ABSTRACT
The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.
Subject(s)
Acyltransferases/chemistry , Antigens, Bacterial/chemistry , Cysteine/chemistry , Enzyme Inhibitors/chemistry , Acyltransferases/genetics , Acyltransferases/metabolism , Allosteric Regulation , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Azoles/chemistry , Azoles/pharmacology , Biocatalysis/drug effects , Catalytic Domain , Chloromercuribenzoates/chemistry , Chloromercuribenzoates/pharmacology , Crystallography, X-Ray , Cysteine/genetics , Cysteine/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Hydrogen Bonding/drug effects , Iodoacetamide/chemistry , Iodoacetamide/pharmacology , Isoindoles , Models, Molecular , Molecular Structure , Mutation , Organoselenium Compounds/chemistry , Organoselenium Compounds/pharmacology , Protein Conformation , Protein Structure, SecondaryABSTRACT
The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen (EBS) as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that EBS binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of EBS shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antigens, Bacterial/metabolism , Azoles/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Organoselenium Compounds/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Antigens, Bacterial/genetics , Azoles/chemistry , Isoindoles , Membrane Proteins , Models, Molecular , Molecular Structure , Mutation , Mycobacterium tuberculosis/genetics , Organoselenium Compounds/chemistry , Protein Binding , Protein Conformation , Saccharomyces cerevisiae ProteinsABSTRACT
Tuberculosis (TB) is a global health threat with nearly 500 000 new cases of multidrug-resistant TB estimated to occur every year, so new drugs are desperately needed. A number of current antimycobacterial drugs work by interfering with the biosynthesis of key components of the mycolylarabinogalactan (mAG). In light of this observation, other enzymes involved in the synthesis of the mAG should also serve as targets for antimycobacterial drug development. One potential target is the Antigen 85 (Ag85) complex, a family of mycolyltransferases that are responsible for the transfer of mycolic acids from trehalose monomycolate (TMM) to the arabinogalactan. Virtual thiophenyl-arabinoside conjugates were docked to antigen Ag85C (PDB code: 1va5 ) using Glide. Compounds with good docking scores were synthesized by a Gewald synthesis followed by linking to 5-thioarabinofuranosides. The resulting thiophenyl-thioarabinofuranosides were assayed for inhibition of mycoyltransferase activity using a 4-methylumbelliferyl butyrate fluorescence assay. The conjugates showed K(i) values ranging from 18.2 to 71.0 µM. The most potent inhibitor was soaked into crystals of Mycobacterium tuberculosis antigen 85C and the structure of the complex determined. The X-ray structure shows the compound bound within the active site of the enzyme with the thiophene moiety positioned in the putative α-chain binding site of TMM and the arabinofuranoside moiety within the known carbohydrate-binding site as exhibited for the Ag85B-trehalose crystal structure. Unexpectedly, no specific hydrogen bonding interactions are being formed between the arabinofuranoside and the carbohydrate-binding site of the active site suggesting that the binding of the arabinoside within this structure is driven by shape complementarily between the arabinosyl moiety and the carbohydrate binding site.
