A template guided approach to generating cell permeable inhibitors of Staphylococcus aureus biotin protein ligase

Inhibitors of biotin protein ligase (BPL) are novel antimicrobial compounds with the potential to treat infections caused by bacteria resistant to current antibiotics. A novel BPL inhibitor (12, K_i 1.4 μM) was synthesized from biotin acetylene and an azide-functionalized analogue of fluorescent nitrobenzofurazan by Cu(I) catalysed cycloaddition and also by template guided synthesis using wild-type BPL from Staphylococcus aureus. LC/HRMS-based detection provides improved sensitivity over previous reports using a mutant BPL, with demonstrated applicability to other BPLs. Super-imaging fluorescence microscopy demonstrated the accumulation of 12 in the cytoplasm of S. aureus, but not Escherichia coli. This novel fluorescent probe can be used to gain new insights into the mechanism of uptake, efflux and metabolism of BPL inhibitors in S. aureus.     

Natural product growth inhibitors of Mycobacterium tuberculosis

This review covers natural products (secondary metabolites) with reported growth inhibitory activity towards Mycobacterium tuberculosis or related organisms. Such compounds have been isolated from a variety of sources including terrestrial and marine plants and animals, and microorganisms, with the express intent of identifying novel scaffolds for the development of new antituberculosis agents. The literature from January 2003 to December 2005 (inclusive) is reviewed and 146 references to 353 compounds are cited. The compounds are presented in order of chemical type, namely lipids/fatty acids and simple aromatics, phenolics and quinones, peptides, alkaloids, terpenes (monoterpenoids, diterpenes, sesquiterpenes and triterpenes), steroids and miscellaneous structures.     

Structure-based discovery of novel inhibitors of Mycobacterium tuberculosis CYP121 from Indonesian natural products

Tuberculosis (TB) continues to be a serious global health threat with the emergence of multidrug-resistant tuberculosis (MDR-TB) and extremely drug-resistant tuberculosis (XDR-TB). There is an urgent need to discover new drugs to deal with the advent of drug-resistant TB variants. This study aims to find new M. tuberculosis CYP121 inhibitors by the screening of Indonesian natural products using the principle of structure-based drug design and discovery. In this work, eight natural compounds isolated from Rhoeo spathacea and Pluchea indica were selected based on their antimycobacterial activity. Derivatives compound were virtually designed from these natural molecules to improve the interaction of ligands with CYP121. Virtual screening of ligands was carried out using AutoDock Vina followed by 50 ns molecular dynamics simulation using YASARA to study the inhibition mechanism of the ligands. Two ligands, i.e., kaempferol (KAE) and its benzyl derivative (KAE3), are identified as the best CYP121 inhibitors based on their binding affinities and adherence to the Lipinski’s rule. Results of molecular dynamics simulation indicate that KAE and KAE3 possess a unique inhibitory mechanism against CYP121 that is different from GGJ (control ligand). The control ligand alters the overall dynamics of the receptor, which is indicated by changes in residue flexibility away from CYP121 binding site. Meanwhile, the dynamic changes caused by the binding of KAE and KAE3 are isolated around the binding site of CYP121. These ligands can be developed for further potential biological activities.     

Mycobacterium tuberculosis serine/threonine protein kinases: structural information for the design of their specific ATP-competitive inhibitors

In the last decades, human protein kinases (PKs) have been relevant as targets in the development of novel therapies against many diseases, but the study of Mycobacterium tuberculosis PKs (MTPKs) involved in tuberculosis pathogenesis began much later and has not yet reached an advanced stage of development. To increase knowledge of these enzymes, in this work we studied the structural features of MTPKs, with focus on their ATP-binding sites and their interactions with inhibitors. PknA, PknB, and PknG are the most studied MTPKs, which were previously crystallized; ATP-competitive inhibitors have been designed against them in the last decade. In the current work, reported PknA, PknB, and PknG inhibitors were extracted from literature and their orientations inside the ATP-binding site were proposed by using docking method. With this information, interaction fingerprints were elaborated, which reveal the more relevant residues for establishing chemical interactions with inhibitors. The non-crystallized MTPKs PknD, PknF, PknH, PknJ, PknK, and PknL were also studied; their three-dimensional structural models were developed by using homology modeling. The main characteristics of MTPK ATP-binding sites (the non-crystallized and crystallized MTPKs, including PknE and PknI) were accounted; schemes of the main polar and nonpolar groups inside their ATP-binding sites were constructed, which are suitable for a major understanding of these proteins as antituberculotic targets. These schemes could be used for establishing comparisons between MTPKs and human PKs in order to increase selectivity of MTPK inhibitors. As a key tool for guiding medicinal chemists interested in the design of novel MTPK inhibitors, our work provides a map of the structural elements relevant for the design of more selective ATP-competitive MTPK inhibitors.     

Syntheses of mycobactin analogs as potent and selective inhibitors of Mycobacterium tuberculosis

Three analogs of mycobactin T, the siderophore secreted by Mycobacterium tuberculosis (Mtb) were synthesized and screened for their antibiotic activity against Mtb H(37)Rv and a broad panel of Gram-positive and Gram-negative bacteria. The synthetic mycobactins were potent (MIC(90) 0.02-0.88 μM in 7H12 media) and selective Mtb inhibitors, with no inhibitory activity observed against any other of the microorganisms tested. The maleimide-containing analog 40 represents a versatile platform for the development of mycobactin-drug conjugates, as well as other applications.     

Homology modeling and molecular dynamic simulation of UDP-N-acetylmuramoyl-l-alanine-d-glutamate ligase (MurD) from Mycobacterium tuberculosis H37Rv using in silico approach

The present study aimed to identify the prospective inhibitors of MurD, a cytoplasmic enzyme that catalyzes the addition of d-glutamate to the UDP-N-acetylmuramoyl-l-alanine nucleotide precursor in Mycobacterium tuberculosis (MTB), using virtual screening, docking studies, pharmacokinetic analysis, Molecular Dynamic (MD) simulation, and Molecular Mechanics Generalized Born and Surface Area (MM-GBSA) analyses. The three dimensional (3D) structure was determined based on the homology technique using a template from Streptococcus agalactiae. The modeled structure had three binding sites, namely; substrate binding site (Val18, Thr19, Asp39, Asp40, Gly75, Asn147, Gln171 and His192), the ATP binding site (Gly123, Lys124, Thr125, Thr126, Glu166, Asp283, and Arg314) and the glutamic acid binding site (Arg382, Ser463, and Tyr470). These residues mentioned above play a critical role in the catalytic activity of the enzyme, and their inhibition could serve as a stumbling block to the normal function of the enzyme. A total of 10,344 obtained from virtual screened of Zinc and PubChem databases. These compounds further screened for Lipinski rule of five, docking studies and pharmacokinetic analysis. Four compounds with good binding energies (ZINC11881196 = −10.33 kcal/mol, ZINC12247644 = −8.90 kcal/mol, ZINC14995379 =−8.42 kcal/mol, and PubChem6185 = −8.20 kcal/mol), better than the binding energies of the ATP (−2.31 kcal/mol) and the ligand with known IC₅₀, Aminothiazole (−7.11 kcal/mol) were selected for the MD simulation and MM-GBSA analyses. The result of the analyses showed that all the four ligands formed a stable complex and had the binding free energies better than the binding energy of ATP. Therefore, these ligands considered as suitable prospective inhibitors of the MurD after experimental validation.     

Computational studies on N-phenyl pyrrole derivatives as MmpL3 inhibitors in Mycobacterium tuberculosis

The fight against tuberculosis (TB) is a time immemorial one and the emergence of new drug resistant strains of Mycobacterium tuberculosis keeps throwing new challenges to the scientific community immersed in finding mechanisms to control this dreaded disease. Computer aided drug designing (CADD) is one of the several approaches that can assist in identifying the potent actives against Mycobacterium. In this work, a series of 109 known Mycobacterial membrane proteins large 3 (MmpL3) inhibitors were pooled and atom based 3D QSAR analysis was performed to understand the structural features essential for inhibitory activity against the MmpL3, known to be a key player in transporting substances critical for cell wall integrity of Mycobacterium. The data set employed was randomly split into training set and test set molecules. The training set of 74 molecules was used to derive CoMFA and CoMSIA models that were statistically reliable (CoMFA: q²_loo = 0.53; r²_ncv = 0.93 and CoMSIA: q²_loo = 0.60; r²_ncv = 0.93). The derived models also exhibited good external predictive ability (CoMFA: r²_pred = 0.78 and CoMSIA: r²_pred = 0.79). The results are quite encouraging and information derived from these analyses was applied to design new molecules. The designed molecule showed appreciable predicted activity values and reasonably good ADMET profile. The strategy used in designing new molecules can be pursued in the hunt for new chemical entities targeting MmpL3, expanding the existing arsenal against TB.     

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.     

Total Synthesis of a Mycolic Acid from Mycobacterium tuberculosis

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters (“cord factor”) form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram‐scale synthesis of four stereo‐isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn‐methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material.     

Total synthesis of enantiopure beta-D-mannosyl phosphomycoketides from Mycobacterium tuberculosis

The first stereoselective total synthesis of a beta-d-mannosyl phosphomycoketide is reported. To introduce the stereogenic centers in the chain, three linear chiral building blocks were prepared using two different asymmetric catalytic conjugate addition protocols. Coupling of the various linear fragments was affected using a Julia-Kocienski sequence. This approach constitutes a general and convergent method for the construction of saturated oligoisoprenoid chains of any length and stereochemistry. In addition, an alternative approach for the formation of the difficult beta-mannosyl phosphate linkage was shown to be successful. Biological evalutation of the all-S compound revealed that its antigenic potency for T cells is identical to that of the natural product. This result implies that the fine structure of the lipid part has a strong influence on biological activity and that the T cell response is discriminating between different stereoisomers.     

Structural characterization of lipoarabinomannans from Mycobacterium tuberculosis and Mycobacterium smegmatis by ESI mass spectrometry

Structural aspects of lipoarabinomannans (LAM) from Mycobacterium tuberculosis and Mycobacterium smegmatis were investigated by using mild acid hydrolysis in combination with Fourier-transform ion cyclotron resonance (FT-ICR), and quadrupole ion trap mass spectrometry. Exact mass measurements with less than 2.5 ppm mass error confirmed the presence of a series of arabinose oligomers (Ara(n); n = 2-7) as the major components observed following mild acid hydrolysis of both M. tuberculosis and M. smegmatis LAM. However, the mass spectrum of the resulting LAM extract also revealed a highly-abundant distribution of ions that exact mass measurements identified as mannose-linked arabinose species, Ara(n)Man(m) + Na+ (n = 1-6; m = 1-3). The observed mannose caps were linked to arabinose species as mono-, di-, and trimannose units, and the ratio of the mono-, di-, and trimannose caps was determined to be 1.00:9.00:1.15, respectively, different from previous reports. Analysis of the linkage of lithiated arabinose trimer standards was accomplished with MS3 experiments and the information generated was used to identify linkages of arabinose trimers generated by mild acid hydrolysis of M. tuberculosis and M. smegmatis LAM. The MS3 spectra confirmed the linkage of arabinose trimers from M. smegmatis and M. tuberculosis LAM as predominantly alpha(1 --> 5), alpha(1 --> 5).     

Structure-based design,synthesis of novel inhibitors of Mycobacterium tuberculosis FabH as potential anti-tuberculosis agents

Mycobacterium tuberculosis FabH,an essential enzyme in mycolic acids biosynthetic pathway,is an attractive target for novel anti-tuberculosis agents.Structure-based design,synthesis of novel inhibitors of mtFabH was reported in this paper.A novel scaffold structure was designed,and 12 candidate compounds that displayed favorable binding with the active site were identified and synthesized.     

Design, synthesis, and biological evaluation of beta-ketosulfonamide adenylation inhibitors as potential antitubercular agents

[reaction: see text] The antitubercular nucleoside antibiotics 1 and 2 were recently described that inhibit the adenylate-forming enzyme MbtA and disrupt biosynthesis of the virulence-conferring siderophore known as mycobactin in Mycobacterium tuberculosis. Herein, we report efforts to refine this inhibitor scaffold by replacing the labile acylsulfamate linkage (highlighted) with the more chemically robust beta-ketosulfonamide linkage of 3 and 4.     

The methyl-branched fortifications of Mycobacterium tuberculosis

Mycobacterium tuberculosis continues to be the predominant global infectious agent, annually killing over three million people. Recommended drug regimens have the potential to control tuberculosis, but lack of adherence to such regimens has resulted in the emergence of resistant strains. Mycobacterium tuberculosis has an unusual cell envelope, rich in unique long-chain lipids, that provides a very hydrophobic barrier to antibiotic access. Such lipids, however, can be drug targets, as exemplified by the action of the front-line drug isoniazid on mycolic acid biosynthesis. A number of these lipids are potential key virulence factors and their structures are based on very characteristic methyl-branched long-chain acids and alcohols. This review details the history, structure, and genetic aspects of the biosynthesis of these methyl-branched components, good examples of which are the phthiocerols and the mycocerosic and mycolipenic acids.     

Towards the proteome of Mycobacterium tuberculosis

Human tuberculosis is caused by the intracellular pathogen Mycobacterium tuberculosis. Sequencing of the genome of M. tuberculosis strain H37Rv has predicted 3924 open reading frames, and enabled identification of proteins from this bacterium by peptide mass fingerprinting. Extracellular proteins from the culture medium and proteins in cellular extracts were examined by two-dimensional gel electrophoresis using immobilized pH gradient technology. By mass spectrometry and immunodetection, 49 culture filtrate proteins and 118 lysate proteins were identified, 83 of which were novel. To date, 288 proteins have been identified in M. tuberculosis proteome studies, and a list is presented which includes all identified proteins (available at http://www.ssi.dk/publichealth/tbimmun). The information obtained from the M. tuberculosis proteome so far is discussed in relation to the information obtained from the complete genome sequence.     

The absence of proximal strain in the truncated hemoglobins from Mycobacterium tuberculosis

HbN and HbO are two truncated hemoglobins from Mycobacterium tuberculosis. Resonance Raman spectra of the deoxy derivatives of these two homodimeric hemoglobins indicate that there is no proximal strain imposed by intersubunit interactions on the proximal iron-histidine bond as that observed in the tetrameric human hemoglobin. In addition, with nanosecond laser flash photolysis, it was concluded that movement along the Fe-His bond following the dissociation of CO does not trigger a quaternary structural transition in these two hemoglobins.