Characterization of competitive inhibitors for the transferase activity of Pseudomonas earuginosa 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 μM for NAP and CMP12, respectively. It was demonstrated that NAP was a competitive inhibitor of the ADPRT reaction for the NAD⁺ substrate with a K_i of 45 ± 5 nM, which was in good agreement with the dissociation constant determined independently (K_D = 56 ± 6 nM). The IC₅₀ value for NAP was 87 ± 12 nM, which strongly correlated with the K_i and K_D 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.