Transition State Analogues of Plasmodium falciparum and Human Orotate Phosphoribosyltransferases

• Published in: The Journal of biological chemistry Vol. 288; no. 48; pp. 34746 - 34754
• Main Authors: Zhang, Yong, Evans, Gary B., Clinch, Keith, Crump, Douglas R., Harris, Lawrence D., Fröhlich, Richard F.G., Tyler, Peter C., Hazleton, Keith Z., Cassera, María B., Schramm, Vern L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29.11.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Antimalarials - chemistry; Binding Sites; Biosynthesis; Enzyme Catalysis; Enzymology; Humans; Hydrogen Bonding; Inhibitor Design; Kinetics; Malaria; Malaria - drug therapy; Malaria - enzymology; Malaria - parasitology; Metabolic Networks and Pathways; N-Ribosyltransferases; Nucleosides; Orotate Phosphoribosyltransferase - chemistry; Orotate Phosphoribosyltransferase - genetics; Orotate Phosphoribosyltransferase - metabolism; Orotidine 5′-Monophosphate; Plasmodium; Plasmodium falciparum - chemistry; Plasmodium falciparum - enzymology; Plasmodium falciparum - metabolism; Protein Conformation; Pyrimidine; Pyrimidines - biosynthesis; Pyrimidines - chemistry; Pyrrolidines - pharmacology; Substrate Specificity; Plasmodium; Inhibitor Design; Malaria; N-Ribosyltransferases; Enzyme Catalysis; Orotidine 5′-Monophosphate; Biosynthesis; Pyrimidine
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.521955

The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (Kd) as low as 80 nm. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5′-phosphate bound to OPRTs with Kd values near 40 nm. Analogues designed with a C5-pyrimidine carbon–carbon bond to ribocation mimics gave Kd values in the range of 80–500 nm. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5′-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum. Background: Human and malaria orotate phosphoribosyltransferases (OPRTs) in the de novo pyrimidine biosynthesis pathway are characterized by ribocation transition states with fully dissociated leaving orotate groups. Results: Transition state analogues with varied ribocation substitutions, different linkers, and distinct leaving groups exhibited nanomolar binding affinities for the OPRT enzymes. Conclusion: Components crucial for inhibitor binding to the OPRT active sites have been identified, and powerful inhibitors were characterized. Significance: Transition state analogues of OPRTs provide new insights into the nature of potential antimalarials and anticancer agents.