Peptides corresponding to the heptad repeat motifs in the transmembrane protein (gp41) of human immunodeficiency virus type 1 elicit antibodies to receptor-activated conformations of the envelope glycoprotein.

Two heptad repeat regions in the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane subunit (gp41) self-assemble into a six-helix bundle structure that is critical for virus entry. Immunizations with peptides corresponding to these regions generated antibodies specific to the receptor-activated conformations of gp41.

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.

Design, synthesis, and evaluation of conformationally constrained tongs, new inhibitors of HIV-1 protease dimerization.

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.

Stereoselective synthesis of peptidyl trifluoromethyl alcohols and ketones: inhibitory potency against human leucocyte elastase, cathepsin G, porcine pancreatic elastase and HIV-1 protease.

New fluorinated inhibitors have been designed to target two major proteases-human leucocyte elastase and HIV-1 protease. Two series of beta-peptidyl trifluoromethyl alcohols (TFMAs) Z-L-Val-NH-*CH(Y)*CH(OH)-CF3, where *C is the chiral centre, varied in the nature of the substituent Y, a phenylethyl [-(CH2)2-C6H5] or an isopropyl [-CH(CH3)2] group. These TFMAs were first synthesized as two pairs of the syn and anti diastereoisomers. The inhibitory effects of these mixtures were then assessed on three serine proteases chosen on the basis of the aromatic and aliphatic nature of the substituents-human leucocyte elastase (HLE), human cathepsin G (HCG) and porcine pancreatic elastase (PPE). In the presence of detectable inhibition, each epimer at C2 was separated to determine its inhibition constant (Ki) towards HLE, HCG and PPE. The stereoisomerically pure TFMAs were then oxidized into peptidyl trifluoromethyl ketones (TFMKs) for similar inhibition assays. The absolute configuration of the compounds remained unknown. One epimer at C2 of each syn and anti TFMA with the phenylethyl substituent behaved as a competitive inhibitor towards HLE and HCG with inhibition constants below the millimolar range, whereas their TFMK counterparts were non-inhibitors. In the second series, the two ketones inhibited both elastases with Ki values in the micromolar range, whereas only the syn TFMA was active towards HLE (Ki = 5.65 x 10(-4)M). The tested compounds also had structural properties compatible with recognition by HIV-1 protease. The inhibition of the enzyme was observed with TFMK only (IC50 = 15-200 microM). The phenylethyl substituent promoted inhibition by a factor of 10 (IC50 = 15 microM) compared with the isopropyl substituent (IC50 = 200 microM) leading to selective inhibition of HIV-1 protease. Isomerically pure TFMKs were more potent towards HLE than the alcohols from which they were obtained. However, an enantiomerically pure TFMA selectively inhibited HLE unlike its TFMK analogue which also inhibited PPE. This last result together with the selective inhibition of HIV-1 protease by TFMK with a phenylethyl substituent might be relevant to the design of specific HLE and HIV-1 inhibitors as therapeutic agents.

Synthesis of N-glyoxylyl peptides and their in vitro evaluation as HIV-1 protease inhibitors.

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-Ile-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.

[Synthetic inhibitors targeting serine and aspartic acid proteases]. / Inhibiteurs synthétiques à visée pharmacologique ciblant les protéases à sérine et à acides aspartiques.

The interaction of novel series of synthetic inhibitors with various serine proteases (leukocyte elastase, thrombin, cathepsin G, chymotrypsin, plasminogen activators and plasmin) and an aspartic protease (HIV-1 protease) were studied. Various aspects were analyzed: mechanism of action, structure-activity relationships, and in some cases, molecular modelling and biological evaluation. Functionalized cyclopeptides and N-aryl azetidin-2-ones behaved as suicide substrates acting specifically on trypsin-like proteases (thrombin or urokinase) and elastases, respectively. Novel hydrazinopeptides acted as reversible inhibitors of elastases. Coumarin derivatives inactivated very efficiently chymotrypsin-like proteases (k(inact)/K(I) = 760,000 M(-1) .s(-1)). Inhibitors of HIV-1 protease acting either as inactivators or dimerization inhibitors are under investigation. The inhibitors described above are useful for elucidating the biological roles of the target enzymes and constitute potential drugs.

The HimA and HimD subunits of integration host factor can specifically bind to DNA as homodimers.

Integration host factor (IHF) is a heterodimeric protein from Escherichia coli which specifically binds to an asymmetric consensus sequence. We have isolated the individual subunits of IHF, HimA and HimD, and show that an active IHF protein can be reconstituted from these subunits. The HimA and HimD polypeptides alone are capable of specifically recognizing the same ihf sequence. The mobilities of the protein-DNA complexes in a gel-retardation assay suggest that the proteins bind as homodimers. The stability of the HimD-DNA complex is approximately 100-fold lower than that of the IHF-DNA complex. The HimA-DNA complex is even less stable and is only observed when a large excess of HimA is used. This instability is possibly due to the inability of HimA to form stable homodimers. By domain swapping between HimA and HimD, we have constructed an IHF fusion protein which has the putative DNA-binding domains of only HimA. This fusion protein forms stable dimers and makes specific protein-DNA complexes with a high efficiency. A comparable fusion protein with only the DNA-binding domains of HimD forms less stable complexes, suggesting that sequence-specific contacts between IHF and the ihf consensus are mainly provided by the HimA subunit.