Host cell factor requirement for hepatitis C virus enzyme maturation.

The cellular chaperone, HSP90, is identified here as an essential factor for the activity of NS2/3 protease of hepatitis C virus. The cleavage activity of NS2/3 protease synthesized in reticulocyte lysate is ATP-dependent, as evidenced by ATP depletion experiments and inhibition with nonhydrolyzable ATP analogs. Geldanamycin and radicicol, ATP-competitive inhibitors of the chaperone HSP90, also inhibit the cleavage of in vitro-synthesized NS2/3. Furthermore, these HSP90 inhibitors prevent NS2/3 cleavage when the protease is expressed in mammalian cells. The physical association of NS2/3 with HSP90 is demonstrated by immunoprecipitation. Thus, by way of a chaperone/folding activity, an HSP90-containing complex is required for maturation of the polyprotein that encodes the enzymes essential for hepatitis C virus replication.

Identification of MK-944a: a second clinical candidate from the hydroxylaminepentanamide isostere series of HIV protease inhibitors.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

The herpesvirus proteases as targets for antiviral chemotherapy.

Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure-activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

Inhibition of hepatitis C virus NS2/3 processing by NS4A peptides. Implications for control of viral processing.

The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3. The minimal protein region necessary to catalyze this cleavage includes most of NS2 and the N-terminal one-third of NS3. Autocleavage reactions using NS2/3 protein translated in vitro are used here to investigate the inhibitory potential of peptides likely to affect the reaction. Peptides representing the cleaved sequence have no effect upon reaction rates, and the reaction rate is insensitive to dilution. Both results are consistent with prior suggestions that the NS2/3 cleavage is an intramolecular reaction. Surprisingly, peptides containing the 12-amino acid region of NS4A responsible for binding to NS3 inhibit the NS2/3 reaction with K(i) values as low as 3 microM. Unrelated peptide sequences of similar composition are not inhibitory, and neither are peptides containing incomplete segments of the NS4A region that binds to NS3. Inhibition of NS2/3 by NS4A peptides can be rationalized from the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavage point) as suggested by the known three-dimensional structure of the NS3 protease domain (Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., Steinkuhler, C., Tomei, L., De Francesco, R., Kuo, L. C., and Chen, Z. (1998) Protein Sci. 7, 837-847). These findings may imply a sequential order to proteolytic maturation events in hepatitis C virus.

Dimerization and activation of the herpes simplex virus type 1 protease.

The quaternary state of the herpes simplex virus type 1 (HSV-1) protease has been analyzed in relation to its catalytic activity. The dependence of specific activity upon enzyme concentration indicated that association of the 27-kDa subunits strongly increased activity. Size-exclusion chromatography identified the association as a monomer-dimer equilibrium. Isolation of monomeric and dimeric species from a size-exclusion column followed by immediate assay identified the dimer as the active form of the enzyme. Activation of the protease by antichaotropic cosolvents correlated with changes in the monomer-dimer equilibrium. Thus, dimerization of the enzyme was enhanced in solvents containing glycerol or the anions citrate or phosphate. These are substances previously identified as activators of HSV-1 protease (Hall, D. L., and Darke, P. L. (1995) J. Biol. Chem. 270, 22697-22700). The relative potencies of these cosolvents as enzyme activators correlated with their efficiency in promoting dimerization. Under all solvent conditions examined, the dependence of specific activity upon enzyme concentration was consistent with a kinetic model in which only the dimer is active. Dissociation constants for the HSV-1 protease dimer determined with this model at 15 degrees C, pH 7.5, were 964 and 225 nM in 20% glycerol with 0.2 and 0.5 M citrate present, respectively. The activation of the HSV-1 protease by antichaotropic cosolvents was hereby shown to be similar in nature to the activation of the other well characterized herpesvirus protease, that from human cytomegalovirus.

Nonpeptidal P2 ligands for HIV protease inhibitors: structure-based design, synthesis, and biological evaluation.

Design and synthesis of nonpeptidal bis-tetrahydrofuran ligands based upon the X-ray crystal structure of the HIV-1 protease-inhibitor complex 1 led to replacement of two amide bonds and a 10 pi-aromatic system of Ro 31-8959 class of HIV protease inhibitors. Detailed structure-activity studies have now established that the position of ring oxygens, ring size, and stereochemistry are all crucial to potency. Of particular interest, compound 49 with (3S,3aS,6aS)-bis-Thf is the most potent inhibitor (IC50 value 1.8 +/- 0.2 nM; CIC95 value 46 +/- 4 nM) in this series. The X-ray structure of protein-inhibitor complex 49 has provided insight into the ligand-binding site interactions. As it turned out, both oxygens in the bis-Thf ligands are involved in hydrogen-bonding interactions with Asp 29 and Asp 30 NH present in the S2 subsite of HIV-1 protease. Stereoselective routes have been developed to obtain these novel ligands in optically pure form.

Elucidation of basic mechanistic and kinetic properties of influenza endonuclease using chemically synthesized RNAs.

Influenza virus utilizes a unique mechanism for initiating the transcription of viral mRNA. The viral transcriptase ribonucleoprotein complex hydrolyzes host cell transcripts containing the cap 1 structure (m7GpppG(2'-OMe)-) to generate a capped primer for viral mRNA transcription. Basic aspects of this viral endonuclease reaction are elucidated in this study through the use of synthetic, radiolabeled RNA substrates and substrate analogs containing the cap 1 structure. Unlike most ribonucleases, this viral endonuclease is shown to catalyze the hydrolysis of the scissile phosphodiester, resulting in 5'-phosphate- and 3'-hydroxyl-containing fragments. Nevertheless, the 2'-OH adjacent to the released ribosyl 3'-OH is shown to be important for catalysis. In addition, while the endonuclease steady-state turnover rate is measured to be 2 h(-1), phosphodiester bond hydrolysis is not rate-limiting. The direct generation of a free 3'-OH and the subsequent slow release of this product are consistent with the viral need for efficient use of the capped primer in subsequent reactions of the influenza transcriptase complex.

Active human cytomegalovirus protease is a dimer.

The quaternary state of the human cytomegalovirus (hCMV) protease has been analyzed in relation to its catalysis of peptide hydrolysis. Based on results obtained from steady state kinetics, size exclusion chromatography, and velocity sedimentation, the hCMV protease exists in a monomer-dimer equilibrium. Dimerization of the protease is enhanced by the presence of glycerol and high concentrations of enzyme. Isolation of monomeric and dimeric species eluted from a size exclusion column, followed by immediate assay, identifies the dimer as the active species. Activity measurements conducted with a range of enzyme concentrations are also consistent with a kinetic model in which only the dimeric hCMV protease is active. Using this model, the dissociation constant of the protease is 6.6 microM in 10% glycerol and 0.55 microM in 20% glycerol at 30 degrees C and pH 7.5.

Activation of the herpes simplex virus type 1 protease.

The catalytic efficiency of the mature HSV-1 protease has been examined as a function of solvent composition. With the peptide substrate HTYLQASEKFKMWG-amide, the specificity constant (kcat/Km) at pH 7.5 for cleavage is 5.2 M-1 s-1. This value increases to 38 M-1 s-1 when 25% glycerol is present in the reaction mixture. It was found that glycerol activation is but one case of the general phenomenon of HSV-1 protease activation by kosmotropes, or water structure-forming cosolvents. For example, an 860-fold increase in the protease activity (kcat/Km = 4500 M-1 s-1) occurs in the presence of 0.8 M sodium citrate. Similarly, the presence of 0.8 M sodium phosphate activates the catalytic efficiency by 420-fold (kcat/Km = 2200 M-1 s-1). The extent of HSV-1 protease activation by various anions correlates with the Hofmeister series. Both the susceptibility to proteolysis by trypsin and the protein fluorescence spectra of the HSV-1 protease change in the presence of activating solvents, suggesting a conformational change accompanying activation.

Crystal structure at 1.9-A resolution of human immunodeficiency virus (HIV) II protease complexed with L-735,524, an orally bioavailable inhibitor of the HIV proteases.

L-735,524 is a potent, orally bioavailable inhibitor of human immunodeficiency virus (HIV) protease currently in a Phase II clinical trial. We report here the three-dimensional structure of L-735,524 complexed to HIV-2 protease at 1.9-A resolution, as well as the structure of the native HIV-2 protease at 2.5-A resolution. The structure of HIV-2 protease is found to be essentially identical to that of HIV-1 protease. In the crystal lattice of the HIV-2 protease complexed with L-735,524, the inhibitor is chelated to the active site of the homodimeric enzyme in one orientation. This feature allows an unambiguous assignment of protein-ligand interactions from the electron density map. Both Fourier and difference Fourier maps reveal clearly the closure of the flap domains of the protease upon L-735,524 binding. Specific interactions between the enzyme and the inhibitor include the hydroxy group of the hydroxyaminopentane amide moiety of L-735,524 ligating to the carboxyl groups of the essential Asp-25 and Asp-25' enzymic residues and the amide oxygens of the inhibitor hydrogen bonding to the backbone amide nitrogen of Ile-50 and Ile-50' via an intervening water molecule. A second bridging water molecule is found between the amide nitrogen N2 of L-735,524 and the carboxyl oxygen of Asp-29'. Although other hydrogen bonds also add to binding, an equally significant contribution to affinity arises from hydrophobic interactions between the protease and the inhibitor throughout the pseudo-symmetric S1/S1', S2/S2', and S3/S3' regions of the enzyme. Except for its pyridine ring, all lipophilic moieties (t-butyl, indanyl, benzyl, and piperidyl) of L-735,524 are rigidly defined in the active site.

L-735,524: the design of a potent and orally bioavailable HIV protease inhibitor.

A series of HIV protease inhibitors possessing a hydroxylaminepentanamide transition state isostere have been developed. Incorporation of a basic amine into the backbone of the L-685,434 (2) series provided antiviral potency combined with a highly improved pharmacokinetic profile in animal models. Guided by molecular modeling and an X-ray crystal structure of the inhibited enzyme complex, we were able to design L-735,524. This compound is potent and competitively inhibits HIV-1 PR and HIV-2 PR with Ki values of 0.52 and 3.3 nM, respectively. It also stops the spread of the HIV-1IIIb-infected MT4 lymphoid cells at concentrations of 25-50 nM. To date, numerous HIV-PR inhibitors have been reported, but few have been studied in humans because they lack acceptable oral bioavailability. L-735,524 is orally bioavailable in three animals models, using clinically acceptable formulations, and is currently in phase II human clinical trials.

Purification of active herpes simplex virus-1 protease expressed in Escherichia coli.

Assembly of viral capsids for replication of herpes simplex virus requires the proteolytic processing of the assembly protein ICP35. The protease responsible for this process is encoded within the 635-amino acid open reading frame of the UL26 gene of the virus. A simple purification scheme is given in this report for the native, mature form of the protease expressed in Escherichia coli. The scheme allows the preparation of milligram quantities of purified enzyme for elucidation of kinetic mechanism as well as for structural studies. Utilizing a 13-residue peptide substrate representing the natural cleavage site that releases the protease, kcat and Km values of the purified native enzyme are 2.0 min-1 and 0.88 mM, respectively. Thus, peptide cleavage is less efficient than reported for other viral proteases. The possibility exists that viral or cellular factors are involved in vivo for activation of the protease for herpes capsid maturation.

The development of cyclic sulfolanes as novel and high-affinity P2 ligands for HIV-1 protease inhibitors.

Design and synthesis of a novel series of protease inhibitors incorporating conformationally constrained cyclic ligands for the S2-substrate binding site of HIV-1 protease is described. We recently reported urethanes of 3-tetrahydrofuranyl as P2 ligands for HIV-1 protease inhibitors. Subsequently, we have found that the urethane of 3(S)-hydroxysulfolane further increased the in vitro potency of these inhibitors. Furthermore, introduction of a small 2-alkyl group cis to the 3-hydroxyl group of either heterocyclic system further enhanced enzyme affinity. The cis-2-isopropyl group thus far offered optimum enhancement of the inhibitory properties. This led to the discovery of inhibitor 43 (IC50 3.5 nM, CIC95 50 +/- 14 nM) of comparable in vitro antiviral potency to the current clinical candidate 1 (Ro 31-8959) but of reduced molecular weight due to the exclusion of the P3 quinoline ligand. Also, it has been demonstrated that the octahydropyrindene derivative 34 is an effective replacement of the P1' decahydroisoquinoline derivative.

L-735,524: an orally bioavailable human immunodeficiency virus type 1 protease inhibitor.

To date, numerous inhibitors of the human immunodeficiency virus type 1 protease have been reported, but few have been studied extensively in humans, primarily as a consequence of poor oral bioavailability in animal models. L-735,524 represents a class of human immunodeficiency virus type 1 protease inhibitors, termed hydroxyaminopentane amides, that incorporate a basic amine into the hydroxyethylene inhibitor backbone. L-735,524 is a potent inhibitor of virus replication in cell culture and inhibits the protease-mediated cleavage of the viral precursor polyproteins that results in the production of noninfectious progeny viral particles. The compound is effective against viruses resistant to reverse transcriptase inhibitors and is synergistically active when used in combination with reverse transcriptase inhibitors. Most importantly, L-735,524 exhibits good oral bioavailability and plasma pharmacokinetic profiles in two species of laboratory animals by using clinically acceptable formulations. Accordingly, the compound was selected for evaluation of safety and pharmacokinetic studies in humans.

Stability of dimeric retroviral proteases.

The determination of dimer stabilities for the retroviral proteases has proved more challenging than anticipated, but it is a tractable problem when careful attention is made to potential interferences. For investigations of retroviral proteases not yet characterized, the fundamentally rigorous sedimentation equilibrium and other biophysical techniques may yet provide useful Kd values. They are preferable to the indirect methods emphasized in this chapter but nevertheless should be coupled with basic considerations such as recovery of activity at the end of an experiment and the relevance of values obtained to other situations. In the likely event that nanomolar Kd values are encountered in new investigations, the assay techniques provide the most readily available methods for many laboratories. Because these methods are sensitive to anything that affects enzyme activity, the use of complementary methods to verify dimerization constants is imperative. Inactivating reactions not due to monomer formation should be explored, and the potential impact of those reactions on the constants being measured should be estimated. Most of the Kd and dimerization rate data available for retroviral proteases are obtained with the HIV-1 protease, with each investigator choosing methods and solvent conditions different from the others. The confusing diversity of results should be the impetus for a direct comparison of methods for the identification of the sources of differences. If more comprehensive and rigorous measures of the kinetics and thermodynamics of subunit aggregation are obtained, they might be coupled with the large volume of detailed structural data accumulating for this class of protein to provide insights into more general problems of protein-folding chemistry.

Dissociation and association of the HIV-1 protease dimer subunits: equilibria and rates.

The kinetics and equilibrium properties were investigated for the interconversion between the active dimer of human immunodeficiency virus 1 (HIV-1) protease and its inactive monomeric subunits. The equilibrium dissociation constant (Kd) of the dimeric protease as well as the monomer association rate were obtained by monitoring the fluorescence change of an active-site-directed fluorescent probe (L-737244) upon its binding to the protease. The Kd of the HIV-1 protease is strongly pH dependent. At pH 5.5 where the enzyme is most active catalytically, the extrapolated values of Kd are 0.75 and 3.4 nM at 30 and 37 degrees C, respectively. The rate constant for HIV-1 monomer association, approximately 4 x 10(5) M-1 s-1, is within the range commonly observed for protein-protein interactions. Dimer dissociation was further scrutinized in the presence of an inactive, point mutant form of the enzyme. As a result of subunit exchange between the native and mutant enzymes and the formation of an inactive heterodimer, there was a time-dependent decrease in the activity of the native protease. Enzyme activity could be reinstated with the addition of an active-site-directed inhibitor (L-365862) which selectively binds active dimers. The rate of dimer dissociation was found to also decrease with pH. At pH 5.5 and 30 degrees C, the half-life for subunit dissociation is about 0.5 h. The slow dissociation, coupled with the high stability for dimer association, attests to the importance of allowing sufficient time for dimer-monomer equilibration in kinetic assays in order to avoid reaching erroneous conclusions in studies of dimer dissociation.

Potent HIV protease inhibitors: the development of tetrahydrofuranylglycines as novel P2-ligands and pyrazine amides as P3-ligands.

A series of protease inhibitors bearing constrained unnatural amino acids at the P2-position and novel heterocycles at the P3-position of compound 1 (Ro 31-8959) were synthesized, and their in vitro enzyme inhibitory and antiviral activities were evaluated. Replacement of P2-asparagine of compound 1 with (2S,3'R)-tetrahydrofuranylglycine resulted in improvement in enzyme inhibitory as well as antiviral potencies (compound 23). Interestingly, incorporation of (2S,3'S)-tetrahydrofuranylglycine at the P2-position proved to be less effective. The resulting compound 24 was 100-fold less potent than the 2S,3R-isomer (compound 23). This stereochemical preference indicated a hydrogen-bonding interaction between the tetrahydrofuranyl oxygen and the residues of the S2-region of the enzyme active site. Furthermore, replacement of P3-quinolinoyl ligand of 1 with various novel heterocycles resulted in potent inhibitors of HIV proteases. Of particular interest, compound 2 with (2S,3'R)-tetrahydrofuranylglycine at P2 and pyrazine derivative at P3 is one of the most potent inhibitors of HIV-1 (IC50 value 0.07 nM) and HIV-2 (IC50 value 0.18 nM) proteases. Another important result in this series is the identification of compound 27 in which the P2-P3-amide carbonyl has been removed. The resulting compound 27 has exhibited improvement in antiviral potency while retaining the enzyme inhibitory potency similar to compound 1.