The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

Gavalda, Sabine; Léger, Mathieu; Rest, Benoı̂t van der; Stella, Alexandre; Bardou, Fabienne; Montrozier, Henri; Chalut, Christian; Burlet‐Schiltz, Odile; Marrakchi, Hédia; Daffé, Mamadou; Quémard, Annaık̈

doi:10.1074/jbc.m109.006940

Cited by 106 publications

(123 citation statements)

References 26 publications

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“…11 FadD32 acts in concert with Pks13 and activates the very long meromycolic acid (C 50 -C 60 ) prior to its condensation with a C 24 -C 26 fatty acid, 12 which itself is activated by the AccD4-containing acyl-CoA carboxylase ACCase, to yield, upon reduction, mycolic acids (Fig. 1A).…”

Section: Introductionmentioning

confidence: 99%

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

et al. 2013

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FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of T m (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

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Section: Introductionmentioning

confidence: 99%

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

et al. 2013

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“…Overall, the M. marinum FadD29-PpsA in vitro system appears to be competent to catalyze PHPA activation, loading, extension, and reduction in line with the model proposed in Fig. 3 (37,52), a FadD32-Pks13 partnership that takes place during mycolic acid biosynthesis (52,61,62), and a FadD30-Pks6 partnership believed to be required for production of novel polar lipids (52,63). To our knowledge, however, the partially overlapping PGL/PDIM biosynthetic pathways provide the first example of two distinct acyl-AMP ligases (i.e., FadD29 and FadD26) loading the same type I PKS (i.e., PpsA) with two alternate starter units (i.e., PHPAs and fatty acids).…”

Section: Resultsmentioning

confidence: 69%

Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

et al. 2015

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dPhenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathwaydedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids. Mycobacterial infections are responsible for devastating morbidity and mortality worldwide (1-5). A critical player in the ability of the mycobacteria to produce disease is a formidable cell envelope believed to be responsible for the intrinsic resilience of the mycobacteria to inhospitable environments, antimicrobial agents, and host immune defenses (6-12). Among the unique components found in the cell envelope of several pathogenic mycobacteria are two structurally related families of glycosylated and nonglycosylated lipids commonly referred to as phenolic glycolipids (PGLs) and phthiocerol dimycocerosates (PDIMs), respectively (for a review, see reference 13). PGLs and PDIMs have unusual lipid scaffolds consisting of ␤-diol-containing, long-chain, aliphatic polyketides esterified with long-chain, multimethylbranched fatty acids onto the diol functionality (Fig. 1). PGLs and PDIMs are found in several mycobacterial pathogens (e.g., Mycobacterium tuberculosis strains, Mycobacterium bovis, Mycobacterium leprae, and Mycobacterium marinum) and thought to be constituents of the characteristic mycobacterial outer membrane. Multiple lines of investigation have provided considerable support for the idea that PGLs and PDIMs are implicated in virulence via co...

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“…Esta enzima atua em associação com a Pks13 para ativar o ácido meromicólico antes da sua condensação com a porção C24-26 do ácido graxo. 59 O operon fadD32-pks13-accD4 é essencial para a viabilidade do MTB, e estudos de silenciamento de genes apontaram o operon fadD32 e a enzima FadD32 como alvos potenciais na micobactéria. 60 Recentemente, foi reportada a síntese de quarenta derivados de cumarina com potente atividade e seletividade contra o MTB.…”

Section: Alvos Envolvidos Com a Biossíntese Da Parede Celularunclassified

Potenciais alvos moleculares para o desenvolvimento de novos fármacos antituberculose

2017

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POTENTIAL MOLECULAR TARGETS FOR ANTITUBERCULOSIS DRUG DISCOVERY. Tuberculosis (TB) is an infectious disease caused by mycobacteria from the Mycobacterium genus, mainly by Mycobacterium tuberculosis (MTB). The World HealthOrganization (WHO) aims to reduce the number of TB cases worldwide in the coming years. Nevertheless, the increasing number of multidrug-resistant (MDR-TB) and extensive-drug resistance (XDR-TB) strains, and the ineffectiveness of the current treatment in latent tuberculosis are challenges to be overcome. In this review, we will demonstrate the recent advances in the tuberculosis drug discovery, focusing the research of new molecular targets in the Mycobacterium tuberculosis. Among the promising targets described herein, we highlight those, which act in different pathways in the mycobacteria, such as energy metabolism, cell wall biosynthesis, DNA synthesis, iron metabolism and transport through membranes. Furthermore, bioactive compounds discovered using phenotypic assays screening and validated through genetic approaches are also presented.Keywords: medicinal chemistry; tuberculosis; Mycobacterium tuberculosis; molecular targets; new drugs. INTRODUÇÃOA tuberculose (TB) é uma doença infecciosa causada por micobactérias do complexo Mycobacterium, no entanto, a espécie responsável pelo maior número de casos de morbidade e mortalidade é o Mycobacterium tuberculosis (MTB). 1 Ocupando o ranking mundial como a principal causa de mortes causadas por doenças infecciosas, nos últimos anos a tuberculose ultrapassou, em número de casos, a infecção causada pelo vírus da imunodeficiência humana (HIV). 2,3 O último levantamento realizado em 2015 pela Organização Mundial da Saúde (OMS) apontou 9.6 milhões de novos casos no mundo e 1.5 milhões de mortes causadas pela doença. 3 Somado a esses dados, o surgimento e aumento de cepas de M. tuberculosis multirresistente aos fármacos (MDR-TB) e extensivamente resistente aos fármacos (XDR-TB) vêm alarmando as autoridades de todo o mundo. Essas formas de tuberculose apresentam baixas taxas de cura e maiores taxas de mortalidade devido às dificuldades de tratamento. 4 Além disso, já foram relatados na clínica casos de tuberculose totalmente resistente aos fármacos (TDR-TB). 5,6 Ao longo dos últimos anos, é possível constatar algumas evoluções no desenvolvimento de compostos candidatos a fármacos que possam atuar contra a TB. 7 Após um intervalo de mais de 50 anos sem novos medicamentos para o tratamento da TB, a bedaquilina foi aprovada em 2012 pela agência norte-americana U.S. Food and Drug Administration (FDA) para o tratamento de MDR-TB. Atualmente, diversos candidatos a fármacos estão em estudos clínicos, [8][9][10][11][12] 39 Atualmente, diversos métodos e estratégias são utilizados para o desenvolvimento de novos fármacos contra a TB, como por exemplo: 1) abordagens genéticas para identificação de novos alvos; 2) ensaios de triagem em larga escala utilizando a micobactéria como plataforma; 3) ferramentas de biologia estrutural e triagem virtual e; 4) modificações ...

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The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

Cited by 106 publications

References 26 publications

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

Potenciais alvos moleculares para o desenvolvimento de novos fármacos antituberculose

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