A direct, real-time, spectrophotometric assay for measuring ENPP1-catalyzed cGAMP hydrolysis.

The ectonucleotidase ENPP1 is the major extracellular hydrolase of the innate immune system activator 2'3'-cyclic GMP-AMP (cGAMP). Tumors that overexpress ENPP1 and rapidly degrade cGAMP avoid immune surveillance in the tumor microenvironment and are highly resistant to cancer immunotherapy. Inhibition of cGAMP degradation by ENPP1 has emerged as a promising strategy to improve cancer therapies. A direct, real-time assay of ENPP1 enzymatic activity would benefit quantitative evaluation of candidate ENPP1 inhibitors. The nonphysiological substrate p-nitrophenyl 5'-thymidine monophosphate is commonly used for this purpose, as it offers a readily detectable colorimetric readout that can be evaluated in real time. However, compounds that potently inhibit p-nitrophenyl 5'-thymidine monophosphate hydrolysis can weakly inhibit ENPP1 with physiological nucleotide substrates, highlighting the importance of testing ENPP1 inhibitors with native substrates (i.e., cGAMP). No direct, real-time assays for cGAMP hydrolysis are established. Here, we present a real-time, spectrophotometric assay to monitor ENPP1-catalyzed cGAMP hydrolysis. The increase in extinction coefficient associated with conversion of substrate(s) to AMP and GMP products is used to convert time courses of absorbance change to rates of product formation. Time courses of GMP product formation generated from the absorbance change superimpose with those generated by the direct measurement of GMP product concentration via chemical quench-flow and HPLC analysis. ENPP1 inhibition by the nonhydrolyzable ATP analog, α,β-methylene-ATP, yields an inhibition constant (K