We have designed, synthesized, and evaluated the inhibitory activity and metabolic stability of new peptidomimetic molecular tongs based on a naphthalene scaffold for inhibiting HIV-1 protease dimerization. Peptidomimetic motifs were inserted into one peptidic strand to make it resistant to proteolysis. The peptidic character of the molecular tongs can be decreased without changing the way they inhibit dimerization. Mutated HIV-1 proteases are also vulnerable to dimerization inhibitors, and the multimutated protease ANAM-11 is twice as sensitive to the inhibitor compared to wild-type protease. Thus, the metabolic stability of antidimeric molecular tongs can be increased without compromising their ability to inhibit wild-type and mutated HIV-1 proteases in vitro.

With the view to deliver anti-HIV nucleoside and nucleoside-monophosphate (MP) analogues specifically into HIV-infected cells, we synthesized a series of ester and phosphoramidate peptide conjugates of zidovudine (AZT) and of AZT-MP, respectively, wherein the peptide sequences derive from a HIV-protease (PR) hydrolysable substrate. Their in vitro stability with respect to hydrolysis, anti-HIV activity and cytotoxicity, and ability to inhibit the HIV-PR activity were investigated. Concerning the ester AZT-peptide conjugates, their antiviral activity level in thymidine kinase-expressing (TK+) CEM-SS and MT-4 cells was in most cases closely correlated to their hydrolysis rate: the faster the hydrolysis, the closer the anti-HIV activity to that of AZT. None of them was a HIV-PR substrate, indicating that their antiviral activity was not related to their intracellular hydrolysis by this enzyme. None of them inhibited HIV in TK-deficient (TK-) CEM cells, demonstrating that they probably act as prodrugs of AZT. Most of the phosphoramidate peptide conjugates of AZT-MP were rapidly degraded in a physiological buffer into several metabolites including AZT. Their anti-HIV activity in TK+ CEM-SS and MT-4 cells was much lower than that of AZT, indicating that only low amounts of AZT or AZT-MP were released into cells during incubation. Antiviral activities measured on TK- CEM cells for some phosphoramidates suggest that low amounts of AZT-MP could be released intracellularly. However, this AZT-MP release was not initiated by a HIV-PR hydrolysis, as no evidence for peptide cleavage was obtained by HPLC analysis of one representative compound after incubation with HIV-PR.

New "molecular tongs" based on naphthalene and quinoline scaffolds linked to two peptidic strands were synthesized. They were designed to prevent dimerization of HIV-1 protease by targeting the antiparallel beta-sheet involving N- and C-termini of each monomer. Compared to "molecular tongs" previously described (Bouras, A.; Boggetto, N.; Benatalah, Z.; de Rosny, E.; Sicsic, S.; Reboux-Ravaud, M. J. Med. Chem. 1999, 42, 957-962), two main different structural features were introduced: positively charged quinoline as a new scaffold and two peptidic strands displaying different sequences. Seventeen new "molecular tongs" with dipeptidic or tripeptidic strands were synthesized. These molecules were assayed on HIV-1 protease using the Zhang kinetic technique. Eleven molecules behaved as pure dimerization inhibitors, mostly at the submicromolar range. Compared to a naphthalene scaffold, the quinoline one was shown in several cases to favor dimerization inhibition. The simplified hydrophobic Val-Leu-Val-OMe strand was confirmed as particularly favorable. The C-terminal analogue strand Thr-Leu-Asn-OMe was shown to be the best one for inducing dimerization inhibition (K(id) of 80 nM for compound 30). The mechanism of inhibition was ascertained using ANS binding and gel filtration. Experimental results are in agreement with the dissociation of the HIV-1 protease dimeric form in the presence of the synthesized molecular tongs.

Original inhibitors of HIV-1 protease based on a chiral bicyclic guanidinium scaffold linked to short peptidic mimics of the terminal protease sequences and to a lipophilic group were designed. These inhibitors prevent dimerization of the native protease by an interfacial structure at the highly conserved antiparallel beta-strand involving both the N and C termini that substantially account for dimerization. The preorganized guanidinium spacer introduces additional electrostatic hydrogen-bonding interactions with the C-terminal Phe-99 carboxylate. Lipophilic residues linked to side chains and the guanidinium scaffold are essential for dimerization inhibition as ascertained by Zhang kinetics (4, K-id = 290 nM; 6 or 6', K-id = 150 nM; 8, K-id = 400 nM) combined with a circular dichroism study on the enzyme thermal stability. Remarkably, less hydrophobic compounds result in mixed dimerization (1a and 3) or active site inhibitors (5). Removal of the guanidinium hydrophobic groups leads to less active or inactive ligands.

The aminoxy acids NH 2 O–C– α HRCO– 2 H are much more easily obtained in the enantiomerically pure form than the analogous hydrazino acids NH 2 NH–C– α HRCO– 2 H, and it has been shown that the isosteric amidoxy ψ[CO–NH–O] and hydrazide ψ[CO–NH–NH] amide surrogates induce two quite similar γ‐like folded structures. An aminoxy acid can also be N ‐coupled to a peptide aldehyde to give the aldoxime ψ[CHNO–O] link or to a peptide ketone to form the ketoxime ψ[CRN O] link. The former can be further reduced into the hydroxylamine ψ[CH 2 NH–O] link which gives rise to reduced amidoxy peptides. The structural properties induced by these amide surrogates were studied, using IR and NMR spectroscopy, paying particular attention to the Z/E ‐isomerism of the oxime link. In order to investigate their inhibitory potency, the three amide surrogates were introduced in the Pro 3 ‐Val 4 and Val 4 ‐Ala 5 position of Z‐Ala 1 ‐Ala 2 ‐Pro 3 ‐Val 4 ‐Ala 5 ‐Ala 6 ‐NHiPr, a substrate which is cleaved in the Val 4 ‐Ala 5 position by human leukocyte elastase (HLE). The [Val 4 ψ[CONH–O–]Ala 5 ] analogue was still a substrate, while the [Pro 3 ψ[CONH–O–]Val 4 ] and [Val 4 ψ[CHN O –]Ala 5 ] pseudopeptides acted as HLE competitive inhibitors. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.

The structure of new lipopeptides targeting the enzymic dimer interface have been rationally improved resulting in dimerization inhibitors of the human immunodeficiency virus 1 protease (Kid=5nM for the best inhibitor). The contribution of each amino acid in inhibitory 3-mer lipopeptides was analyzed demonstrating that the C-terminal amino acid residue may preferably be replaced by thyroxine and thyronine. The negative charge of Glu is not essential. Lengthening of the peptidic chain may lead to a decrease of efficiency and a change in the mechanism (competitive inhibition instead of dimerization inhibition). The N-terminal blocking group can be replaced by 2-aminopalmitic acid. The mechanism of inhibition has been ascertained using Zhang’s kinetic analysis combined with a physical method based on binding of 1-anilino-8-naphtalene sulfonate to enzyme. By targeting the hydrophobic pocket and the interface antiparallel β-sheet found relatively free of mutations in contrary to the active site, these efficient dimerization inhibitors may provide a way of overcoming the drug resistances observed with therapeutic antiproteases that bind to the active site.

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.

The use of metal-organic complexes is a potentially fruitful approach for the development of novel enzyme inhibitors. They hold the attractive promise of forming stronger attachments with the target by combining the co-ordination ability of metals with the unique stereoelectronic properties of the ligand. We demonstrated that this approach can be successfully used to inhibit the protease of the human immunodeficiency virus (type 1). Several ligands bearing substituents designed to interact with the catalytic site of the enzyme when complexed to Cu2+ were synthesised. The inhibition pattern of the resulting copper(II) complexes was analysed. We showed that the copper(II) complex of N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide (C1) interacts with the active site of the enzyme leading to competitive inhibition. On the other hand, N2-pyridine-amide ligands and oxazinane carboxamide ligand were found to be poor chelators of the cupric ion under the enzymatic assay conditions. In these cases, the observed inhibition was attributed to released cupric ions which react with cysteine residues on the surface of the protease. While unchelated metal cations are not likely to be useful agents, metal chelates such as C1 should be considered as promising lead compounds for the development of targeted drugs.

La protease dimerique du VIH (PR) intervient au cours de la derniere etape de la replication virale et assure la maturation du virion. Elle clive specifiquement les precurseurs polyproteiques p55gag et p160gag-pol et libere ainsi les enzymes virales et les proteines structurales. De nombreux travaux demontrent qu'il s'agit d'une cible importante dans la lutte contre le sida. Six inhibiteurs de la PR sont a l'heure actuelle couramment utilises en therapeutique humaine en association avec des inhibiteurs de la transcriptase inverse (traitements antiretroviraux hautement actifs ou Haart). Ces inhibiteurs de la PR sont des analogues de l'etat de transition qui ciblent le site actif. Pour contrer les resistances a ces antiproteases et optimiser leur efficacite inhibitrice, des modifications structurales sont introduites afin d'aboutir a des inhibiteurs de deuxieme generation. Une strategie alternative plus recente consiste a developper des inhibiteurs de la dimerisation, molecules empechant la formation de l'enzyme dimerique active et qui ciblent le feuillet beta antiparallele forme entre les extremites C- et N-terminales des monomeres. Enfin, la formation d'heterodimeres inactifs aboutissant a la formation de particules immatures est exploree.

La protease dimerique du VIH (PR) intervient au cours de la derniere etape de la replication virale et assure la maturation du virion. Elle clive specifiquement les precurseurs polyproteiques p55gag et p160gag-pol et libere ainsi les enzymes virales et les proteines structurales. De nombreux travaux demontrent qu'il s'agit d'une cible importante dans la lutte contre le sida. Six inhibiteurs de la PR sont a l'heure actuelle couramment utilises en therapeutique humaine en association avec des inhibiteurs de la transcriptase inverse (traitements antiretroviraux hautement actifs ou Haart). Ces inhibiteurs de la PR sont des analogues de l'etat de transition qui ciblent le site actif. Pour contrer les resistances a ces antiproteases et optimiser leur efficacite inhibitrice, des modifications structurales sont introduites afin d'aboutir a des inhibiteurs de deuxieme generation. Une strategie alternative plus recente consiste a developper des inhibiteurs de la dimerisation, molecules empechant la formation de l'enzyme dimerique active et qui ciblent le feuillet beta antiparallele forme entre les extremites C- et N-terminales des monomeres. Enfin, la formation d'heterodimeres inactifs aboutissant a la formation de particules immatures est exploree.

The active form of human immunodeficiency virus type 1 protease (HIV-1 PR) is a homodimeric structure in which two subunits are linked through a two-stranded antiparallel beta-sheet consisting of the N- and C-termini of each monomer. To inhibit the dimerization process or disrupt the dimeric interface leading to inactive enzyme, conformationally constrained "molecular tongs" have been designed and synthesized to interfere with one monomer end in a beta-sheet fashion. These molecules are based on two peptidic strands attached to an aromatic scaffold. Inhibitions (submicromolar range) were obtained with molecular tongs containing tripeptidic or tetrapeptidic arms attached to a pyridinediol- or naphthalenediol-based scaffold (Kid = 0.56-4.5 microM at pH 4.7 and 30 degrees C). Kinetic studies are in agreement with an interface inhibition mechanism.

A series of novel synthetic peptides containing an N-terminal glyoxylyl function (CHOCO-) have been tested as inhibitors of HIV-1 protease. The N-glyoxylyl peptide CHOCO-Pro-He-Val-NH2, which fulfills the specificity requirements of the MA/CA protease cleavage site together with the criteria of transition state analogue of the catalyzed reaction, was found to be a moderate competitive inhibitor although favorable interactions were visualized between its hydrated form and the catalytic aspartates using molecular modeling. Increasing the length of the peptide sequence led to compounds acting only as substrates.

N-aryl-3,3-difluoroazetidin-2-ones featured by a latent electrophilic methylene quinoniminium function have been synthesized and evaluated as inhibitors of human leucocyte elastase. To promote hydrophobic interactions with the enzyme, to increase the rates of beta-lactam ring opening and of benzylic group departure, or to induce hydrosolubility, these compounds incorporate on their aromatic ring either an alkyl moiety, a methoxy substituent or a carboxylic group. Some of these beta-lactams proved to be good inactivators of human leucocyte elastase.