Spontaneous migration of cellular aggregates from giant keratocytes to running spheroids.

Despite extensive knowledge on the mechanisms that drive single-cell migration, those governing the migration of cell clusters, as occurring during embryonic development and cancer metastasis, remain poorly understood. Here, we investigate the collective migration of cell on adhesive gels with variable rigidity, using 3D cellular aggregates as a model system. After initial adhesion to the substrate, aggregates spread by expanding outward a cell monolayer, whose dynamics is optimal in a narrow range of rigidities. Fast expansion gives rise to the accumulation of mechanical tension that leads to the rupture of cell-cell contacts and the nucleation of holes within the monolayer, which becomes unstable and undergoes dewetting like a liquid film. This leads to a symmetry breaking and causes the entire aggregate to move as a single entity. Varying the substrate rigidity modulates the extent of dewetting and induces different modes of aggregate motion: "giant keratocytes," where the lamellipodium is a cell monolayer that expands at the front and retracts at the back; "penguins," characterized by bipedal locomotion; and "running spheroids," for nonspreading aggregates. We characterize these diverse modes of collective migration by quantifying the flows and forces that drive them, and we unveil the fundamental physical principles that govern these behaviors, which underscore the biological predisposition of living material to migrate, independent of length scale.

Dimer disruption and monomer sequestration by alkyl tripeptides are successful strategies for inhibiting wild-type and multidrug-resistant mutated HIV-1 proteases.

Wild-type and drug-resistant mutated HIV-1 proteases are active as dimers. This work describes the inhibition of their dimerization by a new series of alkyl tripeptides that target the four-stranded antiparallel beta-sheet formed by the interdigitation of the N- and C-monomer ends of each monomer. Analytical ultracentrifugation was used to give experimental evidence of their mode of action that is disruption of the active homodimer with formation of inactive monomer-inhibitor complexes. The minimum length of the alkyl chain needed to inhibit dimerization was established. Sequence variations led to a most potent HIV-PR dimerization inhibitor: palmitoyl-Leu-Glu-Tyr (Kid = 0.3 nM). Insertion of d-amino acids at the first two positions of the peptide moiety increased the inhibitor resistance to proteolysis without abolishing the inhibitory effect. Molecular dynamics simulations of the inhibitor series complexed with wild-type and mutated HIV-PR monomers corroborated the kinetic data. They suggested that the lipopeptide peptide moiety replaces the middle strand in the highly conserved intermolecular four-stranded beta-sheet formed by the peptide termini of each monomer, and the alkyl chain is tightly grasped by the active site groove capped by the beta-hairpin flap in a "superclosed" conformation. These new inhibitors were equally active in vitro against both wild-type and drug-resistant multimutated proteases, and the model suggested that the mutations in the monomer did not interfere with the inhibitor.

New constrained "molecular tongs" designed to dissociate HIV-1 protease dimer.

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.

Thyroxine-derivatives of lipopeptides: bifunctional dimerization inhibitors of human immunodeficiency virus-1 protease.

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 (K(id)=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 beta-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.