Rapid synthesis and in situ screening of potent HIV-1 protease dimerization inhibitors

A library of dimerization inhibitors of HIV-1 protease is described based on crosslinked interfacial peptides. The 54 component library was designed to contain two modifications to the starting structure, one each in the Northern and Southern fragments. A rapid synthesis and in situ screening method in microtiter plates was developed to facilitate the generation and evaluation of the library members. More than 90% of the doubly modified agents were more potent than their respective singly mutated parent compounds, and five of the most potent dimerization inhibitors of HIV-1 protease described to date were identified. The free energy of binding for the combined two modifications was generally found to be additive, demonstrating the predictive value of earlier libraries.     

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).     

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.     

Possible allosteric interactions of monoindazole-substituted P2 cyclic urea analogues with wild-type and mutant HIV-1 protease

Our ongoing efforts to understand the difference in the binding pattern of HIV-1 protease inhibitor (HIVPI) with the wild-type and mutant HIV-1 protease (HIVPR) and to provide mechanistic insight are continued further. We report here the results of a recent quantitative structure-activity relationship (QSAR) study on monoindazole-substituted P2 analogues of cyclic urea HIVPIs. The QSAR models revealed an inverted parabolic relationship between biological activity and calculated molar refractivity (CMR). That is, biological activity first decreases with increase in CMR and at a certain minimum point (inversion point) it suddenly changes and increases with further increase in CMR. CMR is a measure of volume-dependent-polarizability and is an indication of the polar interactions between ligand and receptor. The results seem to be best rationalized by larger molecules inducing a change in a receptor unit that allows for a new mode of interaction. Similar QSAR models were also observed for the biological activity of these molecules tested against a panel of mutant viruses including mutant strains with single amino acid substitution (I84V), double amino acid substitutions (I84V/V82F), and multiple amino acid changes corresponding to mutations observed in clinical isolates of patients treated with Ritonavir((R)). Interestingly the inversion points for these mutant strains were found larger than for wild-type. The subtle but significant difference in the inversion point indicates change in the shape and size of the binding pocket. Earlier QSAR studies have shown that the correlation of biological activity with an inverted parabola is an indicative of the 'allosteric interaction' of the ligands with the receptor. This report presents a detail analysis of these observations.     

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.     

Sphingosine kinase 1 (SK1) allosteric inhibitors that target the dimerization site

The sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) signaling pathway is a crucial target for numerous human diseases from cancer to cardiovascular diseases. However, available SK1 inhibitors that target the active site suffer from poor potency, selectivity and pharmacokinetic properties. The selectivity issue of the kinases, which share a highly-conserved ATP-pocket, can be overcome by targeting the less-conserved allosteric sites. SK1 is known to function minimally as a dimer; however, the crystal structure of the SK1 dimer has not been determined. In this study, a template-based algorithm implemented in PRISM was used to predict the SK1 dimer structure and then the possible allosteric sites at the dimer interface were determined via SiteMap. These sites were used in a virtual screening campaign that includes an integrated workflow of structure-based pharmacophore modeling, virtual screening, molecular docking, re-screening of common scaffolds to propose a series of compounds with different scaffolds as potential allosteric SK1 inhibitors. Finally, the stability of the SK1-ligand complexes was analyzed by molecular dynamics simulations. As a final outcome, ligand 7 having a 4,9-dihydro-1H-purine scaffold and ligand 12 having a 2,3,4,9-tetrahydro-1H-β-carboline scaffold were found to be potential selective inhibitors for SK1.     

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.     

HIV-1 integrase inhibition of biscoumarin analogues

Nineteen biscoumarins bearing free and modified hydroxyl substituents at benzoyloxyphenyl linker have been synthesized by multiple step synthesis. Among these biscoumarins, thirteen were found to be active molecules against HIV-1 integrase (HIV-1 IN). The structure-activity relationship of the nineteen compounds on HIV IN may be useful for the design of potent therapeutic agents.     

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.     

药效团检索设计新的HIV-1蛋白酶抑制剂

通过对自建的未开发化合物三维结构库进行药效团检索,得到了4个对HIV-1蛋白酶抑制活化的化合物,通过构象分析发现包含药效团的构象处于优势构象,而且4个结构都含有带两个邻位羟基的苯环和一个间位羰基的药效团以及公共子结构。通过计算发现它们的疏水参数都很小。在考虑满足包含药效团的结构特征和有适中的疏水参数两个因素的前提下,设计出了新的具有潜在抑制HIV-1蛋白酶活性的化合物。它们的结构都比检索得到的四个化合物更为简单,因此易于合成。     

An electrochemical approach for the detection of HIV-1 protease

An electrochemical biosensor with a new bioorganometallic approach for the detection of human immunodeficiency virus (HIV) type 1 protease (HIV-1 PR) using surface-bound ferrocenoyl (Fc)-pepstatin conjugates is presented.     

Mass-dependent bond vibrational dynamics influence catalysis by HIV-1 protease

Protein motions that occur on the microsecond to millisecond time scale have been linked to enzymatic rates observed for catalytic turnovers, but not to transition-state barrier crossing. It has been hypothesized that enzyme motions on the femtosecond time scale of bond vibrations play a role in transition state formation. Here, we perturb femtosecond motion by substituting all nonexchangeable carbon, nitrogen, and hydrogen atoms with (13)C, (15)N, and (2)H and observe the catalytic effects in HIV-1 protease. According to the Born-Oppenheimer approximation, isotopic substitution alters vibrational frequency with unchanged electrostatic properties. With the use of a fluorescent peptide to report on multiple steps in the reaction, we observe significantly reduced rates in the heavy enzyme relative to the light enzyme. A possible interpretation of our results is that there exists a dynamic link between mass-dependent bond vibrations of the enzyme and events in the reaction coordinate.     

Specificity rule discovery in HIV-1 protease cleavage site analysis

Several machine learning algorithms have recently been applied to modeling the specificity of HIV-1 protease. The problem is challenging because of the three issues as follows: (1) datasets with high dimensionality and small number of samples could misguide classification modeling and its interpretation; (2) symbolic interpretation is desirable because it provides us insight to the specificity in the form of human-understandable rules, and thus helps us to design effective HIV inhibitors; (3) the interpretation should take into account complexity or dependency between positions in sequences. Therefore, it is necessary to investigate multivariate and feature-selective methods to model the specificity and to extract rules from the model. We have tested extensively various machine learning methods, and we have found that the combination of neural networks and decompositional approach can generate a set of effective rules. By validation to experimental results for the HIV-1 protease, the specificity rules outperform the ones generated by frequency-based, univariate or black-box methods.     

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.     

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.     

Synthesis and HIV-1 integrase inhibition activity of some N-arylindoles

Eight simple N-arylindoles were designed, synthesized and evaluated as human immunodeficiency virus (HIV)-1 integrase inhibitors in vitro for the first time. Among these compounds, 3b, 3e and 3g demonstrated significant anti-HIV-1 integrase activity. Especially 3b showed the highest anti-HIV-1 integrase activity with EC50 value of 7.88 microg/ml and TI value of 24.61. Meantime, some structure-activity relationships were also observed and will provide a new lead for design and discovery of more potent N-arylindoles as HIV-1 integrase inhibitors.