Daniel W. Norbeck

Ordered accumulation of mutations in HIV protease confers resistance to ritonavir

Analysis of the HIV protease gene from the plasma of HIV–infected patients revealed substitutions at nine different codons selected in response to monotherapy with the protease inhibitor ritonavir. Mutants at valine–82, although insufficient to confer resistance, appeared first in most patients. Significant phenotypic resistance required multiple mutations in HIV protease, which emerged subsequently in an ordered, stepwise fashion. The appearance of resistance mutations was delayed in patients with higher plasma levels of ritonavir. Early mutants retained susceptibility to structurally diverse protease inhibitors, suggesting that dual protease inhibitor therapy might increase the duration of viral suppression.

Antiviral and pharmacokinetic properties of C2 symmetric inhibitors of the human immunodeficiency virus type 1 protease.

Specific processing of the human immunodeficiency virus (HIV) gag and gag-pol polyprotein gene products by the HIV protease is essential for the production of mature, infections progeny virions. Inhibitors of HIV protease block this maturation and thus prohibit the spread of HIV in vitro. Previously, we reported a series of novel, symmetric inhibitors of HIV protease designed to match the C2 symmetric structure of the active site of the enzyme. In response to the poor aqueous solubility of those lead compounds, we designed a series of analogs with substantially improved (greater than 10(4) fold) solubility. These inhibitors showed anti-HIV activity in H9 and MT4 cells at 0.05 to 10 microM, and in most cases, they were noncytotoxic at concentrations in excess of 100 microM. Further examination of one inhibitor (A-77003) revealed broad-spectrum activity against both HIV types 1 and 2, including azidothymidine-resistant HIV, in a variety of transformed and primary human cell lines. After administration of the inhibitors to rats, short half-lives and, with two notable exceptions, moderate oral bioavailability were observed. Additional pharmacokinetic studies in dogs and monkeys revealed the potential utility of A-77003 as an intravenous anti-HIV agent.

(±)-Dioxolane-T ((±)-1-[(2β,4β)-2-(hydroxymethyl)-4-dioxolanyl]thymine). A new 2′,3′-dideoxynucleoside prototype with in vitro activity against HIV

Abstract A novel analogue of 3′-deoxythymidine, in which the 3′-carbon is replaced by oxygen, was synthesized in 5 steps from benzyloxyacetaldehyde dimethyl acetal and (±)-methyl glycerate. In ATH8 cells, this analogue showed significant inhibition of the infectivity and cytopathic effect of HIV at a concentration of 20 uM, while the growth of the uninfected control cells was not affected by concentrations as high as 200 uM. X-ray crystallographic analysis confirmed the assignment of stereochemistry and established a3T4 type conformation of the dioxolane ring.

X-ray Crystallographic Structure of ABT-378 (Lopinavir) Bound to HIV-1 Protease

The crystal structure of ABT-378 (lopinavir), bound to the active site of HIV-1 protease is described. A comparison with crystal structures of ritonavir, A-78791, and BILA-2450 shows some analogous features with previous reported compounds. A cyclic urea unit in the P(2) position of ABT-378 is novel and makes two bidentate hydrogen bonds with Asp 29 of HIV-1 protease. In addition, a previously unreported shift in the Gly 48 carbonyl position is observed. A discussion of the structural features responsible for its high potency against wild-type HIV protease is given along with an analysis of the effect of active site mutations on potency in in vitro assays.

Chapter 15. HIV Protease Inhibitors

Publisher Summary The basic hypothesis that specific antiretroviral agents should ameliorate the course of HIV infection is now supported, by clinical trials, with nucleoside-based inhibitors of the viral reverse transcriptase. Unfortunately, the most widely used of these agents—azidothymidine (AZT) and dideoxyinosine (DDI)—can have significant toxicity and the mutations in the viral target can lead to drug resistance. While nonnucleoside inhibitors of the reverse transcriptase may overcome some of these problems, inhibitors of other essential viral proteins should also be useful, either as single agents or in combination. Just a few years after the sequence of the HIV genome became available, inhibitors of such a protein, the HIV protease, were close to being used in the experimental AIDS therapy. Several factors contributed to this rapid progress. Sequence homologies of HIV-1, with previously studied retroviruses, supported the existence of the protease enzyme and the presence of a conserved Asp–Thr–Gly sequence advocated membership in the aspartyl protease class. This classification, coupled with homology-based predictions of substrate sequences, meant that the strategies developed for the design of inhibitors of renin, an aspartic proteinase, involved in the regulation of blood pressure, would be directly applicable to the HIV protease. This chapter discusses viral protease, the HIV-1 protease, role of the HIV protease, the viral life cycle, in vitro assays for protease inhibition, substrate specificities, design of protease inhibitors, antiviral activities of protease inhibitors, and their future prospects. The chapter also discusses the subsite specificity in the substrates of HIV protease and peptide-based inhibitors of the HIV protease.

Design of orally bioavailable, symmetry-based inhibitors of HIV protease.

A series of novel inhibitors of HIV-1 protease with excellent oral bioavailability is described. Differential acylation of the two amino groups of symmetry-based diamine core groups 2-5 led to unsymmetrically substituted inhibitors 17-43, many of which inhibited HIV protease at subnanomolar concentrations. Anti-HIV activity in vitro was observed at 0.1-1 microM. A systematic evaluation of the pharmacokinetic behavior of these inhibitors in rats identified the influence of aqueous solubility, molecular size and hydrogen-bonding functionality. Compound 30 (A-80987) was selected for further evaluation based on a favorable Cmax/ ED50 ratio (> 20) and half-life (> 2 h).

Novel α- and β-Amino Acid Inhibitors of Influenza Virus Neuraminidase

ABSTRACT In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 α- and β-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited Ki values in the 50 μM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the α-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the β-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the α- or β-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.

Potent HIV-1 protease inhibitors with antiviral activities in vitro

A series of novel difluoroketones with low molecular weight (less than 600 m.u.) and which are potent inhibitors of the HIV-1 protease (IC50 = 1.0 to 21 nM) were synthesized. These compounds also exhibited antiviral activity by inhibition of the cytopathic effect of HIV-1(3)B in MT-4 cells in vitro.

Synthesis and antiviral activities of the major metabolites of the HIV protease inhibitor ABT-378 (Lopinavir).

The HIV protease inhibitor ABT-378 (Lopinavir) is metabolized rapidly and extensively by CYP-3A4 catalyzed oxidation. Three of the major metabolites identified were synthesized and their antiviral (HIV) activities determined.

Pseudo-symmetrical difluoroketones: Highly potent and specific inhibitors of HIV-1 protease

A series of novel, pseudo‐symmetrical difluoroketones which are highly potent inhibitors of the HIV‐1 protease (IC50 = 1.55‐0.02 nM) were synthesized. These compounds also possess good antiviral activity by inhibition of the cytopathic effect of HIV‐13B in MT‐4 cells in vitro.