In Vitro Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Increased Resistance to ABT-378, a Novel Protease Inhibitor

ABT-378, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which is significantly more active than ritonavir in cell culture, is currently under investigation for the treatment of AIDS. Development of viral resistance to ABT-378 in vitro was studied by serial passage of HIV-1 (pNL4-3) in MT-4 cells. Selection of viral variants with increasing concentrations of ABT-378 revealed a sequential appearance of mutations in the protease gene: I84V-L10F-M46I-T91S-V32I-I47V. Further selection at a 3.0 μM inhibitor concentration resulted in an additional change at residue 47 (V47A), as well as reversion at residue 32 back to the wild-type sequence. The 50% effective concentration of ABT-378 against passaged virus containing these additional changes was 338-fold higher than that against wild-type virus. In addition to changes in the protease gene, sequence analysis of passaged virus revealed mutations in the p1/p6 (P₁′ residue Leu to Phe) and p7/p1 (P₂ residue Ala to Val) gag proteolytic processing sites. The p1/p6 mutation appeared in several clones derived from early passages and was present in all clones obtained from passage P11 (0.42 μM ABT-378) onward. The p7/p1 mutation appeared very late during the selection process and was strongly associated with the emergence of the additional change at residue 47 (V47A) and the reversion at residue 32 back to the wild-type sequence. Furthermore, this p7/p1 mutation was present in all clones obtained from passage P17 (3.0 μM ABT-378) onward and always occurred in conjunction with the p1/p6 mutation. Full-length molecular clones containing protease mutations observed very late during the selection process were constructed and found to be viable only in the presence of both the p7/p1 and p1/p6 cleavage-site mutations. This suggests that mutation of these gag proteolytic cleavage sites is required for the growth of highly resistant HIV-1 selected by ABT-378 and supports recent work demonstrating that mutations in the p7/p1/p6 region play an important role in conferring resistance to protease inhibitors (L. Doyon et al., J. Virol. 70:3763–3769, 1996; Y. M. Zhang et al., J. Virol. 71:6662–6670, 1997).     

Crystal structures of multidrug-resistant HIV-1 protease in complex with two potent anti-malarial compounds

Two potent inhibitors (compounds 1 and 2) of malarial aspartyl protease, plasmepsin-II, were evaluated against wild type (NL4-3) and multidrug-resistant clinical isolate 769 (MDR) variants of human immunodeficiency virus type-1 (HIV-1) aspartyl protease. Enzyme inhibition assays showed that both 1 and 2 have better potency against NL4-3 than against MDR protease. Crystal structures of MDR protease in complex with 1 and 2 were solved and analyzed. Crystallographic analysis revealed that the MDR protease exhibits a typical wide-open conformation of the flaps (Gly48 to Gly52) causing an overall expansion in the active site cavity, which, in turn caused unstable binding of the inhibitors. Due to the expansion of the active site cavity, both compounds showed loss of direct contacts with the MDR protease compared to the docking models of NL4-3. Multiple water molecules showed a rich network of hydrogen bonds contributing to the stability of the ligand binding in the distorted binding pockets of the MDR protease in both crystal structures. Docking analysis of 1 and 2 showed a decrease in the binding affinity for both compounds against MDR supporting our structure-function studies. Thus, compounds 1 and 2 show promising inhibitory activity against HIV-1 protease variants and hence are good candidates for further development to enhance their potency against NL4-3 as well as MDR HIV-1 protease variants.     

Novel lopinavir analogues incorporating heterocyclic replacements of six-member cyclic urea--synthesis and structure-activity relationships

The HIV protease inhibitor ABT-378 (lopinavir) has a six-member cyclic urea in the P-2 position. A series of analogues in which the six-member cyclic urea is replaced by various heterocycles was synthesized and the structure-activity relationships explored.     

Synthesis and structure-activity relationships of a novel series of HIV-1 protease inhibitors encompassing ABT-378 (Lopinavir)

The HIV protease inhibitor ABT-378 (Lopinavir) has a 2,6-dimethylphenoxyacetyl group in the P-2' position. Analogues in which this group is replaced with various substituted phenyl or heteroaryl groups were synthesized and the structure-activity relationships explored.     

Discovery of potent HIV-1 protease inhibitors incorporating sulfoximine functionality

Based on the unique property of sulfoximine and the homodimeric C(2) structural symmetry of HIV-1 protease, a novel class of sulfoximine-based pseudosymmetric HIV-1 protease inhibitors was designed and synthesized. The sulfoximine moiety was demonstrated to be important for HIV-1 protease inhibitor potency. The most active stereoisomer (2S,2'S) displays a potency of 2.5 nM (IC(50)) against HIV-1 protease and an anti-HIV-1 activity of 408 nM (IC(50)). A possible mode of action is proposed.     

High resolution crystal structures of HIV-1 protease with a potent non-peptide inhibitor (UIC-94017) active against multi-drug-resistant clinical strains

The compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetics that is chemically related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum potent inhibitor active against HIV-1 clinical isolates with minimal cytotoxicity. We have determined the high-resolution crystal structures of UIC-94017 in complexes with wild-type HIV-1 protease (PR) and mutant proteases PR(V82A) and PR(I84V) that are common in drug-resistant HIV. The structures were refined at resolutions of 1.10-1.53A. The crystal structures of PR and PR(I84V) with UIC-94017 ternary complexes show that the inhibitor binds to the protease in two overlapping positions, while the PR(V82A) complex had one ordered inhibitor. In all three structures, UIC-94017 forms hydrogen bonds with the conserved main-chain atoms of Asp29 and Asp30 of the protease. These interactions are proposed to be critical for the potency of this compound against HIV isolates that are resistant to multiple protease inhibitors. Other small differences were observed in the interactions of the mutants with UIC-94017 as compared to PR. PR(V82A) showed differences in the position of the main-chain atoms of residue 82 compared to PR structure that better accommodated the inhibitor. Finally, the 1.10A resolution structure of PR(V82A) with UIC-94017 showed an unusual distribution of electron density for the catalytic aspartate residues, which is discussed in relation to the reaction mechanism.     

Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

The success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.     

Adaptability and flexibility of HIV-1 protease.

Even though more than 200 three-dimensional structures of HIV-1 protease complexed to a variety of inhibitors are available in the Protein Data Bank; very few structures of unliganded protein have been determined. We have recently solved structures of unliganded HIV-1 protease tethered dimer mutants to resolutions of 1.9 A and 2.1 A, and have found that the flaps assume closed-flap conformation even in the absence of any bound ligand. We report comparison of the unliganded closed-flap structure with structures of HIV-1 protease inhibitor complexes with a view to accurately identifying structural changes that the ligand can induce on binding to HIV-1 protease in the crystal. These studies reveal that the least flexible region present in the active site of HIV-1 protease need not also be the least adaptable to external stress, thus highlighting the conceptual difference between flexibility and adaptability of proteins in general.     

Studies on flexibility and binding affinity of Asp25 of HIV-1 protease mutants

We have investigated and highlighted the behavior of binding residue, Asp25 by computational analysis, which play an important role in understanding docking process with drug molecule, Ritonavir (Norvir^®) and the flexibility nature of the Human Immunodeficiency Virus-1 (HIV-1) protease enzyme. It is well known that Ritonavir is a potent and a selective HIV-1 protease inhibitor. Molecular dockings were performed in order to gain insights regarding the binding mode of this inhibitor. In our analysis, we observed Ritonavir had different rank orders of scores against different mutant of this enzyme. Asp25 of the enzyme was found to be the active site for all the mutants. The results clearly suggest that Ritonavir is not able to appropriately bind at the active site of each HIV-1 protease mutant due to RMSD difference of the amino acid (Asp) at the position 25 of all mutants. These findings support the concept that 3D space of active site is a qualitative assessment for binding affinity of inhibitor with an enzyme. The investigation on the flexibility nature of Asp25 by normal mode analysis, show that binding residue posses less flexibility due to its solvation potential. The overall analysis of our study brings clarity to the binding behavior with respect to the different mutants with Ritonavir on the basis RMSD and also on the flexible nature of HIV-1 protease enzyme with respect to Asp25 position.     

Crystal structure of lysine sulfonamide inhibitor reveals the displacement of the conserved flap water molecule in human immunodeficiency virus type 1 protease

Human immunodeficiency virus type 1 (HIV-1) protease has been continuously evolving and developing resistance to all of the protease inhibitors. This requires the development of new inhibitors that bind to the protease in a novel fashion. Most of the inhibitors that are on the market are peptidomimetics, where a conserved water molecule mediates hydrogen bonding interactions between the inhibitors and the flaps of the protease. Recently a new class of inhibitors, lysine sulfonamides, was developed to combat the resistant variants of HIV protease. Here we report the crystal structure of a lysine sulfonamide. This inhibitor binds to the active site of HIV-1 protease in a novel manner, displacing the conserved water and making extensive hydrogen bonds with every region of the active site.     

Evaluation of homology modeling of HIV protease

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

Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV-1 protease inhibitors.

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 A. The peptidomimetic inhibitor Boc-Phe-Psi[CH2CH2NH]-Phe-Glu-Phe-NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nm and it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors. The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only. The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere. A comparison with other inhibitors of this series containing isostere hydroxyl group in R or S configuration shows different ways of accommodation of inhibitor in the active site. Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution.     

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.     

Synthesis and antiviral activities of the major metabolites of the HIV protease inhibitor ABT-378 (Lopinavir)

The HIV protease inhibitor ABT-378 (Lopinavir) is metabolized rapidly and extensively by CYP-3A4 catalyzed oxidation. Three of the major metabolites identified were synthesized and their antiviral (HIV) activities determined.     

Predicting structural effects in HIV-1 protease mutant complexes with flexible ligand docking and protein side-chain optimization

We present a computational approach for predicting structures of ligand-protein complexes and analyzing binding energy landscapes that combines Monte Carlo simulated annealing technique to determine the ligand bound conformation with the dead-end elimination algorithm for side-chain optimization of the protein active site residues. Flexible ligand docking and optimization of mobile protein side-chains have been performed to predict structural effects in the V32I/I47V/V82I HIV-1 protease mutant bound with the SB203386 ligand and in the V82A HIV-1 protease mutant bound with the A77003 ligand. The computational structure predictions are consistent with the crystal structures of these ligand-protein complexes. The emerging relationships between ligand docking and side-chain optimization of the active site residues are rationalized based on the analysis of the ligand-protein binding energy landscape. Proteins 33:295–310, 1998. © 1998 Wiley-Liss, Inc.     

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.     

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.     

Design and synthesis of potent HIV-1 protease inhibitors incorporating hydroxyprolinamides as novel P2 ligands

A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different hydroxyprolinamide P2 ligands were designed and synthesized. Variation of substitutions at the P2 significantly affected the enzyme inhibitory potency of the inhibitors. Compounds 2a and 2d showed excellent enzyme inhibitory activity with IC₅₀ values in the nanomolar range. An active site binding model for inhibitors 2a and 2d was suggested based upon the computational-docking results of the ligand with HIV-1 protease. This model offers molecular insights regarding ligand-binding site interactions of the hydroxyprolinamide-derived novel P2-ligand.     

Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition but, due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be aided greatly by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.     

Crystal structure of an in vivo HIV-1 protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance

Saquinavir is a widely used HIV-1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV-1 viral enzymes. Three HIV-1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these mutants demonstrate a 13.5-, 3-, and 419-fold increase in Ki values, respectively, compared to the wild-type enzyme (Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364-12370). To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV-1 protease crystal structure of the G48V/L90M double mutant in complex with saquinavir at 2.6 A resolution. This mutant complex is compared with that of the wild-type enzyme bound to the same inhibitor (Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340-3342). Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main-chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S1/S1' substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild-type enzyme and therefore may facilitate catalysis by the resistant mutant.