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.

Design, activity, and 2.8 A crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease.

A two-fold (C2) symmetric inhibitor of the protease of human immunodeficiency virus type-1 (HIV-1) has been designed on the basis of the three-dimensional symmetry of the enzyme active site. The symmetric molecule inhibited both protease activity and acute HIV-1 infection in vitro, was at least 10,000-fold more potent against HIV-1 protease than against related enzymes, and appeared to be stable to degradative enzymes. The 2.8 angstrom crystal structure of the inhibitor-enzyme complex demonstrated that the inhibitor binds to the enzyme in a highly symmetric fashion.

A novel prodrug of an impermeant inhibitor of 3-deoxy-D-manno-2-octulosonate cytidylyltransferase has antibacterial activity.

Although 8-amino-2,6-anhydro-3,8-dideoxy-D-glycero-D-talo-octonic acid (2) is a potent inhibitor of 3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase), it is unable to reach its cytoplasmic target and is therefore inactive as an antibacterial agent. However, esterification of 2 with 8-(hydroxymethyl)-1-naphthyl methyl disulfide (8) generates a prodrug (12), which gains entry into bacterial cells. Intracellular reduction of the disulfide leads to a rapid, intramolecular, displacement of the acid 2, which then inhibits the growth of Gram-negative bacteria by interfering with the biosynthesis of lipopolysaccharide.

Discovery of ritonavir, a potent inhibitor of HIV protease with high oral bioavailability and clinical efficacy.

The structure-activity studies leading to the potent and clinically efficacious HIV protease inhibitor ritonavir are described. Beginning with the moderately potent and orally bioavailable inhibitor A-80987, systematic investigation of peripheral (P3 and P2') heterocyclic groups designed to decrease the rate of hepatic metabolism provided analogues with improved pharmacokinetic properties after oral dosing in rats. Replacement of pyridyl groups with thiazoles provided increased chemical stability toward oxidation while maintaining sufficient aqueous solubility for oral absorption. Optimization of hydrophobic interactions with the HIV protease active site produced ritonavir, with excellent in vitro potency (EC50 = 0.02 microM) and high and sustained plasma concentrations after oral administration in four species. Details of the discovery and preclinical development of ritonavir are described.

A novel, picomolar inhibitor of human immunodeficiency virus type 1 protease.

The design, synthesis, and molecular modeling studies of a novel series of azacyclic ureas, which are inhibitors of human immunodeficiency virus type 1 (HIV-1) protease that incorporate different ligands for the S1', S2, and S2' substrate-binding sites of HIV-1 protease are described. The synthesis of this series is highly flexible in the sense that the P1', P2, and P2' residues of the inhibitors can be changed independently. Molecular modeling studies on the phenyl ring of the P2 and P2' ligand suggested incorporation of hydrogen-bonding donor/acceptor groups at the 3' and 4-positions of the phenyl ring should increase binding potency. This led to the discovery of compound 7f (A-98881), which possesses high potency in the HIV-1 protease inhibition assay and the in vitro MT-4 cell culture assay (Ki = approximately 5 pM and EC50 = 0.002 microM). This compares well with the symmetrical cyclic urea 1 pioneered at DuPont Merck.

A C2 symmetry-based HIV protease inhibitor, A77003, irreversibly inhibits infectivity of HIV-1 in vitro.

A C2 symmetry-based HIV protease inhibitor, A77003, exerts potent antiviral activity against a wide spectrum of HIV isolates in vitro. In this study, we asked whether A77003 could cause irreversible conformational changes to HIV-1, whether the amounts of viral RNA and p24 capsid protein per virion were altered, and how the infectivity of the virus produced in the presence of the drug was affected. We found that the number of viral particles and per-virion viral RNA content of the virus produced in the presence of A77003 did not significantly differ from those of the virus produced in the absence of the drug, whereas significant morphological changes were observed as assessed by transmission electron microscopy. However, the virus produced in the presence of A77003 contained substantially less p24gag protein per virion particle as compared to those produced in the absence of the drug or in the presence of AZT. Virions produced in the presence of A77003 showed up to 50-fold less infectious capability in subsequent tissue culture than control virions produced in the absence of drug or in the presence of AZT. This reduction in infectivity was maintained for at least 10 days in culture. The present data suggest that A77003 impairs HIV-1 protease-mediated Gag processing, interferes with the assembly and maturation of the virus, and leads to an irreversible loss of the infectivity of the virus, although a low but positive level of reversion to infectivity during the 10-day assay occurs. These features of A77003 (and perhaps similar HIV protease inhibitors as well) anti-HIV activity should represent desirable properties for antiviral therapy of AIDS and related diseases.

Human immunodeficiency virus type 1 virions composed of unprocessed Gag and Gag-Pol precursors are capable of reverse transcribing viral genomic RNA.

The structural proteins and enzymes of the human immunodeficiency virus type 1 core are translated as part of two polyprotein precursors, Gag and Gag-Pol, which are cleaved by a virally encoded protease. Viruses grown in the presence of inhibitors of the protease contain core particles that are aberrantly assembled, and upon infection of susceptible cells, they do not synthesize viral DNA. Through the use of a proteinase inhibitor (A77003), we determined that the viral reverse transcriptase can efficiently synthesize viral DNA as part of the unprocessed Gag-Pol precursor. We also found that the stabilities of core particles composed of unprocessed precursors were considerably enhanced. These observations suggest that for viruses composed of unprocessed precursors, replication is interrupted before the reverse transcription step.

Selection and analysis of human immunodeficiency virus type 1 variants with increased resistance to ABT-538, a novel protease inhibitor.

Inhibitors of the human immunodeficiency virus protease represent a promising new class of antiretroviral drugs for the treatment of AIDS. We now report the in vitro selection of viral variants with decreased sensitivity to a symmetry-based protease inhibitor, ABT-538, currently being tested in clinical trials. Molecular characterization of the variants shows that an isoleucine-to-valine substitution at position 84 results in a substantial decrease in sensitivity to the drug. Moreover, an additional mutation at position 82, valine to phenylalanine, further decreases viral susceptibility to ABT-538. Three-dimensional analysis of the protease-drug complex provides a structural explanation for the relative drug resistance induced by these two mutations. These findings emphasize the importance of closely monitoring patients receiving ABT-538 for the emergence of viral resistance and provide information that may prove useful in designing the next generation of protease inhibitors.

Effects of purine nucleoside analogues with a cyclobutane ring and erythromycin A oxime derivatives on duck hepatitis B virus replication in vivo and in cell culture and HIV-1 in cell culture.

The effects on duck hepatitis B virus (DHBV) replication of specific analogues of two classes of chemical compounds not previously tested against hepadnaviruses are described. One is erythromycin A-9-methyloxime (EMO) and other oxime derivatives of erythromycin A, and the other is purine nucleoside analogues (cyclobut A and cyclobut G) with cyclobutane rings. Viral replication was assessed by measuring serum levels of DHBV DNA in infected ducklings and DHBV DNA in infected primary duck hepatocyte cultures. Administration of EMO 15 mg/kg of body weight IM to infected ducklings resulted in a rapid fall in DHBV DNA levels during therapy and a return to pretreatment levels after EMO administration was stopped. There was local toxicity at injection sites with muscle necrosis in some animals. When 100 mg/kg EMO was administered by gastric tube no such viral response was observed. The difference in virus response to EMO 15mg/kg IM and 100 mg/kg by gastric tube was not due to failure to achieve comparable blood and tissue levels of EMO administered by the different routes. The results suggest an indirect effect dependent on IM injection of EMO rather than a direct antiviral effect of the compound. Administration of cyclobut G or cyclobut A at 70 mg/kg IM led to a rapid reduction of DHBV DNA to undetectable levels in serum, and in only 1 of 4 animals did DHBV DNA became detectable again within 10 days after stopping the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles.

The production of infectious particles by human immunodeficiency virus type 1 is dependent on the accurate cleavage of its Gag and Gag/Pol precursors by a virally encoded protease. In the absence of protease activity, morphologically abnormal particles which are noninfectious are formed. Recently, inhibitors of the protease of human immunodeficiency virus type 1 have been developed as potential therapeutic agents. We have examined the basis for the loss of infectivity at the limiting inhibitor concentrations that are likely to be achieved in clinical settings. We found that subtle defects in processing are correlated with profound deficits in infectivity. Further, we correlated this partially disrupted processing with an altered virion morphology. These data suggest that accurate and complete processing is essential to the formation of infectious, morphologically normal virions and that the pathway by which these precursors are processed and assembled is sensitive to partial inhibition of the protease by an inhibitor disproportionate to the effect of the inhibitor on the viral protease itself.

In vitro inhibition of human immunodeficiency virus (HIV) type 1 replication by C2 symmetry-based HIV protease inhibitors as single agents or in combinations.

C2 symmetry-based human immunodeficiency virus (HIV) protease inhibitors were examined in vitro as single agents or in combination with 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine for activity against HIV type 1 (HIV-1). Ten C2 symmetry-based or pseudo-C2 symmetry-based HIV protease inhibitors were active against a laboratory strain (HIV-1IIIB) in the HIV-1 cytopathic effect inhibition assay. Three inhibitors, A75925, A76928, and A77003, selected to represent a range of aqueous solubility and antiviral activity, were active against four different HIV-1 strains tested. These three inhibitors exhibited a significant inhibition of the cytopathic effect of HIV-1 against the CD4+ ATH8 cell line, with 90% inhibitory concentrations ranging from 0.1 to 4 microM. Cellular toxicity was negligible at up to 20 microM. Furthermore, they completely inhibited the replication of monocytotropic strain HIV-1Ba-L in purified monocytes and macrophages at 0.75 to 2 microM. Potent inhibitory activity against a primary HIV-1 isolate and an AZT-resistant HIV-1 variant was also observed for all three inhibitors in phytohemagglutinin-activated peripheral blood mononuclear cells. When these three HIV protease inhibitors and AZT or 2',3'-dideoxyinosine were used in combinations against a primary HIV isolate in phytohemagglutinin-activated peripheral blood mononuclear cells and the results were analyzed with the COMBO program package, their antiviral activities were identified to be synergistic in some cases and additive in others. The present data warrant further investigations of these compounds as potential antiviral agents for the therapy of HIV infections.

Human serum alpha 1 acid glycoprotein reduces uptake, intracellular concentration, and antiviral activity of A-80987, an inhibitor of the human immunodeficiency virus type 1 protease.

The therapeutic utility of a human immunodeficiency virus type 1 (HIV-1) protease inhibitor may depend on its intracellular concentration, which is a property of its uptake, metabolism, and/or efflux. Previous studies in our laboratory indicated that the addition of alpha 1 acid glycoprotein (alpha 1 AGP) to the medium markedly increased the amount of the drug required to limit infection in vitro. In this study, physiologically relevant concentrations of alpha 1 AGP and a radiolabeled inhibitor, A-80987, were used to determine both the uptake and activity of the agent in HIV-1-infected human peripheral blood mononuclear cells and cell lines. Both the uptake and efflux of 14C-labeled A-80987 were rapid (t1/2, < 5 min). Uptake of the drug was linearly dependent on the concentration but insensitive to the metabolic inhibitors KF, sodium cyanide, or CCCP (carbonyl cyanide m-chlorophenyl hydrazone). The amount of A-80987 which entered the cells was inversely proportional to the concentration of alpha 1 AGP (r2, 0.99) and directly proportional to the amount of extracellular non-protein-bound drug (r2, 0.99). Most importantly, the antiviral activity of the drug in HIV-1-infected peripheral blood mononuclear cells and MT-2 cells was directly related to the amount of intracellular A-80987. This study demonstrates that A-80987 binds to alpha 1 AGP, resulting in a free fraction below 10%. Cellular uptake of A-80987 is proportionally decreased in the presence of alpha 1 AGP, which results in less-than-expected antiviral activity. Importantly, we demonstrate for the first time that the inhibition of HIV protease is highly correlated with the amount of intracellular inhibitor.

Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides.

The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in an Escherichia coli strain which also overexpressed the rare argU tRNA. The protease was isolated from inclusion bodies, refolded, and exhibited a kcat/Km = 3280 M-1 s-1 using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides. Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease with kinact/Kinact values as high as 23,400 M-1 s-1. The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to the kinact/Kinact rate constants for bromomethylketonehydrazide inhibition of 3C protease. Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145-154, a region which contains the active site cysteine-148 residue. The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture.

In vitro selection and characterization of human immunodeficiency virus type 1 variants with increased resistance to ABT-378, a novel protease inhibitor.

ABT-378, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which is significantly more active than ritonavir in cell culture, is currently under investigation for the treatment of AIDS. Development of viral resistance to ABT-378 in vitro was studied by serial passage of HIV-1 (pNL4-3) in MT-4 cells. Selection of viral variants with increasing concentrations of ABT-378 revealed a sequential appearance of mutations in the protease gene: I84V-L10F-M46I-T91S-V32I-I47V. Further selection at a 3.0 microM inhibitor concentration resulted in an additional change at residue 47 (V47A), as well as reversion at residue 32 back to the wild-type sequence. The 50% effective concentration of ABT-378 against passaged virus containing these additional changes was 338-fold higher than that against wild-type virus. In addition to changes in the protease gene, sequence analysis of passaged virus revealed mutations in the p1/p6 (P1' residue Leu to Phe) and p7/p1 (P2 residue Ala to Val) gag proteolytic processing sites. The p1/p6 mutation appeared in several clones derived from early passages and was present in all clones obtained from passage P11 (0.42 microM ABT-378) onward. The p7/p1 mutation appeared very late during the selection process and was strongly associated with the emergence of the additional change at residue 47 (V47A) and the reversion at residue 32 back to the wild-type sequence. Furthermore, this p7/p1 mutation was present in all clones obtained from passage P17 (3.0 microM ABT-378) onward and always occurred in conjunction with the p1/p6 mutation. Full-length molecular clones containing protease mutations observed very late during the selection process were constructed and found to be viable only in the presence of both the p7/p1 and p1/p6 cleavage-site mutations. This suggests that mutation of these gag proteolytic cleavage sites is required for the growth of highly resistant HIV-1 selected by ABT-378 and supports recent work demonstrating that mutations in the p7/p1/p6 region play an important role in conferring resistance to protease inhibitors (L. Doyon et al., J. Virol. 70:3763-3769, 1996; Y. M. Zhang et al., J. Virol. 71:6662-6670, 1997).

Genotypic changes in human immunodeficiency virus type 1 associated with loss of suppression of plasma viral RNA levels in subjects treated with ritonavir (Norvir) monotherapy.

Ten subjects received 600 to 1,200 mg of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor ritonavir per day. Following 2 weeks of therapy, plasma HIV RNA levels decreased by a mean of 1. 57 (range, 0.89 to 1.96) log units. With continued therapy, HIV RNA levels began to rise in eight subjects. The initial rise in plasma RNA levels was temporally associated with the development and quantitative increase in the V82 resistance mutation. Doubling times of the V82A mutant virus were estimated to be 2.4 to 4.8 days. An L63P/A mutation was commonly present at baseline even in subjects with a durable virologic response. The concomitant acquisition of an L63P/A mutation with the V82A/F mutation at the time when plasma RNA levels rebounded suggests a role for the L63P/A mutation in improving the fitness of the V82A/F mutation. Subsequent additional genotypic changes at codons 54 and 84 were often associated with further increases in plasma RNA levels. Ongoing viral replication in the presence of drugs resulted in the appearance of additional genotypic changes, including the L90M saquinavir resistance mutation, and decreased phenotypic susceptibility. The relative fitness of the protease V82A ritonavir resistance mutation and reverse transcriptase T215Y/F zidovudine resistance mutation following drug withdrawal were estimated to be 96 to 98% that of the wild type. Durability of the virologic response was associated with plasma RNA levels at the nadir. A virologic response beyond 60 days was not observed unless plasma HIV RNA levels were suppressed below 2,000 copies/ml, consistent with estimates from V82A doubling times for selection of a single resistance mutation to dominate the replicating population.

Cyclobut-A and cyclobut-G, carbocyclic oxetanocin analogs that inhibit the replication of human immunodeficiency virus in T cells and monocytes and macrophages in vitro.

Two newly synthesized carbocyclic oxetanocin analogs, (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine (cyclobut-A) and (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine (cyclobut-G) were tested for activity against the infectivity of human immunodeficiency virus (HIV) in vitro. A number of other carbocyclic oxetanocin analogs failed to exert good antiretroviral effects. Both cyclobut-A and cyclobut-G protected CD4+ ATH8 cells against the infectivity and cytopathic effect of HIV type 1 (HIV-1) and suppressed proviral DNA synthesis in ATH8 cells exposed to HIV-1 in vitro at concentrations of 50 to 100 microM. These compounds also inhibited the in vitro infectivity of another human pathogenic retrovirus, HIV-2. Furthermore, both compounds completely suppressed the replication of a monocytotropic strain of HIV-1 in monocytes and macrophages at concentrations as low as 0.5 microM, as assessed by inhibition of HIV-1 p24 gag protein production. We also found that 2'-deoxyguanosine readily reversed the antiretroviral activity of cyclobut-G in our system, whereas the activity of cyclobut-A was hardly reversed by 2'-deoxyadenosine or 2'-deoxycytidine. We noted, however, that these compounds inhibited the proliferation of peripheral blood mononuclear cells at concentrations of greater than or equal to 100 microM in vitro. Although both cyclobut-A and cyclobut-G appear to have a certain level of in vitro toxicity, our observations may have theoretical and clinical implications in understanding the structure-activity relationships of antiretroviral agents active against HIV.

Novel alpha- and beta-amino acid inhibitors of influenza virus neuraminidase.

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 alpha- and beta-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited K(i) values in the 50 microM 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 alpha-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the beta-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 alpha- or beta-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.