Crystal structure of a novel synthetic inhibitor of HIV-1 protease

The crystal structure of a novel non-peptidic HIV-1 protease inhibitor derived by simple solid-state dimerization of 4-aryl-1,4-dihydropyridines, reveals a strained central cage and the conformation of its phenyl, benzyl, and hydroxymethylene substituents. The polycyclic cage includes two nearly flat cyclobutane rings and four fused piperidine rings in boat conformations. The cage geometry reveals two unexpected features, namely marked distortions of the valence angles in every second piperidine and a shortening of one of the cyclobutane bonds. The molecule displays exact centrosymmetry, but the central cage and the hydroxymethylene substituents also approximate the C₂-symmetry of the target enzyme. The two independent hydroxyl groups are involved in intermolecular hydrogen bonding, one as a donor, the other as an acceptor. The disposition of the hydroxyl groups in the molecular framework is compatible with the dual role of the inhibitor in the active-site cavity of HIV-1 protease, whereby one OH group is hydrogen-bonded to the catalytic aspartates, whereas another one provides an interface to the locked flaps of the enzyme.     

Structure-based QSAR analysis of a set of 4-hydroxy-5,6-dihydropyrones as inhibitors of HIV-1 protease: an application of the receptor-dependent (RD) 4D-QSAR formalism

Receptor-dependent (RD) 4D-QSAR models were constructed for a set of 39 4-hydroxy-5,6-dihydropyrone analogue HIV-1 protease inhibitors. The receptor model used in this QSAR analysis was derived from the HIV-1 protease (PDB ID ) crystal structure. The bound ligand in the active site of the enzyme, also a 4-hydroxy-5,6-dihydropyrone analogue, was used as the reference ligand for docking the data set compounds. The optimized RD 4D-QSAR models are not only statistically significant (r(2) = 0.86, q(2) = 0.80 for four- and greater-term models) but also possess reasonable predictivity based on test set predictions. The proposed "active" conformations of the docked analogues in the active site of the enzyme are consistent in overall molecular shape with those suggested from crystallographic studies. Moreover, the RD 4D-QSAR models also "capture" the existence of specific induced-fit interactions between the enzyme active site and each specific inhibitor. Hydrophobic interactions, steric shape requirements, and hydrogen bonding of the 4-hydroxy-5,6-dihydropyrone analogues with the HIV-1 protease binding site model dominate the RD 4D-QSAR models in a manner again consistent with experimental conclusions. Some possible hypotheses for the development of new lead HIV-1 protease inhibitors can be inferred from the RD 4D-QSAR models.     

Large-scale motions and electrostatic properties of furin and HIV-1 protease

We present a comparative study between two members of serine and aspartic proteases complexed with a peptide substrate. The same computational setup is used to characterize the structural, electrostatic, and electronic properties for the Michaelis complex of furin, a serine protease, and of the aspartic protease from HIV-1. In both cases plane-wave density functional theory (PW-DFT) and empirical force-field-based molecular dynamics calculations are used. For furin, calculations are extended to the complex with the intermediate of the first step of the reaction. Comparisons are also made with results from recent PW-DFT investigations on both families of enzymes and with the same chemical groups in an aqueous environment. It is found that the substrate carbonyl group is more polarized in the furin complex than in the HIV-1 protease one. A further difference regards the large-scale motions of the complexes as a whole and local conformational fluctuations at the active site. The global and local fluctuations are well coupled for HIV-1 protease but not for furin. Thus, despite some chemical analogies in the first step of the reaction mechanism, furin and HIV-1 protease complexes appear to be characterized by a different interplay of electrostatics and conformational fluctuations.     

Heterocyclic HIV-1 protease inhibitors

[formula: see text] A series of simple heterocyclic HIV-1 protease inhibitors were developed on the basis of size, shape, and electronic complementarity to the active site of the enzyme. The C2-symmetric heterocycles do not contain a transition-state isostere nor are they active site directed irreversible inhibitors; thus, they represent the success of a new design strategy. The first generation heterocycles inhibit the protease in the micromolar range, whereas control compounds show no bioactivity at the same concentrations.     

Hydrophobic core flexibility modulates enzyme activity in HIV-1 protease

Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.     

Switching between allosteric and dimerization inhibition of HIV-1 protease

Refining the functional groups on a phenethylamine moiety within an inhibitor of HIV-1 protease led to a switch in the mechanism of inhibition from competitive and allosteric to dimerization inhibition. Phenylether extensions to the phenethylamine group led to agents that target the dimerization interface of HIV-1 protease with high potency.     

Computational design of novel cyclic urea as HIV-1 protease inhibitor

A series of novel cyclic urea molecules 5,6-dihydroxy-1,3-diazepane-2,4,7-trione as HIV-1 protease inhibitors were designed using computational techniques. The designed molecules were compared with the known cyclic urea molecules by performing docking studies, calculating their ADME (Absorption, Distribution, Metabolism, and Excretion) properties and protein ligand interaction energy. These novel molecules were designed by substituting the P ₁/P′ ₁ positions (4 ^th and 7 ^th position of 1, 3-diazepan-2-one) with double bonded oxygens. This reduces the molecular weight and increases the bioavailability, indicating better ADME properties. The docking studies showed good binding affinity towards HIV-1 protease. The biological activity of these inhibitors were predicted by a model equation generated by the regression analysis between biological activity (log 1/K ⁱ ) of known inhibitors and their protein ligand interaction energy. The synthetic studies are in progress.      

Hydrogen bonding in molecular recognition by HIV-1 protease

Molecular recognition of the cleavage sites of the substrates by HIV-1 protease is analyzed in terms of hydrogen bonding. Crystal structures of an inactive enzyme complexed with six different substrates were used as reference structures. Applying molecular mechanics calculations it can be shown that the interaction energies between the real substrate and the enzyme are larger than with other peptides. From the analysis, it can be concluded that water molecules are essential in the recognition process. Moreover, the hydrogen bonds between the protease and various substrates are characterized in detail.     

The influence of macromolecular crowding on HIV-1 protease internal dynamics

High macromolecular concentrations, or crowded conditions, have been shown to affect a wide variety of molecular processes, including diffusion, association and dissociation, and protein folding and stability. Here, we model the effect of macromolecular crowding on the internal dynamics of a protein, HIV-1 protease, using Brownian dynamics simulations. HIV-1 protease possesses a pair of flaps which are postulated to open in the early stages of its catalytic mechanism. Compared to low concentrations, close-packed concentrations of repulsive crowding agents are found to significantly reduce the fraction of time that the protease flaps are open. Macromolecular crowding is likely to have a major effect on in vivo enzyme activity, and may play an important regulatory role in the viral life cycle.     

Structural exploration of hydroxyethylamines as HIV-1 protease inhibitors: new features identified

The current study deals with chemometric modelling strategies (Naïve Bayes classification, hologram-based quantitative structure–activity relationship (HQSAR), comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA)) to explore the important features of hydroxylamine derivatives for exerting potent human immunodeficiency virus-1 (HIV-1) protease inhibition. Depending on the statistically validated reliable and robust quantitative structure–activity relationship (QSAR) models, important and crucial structural features have been identified that may be responsible for enhancing the activity profile of these hydroxylamine compounds. Arylsulfonamide function along with methoxy or fluoro substitution is important for enhancing activity. Bulky steric substitution at the sulfonamide nitrogen disfavours activity whereas smaller hydrophobic substitution at the same position is found to be favourable. Apart from the crucial oxazolidinone moiety, pyrrolidine, cyclic urea and methyl ester functions are also responsible for increasing the HIV-1 protease inhibitory profile. Observations derived from these modelling studies may be utilized further in designing promising HIV-1 protease inhibitors of this class.     

Chemical modification of oleanene type triterpenes and their inhibitory activity against HIV-1 protease dimerization

Oleanolic acid derivatives with different lengths of 3-O-acidic acyl chains were synthesized and evaluated for their inhibitory activity against HIV-1 protease. The lengths of the acidic chains were optimized to 6 and 8 carbons. Changing a 3-ester bond to an amide bond or dimerization of the triterpenes retained their inhibitory activity against HIV-1 protease. Introduction of an additional acidic chain to C-28 of oleanolic acid increased the inhibitory activity appreciably, though a derivative with only one acidic chain linked at C-28 also showed potent activity against HIV-1 protease. The inhibitory mechanism was proved directly by size exclusion chromatography to be inhibition of dimerization of the enzyme polypeptides. The ester bonds of the triterpene derivatives were found to be stable to lipase under mild alkaline conditions.     

High-speed synthesis of potent C2-symmetric HIV-1 protease inhibitors by in-situ aminocarbonylations

Two novel series of C2-symmetric HIV-1 protease inhibitors were synthesized by microwave-promoted, palladium-catalyzed aminocarbonylations of the o-iodo- and m-bromobenzyloxy P1/P1' substituted core structures. Molybdenum hexacarbonyl was used as a convenient solid source of carbon monoxide in these transformations. After the initial high-speed library generation, biological testing identified highly active HIV-1 protease inhibitors. Selected ortho- and meta-decorated inhibitors were subsequently resynthesized on a larger scale and retested for their affinity toward HIV-1 protease, showing micromolar to low nanomolar inhibition. The discovery of highly active inhibitors containing large phenyl amide ortho substituents in the P1/P1' positions indicates that larger groups than previously believed are tolerated in this part of the S1/S1' pocket.     

Polyoxometalate HIV-1 protease inhibitors. A new mode of protease inhibition.

Nb-containing polyoxometalates (POMs) of the Wells-Dawson class inhibit HIV-1 protease (HIV-1P) by a new mode based on kinetics, binding, and molecular modeling studies. Reaction of alpha(1)-K(9)Li[P(2)W(17)O(61)] or alpha(2)-K(10)[P(2)W(17)O(61)] with aqueous H(2)O(2) solutions of K(7)H[Nb(6)O(19)] followed by treatment with HCl and KCl and then crystallization affords the complexes alpha(1)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(1)()1) and alpha(2)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(2)()1) in 63 and 86% isolated yields, respectively. Thermolysis of the crude peroxoniobium compounds (72-96 h in refluxing H(2)O) prior to treatment with KCl converts the peroxoniobium compounds to the corresponding polyoxometalates (POMs), alpha(1)-K(7)[P(2)W(17)NbO(62)] (alpha(1)()2) and alpha(2)-K(7)[P(2)W(17)NbO(62)] (alpha(2)()2), in moderate yields (66 and 52%, respectively). The identity and high purity of all four compounds were confirmed by (31)P NMR and (183)W NMR. The acid-induced dimerization of the oxo complexes differentiates sterically between the cap (alpha(2)) site and the belt (alpha(1)) site in the Wells-Dawson structure (alpha(2)()2 dimerizes in high yield; alpha(1)()2 does not). All four POMs exhibit high activity in cell culture against HIV-1 (EC(50) values of 0.17-0.83 microM), are minimally toxic (IC(50) values of 50 to >100 microM), and selectively inhibit purified HIV-1 protease (HIV-1P) (IC(50) values for alpha(1)()1, alpha(2)()1, alpha(1)()2, and alpha(2)()2 of 2.0, 1.2, 1.5, and 1.8 microM, respectively). Thus, theoretical, binding, and kinetics studies of the POM/HIV-1P interaction(s) were conducted. Parameters for [P(2)W(17)NbO(62)](7)(-) were determined for the Kollman all-atom (KAA) force field in Sybyl 6.2. Charges for the POM were obtained from natural population analysis (NPA) at the HF/LANL2DZ level of theory. AutoDock 2.2 was used to explore possible binding locations for the POM with HIV-1P. These computational studies strongly suggest that the POMs function not by binding to the active site of HIV-1P, the mode of inhibition of all other HIV-1P protease inhibitors, but by binding to a cationic pocket on the "hinge" region of the flaps covering the active site (2 POMs and cationic pockets per active homodimer of HIV-1P). The kinetics and binding studies, conducted after the molecular modeling, are both in remarkable agreement with the modeling results: 2 POMs bind per HIV-1P homodimer with high affinities (K(i) = 1.1 +/- 0.5 and 4.1 +/- 1.8 nM in 0.1 and 1.0 M NaCl, respectively) and inhibition is noncompetitive (k(cat) but not K(m) is affected by the POM concentration).     

Structural and electronic properties of new fullerene derivatives and their possible application as HIV-1 protease inhibitors

Density functional theory (DFT) calculations have been carried out at the hybrid Becke 3-Lee–Yang–Parr; B3LYP/3-21G** level of theory to study two series of hydroxy-chalca-acetic acid-(4-pyrrolidin-1-yl-phenyl) ester [C₆₀-C₂H₄N-(4-XCOCH₂OH)C₆H₄] and hydroxy-chalcoacetic acid-[2-(2-hydroxy-acetylchalcanyl)-4-pyrrolidin-1-yl-phenyl] ester[C₆₀-C₂H₄N-(3,4-XCOCH₂OH)C₆H₄]. The X atom is O, S or Se for the two series. The vibrational spectra, physical, chemical, thermodynamics and Quantitative Structure Activity Relationship (QSAR) properties of the studied molecules are calculated and discussed. We have evaluated these molecules as HIV-1 protease inhibitors based on the hydrogenation interaction between the hydroxymethylcarbonyl (HMC) groups and the two aspartic acid of the HIV-1 protease active site. Results show that some of the investigated fullerene-based derivatives can be considered promising as HIV-1 protease inhibitors.     

Potent HIV-1 protease inhibitors incorporating meso-bicyclic urethanes as P2-ligands: structure-based design, synthesis, biological evaluation and protein-ligand X-ray studies

Recently, we designed a series of novel HIV-1 protease inhibitors incorporating a stereochemically defined bicyclic fused cyclopentyl (Cp-THF) urethane as the high affinity P2-ligand. Inhibitor with this P2-ligand has shown very impressive potency against multi-drug-resistant clinical isolates. Based upon the -bound HIV-1 protease X-ray structure, we have now designed and synthesized a number of meso-bicyclic ligands which can conceivably interact similarly to the Cp-THF ligand. The design of meso-ligands is quite attractive as they do not contain any stereocenters. Inhibitors incorporating urethanes of bicyclic-1,3-dioxolane and bicyclic-1,4-dioxane have shown potent enzyme inhibitory and antiviral activities. Inhibitor (K(i) = 0.11 nM; IC(50) = 3.8 nM) displayed very potent antiviral activity in this series. While inhibitor showed comparable enzyme inhibitory activity (K(i) = 0.18 nM) its antiviral activity (IC(50) = 170 nM) was significantly weaker than inhibitor . Inhibitor maintained an antiviral potency against a series of multi-drug resistant clinical isolates comparable to amprenavir. A protein-ligand X-ray structure of -bound HIV-1 protease revealed a number of key hydrogen bonding interactions at the S2-subsite. We have created an active model of inhibitor based upon this X-ray structure.     

Structural optimization of cyclic sulfonamide based novel HIV-1 protease inhibitors to picomolar affinities guided by X-ray crystallographic analysis

Synthesis and HIV-1 protease inhibitory activity of compound 5 based on the structure of a novel cyclic sulfonamide pharmacophore has been recently disclosed from our group. X-ray crystallographic structure of 5 when bound to the HIV-1 protease defined its binding mode. The importance of the geometry of the substitution at C4–Me (S configuration) was emphasized. In the present paper we wish to disclose the design of novel inhibitors 47 and 48 based on the X-ray structure of compound 5 bound to the HIV-1 protease, their synthesis and activity against HIV-1 protease. By making changes at the C4 position and the carbamate linkage the above compounds 47 and 48 were found to be approximately one hundred fold more active compared to 5 and their K_i values are in the picomolar range. An unusual observation regarding the activity and geometry was made with compounds 51 and 52. X-ray results demonstrate that 48 and 52 bind to the same binding pocket with simultaneous change in the conformation of the cyclic sulfonamide ring.     

Accurate prediction of protonation state as a prerequisite for reliable MM-PB(GB)SA binding free energy calculations of HIV-1 protease inhibitors

Binding free energies were calculated for the inhibitors lopinavir, ritonavir, saquinavir, indinavir, amprenavir, and nelfinavir bound to HIV-1 protease. An MMPB/SA-type analysis was applied to conformational samples from 3 ns explicit solvent molecular dynamics simulations of the enzyme-inhibitor complexes. Binding affinities and the sampled conformations of the inhibitor and enzyme were compared between different HIV-1 protease protonation states to find the most likely protonation state of the enzyme in the complex with each of the inhibitors. The resulting set of protonation states leads to good agreement between calculated and experimental binding affinities. Results from the MMPB/SA analysis are compared with an explicit/implicit hybrid scheme and with MMGB/SA methods. It is found that the inclusion of explicit water molecules may offer a slight advantage in reproducing absolute binding free energies while the use of the Generalized Born approximation significantly affects the accuracy of the calculated binding affinities.     

Synthesis of novel HIV-1 protease inhibitors based on carbohydrate scaffolds

The synthesis of peptidomimetic inhibitors of HIV-1 protease based on 6-deoxy-6-amino-β-d-glucopyranoside and 6-deoxy-6-amino-β-d-mannopyranoside scaffolds has been achieved. The inhibitors had IC₅₀ values in the micromolar range. The results provide a platform for the development of more potent carbohydrate based inhibitors of HIV-1 and other aspartic proteases.