Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance

Multidrug resistance continues to be a barrier to the effectiveness of highly active antiretroviral therapy in the treatment of human immunodeficiency virus 1 (HIV-1) infection. Darunavir (DRV) is a highly potent protease inhibitor (PI) that is oftentimes effective when drug resistance has emerged against first-generation inhibitors. Resistance to darunavir does evolve and requires 10–20 amino acid substitutions. The conformational landscapes of six highly characterized HIV-1 protease (PR) constructs that harbor up to 19 DRV-associated mutations were characterized by distance measurements with pulsed electron double resonance (PELDOR) paramagnetic resonance spectroscopy, namely double electron–electron resonance (DEER). The results show that the accumulated substitutions alter the conformational landscape compared to PI-naïve protease where the semi-open conformation is destabilized as the dominant population with open-like states becoming prevalent in many cases. A linear correlation is found between values of the DRV inhibition parameter Ki and the open-like to closed-state population ratio determined from DEER. The nearly 50% decrease in occupancy of the semi-open conformation is associated with reduced enzymatic activity, characterized previously in the literature.

Safety and Pharmacokinetics of Brecanavir, a Novel Human Immunodeficiency Virus Type 1 Protease Inhibitor, following Repeat Administration with and without Ritonavir in Healthy Adult Subjects

ABSTRACT Brecanavir (BCV) is a novel, potent protease inhibitor in development for the treatment of human immunodeficiency virus (HIV-1) infection with low nM in vitro 50% inhibitory concentrations (IC50s) against many multiprotease inhibitor resistant viruses. This study was a double-blind, randomized, placebo-controlled repeat-dose escalation to evaluate the safety, tolerability, and pharmacokinetics of BCV, with or without ritonavir (RTV), in 68 healthy subjects. Seven sequential cohorts (n = 10) received BCV (50 to 600 mg) in combination with 100 mg RTV (every 12 h [q12h] or q24h) or alone at 800 mg q12h for 15 days. BCV alone or in combination with RTV was well tolerated, with no serious adverse events reported. The most common drug-related adverse event was headache. BCV was readily absorbed with median time to maximum concentration of drug in serum values ranging from 2.5 to 5.0 h postdose following single- and repeat-dose administration of BCV alone and BCV with RTV 100 mg. Geometric mean BCV accumulation ratios ranged from 1.4 to 1.56 following BCV-RTV q24h regimens and from 1.84 to 4.93 following BCV q12h regimens. BCV steady state was generally achieved by day 13 in all groups. All day 15 BCV-RTV trough concentration values in q12h regimens reached or surpassed the estimated protein-binding corrected in vitro IC50 target BCV concentration of 28 ng/ml for highly resistant isolates. The pharmacokinetic and safety profile of BCV-RTV supports continued investigation in HIV-1-infected subjects.

ABT-378, a Highly Potent Inhibitor of the Human Immunodeficiency Virus Protease

ABSTRACT The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 μM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, ≤0.06 μM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC50 in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS.

Message of nexin 1, a serine protease inhibitor, is accumulated in the follicular papilla during anagen of the hair cycle

A group of specialized mesenchymal cells located at the root of the mammalian hair follicle, known as the follicular or dermal papillary cells, are involved in regulating the hair cycle, during which keratinocytes of the lower follicle undergo proliferation, degeneration and regrowth. Using the arbitrarily primed-PCR approach, we have identified a 1.3 kb messenger RNA that is present in large quantities in cultured rat follicular papillary cells, but not in skin fibroblasts. This mRNA encodes nexin 1, a potent protease inhibitor that can inactivate several growth-modulating serine proteases including thrombin, urokinase and tissue plasminogen activator. In situ hybridization showed that nexin 1 message is accumulated in the follicular papilla cells of anagen follicles, but is undetectable in keratinocytes or other skin mesenchymal cells. In addition, nexin 1 message level varies widely among several immortalized rat vibrissa papillary cell lines, and these levels correlate well with the reported abilities of these cell lines to support in vivo follicular reconstitution. These results suggest a possible role of nexin 1 in regulating hair follicular growth.