L. A. Basso

Drugs that Inhibit Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

Tuberculosis resurged in the late 1980s and now kills more than 2 million people a year. The reemergence of tuberculosis as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, and the proliferation of multidrug- resistant (MDR) strains have created much scientific interest in developing new antimycobacterial agents to both treat Mycobacterium tuberculosis strains resistant to existing drugs, and shorten the duration of short-course treatment to improve patient compliance. Bacterial cell-wall biosynthesis is a proven target for new antibacterial drugs. Mycolic acids, which are key components of the mycobacterial cell wall, are α-alkyl, β-hydroxy fatty acids, with a species-dependent saturated “short” arm of 20-26 carbon atoms and a “long” meromycolic acid arm of 50-60 carbon atoms. The latter arm is functionalized at regular intervals by cyclopropyl, α-methyl ketone, or α-methyl methylethers groups. The mycolic acid biosynthetic pathway has been proposed to involve five distinct stages: (i) synthesis of C20 to C26 straight-chain saturated fatty acids to provide the α-alkyl branch; (ii) synthesis of the meromycolic acid chain to provide the main carbon backbone, (iii) modification of this backbone to introduce other functional groups; (iv) the final Claisen-type condensation step followed by reduction; and (v) various mycolyltransferase processes to cellular lipids. The drugs shown to inhibit mycolic acid biosynthesis are isoniazid, ethionamide, isoxyl, thiolactomycin, and triclosan. In addition, pyrazinamide was shown to inhibit fatty acid synthase type I which, in turn, provides precursors for fatty acid elongation to long-chain mycolic acids by fatty acid synthase II. Here we review the biosynthesis of mycolic acids and the mechanism of action of antimicrobial agents that act upon this pathway. In addition, we describe molecular modeling studies on InhA, the bona-fide target for isoniazid, which should improve our understanding of the amino acid residues involved in the enzymes mechanism of action and, accordingly, provide a rational approach to the design of new drugs.

Shikimate kinase: A potential target for development of novel antitubercular agents

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

Enoyl Reductases as Targets for the Development of Anti-Tubercular and Anti-Malarial Agents

Tuberculosis (TB) and Malaria are neglected diseases, which continue to be major causes of morbidity and mortality worldwide, killing together around 5 million people each year. Mycolic acids, the hallmark of mycobacteria, are high-molecular-weight alpha-alkyl, beta-hydroxy fatty acids. Biochemical and genetic experimental data have shown that the product of the M. tuberculosis inhA structural gene (InhA) is the primary target of isoniazid mode of action, the most prescribed anti-tubercular agent. InhA was identified as an NADH-dependent enoyl-ACP(CoA) reductase specific for long-chain enoyl thioesters and is a member of the Type II fatty acid biosynthesis system, which elongates acyl fatty acid precursors of mycolic acids. M. tuberculosis and P. falciparum enoyl reductases are targets for the development of anti-tubercular and antimalarial agents. Here we present a brief description of the mechanism of action of, and resistance to, isoniazid. In addition, data on inhibition of mycobacterial and plasmodial enoyl reductases by triclosan are presented. We also describe recent efforts to develop inhibitors of M. tuberculosis and P. falciparum enoyl reductase enzyme activity.

Analysis of select members of the E26 (ETS) transcription factors family in colorectal cancer

The E-twenty-six (ETS) family of transcription factors is known to act as positive or negative regulators of the expression of genes that are involved in diverse biological processes, including those that control cellular proliferation, differentiation, hematopoiesis, apoptosis, metastasis, tissue remodeling, and angiogenesis. Identification of target gene promoters of normal and oncogenic transcription factors provides new insights into the regulation of genes that are involved in the control of normal cell growth and differentiation. The aim of the present investigation was to analyze the differential expression of 11 ETS (ELF-3, ESE3, ETS1, ETV3, ETV4, ETV6, NERF, PDEF, PU1, Spi-B, and Spi-C) as potential markers for prognostic of colorectal cancer. A series of paired tissue biopsies consisting of a tumor and a non-affected control sample were harvested from 28 individuals suffering from diagnosed colorectal lesions. Total RNA was isolated from the samples, and after reverse transcription, differential expression of the select ETS was carried out through real-time polymerase chain reaction. Tumor staging as determined by histopathology was carried out to correlate the degree of tumor invasiveness with the expression of the ETS genes. The results demonstrated a different quantitative profile of expression in tumors and normal tissues. ETV4 was significantly upregulated with further increase in the event of lymph node involvement. PDEF and Spi-B presented downregulation, which was more significant when lymph node involvement was present. These findings were supported by immunohistochemistry of tumoral tissues. The results suggest that select ETS may serve as potential markers of colorectal cancer invasiveness and metastasis.

Molecular models of tryptophan synthase from Mycobacterium tuberculosis complexed with inhibitors

The development of new therapies against infectious diseases is vital in developing countries. Among infectious diseases, tuberculosis is considered the leading cause of death. A target for development of new drugs is the tryptophan pathway. The last enzyme of this pathway, tryptophan synthase (TRPS), is responsible for conversion of the indole 3-glycerol phosphate into indol and the condensation of this molecule with serine-producting tryptophan. The present work describes the molecular models of TRPS from Mycobacterium tuberculosis (MtTRPS) complexed with six inhibitors, the indole 3-propanol phosphate and five arylthioalkyl-phosphonated analogs of substrate of the α-subunit. The molecular models of MtTRPS present good stereochemistry, and the binding of the inhibitors is favorable. Thus, the generated models can be used in the design of more specific drugs against tuberculosis and other infectious diseases.

Shikimate Kinase (EC 2.7.1.71) from Mycobacterium tuberculosis: Kinetics and Structural Dynamics of a Potential Molecular Target for Drug Development

The enzymes of the shikimate pathway represent potential molecular targets for the development of non-toxic antimicrobial agents and anti-parasite drugs. One of the most promising of these enzymes is shikimate kinase (EC 2.7.1.71), which is responsible for the fifth step in the shikimate pathway. This enzyme phosphorylates shikimic acid to yield shikimate-3-phosphate, using ATP as a substrate. In this work, the conformational dynamics of the shikimate kinase from Mycobacterium tuberculosis was investigated in its apostate in solution. For this study, the enzyme was subjected to a gradient of temperatures from 15°C to 45°C in the presence or absence of deuterium oxide, and the amide H/D exchange was monitored using ESI-mass spectrometry. We observed: i) the phosphate binding domain in the apo-enzyme is fairly rigid and largely protected from solvent access, even at relatively high temperatures; ii) the shikimate binding domain is highly flexible, as indicated by the tendency of the apo-enzyme to exhibit large conformational changes to permit LID closure after the shikimate binding; iii) the nucleotide binding domain is initially conformationally rigid, which seems to favour the initial orientation of ADP/ATP, but becomes highly flexible at temperatures above 30°C, which may permit domain rotation; iv) part of the LID domain, including the phosphate binding site, is partially rigid, while another part is highly flexible and accessible to the solvent.

Structural bioinformatics study of PNP from Listeria monocytogenes.

This work describes for the first time a model of Purine Nucleoside Phosphorylase from Listeria monocytogenes (LmPNP). We modeled the complexes of LmPNP with ligands in order to determine the structural basis for specificity. Comparative analysis of the model of LmPNP allowed identification of structural features responsible for ligand affinities.