Resistance mechanism revealed by crystal structures of unliganded nelfinavir-resistant HIV-1 protease non-active site mutants N88D and N88S

Nelfinavir is an inhibitor of HIV-1 protease, and is used for treatment of patients suffering from HIV/AIDS. However, treatment results in drug resistant mutations in HIV-1 protease. N88D and N88S are two such mutations which occur in the non-active site region of the enzyme. We have determined crystal structures of unliganded N88D and N88S mutants of HIV-1 protease to resolution of 1.65 Å and 1.8 Å, respectively. These structures refined against synchrotron data lead to R-factors of 0.1859 and 0.1780, respectively. While structural effects of N88D are very subtle, the mutation N88S has caused a significant conformational change in D30, an active site residue crucial for substrate and inhibitor binding.     

The L33F darunavir resistance mutation acts as a molecular anchor reducing the flexibility of the HIV-1 protease 30s and 80s loops

HIV-1 protease (PR) is a 99 amino acid protein responsible for proteolytic processing of the viral polyprotein – an essential step in the HIV-1 life cycle. Drug resistance mutations in PR that are selected during antiretroviral therapy lead to reduced efficacy of protease inhibitors (PI) including darunavir (DRV). To identify the structural mechanisms associated with the DRV resistance mutation L33F, we performed X-ray crystallographic studies with a multi-drug resistant HIV-1 protease isolate that contains the L33F mutation (MDR769 L33F). In contrast to other PR L33F DRV complexes, the structure of MDR769 L33F complexed with DRV reported here displays the protease flaps in an open conformation. The L33F mutation increases noncovalent interactions in the hydrophobic pocket of the PR compared to the wild-type (WT) structure. As a result, L33F appears to act as a molecular anchor, reducing the flexibility of the 30s loop (residues 29–35) and the 80s loop (residues 79–84). Molecular anchoring of the 30s and 80s loops leaves an open S1/S1′ subsite and distorts the conserved hydrogen-bonding network of DRV. These findings are consistent with previous reports despite structural differences with regards to flap conformation.     

Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3

The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.     

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography

Recombinant wild-type protease of human immunodeficiency virus, type [(HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/μg. Two proteolytically inactive HIV-1 mutant proteases (Arg-87 → Lys; Asn-88 → Glu) were found to bind to pepstatin A agarose, and they were purified as the wild-type protease. A third mutant protease (Arg-87 → Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.     

The higher barrier of darunavir and tipranavir resistance for HIV-1 protease

Darunavir and tipranavir are two inhibitors that are active against multi-drug resistant (MDR) HIV-1 protease variants. In this study, the invitro inhibitory efficacy was tested against a MDR HIV-1 protease variant, MDR 769 82T, containing the drug resistance mutations of 46L/54V/82T/84V/90M. Crystallographic and enzymatic studies were performed to examine the mechanism of resistance and the relative maintenance of potency. The key findings are as follows: (i) The MDR protease exhibits decreased susceptibility to all nine HIV-1 protease inhibitors approved by the US Food and Drug Administration (FDA), among which darunavir and tipranavir are the most potent; (ii) the threonine 82 mutation on the protease greatly enhances drug resistance by altering the hydrophobicity of the binding pocket; (iii) darunavir or tipranavir binding facilitates closure of the wide-open flaps of the MDR protease; and (iv) the remaining potency of tipranavir may be preserved by stabilizing the flaps in the inhibitor-protease complex while darunavir maintains its potency by preserving protein main chain hydrogen bonds with the flexible P2 group. These results could provide new insights into drug design strategies to overcome multi-drug resistance of HIV-1 protease variants.     

1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin

Under the selection pressure of drugs, mutations appear in HIV-1 protease even at the sites, which are conserved in the untreated individuals. Cysteine 95 is a highly conserved residue and is believed to be involved in regulation of HIV-1 protease. In some of the virus isolates from patients undergoing heavy treatment with anti-HIV protease drugs, C95F mutation has appeared. The present study reports 1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin. It is found that in this mutant, dimer interface has become more rigid and that the packing at the interface of terminal and core domains is altered. These alterations may be relevant to C95F mutation conferring drug resistance to HIV-1 protease.     

Exploration of the effects of sequence variations between HIV-1 and HIV-2 proteases on their three-dimensional structures

Abstract

HIV protease inhibitors (PIs) approved by the FDA (US Food and Drug Administration) are a major class of antiretroviral. HIV-2 protease (PR2) is naturally resistant to most of them as PIs were designed for HIV-1 protease (PR1). In this study, we explored the impact of amino-acid substitutions between PR1 and PR2 on the structure of protease (PR) by comparing the structural variability of 13 regions using 24 PR1 and PR2 structures complexed with diverse ligands. Our analyses confirmed structural rigidity of the catalytic region and highlighted the important role of three regions in the conservation of the catalytic region conformation. Surprisingly, we showed that the flap region, corresponding to a flexible region, exhibits similar conformations in PR1 and PR2. Furthermore, we identified regions exhibiting different conformations in PR1 and PR2, which could be explained by the intrinsic flexibility of these regions, by crystal packing, or by PR1 and PR2 substitutions. Some substitutions induce structural changes in the R2 and R4 regions that could have an impact on the properties of PI-binding site and could thus modify PI binding mode. Substitutions involved in structural changes in the elbow region could alter the flexibility of the PR2 flap regions relative to PR1, and thus play a role in the transition from the semi-open form to the closed form, and have an impact on ligand binding. These results improve the understanding of the impact of sequence variations between PR1 and PR2 on the natural resistance of HIV-2 to commercially available PIs.

Communicated by Ramaswamy H. Sarma     

HIV-1 protease inhibition potential of functionalized polyoxometalates

Polyoxometalates (POMs) are interesting biomedical agents due to their versatile anticancer and antiviral properties, such as remarkable anti-HIV activity. Although POMs are tunable and easily accessible inorganic drug prototypes in principle, their full potential can only be tapped by enhancing their biocompatibility, for example, through organic functionalization. We have therefore investigated the HIV-1 protease inhibition potential of functionalized Keggin- and Dawson-type POMs with organic side chains. Their inhibitory performance was furthermore compared to other POM types, and the buffer dependence of the results is discussed. In addition, chemical shift mapping NMR experiments were performed to exclude POM-substrate interactions. Whereas the introduction of organic side chains into POMs is a promising approach in principle, the influence of secondary effects on the reaction system also merits detailed investigation.     

Molecular dynamics of HIV-1 protease.

Molecular dynamics simulations have been carried out based on the GROMOS force field on the aspartyl protease (PR) of the human immunodeficiency virus HIV-1. The principal simulation treats the HIV-1 PR dimer and 6990 water molecules in a hexagonal prism cell under periodic boundary conditions and was carried out for a trajectory of 100 psec. Corresponding in vacuo simulations, i.e., treating the isolated protein without solvent, were carried out to study the influence of solvent on the simulation. The results indicate that including waters explicitly in the simulation results in a model considerably closer to the crystal structure than when solvent is neglected. Detailed conformational and helicoidal analysis was performed on the solvated form to determine the exact nature of the dynamical model and the exact points of agreement and disagreement with the crystal structure. The calculated dynamical model was further elucidated by means of studies of the time evolution of the cross-correlation coefficients for atomic displacements of the atoms comprising the protein backbone. The cross-correlation analysis revealed significant aspects of structure originating uniquely in the dynamical motions of the molecule. In particular, an unanticipated through-space, domain-domain correlation was found between the mobile flap region covering the active site and a remote regions of the structure, which collectively act somewhat like a molecular cantilever. The significance of these results is discussed with respect to the inactivation of the protease by site-specific mutagenesis, and in the design of inhibitors.     

High-performance liquid chromatographic assay of a new HIV-1 protease inhibitor, LB71350, in the plasma of dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of LB71350 in the plasma of dogs. The analyte was deproteinized with 1.5 volumes of methanol and 0.5 volumes of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a stepwise gradient mixture of acetonitrile and 0.2% triethylamine–HCl with a flow-rate of 1 ml/min and detection at UV 245 nm. The proportion of acetonitrile was kept at 52% for the first 6 min, increased to 100% for the next 0.5 min, kept at 100% for the next 2 min, decreased to 52% for the next 0.5 min, and finally kept at 52% for the next 7 min. The retention time of LB71350 was 6.9 min. The calibration was linear over the concentration range of 0.1–100 mg/l for dog plasma (r>0.997) and the limit of quantitation was 0.1 mg/l using 0.1 ml plasma. The quality control samples were reproducible with acceptable accuracy and precision at 0.1, 1, 10 and 100 mg/l concentrations. The within-day recovery (n=5) was 90.2–93.9%, the between-day recovery (n=5) was 89.5–93.5%, and the absolute between-day recovery (n=5) was 77–81%. The within-day precision (n=5) and between-day precision (n=5) were 2.59–5.82% and 3.17–4.55%, respectively. No interferences from endogenous substances were observed. Taken together, the above HPLC assay method by deproteinization and UV detection was suitable for the determination of LB71350 in the preclinical pharmacokinetics.     

Adaptive inhibitors of the HIV-1 protease

A significant obstacle to the efficacy of drugs directed against viral targets is the presence of amino acid polymorphisms in the targeted molecules. Amino acid polymorphisms may occur naturally due to the existence of variations within and between viral strains or as the result of mutations associated with drug resistance. An ideal drug will be one that is extremely effective against a primary target and maintains its effectiveness against the most important variations of the target molecule. A drug that simultaneously inhibits different variants of the target will lead to a faster suppression of the virus, retard the appearance of drug-resistant mutants and provide more efficacious and, in the long range, more affordable therapies. Drug molecules with the ability to inhibit several variants of a target with high affinity have been termed adaptive drugs (Nat. Biotechnol. 20 (2002) 15; Biochemistry 42 (2003) 8459; J. Cell. Biochem. S37 (2001) 82). Current drug design paradigms are predicated upon the lock-and-key hypothesis, which emphasizes shape complementarity as a way to attain specificity and improved binding affinity. Shape complementarity is accomplished by the introduction of conformational constraints in the drug molecule. While highly constrained molecules do well against a unique target, they lack the ability to adapt to target variations like those originating from naturally occurring polymorphisms or drug-resistant mutations. Targeting an array of closely related targets rather than a single one while still maintaining selectivity, requires a different approach. A plausible strategy for designing high affinity adaptive inhibitors is to engineer their most critical interactions (for affinity and specificity) with conserved regions of the target while allowing for adaptability through the introduction of flexible asymmetric functionalities in places facing variable regions of the target. The fundamental thermodynamics and structural principles associated with this approach are discussed in this chapter.     

Role of conformational fluctuations in the enzymatic reaction of HIV-1 protease

The emergence of compensatory drug-resistant mutations in HIV-1 protease challenges the common view of the reaction mechanism of this enzyme. Here, we address this issue by performing classical and ab initio molecular dynamics simulations (MD) on a complex between the enzyme and a peptide substrate. The classical MD calculation reveals large-scale protein motions involving the flaps and the cantilever. These motions modulate the conformational properties of the substrate at the cleavage site. The ab initio calculations show in turn that substrate motion modulates the activation free energy barrier of the enzymatic reaction dramatically. Thus, the catalytic power of the enzyme does not arise from the presence of a pre-organized active site but from the protein mechanical fluctuations. The implications of this finding for the emergence of drug-resistance are discussed.     

HIV-1 protease regulation: The role of the major homology region and adjacent C-terminal capsid sequences

The maturation of human immunodeficiency type-1 virions is accomplished through the proteolytic processing of Gag and GagPol precursor proteins by the viral protease (PR). Since virions must be assembled at the cell surface from uncleaved precursor molecules, intracellular activation of PR must be inhibited. We have previously developed a system where the intracellular activity of PR, associated with GagPol, was inhibited by the expression of Gagin trans. The disproportionate synthesis of Gag inhibits the activation of PR in the cytoplasm. Sequences capable of mediating this inhibition were localized to capsid. In this communication, the region of HIV-1 capsid capable of mediating inhibition was further defined and shown to require the major homology region of capsid within Gag.     

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.     

Discovery of diarylpyridine derivatives as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

Two series (4 and 5) of diarylpyridine derivatives were designed, synthesized, and evaluated for anti-HIV-1 activity. The most promising compound, 5e, inhibited HIV-1 IIIB, NL4-3, and RTMDR1 with low nanomolar EC₅₀ values and selectivity indexes of >10,000. The results of this study indicate that diarylpyridine can be used as a novel scaffold to derive a new class of potent NNRTIs, active against both wild-type and drug-resistant HIV-1 strains.     

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

Molecular dynamics studies on HIV-1 protease: a comparison of the flap motions between wild type protease and the M46I/G51D double mutant

The emergence of drug-resistant mutants of HIV-1 is a tragic effect associated with conventional long-treatment therapies against acquired immunodeficiency syndrome. These mutations frequently involve the aspartic protease encoded by the virus; knowledge of the molecular mechanisms underlying the conformational changes of HIV-1 protease mutants may be useful in developing more effective and longer lasting treatment regimes. The flap regions of the protease are the target of a particular type of mutations occurring far from the active site. These mutations modify the affinity for both substrate and ligands, thus conferring resistance. In this work, molecular dynamics simulations were performed on a native wild type HIV-1 protease and on the drug-resistant M46I/G51D double mutant. The simulation was carried out for a time of 3.5 ns using the GROMOS96 force field, with implementation of the SPC216 explicit solvation model. The results show that the flaps may exist in an ensemble of conformations between a “closed” and an “open” conformation. The behaviour of the flap tips during simulations is different between the native enzyme and the mutant. The mutation pattern leads to stabilization of the flaps in a semi-open configuration.     

Structural evidence for effectiveness of darunavir and two related antiviral inhibitors against HIV-2 protease

No drug has been targeted specifically for HIV-2 (human immunodeficiency virus type 2) infection despite its increasing prevalence worldwide. The antiviral HIV-1 (human immunodeficiency virus type 1) protease (PR) inhibitor darunavir and the chemically related GRL98065 and GRL06579A were designed with the same chemical scaffold and different substituents at P2 and P2′ to optimize polar interactions for HIV-1 PR (PR1). These inhibitors are also effective antiviral agents for HIV-2-infected cells. Therefore, crystal structures of HIV-2 PR (PR2) complexes with the three inhibitors have been solved at 1.2-Å resolution to analyze the molecular basis for their antiviral potency. Unusually, the crystals were grown in imidazole and zinc acetate buffer, which formed interactions with the PR2 and the inhibitors. Overall, the structures were very similar to the corresponding inhibitor complexes of PR1 with an RMSD of 1.1 Å on main-chain atoms. Most hydrogen-bond and weaker C-H…O interactions with inhibitors were conserved in the PR2 and PR1 complexes, except for small changes in interactions with water or disordered side chains. Small differences were observed in the hydrophobic contacts for the darunavir complexes, in agreement with relative inhibition of the two PRs. These near-atomic-resolution crystal structures verify the inhibitor potency for PR1 and PR2 and will provide the basis for the development of antiviral inhibitors targeting PR2.