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Reversible tetrapeptide-based compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease. Inhibition of viral replicon RNA production in Huh-7 cells has also been demonstrated. We show herein that the inclusion of hydrogen bond donors on the P4 capping group of tetrapeptide-based inhibitors result in increased binding potency to the NS3.4A protease. The capping groups also impart significant effects on the pharmacokinetic profile of these inhibitors.

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3·4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3·4A protease inhibitor, BILN 2061 (Lin, C., Lin, K., Luong, Y.-P., Rao, B. G., Wei, Y.-Y., Brennan, D. L., Fulghum, J. R., Hsiao, H.-M., Ma, S., Maxwell, J. P., Cottrell, K. M., Perni, R. B., Gates, C. A., and Kwong, A. D. (2004) J. Biol. Chem. 279, 17508-17514). Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala156 with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3·4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3·4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3·4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp168 are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala156 remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.

The α-ketoamide warhead (e.g., 15) was found to be a practical replacement for aliphatic aldehydes in a series of HCV NS3·4A protease inhibitors. Structure–activity relationships and prime side optimization are discussed.

Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3·4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P1 and P2 inhibitor positions is discussed.

The benzothiophene anti-estrogen, raloxifene [LY156758; (6-hydroxy-2-(4-hydroxyphenyl) benzo(b)thien-3-yl)(4-(2-1-piperidinyl)ethoxy)phenyl methanone hydrochloride] has selective estrogen pharmacological antagonist activity in female rats. The present studies were done in the male rat to assess activity of raloxifene related to inhibition of prostatic growth and effects on the hypothalamic-pituitary-gonadal axis. Raloxifene did not compete for binding of the androgen, [3H]-methyltrienolone (R1881) in cytosolic extracts of ventral prostate. Similarly, the compound did not inhibit prostatic 5 alpha-reductase or testicular 17 alpha-hydroxy/C17,20-lyase activities. Raloxifene had no effect on the ventral prostatic uptake of [3H]-R1881 in vivo. Administration of estradiol to castrated male rats stimulated fourfold increases of in vitro ventral prostatic binding of [3H]-R1881. Raloxifene was devoid of agonist activity in castrated animals, because the compound had no stimulatory effect on prostatic androgen receptor binding activity. When raloxifene was coadministered with estradiol, the compound markedly antagonized the estrogen-induced increase of prostatic [3H]-R1881 binding, confirming its antiestrogenic properties in male rats. Serum prolactin was also elevated significantly (P < 0.05) with a single injection of raloxifene (20.0 mg/kg). In these same animals, serum FSH was significantly (P < 0.05) decreased by one dose (10.0 mg/kg) of the compound. Luteinizing hormone levels in castrated male rats were unaffected by raloxifene administration. Raloxifene treatment of castrated males significantly (P < 0.05) antagonized the stimulatory response of the ventral prostate (VP) to exogenous androgens in a dose-dependent manner. Raloxifene treatment of intact male rats for 14 and 28 days produced significant (P < 0.05) dose-dependent regression of the VP and seminal vesicles (SV). The VP regressive responses to raloxifene were associated with a decline in serum testosterone levels. Histological analysis of the VPs in raloxifene-treated rats was consistent with an androgen-deprived state. These findings support the contention that raloxifene is a pure estrogen antagonist and a physiological antagonist of androgen action in male rats. These pharmacological properties provide support for further structure-activity and mechanistic investigations with benzothiophenes in the medical management of prostatic neoplasia.

LY207320 is an in vitro inhibitor (estimated IC50 = 0.06 microM) of steroid 5 alpha-reductase that catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT). In contrast, LY207320 was only moderately active against rat prostatic 5 alpha-reductase in vivo (32% inhibition at 50.0 mg/kg single dose). LY207320 did, however, inhibit the in vivo uptake of [3H]-T by the prostate. The antiprostatic and endocrine effects of this agent were evaluated following daily (21 days) administration to castrated, androgen-supplemented castrate, and intact rats. LY207320, which has modest progestational competitive binding activity, does not bind to rat prostatic androgen or uterine estrogen cytosolic receptors. In the castrated male rat, subcutaneously (s.c.) administered LY207320 had no androgen agonist activity, as evidenced by a lack of accessory sex organ weight gains. Administration of s.c. LY207320 to intact rats for 21 days at doses greater than 5.0 mg/kg-day produced significant (P0.05) reductions of seminal vesicle and ventral prostatic weights (maximal regression = -65% and -40% from control values, respectively at 50.0 mg/kg-day). The compound had no regressive activity on male accessory sex organs when administered orally. LY207320 did not alter circulating prolactin, LH, or corticosterone levels, but at high doses (or = 50.0 mg/kg-day), lowered circulating T[-67% from intact control levels (P0.05)]. Histological analysis of the rat ventral prostates (RVPs) in LY207320-treated rats was consistent with an androgen-deprived state. Decreased circulating androgens and prostatic regression are associated with inhibition of testicular 17 alpha-hydroxy/C17,20-lyase enzyme activity (IC50 = 0.06 microM). These findings support the contention that LY207320 is a physiological antagonist of androgen action in male rats, and that its effects are mediated primarily through inhibition of testicular androgen production rather than accessory sex organ 5 alpha-reductase.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.