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1.
BMC Microbiol ; 17(1): 122, 2017 05 25.
Article in English | MEDLINE | ID: mdl-28545531

ABSTRACT

BACKGROUND: Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors. RESULTS: We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 µM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 µg/ml. These compounds have no activity on horseradish peroxidase and D-amino acid oxidase at a concentration of 50 µM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 µM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 µM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase. CONCLUSIONS: Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.


Subject(s)
Aeromonas hydrophila/drug effects , Alanine Racemase/drug effects , Anti-Bacterial Agents/pharmacology , Drug Discovery , Aeromonas hydrophila/enzymology , Aeromonas hydrophila/growth & development , Anti-Bacterial Agents/chemistry , Catalytic Domain/drug effects , Cell Survival/drug effects , D-Amino-Acid Oxidase/drug effects , Drug Evaluation, Preclinical , Enzyme Assays , HeLa Cells/drug effects , Homogentisic Acid/antagonists & inhibitors , Homogentisic Acid/chemistry , Horseradish Peroxidase/drug effects , Humans , Hydroquinones/antagonists & inhibitors , Hydroquinones/chemistry , Inhibitory Concentration 50 , Kinetics , Microbial Sensitivity Tests , Molecular Docking Simulation/methods , Patulin/antagonists & inhibitors , Patulin/chemistry
2.
FEBS J ; 273(3): 504-12, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16420474

ABSTRACT

The flavoenzyme d-amino acid oxidase from Rhodotorula gracilis is a homodimeric protein whose dimeric state has been proposed to occur as a result of (a) the electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3'' of the other monomer, and (b) the interaction of residues (e.g. Trp243) belonging to the two monomers at the mixed interface region. The role of Trp243 was investigated by substituting it with either tyrosine or isoleucine: both substitutions were nondisruptive, as confirmed by the absence of significant changes in catalytic activity, but altered the tertiary structure (yielding a looser conformation) and decreased the stability towards temperature and denaturants. The change in conformation interferes both with the interaction of the coenzyme to the apoprotein moiety (although the kinetics of the apoprotein-FAD complex reconstitution process are similar between wild-type and mutant D-amino acid oxidases) and with the interaction between monomers. Our results indicate that, in the folded holoenzyme, Trp243 is situated at a position optimal for increasing the interactions between monomers by maximizing van der Waals interactions and by efficiently excluding solvent.


Subject(s)
D-Amino-Acid Oxidase/chemistry , Rhodotorula/enzymology , Tryptophan/chemistry , D-Amino-Acid Oxidase/drug effects , Enzyme Activation , Enzyme Stability/drug effects , Kinetics , Models, Molecular , Mutation , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Static Electricity , Structure-Activity Relationship , Temperature , Time Factors , Urea/pharmacology
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