L-735,524: the design of a potent and orally bioavailable HIV protease inhibitor.

A series of HIV protease inhibitors possessing a hydroxylaminepentanamide transition state isostere have been developed. Incorporation of a basic amine into the backbone of the L-685,434 (2) series provided antiviral potency combined with a highly improved pharmacokinetic profile in animal models. Guided by molecular modeling and an X-ray crystal structure of the inhibited enzyme complex, we were able to design L-735,524. This compound is potent and competitively inhibits HIV-1 PR and HIV-2 PR with Ki values of 0.52 and 3.3 nM, respectively. It also stops the spread of the HIV-1IIIb-infected MT4 lymphoid cells at concentrations of 25-50 nM. To date, numerous HIV-PR inhibitors have been reported, but few have been studied in humans because they lack acceptable oral bioavailability. L-735,524 is orally bioavailable in three animals models, using clinically acceptable formulations, and is currently in phase II human clinical trials.

A series of potent HIV-1 protease inhibitors containing a hydroxyethyl secondary amine transition state isostere: synthesis, enzyme inhibition, and antiviral activity.

A series of HIV-1 protease inhibitors containing a novel hydroxyethyl secondary amine transition state isostere has been synthesized. The compounds exhibit a strong preference for the (R) stereochemistry at the transition state hydroxyl group. Molecular modeling studies with the prototype compound 11 have provided important insights into the structural requirements for good inhibitor-active site binding interaction. N-Terminal extension of 11 into the P2-P3 region led to the discovery of 19, the most potent enzyme inhibitor in the series (IC50 = 5.4 nM). 19 was shown to have potent antiviral activity in cultured MT-4 human T-lymphoid cells. Comparison of analogs of 19 with analogs of 1 (Ro31-8959) demonstrates that considerably different structure-activity relationships exist between these two subclasses of hydroxyethylamine HIV-protease inhibitors.

Synthesis and antiviral activity of a series of HIV-1 protease inhibitors with functionality tethered to the P1 or P1' phenyl substituents: X-ray crystal structure assisted design.

By tethering of a polar hydrophilic group to the P1 or P1' substituent of a Phe-based hydroxyethylene isostere, the antiviral potency of a series of HIV protease inhibitors was improved. The optimum enhancement of anti-HIV activity was observed with the 4-morpholinylethoxy substituent. The substituent effect is consistent with a model derived from inhibitor docked in the crystal structure of the native enzyme. An X-ray crystal structure of the inhibited enzyme determined to 2.25 A verifies the modeling predictions.

Nonpeptidal P2 ligands for HIV protease inhibitors: structure-based design, synthesis, and biological evaluation.

Design and synthesis of nonpeptidal bis-tetrahydrofuran ligands based upon the X-ray crystal structure of the HIV-1 protease-inhibitor complex 1 led to replacement of two amide bonds and a 10 pi-aromatic system of Ro 31-8959 class of HIV protease inhibitors. Detailed structure-activity studies have now established that the position of ring oxygens, ring size, and stereochemistry are all crucial to potency. Of particular interest, compound 49 with (3S,3aS,6aS)-bis-Thf is the most potent inhibitor (IC50 value 1.8 +/- 0.2 nM; CIC95 value 46 +/- 4 nM) in this series. The X-ray structure of protein-inhibitor complex 49 has provided insight into the ligand-binding site interactions. As it turned out, both oxygens in the bis-Thf ligands are involved in hydrogen-bonding interactions with Asp 29 and Asp 30 NH present in the S2 subsite of HIV-1 protease. Stereoselective routes have been developed to obtain these novel ligands in optically pure form.

Identification of MK-944a: a second clinical candidate from the hydroxylaminepentanamide isostere series of HIV protease inhibitors.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

Yeast-expressed p55 precursor core protein of human immunodeficiency virus type 1 does not elicit protective immunity in chimpanzees.

Yeast-expressed p55 precursor core protein of human immunodeficiency virus type 1 (HIV-1) was used to immunize chimpanzees. The animals developed high titers of antibodies to p55 as well as to the p24 and p17 mature cleavage products of the core precursor. Virus-neutralizing antibodies were not elicited. The induced immune responses did not prevent establishment of HIV-1 infection following challenge of one immunized chimpanzee with live virus.

The genetic and functional basis of HIV-1 resistance to nonnucleoside reverse transcriptase inhibitors.

The nonnucleoside reverse transcriptase (RT) inhibitors are structurally diverse compounds that are specific inhibitors of the human immunodeficiency virus type 1 RT enzyme. The compounds are largely functionally identical and bind to a common site in the enzyme. HIV-1 variants that exhibit reduced susceptibility to these inhibitors have been derived in cell culture and, more recently, from HIV-1-infected patients undergoing experimental therapy. The variants express amino acid substitutions at RT positions that apparently interact directly with the inhibitors. Effects of specific substitutions at these positions vary among the compounds, suggesting subtle differences in how the compounds physically interact with the enzyme.

The immune response to poliovirus-specific synthetic peptides: effects of adjuvants and test animal species.

Carrier protein conjugates of five synthetic peptides containing amino acid sequences specific to capsid proteins VP1 and VP2 of poliovirus type 1 were tested for their abilities to elicit an immune response in the presence of either of two adjuvants and in several animal species. Freund's adjuvant induced significantly higher level anti-peptide antibody titers than A1(OH)3. However, no difference was noted between the two adjuvants in their abilities to aid in the induction of cross-reactive virus neutralizing antibody. The latter antibody was more readily produced by rabbits than by guinea pigs in spite of equivalent anti-peptide titers. Rats failed to produce neutralizing antibodies and their anti-peptide antibody levels were generally lower. The significance of these results for studies involving the development of synthetic peptide immunogens is discussed.

A microtiter cell-culture assay for the determination of anti-human immunodeficiency virus neutralizing antibody activity.

A microtiter cell-culture assay is described for measuring neutralizing antibody activity directed against the human immunodeficiency virus (HIV). The assay relies upon inhibition of HIV-mediated cell killing of infected MT-4 lymphoid cells. The assay exhibits comparable sensitivity to two other methods used for such measurements, is relatively rapid, may be adapted for screening large numbers of samples and involves minimal handling of infectious virus.

In vivo selection of HIV-1 variants with reduced susceptibility to the protease inhibitor L-735,524 and related compounds.

L-735,524 is a potent inhibitor of the HIV-1 protease. In cell culture, the compound interferes with virus replication by causing production of noninfectious immature viral particles containing an unprocessed gag-pol polyprotein. Initial human clinical studies demonstrated that treatment with the inhibitor caused circulating viral levels to decline and this decline was associated with increases in the CD4 count of varying magnitude. However, in most patients, antiviral activity is lost as viral variants with reduced susceptibility to the inhibitor are selected. The resistant phenotype appears to require an amino acid substitution at protease codon 82. However, this amino acid alteration alone is insufficient for expression of the resistance phenotype. Co-expression of various additional alterations seems to be required, but the nature of these additional substitutions differs among resistant isolates. HIV-1 variants, cross-resistant to a panel of structurally diverse protease inhibitors, were isolated from patients following prolonged L-735,524 therapy.

Antigenic conservation and divergence between the viral-specific proteins of poliovirus type 1 and various picornaviruses.

Immuneprecipitation analyses of various picornavirus-infected cell lysates were performed using antisera to poliovirus type 1-specific structural and nonstructural proteins. The results showed differing patterns of antigenic conservation and divergence. However, the VP3 and 2C polypeptides were strongly antigenically conserved among the large majority of these viruses. This conservation was especially notable given the degree of divergence exhibited by the other viral proteins and may be due to environmental pressure exerted by interaction with the host cell. The results, furthermore, allowed for an analysis of the evolutionary relationship of the tested viruses. This analysis showed a particularly strong antigenic relationship between the proteins of the poliovirus group and coxsackievirus A21 as well as a weaker, but significant, relationship with coxsackieviruses B1 and B3.

Vero cell-expressed Epstein-Barr virus (EBV) gp350/220 protects marmosets from EBV challenge.

The Epstein-Barr virus (EBV) major membrane antigen, gp350/220, was purified from expressing, genetically engineered Vero cells. The antigen, formulated either with alum or Freund's adjuvant, was inoculated into EBV infection-susceptible marmosets. After several injections, most of the marmosets developed anti-gp350/220 antibodies, and several exhibited virus-neutralizing activity. The immune response elicited by the alum-absorbed antigen proved to be protective upon virus challenge of the inoculated animals. Protection did not correlate with the presence of neutralizing antibodies.

Drug resistance and predicted virologic responses to human immunodeficiency virus type 1 protease inhibitor therapy.

The extent to which human immunodeficiency virus (HIV) type 1 drug resistance compromises therapeutic efficacy is intimately tied to drug potency and exposure. Most HIV-1 protease inhibitors maintain in vivo trough levels above their human serum protein binding-corrected IC(95) values for wild-type HIV-1. However, these troughs are well below corrected IC(95) values for protease inhibitor-resistant viruses from patients experiencing virologic failure of indinavir and/or nelfinavir. This suggests that none of the single protease inhibitors would be effective after many cases of protease inhibitor failure. However, saquinavir, amprenavir, and indinavir blood levels are increased substantially when each is coadministered with ritonavir, with 12-h troughs exceeding corrected wild-type IC(95) by 2-, 7-, and 28-79-fold, respectively. These indinavir and amprenavir troughs exceed IC(95) for most protease inhibitor-resistant viruses tested. This suggests that twice-daily indinavir-ritonavir and, to a lesser extent, amprenavir-ritonavir may be effective for many patients with viruses resistant to protease inhibitors.