Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Biochim Biophys Acta ; 1854(10 Pt A): 1338-50, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25988243

ABSTRACT

Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 µg/ml and 6.24 µg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.


Subject(s)
Acetolactate Synthase/antagonists & inhibitors , Antitubercular Agents/chemistry , Aptamers, Nucleotide/chemistry , Bacterial Proteins/antagonists & inhibitors , DNA, Single-Stranded/chemistry , Enzyme Inhibitors/chemistry , Mycobacterium tuberculosis/drug effects , Acetolactate Synthase/chemistry , Acetolactate Synthase/genetics , Animals , Antitubercular Agents/metabolism , Antitubercular Agents/pharmacology , Aptamers, Nucleotide/biosynthesis , Aptamers, Nucleotide/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Cell Survival/drug effects , DNA, Single-Stranded/biosynthesis , DNA, Single-Stranded/pharmacology , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Library , Macrophages/cytology , Macrophages/drug effects , Mice , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Sequence Data , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/growth & development , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , SELEX Aptamer Technique
2.
Eur J Med Chem ; 94: 298-305, 2015 Apr 13.
Article in English | MEDLINE | ID: mdl-25771108

ABSTRACT

Acetohydroxyacid synthase (AHAS) catalyzes the first essential biosynthetic step of branched-chain amino acids and is a biologically safe target against Mycobacterium tuberculosis (MTB). In our previous research, we used virtual screening to identify some novel AHAS inhibitors as potent antituberculosis agents. In this study, we synthesized twenty-four additional quinazolinone benzoates and explored their antitubercular activity. Five of these compounds displayed significant MTB-AHAS inhibition and their IC50 values were determined to be in the range of 6.50 µM-12.08 µM. Importantly, these compounds also exhibited strong in vitro activity (MICs in the range of 2.5-10 mg/L) and intracellular activity against clinically isolated extensively drug-resistant strains of M. tuberculosis. Taken together, these results indicated that the quinazolinone benzoate compounds should be regarded as promising lead compounds for the development of potent antituberculosis drugs with a novel mode of action.


Subject(s)
Acetolactate Synthase/antagonists & inhibitors , Antitubercular Agents/pharmacology , Benzoates/pharmacology , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/drug effects , Quinazolinones/pharmacology , Acetolactate Synthase/metabolism , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Benzoates/chemical synthesis , Benzoates/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Quinazolinones/chemical synthesis , Quinazolinones/chemistry , Structure-Activity Relationship
3.
Int J Biol Macromol ; 72: 104-9, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25128823

ABSTRACT

Catabolic acetolactate synthase (cALS) from Enterococcus faecalis is a FAD-independent enzyme, which catalyzes the condensation of two molecules of pyruvate to produce acetolactate. Mutational and kinetic analyses of variants suggested the importance of H111, Q112, and Q411 residues for catalysis in cALS. The wild-type and variants were expressed as equally soluble proteins and co-migrated to a size of 60 kDa on SDS-PAGE. Importantly, H111 in cALS, which is widely present as phenylalanine in many other ThDP-dependent enzymes, plays a crucial role in substrate binding. Interestingly, the H111 variants, H111R and H111F, demonstrated altered specific activity of H111 variants with 17- and 26-fold increases in Km, respectively, compared to wild-type cALS. Furthermore, Q112 variants, Q112E, Q112N, and Q112V, exhibited significantly lower specific activity with 70-, 15-, and 10-fold higher Ks for ThDP, respectively. In the case of Q411, the variant Q411E showed a 10-fold rise in Km and a 20-fold increase in Ks for ThDP. Further, the molecular docking results indicated that the binding mode of ThDP was slightly affected in the variants of cALS. Based on these results, we suggest that H111 plays a role in substrate binding, and further suggest that Q112 and Q411 might be involved in ThDP binding of cALS.


Subject(s)
Acetolactate Synthase/genetics , Amino Acids/genetics , Enterococcus faecalis/enzymology , Flavin-Adenine Dinucleotide/metabolism , Mutagenesis, Site-Directed/methods , Acetolactate Synthase/chemistry , Acetolactate Synthase/isolation & purification , Amino Acid Sequence , DNA Mutational Analysis , Electrophoresis, Polyacrylamide Gel , Kinetics , Molecular Docking Simulation , Molecular Sequence Data , Pyruvic Acid/metabolism , Sequence Alignment , Spectrometry, Fluorescence , Structural Homology, Protein , Thiamine Pyrophosphate/metabolism , Tryptophan/metabolism
4.
Appl Biochem Biotechnol ; 174(2): 793-802, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25096391

ABSTRACT

Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica subsp. enterica ser. typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. The development of an early and ultra-sensitive detection system is the first critical step in controlling this disease. To accomplish this, we used the cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. A total of 12 target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX under stringent selection conditions, and negative selection further enhanced the selectivity among these aptamers. Aptamer specificity was investigated using the gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar to submicromolar levels. Furthermore, individual aptamers were conjugated onto polyvalent directed aptamer polymer, which led to 100-fold increase in binding affinity compared to the individual aptamers alone. Taken together, this study reports the identification of higher affinity and specificity ssDNA aptamers (30mer), which may be useful as capture and detection probes in biosensor-based detection systems for salmonellosis.


Subject(s)
Aptamers, Nucleotide/chemistry , DNA, Single-Stranded/chemistry , Salmonella enteritidis/isolation & purification , Salmonella typhimurium/isolation & purification , Base Sequence , DNA Primers , Limit of Detection , Polymerase Chain Reaction , SELEX Aptamer Technique
5.
Enzyme Microb Technol ; 58-59: 52-9, 2014 May 10.
Article in English | MEDLINE | ID: mdl-24731825

ABSTRACT

Mycobacterium tuberculosis AHAS is a potential target for the development of novel anti-tuberculosis agents. Silico analysis showed that conserved His84 and Gln86 residues lie in the catalytic dimer interface of M. tuberculosis AHAS. Mutational analyses of these invariants led to significant reduction in their activity with reduced affinity toward the substrate. Importantly, mutation of Gln86 to Trp abolished complete activity. Further, molecular dynamics simulation studies suggested that these residues are likely to play a key role in maintaining the Glu85 side chain in the required geometry with N1' atom of ThDP during catalysis. In addition, substitution of essential Glu85 by Ala, Asp, and Gln led to severe drop in catalytic activity with reduced affinity toward ThDP confirming its catalytic role in M. tuberculosis AHAS.


Subject(s)
Acetolactate Synthase/chemistry , Bacterial Proteins/chemistry , Mycobacterium tuberculosis/enzymology , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Catalysis , Conserved Sequence , Dimerization , Escherichia coli/metabolism , Glutamic Acid/chemistry , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Thiamine Pyrophosphate/metabolism
6.
Enzyme Microb Technol ; 53(4): 243-9, 2013 Sep 10.
Article in English | MEDLINE | ID: mdl-23931689

ABSTRACT

Acetohydroxyacid synthase (AHAS) of Mycobacterium tuberculosis is a promising target for the development of anti-tuberculosis agents. With the absence of an available bacterial AHAS crystal structure, that of M. tuberculosis, site-directed mutagenesis has been a useful tool for determining its structural and functional features. In this study, a highly conserved proline residue (P126 of M. tuberculosis AHAS) was selected, and the possible role was evaluated by site-directed mutagenesis. P126 was replaced by valine, threonine, alanine, and glutamate to yield P126V, P126T, P126A, and P126E, respectively. All variants were expressed in their soluble forms in Escherichia coli and purified to near homogeneity. The molecular mass (SDS-PAGE) of the purified variants was ∼68 kDa, which is similar to that of wild-type AHAS. The P126V, P126T, and P126A variants exhibited significantly lower activity than wild-type AHAS, whereas P126E was inactive under the tested assay conditions. Furthermore, the P126V and P126T variants showed a significantly decreased preference toward pyruvate and ThDP as substrate and cofactor respectively, whereas the P126A showed similar kinetics to that of wild-type AHAS. Like in AHAS from yeast Saccharomyces cerevisiae (PDB ID: 1N0H), residue P126 is located in the ThDP binding pocket of M. tuberculosis AHAS homology model. Collectively, these results suggest that the conserved P126 plays a significant role in the ThDP binding of M. tuberculosis AHAS.


Subject(s)
Acetolactate Synthase/metabolism , Mycobacterium tuberculosis/enzymology , Thiamine Pyrophosphate/metabolism , Acetolactate Synthase/chemistry , Acetolactate Synthase/genetics , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain/genetics , Conserved Sequence , Genes, Bacterial , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mycobacterium tuberculosis/genetics , Proline/chemistry , Protein Conformation , Sequence Homology, Amino Acid , Structural Homology, Protein
7.
Biochimie ; 95(7): 1411-21, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23523771

ABSTRACT

Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a Km of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The Kd for thiamine diphosphate (ThDP) was 89.92 ± 17.9 µM, as determined by fluorescence quenching. The cofactor activation constants (Ks) for ThDP and (Kc) for Mg(2+) were 0.6 ± 0.1 and 560.8 ± 7.4 µM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC50 values of 1.19 µM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 µg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (-7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.


Subject(s)
Acetolactate Synthase/antagonists & inhibitors , Bacterial Proteins/antagonists & inhibitors , Pseudomonas aeruginosa/enzymology , Acetolactate Synthase/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Hydrogen Bonding , Hydrogen-Ion Concentration , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Temperature
SELECTION OF CITATIONS
SEARCH DETAIL
...