Insights into the DNA and RNA Interactions of Human Topoisomerase III Beta Using Molecular Dynamics Simulations.

Human topoisomerase III beta (hTOP3B) is the only topoisomerase in the human cell that can act on both DNA and RNA substrates. Recent findings have emphasized the physiological importance of hTOP3B and consolidated it as a valuable drug target for antiviral and anticancer therapeutics. Although type IA topoisomerases of different organisms have been studied over the years, the step-by-step interaction of hTOP3B and nucleic acid substrates is still not well understood. Due to the lack of hTOP3B-RNA structures as well as DNA/RNA covalent complexes, computational investigations have been limited. In our study, we utilized molecular dynamics (MD) simulations to study the interactions between hTOP3B and nucleic acids to get a closer look into the residues that play a role in binding DNA or RNA and facilitate catalysis, along with the differences and similarities when hTOP3B interacts with DNA compared to RNA. For this, we generated multiple models of hTOP3B complexed with DNA and RNA sequences using the hTOP3B crystal structure and 8-mer single-stranded DNA and RNA sequences. These models include both covalent and noncovalent complexes, which are then subjected to MD simulations and analyzed. Our findings highlight the complexes' stability, sequence preference, and interactions of the binding pocket residues with different nucleotides. Our work demonstrates that hTOP3B forms stable complexes with both DNA and RNA and provides a better understanding of the enzyme's interaction with different nucleic acid substrate sequences.

The natural history and genotype-phenotype correlations of TMPRSS3 hearing loss: an international, multi-center, cohort analysis.

TMPRSS3-related hearing loss presents challenges in correlating genotypic variants with clinical phenotypes due to the small sample sizes of previous studies. We conducted a cross-sectional genomics study coupled with retrospective clinical phenotype analysis on 127 individuals. These individuals were from 16 academic medical centers across 6 countries. Key findings revealed 47 unique TMPRSS3 variants with significant differences in hearing thresholds between those with missense variants versus those with loss-of-function genotypes. The hearing loss progression rate for the DFNB8 subtype was 0.3 dB/year. Post-cochlear implantation, an average word recognition score of 76% was observed. Of the 51 individuals with two missense variants, 10 had DFNB10 with profound hearing loss. These 10 all had at least one of 4 TMPRSS3 variants predicted by computational modeling to be damaging to TMPRSS3 structure and function. To our knowledge, this is the largest study of TMPRSS3 genotype-phenotype correlations. We find significant differences in hearing thresholds, hearing loss progression, and age of presentation, by TMPRSS3 genotype and protein domain affected. Most individuals with TMPRSS3 variants perform well on speech recognition tests after cochlear implant, however increased age at implant is associated with worse outcomes. These findings provide insight for genetic counseling and the on-going design of novel therapeutic approaches.

Minor electrostatic changes robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

miRNA-211 maintains metabolic homeostasis in medulloblastoma through its target gene long-chain acyl-CoA synthetase 4.

The prognosis of childhood medulloblastoma (MB) is often poor, and it usually requires aggressive therapy that adversely affects quality of life. microRNA-211 (miR-211) was previously identified as an important regulator of cells that descend from neural cells. Since medulloblastomas primarily affect cells with similar ontogeny, we investigated the role and mechanism of miR-211 in MB. Here we showed that miR-211 expression was highly downregulated in cell lines, PDXs, and clinical samples of different MB subgroups (SHH, Group 3, and Group 4) compared to normal cerebellum. miR-211 gene was ectopically expressed in transgenic cells from MB subgroups, and they were subjected to molecular and phenotypic investigations. Monoclonal cells stably expressing miR-211 were injected into the mouse cerebellum. miR-211 forced expression acts as a tumor suppressor in MB both in vitro and in vivo, attenuating growth, promoting apoptosis, and inhibiting invasion. In support of emerging regulatory roles of metabolism in various forms of cancer, we identified the acyl-CoA synthetase long-chain family member (ACSL4) as a direct miR-211 target. Furthermore, lipid nanoparticle-coated, dendrimer-coated, and cerium oxide-coated miR-211 nanoparticles were applied to deliver synthetic miR-211 into MB cell lines and cellular responses were assayed. Synthesizing nanoparticle-miR-211 conjugates can suppress MB cell viability and invasion in vitro. Our findings reveal miR-211 as a tumor suppressor and a potential therapeutic agent in MB. This proof-of-concept paves the way for further pre-clinical and clinical development.

Macromolecular crowding potently stimulates DNA supercoiling activity of Mycobacterium tuberculosis DNA gyrase.

Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic reactions. Studying these reactions in vitro, using agents such as polyetthylene glycols (PEGs) and polyvinyl alcohols (PVAs) to mimic intracellular crowding conditions, is essential due to the notable differences from enzyme behaviors observed in diluted aqueous solutions. In this article, we studied Mycobacterium tuberculosis (Mtb) DNA gyrase under macromolecular crowding conditions by incorporating PEGs and PVAs into the DNA supercoiling reactions. We discovered that high concentrations of potassium glutamate, glycine betaine, PEGs, and PVA substantially stimulated the DNA supercoiling activity of Mtb DNA gyrase. Steady-state kinetic studies showed that glycine betaine and PEG400 significantly reduced the KM of Mtb DNA gyrase and simultaneously increased the Vmax or kcat of Mtb DNA gyrase for ATP and the plasmid DNA molecule. Molecular dynamics simulation studies demonstrated that PEG molecules kept the ATP lid of DNA gyrase subunit B in a closed or semiclosed conformation, which prevented ATP molecules from leaving the ATP-binding pocket of DNA gyrase subunit B. The stimulation of the DNA supercoiling activity of Mtb DNA gyrase by these molecular crowding agents likely results from a decrease in water activity and an increase in excluded volume.

Multi-functional auto-fluorescent nanogels for theranostics.

Here in the present article, the state of art for nanotechnology-enabled nanogel theranostics and the upcoming concepts in nanogel-based therapeutics are summarized. The benefits, innovation, and prospects of nanogel technology are also briefly presented.

The oncogenic circular RNA circ_63706 is a potential therapeutic target in sonic hedgehog-subtype childhood medulloblastomas.

Medulloblastoma (MB) develops through various genetic, epigenetic, and non-coding (nc) RNA-related mechanisms, but the roles played by ncRNAs, particularly circular RNAs (circRNAs), remain poorly defined. CircRNAs are increasingly recognized as stable non-coding RNA therapeutic targets in many cancers, but little is known about their function in MBs. To determine medulloblastoma subgroup-specific circRNAs, publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify circRNAs that differentiate between MB subgroups. circ_63706 was identified as sonic hedgehog (SHH) group-specific, with its expression confirmed by RNA-FISH analysis in clinical tissue samples. The oncogenic function of circ_63706 was characterized in vitro and in vivo. Further, circ_63706-depleted cells were subjected to RNA-seq and lipid profiling to identify its molecular function. Finally, we mapped the circ_63706 secondary structure using an advanced random forest classification model and modeled a 3D structure to identify its interacting miRNA partner molecules. Circ_63706 regulates independently of the host coding gene pericentrin (PCNT), and its expression is specific to the SHH subgroup. circ_63706-deleted cells implanted into mice produced smaller tumors, and mice lived longer than parental cell implants. At the molecular level, circ_63706-deleted cells elevated total ceramide and oxidized lipids and reduced total triglyceride. Our study implicates a novel oncogenic circular RNA in the SHH medulloblastoma subgroup and establishes its molecular function and potential as a future therapeutic target.

Potent Inhibition of Bacterial DNA Gyrase by Digallic Acid and Other Gallate Derivatives.

Bacterial DNA gyrase, an essential enzyme, is a validated target for discovering and developing new antibiotics. Here we screened a pool of polyphenols and discovered that digallic acid is a potent DNA gyrase inhibitor. We also found that several food additives based on gallate, such as dodecyl gallate, potently inhibit bacterial DNA gyrase. Interestingly, the IC50 of these gallate derivatives against DNA gyrase is correlated with the length of hydrocarbon chain connecting to the gallate. These new bacterial DNA gyrase inhibitors are ATP competitive inhibitors of DNA gyrase. Our results also show that digallic acid and certain gallate derivatives potently inhibit E. coli DNA topoisomerase IV. Several gallate derivatives have strong antimicrobial activities against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). This study provides a solid foundation for the design and synthesis of gallate-based DNA gyrase inhibitors that may be used to combat antibacterial resistance.

Potential Autoimmunity Resulting from Molecular Mimicry between SARS-CoV-2 Spike and Human Proteins.

Molecular mimicry between viral antigens and host proteins can produce cross-reacting antibodies leading to autoimmunity. The coronavirus SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. Thus, we computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative high autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins, such as PRKG1 involved in platelet activation and calcium regulation, and tropomyosin, which is linked to cardiac disease. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions.

AT-hook peptides bind the major and minor groove of AT-rich DNA duplexes.

The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as 'AT-hooks', are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three 'AT-hook' peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, 'key-locked' conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.

Cysteine Mutations in the Ebolavirus Matrix Protein VP40 Promote Phosphatidylserine Binding by Increasing the Flexibility of a Lipid-Binding Loop.

Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys311 has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys311 and Cys314 residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS.

Structural insights into the repair mechanism of AGT for methyl-induced DNA damage.

Methylation induced DNA base-pairing damage is one of the major causes of cancer. O6-alkylguanine-DNA alkyltransferase (AGT) is considered a demethylation agent of the methylated DNA. Structural investigations with thermodynamic properties of the AGT-DNA complex are still lacking. In this report, we modeled two catalytic states of AGT-DNA interactions and an AGT-DNA covalent complex and explored structural features using molecular dynamics (MD) simulations. We utilized the umbrella sampling method to investigate the changes in the free energy of the interactions in two different AGT-DNA catalytic states, one with methylated GUA in DNA and the other with methylated CYS145 in AGT. These non-covalent complexes represent the pre- and post-repair complexes. Therefore, our study encompasses the process of recognition, complex formation, and separation of the AGT and the damaged (methylated) DNA base. We believe that the use of parameters for the amino acid and nucleotide modifications and for the protein-DNA covalent bond will allow investigations of the DNA repair mechanism as well as the exploration of cancer therapeutics targeting the AGT-DNA complexes at various functional states as well as explorations via stabilization of the complex.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.

Covalent Complex of DNA and Bacterial Topoisomerase: Implications in Antibacterial Drug Development.

A topoisomerase-DNA transient covalent complex can be a druggable target for novel topoisomerase poison inhibitors that represent a new class of antibacterial or anticancer drugs. Herein, we have investigated molecular features of the functionally important Escherichia coli topoisomerase I (EctopoI)-DNA covalent complex (EctopoIcc) for molecular simulations, which is very useful in the development of new antibacterial drugs. To demonstrate the usefulness of our approach, we used a model small molecule (SM), NSC76027, obtained from virtual screening. We examined the direct binding of NSC76027 to EctopoI as well as inhibition of EctopoI relaxation activity of this SM via experimental techniques. We then performed molecular dynamics (MD) simulations to investigate the dynamics and stability of EctopoIcc and EctopoI-NSC76027-DNA ternary complex. Our simulation results show that NSC76027 forms a stable ternary complex with EctopoIcc. EctopoI investigated here also serves as a model system for investigating a complex of topoisomerase and DNA in which DNA is covalently attached to the protein.

Conformational Flexibility of the Protein-Protein Interfaces of the Ebola Virus VP40 Structural Matrix Filament.

The Ebola virus (EBOV) is a virulent pathogen that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV transformer protein VP40 plays crucial roles in viral assembly and budding at the plasma membrane of infected cells. One of VP40's roles is to form the long, flexible, pleomorphic filamentous structural matrix for the virus. Each filament contains three unique interfaces: monomer NTD-NTD to form a dimer, dimer-to-dimer NTD-NTD oligomerization to form a hexamer, and end-to-end hexamer CTD-CTD to build the filament. However, the atomic-level details of conformational flexibility of the VP40 filament are still elusive. In this study, we have performed explicit-solvent, all-atom molecular dynamic simulations to explore the conformational flexibility of the three different interface structures of the filament. Using dynamic network analysis and other calculational methods, we find that the CTD-CTD hexamer interface with weak interdomain amino acid communities is the most flexible, and the NTD-NTD oligomer interface with strong interdomain communities is the least flexible. Our study suggests that the high flexibility of the CTD-CTD interface may be essential for the supple bending of the Ebola filovirus, and such flexibility may present a target for molecular interventions to disrupt the Ebola virus functioning.

Molecular mechanisms of pore formation and membrane disruption by the antimicrobial lantibiotic peptide Mutacin 1140.

The emergence of antibiotic-resistance is a major concern to global human health and identification of novel antibiotics is critical to mitigate the threat. Mutacin 1140 (MU1140) is a promising antimicrobial lanthipeptide and is effective against Gram-positive bacteria. Like nisin, MU1140 targets and sequesters lipid II and interferes with its function, which results in the inhibition of bacterial cell wall synthesis, and leads to bacteria cell lysis. MU1140 contains a structurally similar thioether cage for binding the lipid II pyrophosphate as for nisin. In addition to lipid II binding, nisin is known to form membrane pores. Membrane pore formation and membrane disruption is a common mode of action for many antimicrobial peptides, including gallidermin, a lantibiotic peptide with similar structural features as MU1140. However, whether and how MU1140 and its variants can form permeable membrane pores remains to be demonstrated. In this work, we explored the potential mechanisms of membrane pore formation by performing molecular simulations of the MU1140-lipid II complex in the bacterial membrane. Our results suggest that MU1140-lipid II complexes are able to form water permeating membrane pores. We find that a single chain of MU1140 complexed with lipid II in the transmembrane region can permeate water molecules across the membrane via a single-file water transport mechanism. The ordering of the water molecules in the single-file chain region as well as the diffusion behavior is similar to those observed in other biological water channels. Multiple complexes of MU1140-lipid II in the membrane showed enhanced permeability for the water molecules, as well as a noticeable membrane distortion and lipid relocation, suggesting that a higher concentration of MU1140 assembly in the membrane can cause significant disruption of the bacterial membrane. These investigations provide an atomistic level insight into a novel mode of action for MU1140 that can be exploited to develop optimized peptide variants with improved antimicrobial properties.

A cylindrical assembly model and dynamics of the Ebola virus VP40 structural matrix.

The Ebola filovirus causes severe hemorrhagic fever with a high fatality rate in humans. The primary structural matrix protein VP40 displays transformer-protein characteristics and exists in different conformational and oligomeric states. VP40 plays crucial roles in viral assembly and budding at the plasma membrane of the infected cells and is capable of forming virus-like particles without the need for other Ebola proteins. However, no experimental three-dimensional structure for any filovirus VP40 cylindrical assembly matrix is currently available. Here, we use a protein-protein docking approach to develop cylindrical assembly models for an Ebola virion and also for a smaller structural matrix that does not contain genetic material. These models match well with the 2D averages of cryo-electron tomograms of the authentic virion. We also used all-atom molecular dynamics simulations to investigate the stability and dynamics of the cylindrical models and the interactions between the side-by-side hexamers to determine the amino acid residues that are especially important for stabilizing the hexamers in the cylindrical ring configuration matrix assembly. Our models provide helpful information to better understand the assembly processes of filoviruses and such structural studies may also lead to the design and development of antiviral drugs.

Differentiating Parallel and Antiparallel DNA Duplexes in the Gas Phase Using Trapped Ion Mobility Spectrometry.

Deoxyribonucleic acids can form a wide variety of structural motifs which differ greatly from the typical antiparallel duplex stabilized by Watson-Crick base pairing. Many of these structures are thought to occur in vivo and may have essential roles in the biology of the cell. Among these is the parallel-stranded duplex-a structural motif in which DNA strands associate in a head-to-head fashion with the 5' ends at the same end of the duplex-which is stabilized by reverse Watson-Crick base pairing. In this study, parallel- and antiparallel-stranded DNA duplexes formed from two different 12-mer oligonucleotides were studied using native electrospray ionization combined with trapped ion mobility spectrometry and mass spectrometry. The DNA duplex charge plays an important role in the gas-phase mobility profile, with a more compact form in negative mode than in positive mode (ΔΩ ≈ 100 Å2 between -4 and +4). Despite sequence mismatches, homo- and hetero-DNA duplexes were formed in solution and transfer to the gas phase, where a more compact structure was observed for the parallel compared to the antiparallel duplexes (ΔΩ ≈ 50 Å2), in good agreement with theoretical calculations. Theoretical studies suggest that a reduction (or compaction) along the helical axis of the parallel and antiparallel DNA duplexes is observed upon transfer to the gas phase.

A dominant variant in the PDE1C gene is associated with nonsyndromic hearing loss.

Identification of genes with variants causing non-syndromic hearing loss (NSHL) is challenging due to genetic heterogeneity. The difficulty is compounded by technical limitations that in the past prevented comprehensive gene identification. Recent advances in technology, using targeted capture and next-generation sequencing (NGS), is changing the face of gene identification and making it possible to rapidly and cost-effectively sequence the whole human exome. Here, we characterize a five-generation Chinese family with progressive, postlingual autosomal dominant nonsyndromic hearing loss (ADNSHL). By combining population-specific mutation arrays, targeted deafness genes panel, whole exome sequencing (WES), we identified PDE1C (Phosphodiesterase 1C) c.958G>T (p.A320S) as the disease-associated variant. Structural modeling insights into p.A320S strongly suggest that the sequence alteration will likely affect the substrate-binding pocket of PDE1C. By whole-mount immunofluorescence on postnatal day 3 mouse cochlea, we show its expression in outer (OHC) and inner (IHC) hair cells cytosol co-localizing with Lamp-1 in lysosomes. Furthermore, we provide evidence that the variant alters the PDE1C hydrolytic activity for both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Collectively, our findings indicate that the c.958G>T variant in PDE1C may disrupt the cross talk between cGMP-signaling and cAMP pathways in Ca2+ homeostasis.

Discovery of novel bacterial topoisomerase I inhibitors by use of in silico docking and in vitro assays.

Topoisomerases are important targets for antibacterial and anticancer therapies. Bacterial topoisomerase I remains to be exploited for antibiotics that can be used in the clinic. Inhibitors of bacterial topoisomerase I may provide leads for novel antibacterial drugs against pathogens resistant to current antibiotics. TB is the leading infectious cause of death worldwide, and new TB drugs against an alternative target are urgently needed to overcome multi-drug resistance. Mycobacterium tuberculosis topoisomerase I (MtbTopI) has been validated genetically and chemically as a TB drug target. Here we conducted in silico screening targeting an active site pocket of MtbTopI. The top hits were assayed for inhibition of MtbTopI activity. The shared structural motif found in the active hits was utilized in a second round of in silico screening and in vitro assays, yielding selective inhibitors of MtbTopI with IC50s as low as 2 µM. Growth inhibition of Mycobacterium smegmatis by these compounds in combination with an efflux pump inhibitor was diminished by the overexpression of recombinant MtbTopI. This work demonstrates that in silico screening can be utilized to discover new bacterial topoisomerase I inhibitors, and identifies a novel structural motif which could be explored further for finding selective bacterial topoisomerase I inhibitors.