Structural, computational, docking and biological studies of a triaminopyrimidine caspase-1 inhibitor

•The X-ray structure of a nanomolar inhibitor of caspase-1 was obtained.•The inhibitor showed protonation at multiple sites in solution.•An X-ray structure of the protonated inhibitor was obtained.•DFT studies supported the protonation sites.•Docking showed that the protonated inhibitors bind well to caspase-1. In order to more fully characterize the binding properties and activities of a potent, low nanomolar IC50 (half maximal inhibitory concentration) inhibitor of caspase-1, CK-1–41, a series of experiments were designed to probe its structure and function. To accomplish this, larger quantities of the molecule were synthesized in an efficient manner using a microwave method, in only 30 min. The structure of the inhibitor was determined by X-ray crystallography in its neutral and protonated form yielding useful structural information. The protonation site in the X-ray structure was found to be the trialkylamine basic site, but nuclear magnetic resonance (NMR) H/D exchange experiments shows that protonation at the triaminopyrimidine sp2 C is also accessible in solution. Density Functional Theory (DFT) calculations were performed and supported the experimental H/D exchange results. Docking studies with several protonated forms of the inhibitor with caspase-1 revealed the compound binds favorably in an allosteric site, with the strongest binding from the protonated form where the proton is bound to the sp2 C. Due to the involvement of caspase-1 in inflammatory cancers, CK-1–41 was screened against a panel of cancer cell lines displaying modest activity against some of the cell lines tested. These studies further clarify the structure and biological function of the representative member of the triaminopyrimidine inhibitors of caspase-1. [Display omitted]