Disruption of the HIV-1 protease dimer with interface peptides: structural studies using NMR spectroscopy combined with [2-(13)C]-Trp selective labeling

HIV-1 protease (HIV-1 PR), which is encoded by retroviruses, is required for the processing of gag and pol polyprotein precursors, hence it is essential for the production of infectious viral particles. In vitro inhibition of the enzyme results in the production of progeny virions that are immature and noninfectious, suggesting its potential as a therapeutic target for AIDS. Although a number of potent protease inhibitor drugs are now available, the onset of resistance to these agents due to mutations in HIV-1 PR has created an urgent need for new means of HIV-1 PR inhibition. Whereas enzymes are usually inactivated by blocking of the active site, the structure of dimeric HIV-1 PR allows an alternative inhibitory mechanism. Since the active site is formed by two half-enzymes, which are connected by a four-stranded antiparallel beta-sheet involving the N- and C- termini of both monomers, enzyme activity can be abolished by reagents targeting the dimer interface in a region relatively free of mutations would interfere with formation or stability of the functional HIV-1 PR dimer. This strategy has been explored by several groups who targeted the four-stranded antiparallel beta-sheet that contributes close to 75% of the dimerization energy. Interface peptides corresponding to native monomer N- or C-termini of several of their mimetics demonstrated, mainly on the basis of kinetic analyses, to act as dimerization inhibitors. However, to the best of our knowledge, neither X-ray crystallography nor NMR structural studies of the enzyme-inhibitor complex have been performed to date. In this article we report a structural study of the dimerization inhibition of HIV-1 PR by NMR using selective Trp side chain labeling.     

Atomistic simulations of the HIV-1 protease folding inhibition

Biochemical experiments have recently revealed that the p-S8 peptide, with an amino-acid sequence identical to the conserved fragment 83-93 (S8) of the HIV-1 protease, can inhibit catalytic activity of the enzyme by interfering with protease folding and dimerization. In this study, we introduce a hierarchical modeling approach for understanding the molecular basis of the HIV-1 protease folding inhibition. Coarse-grained molecular docking simulations of the flexible p-S8 peptide with the ensembles of HIV-1 protease monomers have revealed structurally different complexes of the p-S8 peptide, which can be formed by targeting the conserved segment 24-34 (S2) of the folding nucleus (folding inhibition) and by interacting with the antiparallel termini β-sheet region (dimerization inhibition). All-atom molecular dynamics simulations of the inhibitor complexes with the HIV-1 PR monomer have been independently carried out for the predicted folding and dimerization binding modes of the p-S8 peptide, confirming the thermodynamic stability of these complexes. Binding free-energy calculations of the p-S8 peptide and its active analogs are then performed using molecular dynamics trajectories of the peptide complexes with the HIV-1 PR monomers. The results of this study have provided a plausible molecular model for the inhibitor intervention with the HIV-1 PR folding and dimerization and have accurately reproduced the experimental inhibition profiles of the active folding inhibitors.     

Inhibition of HIV-1 protease by short peptides derived from the terminal segments of the protease.

The active HIV-1 protease is a homodimeric enzyme. A beta-sheet consisting of N- and C-terminal segments provides the main driving force for dimerization of the inactive protomers. Several short peptides with sequences derived from the N- and C-termini of the protease were tested for inhibition of protease activity and for inhibition of HIV-1 replication in lymphocytes. Medium inhibitory activity was found with each of the peptides in the enzyme test and no inhibition of the lymphocytes was found up to 200 micrograms/ml. The enzyme tests indicate that HIV-1 protease is the target of the inhibitory action. Synergistic action could not be found with pairs of the peptides derived from the two different termini. Prolonged incubation with one of the peptides increased inhibition indicating a slow dissociation of the protease dimers. No cytotoxic effect of the inhibitors could be found below 200 micrograms/ml.     

Yeast two-hybrid assay for examining human immunodeficiency virus protease heterodimer formation with dominant-negative inhibitors and multidrug-resistant variants

The yeast two-hybrid assay was used to study the dimerization of engineered and naturally occurring variants of human immunodeficiency virus (HIV) protease (PR) monomers. Defective monomers that were previously shown to exhibit a dominant-negative (D-N) effect in cultured mammalian cells were tested for their ability to interact in the two-hybrid assay. Similarly, monomers with dimer-interface substitutions and monomers harboring in vivo selected mutations that confer multidrug resistance (mdr) in an AIDS patient were tested for interaction in yeast. Dimer formation between wt monomers with catalytic aspartates was not detected in yeast, whereas the dimerization of PR monomers harboring the acid active site substitution D25N was readily demonstrated. The use of inactive monomers harboring the D25N substitution as a genetic background for studying additional HIV PR mutations allowed for the probing of interactions between monomers with mdr-associated mutations with those based on the HIV-1 HXB2R sequence. The HTLVIII/HIV-1 HXB2R clone has been the basis for a large number of HIV-related plasmids, primers, antibodies, and other specific reagents throughout the HIV research community. The results of our assay suggest that HXB2R-based D-N PR inhibitors associate with variant monomers based on the recently obtained nucleotide sequence from an AIDS patient with a multidrug-resistant virus. These results further encourage the use of D-N PR inhibitors as antiviral agents which may complement existing small-molecule combination therapies.     

Sidechain-linked inhibitors of HIV-1 protease dimerization

There is a great need for alternative modes of inhibition for the design of anti-HIV therapies, due to the increased resistance of HIV to currently approved drugs. A novel strategy for generating potent dimerization inhibitors of HIV-1 protease is described based on sidechain-linked interfacial peptides. In a number of cases the activity of these agents against HIV-1 protease was found to be among the most potent reported, with inhibitory constants in the low nM range.     

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.     

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.     

Special considerations in the purification of the GM3 ganglioside-forming enzyme, CMP-sialic acid:lactosylceramide alpha 2-3 sialyltransferase (SAT-1): effects of protease inhibitors on rat hepatic SAT-1 activity

Co-purification of an endogenous proteolytic activity has been proposed as the cause for the size heterogeneity of sialyltransferases. Reported herein are results on the effects of various protease inhibitors, sulfhydryl-reducing agents and antimicrobial agents on SAT-1 activity. Addition of protease inhibitors to immunoaffinity-purified rat liver SAT-1 dramatically affects its activity. All protease inhibitors examined, with the exception of PMSF, inhibited the purified enzyme. The most inhibitory were the cysteine (thiol) protease inhibitors. This effect is less spectacular when the effect of these inhibitors was studied on SAT-1 activity in Golgi-enriched microsomes, although the inhibition was greatest by the cysteine protease inhibitors. One dramatic effect, found in both cases, was the apparent activation of SAT-1 activity in the presence of beta-mercaptoethanol.     

Polyarginine inhibits gp160 processing by furin and suppresses productive human immunodeficiency virus type 1 infection

Correct endoproteolytic maturation of gp160 is essential for the infectivity of human immunodeficiency virus type 1. This processing of human immunodeficiency virus-1 envelope protein, gp160, into gp120 and gp41 has been attributed to the activity of the cellular subtilisin-like proprotein convertase furin. The prototypic furin recognition cleavage site is Arg-X-Arg/Lys-Arg. Arg-Arg-Arg-Arg-Arg-Arg or longer iterations of polyarginine have been shown to be competitive inhibitors of substrate cleavage by furin. Here, we tested polyarginine for inhibition of productive human immunodeficiency virus-1-infection in T-cell lines, primary peripheral blood mononuclear cells, and macrophages. We found that polyarginine inhibited significantly human immunodeficiency virus-1 replication at concentrations that were benign to cell cultures ex vivo and mice in vivo. Using a fluorogenic assay, we demonstrated that polyarginine potently inhibited substrate-specific proteolytic cleavage by furin. Moreover, we verified that authentic processing of human immunodeficiency virus-1 gp160 synthesized in human cells from an infectious human immunodeficiency virus-1 (HIV-1) molecular clone was effectively blocked by polyarginine. Taken together, our data support that inhibitors of proteolytic processing of gp160 may be useful for combating human immunodeficiency virus-1 and that polyarginine represents a lead example of such inhibitors.     

A unidirectional crosslinking strategy for HIV-1 protease dimerization inhibitors

A novel strategy to identify potent HIV-1 protease dimerization inhibitors was developed using 12-aminododecanoic acid as a tether to crosslink interfacial peptides. The directionality of the southern peptide was changed from N-->C to C-->N as compared to previously reported inhibitors. The terminal amine of the southern peptide and side chains were further diversified to find essential functional groups for dimerization inhibition of HIV-1 protease.     

Design and synthesis of new inhibitors of HIV-1 protease dimerization with conformationally constrained templates.

To inhibit the HIV-1 protease dimerization necessary to exhibit enzymatic activity, we synthesized and evaluated a new beta-sheet peptide (compound 1), containing 4-(2-aminoethyl)-6-dibenzofuranpropionic acid as a conformationally restricted linker and a non-peptidic beta-strand mimetic, 2-[3-([2-[(9-fluorenylmethoxy)carbonyl]hydrazino]carbonyl)-4-methoxyanilino]-2-oxoacetic acid (Fmoc-Hao-OH, compound 2). Kinetic analysis showed that compound 1 inhibited the dimerization of HIV-1 protease by a dissociative mechanism with a K(id) value of 5.4 microM at 37 degrees C (pH 5.0). However, compound 2 showed a small shift in the slope of the lines in the Zhang-Poorman plot (K(id)=9.1 microM), suggesting that compound 2 inhibits the dimerization of HIV-1 PR not only through a dissociative mechanism but also through an active-site directed mechanism partly. This is the first study of a non-peptidic inhibitor of HIV-1 protease dimerization.     

Genetic selection for dissociative inhibitors of designated protein-protein interactions

Many biological processes rely on protein-protein interactions. These processes include signal transduction, cell cycle regulation, gene regulation, and viral assembly and replication. Moreover, many proteins and enzymes manifest their function as oligomers. We describe here an efficient means to sift through large combinatorial libraries and identify molecules that block the interaction of target proteins in vivo. The power of this approach is demonstrated by the identification of nine-residue peptides from a combinatorial library that inhibit the intracellular dimerization of HIV-1 protease. Fewer than 1 in 106 peptides do so. In vitro biochemical analyses of one such peptide demonstrate that it acts by dissociating HIV-1 protease into monomers, which are inactive catalysts. Inhibition is enhanced further by dimerizing the peptide. This approach enables the facile identification of new molecules that control cellular processes.     

Loss of protease dimerization inhibition activity of darunavir is associated with the acquisition of resistance to darunavir by HIV-1

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV expression assay. All recombinant HIV(NL4-3)-based clones with such a protease dimerization-disrupting substitution failed to replicate. A highly DRV-resistant in vitro-selected HIV variant and clinical HIV strains isolated from AIDS patients failing to respond to DRV-containing antiviral regimens typically had the V32I, L33F, I54M, and I84V substitutions in common in protease. None of up to 3 of the 4 substitutions affected DRV's protease dimerization inhibition, which was significantly compromised by the four combined substitutions. Recombinant infectious clones containing up to 3 of the 4 substitutions remained sensitive to DRV, while a clonal HIV variant with all 4 substitutions proved highly resistant to DRV with a 205-fold 50% effective concentration (EC(50)) difference compared to HIV(NL4-3). The present data suggest that the loss of DRV activity to inhibit protease dimerization represents a novel mechanism contributing to HIV resistance to DRV. The finding that 4 substitutions in PR are required for significant loss of DRV's protease dimerization inhibition should at least partially explain the reason DRV has a high genetic barrier against HIV's acquisition of DRV resistance.     

Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode

The main protease (M^pro) plays a vital role in proteolytic processing of the polyproteins in the replicative cycle of SARS coronavirus (SARS-CoV). Dimerization of this enzyme has been shown to be indispensable for trans-cleavage activity. However, the auto-processing mechanism of M^pro, i.e. its own release from the polyproteins through autocleavage, remains unclear. This study elucidates the relationship between the N-terminal autocleavage activity and the dimerization of “immature” M^pro. Three residues (Arg4, Glu290, and Arg298), which contribute to the active dimer conformation of mature M^pro, are selected for mutational analyses. Surprisingly, all three mutants still perform N-terminal autocleavage, while the dimerization of mature protease and trans-cleavage activity following auto-processing are completely inhibited by the E290R and R298E mutations and partially so by the R4E mutation. Furthermore, the mature E290R mutant can resume N-terminal autocleavage activity when mixed with the “immature” C145A/E290R double mutant whereas its trans-cleavage activity remains absent. Therefore, the N-terminal auto-processing of M^pro appears to require only two “immature” monomers approaching one another to form an “intermediate” dimer structure and does not strictly depend on the active dimer conformation existing in mature protease. In conclusion, an auto-release model of M^pro from the polyproteins is proposed, which will help understand the auto-processing mechanism and the difference between the autocleavage and trans-cleavage proteolytic activities of SARS-CoV M^pro.     

An autoregulated dual-function antitat gene for human immunodeficiency virus type 1 gene therapy.

One approach to gene therapy for AIDS is to block the replication of human immunodeficiency virus type 1 (HIV-1) by inhibiting that tat gene, whose product activates the expression of all HIV-1 genes. To accomplish this, we constructed an antitat gene expressing an RNA with dual (polymeric TAR and antisense-tat) function in an attempt to both sequester Tat protein and block its translation from mRNA. A minigene consisting of the antitat gene driven by the HIV-1 long terminal repeat was inserted into a double-copy retrovirus vector, such that antitat expression would be upregulated only in HIV-1-infected cells. After transduction of a T-lymphocytic cell line (Molt-3) the antitat gene inhibited HIV-1 replication. This inhibition was inversely correlated with the virus infections dose. Virus replication was also inhibited for 5 months in two different T-cell lines after they had been infected at a high multiplicity of infection, suggesting that the antitat gene may be effective over long periods. Importantly, antitat blocked the replication and the cytopathic effect of HIV-1 in human peripheral blood mononuclear cells and led to as much as 4,000-fold inhibition of the replication of an HIV-1 field isolate as well as HIV-1 prototypes maintained in culture. These results suggest that antitat gene therapy has potential use for blocking HIV-1 replication in infected individuals.     

Inhibition of HIV-1 integration protein by aurintricarboxylic acid monomers, monomer analogs, and polymer fractions.

Several aurintricarboxylic acid (ATA) monomers, monomer analogs, and polymer fractions have been tested as inhibitors of HIV-1 integration protein (IN). Both of the ATA monomers and all of the ATA polymer fractions inhibited a selective DNA cleavage reaction catalyzed by IN. The ATA monomer analogs were inactive or had low activity. The activities of the substances as inhibitors of HIV IN correlated in a positive way with their activities as inhibitors of the cytopathic effect of HIV-1 in CEM and HIV-2 in MT4 cells. These results suggest that inhibition of HIV IN may contribute to the antiviral activity of the ATA monomers and monomer analogs in cell culture.