HIV-1 Protease Enzyme Research Articles

4-Hydroxy-5,6-Dihydro-2 H-pyran-2-ones. 3. Bicyclic and hetero-aromatic ring systems as 3-position scaffolds to bind to S 1′ and S 2′ of the HIV-1 protease enzyme

5,6-Dihydro-2 H-pyran-2-ones are potent inhibitors of HIV-1 protease, which bind to the S 1, S 2, S 1′, and S 2′ pockets and have a unique binding mode with the catalytic aspartyl groups and the flap region of the enzyme. Efforts to explore 3-position heterocyclic scaffolds that bind to the S 1′ and S 2′ pockets have provided a number of selected analogs that display high HIV-1 protease inhibitory activity.

Nelfinavir mesylate: a protease inhibitor.

To review the clinical pharmacology, pharmacokinetics, efficacy, adverse effects, drug interactions, and dosage guidelines of nelfinavir mesylate. A MEDLINE search restricted to English-language literature from January 1966 to February 1998 and an extensive review of journals was conducted to prepare this article. MeSH headings included protease inhibitors, nelfinavir mesylate, and AG1343. Abstracts presented at meetings and data submitted to the Food and Drug Administration (FDA) were also reviewed. The data on efficacy, pharmacokinetics, adverse effects, and drug interactions were obtained from in vitro studies, as well as open-label and controlled trials. Nelfinavir inhibits HIV protease enzyme resulting in formation of immature and noninfectious virions. In combination with nucleoside reverse transcriptase inhibitors, nelfinavir is effective in reducing the viral load below the quantifiable limit (< 500 copies/mL) and increasing the mean CD4+ cell count. This antiviral effect is sustained at least over 21 months. The bioavailability of nelfinavir ranges from 20% to 80%, and it increases when nelfinavir is administered with food. Following multiple dosing of nelfinavir 750 mg three times daily, maximum concentration at steady-state was 3-4 micrograms/mL and minimum concentration was 1-3 micrograms/mL. The elimination half-life for nelfinavir ranges from three to five hours. Nelfinavir is primarily metabolized in the liver by the cytochrome P450 isoenzymes and excreted in the feces. Current dosing recommendations are 750 mg three times daily for adults and adolescents and 20-30 mg/kg/dose three times daily for children aged 2-13 years. Studies of twice-daily regimens in adults are being conducted and are promising. Use of nelfinavir as salvage therapy is also being studied. Some of the commonly reported adverse events of nelfinavir are diarrhea, nausea, vomiting, and abdominal pain. Despite the limited published data, the FDA has approved nelfinavir in combination therapy for the treatment of HIV infection. The choice of antiretroviral (ARV) regimens should be made based on the risk of disease progression as indicated by HIV RNA concentrations and CD4+ cell counts, patients' previous ARV experiences and responses, concomitant drug therapy, compliance history, underlying disease states, and adverse reaction history.

Comparison of azacyclic urea A-98881 as HIV-1 protease inhibitor with cage dimeric N-benzyl 4-(4-methoxyphenyl)-1,4-dihydropyridine as representative of a novel class of HIV-1 protease inhibitors: a molecular modeling study.

The functional groups of cage dimeric N-alkyl substituted 3,5-bis(hydroxymethyl)-4-(4-methoxyphenyl)-1,4-dihydropyridines are similar to those of cyclic and azacyclic ureas that are potent inhibitors of HIV-1 protease of the dihydroxyethylene- and hydroxyethylene type, respectively. In the following study the conformity of common functional groups is investigated concerning their orientation in space as well as in the enzyme HIV-1 protease. Starting from X-ray crystal data of the centrosymmetric cage dimeric N-benzyl derivative with ester groups, the derivative with hydroxymethylene groups was built and a systematic conformational search was performed for the conformationally important torsion angles considering electrostatic and van der Waals interactions. From the huge number of conformations those comprising centrosymmetrical and C2-symmetrical energy minima were selected and minimized. The three remaining conformers were fitted to the azacyclic urea A-98881 selected from the HIV-1 protease enzyme-inhibitor complex using the centroids of the corresponding aromatic residues and additionally by the field fit option of the Advanced CoMFA module of SYBYL. Interestingly, the energetically most favourable one, which, additionally, possesses C2-symmetry like the active site cavity of HIV-1 protease, showed the best fit. Comparing the electrostatic potential (EP) of the latter with the EP of A-98881 the aromatic residues show excellent accordance. Slight differences in the extent of the EP were found in the areas of the hydroxymethylene groups of the cage dimer and the single hydroxy group as well as the urea carbonyl group of A-98881, respectively. In order to compare the binding possibilities to the enzyme HIV-1 protease for the cage dimer and A-98881, their interaction fields with certain probes (CH3 for alkyl, NHamide, and carbonyl, O- of COO-), representing the decisive functional groups of the active site, have been calculated using GRID and projected into the enzyme placing the structures according to the position of A-98881 in the enzyme-inhibitor complex. The strongest calculated fields of the O- probe were found near Asp 25 for both structures. Another respective conformity consists in the overlap of the fields for the NHamide probe near Ile 50 and 50' for the investigated cage dimer and A-98881.

Inhibiting the Dimerization of HIV-1 Protease

Disrupting protein-protein interaction in multi-subunit enzymes is currently an underutilized mode of inhibition, and little information exists to assist the researcher in the design of agents to effect such a change within the enzyme. This work seeks to address these limitations by providing a general strategy for the design of dimerization inhibitors of the therapeutically significant enzyme HIV-1 protease.

A Convergent, Scalable Synthesis of HIV Protease Inhibitor PNU-140690.

PNU-140690, an inhibitor of the HIV protease enzyme undergoing clinical evalution as a chemotherapeutic agent for treatment of AIDS, was synthesized by a convergent approach amenable to large-scale preparation in a pilot plant environment. The key step is the aldol addition of nitroaromatic ester (+)-8 to aldehyde 19e. The two stereocenters present in the target molecule were each set independently by resolution of enantiomers. Intermediates along the synthetic routes were chosen to maximize opportunities for isolation and purification by crystallization.

Design and Synthesis of a Novel Series of Nonpeptidic HIV‐1 Protease Inhibitors*

Using molecular modelling and the X-ray crystal structures of peptide, (1-(naphthoxyacetyl)-L-histidyl-5(S)-amino-6-cyclohexyl-3(R),4(R)-dihydroxy-2(R)-isopropylhexanoyl-L-isoleucine N-(2-pyridylmethyl)amide (U-75875) and nonpeptide-derived inhibitors, phenprocoumon (3-(α-ethylbenzyl)-4-hydroxycoumarin, warfarin (3-(α-(2-oxopropyl) benzyl)-4-hydroxycoumarin), 4-hydroxy-3-(benzylsulphanyl)6-phenylpyran-2-one and 3-phenoxypropyl-4-hydroxycoumarin bound to HIV-1 protease, we designed and synthesized potential nonpeptidic inhibitors that incorporate S1′ S1 and S2 binding sites of HIV-1 protease. Using an interactive docking procedure, the coumarin and chromone series of compounds were docked into the active site of the enzyme and their binding modes were analysed by molecular dynamics simulations for 50 ps. The results helped to identify the 4-hydroxycoumarin-3-yl styryl ketones 4a–f, 4-hydroxycoumarin-3-yl phenyl ketones 4g and 4h and 3-hydroxybenzopyrans 8a and 8b as HIV-1 protease inhibitors. These compounds were synthesized and tested for HIV-1 protease inhibitory activity and in-vitro antiviral activity in the CEM-SS cell line assay. The most active compounds, 4-hydroxy-2-oxo-2H-coumarin-3-yl (un)substituted styryl ketones, 4a and 4d, exhibited 33 and 47-9% inhibition of HIV-1 protease at 10 μM concentration, respectively. All compounds exhibited poor antiviral activity, and no correlation was found between in-vitro HIV-1 protease enzyme inhibitory activity and antiviral activity.

Protease inhibitors in patients with HIV disease. Clinically important pharmacokinetic considerations.

Since its introduction in 1987, zidovudine monotherapy has been the treatment of choice for patients with HIV infection. Unfortunately it has been established that the beneficial effects of zidovudine are not sustained due to the development of resistant viral strains. This has led to the strategy of combination therapy, and in 1995 treatment with zidovudine plus didanosine, or zidovudine plus zalcitabine, was demonstrated to be more effective than zidovudine monotherapy in preventing disease progression and reducing mortality in patients with HIV disease. Recent work demonstrates an even greater antiviral effect from triple therapy with 2 nucleosides, zidovudine plus zalcitabine with the addition of saquinavir, a new protease inhibitor drug. The HIV protease enzyme is responsible for the post-translational processing of gag and gag-pol polyprotein precursors, and its inhibition by drugs such as saquinavir, ritonavir, indinavir and VX-478 results in the production of non-infectious virions. As resistance may also develop to the protease inhibitors they may be used in combination, and future strategies may well include quadruple therapy with 2 nucleoside analogues plus 2 protease inhibitors. Administration of protease inhibitors alone or in combination with other drugs does raise a number of important pharmacokinetic issues for patients with HIV disease. Some protease inhibitors (e.g. saquinavir) have kinetic profiles characterised by reduced absorption and a high first pass effect, resulting in poor bioavailability which may be improved by administrating with food. Physiological factors including achlorhydria, malabsorption and hepatic dysfunction may influence the bioavailability of protease inhibitors in HIV disease. Protease inhibitors are very highly bound to plasma proteins (> 98%), predominantly to alpha 1-acid glycoprotein. This may influence their antiviral activity in vitro and may also predispose to plasma protein displacement interactions. Such interactions are usually only of clinical relevance if the metabolism of the displaced drug is also inhibited. This is precisely the situation likely to pertain to the protease inhibitors, as ritonavir may displace other protease inhibitor drugs, such as saquinavir, from plasma proteins and inhibit their metabolism. Protease inhibitors are extensively metabolised by the cytochrome P450 (CYP) enzymes present in the liver and small intestine. In vitro studies suggest that the most influential CYP isoenzyme involved in the metabolism of the protease inhibitors is CYP3A, with the isoforms CYP2C9 and CYP2D6 also contributing. Ritonavir has an elimination half-life (t1/2 beta) of 3 hours, indinavir 2 hours and saquinavir between 7 and 12 hours. Renal elimination is not significant, with less than 5% of ritonavir and saquinavir excreted in the unchanged form. As patients with HIV disease are likely to be taking multiple prolonged drug regimens this may lead to drug interactions as a result of enzyme induction or inhibition. Recognised enzyme inducers of CYP3A, which are likely to be prescribed for patients with HIV disease, include rifampicin (rifampin) [treatment of pulmonary tuberculosis], rifabutin (treatment and prophylaxis of Mycobacterium avium complex), phenobarbital (phenobarbitone), phenytoin and carbamazepine (treatment of seizures secondary to cerebral toxoplasmosis or cerebral lymphoma). These drugs may reduce the plasma concentrations of the protease inhibitors and reduce their antiviral efficacy. If coadministered drugs are substrates for a common CYP enzyme, the elimination of one or both drugs may be impaired. Drugs which are metabolised by CYP3A and are likely to be used in the treatment of patients with HIV disease include the azole antifungals, macrolide antibiotics and dapsone; therefore, protease inhibitors may interact with these drugs. (ABSTRACT TRUNCATED)

Aromatic P 1 replacements for the highly potent HIV-1 protease inhibitor CRIXIVAN ®

A series of analogs of CRIXIVAN ® containing various aromatic P 1 ligands were prepared and evaluated for inhibition of the HIV-1 protease enzyme. These new compounds were found to be effective inhibitors at nanomolar concentrations in the in vitro enzyme inhibition assay as well as the whole cell assay.

Saquinavir. A review of its pharmacology and clinical potential in the management of HIV infection.

Saquinavir is a peptide derivative which inhibits the HIV protease enzyme, preventing post-translational processing of viral polyproteins. It was the first agent of its class to become available for the treatment of HIV infection. Well controlled studies have assessed the effects of saquinavir, when used alone or in combination with reverse transcriptase inhibitors, in patients with advanced HIV infection. Analysis of CD4+ cell counts and measures of viral load in the ACTG 229 study suggest that triple therapy with saquinavir, zidovudine and zalcitabine is more effective than double therapy with saquinavir plus zidovudine or zidovudine plus zalcitabine in patients who have previously received long term treatment with zidovudine. Similar assessments from a small study in previously untreated patients suggest that double therapy with saquinavir plus zidovudine is more effective than monotherapy with either agent. Combination therapy with saquinavir and zalcitabine significantly reduced the time to the first AIDS-defining event or death, and the time to death, compared with zalcitabine alone, according to data from a large, long term study (NV 14256) in patients with advanced HIV infection who had previously received zidovudine. Saquinavir is generally well tolerated, either as monotherapy or in combination with reverse transcriptase inhibitors; no change in tolerability profile was reported when saquinavir was added to treatment with nucleoside analogues. In vitro and clinical studies have documented the emergence of saquinavir-resistant HIV strains. Although the possible impact of resistance on the clinical efficacy of saquinavir has yet to be fully characterised, genotypic and phenotypic resistance appear to develop slowly during treatment with saquinavir, and the drug does not appear to have a significant effect on the incidence of mutations associated with cross-resistance to other protease inhibitors. Thus, laboratory and clinical results suggest that saquinavir in combination with reverse transcriptase inhibitors is effective in the treatment of advanced HIV infection. Initial data on the effects of saquinavir on disease progression and mortality are promising, and the apparent absence of mutations conferring cross-resistance to other protease inhibitors is a potentially valuable clinical feature. Additional data on disease progression, mortality and viral resistance, together with information on relative efficacy (in comparison with other protease inhibitors), will need to be assessed before the clinical value of saquinavir can be fully determined. Nevertheless, saquinavir represents a valuable new pharmacological option for the treatment of HIV infection and is likely to be a useful component of combined therapy with reverse transcriptase inhibitors and/or other protease inhibitors.

Current knowledge and future prospects for the use of HIV protease inhibitors.

The HIV protease (or proteinase) enzyme is an essential component of the replicative cycle of HIV, performing the post-transitional processing of the gag and gag-pol gene products into the functional core proteins and viral enzymes. Inhibition of this enzyme leads to production of immature noninfectious viral progeny, and hence prevention of further rounds of infection. Structurally, the enzyme is a homodimer consisting of two identical 99 amino acid chains. HIV protease is a member of the aspartic protease family but is structurally dissimilar to human aspartic proteases such as renin, gastricsin and cathepsin D and E, suggesting the possibility of creating inhibitors with a wide therapeutic index. At least 6 inhibitors of HIV protease are currently in clinical development: saquinavir, indinavir, ritonavir, nelfinavir (AG-1343), KNI-272 and VX-478, the first four of which have shown antiretroviral activity and acceptable tolerability in initial phase I/II clinical trials. Resistance or reduced sensitivity to the leading protease inhibitors has been reported in vivo and appears to be associated with loss of therapeutic effect. However, resistance patterns appear to be distinct. Treatment for 1 year with indinavir has been reported to lead to selection of virus in 4 patients, which was cross-resistant to all other leading protease inhibitors. On the other hand, a larger series of clinical isolates from patients receiving saquinavir alone or in combination with zidovudine for up to 3 years did not lead to virus cross-resistant to either indinavir or ritonavir. This suggests that care should be exercised in designing the sequence of protease usage. Additionally, differing resistance patterns may be used to select combinations of protease inhibitors in future trials. Data from studies combining protease inhibitors with nucleoside analogues suggest value in terms of larger and more prolonged virological and immunological marker responses than are observed with single agent therapy, and this is likely to be the primary role for protease inhibitors; both in initial combinations for patients commencing therapy and as add-in therapies for patients previously treated with antiretrovirals. However, in vitro and animal pharmacokinetic studies also give evidence of the possibility of combining protease inhibitors, potentially leading to improved bioavailability, antiviral synergy and delay in emergence of viral resistance.

Cyclic urea HIV protease inhibitors containing alkynyl- and alkenyl-tethered heterocycles in the P2 region

A series of cyclic urea HIV protease inhibitors containing alkynyl-and alkenyl-tethered heterocycles in the P2 region were synthesized. The alkynyl compounds exhibited poor activity against the HIV protease enzyme. However, reduction of the triple bond afforded trans-alkenyl derivatives ( XU348 and XU430) which displayed superb potency (sub-nanomolar K i).

Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected

The D and L forms of the enzyme HIV-1 protease have been prepared by total chemical synthesis. The two proteins had identical covalent structures. However, the folded protein-enzyme enantiomers showed reciprocal chiral specificity on peptide substrates. That is, each enzyme enantiomer cut only the corresponding substrate enantiomer. Reciprocal chiral specificity was also evident in the effect of enantiomeric inhibitors. These data imply that the folded forms of the chemically synthesized D- and L-enzyme molecules are mirror images of one another in all elements of the three-dimensional structure. Enantiomeric proteins are expected to display reciprocal chiral specificity in all aspects of their biochemical interactions.

Direct observation of a ternary complex between the dimeric enzyme HIV-1 protease and a substrate-based inhibitor

Direct observation of a ternary complex between the dimeric enzyme HIV-1 protease and a substrate-based inhibitor