Current and Novel Inhibitors of HIV Protease

Abbreviations

1.Introduction

Figure 1. Trends in annual rates of death due to 7 leading causes among persons 25-44 years old in the United States during period 1987-2004. Dramatic decrease in the rate of death due to AIDS coincides with the introduction of HIV protease inhibitors (source: National Vital Statistics, Centers for Disease Control and Prevention, Atlanta).

Figure 2. The three-dimensional crystal structure of HIV PR dimer depicting mutations associated with resistance to clinically used protease inhibitors [7]. Mutated residues are represented with their Cα atoms (spheres) and colored in the shades of red and blue for major and minor mutations, respectively. For major mutations, the semi-transparent solvent accessible surface is also shown in red. Active site aspartates and PI darunavir bound to the active site are represented in stick models. The figure was generated using the structure of highly mutated patient derived HIV-1 PR (PDB code 3GGU [8]) and program PyMol [9].

Table 1. Mutations in the protease gene associated with resistance to PIs ^a.

2. Inhibitors currently used in clinical practice: structure, antiviral activity, side-effects, and pharmaceutical boosting

Table 2. Overview of the inhibitors of HIV protease approved for clinical use with their dosage, side effects, and the position in the present therapeutic arsenal, elaborated with regard to the recommendations of the US Department of Health and Human Services [Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-Infected adults and adolescents. Department of Health and Human Services. November 3, 2008; 1-139. Available online: http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf. (Accessed 12 September 2009)]

2.1. The first generation HIV protease inhibitors

The design of the first protease inhibitor saquinavir (SQV, Ro-31-8959, marketed by Hoffman-La Roche), which was approved by the FDA in December 1995 under the commercial name Invirase®, was based on rather unusual specificity of HIV protease for cleavage of the Phe-Pro and Tyr-Pro bond (Figure 3a). The scissile bonds were replaced by a hydroxyethylamine isostere [35]. Despite its high in vitro antiviral activity, the clinical efficacy of this drug was significantly reduced due to the insufficient bioavailability (less than 4%) and intense hepatic metabolism. In order to improve the bioavailability of SQV, a novel formulation of the drug in soft capsules was developed and marketed under the name of Fortovase® (Hoffman-La Roche) [36]. Subsequently, SQV was co-formulated with with ritonavir as a booster. As of February 15, 2006, the sales of Fortovase were discontinued in the United States because of poor drug tolerance; Invirase boosted by ritonavir remained the only drug formulation of SQV currently available on the market.

Typical primary resistance-conferring mutations selected under the pressure of SQV are G48V or L90M. The most common adverse reactions reported in patients receiving SQV were gastrointestinal disturbances, such as diarrhea, nausea, or abdominal discomfort [37].

Ritonavir (RTV, ABT-538) was designed by Abbot laboratories and gained FDA approval in 1996 under the name Norvir® [38]. The drug design process originally concentrated on C-2 symmetric molecules. However, the structures of the inhibitor/enzymes complexes proved that even the most active C-2 symmetric compound loses symmetry when bound to the active site of the enzyme. Therefore, the final structure of the inhibitor is asymmetrical (Figure 3b), which also enables improvement of bioavailability by different modifications of the end groups. Moreover, C-2 symmetric compounds would be more sensitive to PR resistance development since every mutation in PR would affect the inhibitor binding twice.

RTV inhibits both HIV-1 and HIV-2 proteases. In vivo, the major observed mutations decreasing the susceptibility of enzyme to RTV are I84V and V82A. Ritonavir was originally approved as an antiretroviral drug, but due to frequent occurrence of side effects (such as gastrointestinal symptoms like nausea, abdomenalgia and diarrhea) and high cross-resistance with other PIs, its use as a HIV-protease inhibitor was gradually abandoned. Unexpectedly, RTV has proven to be a potent inhibitor of cytochrome P-450 3A4, the microsomal enzyme responsible for the bulk of metabolism of other PIs (as well as other xenobiotics, see above). Therefore, ritonavir is currently used almost exclusively as a pharmacokinetic boosting agent which increases the plasma concentration of other PIs and thus prolongs their therapeutic effects [39].

Indinavir (IDV, MK-639, L-735,524), manufactured by Merck & Co, was approved in 1996 under the trade name Crixivan® [40]. Design of this compound possessing a hydroxyaminopentanamide transition state isostere was based on the framework of previously developed renin inhibitors. Physical properties, i.e., solubility and lipophilicity, of optimized molecules were further improved using molecular modeling and X-ray crystal structure analysis of the inhibitor/enzyme complexes (Figure 3c). Although indinavir used to be among the most widely clinically used first-generation PIs, at present it has been almost completely abandoned due to toxicity and frequent side effects. Nephrolithiasis is a typical side effect of IDV caused by disturbances of the drug solubility in urine. The compound is more soluble in acidic urine due to the presence of the basic polar groups. With the increase of the actual pH (>5.5), IDV tends to crystallize in kidneys [41]. Another typical side effect of IDV is the lipodystrophy syndrome, characterized by maldistribution of body fat (accumulation in abdominal area – “crix belly” and “buffalo hump”) [42].

The first protease inhibitor which could be used by adults as well as by children was nelfinavir (NFV, AG-1343), approved in 1997 and marketed as Viracept®. Developed by Agouron Pharmaceuticals by iterative protein co-crystal structure analysis, it was the first PI which structure has been optimized by Monte Carlo simulations [43] (Figure 3d). In more than a half of the patients, resistance conferring mutation D30N is selected, often accompanied by N88D. The other possible pathway leading to NFV resistance involves the selection of L90M mutation. Interestingly, these two pathways seem to be mutually exclusive [44]. The therapeutic use of NFV is currently limited because of the significantly lower efficacy compared to the recently introduced second generation PIs. The major adverse effect of NFV is gastrointestinal toxicity characterized by diarrhea, often of a severe grade. Other common side effects are metabolic disturbances, such as lipodystrophy and hyperlipidemia [45].

Amprenavir (APV, 141W94 or VX-478) from Vertex Pharmaceuticals and GlaxoSmithKline has been marketed under the name Agenerase® since 1999 [46]. Its central part is derived from SQV, but the P2 end group is replaced by tetrahydrofurancarbamate and the P1´-P2´ moiety by a sulfonamide derivative (Figure 3e). This structure has some advantages over SQV such as fewer chiral centers, easier synthesis, and higher oral bioavailability. Mutations which result in a decrease of susceptibility to Agenerase® occur mainly in positions M46I/L, I47V, I50L, I54L/V, and I84V. Mutations conferring resistance to APV were also observed in the p1/p6 of the Gag substrate cleavage site, at positions L449 and P453 [47]. The adverse events of APV are mainly gastrointestinal, such as loose stools and abdominal pain; skin rash is also relatively common. APV administration could also lead to hypertriglyceridemia and hypercholesterolemia [48-50].

In order to further improve the safety, pharmacokinetics, and therapeutic potency of amprenavir, fosamprenavir (FPV,fAPV, GW 433908, VX-175), a hydrophilic phosphate ester prodrug of amprenavir, was developed by Vertex Laboratories and GlaxoSmithKline [51] (Figure 3f, FPV is quickly converted to APV by host phosphatases during absorption in the gut. Several mutations, namely V32I, I47V/A, I50V, I54L/M, L76V and I84 V/A, are relative contraindications to the use of FPV. The side effects of FPV are comparable to those of APV [52].

Figure 3.

Figure 3. Chemical structures of the first generation HIV protease inhibitors.

2.2. The second generation HIV protease inhibitors

The second generation inhibitors were designed to inhibit HIV PR species resistant to the inhibitors of the first generation and to introduce well tolerated drugs with minimized side effects and simple once-daily dosing, which may improve patients´ adherence to the treatment.

Lopinavir (LPV, ABT-378) is the second generation inhibitor most widely used in drug-naïve patients. Developed by Abbott, it has been marketed in a co/formulation with ritonavir under the name Kaletra® since 2000. LPV was designed to inhibit resistant PR species that contain the common mutation V82A. The molecular design of LPV was based on the structure of ritonavir and began by eliminating the P3 isopropylthiazolyl group of RTV interacting with the V82 residue of the wild-type PR. Further replacement of the thiazolylmethoxycarbonyl moiety in the P2’ with dimethylphenoxyacetyl group yielded LPV (Figure 4a). The core P1-P1’ positions are thus occupied by the same hydroxyethylene peptidomimetic as in RTV [53].

At present, Kaletra® is the first choice PI for an initial antiretroviral regimen. The development of resistance is associated with accumulation of mutations occurring at nine to eleven positions (“lopinavir mutation score”) [54]. Furthermore, a single mutation I47A, contributing to the reduced sensitivity of HIV-1 [55-57] and also HIV-2 [58] protease to the LPV, has been reported [59]. Nevertheless, this mutation compromises the replication capacity of the corresponding virus ([58] and the citations therein), thus explaining its low prevalence in the HIV-positive patients. The side effects of LPV are similar to those of other PIs. The major problems are gastrointestinal events, such as abdominal pain, gaseous symptoms or diarrhea, and metabolic disturbances like hypercholesterolemia and hypertriglyceridemia [60].

The structure-based design of atazanavir ( ATV, CGP 73547, BMS-232632) was part of a complex exploration of azadipeptide analogues [61] (Figure 4b). Originally developed by Ciba-Geigy and marketed since 2003 under the trade name Reyataz® by Bristol-Myers Squibb, atazanavir was the first PI that was compatible with once-daily dosing. The compound shows the least binding to the serum proteins, minimal effect on insulin-stimulated glucose uptake and appeared to be less likely to cause lipodystrophy and high level of cholesterol as side effects. The main mutations contributing to the resistance are I50L, I84V, and N88S. In patients treated with ATV, a prominent indirect hyperbilirubinaemia frequently occurs, often leading to clinically apparent jaundice. This condition is, indeed, not dangerous, but may have an undesirable cosmetic effect, which may discourage the patient from the therapy [62].

A unique compound was designed at Pharmacia & Upjohn and later introduced by Boehringer-Ingelheim. Tipranavir (TPV, PNU-140690), approved under the name Aptivus® in 2005, is a non-peptidic inhibitor, belonging to the 4-hydroxy-5,6-dihydro-2-pyrone sulfonamides [63] (Figure 4c). Despite its structural differences from the peptidic inhibitors, its interactions with HIV PR are similar in many respects, as shown by its crystal structure [64]. Nevertheless, TPV shows considerable activity against numerous PI-resistant HIV strains [65]. As many as 16–20 mutations in the protease gene are needed for significant decrease of the HIV PR susceptibility [66]. In addition to the gastrointestinal and metabolic disturbances that are common for the whole class, the main limitation of the drug is its hepatotoxicity [67]. Occasionally, intracranial hemorrhaging has also been reported [68]. Since it is a CYP450 3A4 inducer, TPV requires a double dosage of RTV as a pharmacokinetic booster [69].

The most recently (2006) approved nonpeptidic HIV protease inhibitor is darunavir (DRV, TMC-114, UIC-94017, trade name Prezista®) [70]. Developed by Tibotec BVBA, formerly Tibotec-Virgo NV, it is a structural homolog of TMC-126 (UIC-94003). Both compounds are chemically similar to APV, but differ by the presence of a bis-tetrahydrofuranyl moiety, (Figure 4d), which forms a crucial hydrogen bonding interactions with the main chain of Asp29 and Asp30 in the PR. These polar interactions with the backbone atoms in the PR active site enable DRV to mimic the conserved hydrogen bonds of the natural substrates [71]. Its broad specificity against mutated, highly resistant PR species could be interpreted by its ability to fit to the proposed ”substrate envelope” within the active site [72,73]. Recently, ultra-high resolution crystal structures revealed that DRV binds not only to the active-site cavity, but can also occupy a surface pocket formed by one of the flaps (flexible loops covering the enzyme active site) [74]. Enzyme kinetic experiments further confirmed the notion that both DRV and APV have a second binding site outsite the active cleft [75]. An assay based on fluorescent resonance energy transfer suggests that DRV has a dual mechanism of inhibition, as it might also be a potential PR dimerization inhibitor [76].

Even though DRV is chemically related to APV, it binds nearly 100 times more tightly than APV and 1,000 times more tightly than SQV, IDV, RTV and NFV to the wild-type HIV-1 protease [77,78]. In clinical studies with treatment experienced and naïve individuals, patients developing darunavir resistance tend to have a high number of PI resistance-associated mutations. Thus, the genetic barrier to the development of resistance, a measure of the difficulty for the virus to escape from the selective pressure of the drug by developing mutations, is very high for DRV. The substitutions suggested to be critical for the development of DRV resistance were V11I, V32I, L33F, I47V, I50V, I54L/M, G73S, T74P, L76V, I84V, and L89V [79-81]. Grantz Saskova et al. have recently characterized a virus strain isolated from HIV-positive patient under prolonged treatment by DRV that contained as many as 22 mutations in the PR region and exhibited pronounced DRV resistance [8].

The high genetic barrier to the development of resistance, its better clinical efficacy against multidrug-resistant HIV variants together with favorable tolerability, safety, and once-daily regime, make DRV one of the preferred therapeutic option both for drug-naïve and for highly treatment-experienced patients. DRV is generally well tolerated, but the main adverse effects are common with the other members of the class, such as gastrointestinal symptoms and lipid abnormalities [82].

Figure 4. Chemical structures of the second generation HIV protease inhibitors

3. Inhibitors of HIV protease in the pipeline

Figure 5. Chemical structures of inhibitors HIV protease in the pipeline.

4. Other non-peptidic HIV protease active site inhibitors

Figure 6. Chemical structure of DMP450 (Mozenavir (DuPont).

Figure 7. Crystal structure of metallacarborane inhibitor bound to HIV PR. (a) Two metallacarborane clusters bind to the flap-proximal part of the active site. The HIV PR is represented by a ribbon diagram and colored by rainbow from blue to red (N- to C- termini), the atoms of the metallacarborane cluster are represented by spheres and colored orange for boron atoms, gray for carbon atoms, and blue for cobalt. The structural formula is depicted in (b). Hydrogens are omitted for clarity.

5. Alternative HIV protease inhibitors targeting functional domains outside the enzyme active site

5.1. HIV PR dimerization inhibitors

HIV PR is only active as a dimer in which each of the two catalytic aspartates is contributed by one monomer. The determination of the dimer dissociation constant (K_d)has been the goal of considerable efforts of many groups, but its value differs substantially depending on the various techniques and experimental conditions (reviewed in [110]). The reported K_d values from kinetic studies are typically of the order of 10⁻⁷ to 10⁻⁹ M, whereas the values derived from sedimentation analysis are on average three orders of magnitude lower [111-117]. It was already suggested in 1990 that blocking the dimerization of the protease monomers could be an effective means for inactivating the enzyme [118]. The crystal structure of HIV-1 protease [119] shows that the enzyme dimerization interface is formed by the β-hairpin of the two flaps, catalytic triad, helices (residues 84-93) interacting with residues 4-10, and N- and C-terminal β-strands (Figure 8a). A four-stranded antiparallel β-sheet composed of the two N‑termini (residues 1-4) interdigitating the two C-termini (residues 96-99) (Figure 8b) contributes to 75% of the stabilizing energy [120]. This dimeric interface is conserved among most HIV-1 isolates and drug-resistant variants (see Figure 2) and thus represents an attractive target for development of ligands preventing dimerization.

Early studies demonstrated that interface peptides reproducing the native sequence of C-terminal and N-terminal fragments act as HIV PR dimerization inhibitors, although in micromolar concentration [112,121,122]. Connection of the N- and C- terminal peptides with flexible linkers [123,124] or more rigid scaffolds (“molecular tongs“) [125,126] further increased their inhibitory potency. A recent study describes novel interfacial peptides tethered through their side chains, with inhibitory potencies against the wild-type HIV PR being in low nanomolar range [127].

Figure 8. HIV PR dimerization interface. (a) The overall structure of the HIV PR dimer with an inhibitor bound in the active site. Monomers are colored blue and red, respectively. Regions involved in creation of a dimeric interface are highlighted by darker shades and indicated by residue numbers. (b) A detail of the four-stranded antiparallel β-sheet formed by interdigitation of C- and N-terminal strands. Monomers are colored blue and red, respectively. A hydrogen bonding network is represented by green dashed lines. The figure was generated using the structure of highly mutated, patient derived HIV-1 PR (PDB code 3GGU [8]) and program PyMol [9].

Figure 8.

Modification of the termini of an interfacial peptide by attachment of a lipophilic group and alkyl chains (e.g., palmitoyl) improves both the inhibitory capacity and the specificity [128,129]. Currently, the most potent lipopeptide inhibitors, containing the minimal peptide sequence Leu-Glu-Tyr modified on the N-terminus by palmitoyl, attained sub-nanomolar K_i values for the in vitro inhibition of the wild type and drug-resistant mutant variants [130]. Irreversible inhibitors, in which the interface peptide is able to covalently modify the protein dimeric interface through a disulfide bridge with Cys95, have also been described [131].

Interesting macromolecular inhibitors targeting dimerization domains are fusions of the N-terminal HIV-1 PR peptide with a cell permeable domain from HIV-1 Tat [132,133]. Also, an antibody recognizing the N terminus of HIV PR (residues 1-7), which inhibits activity of both HIV-1 and HIV-2 proteases with K_i values in low nano-molar range [134,135], has been reported. The use of the antibody as an anti-HIV drug is rather limited, nevertheless its structure [136,137] might be used as a lead in design of low-molecular mimics.

There has been a significant effort to develop dimerization inhibitors of HIV PR and characterize their binding on a structural level [138]. Also, there is great interest to develop inhibitors targeting protein-protein interfaces in enzymes and other proteins. Several protein–protein interfaces indeed became targets for successful drug development, e.g., HIV proteins and intracellular co-factors [139], HIV capsid assembly (reviewed in [140]) or herpes virus protease dimerization [141]. These advances might positively boost the development of new HIV PR dimerization inhibitors [142].

5.2. Inhibitors targeting HIV PR flaps

Residues 43-58 of HIV PR form an anti-parallel β-hairpin referred to as “the flap”. Two flaps within a dimer cover the active site cavity and their movement is crucial for binding and release of protease substrate. The conformation behavior of this glycine rich region has been extensively studied [143,144] and the most populated states are closed and semi-open. The closed state is found when a substrate or peptidomimetic inhibitor is bound to the active site (Figure 9b) while the semi-open conformation is most prevalent in the enzyme apo-form (Figure 9a). Since all currently approved FDA PIs target the closed conformation, developing of inhibitors targeted to the open flap conformation with a different binding mode might be an alternative to circumvent the cross-resistance.

Recent examples of inhibitors targeting the open-flap conformation are metallacarborane-based [101] and pyrrolidine-based [146] compounds. The metallacarboranes described earlier in this review bind to the flap-proximal part of the enzyme active site without interaction with the catalytic aspartates, while the pyrrolidine diester inhibitors target the catalytic dyad.

The region preceding the flap in the sequence (residues 34-42) is called the flap elbow (or the hinge). It folds into a surface exposed loop with no particular secondary structure which undergoes substantial change during flap opening and closing (Figure 9). With the exception of an insertion in position 35 [3], no resistance mutations are associated with this region (Figure 2, Table 1). The flap elbow thus might represent a promising drug target.

A monoclonal antibody recognizing an epitope corresponding to residues 36-46 inhibits the activity of HIV PR with K_i value in a nanomolar range [147]. The proposed inhibition mechanism based on the crystal structure of the antibody fragment in complex with the 36-46 epitope peptide postulates that antibody binding prevents flap closure over the active site [148]. An example of another inhibitory compound with predicted affinity toward the flap elbow are Nb-containing polyoxometalates [97], mentioned above. They inhibit HIV PR with K_i values in a low-nanomolar range and exhibit a non-competitive mode with stoichiometry 2 inhibitors to 1 PR dimer. Computational studies suggested that these compounds bind to a cationic pocket formed by residues Lys41, Lys43, and Lys55.

Figure 9. HIV PR flap conformations. (a) Overall structure of the apo-form of the HIV PR. The flaps (residues 43-58) in semi-open conformation are highlighted in red, residues 37-42, so called flap elbows are also indicated. The figure was generated using the structure of free HIV-1 PR (PDB code 1HHP [145]) and program PyMol [9]. (b) Overall structure of the HIV PR with flaps (in dark green) in closed conformation. Residues 37-42, so called flap elbows are also indicated. Inhibitor bound in the enzyme active site is omitted from the figure. The figure was generated using the structure of a highly mutated patient derived HIV-1 PR (PDB code 3GGU [8]) and program PyMol [9].

6. Conclusions

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