Summary /

The binding mechanism of ML132 is studied through molecular modeling using 2HBQ, a co-crystal of caspase 1 and Z-VAD-FMK (Romanowski 2004, Wilson 1994, Okamoto 1999).  The presumption of a covalent reversible mechanism of inhibition when building a model for binding the probe is used. The nitrile carbon was therefore held at a proximal distance (2.6 Å) from the catalytic cysteine residue (C285) by constraint docking, and flexibility was granted to the remainder of the small molecule to achieve an optimal binding pose using FRED12. The results demonstrate complementarity between the peptidic fragment of ML132 and the peptide binding domain of caspase 1. Key interactions were noted for the acid moiety and arginine residues 341 and 179 in similar fashion to other Asp containing small molecule caspase 1 inhibitors. While direct interrogation of a covalent interaction between the nitrile and C285 was not pursued in our model, this representation does illustrate the open binding cavity that accommodates the tetrahedral intermediate that forms as a result of covalent binding with aldehyde based inhibitors (a mimetic of the hemithiolacetal intermediate associated with transition state 1 (TS1) during proteolysis). In contrast, covalent interactions between a thiol and a nitrile form a thioimidate intermediate that mimics transition state 2 (TS2) of an enzymatic proteolytic event between a cysteine proteases and a substrate. Ménard and coworkers examined aldehyde and nitrile inhibitors of papain and found that the thioimidate intermediate engages the oxyanion hole interaction in a manner that more closely mimics the natural process of hydrolysis during proteolysis13. This may have consequences for both the binding affinity of nitrile-based cysteine proteases inhibitors and their ultimate resolution through hydrolysis of the thioimidate intermediate.

Figure 1. Molecular model (ribbon and space filling) of ML132 bound to Caspase 1.