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.     

Molecular docking and 3D-QSAR studies of HIV-1 protease inhibitors

HIV-1 protease is an obligatory enzyme in the replication process of the HIV virus. The abundance of structural information on HIV-1PR has made the enzyme an attractive target for computer-aided drug design strategies. The daunting ability of the virus to rapidly generate resistant mutants suggests that there is an ongoing need for new HIV-1PR inhibitors with better efficacy profiles and reduced toxicity. In the present investigation, molecular modeling studies were performed on a series of 54 cyclic urea analogs with symmetric P2/P2′ substituents. The binding modes of these inhibitors were determined by docking. The docking results also provided a reliable conformational superimposition scheme for the 3D-QSAR studies. To gain insight into the steric, electrostatic, hydrophobic and hydrogen-bonding properties of these molecules and their influence on the inhibitory activity, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed. Two different alignment schemes viz. receptor-based and atom-fit alignment, were used in this study to build the QSAR models. The derived 3D-QSAR models were found to be robust with statistically significant r ² and r ² _pred values and have led to the identification of regions important for steric, hydrophobic and electronic interactions. The predictive ability of the models was assessed on a set of molecules that were not included in the training set. Superimposition of the 3D-contour maps generated from these models onto the active site of enzyme provided additional insight into the structural requirements of these inhibitors. The CoMFA and CoMSIA models were used to design some new inhibitors with improved binding affinity. Pharmacokinetic and toxicity predictions were also carried out for these molecules to gauge their ADME and safety profile. The computational results may open up new avenues for synthesis of potent HIV-1 protease inhibitors.     

Dimerization inhibitors of HIV-1 protease

By targeting the highly conserved antiparallel beta-sheet formed by the interdigitation of the N- and C-terminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance observed with current active-site directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N- and C-terminal mimetics, lipopeptides and cross-linked interface peptides.     

Lipopeptides as dimerization inhibitors of HIV-1 protease.

In AIDS therapy, attempts have been made to inhibit the virus-encoded enzymes, e.g. HIV-1 protease, using active site-directed inhibitors. This approach is questionable, however, due to virus mutations and the high toxicity of the drugs. An alternative method to inhibit the dimeric HIV protease is the targeting of the interface region of the protease subunits in order to prevent subunit dimerization and enzyme activity. This approach should be less prone to inactivation by mutation. A list of improved 'dimerization inhibitors' of HIV-1 protease is presented. The main structural features are a short 'interface' peptide segment, including non-natural amino acids, and an aliphatic N-terminal blocking group. The high inhibitory power of some of the lipopeptides [e.g. palmitoyl-Tyr-Glu-Leu-OH, palmitoyl-Tyr-Glu-(L-thyronine)-OH, palmitoyl-Tyr-Glu-(L-biphenyl-alanine)-OH] with low nanomolar Ki values in the enzyme test suggests that mimetics with good bio-availability can be derived for AIDS therapy.     

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.     

Novel pseudosymmetric inhibitors of HIV-1 protease

Compounds containing the easily accessible Phe[CH(OH)CH₂N(NH)Phe dipeptide isostere as a non-hydrolyzable replacement of the scissile amide bond in the natural substrate are potent inhibitors of HIV-1 protease. The expected symmetric binding pattern of the most potent inhibitor in this series (CGP 53280, IC₅₀ = 9 nM) is illustrated by the X-ray analysis performed with the corresponding enzyme-inhibitor complex.     

Sidechain-linked inhibitors of HIV-1 protease dimerization

There is a great need for alternative modes of inhibition for the design of anti-HIV therapies, due to the increased resistance of HIV to currently approved drugs. A novel strategy for generating potent dimerization inhibitors of HIV-1 protease is described based on sidechain-linked interfacial peptides. In a number of cases the activity of these agents against HIV-1 protease was found to be among the most potent reported, with inhibitory constants in the low nM range.     

Macromolecular inhibitors of HIV-1 protease. Characterization of designed heterodimers

Defective variants of human immunodeficiency virus type 1 (HIV-1) protease (HIV PR) have been engineered to inhibit wild-type (wt) HIV PR activity. These variants were designed to promote the formation of heterodimers and to destabilize the formation of inactive variant homodimers of HIV-1 protease through substitutions at Asp-25, Ile-49, and Gly-50 (Babé, L. M., Rosé, J., and Craik, C. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10069-10073; McPhee, F., Good, A. C., Kuntz, I. D., and Craik, C. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11477-11481). The mechanism of action of these dominant-negative inhibitors was established using recombinantly expressed defective monomers. The defective monomers were refolded in vitro in the presence of wt HIV PR and showed dose-dependent inhibition of proteolytic activity. This inhibition was shown to result from the formation of inactive heterodimers between defective and wt HIV PR monomers. Heterodimer formation was detected by (i) isolating refolded, inactive heterodimers using histidine-tagged defective monomers and (ii) isolating heterodimers from bacteria coexpressing both wt and defective variants of HIV PR. Single-chain variants of HIV PR, in which the C terminus of the wt HIV PR monomer was covalently tethered to the N terminus of the defective monomer, were also expressed and analyzed. Thermal denaturation of these single-chain heterodimers using differential scanning calorimetry revealed a 1.5-7.2 degrees C greater thermal stability than single-chain wt HIV PR. The thermodynamic trend shown by these three variants mirrors their relative inhibition in provirus transfection assays. These data support the model that the effects seen both in tissue culture and in vitro arise from an increase in stability conferred on these heterodimers by interface mutations and identifies heterodimer formation as their mechanism of inhibition.     

Synthesis of macrocyclic trypanosomal cysteine protease inhibitors

The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.     

Molecular mechanics analysis of inhibitor binding to HIV-1 protease.

Crystallographic structures of HIV protease with three different peptide-mimetic inhibitors were subjected to energy minimization using molecular mechanics, the minimized structures analyzed and the inhibitor binding energies calculated. Partial charge assignment for the hydrogen bonded catalytic aspartic acids, Asp25 and -25', was in good agreement with charge calculations using semi-empirical molecular orbital methods. Root mean square deviations on minimization were small and similar for both subunits in the protease dimer. The surface loops, which had the largest B factors, changed most on minimization; the hydrophobic core and the inhibitor binding site showed little change. The distance-dependent dielectric of D(r) = 4r was found to be preferable to D(r) = r. Distance restraints were applied for the intermolecular hydrogen bonds to maintain the conformation of the inhibitor binding site. Using the dielectric of D(r) = 4r, the calculated interaction energy of the three inhibitors with the protease ranged from -53 to -56 kcal/mol. The psi groups of the inhibitors were changed to add or remove a 'transition state analogue' hydroxyl group, and the loss in energy on the removal of this group was calculated to be 0.9-1.7 kcal/mol. This would represent 19-36% of the total measured difference in binding energy between the inhibitors JG365 and MVT-101.     

Crucial amides for dimerization inhibitors of HIV-1 protease

An inhibitor based on crosslinked peptides from the interfacial region of HIV-1 protease, previously shown to act by dimerization inhibition, was modified by N-methylation to ascertain the importance of the amide hydrogens on inhibition. The effects of N-methylation on HIV-1 protease inhibition, as well as the effects on degradation by proteases are described.     

Peptide substrates and inhibitors of the HIV-1 protease

Oligopeptides containing the consensus retroviral protease cleavage sequence Ser/Thr-X-Y-Tyr/Phe-Pro are substrates for purified recombinant HIV-1 protease with Km's in the millimolar range. The minimum sequence containing the consensus pentapeptide which serves as a good substrate is a heptapeptide spanning the P4-P3' residues. Substitution of reduced Phe-Pro or Tyr-Pro dipeptide isosteres or the statine analog 3-hydroxy-4-amino-5-phenylpentanoic acid for the scissile dipeptide afforded inhibitors of HIV-1 protease with Ki values in the micromolar range, three orders of magnitude better in affinity than the corresponding substrates. Inhibitors of HIV-1 protease may provide a novel and potentially useful therapeutic approach to the treatment of acquired immune deficiency syndrome (AIDS).     

Kinetic and thermodynamic characterization of HIV-1 protease inhibitors

Interaction kinetic and thermodynamic analyses provide information beyond that obtained in general inhibition studies, and may contribute to the design of improved inhibitors and increased understanding of molecular interactions. Thus, a biosensor-based method was used to characterize the interactions between HIV-1 protease and seven inhibitors, revealing distinguishing kinetic and thermodynamic characteristics for the inhibitors. Lopinavir had fast association and the highest affinity of the tested compounds, and the interaction kinetics were less temperature-dependent as compared with the other inhibitors. Amprenavir, indinavir and ritonavir showed non-linear temperature dependencies of the kinetics. The free energy, enthalpy and entropy (DeltaG, DeltaH, DeltaS) were determined, and the energetics of complex association (DeltaG(on), DeltaH(on), DeltaS(on)) and dissociation (DeltaG(off), DeltaH(off), DeltaS(off)) were resolved. In general, the energetics for the studied inhibitors was in the same range, with the negative free energy change (DeltaG < 0) due primarily to increased entropy (DeltaS > 0). Thus, the driving force of the interaction was increased degrees of freedom in the system (entropy) rather than the formation of bonds between the enzyme and inhibitor (enthalpy). Although the DeltaG(on) and DeltaG(off) were in the same range for all inhibitors, the enthalpy and entropy terms contributed differently to association and dissociation, distinguishing these phases energetically. Dissociation was accompanied by positive enthalpy (DeltaH(off) > 0) and negative entropy (DeltaS(off) < 0) changes, whereas association for all inhibitors except lopinavir had positive entropy changes (DeltaS(on) > 0), demonstrating unique energetic characteristics for lopinavir. This study indicates that this type of data will be useful for the characterization of target-ligand interactions and the development of new inhibitors of HIV-1 protease.     

Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-l-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.     

HIV-1 protease inhibitors with picomolar potency against PI-resistant HIV-1 by modification of the P1' substituent

Transposition of the pyridyl nitrogen from the P(3) substituent to the P(1)' substituent in HIV-1 protease inhibitors (PI) affords compounds such as 3 with an improved inhibitory profile against multiple P450 isoforms. These compounds also displayed increased potency, with 3 inhibiting viral spread (CIC(95)) at <8 nM for every strain of PI-resistant HIV-1 tested. The poor to modest bioavailability of these compounds may correlate in part to their aqueous solubility.     

Beta-lactam compounds as apparently uncompetitive inhibitors of HIV-1 protease

Compounds of a combinatorial monocyclic beta-lactam library were found to be apparently uncompetitive inhibitors of HIV-1 protease, providing lead compounds for a new class of HIV protease inhibitors.     

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.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.