Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation.

The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.     

Human immunodeficiency virus type 1 and type 2 protease monomers are functionally interchangeable in the dimeric enzymes.

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 proteases are dimers of identical subunits. We made a construct for the expression of recombinant one-chain HIV-2 protease dimer, which, like the previously described one-chain HIV-1 protease dimer, is fully active. The constructs for the one-chain dimers of HIV-1 and HIV-2 proteases were modified to produce hybrid one-chain dimers consisting of both HIV-1 and HIV-2 protease monomers. Although the monomers share only 47.5% sequence identity, the hybrid one-chain dimers are fully active, suggesting that the folding of both HIV-1 and HIV-2 protease monomers is functionally similar.     

Grafting segments from the extracellular surface of CCR5 onto a bacteriorhodopsin transmembrane scaffold confers HIV-1 coreceptor activity

Components from the extracellular surface of CCR5 interact with certain macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1) to mediate viral fusion and entry. To mimic these viral interacting site(s), the amino-terminal and extracellular loop segments of CCR5 were linked in tandem to form concatenated polypeptides, or grafted onto a seven-transmembrane bacteriorhodopsin scaffold to generate several chimeras. The chimera studies identified specific regions in CCR5 that confer HIV-1 coreceptor function, structural rearrangements in the transmembrane region that may modulate this activity, and a role for the extracellular surface in folding and assembly. Methods developed here may be applicable to the dissection of functional domains from other seven-transmembrane receptors and form a basis for future structural studies.     

A folding inhibitor of the HIV-1 protease

Because the human immunodeficiency virus type 1 protease (HIV-1-PR) is an essential enzyme in the viral life cycle, its inhibition can control AIDS. The folding of single-domain proteins, like each of the monomers forming the HIV-1-PR homodimer, is controlled by local elementary structures (LES, folding units stabilized by strongly interacting, highly conserved, as a rule hydrophobic, amino acids). These LES have evolved over myriad generations to recognize and strongly attract each other, so as to make the protein fold fast and be stable in its native conformation. Consequently, peptides displaying a sequence identical to those segments of the monomers associated with LES are expected to act as competitive inhibitors and thus destabilize the native structure of the enzyme. These inhibitors are unlikely to lead to escape mutants as they bind to the protease monomers through highly conserved amino acids, which play an essential role in the folding process. The properties of one of the most promising inhibitors of the folding of the HIV-1-PR monomers found among these peptides are demonstrated with the help of spectrophotometric assays and circular dichroism spectroscopy.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

Crucial stages of protein folding through a solvable model: predicting target sites for enzyme-inhibiting drugs

An exactly solvable model based on the topology of a protein native state is applied to identify bottlenecks and key sites for the folding of human immunodeficiency virus type 1 (HIV-1) protease. The predicted sites are found to correlate well with clinical data on resistance to Food and Drug Administration-approved drugs. It has been observed that the effects of drug therapy are to induce multiple mutations on the protease. The sites where such mutations occur correlate well with those involved in folding bottlenecks identified through the deterministic procedure proposed in this study. The high statistical significance of the observed correlations suggests that the approach may be promisingly used in conjunction with traditional techniques to identify candidate locations for drug attacks.     

Small-molecule inhibition of human immunodeficiency virus type 1 replication by specific targeting of the final step of virion maturation

Despite the effectiveness of currently available human immunodeficiency virus type 1 (HIV-1) therapies, a continuing need exists for new drugs to treat HIV-1 infection. We investigated the mechanism by which 3-O-[3',3'-dimethylsuccinyl]-betulinic acid (DSB) inhibits HIV-1 replication. DSB functions at a late stage of the virus life cycle but does not inhibit the HIV-1 protease in vitro or interfere with virus assembly or release. DSB specifically delays the cleavage of Gag between the capsid (CA) and p2, resulting in delayed formation of the mature viral core and reduced HIV-1 infectivity. Replication of simian immunodeficiency virus (SIV) was resistant to DSB; however, a chimeric SIV carrying CA-p2 sequences from HIV-1 was inhibited by the drug, indicating that susceptibility to DSB maps to the CA-p2 region of the HIV-1 Gag protein. A single point mutation at the CA-p2 cleavage site of HIV-1 conferred strong resistance to DSB, confirming the target of the drug. HIV-1 strains that are resistant to a variety of protease inhibitors were sensitive to DSB. These findings indicate that DSB specifically protects the CA-p2 cleavage site from processing by the viral protease during virion maturation, thereby revealing a novel mechanism for pharmacologic inhibition of HIV-1 replication.     

The three-dimensional structure of the aspartyl protease from the HIV-1 isolate BRU.

The crystal structure of the aspartyl protease encoded by the gene pol of the human immunodeficiency virus (HIV-1, isolate BRU) has been determined to 2.7 A resolution. The enzyme, expressed as an insoluble denatured polypeptide in inclusion bodies of Escherichia coli has been renatured and crystallized. It differs by several amino acid replacements from the homologous enzymes of other HIV-1 isolates. A superposition of the C alpha-backbone of the BRU protease with that of the SF2 protease gives a roots mean square positional difference of 0.45 A. Thus, neither the denaturation/renaturation process nor the amino acid replacements have a noticeable effect on the three-dimensional structure of the BRU protease or on the detailed conformation of the catalytic site, which is very similar to that of other aspartyl proteases.     

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.     

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases

The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF(2)-MazE(2)-MazF(2) heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.     

Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease

A protein corresponding to the putative protease of the human immunodeficiency virus 1 (HIV-1) has been prepared by total chemical synthesis. This 99 residue synthetic enzyme showed specific proteolytic activity on fragments of the natural gag precursor and on synthetic peptide substrates, two of which released fragments corresponding to the N terminus and C terminus of the protease molecule itself. The observed substrate specificity was not restricted to cleavage at Phe/Tyr-Pro bonds. Inhibition studies provided direct evidence that the HIV-1 protease belongs to the family of aspartic proteases. The availability of the HIV-1 protease as a defined molecular species has important implications for the design of specific inhibitors that do not interfere with the host cell metabolism as a possible route to antiviral agents against acquired immunodeficiency syndrome (AIDS).     

Suppression of HIV-1 protease inhibitor resistance by phosphonate-mediated solvent anchoring

The introduction of human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) markedly improved the clinical outcome and control of HIV-1 infection. However, cross-resistance among PIs due to a wide spectrum of mutations in viral protease is a major factor limiting their broader clinical use. Here we report on the suppression of PI resistance using a covalent attachment of a phosphonic acid motif to a peptidomimetic inhibitor scaffold. The resulting phosphonate analogs maintain high binding affinity to HIV-1 protease, potent antiretroviral activity, and unlike the parent molecules, display no loss of potency against a panel of clinically important PI-resistant HIV-1 strains. As shown by crystallographic analysis, the phosphonate moiety is highly exposed to solvent with no discernable interactions with any of the enzyme active site or surface residues. We term this effect "solvent anchoring" and demonstrate that it is driven by a favorable change in the inhibitor binding entropy upon the interaction with mutant enzymes. This type of thermodynamic behavior, which was not found with the parent scaffold fully buried in the enzyme active site, is a result of the increased degeneracy of inhibitor binding states, allowing effective molecular adaptation to the expanded cavity volume of mutant proteases. This strategy, which is applicable to various PI scaffolds, should facilitate the design of novel PIs and potentially other antiviral therapeutics.     

Serpin mechanism of hepatitis C virus nonstructural 3 (NS3) protease inhibition: induced fit as a mechanism for narrow specificity

Hepatitis C virus (HCV) nonstructural 3 (NS3) serine protease disrupts important cellular antiviral signaling pathways and plays a pivotal role in the proteolytic maturation of the HCV polyprotein precursor. This recent discovery has fostered the search for NS3 protease inhibitors. However, the enzyme's unusual induced fit behavior and peculiar molecular architecture have imposed considerable obstacles to the development of small molecule inhibitors. In this article, we demonstrate that such unique induced fit behavior and the chymotrypsin-like catalytic domain can provide the structural plasticity necessary to generate protein-based inhibitors of the NS3 protease. We took advantage of the macromolecular scaffold of a Drosophila serpin, SP6, which intrinsically supports chymotrypsin-like enzyme inhibition, to design a novel class of potent and selective inhibitors. We show that altering the SP6 reactive site loop (RSL) resulted in the development of the first effective (K(i) of 34 nm) and selective serpin, SP6(EVC/S), directed at the NS3 protease. SP6(EVC/S) operates as a suicide substrate inhibitor, and its partitioning between the complex-forming and proteolytic pathways for the NS3 protease is HCV NS4A cofactor-dependent and -specific. Once bound to the protease active site, SP6(EVC/S) partitions with equal probability to undergo proteolysis by NS3 at the C-terminal site of the engineered RSL, (P(6))Glu-Ile-(P(4))Val-Met-Thr-(P(1))Cys- downward arrow -(P(1)')Ser, or to form a covalent acyl-enzyme complex characteristic of cognate protease-serpin pairs. Our results also reveal a novel cofactor-induced serpin mechanism of enzyme inhibition that could be explored for developing effective and selective inhibitors of other important induced fit viral proteases of the Flaviviridae family such as the West Nile virus NS3 endoprotease.     

A fast and robust 19F NMR-based method for finding new HIV-1 protease inhibitors

The human immunodeficiency virus (HIV) which encodes, among other indispensable enzymes, an aspartic protease that is essential for viral maturation and replication. Numerous inhibitors of the protease have been developed. However, the eventual resistance of HIV-1 to these drugs implies a continuous battle to develop new inhibitors. Proposed herein is a robust, fast, and reliable method employing (19)F NMR for the evaluation of the inhibitory activity of new compounds against HIV-1 protease.     

The folding and dimerization of HIV-1 protease: evidence for a stable monomer from simulations

HIV-1 protease (PR) is a major drug target in combating AIDS, as it plays a key role in maturation and replication of the virus. Six FDA-approved drugs are currently in clinical use, all designed to inhibit enzyme activity by blocking the active site, which exists only in the dimer. An alternative inhibition mode would be required to overcome the emergence of drug-resistance through the accumulation of mutations. This might involve inhibiting the formation of the dimer itself. Here, the folding of HIV-1 PR dimer is studied with several simulation models appropriate for folding mechanism studies. Simulations with an off-lattice Gō-model, which corresponds to a perfectly funneled energy landscape, indicate that the enzyme is formed by association of structured monomers. All-atom molecular dynamics simulations strongly support the stability of an isolated monomer. The conjunction of results from a model that focuses on the protein topology and a detailed all-atom force-field model suggests, in contradiction to some reported equilibrium denaturation experiments, that monomer folding and dimerization are decoupled. The simulation result is, however, in agreement with the recent NMR detection of folded monomers of HIV-1 PR mutants with a destabilized interface. Accordingly, the design of dimerization inhibitors should not focus only on the flexible N and C termini that constitute most of the dimer interface, but also on other structured regions of the monomer. In particular, the relatively high phi values for residues 23-35 and 79-87 in both the folding and binding transition states, together with their proximity to the interface, highlight them as good targets for inhibitor design.     

Narrow substrate specificity and sensitivity toward ligand-binding site mutations of human T-cell Leukemia virus type 1 protease

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a number of human diseases; therefore, its protease is a potential target for chemotherapy. To compare the specificity of HTLV-1 protease with that of human immunodeficiency virus type 1 (HIV-1) protease, oligopeptides representing naturally occurring cleavage sites in various retroviruses were tested. The number of hydrolyzed peptides as well as the specificity constants suggested a substantially broader specificity of the HIV protease. Amino acid residues of HTLV-1 protease substrate-binding sites were replaced by equivalent ones of HIV-1 protease. Most of the single and multiple mutants had altered specificity and a dramatically reduced folding and catalytic capability, suggesting that mutations are not well tolerated in HTLV-1 protease. The catalytically most efficient mutant was that with the flap residues of HIV-1 protease. The inhibition profile of the mutants was also determined for five inhibitors used in clinical practice and inhibitor analogs of HTLV-1 cleavage sites. Except for indinavir, the HIV-1 protease inhibitors did not inhibit wild type and most of the mutant HTLV-1 proteases. The wild type HTLV-1 protease was inhibited by the reduced peptide bond-containing substrate analogs, whereas the mutants showed various degrees of weakened binding capability. Most interesting, the enzyme with HIV-1-like residues in the flap region was the most sensitive to the HIV-1 protease inhibitors and least sensitive to the HTLV-1 protease inhibitors, indicating that the flap plays an important role in defining the specificity differences of retroviral proteases.     

Evaluation of homology modeling of HIV protease

The model of human immunodeficiency virus (HIV-1) protease which was based on the crystal structure of Rous sarcoma virus (RSV) protease has been compared to the recently determined crystal structure of chemically synthesized HIV-1 protease. The overall difference between the model and crystal structure was 1.4 A root mean square (rms) deviation for 86 superimposed C alpha atoms. The position of the flexible flap differs in the model and six residues at the amino terminus were incorrectly placed. With these exceptions, all atoms of the model and crystal structure agree to 2.1 A rms deviation. The conformation of some surface bends in the model agrees less well with the crystal structure. Identical amino acids in RSV and HIV proteases were modeled more reliably than different types of amino acids. The amino acids which form the substrate binding site were modeled most accurately to 1.2 A rms deviation for all atoms compared to the crystal structure. This suggests that functionally significant regions of related proteins can be modeled with high accuracy. The model gave correct predictions for residues making interactions with the substrate, and therefore could be used to design inhibitors. The model based on the RSV protease structure is more similar to the experimental structure than are previous models based on the structures of non-viral aspartic proteases.