Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the bhydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors

New Export Pathway in Plasmodium falciparum -Infected Erythrocytes: Role of the Parasite Group II Chaperonin, PfTRiC

International audienceThe export of numerous proteins to the plasma membrane of its host erythrocyte is essential for the virulence and survival of the malaria parasite Plasmodium falciparum. The Maurer's clefts, membrane structures transposed by the parasite in the cytoplasm of its host erythrocyte, play the role of a marshal platform for such exported parasite proteins. We identify here the export pathway of three resident proteins of the Maurer's clefts membrane: the proteins are exported as soluble forms in the red cell cytoplasm to the Maurer's clefts membrane in association with the parasite group II chaperonin (PfTRIC), a chaperone complex known to bind and address a large spectrum of unfolded proteins to their final location. We have also located the domain of interaction with PfTRiC within the amino‐terminal domain of one of these Maurer's cleft proteins, PfSBP1. Because several Maurer's cleft membrane proteins with different export motifs seem to follow the same route, we propose a general role for PfTRiC in the trafficking of malarial parasite proteins to the host erythrocyte

Susceptibility and genetic mutations associated to TAC resistance in <i>M. tuberculosis</i> strains selected on high concentrations of TAC or SRI-224.

<p>Susceptibility and genetic mutations associated to TAC resistance in <i>M. tuberculosis</i> strains selected on high concentrations of TAC or SRI-224.</p

Dose-response effects of TAC and related analogues on mycolic acid biosynthesis in <i>M. tuberculosis.</i>

<p>The inhibitory effect on the incorporation of [2-¹⁴C]acetate was assayed by labeling in the presence of increasing concentrations of TAC, <b>15 </b>or <b>16</b>. The corresponding fatty acid methyl esters (FAME) and mycolic acid methyl esters (MAME) were extracted and equal counts were loaded onto a TLC plate. (<b>A</b>) <b>1D TLC profile of MAMEs.</b> Radiolabeled lipids (40,000 cpm) were resolved with hexane/ethyl acetate (19/1, v/v, 2 runs) and exposed overnight to a film. (<b>B</b>) <b>2D TLC profile of MAMEs.</b> Radiolabeled lipids (30, 000 cpm) were resolved with hexane/ethyl acetate (19/1, v/v, 2 runs) in the first dimension and petroleum ether/diethyl ether (17/3, v/v, 3 runs) in the second direction on 10% silver nitrate-impregnated plates and exposed for two days to a film. Α, α-mycolates; keto, ketomycolates, methoxy, methoxymycolates. Arrowheads indicate positions of the unsaturated mycolic acid species. OAME, oleic acid methyl ester.</p

Mycolic acid profile of the parental <i>M. tuberculosis</i> strain and independent TAC-resistant derived mutants.

<p>FAMEs and MAMEs were extracted from exponentially growing cultures and resolved by single dimension TLC in hexane/ethyl acetate (19/1, v/v) prior to visualization using molybdophosphoric acid and charring. α, α-mycolic acids; methoxy, methoxy-mycolic acids; keto, keto-mycolic acids.</p

Oligonucleotides used in this study.

<p>F and R stand for forward and reverse, respectively.</p

Structures of chemical analogues of thiacetazone and their corresponding minimum inhibitory concentrations in <i>M. tuberculosis</i>.

<p>Shaded lanes highlight the more efficient analogues.</p

MIC of TAC and its related analogues <b>15</b>, <b>16</b> and <b>17</b> against recombinant <i>M. tuberculosis</i> strains overexpresssing the different dehydratase complexes^a.

a<p>MIC₉₉ expressed in µg/ml was determined by dilution on solid agar medium 7H10 supplemented with OADC.</p

<i>HadAB</i> and <i>hadBC</i> expression levels in TAC-resistant <i>M. tuberculosis</i> mutants.

<p>The parental <i>M. tuberculosis</i> strain as well as MTTR3, MTTR13 or MTTR18 were grown to mid-log phase. Total RNA was isolated from three independent replicates and the levels of <i>hadAB </i><b>(A)</b> and <i>hadBC </i><b>(B)</b> transcripts relative to those of the s<i>igA</i> gene were measured by quantitative reverse transcription-PCR. Recombinant strains carrying the pVV16-<i>hadAB</i> or pVV16-<i>hadBC</i> constructs were included as positive controls of HadAB and HadBC overexpression, respectively.</p

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International audienceLe site de référence du Partenariat européen d'innovation pour un vieillissement actif et en bonne santé MACVIA-LR (contre les maladies chroniques pour un vieillissement en bonne santé en Languedoc-Roussillon