By using a structure-based computer-assisted search, we have found a butyrophenone derivative that is a selective inhibitor of the human immunodeficiency virus 1 (HIV-1) protease. The computer program creates a negative image of the active site cavity using the crystal structure of the HIV-1 protease. This image was compared for steric complementarity with 10,000 molecules of the Cambridge Crystallographic Database. One of the most interesting candidates identified was bromperidol. Haloperidol, a closely related compound and known antipsychotic agent, was chosen for testing. Haloperidol inhibits the HIV-1 and HIV-2 proteases in a concentration-dependent fashion with a K1 of -100 ILM. It is highly selective, having little inhibitory effect on pepsin activity and no effect on renin at concentrations as high as 5 mM. The hydroxy derivative of haloperidol has a similar effect on HIV-1 protease but a lower potency against the HIV-2 enzyme. Both haloperidol and its hydroxy derivative showed activity against maturation of viral polypeptides in a cell assay system. Although this discovery holds promise for the generation of nonpeptide protease inhibitors, we caution that the serum concentrations of haloperidol in normal use as an antipsychotic agent are <10 ng/ml (0.03 IAM). Thus, concentrations required to inhibit the HIV-1 protease are >1000 times higher than the concentrations normally used. Haloperidol is highly toxic at elevated doses and can be life-threatening. Haloperidol is not useful as a treatment for AIDS but may be a useful lead compound for the development of an antiviral pharmaceutical.

Chronic hepatitis B virus (HBV) infection, which occurs in 1 to 5% of adult infections and up to 30% of pediatric infections, is characterized by high levels of viral replication averaging a daily production of about 10 11 viral particles, hepatic inflammation, necrosis, and ultimately liver failure (36). An estimated 350 million individuals are classified as chronically HBV infected, with the highest concentrations of infection occurring in large parts of Asia and Africa (23).Chronic HBV can be treated with nucleoside analogues that inhibit polymerase activity. Lamivudine was the first licensed polymerase inhibitor, and it results in significant suppression of HBV DNA levels. However, this response, similar to the loss of hepatitis B virus e antigen, is often not sustained upon discontinuation of treatment (11,28). The emergence of viral resistance in 15 to 20% of treated patients per year clearly pinpoints the limitations of this treatment.Newer drugs such as adefovir dipivoxil, entecavir, and telbivudine can result in less resistance, increased suppression of DNA levels, or in somewhat higher levels of hepatitis B virus e antigen loss. Real long-term treatment data with these drugs are, however, limited, and it is unclear how well these responses would be sustained if therapy were withdrawn.

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N-and C-terminal residues of both monomers, which form a four-stranded 6-sheet. Peptides mimicking one 6-strand (residues 95-99), or two &strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIVl) interface were shown to inhibit the HIVl and 2 proteases (PRs) with ICso's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIVl P R dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIVl PR. Both one-and two-strand peptides alter the monomer/dimer equilibrium of both HIVl and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIVl and H1V2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one-and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.Keywords: aspartyl protease; cross-linking; dimer interface; dissociation; enzyme inhibition; HIVl protease; HIV2 protease; peptide; refolding; tethered dimer Dimerization is required to create a symmetric active site for aspartyl proteases (PRs) encoded by retroviruses. The crystal structures of the human immunodeficiency virus 1 (HIVl) (Navia et al., 1989;Wlodawer et al., 1989) and Rous sarcoma virus (RSV) PRs confirmed the homodimeric nature of these viral enzymes and also defined the regions of interaction between the monomers. The extensive hydrophobic interface present in these PR dimers is dominated by a n antiparallel P-sheet formed by the interdigitation of the N-terminal (residues 1-4 in HIVl and 1-5 in RSV) and the C-terminal (residues 96-99 in HIVl and 119-124 in RSV) 0-strands of each monomer ( Fig. 1 and kinemages). These interactions appear to be a major stabilizing force in the enzyme,

in the design of antiviral agents. This focus has resulted in a wealth of new information on the structure and function of these enzymes.Several viral proteases will be described here that exemplify the diversity of structures and biological func-