Using a combination of iterative structure-based design and an analysis of oral pharmacokinetics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor (Ki = 2 nM) and antiviral agent (HIV-1 ED50 = 14 nM). An X-ray cocrystal structure of the enzyme-AG1343 complex reveals how the novel thiophenyl ether and phenol-amide substituents of the inhibitor interact with the S1 and S2 subsites of HIV-1 protease, respectively. In vivo studies indicate that AG1343 is well absorbed orally in a variety of species and possesses favorable pharmacokinetic properties in humans. AG1343 (Viracept) has recently been approved for marketing for the treatment of AIDS.

Proviral integration is essential for HIV‐1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN‐mutant HIV‐1 molecular clones are complemented in trans by Vpr–IN fusion proteins, thereby enabling the study of IN function in replicating viruses. Using this approach we found that (i) Vpr‐linked IN is efficiently packaged into virions independent of the Gag–Pol polyprotein, (ii) fusion proteins containing a natural RT/IN processing site are cleaved by the viral protease and (iii) only the cleaved IN protein complements IN‐defective HIV‐1 efficiently. Vpr‐mediated packaging restored IN function to a wide variety of IN‐deficient HIV‐1 strains including zinc finger, catalytic core and C‐terminal domain mutants as well as viruses from which IN was completely deleted. Furthermore, trans complemented IN protein mediated a bona fide integration reaction, as demonstrated by the precise processing of proviral ends (5′‐TG…CA‐3′) and the generation of an HIV‐1‐specific (5 bp) duplication of adjoining host sequences. Intragenic complementation between IN mutants defective in different protein domains was also observed, thereby providing the first evidence for IN multimerization in vivo.

Noncoded D-amino acids have been designed to replace the quinaldic amide-asparaginyl moiety (P2/P3 ligand) found in several potent human immunodeficiency virus (HIV) protease inhibitors such as LY289612. The substituted nitrogen, optimally an N-methanesulfonyl moiety, served as a CH2CONH2 (asparagine side chain mimic), while the amino acid side chain became the backbone and P3 ligand of these novel inhibitors. Compounds derived from S-aryl-D-cysteine proved to be potent HIV protease inhibitors which also exhibited potent whole cell antiviral activity. Oxidation of the cysteines to the sulfoxide or sulfone oxidation states resulted in significant improvements in potency. For example, the compound derived from N-(methyl-sulfonyl)-2-S-naphthylcysteine sulfone, 17c, was a 3.5 nM inhibitor of HIV protease which inhibited the spread of virus in MT4 cells with an IC50 = 4.3 nM. Compounds 17c,g,i were found to be orally bioavailable in a rat model.

Using the hydroxyethylamine isosteres 1 and 2 containing the novel cis-octahydrothienopyridine moiety, substantial enhancement of binding potencies for HIV-1 protease inhibitors which incorporate carbamate linked heterocyclic P2 ligands has been realized. This increase in binding has led to a very potent antiviral compound (LY326188). The pharmacokinetics of selected derivatives are detailed in this report.

We have created a temperature-sensitive (ts) mutant of human immunodeficiency virus type 1, using the technique of charge-cluster-to-alanine scanning mutagenesis to introduce specific changes into the integrase coding region. In the ts mutant virus, the lysine at amino acid 136 and the glutamic acid at amino acid 138 of integrase have been replaced with alanines (K136A/E138A). When K136A/E138A is synthesized at 35 degrees C, it replicates to a similar degree as wild-type virus during infection of CEM cells at 35 degrees C on the basis of syncytium formation, levels of core antigen, and reverse transcriptase activity. However, during infection at the nonpermissive temperature of 39.5 degrees C, K136A/E138A is capable of only one round of integration. Mutant virions formed at 39.5 degrees C do not integrate but are indistinguishable from wild-type virions when scored for activity of reverse transcriptase and correct expression and processing of Gag and Pol proteins. We demonstrate that the defect responsible for the ts phenotype of K136A/E138A is localized to a step after proviral formation and integrase protein synthesis but prior to particle maturation. It is the temperature at which the K136A/E138A virion is synthesized, not the temperature at which infection occurs, which determines the ability of the virus to integrate.

Using the X-ray crystal structure of the inhibitor 1 complexed to HIV-1 protease, a new series of HIV-1 protease inhibitors was developed incorporating substituted isophthalic acid derivatives as amino acid surrogates. Through iterative structure-based design, the lead compound 2 was optimized to produce a variety of non-peptide HIV-1 protease inhibitors with significant antiviral activity. In contrast to 1, several members of this series exhibit significant oral absorption in animals.

Using information obtained from the co-crystal structure of an initial peptidomimetic lead complexed with HIV-1 protease, a series of inhibitors was constructed with substituents designed to span from the P1 to the P3 pockets of the enzyme. In accord with prediction, systematic extension of the P1 substituent with large, lipophilic groups leads to enhancements in binding potencies for this class of inhibitors. Surprisingly, inhibitors with large substituents at both P1 and P3 are also well-tolerated by the enzyme, providing compounds with subnanomolar binding affinities for HIV-1 protease.

To identify the minimal structural elements necessary for biological activity, the rigid tricyclic nucleus of the known human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor tetrahydroimidazobenzodiazepinthione was subjected to systematic bond disconnection to obtain simpler structures. A rational selection and testing of modeled analogs containing these potential pharmacophoric moieties led to the discovery of a new series of nonnucleoside inhibitors of RT. The lead compound of this new PETT series of nonnucleoside RT inhibitors, N-(2-phenylethyl)-N'-(2-thiazolyl)thiourea (LY73497), was found to inhibit HIV-1 but not HIV-2 or simian immunodeficiency virus in cell culture at micromolar concentrations. This derivative was also found to inhibit HIV-1 RT. Through an integrated effort involving synthesis and molecular modeling, compounds with nanomolar potency against HIV-1 in cell culture were developed. In these studies, LY300046-HCl was identified as a potent nonnucleoside inhibitor of HIV-1 RT possessing favorable pharmacokinetic properties.

Replacement of the decahydroisoquinoline group contained in Ro 31-8959 by a cis-octahydrothienopyridine moiety has provided a high affinity hydroxyethylamine isostere for use in HIV-1 protease inhibitors. Further gains in potency have been realized by incorporation of a sulfur atom into the P1 benzyl group. Modification by a key P2 ligand provided LY316440, a potent, orally absorbed inhibitor of HIV-1 protease.

Novel noncoded D-amino acids have been combined with decahydroisoquinoline, octahydrothienopyridine, and urea hydroxyethylamine isosteres to provide potent HIV-1 protease inhibitors with excellent HIV-1 antiviral activity. LY314613 shows a promising combination of potency and oral bioavailability. Trends in the SAR and comparisons to other isostere derivatives will be discussed.

Phenylethylthiazolylthiourea (PETT) derivatives have been identified as a new series of non-nucleoside inhibitors of HIV-1 RT. Structure-activity relationship studies of this class of compounds resulted in the identification of N-[2-(2-pyridyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea hydrochloride (trovirdine; LY300046.HCl) as a highly potent anti-HIV-1 agent. Trovirdine is currently in phase one clinical trials for potential use in the treatment of AIDS. Extension of these structure-activity relationship studies to identify additional compounds in this series with improved properties is ongoing. A part of this work is described here. Replacement of the two aromatic moieties of the PETT compounds by various substituted or unsubstituted heteroaromatic rings was investigated. In addition, the effects of multiple substitution in the phenyl ring were also studied. The antiviral activities were determined on wild-type and constructed mutants of HIV-1 RT and on wild-type HIV-1 and mutant viruses derived thereof, Ile100 and Cys181, in cell culture assays. Some selected compounds were determined on double-mutant viruses, HIV-1 (Ile 100/Asn103) and HIV-1 (Ile100/Cys181). A number of highly potent analogs were synthesized. These compounds displayed IC50's against wild-type RT between 0.6 and 5 nM. In cell culture, these agents inhibited wild-type HIV-1 with ED50's between 1 and 5 nM in MT-4 cells. In addition, these derivatives inhibited mutant HIV-1 RT (Ile 100) with IC50's between 20 and 50 nM and mutant HIV-1 RT (Cys 181) with IC50's between 4 and 10 nM, and in cell culture they inhibited mutant HIV-1 (Ile100) with ED50's between 9 and 100 nM and mutant HIV-1 (Cys181) with ED50's between 3 and 20 nM.

Integrase is the only viral protein necessary for integration of retroviral DNA into chromosomal DNA of the host cell. Biochemical analysis of human immunodeficiency virus type 1 (HIV-1) integrase with purified protein and synthetic DNA substrates has revealed extensive information regarding the mechanism of action of the enzyme, as well as identification of critical residues and functional domains. Since in vitro reactions are carried out in the absence of other viral proteins and they analyze strand transfer of only one end of the donor substrate, they do not define completely the process of integration as it occurs during the course of viral infection. In an effort to further understand the role of integrase during viral infection, we initially constructed a panel of 24 HIV-1 mutants with specific alanine substitutions throughout the integrase coding region and analyzed them in a human T-cell line infection. Of these mutant viruses, 12 were capable of sustained viral replication, 11 were replication defective, and 1 was temperature sensitive for viral growth. The replication defective viruses express and correctly process the integrase and Gag proteins. Using this panel of mutants and an additional set of 18 mutant viruses, we identified nine amino acids which, when replaced with alanine, destroy integrase activity. Although none of the replication-defective mutants are able to integrate into the host genome, a subset of them with alterations in the catalytic triad are capable of Tat-mediated transactivation of an indicator gene linked to the viral long terminal repeat promoter. We present evidence that integration of the HIV-1 provirus is essential not only for productive infection of T cells but also for virus passage in both cultured peripheral blood lymphocytes and macrophage cells.