Inhibition of 3(17).beta.-hydroxysteroid dehydrogenase from Pseudomonas testosteroni by steroidal A ring fused pyrazoles

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Abstract
Several 2,3- and 3,4-steroidal fused pyrazoles have been investigated as potential inhibitors of NAD(P)H-dependent steroid oxidoreductases. These compounds are proven to be potent, specific inhibitors for 3(17).beta.-hydroxysteroid dehydrogenase from Pseudomonas testosteroni with Ki values of 6-100 nM. In contrast, the activities of 3.alpha.,20.beta.-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, steroid 5.alpha.-reductase from rat prostate, and 3.alpha.-hydroxysteroid dehydrogenase from rat liver were unaffected by micromolar concentrations of these compounds. Product and dead-end inhibition studies indicate an ordered association to the .beta.-dehydrogenase with the cofactor binding prior to substrate or inhibitor. From the results of double inhibition experiments, it is proposed that inhibition occurs through formation of an enzyme.sbd.NAD+.sbd.inhibitor ternate. On the basis of pH profiles of Vm/Km, Vm, and 1/Ki and of absorbance difference spectra, a hypothetical mechanism of inhibition by the steroidal pyrazoles, drawn by analogy from the inhibition of liver alcohol dehydrogenase by alkylpyrazoles [Theorell, H., and Yonetani, T. (1963) Biochem. Z. 338, 537-553; Andersson, P., Kvassman, J.K., Lindstrom, A. Olden, B., and Pettersson, G. (1981) Eur. J. Biochem. 113, 549-554], is reconsidered. The pH studies and enzyme modification experiments by diethyl pyrocarbonate suggest the involvement of histidine in binding of the inhibitor. A modified proposal for the structure of the enzyme.sbd.NAD+.sbd.steroidal pyrazole complex is proposed. In this ternate, the inhibitor bridges an enzymatic histidine and the nicotinamide ring of the NAD+ through two hydrogen bonds: one formed between the lone pair of electrons of an imidazole nitrogen and the pyrazole N.sbd.H with the second between the lone pair of electrons of the second pyrazole nitrogen and the electropositive cofactor.