Bioorg. Med. Chem. Lett. 76 (2022) 129008 Available online 27 September 2022 0960-894X/© 2022 Elsevier Ltd. All rights reserved. Identifcation of a new and diverse set of Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) inhibitors using structure-based virtual screening: Experimental validation and molecular dynamics studies Prateek Raj a , Karthik Selvam a , Koyel Roy b , Shailesh Mani Tripathi c , Sharyu Kesharwani c , Balasubramanian Gopal a , Umesh Varshney b, d , Sandeep Sundriyal c, * a Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India b Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India c Department of Pharmacy, Birla Institute of Technology and Science Pilani, Pilani Campus, Rajasthan 333031, India d Jawaharlal Nehru Centre for Advanced Scientifc Research, Jakkur, Bangalore 560064, India A R T I C L E INFO Keywords: Mycobacterium Uracil-DNA glycosylase Molecular docking Virtual screening Molecular dynamics ABSTRACT Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng), a key DNA repair enzyme, represents an attractive target for the design of new antimycobacterial agents. However, only a limited number of weak MtUng inhibitors are reported, primarily based on the uracil ring, and hence, lack diversity. We report the frst structure-based virtual screening (SBVS) using three separate libraries consisting of uracil and non-uracil small molecules, together with the FDA-approved drugs. Twenty diverse virtual hits with the highest predicted binding were procured and screened using a fuorescence-based assay to evaluate their potential to inhibit MtUng. Several of these molecules were found to inhibit MtUng activity at low mM and µM levels, comparable to or better than several other reported Ung inhibitors. Thus, these molecules represent a diverse set of scaffolds for developing next-generation MtUng inhibitors. The most active uracil-based compound 5 (IC 50 = 0.14 mM) was found to be ~ 15-fold more potent than the positive control, uracil. The binding stability and conformation of compound 5 in complex with the enzyme were further confrmed using molecular dynamics simulation. Tuberculosis (TB) is a contagious airborne infection and is one of the leading causes of mortality worldwide. According to World Health Or- ganization (WHO) report, the number of TB deaths has increased glob- ally, from 1.2 million (in 2019) to 1.3 million in 2020. This increased mortality rate has been attributed to the COVID-19 pandemic that dis- rupted the TB diagnostic and treatment services to the patients. 1 Although drugs are available for treating TB, there is a need for improvement because of the limited effectiveness of currently employed therapeutics. The major problems associated with current TB manage- ment are drug resistance, long duration of treatment, and severe side effects. 2 The poor adherence to the treatment course often leads to the emergence of much more diffcult-to-treat multidrug-resistant TB (MDR- TB) strains. The causative agent of TB, Mycobacterium tuberculosis (Mtb), is an intracellular pathogen that infects and multiplies within the host macrophages. To survive within the host, Mtb must maintain its genomic integrity, which is continuously threatened by numerous endogenous and environmental factors. Not surprisingly, Mtb has multiple DNA- repair pathways. 3–5 The enzymes in these pathways recognize and rectify DNA damage and maintain genomic integrity. One such pathway is the base-excision repair pathway (BER) which recognizes and repairs non-bulky single base lesions in DNA. 5,6 . Uracil-DNA glycosylase (UNG/Ung) is an important class of DNA repair enzymes that recognizes and catalyzes uracil excision from single- stranded and double-stranded DNA substrates and initiates the BER pathway. 7,8 Molecular genetics studies have shown the importance of Ung in Mycobacteria. Indeed, mutation rates were substantially elevated in the absence of this enzyme. 9,10 Another study displayed the impor- tance of M. tuberculosis Ung (MtUng) in the survival of bacteria inside the host cell. 11 Therefore, MtUng inhibitors may help treat TB alone or in combination with other antitubercular drugs. MtUng has been well-characterized biochemically and structur- ally. 12,13 The crystal structures of the native enzyme in various forms and in complex with different small molecules, namely, citrate, uracil, and uracil derivatives, are known. These structures comprehensively * Corresponding author. E-mail address: sandeep.sundriyal@pilani.bits-pilani.ac.in (S. Sundriyal). Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl https://doi.org/10.1016/j.bmcl.2022.129008 Received 28 July 2022; Received in revised form 18 September 2022; Accepted 23 September 2022