Inhibition mechanisms and structural complexes of toxins with inhibitors

The mono-ADP-ribosyltransferase (mono-ADPRT), Pseudomonas aeruginosa exotoxin A (ETA), catalyzes the transfer of ADP-ribose from NAD⁺ to its protein substrate.

Our lab has investigated both nonpolar and water-soluble compounds that structurally mimic the nicotinamide moiety of NAD⁺ for their ability to inhibit the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC₅₀ values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with a K_i of 140 nM. We also recently reported the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 Å resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex to diphtheria toxin (a mono-ADPRT) and to PARPs (poly- (ADP-ribosyl) polymerases) shows similarity of the catalytic residues, however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. This work provides insight into the important features required for inhibitors that mimic NAD⁺ and their binding to the mono-ADPRT family of toxins. We are currently characterizing new inhibitors and determining the X-ray structures of these in complex with toxin.

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