Biophysical analysis of the interaction between novel inhibitors and N-acyl ethanolamine acid amidase (NAAA)

• Online Access: http://hdl.handle.net/2047/D20384358

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (NtN) enzyme with a catalytic cysteine residue. The most prominent substrate hydrolyzed is palmitoylethanolamine (PEA), which regulates inflammation. Our inhibitors of NAAA result in increased endogenous levels of PEA and are of interest as potential treatments for inflammatory disorders. We have synthesized two series of inhibitors with a) isothiocyanate and b) cyanamide functional groups that have both been shown to be very potent with low nanomolar IC50 values. These inhibitor classes were both expected to be non-reversible, however, biochemical analysis has indicated that the mechanism of interaction between these two series and the catalytic cysteine residue may differ. We therefore seek to closely characterize the interaction of these inhibitors with the NAAA active site to inform the design of novel analogs. We present efforts towards producing biophysical analyses to observe ligand-protein interactions. This has included stable expression of a construct to produce efficient high yield of the enzyme, MS and NMR analyses, and efforts towards producing a crystal structure model. Combined with biochemical data, our results suggest the isothiocyanate functional group results in a more transient interaction with the catalytic cysteine than the cyanamide functional group.--Author's abstract