Pierangelo Francescato

Human endostatin-derived synthetic peptides possess potent antiangiogenic properties in vitro and in vivo

Pharmacological control of the angiogenic process (i.e., the neovascularization necessary for the growth and progression of tumors and metastases) is considered to be one of the most promising approaches to antineoplastic therapy. Endostatin, a 20-kDa protein derived from collagen XVIII, is one of the first recently discovered endogeneous antiangiogenic substances, but its cell targets and mechanism(s) of action are still unknown. We thought it would be interesting to test whether shorter peptides derived from endostatin might preserve its antiangiogenic activity. Four synthetic peptides corresponding to the sequences 6-49 (I), 50-92 (II), 93-133 (III), and 134-178 (IV) of human endostatin were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis. Fragment I (and fragment IV in the tests performed) was found to be fully biologically active in all of the angiogenesis assays, and sometimes showed even greater potency and efficacy than full-length human endostatin itself.

In silico design of tubulin-targeted antimitotic peptides

Microtubules are polymeric structures formed by the self-assembly of tubulin dimers. The growth and shrinkage of these dynamic arrays have a key role during the cell-proliferation process. This makes tubulin the molecular target of many anticancer drugs currently in use or under clinical trial. Their impressive success is limited by the onset of resistant tumour cells during the treatment, so new resistance-proof molecules need to be developed. Here we use molecular dynamics and free-energy calculations to study the network of interactions that allow microtubule formation. Modelling the protein-protein interface allows us to identify the amino acids responsible for tubulin-tubulin binding and thus to design peptides, which correspond to tubulin subsequences, that interfere with microtubule formation. We show that the application of molecular modelling techniques leads to the identification of peptides that exhibit antitubulin activity both in vitro and in cultured cells.

Local delivery of a synthetic endostatin fragment for the treatment of experimental gliomas

OBJECTIVE:Endostatin is an anti-angiogenic agent that blocks matrix-metalloproteinase-2 and inhibits endothelial cell proliferation. Currently, endostatin is available through recombinant technology, which limits its broader use. In this study, a synthetic endostatin fragment (EF) was analyzed to determine its anti-angiogenic properties when locally delivered by controlled-release polymers and to establish its effect as a treatment for experimental gliomas. METHODS:Cytotoxicity of EF against 9L gliosarcoma and F98 glioma was determined in vitro. EF was loaded into polyanhydride-poly-(bis-[carboxyphenoxy-propane]-sebacic-acid) (pCPP:SA) polymers at increasing concentrations. Pharmacokinetics of the EF/polymer formulations were defined in vitro. Anti-angiogenic properties of the EF/polymer formulations were evaluated in the rat-cornea micropocket assay. Toxicity and efficacy of locally delivered EF polymers either alone or combined with systemic bischloroethylnitrosourea (carmustine) were determined in rats intracranially challenged with 9L gliosarcoma. RESULTS:EF showed scarce cytotoxicity against 9L and F98 in vitro. EF/pCPP:SA formulations showed sustained release by day 19. Mean corneal angiogenesis index 20 days after tumor implantation was 4.5 ± 0.7 for corneas implanted with 40% EF/pCPP:SA compared with controls (8.5 ± 1.3, P = 0.02). Intracranial efficacy studies showed that EF polymers alone did not prolong animal survival. Combination of 40% EF/pCPP:SA polymers with systemic bischloroethylnitrosourea (carmustine) prolonged survival (median survival of 44 d, P = 0.001) and generated 33% long-term survivors. CONCLUSION:Controlled-release polymers can effectively deliver a biologically active EF in a sustained fashion. EF inhibits angiogenesis in vitro and in vivo, and even though EF does not prolong survival as a single agent, it exhibits a synergistic effect when combined with systemic bischloroethylnitrosourea (carmustine) in the intracranial 9L gliosarcoma model.

Computer aided design of FtsZ targeting oligopeptides

FtsZ is a protein involved in the bacterial division process and is thus an emerging target for antibacterial drugs. The network of interactions between FtsZ monomers necessary for exploitation of its biological function are studied here with molecular dynamics and free energy calculations. The results obtained led to the design of FtsZ targeting peptides which exhibited activity against the function of FtsZ in vitro.

New ACE-Inhibitory Peptides from Hemp Seed (Cannabis sativa L.) Proteins

A hemp seed protein isolate, prepared from defatted hemp seed meals by alkaline solubilization/acid precipitation, was subjected to extensive chemical hydrolysis under acid conditions (6 M HCl). The resulting hydrolysate was fractionated by semipreparative RP-HPLC, and the purified fractions were tested as inhibitors of angiotensin converting enzyme (ACE). Mono- and bidimensional NMR experiments and LC-MS analyses led to the identification of four potentially bioactive peptides, i.e. GVLY, IEE, LGV, and RVR. They were prepared by solid-phase synthesis, and tested for ACE-inhibitory activity. The IC50 values were GVLY 16 ± 1.5 μM, LGV 145 ± 13 μM, and RVR 526 ± 33 μM, confirming that hemp seed may be a valuable source of hypotensive peptides.

Purification, sequence determination and synthesis of seminal plasma peptides and synthesis of some of their analogues.

Three peptides were isolated from bovine seminal plasma and purified to homogeneity. The amino acid sequences, as determined by FAB mass spectrometry, are the following: pGlu‐Ala‐ Glu‐Ser‐Asn‐OH, pGlu‐Ala‐Glu‐Ser(PO3H2‐Asn‐OH and pGlu‐Val‐Gly‐Glu‐Ser‐Glu‐Asn‐OH. These three peptides and some of their analogues were synthesized using liquid‐ and solid‐phase techniques. The pentapeptide pGlu‐Ala‐Glu‐Ser‐Asn‐OH showed a remarkable affinity for kinase NII and a strong inhibiting activity in DNA transcription. These findings support the hypothesis that phosphorylated acidic domains of nuclear non‐histone proteins could bind to DNA, thereby controlling transcription.

Esterification of poly(γ-glutamic acid) (γ-PGA) mediated by its tetrabutylammonium salt

Poly(γ-glutamic acid) is a linear anionic biopolymer synthesized by bacterial fermentation from sustainable resources. Being water soluble, biodegradable, edible and non-toxic to humans and the environment, applications of γ-PGA are of interest in a broad range of industrial sectors. However, preparation of γ-PGA derivatives is plagued by several difficulties including its scarce solubility in organic solvents. We here report a γ-PGA derivatization procedure based on the use of its tetrabutylammonium salt. The modified solubility of γ-PGA provided by counterion exchange led to the synthesis of poly(α-ethyl-γ-glutamate), poly(α-benzyl-γ-glutamate) and poly(α-n-butyl-γ-glutamate) under smoother conditions and an almost complete functionalization degree.