Characterization of competitive inhibitors for the transferase activity of Pseudomonas aeruginosa exotoxin A.
A series of small, nonpolar compounds were tested for their ability to inhibit the ADP-ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A. The IC50 values for the compounds tested ranged from 87 nM to 484 microM for NAP and CMP12, respectively. It was demonstrated that NAP was a competitive inhibitor of the ADPRT reaction for the NAD+ substrate with a Ki of 45 +/- 5 nM, which was in good agreement with the dissociation constant determined independently (KD = 56 +/- 6 nM). The IC50 value for NAP was 87 +/- 12 nM, which strongly correlated with the Ki and KD values. Furthermore, NAP was shown to noncovalently associate with the exotoxin A active site using exhaustive dialysis, NMR, and electrospray mass spectrometry. Finally, a computer molecular model using the X-ray structure of the substrate-bound toxin was generated with NAP bound to the active site of exotoxin A at the nicotinamide-binding site. This model is consistent with the X-ray structure of the catalytic domain of poly-ADP-ribose polymerase complexed with 4-amino-naphthalimide (Compound 4) that was included in this study.
Armstrong
Souzan
S
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ont., Canada N1G 2W1.
Li
Jia-He
JH
Zhang
Jie
J
Merrill
A Rod
AR
D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005098:Exotoxins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011485:Protein Binding; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D037521:Virulence Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D005098:Exotoxins; D009243:NAD; D009842:Oligopeptides; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C425904:davunetide