromide (YM155) Causes Oxygen Independent Redox Activated Oxidative DNA Damage. Chem Res Toxicol. 31(7), 612-618.
Project 3: Identification and characterization of covalent inhibitors of SARS-CoV-2 main protease (Mpro)
The current Covid-19 pandemic caused by SARS-CoV-2 has created an unprecedented global health crisis. Although multiple vaccines have already reached the clinics, antiviral drugs are needed for immunocompromised individuals and emerging new strains. The Pfizer Main protease inhibitor, nirmatrelvir, is currently in the clinics. It is marketed as Paxlovid in combination with the 3A4 inhibitor ritonavir which significantly limits the use of other drugs during its administration. There is an urgent need for novel and potent antivirals. The SARS-CoV-2 genome contains two proteases, a papain-like cysteine (Cys) protease (PLPro), and a chymotrypsin-like Cys protease (3CLPro, also known as Main Protease, Mpro) that performs the proteolytic cleavage of the viral polypeptides. The Main protease does not have a human homolog and therefore is a lucrative target. Although a large number of Main protease inhibitors are reported, the development of new synthetic drugs will take years to reach the clinics (Science 2021, 371, 1374–1378; Science 2021, 373, 931–936; Science. 2020 Apr 22 : eabb4489). Hence drug repurposing and herbal products that are already used for human consumption seemed as potential sources.
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We have embarked on the quest of identifying novel Mpro inhibitors. We recently identified and characterized two covalent inhibitors of Mpro from the medicinal herb Withania somnifera (Ashwagandha). The two natural products withanone (win) and withaferin A (wifA) can covalently bind with the active site Cys-145 and the dimerization site Cys-300. Using a cell-based assay we demonstrated that both wifA and win can mitigate the cytotoxic effect of Mpro.
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Chakraborty S, Mallick D, Goswami M, Chakrabarty A, Guengerich FP and Chowdhury G*. The Natural Products Withaferin A and Withanone From the Medicinal Herb Withania somnifera are Covalent Inhibitors of the SARS-CoV-2 Main Protease. J. Nat. Prod. 2022, 85, 2340-2350. Cover Page Article
