Inhibition of cathepsin D by tripeptides containing statine analogs.

Various analogs of statine, a remarkable amino acid component of the protease inhibitor pepstatine, were synthesized and evaluated as tripeptide derivatives for their activity against cathepsin D and HIV-1 protease.

Synthesis and conformational analysis of peptide inhibitors of farnesyltransferase.

Farnesylation of the ras oncogene product by Farnesyl Transferase (FTase) is known to be a critical step in cell transformation leading to uncontrolled proliferation. The peptide CysValTicMet is a potent FTase inhibitor, but its degradation by amino-peptidases and its only weak internalization into cells make it a bad candidate for a future cancer drug. We have prepared improved CysValTicMet analogues using several approaches: (i) amino terminal modifications or introduction of pseudopeptides or non-natural amino acids to increase proteolytic stability, (ii) introduction of hydrophobic aliphatic chains to increase cell internalization and metabolic stability and (iii) transformation into prodrugs. Additionally, we have carried out comparative conformational analysis studies by molecular dynamics of some of the here presented peptides and of our recently described peptidomimetic inhibitors of FTase.

Betulinic acid derivatives: a new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action.

A series of omega-undecanoic amides of lup-20(29)-en-28-oic acid derivatives were synthesized and evaluated for activity in CEM 4 and MT-4 cell cultures against human immunodeficiency virus type 1 (HIV-1) strain IIIB/LAI. The potent HIV inhibitors which emerged, compounds 5a, 16a, and 17b, were all derivatives of betulinic acid (3beta-hydroxylup-20(29)-en-28-oic acid). No activity was found against HIV-2 strain ROD. Compound 5a showed no inhibition of HIV-1 reverse transcriptase activity with poly(C).oligo(dG) as template/primer, nor did it inhibit HIV-1 protease. Additional mechanistic studies revealed that this class of compounds interfere with HIV-1 entry in the cells at a postbinding step.

Sensitive, hydrosoluble, macromolecular fluorogenic substrates for human immunodeficiency virus 1 proteinase.

Hydrosoluble macromolecular fluorogenic substrates specific for the human immunodeficiency virus 1 (HIV-1) proteinase have been prepared. The fluoresceinyl peptide Ftc-epsilon-Ahx-Ser-Phe-Asn-Phe-Pro-Gln-Ile-Thr-(Gly)n, corresponding to the first cleavage site of HIV-1 gag-pol native precursor was linked to a water-soluble neutral (Lys)n derivative. The epsilon-aminohexanoyl residue (epsilon-Ahx) and the glycyl sequence were added in order to improve the stability of the substrate and the accessibility of the cleavage site to the HIV-1 proteinase respectively. This macro-molecular peptidic-substrate conjugate is significantly more water-soluble than the free peptide itself on a substrate molar concentration basis. The assay is based on the quantitative precipitation of the polymeric material by adding propan-2-ol whereas the fluorescent peptide moiety released upon proteolysis remains soluble in the supernatant. The proteinase activity is assessed by measuring the fluorescence of the supernatant. This assay allows the detection of a few fmol of HIV-1 proteinase, even in the presence of cell culture media, plasma or cell lysate and it gives accurate results within a large proteinase concentration range. The hydrosoluble macromolecular substrate is also suitable for determining the HIV-1 proteinase activity using 96-well microplates, allowing us to test accurately and rapidly numerous enzyme samples and/or the potency of new proteinase inhibitors.

Low molecular weight, sequence based, collagenase inhibitors selectively block the interaction between collagenase and TIMP (tissue inhibitor of metalloproteinases).

Sequence-based inhibitors of collagenase bearing an hydroxamate group capable of chelating the active site zinc atom were synthesized and tested. The effect of one of these molecules (RP 59794; Ki about 10(-8) M) on the formation of the TIMP: collagenase complex was also tested. RP 59794 blocks complex formation and can partially dissociate established TIMP: collagenase complexes. It exhibits the same stereospecificity in this activity as in its inhibition of collagenase suggesting that TIMP and RP 59794 both interact with the active site region of collagenase.

[Natural inhibitor of metalloproteinases: structural and functional study]. / Inhibiteur naturel des métalloprotéinases: étude structurale et fonctionnelle.

A potent collagenase inhibitor was purified from cells of calf aorta medial tissue maintained in culture. This molecule was characterized and identified as TIMP (Tissue Inhibitor of Metalloproteinases). Formation of a TIMP--collagenase complex was demonstrated chromatographically using pure TIMP and pure pig synovial cell collagenase. The N-terminal aminoacid sequence of TIMP was determined and, using appropriate oligonucleotide probes the human genes was cloned from a human cDNA bank. This gene was expressed in E. coli, and fully active TIMP was obtained after a denaturation renaturation process. The interest of TIMP as a model for the design of novel collagenase inhibitors is discussed.

[Inhibition of synovial collagenase by actinonin. Study of structure/activity relationship]. / Inhibition de la collagénase synoviale par l'actinonine. Etude de relations structure/activité.

Actinonin is a pseudopeptide antibiotic which inhibits collagenase at micromolar concentrations [10]. In this study, Actinonin analogs were tested to investigate "structure/activity" relationships for this class of molecule. The results indicate that distance between the hydroxamate carbonyl group and that of the first amide bond is important, since increasing this distance by a methylene group, decreases inhibition by a factor of 100. The amide bonds of this inhibitor must be in the peptide bond orientation. Different N terminal amide derivatives and different hydrophobic substituents adjacent to the hydroxamate residue, had little effect on inhibitory activity. However, in this series of molecules, two hydrophobic groups separated by an amide bond seem to be necessary for potent inhibition.

An inhibitor of mammalian collagenase active at micromolar concentrations from an actinomycete culture broth.

Anti-collagenase activity was detected in the culture supernatant of an Actinomycete strain S 4373. The molecule was purified by solvent extraction, medium pressure and high pressure reverse phase chromatography and finally by HPLC gel filtration. The pure product was analyzed by mass spectroscopy and was identified as actinonin, a known pseudopeptide antibiotic. The Ki was determined as 1.4 microM and this value was confirmed using pure synthetic actinonin.