A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation.

A synthetic peptidemimetic substrate of the human immunodeficiency virus 1 (HIV-1) protease with a nonhydrolyzable pseudodipeptidyl insert at the protease cleavage site was prepared. The peptide U-81749 inhibited recombinant HIV-1 protease in vitro (inhibition constant Ki of 70 nanomolar) and HIV-1 replication in human peripheral blood lymphocytes (inhibitory concentration IC50 of 0.1 to 1 micromolar). Moreover, 10 micromolar concentrations of U-81749 significantly inhibited proteolysis of the HIV-1 gag polyprotein (p55) to the mature viral structural proteins p24 and p17 in cells infected with a recombinant vaccinia virus expressing the HIV-1 gag-pol genes. The HIV-1 like particles released from inhibitor-treated cells contained almost exclusively p55 and other gag precursors, but not p24. Incubation of HIV-like particles recovered from drug-treated cultures in drug-free medium indicated that inhibition of p55 proteolysis was at least partially reversible, suggesting that U-81749 was present within the particles.

Nonpeptidic potent HIV-1 protease inhibitors: (4-hydroxy-6-phenyl-2-oxo-2H- pyran-3-yl)thiomethanes that span P1-P2' subsites in a unique mode of active site binding.

Using molecular modeling and the information derived from the X-ray crystal structure of HIV-1 protease (HIV PR) complexed with the pyran-2-one 1, a series of (4-hydroxy-6-phenyl-2-oxo-2H-pyran-3-yl)thiomethanes was designed and analyzed as novel, nonpeptidic inhibitors of HIV PR. Structure-activity studies led to the discovery of inhibitor 19 having (RS)-1-(cyclopentylthio)-3-methylbutyl functionalization at the C-3 position, which exhibited a Kc of 33 nM. A X-ray crystallographic structure of 19 bound to HIV PR showed that structural water-301 (inhibitor-flap-bridging water) was displaced by the inhibitor. Interestingly, the enol moiety of the pyran-2-one formed a hydrogen bond directly with Asp125 and with Asp25 via a bridging water molecule, thus illustrating a unique mode of active site binding by an HIV PR inhibitor. The pendant cyclopentyl and isobutyl groups of 19 occupied the S1' and S2' binding sites, respectively, whereas the 6-phenyl group occupied a region in between the S1 and S3 pockets of HIV PR. Selected compounds were tested for antiviral activity on H9 cells infected with HIV-1IIIb. A correlation between enzymatic activity and antiviral activity was not found in this series. The best antiviral compound in this series, 18, contained (RS)-3-[cyclopentyl(cyclopentylthio)methyl] functionalization at the C-3 position of the pyran-2-one ring and exhibited a CIC50 of 14 microM and TC50 of 70 microM. These studies demonstrate that potent enzyme inhibition can be achieved by inhibitors that span only three subsites.

Inhibitors of the protease from human immunodeficiency virus: design and modeling of a compound containing a dihydroxyethylene isostere insert with high binding affinity and effective antiviral activity.

The peptidomimetic template and the dihydroxyethylene isostere insert that were applied successfully to the design of renin inhibitors have been extended to the related protease from human immunodeficiency virus (HIV). The present report describes the structure-activity study leading to the identification of an inhibitor with a Ki of less than 1 nM for the HIV type-1 protease (compound II). This compound, containing a diol insert, is highly effective in blocking polyprotein processing in in vitro cell culture assays. Results obtained from kinetic analysis, studies of the stereochemistry of the insert, and modeling have led to insights as to the requisites involved in the active site-inhibitor interaction.

Inhibitors of the protease from human immunodeficiency virus: synthesis, enzyme inhibition, and antiviral activity of a series of compounds containing the dihydroxyethylene transition-state isostere.

A number of potential HIV protease inhibitory peptides that contain the dihydroxyethylene isostere were prepared and evaluated for their enzyme binding affinity and antiviral activity in cell cultures. From the template of a previously reported active peptide A, modifications at the N- and C-terminal groups were assessed for potential maintenance of good inhibitory activity of the resulting peptides. Among the active peptides found, peptide XVIII exhibited potent enzyme inhibitory activity. Interestingly, the previously reported, effective 1(S)-amino-2(R)-hydroxyindan C-terminal group for the preparation of very active HIV protease inhibitory peptides could not be applied to the template of peptide XVIII. Molecular modeling of peptide XVIII was studied using the X-ray crystal structure of peptide A as a starting point in order to study the likely conformation of peptide XVIII in the active-site cleft. Relative binding conformations of peptide A and XVIII were obtained, although the reason for poor binding affinity for a number of congeneric peptides in this report was not straightforwardly apparent. More importantly, however, peptide XVIII was found to exhibit more effective antiviral activity in the HIV-1/PBMC assay than the reference peptide A which was previously reported to be approximately equal in efficacy to the reverse transcriptase inhibitor AZT in this assay.

Evaluation of a vitamin-cloaking strategy for oligopeptide therapeutics: biotinylated HIV-1 protease inhibitors.

The outstanding limitations to the oligopeptide as a therapeutic agent are poor oral availability and rapid biliary clearance. To address these concerns a series of eight peptidic HIV-1 protease inhibitors containing the structural segment of the vitamin biotin have been prepared. These have been evaluated with regard to the hypothesis that this vitamin would cloak the peptidic character of these oligopeptides, and thus impart to these inhibitors the potential for absorption and distribution via biotin transporters and receptors. By iterative optimization about a -Cha psi[CH-(OH)CH(OH)]Val- core inhibitory insert, three particularly potent inhibitors (K(i) < or = 10 nM) of the HIV-1 protease were obtained. Although excellent cell culture antiviral activity is observed for other peptidic protease inhibitors of comparable affinity, none in this series exhibited satisfactory antiviral activity. This failure is attributed to the incompatibility of the hydrophilic and hydrogen-bonding biotin segment, with the facile membrane permeability and intracellular access presumably required for antiviral activity. The ability of the biotin to cloak the peptide, and thus render the overall appearance of the conjugate as that of a vitamin, was evaluated. Four of this series were evaluated for recognition by the Caco-2 cell intestinal biotin transporter. None inhibited competitively biotin uptake, indicating a lack of recognition. A vitamin may bind to a specific protein carrier, and thus attain an improved serum profile (by resistance to biliary clearance) and advantageous delivery to cells. Therefore, the serum concentrations of three were evaluated following an iv bolus in a rat model for serum clearance. One of the three protease inhibitors (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-2-(1-methylethyl)-5-[[3-methyl-1-oxo-2-[[5- (hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl)-1- oxopentyl]amino]butyl]amino]-N-[2-methyl-1-[[(2- pyridinylmethyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta (1R*,2R*,3R*),6 alpha]]-) sustained a more than 5-fold increase in serum concentration at all time points relative to the benchmark structure. The remaining two had serum concentrations at least equal to the benchmark, suggestive of improved resistance to clearance. One (L-idonamide,6-cyclohexyl-2,5,6-trideoxy-5-[[2-[[5-(hexahydro-2-ox o-1H- thieno-[3,4-d]imidazol-4-yl)pentyl]thio]benzoyl]amino]-2-(1- methylethyl)-N-[2-methyl-1-[[(2-pyridinyl- methyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta(1R*,2R*),6a alpha]]-) was prepared as a complex with the biotin-binding protein avidin. Avidin may resemble an endogenous serum biotin carrier protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Denaturation/refolding of purified recombinant HIV reverse transcriptase yields monomeric enzyme with high enzymatic activity.

We engineered a prokaryotic expression vector encoding the HIV reverse transcriptase (RT). We grew Escherichia coli JM109 carrying the vector in a 250-liter stirred tank fermentor and purified RT (p66) under native conditions to apparent homogeneity. Purified p66 (greater than or equal to 5 mg/ml) was not stable, and was rapidly processed to its 51 kD derivative (p51), until p66:p51 levels were approximately 1:1. These latter RT preparations were chromatographed as heterodimers and had approximately fivefold higher specific RT enzymatic activities compared with those containing predominantly p66. P66 purified under dilute concentrations (less than or equal to 0.5 mg/ml) was monomeric in solution, resistant to p51 processing for weeks at 4 degrees C, but also had low specific RT enzymatic activities. To attempt the preparation of homogeneous p66 with specific RT enzymatic activities equivalent to p66:p51 heterodimers, purified heterodimers were denatured and p66 was purified and refolded during extensive dialysis (refolded p66). Refolded p66 (less than or equal to 0.5 mg/ml) was monomeric in solution and had identical specific RT enzymatic activities, Km for dTTP, and inhibition by 3'-azido-3'-deoxythymidine triphosphate compared with heterodimeric p66:p51 RT. The data indicates that HIV RT obtained from recombinant E. coli under native conditions is extensively processed at concentrations promoting dimerization. Moreover, RT denaturation and refolding yields apparently homogeneous monomeric p66, with specific RT enzymatic activities equivalent to heterodimeric RT.

Soluble CD4-PE40 is cytotoxic for a transfected mammalian cell line stably expressing the envelope protein of human immunodeficiency virus (HIV-1), and cytotoxicity is variably inhibited by the sera of HIV-1-infected patients.

Sera were obtained from 50 individuals infected with human immunodeficiency virus type 1 or from HIV-1-uninfected individuals before or after vaccination with recombinant gp160. These sera were evaluated for activity antagonistic to the cell-killing activity of the chimeric Pseudomonas exotoxin hybrid protein, sCD4-PE40. For these studies, Chinese hamster ovary (CHO) cells were transfected with a chimeric plasmid encoding the tat, rev, and envelope genes of HIV-1 and a cell line was selected for stable expression of the envelope glycoproteins at the cell surface (CHO-env). Cytotoxicity of sCD4-PE40 for CHO-env in the presence or absence of added human serum was quantitated spectrophometrically following enzymatic reduction of a tetrazolium bromide within the mitochondria of viable cells (MTT assay). Several HIV+ sera inhibited the cytotoxic activity of sCD4-PE40; the antagonist had properties consistent with those of immunoglobulins in that it was heat stable, absorbed by protein A, and reversible by increasing the concentration of sCD4-PE40. Of 15 HIV+ sera which strongly reacted with gp120, 11 (73%) also potently inhibited sCD4-PE40 cytotoxicity, and cytotoxicity was inhibited by sera from some HIV- individuals after, but not before, immunization with gp160. These data suggested a role for antibody to gp120 in the antagonistic activity. However, not all sera with antibody to gp120 antagonized sCD4-PE40 cytotoxicity and high levels of antagonist activity were frequently (40%) found in HIV+ sera lacking immunoblot-detectable antibody to gp120, or antibody to either CD4 or PE40. Grouping of the HIV+ sera according to the patients' absolute number of CD4+ cells revealed that the degree of inhibition of sCD4-PE40 cytotoxicity approached a Gaussian distribution, suggesting that persons with CD4+ cell counts between 200 and 700/mm3 may be more likely to possess significant levels of serum antagonist. This data have implications for the clinical development of sCD4-PE40 or other sCD4-based therapeutics in the management of HIV-1 infection.

A rapid solution immunoassay to quantify binding of the human immunodeficiency virus envelope glycoprotein to soluble CD4.

We developed a particle concentration fluorescent immunoassay to quantify the binding in solution of the human immunodeficiency virus (HIV) external glycoprotein (gp120) to soluble CD4 (sCD4). The assay is rapid (1 hr), quantitative, and requires as little as 0.1 pmole of gp120 per evaluation. We find that gp120, purified from recombinant baculovirus infected insect cells, is suitable for the assay. Moreover, sCD4s obtained either from recombinant E. coli or mammalian cells, consisting of the N-terminal two domains (about 180 amino acids) as well as linked to the active regions of Pseudomonas exotoxin A, bind gp120 similarly.

An ultrasensitive human immunodeficiency virus type 1 protease radioimmuno rate assay with a potential for monitoring blood levels of protease inhibitors in acquired immunodeficiency disease syndrome patients.

The angiotensin I-based peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser yields angiotensin I (Ang I) and Leu-Glu-Glu-Ser upon hydrolysis by the human immunodeficiency virus type 1 (HIV-1) protease, but not by human renin. N-terminal sequencing of the reaction products showed that the HIV-1 protease cleaved exclusively at the Leu-Leu bond. The rate of Ang I formation can be measured by a radioimmunoassay, since the parent peptide has minimal cross reactivity in this assay. The rate of enzymatic hydrolysis is maximal at pH 4.5-5.0 and at an ionic strength of 1 M. At 37 degrees C, 0.1 M Na acetate buffer, pH 5.0, 1 M NaCl, 10% glycerol, 5% ethylene glycol, 1 mg/ml bovine serum albumin, and 3 mM EDTA, the reaction obeys Michaelis-Menten type kinetics with Km = 17.2 +/- 3.5 microM and kcat = 2.30 +/- 0.33 min-1. The activity assay readily quantitates as little as 0.25 nM of HIV-1 protease. The production of Ang I by the HIV-1 protease is inhibited in the presence of a HIV-1 protease inhibitor. The newly discovered substrate is relatively insensitive to human or monkey serum. Therefore, the effect of sera from 20 patients with advanced acquired immunodeficiency disease syndrome (AIDS) on Ang I production in the above assay system was examined. Results of this study indicate that it may be possible to adapt the above Ang I-based system to determine blood levels of HIV-1 protease inhibitors in AIDS patients during clinical trials.

Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain.

A metal binding peptide, hexahistidine, preceding a renin cleavage sequence (Pro-Phe-His-Leu-Val-Ile-His-) was engineered on to the N-terminus of HIV-1 reverse transcriptase (RT). The chimeric protein was expressed in Escherichia coli and characterized after purification by DEAE chromatography and HPLC. Amino-terminal sequencing confirmed the presence of the first 15 amino acids of the chimeric protein. The chimeric exhibited RT activity like that of HIV-1 RT and was cleaved by human renin at the expected site. The potential of a hexa-histidine fusion in the purification of recombinant HIV-1 RT by immobilized metal affinity chromatography (IMAC) on the commonly used resin (IDA-Ni2+) was investigated. The chimeric gene product from a crude E. coli extract was strongly retarded on a immobilized nickel column, while most of the contaminating E. coli proteins were eliminated after elution with 20-35 mM imidazole. The bound chimeric protein was eluted with 300 mM imidazole and appeared predominantly as a single band on an SDS-polyacrylamide gel. The remarkable specificity of this affinity tail was further demonstrated by separating the chimeric protein from HIV-1 RT in a crude extract prepared by mixing extracts from cells expressing HIV-1 RT and the hexahistidine recombinant chimeric protein. The usefulness of a enzymatically cleavable metal binding peptide in the rapid purification and production of HIV-1 RT without proteolysis to a heterodimer is discussed.

Purification of an active monomeric recombinant HIV-1 trans-activator.

A method for the purification of a truncated, biologically active human immunodeficiency virus type 1 (HIV-1) trans-activator (rTAT) from recombinant Escherichia coli is reported here. The purification steps utilized include mild extraction (French press), concentration by ammonium sulfate precipitation, chromatography in 8 M urea on an S-Sepharose fast-protein liquid chromatography column, and finally, resolution by C-4 reverse-phase high-performance liquid chromatography. After the final step, the rTAT is dried and stored under salt-free conditions. Amino acid compositional analysis and N-terminal sequence analysis confirm that the purified protein is rTAT. Unlike other methods reported for purification of recombinant HIV-1 trans-activator, our protocol uses urea instead of guanidine HCl. The rTAT is fully soluble in buffered solutions at concentrations exceeding 10 mg/ml, migrates as a single 14 kDa species on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and two-dimensional PAGE gels with a pI of 9.3 +/- 0.3. Additionally, the rTAT migrates as a monomer on size-exclusion chromatography columns under native conditions. Finally, purified rTAT exhibits trans-activator activity when introduced into appropriate reporter cells. Since rTAT is monomeric when tested by gel filtration, and yet exhibits biological activity, we conclude that the method of purification we have utilized is distinct from all other methods reported to date.

An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection.

The activity of the human immunodeficiency virus (HIV) protease is essential for processing of the gag-pol precursor proteins and maturation of infectious virions. We have prepared a peptidomimetic inhibitor, U-75875, that inhibited HIV-1 gag-pol protein processing in an essentially irreversible manner. Noninfectious virus particles produced in the presence of the drug contained gag precursors and were morphologically immature. In human peripheral blood mononuclear cells and in a continuous cell line, U-75875 completely blocked HIV replication; in the latter case, no spread occurred over a period of 4 weeks. U-75875, on a molar basis, was as potent as 3'-azido-3'-deoxythymidine in blocking HIV-1 replication in human lymphocytes and also inhibited HIV-2 and simian immunodeficiency virus proteases, demonstrating that it has broad activity. These results provide further evidence for the therapeutic potential of protease inhibitors in HIV infection.

Discovery and optimization of nonpeptide HIV-1 protease inhibitors.

Several small, achiral nonpeptide inhibitors of HIV-1 protease with low micromolar activity were identified by mass screening of the Parke-Davis compound library. Two of the compounds, structurally similar, were both found to be competitive and reversible inhibitors [compound 1, 4-hydroxy-3-(3-phenoxypropyl)-1-benzopyran-2-one: Ki = 1.0 microM; compound 2, 4-hydroxy-6-phenyl-3-(phenylthio)-pyran-2-one: Ki = 1.1 microM]. These inhibitors were chosen as initial leads for optimization of in vitro inhibitory activity based on molecular modeling and X-ray crystallographic structural data. While improvements in inhibitory potency were small with analogues of compound 1, important X-ray crystallographic structural information of the enzyme-inhibitor complex was gained. When bound, 1 was found to displace H2O301 in the active site while hydrogen bonding to the catalytic Asps and Ile50 and Ile150. The pyranone group of compound 2 was found to bind at the active site in the same manner, with the 6-phenyl and the 3-phenylthio occupying P1 and P1', respectively. The structural information was used to develop design strategies to reach three or four of the internal pockets, P2-P2'. This work led to analogues of diverse structure with high potency (IC50 < 10 nM) that contain either one or no chiral centers and remain nonpeptide. The highly potent compounds possess less anti-HIV activity in cellular assays than expected, and current optimization now focuses on increasing cellular activity. The value of the HIV-1 protease inhibitors described is their potential as better pharmacological agents with a different pattern of viral resistance development, relative to the peptide inhibitors in human clinical trials.

Nonpeptidic HIV protease inhibitors: 4-hydroxy-pyran-2-one inhibitors with functional tethers to P1 phenyl ring to reach S3 pocket of the enzyme.

A systematic study of tethering various groups on 6-phenyl ring of 4-hydroxy-6-phenyl-3-[(2-isopropylphenyl)thio]pyran-2-one was performed to increase the binding affinity with HIV protease. This tethering approach was aimed to fill S3 pocket of the enzyme. Thus, tethering hydrophilic groups resulted in more potent inhibitors. Similarly, various aromatic hydrophobic rings as well as heterocyclic rings were explored as tethering substituents to alter the physical properties as well as to enhance the binding affinity with HIV protease. Inhibitor 24, 4-hydroxy-3-[(2-isopropylphenyl)thio]-6-[4-(3-pyridinylmethoxy+ ++ ) phenyl]-2H-pyran-2-one, was evaluated as a prototypic lead structure to study various physical as well as pharmacological properties of this class of HIV protease inhibitors.