Tricyclic ureas: A new class of HIV-1 protease inhibitors

A new class of tricyclic ureas containing a conformationally constrained proline was designed with the aid of molecular modeling. Efficient stereoselective intermolecular pinacol coupling represented the highlight of the synthesis. These rigid cyclic ureas are active towards HIV-1 protease, with 9 being the most potent compound (Ki = 9 nM) despite interacting with only three side chain binding pockets of HIV protease.     

Synthesis and evaluation of chloromethyl sulfoxides as a new class of selective irreversible cysteine protease inhibitors

The synthesis and biological evaluation of a new class of selective irreversible cysteine protease inhibitors is described. A set of amino acid based chloromethyl sulfoxides was prepared and they were found to inhibit irreversibly the cysteine protease papain. They were selective for cysteine proteases since no inhibition was found for the serine protease chymotrypsin.     

Ionic derivatives of betulinic acid as novel HIV-1 protease inhibitors

Betulinic acid is a natural product possessing abundant and favourable biological activity, including anti-cancer, anti-malarial, anti-inflammatory and anti-HIV properties, while causing minimal toxicity to unaffected cells. The full biological potency of betulinic acid cannot be fully unlocked, however, for a number of reasons, a primary one being its limited solubility in aqueous and biologically pertinent organic media. Aiming to improve the water solubility of betulinic acid without disrupting its structurally related bioactivity, we have prepared different ionic derivatives of betulinic acid. Inhibition bioassays on HIV-1 protease-catalysed peptide hydrolysis indicate significantly improved performance resulting from converting the betulinic acid to organic salt form. Indeed, for one particular cholinium-based derivative, its water solubility is improved more than 100 times and the half maximal inhibitory concentration (IC(50)) value (22 μg mL(-1)) was one-third that of wide-type betulinic acid (60 μg mL(-1)). These encouraging results advise that additional studies of ionic betulinic acid derivatives as a therapeutic solution against HIV-1 infection are warranted.     

Conformational studies of irreversible HIV-1 protease inhibitors containing cis-epoxide as an amide isostere.

We have carried out NMR and molecular modeling studies of peptidomimetic HIV-1 protease inhibitors, LB71116: Qc-Asn-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isopropyl)2 where Qc stands for quinaldic acid and LB71148: Qc-(SMe)Pen(O)2-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isoprop yl)2 where (SMe)Pen(O)2 stands for S-methyl-S-dioxo-penicillamine. Through conformational calculations and NMR data analysis, we have obtained preferred conformations of the two inhibitors in solution. To our knowledge, this work is one of the first extensive conformational studies of peptidomimetics containing cis-epoxide amide isostere. The resulting preferred conformations contain extended structures. In these conformations, the psi of Phe(cep) is maintained about 130 degrees and the phi angle of (cep)Gly prefers +/- 150 degrees [where Phe(cep) and (cep)Gly are the residues generated by the replacement of the Phe-Gly peptide bond with cis-epoxide]. Two conformations were commonly observed in the preferred conformations of each inhibitor. Through restrained molecular dynamics simulating the hydrogen bond formation between our inhibitor and a water molecule ('flap water'), one of the conformations is assumed as the conformation which can bind to the enzyme without large conformational changes. Recently, we had the opportunity to compare the selected preferred conformation with the binding conformation of LB71116 observed from the X-ray studies of the complex between LB71116 and HIV-1 protease. These two conformations are surprisingly similar to each other. Thus, we can explain high activity and selectivity of our inhibitors to the HIV-1 protease by the similarity between the preferred conformations in solution and the binding conformation.     

Synthesis of a new class of HIV-1 inhibitors

A new family of molecules potentially inhibitors of the HIV-1 Tat-TAR complex was prepared. These compounds are constituted by dinucleotide analogs (PNA dimer) bound, through a linker, to an arginine residue. In this series, several molecules inhibit viral development in cell culture with a micromolar IC50 and without cellular toxicity until 200 microM concentration.     

Evaluation of retro-inverso modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster

Effects of retro-inverso (RI) modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster backbone were clarified. Construction of the isoster backbone was achieved by a stereoselective aldol reaction. Four diastereomers with different configurations at the isoster hydroxyl site and the scissile site substituent were synthesized. Inhibitory activities of the new inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor.     

Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.     

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.     

Structure-based prediction of potential binding and nonbinding peptides to HIV-1 protease

HIV-1 protease is a major drug target against AIDS as it permits viral maturation by processing the gag and pol polyproteins of the virus. The cleavage sites in these polyproteins do not have obvious sequence homology or a binding motif and the specificity of the protease is not easily determined. We used various threading approaches, together with the crystal structures of substrate complexes which served as template structures, to study the substrate specificity of HIV-1 protease with the aim of obtaining a better differentiation between binding and nonbinding sequences. The predictions from threading improved when distance-dependent interaction energy functions were used instead of contact matrices. To rank the peptides and properly account for the peptide's conformation in the total energy, the results from using short-range potentials on multiple template structures were averaged. Finally, a dynamic threading approach is introduced which is potentially useful for cases when there is only one template structure available. The conformational energy of the peptide-especially the term accounting for the side chains-was found to be important in differentiating between binding and nonbinding sequences. Hence, the substrate specificity, and thus the ability of the virus to mature, is affected by the compatibility of the substrate peptide to fit within the limited conformational space of the active site groove.     

HIV-1 reproduction is inhibited by peptides derived frm the N- and C-termini of HIV-1 protease.

Two octapeptides derived from the sequence of the N- and C-termini of HIV-1 protease were tested for their ability to inhibit HIV-1 reproduction. Weak inhibitory activity was found with each of the two peptides. It is assumed that HIV-1 protease is the target of the inhibitory action. In spite of the moderate inhibitory activity the results are encouraging since they may be improved by various means.     

(S)-Thiirancarboxylic acid as a reactive building block for a new class of cysteine protease inhibitors

For (S)-thiirancarboxylic acid a second-order rate constant of k2nd = 222 M(-1) min(-1) for the irreversible inhibition of papain was determined. The ethyl and methyl ester do not inhibit the enzyme time-dependently. An improved synthesis of enantiomerically pure thiirancarboxylic acid is described. It is shown that thiirancarboxylates can be substrates for serine proteases (alpha-chymotrypsin) and esterases (pig liver esterase) and even for metallo proteases (thermolysin).     

Design,synthesis and QSAR studies on N-aryl heteroarylisopropanolamines,a new class of non-peptidic HIV-1 protease inhibitors

A series of N-aryl heteroarylisopropanolamines in which an indole or a 3-arylpyrrole moiety was linked to an aryl group through an isopropanolamine linker, were designed and synthesized as potential anti-HIV-1-PR agents. Series was tested for their ability in blocking PR activity. As a rule, indole derivatives of class 1 exhibited more potency than pyrrole analogues of class 2 while tert-butylamide substituents increased anti-PR potency. In fact, bis tert-butylamide 1e showed the highest activity with IC(50)=25 microM. Even if not very potent, a simple class of anti-PR agents, with a facile synthetic pathway was discovered. QSAR studies on isopropanolamines 1 and 2 were performed in comparison with diarylbutanols, a new class of non peptidic anti-PR agents, recently discovered by Agouron Pharmaceuticals. QSAR and CoMFA models based on 30 diarylbutanols used as a training set were developed. The obtained models were used to investigate the binding mode of the newly synthesized derivatives 1 and 2. The results of this study suggest that N-aryl heteroarylisopropanolamines bind to the PR active site similarly to the diarylbutanols of Agouron.     

Inhibition of HIV-1 protease by short peptides derived from the terminal segments of the protease.

The active HIV-1 protease is a homodimeric enzyme. A beta-sheet consisting of N- and C-terminal segments provides the main driving force for dimerization of the inactive protomers. Several short peptides with sequences derived from the N- and C-termini of the protease were tested for inhibition of protease activity and for inhibition of HIV-1 replication in lymphocytes. Medium inhibitory activity was found with each of the peptides in the enzyme test and no inhibition of the lymphocytes was found up to 200 micrograms/ml. The enzyme tests indicate that HIV-1 protease is the target of the inhibitory action. Synergistic action could not be found with pairs of the peptides derived from the two different termini. Prolonged incubation with one of the peptides increased inhibition indicating a slow dissociation of the protease dimers. No cytotoxic effect of the inhibitors could be found below 200 micrograms/ml.     

Evaluation of triazolamers as active site inhibitors of HIV-1 protease

Proteases typically recognize their peptide substrates in extended conformations. General approaches for designing protease inhibitors often consist of peptidomimetics that feature this conformation. Herein we discuss a combination of computational and experimental studies to evaluate the potential of triazole-linked β-strand mimetics as inhibitors of HIV-1 protease activity.     

Novel nonpeptidic inhibitors of HIV-1 protease obtained via a new multicomponent chemistry strategy

Using a newly developed multicomponent chemistry strategy in combination with structure based drug design, a new class of HIV-1 protease inhibitors has been obtained.     

Construction of a human immunodeficiency virus type 1 (HIV-1) library containing random combinations of amino acid substitutions in the HIV-1 protease due to resistance by protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 x 10(5)) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 microM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.     

Biosensor analysis of the interaction of potential dimerization inhibitors with HIV-1 protease

Inhibitors of protein-protein interactions are currently considered as perspective prototypes of a new generation of drugs. The most attractive targets for such inhibitors are the oligomeric enzymes which active sites are formed by amino acid residues from different subunits. HIV-1 protease (HIVp), which is active only as a homodimer form, is the classic example of such enzymes. We have developed a new approach for experimental screening of HIVp dimerization inhibitors. It is based on an original biosensor test-system for differential analysis of interaction of tested substances with HIVp dimers and monomers. Using this test-system we have analyzed the most perspective candidate substances predicted by the method of virtual screening, and also some derivatives of glycyrrhizin, triterpenic and steroid glycosides. In the results of this study we have found one compound, which preferentially interacts with HIVp monomers and inhibits in vitro activity of this enzyme with the IC₅₀ value of about 10^?6 M.     

Substrate analog inhibitors of HIV-1 protease containing phenylnorstatine as a transition state element

Substrates of HIV-1 protease are classified into three groups (A, B and C) based on the amino acid residues present at P1' and P2' sites. Replacement of the scissile amide bond by phenylnorstatine in representative substrate analog sequences from class A, B and C, yielded inhibitors of HIV-1 protease. Of the twelve inhibitors synthesized in this series, class C substrate analog inhibitors are more potent inhibitors (Ki's 3.3-24 microM) than either class A or class B inhibitors. In this series of inhibitors, the (2S,3S) isomer of phenylnorstatine is preferred over the other isomers as a "transition state element" for design of inhibitors of HIV-1 protease.