Abstact

Adenosine monophosphate deaminase 2 (AMPD2) catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate and is believed to play a significant role in nucleotide metabolism, energy homeostasis, and immune oncology. Three primary AMPD isozymes, designated as M (muscle), L (liver), and E (erythrocyte) forms, have been identified. However, due to the high similarity of the catalytic site's amino acid sequence and structural topologies, most reported orthosteric inhibitors exhibit minimal selectivity toward AMPD isozymes. There is therefore a significant need for selective AMPD2 inhibitors as tools for validating the biological roles of AMPD2. In this study, we hypothesized that allosteric AMPD2 inhibitors would show selectivity towards other AMPD isoforms and used an X-ray fragment screening approach to identify these inhibitors. Consequently, we identified compound 5, which showed the capacity to bind to a previously uncharacterized allosteric site. The pharmacophore search based on structural information around 5 and the following X-ray screening also identified 6 and 8, which bind to the same site as 5. The merging of the initial fragment hit 5 with the secondary fragment hit 6 yielded 7, which was further merged with 8, resulting in the successful generation of 9. Moreover, the optimization of 9 led to more potent selective inhibitors 10g and 10h. Our results suggest that the fragment merging method using X-ray screening and pharmacophore searching provides effective opportunities to improve the affinity of the fragment hits. In addition, we believe that these selective compounds could be used as tool compounds for studying the biological roles of AMPD2.