Rational design and synthesis of a novel class of active site-targeted HIV protease inhibitors containing a hydroxymethylcarbonyl isostere. Use of phenylnorstatine or allophenylnorstatine as a transition-state mimic.

A novel class of HIV-1 protease inhibitors containing a hydroxymethylcarbonyl (HMC) isostere were designed from the substrate transition state and synthesized. Phenylnorstatine [Pns; (2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] and the 2S diastereomer, (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid, named allophenylnorstatine (Apns) were effective transition-state mimics, and incorporation of Pns-Pro or Apns-Pro at the P1-P1' site gave potent and specific HIV-1 protease inhibitors. In the inhibitory assays, the chemically synthesized [Ala67,95] HIV-1 protease was used.     

Structure-activity relationship of orally potent tripeptide-based HIV protease inhibitors containing hydroxymethylcarbonyl isostere

We designed and synthesized a new class of peptidomimetic human immunodeficiency virus protease inhibitors containing a unique unnatural amino acid, allophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid], with a hydroxymethylcarbonyl isostere as the active moiety. From a structure-activity relationship study of HIV-1 protease inhibition, enzyme selectivity for other aspartyl proteases, the antiviral activity and pharmacokinetics in rats, 24c (KNI-227) and 24d (KNI-272, our first clinical candidate) were found to be selective and orally potent HIV protease inhibitors. Moreover, an improvement of the pharmacokinetic features of KNI-272 provided two long-lasting and highly bioavailable compounds (24g: JE-2178, 24h: JE-2179).     

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.     

High-speed optimization of inhibitors of the malarial proteases plasmepsin I and II

Four focused libraries targeted for inhibition of the malarial proteases plasmepsin I and II were designed, synthesized, purified, and screened. Selected carboxylic acids and organometallic reactants with diverse physical properties were attached to the hydroxylethylamine scaffold in the P3 and P1' positions to furnish inhibitors with highly improved activity. The concept of controlled and sequential microwave heating was employed for rapid library generation. This combinatorial optimization protocol afforded plasmepsin inhibitors not only with K(i) values in the low nanomolar range, but also with high selectivity versus the human protease cathepsin D. With this class of inhibitory agents, modifications of the P1' substituents resulted in the largest impact on the plasmepsin/cathepsin D selectivity.     

A density functional study of the hydrogen-bond network within the HIV-1 protease catalytic site cleft

The relative energy between two different protonation sites of the Asp25' catalytic site residue is computed and analyzed for various HIV-1 Protease/inhibitor complexes and compared to the wild-type structure. By comparing calculations of negatively charged fragments of gradually increasing size up to 105 atoms we show that correct modeling of the HIV-1 Protease active site requires much larger models than the commonly used acetic acid/acetate moieties. The energy difference between the two proposed protonation sites decreases as the size of the system increases and tends to converge only when the entire catalytic triad of both monomers is taken into account. The importance of the Gly27 backbone amine groups in the stabilization of the negative charge within the catalytic site cleft is revealed. Comparison of the wild-type structure with the structures from various Pr/drug complexes indicates that the HIV-1 protease has a particular catalytic site flexibility.     

KNI-102, a novel tripeptide HIV protease inhibitor containing allophenylnorstatine as a transition-state mimic.

HIV-1 protease inhibitors containing allophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid]-Pro (syn diastereomer) as a transition-state mimic were established to be potent and highly selective. Z-Asn-Apns-Pro-NHBut (KNI-102) is the only tripeptide exhibiting substantial anti-HIV activity and may be of minimum size for potent, selective inhibition of HIV protease. Ready availability due to its simple chemical structure and stability should make it valuable for studies of the development of metabolically stable anti-AIDS drugs.     

Molecular dynamic and free energy studies of primary resistance mutations in HIV-1 protease-ritonavir complexes

To understand the basis of drug resistance of the HIV-1 protease, molecular dynamic (MD) and free energy calculations of the wild-type and three primary resistance mutants, V82F, I84V, and V82F/I84V, of HIV-1 protease complexed with ritonavir were carried out. Analysis of the MD trajectories revealed overall structures of the protein and the hydrogen bonding of the catalytic residues to ritonavir were similar in all four complexes. Substantial differences were also found near the catalytic binding domain, of which the double mutant complex has the greatest impact on conformational changes of the protein and the inhibitor. The tip of the HIV-1 protease flap of the double mutant has the greater degree of opening with respect to that of the others. Additionally, the phenyl ring of Phe82 moves away from the binding pocket S1', and the conformational change of ritonavir subsite P1' consequently affects the cavity size of the protein and the conformational energy of the inhibitor. Calculations of binding free energy using the solvent continuum model were able to reproduce the same trend of the experimental inhibition constant. The results show that the resistance mutants require hydrophobic residues to maintain the interactions in the binding pocket. Changes of the cavity volume correlate well with free energy penalties due to the mutation and are responsible for the loss of drug susceptibility.     

Enhancing protein backbone binding--a fruitful concept for combating drug-resistant HIV

The evolution of drug resistance is one of the most fundamental problems in medicine. In HIV/AIDS, the rapid emergence of drug-resistant HIV-1 variants is a major obstacle to current treatments. HIV-1 protease inhibitors are essential components of present antiretroviral therapies. However, with these protease inhibitors, resistance occurs through viral mutations that alter inhibitor binding, resulting in a loss of efficacy. This loss of potency has raised serious questions with regard to effective long-term antiretroviral therapy for HIV/AIDS. In this context, our research has focused on designing inhibitors that form extensive hydrogen-bonding interactions with the enzyme's backbone in the active site. In doing so, we limit the protease's ability to acquire drug resistance as the geometry of the catalytic site must be conserved to maintain functionality. In this Review, we examine the underlying principles of enzyme structure that support our backbone-binding concept as an effective means to combat drug resistance and highlight their application in our recent work on antiviral HIV-1 protease inhibitors.     

Stereoselective synthesis of 3-aminoindan-1-ones and subsequent incorporation into HIV-1 protease inhibitors

A new method for the stereoselective synthesis of 3-aminoindan-1-ones from triflates of salicylic sulfinyl imines and ethylene glycol vinyl ether has been developed. The reaction sequence starts with a regioselective Heck reaction followed by stereoselective Lewis acid mediated annulation. Acidic cleavage of the sulfinamides produced pure (R)- and (S)-3-aminoindan-1-ones, which were successfully isolated and incorporated into active HIV-1 protease inhibitors.     

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.     

Conformational analysis of TMC114, a novel HIV-1 protease inhibitor

TMC114, a potent novel HIV-1 protease inhibitor, remains active against a broad spectrum of mutant viruses. In order to bind to a variety of mutants, the compound needs to make strong, preferably backbone, interactions and have enough conformational flexibility to adapt to the changing geometry of the active site. The conformational analysis of TMC114 in the gas phase yielded 43 conformers in which five types of intramolecular H-bond interactions could be observed. All 43 conformers were subject to both rigid and flexible ligand docking in the wild-type and a triple mutant (L63P/V82T/I84V) of HIV-1 protease. The largest binding energy was calculated for the conformations that are close to the conformation observed in the X-ray complexes of TMC114 and HIV-1 protease.     

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.     

Design,synthesis, and evaluation of matrix metalloprotease inhibitors bearing cyclopropane-derived peptidomimetics as P1' and P2' replacements

We have previously used trisubstituted cyclopropanes as peptide replacements to induce conformational constraints in known pseudopeptide inhibitors of a number of important enzymes. Cyclopropane-derived peptide mimics are novel in that they are among the few replacements that locally orient the peptide backbone and the amino acid side chain in a predefined manner. Although these dipeptide isosteres have been employed to orient amino acid side chains mimicking the gauche(-) conformation of chi(1)-space, their ability to project the side chains into an anti orientation has not been evaluated. As a first step toward this goal, the conformationally constrained pseudopeptides 8 and 10 and their corresponding flexible analogues 9 and 11 were prepared and tested as inhibitors of matrix metalloproteinases (MMPs). These compounds are analogues of 4 and 5, which were known to be potent MMP inhibitors. The anti orientations of the isopropyl side chain in 8 and the aromatic ring in 10 relative to the peptide backbone substituents on the cyclopropane were predicted to correspond to the known orientations of the P1' and P2' side chains of 5 when bound to MMPs. Hence, 8 and 10 were designed explicitly to probe topological features of the S1' or the S2' binding pockets of the MMPs. They were also designed to explore the importance of the P1'-P2' amide group, which is known to form highly conserved hydrogen bonds in several MMP-inhibitor complexes, and the viability of introducing a retro amide linkage between P2' and P3'. Pseudopeptides 8 and 9 were found to be weak competitive inhibitors of a series of MMPs. Any entropically favorable conformational constraints that were induced by the cyclopropane in 8 were thus overwhelmed by the loss of the hydrogen bonding capability associated with the P1'-P2' amide group. On the other hand, compounds 10 and 11, which contain a P2'-P3' retro amide group, were modest competitive inhibitors of a series of MMPs. The results obtained for 10 and 11 suggest that there may be a loss of hydrogen bonding capability associated with introducing the P2'-P3' retro amide group. However, because the conformationally constrained pseudopeptide 10 was significantly more potent than its flexible analogue 11, trisubstituted cyclopropanes related to 3 may serve as useful rigid dipeptide replacements in some biologically active pseudopeptides.     

Ionization state of the catalytic dyad Asp25/25' in the HIV-1 protease: NMR studies of site-specifically 13C labelled HIV-1 protease prepared by total chemical synthesis

Total chemical synthesis was used to site-specifically (13)C-label active site Asp25 and Asp25' residues in HIV-1 protease and in several chemically synthesized analogues of the enzyme molecule. (13)C NMR measurements were consistent with a monoprotonated state for the catalytic dyad formed by the interacting Asp25, Asp25' side chain carboxyls.     

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.     

Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes

BACKGROUND: The 20S proteasome is a multicatalytic protease complex that exhibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the beta2, beta5 and beta1 subunits, respectively. Modulation of these activities using inhibitors is essential for a better understanding of the proteasome's mechanism of action. Although there are highly selective inhibitors of the proteasome's chymotryptic activity, inhibitors of similar specificity have not yet been identified for the other activities. RESULTS: The X-ray structure of the yeast proteasome reveals that the sidechain of Cys118 of the beta3 subunit protrudes into the S3 subsite of the beta2 active site. The location of this residue was exploited for the rational design of bidentated inhibitors containing a maleinimide moiety at the P3 position for covalent linkage to the thiol group and a carboxy-terminal aldehyde group for hemiacetal formation with the Thr1 hydroxyl group of the active site. Structure-based modelling was used to determine the optimal spacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activity to the beta2 active site. X-ray crystallographic analysis of a yeast proteasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite. CONCLUSIONS: Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes. Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting new biochemical tool.     

Mapping of the active site of proteases in the 1960s and rational design of inhibitors/drugs in the 1990s

For several decades the specificity of proteases has been presented as an active site divided into subsites, using the nomenclature of Schechter & Berger from 1967 (S1, S2... for subsites of the active site; P1, P2... for residues of the substrate occupying the corresponding subsites). At early stages of the research (1960s) it was realized that the size of the active site was larger than expected and important interactions occur in regions remote from the catalytic site. Since the active site was found to be large it was divided into subsites, and a procedure to map it up was developed. The map provides information on the size of the active site (number of subsites), the properties of each subsite (free energy of ligand binding, nature of binding forces, etc.), and it enables rational design of new substrates and inhibitors. Already in 1968 inhibitors with binding constants ten thousand fold higher than available inhibitors, were prepared. The model of a large active site was initially met with strong opposition. Before long, however, predictions of the model (size of the active site, interactions in subsites remote from the catalytic site) were confirmed by X-ray crystallography (1970). During the 1990s proteolytic enzymes received renewed attention in biology and medicine, they became therapeutic targets, and protease inhibitors were successfully applied in the treatment of AIDS and hypertension. The model of large active site divided into subsites, proposed 38 years ago, stood the test of time. This model is still in use in basic research to evaluate enzyme activity, and in pharmaceutical research for the development of inhibitors/drugs.