Impact of DNA ligase inhibition on the nick sealing of polß nucleotide insertion products at the downstream steps of base excision repair pathway.

DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) ß at the downstream steps. We previously demonstrated that a functional interplay between polß and BER ligases is critical for efficient repair, and polß mismatch or oxidized nucleotide insertions confound final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polß+/+ cells more than polß-/- null cells. We showed less efficient ligation after polß nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polß inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polß, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.

NS3 helicase from dengue virus specifically recognizes viral RNA sequence to ensure optimal replication.

The protein-RNA interactions within the flavivirus replication complex (RC) are not fully understood. Our structure of dengue virus NS3 adenosine triphosphatase (ATPase)/helicase bound to the conserved 5' genomic RNA 5'-AGUUGUUAGUCU-3' reveals that D290 and R538 make specific interactions with G2 and G5 bases respectively. We show that single-stranded 12-mer RNA stimulates ATPase activity of NS3, however the presence of G2 and G5 leads to significantly higher activation. D290 is adjacent to the DEXH motif found in SF2 helicases like NS3 and interacts with R387, forming a molecular switch that activates the ATPase site upon RNA binding. Our structure guided mutagenesis revealed that disruption of D290-R387 interaction increases basal ATPase activity presumably as a result of higher conformational flexibility of the ATPase active site. Mutational studies also showed R538 plays a critical role in RNA interactions affecting translocation of viral RNA through dynamic interactions with bases at positions 4 and 5 of the ssRNA. Restriction of backbone flexibility around R538 through mutation of G540 to proline abolishes virus replication, indicating conformational flexibility around residue R538 is necessary for RNA translocation. The functionally critical sequence-specific contacts in NS3 RNA binding groove in subdomain III reveals potentially novel allosteric anti-viral drug targets.

Structure-guided Discovery of a Novel Non-peptide Inhibitor of Dengue Virus NS2B-NS3 Protease.

Dengue fever is a fast emerging epidemic-prone viral disease caused by dengue virus serotypes 1-4. NS2B-NS3 protease of dengue virus is a validated target to develop antiviral agents. A major limitation in developing dengue virus protease inhibitors has been the lack of or poor cellular activity. In this work, we extracted and refined a pharmacophore model based on X-ray crystal structure and predicted binding patterns, followed by a three-dimensional flexible database filtration. These output molecules were screened according to a docking-based protocol, leading to the discovery of a compound with novel scaffold and good cell-based bioactivity that has potential to be further optimized. The discovery of this novel scaffold by combination of in silico methods suggests that structure-guided drug discovery can lead to the development of potent dengue virus protease inhibitors.

Suramin inhibits helicase activity of NS3 protein of dengue virus in a fluorescence-based high throughput assay format.

Dengue fever is a major health concern worldwide. The virus encoded non-structural protein 3 (NS3) is a multifunctional protein endowed with protease, helicase, nucleoside triphosphatase (NTPase) and RNA 5' triphosphatase (RTPase) activities. Helicase activity of NS3 catalyzes the unwinding of double stranded polynucleotides by utilizing the energy released from ATP hydrolysis. As this activity is essential for replication, NS3 helicase represents an attractive drug target for developing a dengue antiviral drug. Here, we report fluorescence based molecular beacon helicase assay using a duplex RNA substrate that contains a fluorophore on the 5' end and a quencher on the 3' end of one of the strands. The assay was optimized with respect to several parameters and adapted to 384-well high-throughput screening format, with an average Z' factor of 0.65. Assay validation with a small diverse set library of 1600 compounds identified, suramin as a significant inhibitor of the helicase activity of NS3. Helicase activity deficient NS3 K199A was used in a counter-screen to identify compounds interfering with the assay. Suramin inhibited DENV (dengue virus) NS3 helicase activity with a Ki of 0.75±0.03µM as a non-competitive inhibitor. The molecular beacon helicase assay together with the counter screen and suramin as a tool compound can be used to identify novel inhibitors of DENV helicase.

A microplate assay for the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins; a vancomycin-neutralizing tripeptide combination prevents penicillin inhibition of peptidoglycan synthesis.

A microplate, scintillation proximity assay to measure the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[(3)H]N-acetylglucosamine in the presence of 40 µM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-D-ala-D-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC50 of 8 nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a 'neutral' combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.

A new plant-derived antibacterial is an inhibitor of efflux pumps in Staphylococcus aureus.

An in-depth evaluation was undertaken of a new antibacterial natural product (1) recently isolated and characterised from the plant Hypericum olympicum L. cf. uniflorum. Minimum inhibitory concentrations (MICs) were determined for a panel of bacteria, including: meticillin-resistant and -susceptible strains of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus; vancomycin-resistant and -susceptible Enterococcus faecalis and Enterococcus faecium; penicillin-resistant and -susceptible Streptococcus pneumoniae; group A streptococci (Streptococcus pyogenes); and Clostridium difficile. MICs were 2-8 mg/L for most staphylococci and all enterococci, but were ≥16 mg/L for S. haemolyticus and were >32 mg/L for all species in the presence of blood. Compound 1 was also tested against Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium but was inactive. The MIC for Mycobacterium bovis BCG was 60 mg/L, and compound 1 inhibited the ATP-dependent Mycobacterium tuberculosis MurE ligase [50% inhibitory concentration (IC(50)) = 75 µM]. In a radiometric accumulation assay with a strain of S. aureus overexpressing the NorA multidrug efflux pump, the presence of compound 1 increased accumulation of (14)C-enoxacin in a concentration-dependent manner, implying inhibition of efflux. Only moderate cytotoxicity was observed, with IC50 values of 12.5, 10.5 and 8.9 µM against human breast, lung and fibroblast cell lines, respectively, highlighting the potential value of this chemotype as a new antibacterial agent and efflux pump inhibitor.

Transplantation of a hydrogen bonding network from West Nile virus protease onto Dengue-2 protease improves catalytic efficiency and sheds light on substrate specificity.

The two-component serine protease of flaviviruses such as Dengue virus (DENV) and West Nile virus (WNV) are attractive targets for inhibitor/therapeutic design. Peptide aldehyde inhibitors that bind to the covalently tethered two-component WNV protease (WNVpro) with 50% inhibitory concentration (IC(50)) at sub-micromolar concentrations, bind the equivalent DENV-2 protease (DEN2pro) with IC(50) of micromolar concentrations at best. Conversely, the protease inhibitor aprotinin binds DEN2pro ∼1000-fold more tightly than WNVpro. To investigate the residues that are crucial for binding specificity differences, a binding-site network of hydrogen bonds was transplanted from WNVpro onto DEN2pro. The transplantations were a combination of single, double and triple mutations involving S79D, S83N and S85Q. The mutant DENV proteases, except those involving S85Q, proved to be more efficient enzymes, as measured by their kinetic parameters. The binding affinities of the mutants to peptide inhibitors however showed only marginal improvement. Protein structure modeling suggests that the negatively charged residue cluster, Glu89-Glu92, of the NS2B cofactor may play an important role in determining substrate/inhibitor-binding specificity. These same residues may also explain why aprotinin binds more tightly to DEN2pro than WNVpro. Our results suggest that structure-based inhibitor design experiments need to explicitly consider/include this C-terminal region whose negative charge is conserved across the four DENV serotypes and also among the flavivirus family of proteases.

An antibacterial from Hypericum acmosepalum inhibits ATP-dependent MurE ligase from Mycobacterium tuberculosis.

In a project to characterise new antibacterial chemotypes from plants, hyperenone A and hypercalin B were isolated from the hexane and chloroform extracts of the aerial parts of Hypericum acmosepalum. The structures of both compounds were characterised by extensive one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and were confirmed by mass spectrometry. Hyperenone A and hypercalin B exhibited antibacterial activity against multidrug-resistant strains of Staphylococcus aureus, with minimum inhibition concentration ranges of 2-128 mg/L and 0.5-128 mg/L, respectively. Hyperenone A also showed growth-inhibitory activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG at 75 mg/L and 100mg/L. Neither hyperenone A nor hypercalin B inhibited the growth of Escherichia coli and both were non-toxic to cultured mammalian macrophage cells. Both compounds were tested for their ability to inhibit the ATP-dependent MurE ligase of M. tuberculosis, a crucial enzyme in the cytoplasmic steps of peptidoglycan biosynthesis. Hyperenone A inhibited MurE selectively, whereas hypercalin B did not have any effect on enzyme activity.

Interaction of N-methyl-2-alkenyl-4-quinolones with ATP-dependent MurE ligase of Mycobacterium tuberculosis: antibacterial activity, molecular docking and inhibition kinetics.

OBJECTIVES: The aim of this study was to comprehensively evaluate the antibacterial activity and MurE inhibition of a set of N-methyl-2-alkenyl-4-quinolones found to inhibit the growth of fast-growing mycobacteria. METHODS: Using the spot culture growth inhibition assay, MICs were determined for Mycobacterium tuberculosis H(37)Rv, Mycobacterium bovis BCG and Mycobacterium smegmatis mc(2)155. MICs were determined for Mycobacterium fortuitum, Mycobacterium phlei, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using microplate dilution assays. Inhibition of M. tuberculosis MurE ligase activity was determined both by colorimetric and HPLC methods. Computational modelling and binding prediction of the quinolones in the MurE structure was performed using Glide. Kinetic experiments were conducted for understanding possible competitive relations of the quinolones with the endogenous substrates of MurE ligase. RESULTS: The novel synthetic N-methyl-2-alkenyl-4-quinolones were found to be growth inhibitors of M. tuberculosis and rapid-growing mycobacteria as well as methicillin-resistant S. aureus, while showing no inhibition for E. coli and P. aeruginosa. The quinolones were found to be inhibitory to MurE ligase of M. tuberculosis in the micromolar range (IC(50) ∼40-200 µM) when assayed either spectroscopically or by HPLC. Computational docking of the quinolones on the published M. tuberculosis MurE crystal structure suggested that the uracil recognition site is a probable binding site for the quinolones. CONCLUSIONS: N-methyl-2-alkenyl-4-quinolones are inhibitors of mycobacterial and staphylococcal growth, and show MurE ligase inhibition. Therefore, they are considered as a starting point for the development of increased affinity MurE activity disruptors.

Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis.

The emergence of total drug-resistant tuberculosis (TDRTB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new anti-mycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 N-terminal residues, the other mutated residues are essential for catalysis. Variations in K(m) values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.

Anti-tubercular screening of natural products from Colombian plants: 3-methoxynordomesticine, an inhibitor of MurE ligase of Mycobacterium tuberculosis.

OBJECTIVES: New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treating resistant forms of tuberculosis. The purpose of this study was to evaluate anti-tubercular activity and selectivity of seven recently isolated natural products from Colombian plants. METHODS: MICs were determined using a liquid medium growth inhibition assay for Mycobacterium tuberculosis H(37)Rv and both solid and liquid media growth inhibition assays for Mycobacterium bovis BCG. Escherichia coli growth inhibition and mammalian macrophage cell toxicity were evaluated to establish the degree of selectivity of the natural product against whole cell organisms. Enzymatic inhibition of ATP-dependent MurE ligase from M. tuberculosis was assayed using a colorimetric phosphate detection method. The most active compound, 3-methoxynordomesticine hydrochloride, was further investigated on M. bovis BCG for its inhibition of sigmoidal growth, acid-fast staining and viability counting analysis. RESULTS: Aporphine alkaloids were found to be potent inhibitors of slow-growing mycobacterial pathogens showing favourable selectivity and cytotoxicity. In terms of their endogenous action, the aporphine alkaloids were found inhibitory to M. tuberculosis ATP-dependent MurE ligase at micromolar concentrations. A significantly low MIC was detected for 3-methoxynordomesticine hydrochloride against both M. bovis BCG and M. tuberculosis H(37)Rv. CONCLUSIONS: Considering all the data, 3-methoxynordomesticine hydrochloride was found to be a potent anti-tubercular compound with a favourable specificity profile. The alkaloid showed MurE inhibition and is considered an initial hit for exploring related chemical space.

ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation.

New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0A resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.