Finasteride is employed in treatment of benign prostatic hyperplasia in man, where its target enzyme is steroid 5α-reductase. It is a novel, potent mechanism-based inhibitor of the human prostate (type 2) isozyme. Although it is accepted as an alternate substrate and is ultimately reduced to dihydrofinasteride, this proceeds through an enzyme-bound NADP−dihydrofinasteride adduct. Finasteride is processed with a second-order rate constant, k i/K i = 1 × 106 M-1 s-1, that approaches k cat/K m for reduction of testosterone, 3 × 106 M-1 s-1, and essentially every catalytic event is lethal (partition ratio ≤ 1.07). The membrane-bound enzyme−inhibitor complex formed from [3H]finasteride appears to release [3H]dihydrofinasteride with a half-life of 1 month at 37 °C (k = (2.57 ± 0.03) × 10-7 s-1), as identified by mass spectroscopy. The intermediate NADP−dihydrofinasteride adduct can be recovered intact by denaturation of the enzyme−inhibitor complex and has been purified. Free in solution, it likewise decomposes to dihydrofinasteride (half-life = 11 days). An extremely potent bisubstrate analog inhibitor, this NADP−dihydrofinasteride adduct binds to the free enzyme with a second-order rate constant equal to k cat/K m for turnover of testosterone and has a dissociation constant K i ≤ 1 × 10-13 M. Finasteride is also a mechanism-based inhibitor of the human skin (type 1) isozyme, but it is processed with a much smaller second-order rate constant, k i/K i = 3 × 103 M-1 s-1, which attenuates its activity against this isozyme in vivo. The mechanism explains the exceptional potency and specificity of finasteride in treatment of benign prostatic hyperplasia, and the concept may have application to other pyridine nucleotide-linked enzymes.
A second open reading frame, fkbD, was found upstream of fkbM in all three aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxylases. Disruption of fkbD had a polar effect on the synthesis of the downstream fkbM gene product and resulted in the formation of 9-deoxo-31-O-demethyl-FK506. This established the product of fkbD as the cytochrome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxylation at position C-9 of the FK506 and FK520 macrolactone ring.The polyketide, immunosuppressant compound FK506 ( Fig. 1) (13) is a 23-membered macrolide with potent antifungal activity produced by several Streptomyces species. FK506 is approximately 100-fold more potent than the structurally unrelated immunosuppressive compound cyclosporin A. Both drugs are important therapeutic agents for the prevention of graft rejection following organ and bone marrow transplantations and for the treatment of autoimmune diseases (22). FK520 (also known as immunomycin and ascomycin) is another immunosuppressive compound similar to FK506 (Fig. 1) in which the allyl group is replaced by an ethyl group at position C-21 of the macrolactone ring (9). Both the antifungal and the immunosuppressive activities of FK520 are approximately one-half of those exhibited by FK506 (9).Through precursor incorporation experiments, Byrne et al. (3) demonstrated that the polyketide portion of FK506 and FK520 is derived, for the most part, from acetate and propionate. Those authors also established the origin of the pipecolate and the cyclohexyl rings to be lysine and shikimic acid, respectively, and demonstrated that the source of the methyl portion of the methoxyl groups at C-13, C-15, and C-31 of FK520 (Fig. 1) is L-methionine.The enzymology of FK506 biosynthesis has also been explored to some extent. The pipecolate-activating enzyme which presumably incorporates pipecolate into the completed polyketide chain has been characterized previously (19). Both 31-O-demethyl-FK520 methyltransferase and 31-O-demethyl-FK506 methyltransferase (FKMT) have been isolated from the producing strains (3, 27). These two enzymes can use each other's substrate interchangeably and methylate the C-31 OH and not the C-13 or C-15 OH group (27).Here, we report the isolation and molecular characterization of two genes involved in the biosynthesis of FK506. One gene, fkbM, encodes FKMT, and the other, fkbD, encodes a cytochrome P-450 9-deoxo-FK506 hydroxylase that catalyzes hydroxylation at C-9. MATERIALS AND METHODSStandard recombinant DNA techniques were performed as described by Sambrook et al. (24).Probe design. N-terminal amino acid sequencing of FKMT from Streptomyces sp. strain MA6858 (27) gave a 39-mer with the sequence SDVVETLRLPNGA TVAHVNAGEAQFLYREIFTDRCYLRH. This peptide sequence was then used to design two nonoverlapping degenerate oligonucleotide probes, P1 and P2, in which inosine was incorporated at the third position of highly degenerate codons (2). P1 corresponded to ...
Activation of  3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to upregulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with  3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus  3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a  3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for  3 agonists in the treatment of human obesity.
MB243 (a 1,3-disubstituted piperazine) is a new, potent, and selective melanocortin receptor subtype-4 agonist with potential application in the treatment of obesity and/or erectile dysfunction. MB243 was observed to covalently bind extensively to liver microsomal proteins from rats and humans. In the presence of glutathione, two thioether adducts were detected in liver microsomal incubations by radiochromatography and LC/MS/MS analysis. These adducts were also formed when bile duct-cannulated rats were dosed with MB243. The two adducts were isolated, and their structures were determined by accurate mass MS/MS and NMR analyses. The proposed structures resulted from a novel contraction of the piperazine ring to yield a substituted imidazoline. A mechanism is proposed, which involves an initial six electron oxidation of the piperazine ring to form a reactive intermediate, which is trapped by glutathione. Hydrolysis of the glutamic acid residue followed by internal aminolysis by the cysteine amino group resulted in opening of the piperazine ring, which is followed by ring closure to an imidazoline. The resulting cysteinyl-glycine conjugate underwent subsequent hydrolysis of the glycine residue. Understanding of the mechanism of bioactivation led to the design of MB243 analogues that exhibited reduced covalent protein binding.
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