Beyond darunavir: recent development of next generation HIV-1 protease inhibitors to combat drug resistance.

We report our recent development of a conceptually new generation of exceptionally potent non-peptidic HIV-1 protease inhibitors that displayed excellent pharmacological and drug-resistance profiles. Our X-ray structural studies of darunavir and other designed inhibitors from our laboratories led us to create a variety of inhibitors incorporating fused ring polycyclic ethers and aromatic heterocycles to promote hydrogen bonding interactions with the backbone atoms of HIV-1 protease as well as van der Waals interactions with residues in the S2 and S2' subsites. We have also incorporated specific functionalities to enhance van der Waals interactions in the S1 and S1' subsites. The combined effects of these structural templates are critical to the inhibitors' exceptional potency and drug-like properties. We highlight here our molecular design strategies to promote backbone hydrogen bonding interactions to combat drug-resistance and specific design of polycyclic ether templates to mimic peptide-like bonds in the HIV-1 protease active site. Our medicinal chemistry and drug development efforts led to the development of new generation inhibitors significantly improved over darunavir and displaying unprecedented antiviral activity against multidrug-resistant HIV-1 variants.

HIV-1 protease has a genetic T-cell adjuvant effect which is negatively regulated by proteolytic activity.

HIV protease (PR) mediates the processing of human immunodeficiency virus (HIV) polyproteins and is necessary for the viral production. Recently, HIV PR was shown to possess both cytotoxic and chaperone like activity. We demonstrate here that HIV PR can serve as a genetic adjuvant that enhances the HIV Env and human papillomavirus (HPV) DNA vaccine-induced T-cell response in a dose-dependent manner, only when codelivered with DNA vaccine. Interestingly, the T-cell adjuvant effects of HIV PR were increased by introducing several mutations that inhibited its proteolytic activity, indicating that the adjuvant properties were inversely correlated with its proteolytic activity. Conversely, the introduction of a mutation in the flap region of HIV PR limiting the access to the core domain of HIV PR inhibited the T-cell adjuvant effect, suggesting that the HIV PR chaperone like activity may play a role in mediating T-cell adjuvant properties. A similar adjuvant effect was also observed in adenovirus vaccine, indicating vaccine type independency. These findings suggest that HIV PR can modulate T-cell responses elicited by a gene-based vaccine positively by inherent chaperone like activity and negatively by its proteolytic activity.

Involvement of HIV-1 protease in virus-induced cell killing.

Acute infection of human CD4+ cells with cytopathic strains of HIV-1 causes rapid cell death. The role played by HIV-1 protease (PR) in virus-induced cell killing was investigated by subjecting C8166 cells to a single round of infection. The presence of HIV-1 PR inhibitor saquinavir from 24h post-infection prevented virus-induced cell lysis. This inhibitor caused only a small reduction in the number of infected cells and in the expression of HIV-1-specific proteins. Moreover, treatments that block HIV-1 reinfection, such as AZT or the anti-CD4 antibody leu3.a, exerted little effect on virus-induced cell death. Thus, the specific inhibition of HIV-1 protease reduced the extent of both necrosis and apoptosis in C8166 cells such that most cells survived HIV-1 infection. Continued treatment of the infected cells with saquinavir led to the progressive suppression of HIV-1 expression; no viral proteins being detected 10 days after primary infection. Notably, reactivation of HIV-1 protease in these cells by removing the saquinavir triggered virus replication and cell lysis. These findings may contribute towards a better understanding of HIV-1 pathogenesis, and emphasise the potential of the virus protease as a key therapeutic target in AIDS treatment.

Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease.

The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we suggest that Vif interacts with the dimerization sites of the viral protease, and that peptide residing at the N terminus of PR abrogates Vif function(s).

Binding of anti-HIV drugs to human serum albumin.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand (i.e., drug) binding capacity. Here, values of the dissociation equilibrium constant (Kd)for the binding of HIV protease and reverse transcriptase inhibitors to HSA are reported. The binding of abacavir, atazanavir,didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir,nevirapine, ritonavir, saquinavir, stavudine, zalcitabine, and zidovudine to the Sudlow site I (i.e., the warfarin cleft) located in the subdomain IIA involves the alteration of the HSA structure around Trp214 and induces intrinsic tryptophan fluorescence quenching. Accordingly, ibuprofen that primarily binds to the Sudlow site II located in the subdomain IIIA does not affect the HSA intrinsic tryptophan fluorescence and the binding of anti-HIV drugs to the Sudlow site 1. Accounting for the physiological concentration of HSA (= 7.0 x 10(-4) M), the average anti-HIV drug concentration in plasma (= 1.0 x 10(-4) M), and Kd values for the binding of anti-HIV drugs to HSA (ranging between 4.4 x 10(-5)M and 3.8 x 10(-4) M), it appears that the fraction of HIV protease and reverse transcriptase inhibitors bound to HSA ranges between 63% and 91%. This represents a significant drawback in the anti-HIV therapy and management, the anti-HIV drug concentration required to achieve 90% protease and reverse transcriptase inhibition in the presence of plasma proteins appears to be at least one order of magnitude higher than that required in their absence.

Organization of focal adhesion plaques is disrupted by action of the HIV-1 protease.

Focal adhesion plaques were severely affected in human embryonic fibroblasts permeabilized with digitonin and incubated in buffer containing the human immunodeficiency virus type 1 protease (HIV-1 PR). A mutant HIV-1 PR (3271 HIV-1 PR) had no effect on focal adhesion plaques. Similar effects were seen with cells microinjected with either HIV-1 PR or 3271 HIV-1 PR. Immunoblots of the human embryonic fibroblasts demonstrated that a number of focal adhesion plaque proteins were specifically cleaved by HIV-1 PR. These included fimbrin, focal adhesion plaque kinase (FAK), talin, and, to a lesser extent, filamin, spectrin and fibronectin. Proteins detected by antibodies to beta 4 integrin and alpha 3 integrin were also cleaved by the HIV-1 PR. Control experiments demonstrated that the effect and protein cleavages described are due to action of the HIV-1 PR and not to the action of endogenous host cell proteases.

Real time NMR monitoring of local unfolding of HIV-1 protease tethered dimer driven by autolysis.

Structural studies in proteases have been hampered because of their inherent autolytic function. However, since autolysis is known to be mediated via protein unfolding, careful monitoring of the autolytic reaction has the potential to throw light on the folding-unfolding equilibria. In this paper we describe real time nuclear magnetic resonance investigations on the tethered dimer construct of the human immunodeficiency virus-1 protease, which have yielded insights into the relative stabilities of several residues in the protein. The residues lying along the active site (bottom, side and top of the active site) and those in helix have lower unfolding free energy values than the other parts of the protein. The residue level stability differences suggest that the protein is well suited to adjust itself in almost all the regions of its structure, as and when perturbations occur, either due to ligand binding or due to mutations.

Amino-terminal polypeptides of vimentin are responsible for the changes in nuclear architecture associated with human immunodeficiency virus type 1 protease activity in tissue culture cells.

Electron microscopy of human skin fibroblasts syringe-loaded with human immunodeficiency virus type 1 protease (HIV-1 PR) revealed several effects on nuclear architecture. The most dramatic is a change from a spherical nuclear morphology to one with multiple lobes or deep invaginations. The nuclear matrix collapses or remains only as a peripheral rudiment, with individual elements thicker than in control cells. Chromatin organization and distribution is also perturbed. Attempts to identify a major nuclear protein whose cleavage by the protease might be responsible for these alterations were unsuccessful. Similar changes were observed in SW 13 T3 M [vimentin(+)] cells, whereas no changes were observed in SW 13 [vimentin(-)] cells after microinjection of protease. Treatment of SW 13 [vimentin(-)] cells, preinjected with vimentin to establish an intermediate filament network, with HIV-1 PR resulted in alterations in chromatin staining and distribution, but not in nuclear shape. These same changes were produced in SW 13 [vimentin(-)] cells after the injection of a mixture of vimentin peptides, produced by the cleavage of vimentin to completion by HIV-1 PR in vitro. Similar experiments with 16 purified peptides derived from wild-type or mutant vimentin proteins and five synthetic peptides demonstrated that exclusively N-terminal peptides were capable of altering chromatin distribution. Furthermore, two separate regions of the N-terminal head domain are primarily responsible for perturbing nuclear architecture. The ability of HIV-1 to affect nuclear organization via the liberation of vimentin peptides may play an important role in HIV-1-associated cytopathogenesis and carcinogenesis.

Design of toxins that can be activated by cell-specific proteases and their potential use in targeted cell killing.

Protein toxins designed to eliminate specific cell types, e.g. disease-associated cells, have mainly made by linking the active domain of the toxin to a protein that only binds to certain cells. A different approach for the construction of toxins capable of killing disease-associated cells is suggested here, based on the knowledge that many of these cells express specific proteases that are not expressed in normal tissue. The construction of toxins that become activated through cleavage by the protease (HIV-1 PR) expressed by the HIV-1 virus is described. These toxins contain a signal for degradation by the N-end rule pathway, which is cleaved off by HIV-1 PR, resulting in increased toxicity. Alternative strategies for the construction of toxins that can be activated by proteases are discussed.

Kynostatin and 17beta-estradiol prevent the apoptotic death of human neuroblastoma cells exposed to HIV-1 protease.

A significant number of adult male patients with acquired immunodeficiency syndrome develop cerebral atrophy and progressive brain disorders such as dementia complex and neuropsychiatric problems. Upon entering the brain via activated macrophages or microglias, the human immunodeficiency type 1 virus (HIV-1) may produce cytotoxic factors such as HIV-1 envelope protein (gp120) and protease. Owing to significant proteolysis of nonviral proteins, the protease derived from HIV-1 may be detrimental to brain cells and neurons. Our results revealed that HIV-1 protease, at nanomolar concentrations, was as potent as gp120 in causing neurotoxicity in human neuroblastoma neurotypic SH-SY5Y cells. As shown by the Oncor ApopTag staining procedure, HIV-1 protease significantly increased the number of apoptotic cells over the serum-free controls. Moreover, HIV-1 protease-induced neurotoxicity was blocked by a selective protease inhibitor, kynostatin (KNI-272). Antioxidants such as 17beta-estradiol, melatonin, and S-nitrosoglutathione also prevented protease-induced neurotoxicity. These findings indicate that oxidative proteolysis may mediate HIV-1 protease-induced apoptosis and the degeneration of neurons and other brain cells. Centrally active protease inhibitors and antioxidants may play an important role in preventing cerebral atrophy and associated dementia complex caused by HIV-1.

Counteracting HIV-1 protease drug resistance: structural analysis of mutant proteases complexed with XV638 and SD146, cyclic urea amides with broad specificities.

The long-term therapeutic benefit of HIV antiretroviral therapy is still threatened by drug-resistant variants. Mutations in the S1 subsite of the protease are the primary cause for the loss of sensitivity toward many HIV protease inhibitors, including our first-generation cyclic urea-based inhibitors DMP323 and DMP450. We now report the structures of the three active-site mutant proteases V82F, I84V, and V82F/I84V in complex with XV638 and SD146, two P2 analogues of DMP323 that are 8-fold more potent against the wild type and are able to inhibit a broad panel of drug-resistant variants [Jadhav, P. K., et al. (1997) J. Med. Chem. 40, 181-191]. The increased efficacy of XV638 and SD146 is due primarily to an increase in P2-S2 interactions: 30-40% more van der Waals contacts and two to four additional hydrogen bonds. Furthermore, because these new interactions do not perturb other subsites in the protease, it appears that the large complementary surface areas of their P2 substituents compensate for the loss of P1-S1 interactions and reduce the probability of selecting for drug-resistant variants.

Cleavage of the complement system C3 component by HIV-1 proteinase.

The C3 factor of the complement system and its C3b fragment are cleaved in vitro by the proteinase of the human immunodeficiency virus, type 1 (HIV PR). The cleavage occurs in the alpha-chain of both substrates at multiple sites yielding a 100 kDa fragment of the C3 alpha-chain and multiple fragments of the C3b alpha-chain. The scissile bonds are: Ala86-Glu87, Leu310-Leu311, His641-Trp642 and Arg649-Ser650. The resulting fragments resemble the physiologically occurring inactive fragments of C3: C3c and C3d, suggesting a possible biological role of the HIV-proteinase in the complement inactivation process.

DNA fragmentation induced by protease activation in p53-null human leukemia HL60 cells undergoing apoptosis following treatment with the topoisomerase I inhibitor camptothecin: cell-free system studies.

We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1 beta converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.

HIV type 1 protease activation of NF-kappa B within T lymphoid cells.

NF-kappa B is a nuclear protein of the rel oncogene family capable of enhancing transcription of several cellular genes, including IL-2 and the IL-2 receptor, and viral genes transcribed from the HIV-1 LTR. It has been reported that HIV-1 protease may cleave the NF-kappa B precursor to its active form in vitro. In this study the effects of HIV protease on NF-kappa B precursor activation were examined in Jurkat T cells by introducing a protease expression vector into the cells. Increased NF-kappa B activity was observed and this increased activity was blocked by a specific inhibitor of the viral protease. Viral transcription, as measured using LTR-CAT assays, was only slightly enhanced in the HIV-protease expressing cells, while secretion of IL-2 and expression of the IL-2 receptor were not affected. The limited activation of NF-kappa B by HIV protease appears unlikely to have a significant effect on virus expression or T cell function.

Cleavage of human and mouse cytoskeletal and sarcomeric proteins by human immunodeficiency virus type 1 protease. Actin, desmin, myosin, and tropomyosin.

HeLa cell actin was cleaved by human immunodeficiency virus type 1 protease when in its soluble, globular form (G-actin). No cleavage of the polymerized, filamentous form of actin (F-actin) was observed when examined by denaturing gel electrophoresis; however, electron microscopy revealed a low level of cleavage of F-actin. Immunoblotting of mouse skeletal and human pectoral muscle myofibrils treated in vitro with human immunodeficiency virus type 1 protease showed that myosin heavy chain, desmin, tropomyosin, and a fraction of the actin were all cleaved. Electron microscopy of these myofibrils demonstrated changes consistent with cleavage of these proteins: Z-lines were rapidly lost, the length of the A bands was shortened, and the thick filaments (myosin filaments) were often laterally frayed such that the structures disintegrated. Nonmuscle myosin heavy chains were also cleaved by this enzyme in vitro. These data demonstrate that this protease can cause alterations in muscle cell ultrastructure in vitro that may be of clinical relevance in infected individuals.

Progression of early steps of human immunodeficiency virus type 1 replication in the presence of an inhibitor of viral protease.

We have evaluated a possible role for human immunodeficiency virus type 1 protease during early steps of replication. For these studies, a specific inhibitor of human immunodeficiency virus protease, Ro31-8959, was used. Synthesis of viral cDNA, its integration into cellular DNA, and its transcription were determined during a one-step, acute infection of MT-4 cells. No consistent difference in any of these parameters was noted between control-infected cultures and those treated with protease inhibitor. However, no infectious progeny virus was produced in treated cultures, and thus spread of infection was severely restricted. Our results do not support an essential activity of viral protease in early steps of replication but are in line with its established role in gag and gag-pol processing and in maturation to infectious progeny virus.

Specific inhibitor of human immunodeficiency virus proteinase prevents the cytotoxic effects of a single-chain proteinase dimer and restores particle formation.

The active form of the retroviral proteinase (PR) is a homodimer of monomeric subunits expressed as integral parts of the viral gag-pol precursor polyproteins, and dimerization of polyproteins is presumed to be important for regulation of PR activity. Expression of a single-chain dimer of the human immunodeficiency virus (HIV) type 1 PR as a component of the viral polyprotein has been shown to prevent particle assembly and viral infectivity (H.-G. Kräusslich, Proc. Natl. Acad. Sci. USA 88:3213-3217, 1991). Ro31-8959, a specific inhibitor of HIV PR, blocked proteolysis of polyproteins containing either wild-type or single-chain dimer PR at the same inhibitor concentration. Different inhibitor concentrations gave three phenotypic effects for the linked PR: at a concentration of 10 nM, cytotoxicity was prevented yet viral polyproteins were almost completely processed and no particles were released. The majority of HIV capsid proteins was found in the soluble cytoplasmic fraction, whereas at a concentration of 1 microM inhibitor most HIV gag proteins were associated with an insoluble fraction. Release of particles consisting of partially processed polyproteins was observed at 100 nM Ro31-8959, and polyprotein processing was blocked at 10 microM. Particles derived from the dimer-containing provirus were noninfectious independently of the inhibitor concentration. Production of infectious HIV after transfection of wild-type provirus was abolished at 100 nM and markedly reduced at 10 nM Ro31-8959.

Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture.

In human skin fibroblasts microinjected with purified human immunodeficiency virus type 1 protease (HIV-1 PR), stress fibers were lost and alterations in nuclear morphology and condensation of nuclear chromatin were observed. Thereafter, the vimentin intermediate filament (IF) network collapsed. No effect was seen on the microtubules. While complicated by loss of affected cells from the substratum, a minimum estimate of the proportion of cells demonstrating these effects is 50%. Observation of single cells demonstrated that these effects were largely irreversible and were steps leading to the death of the HIV-1 PR-injected cells. After microinjection of various dilutions of the HIV-1 PR, it was observed that the changes in nuclear morphology and chromatin condensation were detectable under conditions where little or no effect was observed on both stress fibers and the IF network. Proteins of cells labelled with [35S]methionine and microinjected with either HIV-1 PR or BSA were subjected to two-dimensional gel electrophoresis. The major differences in the gel patterns were a diminution in the amount of vimentin and the appearance of novel products comigrating with cleavage products obtained after treatment of vimentin with HIV-1 PR in vitro. Thus, the HIV-1 PR is capable not only of cleaving IF subunit proteins in vivo, but also can catalyze alterations in other cellular structures.

Antiviral properties of Ro 31-8959, an inhibitor of human immunodeficiency virus (HIV) proteinase.

Ro 31-8959 inhibits the spread of HIV infection and the production of cytopathic effects in cultures of acutely infected cells. IC50 values for these effects are in the range 0.5-6.0 nM and IC90 values are in the range 6.0-30.0 nM. This inhibitor is effective even when added to cultures at a late stage of infection, after syncytia have started to form. Virus antigen, virus particles and virus cytopathic effects can largely be cleared from cultures treated with compound from 3 days until 6 days post infection. In chronically-infected cells, inhibition of virus maturation can be detected after 24 hours' treatment with 10 nM Ro 31-8959. In addition, a significant reduction of the proteolytic processing of p56 to p24 can be demonstrated in these cells with compound at picomolar concentrations. These properties indicate that Ro 31-8959 is highly effective against HIV with the potential to inhibit acute, established acute and chronic infections.

alpha 2-Macroglobulin is cleaved by HIV-1 protease in the bait region but not in the C-terminal inter-domain region.

alpha 2-Macroglobulin is cleaved by human immunodeficiency virus-1 protease. The cleavage site is the Phe684-Tyr685 bond in the "bait region", an exposed part of alpha 2-macroglobulin, creating the "F-form". The methylamine derivative of alpha 2-macroglobulin is also cleaved at the same bond. The homologous chicken ovomacroglobulin does not form an F-form structure with the protease, although, F-form generation by other enzymes is known. This is possibly due to the lack of a suitable cleavage sequence in the corresponding region of ovomacroglobulin. In human alpha 2-macroglobulin, the interdomain segment between the main part of the molecule and the receptor-binding C-terminal domain is not cleaved by the HIV protease although typical cleavage sequences occur. In AIDS, therefore, HIV protease from infected cells in unlikely to interfere with receptor-binding of alpha 2-macroglobulin.