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A Functional Genetic Polymorphism on Human Carbonyl Reductase 1 (CBR1 V88I) Impacts on Catalytic Activity and NADPH Binding Affinity

Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, New York (V.G.-C., S.S.L, J.G.B.); and Department of Structural Biology, Hauptman-Woodward Medical Research Institute and Department of Pharmacology and Therapeutics (D.G.), and Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York (L.P.)

Human carbonyl reductase 1 (CBR1) metabolizes endogenous and xenobiotic substrates such as the fever mediator, prostaglandin E2 (PGE₂), and the anticancer anthracycline drug, daunorubicin. We screened 33 CBR1 full-length cDNA samples from white and black liver donors and performed database analyses to identify genetic determinants of CBR1 activity. We pinpointed a single nucleotide polymorphism on CBR1 (CBR1 V88I) that encodes for a valine-to-isoleucine substitution for further characterization. We detected the CBR1 V88I polymorphism in DNA samples from individuals with African ancestry (p = 0.986, q = 0.014). Kinetic studies revealed that the CBR1 V88 and CBR1 I88 isoforms have different maximal velocities for daunorubicin (V_max CBR1 V88, 181 ± 13 versus V_max CBR1 I88, 121 ± 12 nmol/min · mg, p < 0.05) and PGE₂ (V_max CBR1 V88, 53 ± 7 versus V_max CBR1 I88, 35 ± 4 nmol/min · mg, p < 0.01). Concomitantly, CBR1 V88 produced higher levels of the cardiotoxic metabolite daunorubicinol compared with CBR1 I88 (1.7-fold, p < 0.0001). Inhibition studies demonstrated that CBR1 V88 and CBR1 I88 are distinctively inhibited by the flavonoid, rutin (IC₅₀ CBR1 V88, 54.0 ± 0.4 µM versus IC₅₀ CBR1 I88, 15.0 ± 0.1 µM, p < 0.001). Furthermore, isothermal titration calorimetry analyses together with molecular modeling studies showed that CBR1 V88I results in CBR1 isoforms with different binding affinities for the cofactor NADPH (K_d CBR1 V88, 6.3 ± 0.6 µM versus K_d CBR1 I88, 3.8 ± 0.5 µM). These studies characterize the first functional genetic determinant of CBR1 activity toward relevant physiological and pharmacological substrates.