| Summary: | 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
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