-
João Neres,
Florence Pojer,
Elisabetta Molteni,
Laurent R Chiarelli,
Neeraj Dhar,
Stefanie Boy-Röttger,
Silvia Buroni,
Elizabeth Fullam,
Giulia Degiacomi, Anna Paola Lucarelli, [......],
Giuseppe Zanoni,
Dale E Edmondson,
Edda De Rossi,
Maria Rosalia Pasca,
John D McKinney,
Paul J Dyson,
Giovanna Riccardi,
Andrea Mattevi,
Stewart T Cole,
Claudia Binda
[show abstract]
[hide abstract]
ABSTRACT: The benzothiazinone BTZ043 is a tuberculosis drug candidate with nanomolar whole-cell activity. BTZ043 targets the DprE1 catalytic component of the essential enzyme decaprenylphosphoryl-β-d-ribofuranose-2'-epimerase, thus blocking biosynthesis of arabinans, vital components of mycobacterial cell walls. Crystal structures of DprE1, in its native form and in a complex with BTZ043, reveal formation of a semimercaptal adduct between the drug and an active-site cysteine, as well as contacts to a neighboring catalytic lysine residue. Kinetic studies confirm that BTZ043 is a mechanism-based, covalent inhibitor. This explains the exquisite potency of BTZ043, which, when fluorescently labeled, localizes DprE1 at the poles of growing bacteria. Menaquinone can reoxidize the flavin adenine dinucleotide cofactor in DprE1 and may be the natural electron acceptor for this reaction in the mycobacterium. Our structural and kinetic analysis provides both insight into a critical epimerization reaction and a platform for structure-based design of improved inhibitors.
Science translational medicine 09/2012; 4(150):150ra121. · 7.80 Impact Factor
-
Giulia Manina,
Marco Bellinzoni,
Maria Rosalia Pasca,
João Neres,
Anna Milano,
Ana Luisa De Jesus Lopes Ribeiro,
Silvia Buroni,
Henrieta Skovierová,
Petronela Dianišková,
Katarína Mikušová, [......],
Ahmed Haouz, Anna Paola Lucarelli,
Giulia Degiacomi,
Aurora Piazza,
Laurent R Chiarelli,
Edda De Rossi,
Elena Salina,
Stewart T Cole,
Pedro M Alzari,
Giovanna Riccardi
[show abstract]
[hide abstract]
ABSTRACT: Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl-β-d-ribose 2'-epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro-group to an amino-group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB-BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.
Molecular Microbiology 09/2010; 77(5):1172-85. · 5.01 Impact Factor
-
Giulia Manina,
Marco Bellinzoni,
Maria Rosalia Pasca,
João Neres,
Anna Milano,
Ana Luisa De Jesus Lopes Ribeiro,
Silvia Buroni,
Henrieta Škovierová,
Petronela Dianišková,
Katarína Mikušová, [......],
Ahmed Haouz, Anna Paola Lucarelli,
Giulia Degiacomi,
Aurora Piazza,
Laurent R. Chiarelli,
Edda De Rossi,
Elena Salina,
Stewart T. Cole,
Pedro M. Alzari,
Giovanna Riccardi
[show abstract]
[hide abstract]
ABSTRACT: Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl-β-d-ribose 2′-epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro-group to an amino-group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB-BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.
Molecular Microbiology 07/2010; 77(5):1172 - 1185. · 5.01 Impact Factor
-
Vadim Makarov,
Giulia Manina,
Katarina Mikusova,
Ute Möllmann,
Olga Ryabova,
Brigitte Saint-Joanis,
Neeraj Dhar,
Maria Rosalia Pasca,
Silvia Buroni, Anna Paola Lucarelli, [......],
Vrinda Nandi,
Sowmya Bharath,
Sheshagiri Gaonkar,
Radha K Shandil,
Venkataraman Balasubramanian,
Tanjore Balganesh,
Sandeep Tyagi,
Jacques Grosset,
Giovanna Riccardi,
Stewart T Cole
[show abstract]
[hide abstract]
ABSTRACT: New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.
Science 04/2009; 324(5928):801-4. · 31.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. Moreover, the recent isolation of M. tuberculosis strains resistant to both first- and second-line antitubercular drugs (XDR-TB) threatens to make the treatment of this disease extremely difficult and becoming a threat to public health worldwide. Recently, it has been shown that azoles are potent inhibitors of mycobacterial cell growth and have antitubercular activity in mice, thus favoring the hypothesis that these drugs may constitute a novel strategy against tuberculosis disease. To investigate the mechanisms of resistance to azoles in mycobacteria, we isolated and characterized several spontaneous azoles resistant mutants from M. tuberculosis and Mycobacterium bovis BCG. All the analyzed resistant mutants exhibited both increased econazole efflux and increased transcription of mmpS5-mmpL5 genes, encoding a hypothetical efflux system belonging to the resistance-nodulation-division (RND) family of transporters. We found that the up-regulation of mmpS5-mmpL5 genes was linked to mutations either in the Rv0678 gene, hypothesized to be involved in the transcriptional regulation of this efflux system, or in its putative promoter/operator region.
Tuberculosis (Edinburgh, Scotland) 11/2008; 89(1):84-90. · 2.54 Impact Factor
