Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
Tuberculosis (Edinb) ; 103: 52-60, 2017 03.
Article in English | MEDLINE | ID: mdl-28237034

ABSTRACT

There is a shortage of compounds that are directed towards new targets apart from those targeted by the FDA approved drugs used against Mycobacterium tuberculosis. Topoisomerase I (Mttopo I) is an essential mycobacterial enzyme and a promising target in this regard. However, it suffers from a shortage of known inhibitors. We have previously used computational approaches such as homology modeling and docking to propose 38 FDA approved drugs for testing and identified several active molecules. To follow on from this, we now describe the in vitro testing of a library of 639 compounds. These data were used to create machine learning models for Mttopo I which were further validated. The combined Mttopo I Bayesian model had a 5 fold cross validation receiver operator characteristic of 0.74 and sensitivity, specificity and concordance values above 0.76 and was used to select commercially available compounds for testing in vitro. The recently described crystal structure of Mttopo I was also compared with the previously described homology model and then used to dock the Mttopo I actives norclomipramine and imipramine. In summary, we describe our efforts to identify small molecule inhibitors of Mttopo I using a combination of machine learning modeling and docking studies in conjunction with screening of the selected molecules for enzyme inhibition. We demonstrate the experimental inhibition of Mttopo I by small molecule inhibitors and show that the enzyme can be readily targeted for lead molecule development.


Subject(s)
Antitubercular Agents/pharmacology , DNA Topoisomerases, Type I/metabolism , Drug Design , Machine Learning , Molecular Docking Simulation , Mycobacterium tuberculosis/drug effects , Topoisomerase I Inhibitors/pharmacology , Tuberculosis/drug therapy , Antitubercular Agents/chemistry , Antitubercular Agents/metabolism , Bayes Theorem , Computer-Aided Design , DNA Topoisomerases, Type I/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Targeted Therapy , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/growth & development , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/growth & development , Protein Conformation , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/metabolism , Tuberculosis/microbiology
2.
Antimicrob Agents Chemother ; 59(3): 1549-57, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25534741

ABSTRACT

We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , DNA Topoisomerases, Type I/metabolism , Mycobacterium tuberculosis/drug effects , Small Molecule Libraries/pharmacology , Topoisomerase I Inhibitors/pharmacology , Amino Acid Motifs , Bacterial Proteins/metabolism , Binding Sites/genetics , DNA/genetics , DNA, Bacterial/genetics , Imipramine/pharmacology , Mutation/genetics , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Tuberculosis/drug therapy , Tuberculosis/microbiology
3.
Biochem Biophys Res Commun ; 446(4): 916-20, 2014 Apr 18.
Article in English | MEDLINE | ID: mdl-24642256

ABSTRACT

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme-DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.


Subject(s)
Amsacrine/pharmacology , Antitubercular Agents/pharmacology , DNA Topoisomerases, Type I/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Topoisomerase I Inhibitors/pharmacology , Topoisomerase II Inhibitors/pharmacology , Amsacrine/chemistry , Antitubercular Agents/chemistry , DNA, Bacterial/metabolism , Humans , Molecular Docking Simulation , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/growth & development , Mycobacterium tuberculosis/growth & development , Topoisomerase I Inhibitors/chemistry , Topoisomerase II Inhibitors/chemistry , Tuberculosis/drug therapy , Tuberculosis/microbiology
4.
FASEB J ; 27(8): 3030-8, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23612788

ABSTRACT

Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.


Subject(s)
Antibodies, Monoclonal/pharmacology , Bacterial Proteins/metabolism , DNA Topoisomerases, Type I/metabolism , Topoisomerase Inhibitors/pharmacology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biocatalysis/drug effects , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Cleavage/drug effects , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/immunology , Models, Biological , Models, Molecular , Mutation , Mycobacterium smegmatis/enzymology , Mycobacterium tuberculosis/enzymology , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/genetics , Oligonucleotides/metabolism , Protein Binding/drug effects , Protein Structure, Tertiary , Spectrometry, Fluorescence , Topoisomerase Inhibitors/chemistry , Topoisomerase Inhibitors/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...