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Using recently developed molecular-shape description algorithms, we searched the Available Chemical Directory for known compounds similar in shape to the potent HIV-1 protease inhibitor Merck L-700,417; 15 compounds most similar in shape to the inhibitor were selected for testing in vitro. Four of these inhibited the protease at 100 microM or less and the most active of the four were the naturally occurring pigments biliverdin and bilirubin. Biliverdin and bilirubin inhibited recombinant HIV-1 protease in vitro at pH 7.8 with K1 values of approx. 1 microM, and also inhibited HIV-2 and simian immunodeficiency virus proteases. The related pyrrolic pigments stercobilin, urobilin, biliverdin dimethyl ester and xanthobilirubic acid showed similar inhibitory activity at low micromolar concentrations. Biliverdin, bilirubin and xanthobilirubic acid did not inhibit viral polyprotein processing in cultured cells, but they reduced viral infectivity significantly. At 100 microM, xanthobilirubic acid affected viral assembly, resulting in a 50% decrease in the generation of infectious particles. In contrast, at the same concentrations biliverdin and bilirubin exerted little or no effect on viral assembly but blocked infection of HeLaT4 cells by 50%. These results suggest that bile pigments might be a new class of potential lead compounds for developing protease inhibitors and they raise the question of whether hyperbilirubinaemia can influence the course of HIV infection.

The metabolic fate of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been examined in rat and rabbit liver mitochondrial and rabbit liver microsomal preparations. The mitochondrial preparations rapidly oxidized MPTP, in a pargyline-sensitive reaction, to a polar material that was shown to contain the 1-methyl-4-phenylpyridinium species as the principal product. NADPH-supplemented microsomal preparations converted MPTP to two principal products: 4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine N-oxide. Carbon monoxide and SKF 525A selectively inhibited the oxidation of MPTP to the nor compound, indicating that this N-demethylation reaction is cytochrome P-450 catalyzed. Attempts to trap possible unstable iminium metabolites of MPTP in microsomal incubation mixtures with sodium cyanide led to the isolation of a monocyano adduct that proved to be the N-cyanomethyl derivative. Thus, hepatic mitochondrial and microsomal enzyme systems catalyze the oxidation of MPTP by different pathways, the former leading to the generation of species that may possess neurotoxic properties.

The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is biotransformed by brain monoamine oxidase (MAO) to an unstable dihydropyridinium intermediate that reacts with cyanide ion to form an alpha-cyano-tetrahydropyridine adduct and, in the absence of cyanide ion, undergoes disproportionation to the 1-methyl-4-phenylpyridinium species MPP+ and MPTP. Comparison of the HPLC retention times, diode array UV, and chemical ion mass spectral characteristics of these products with those of synthetic standards led us to propose the 1-methyl-4-phenyl-2,3-dihydropyridinium species 2,3-MPDP+ and 6-cyano-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as tentative structure assignments for the dihydropyridinium metabolite and the cyano adduct, respectively. Results presented in this paper confirm the first assignment and establish that, although the proposed 6-cyano adduct is initially formed, the product that was isolated from the mitochondrial incubation mixtures of MPTP and sodium cyanide actually is the isomeric 2-cyano-1-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine. On the basis of the selective incorporation of deuterium into these products, we provide rational mechanistic interpretations of the disproportionation reaction and the rearrangement of the cyano adducts. These results establish that the MAO-catalyzed bioactivation of MPTP leads to the formation of a variety of reactive molecules that are potentially cytotoxic to nigrostriatal cells.

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