Alessandro Palmioli

First experimental identification of Ras-inhibitor binding interface using a water-soluble Ras ligand

By combining in the same molecule Ras-interacting aromatic moieties and a sugar, we prepared a water-soluble Ras ligand that binds Ras and inhibits guanine nucleotide exchange. With this compound it was possible to determine experimentally by a (15)N-edited HSQC NMR experiment the ligand-Ras binding interface.

Selective cytotoxicity of a bicyclic Ras inhibitor in cancer cells expressing K-RasG13D

Mutation of RAS genes is a critical event in the pathogenesis of different human tumors and in some developmental disorders. Here we present an arabinose-derived bicyclic compound displaying selective cytotoxicity in human colorectal cancer cells expressing K-Ras(G13D), that shows high intrinsic nucleotide exchange rate. We characterize binding of bicyclic compounds by docking and NMR experiments and their inhibitory activity on GEF-mediated nucleotide exchange on wild-type and mutant Ras proteins. We demonstrate that the in vitro inhibition of Ras nucleotide exchange depends on the molar ratio between Ras and its GEF activator, suggesting that the observed in vivo selective effect may depend on biochemical parameters and actual intracellular concentration of the Ras protein and its regulators.

Glucose-derived Ras pathway inhibitors: evidence of Ras-ligand binding and Ras-GEF (Cdc25) interaction inhibition.

Ras proteins act as molecular switches by interconverting between the inactive (GDP-bound) and the active (GTP-bound) states. The conversion of Ras-GDP to Ras-GTP is mediated by the direct interaction of Ras with a family of guanine nucleotide exchange factors (GEFs) that include Sos proteins, RasGRF/Cdc25 and GRP/Cal-DAG-GEF proteins. The RasGTP complex can activate different downstream effectors, the best known of which is the Raf kinase/ErkMAPK cascade that leads to the known cellular effects of Ras proteins, such as ACHTUNGTRENNUNGactivation of cellular growth and proliferation. Up to 30% of human tumors carry a mutated Ras oncogene that encodes a protein whose activation–deactivation cycle is impaired. As a result, Ras is found constitutively in the active (GTP-bound) form and thus causes uncontrolled cellular growth and leads to oncogenesis. 6] With the purpose of developing new selective antitumor drugs, we have synthesized compounds 1–8 derived from the natural sugar d-arabinose (Scheme 1). These compounds were tested in vitro in a quantitative biochemical assay by using human Ras protein (p21) and fluorescently labeled nucleotides. They were shown to be active as inhibitors of the GDP/ GTP nucleotide exchange with a potency in the micromolar range. The same molecules inhibited, in a concentration dependent manner, the growth and proliferation of k-Ras transformed mammalian NIH3T3 cells. NMR binding studies (STD, trNOE) showed that both the benzyl and the phenylhydroxylamine moieties of the inhibitors directly interact with Ras. These finding were confirmed by the observation that molecules lacking either of these groups were totally inactive in the inhibition of nucleotide exchange in Ras. We introduce here monosaccharides 9 and 10 (Scheme 1) that bear the functional groups involved in Ras binding, namely the thiophenyl and the phenyl hydroxylamine groups, linked to a glucopyranose scaffold. The use of glucose as scaffold in the preparation of bioactive and pharmacologically active compounds has known wide application. In order to increase the water solubility of glucose derivatives we planned to link Rasbinding groups in the C1 and C6 positions, and to keep the C2, C3 and C4 hydroxyl groups of the sugar unprotected. The design of these novel compounds was guided by the necessity to present the aromatic moieties with the optimal geometry to bind Ras. Preliminary docking studies showed that both 9 and 10 bound Ras in a similar orientation to that Scheme 1. d-Arabinose-derived Ras inhibitors 1–8 and d-glucose-derived 9 and 10.

Binding properties and biological characterization of new sugar-derived Ras ligands

Since mutations of Ras genes have a great incidence in human tumours, Ras oncoproteins are a major clinical target for the development of anticancer agents. We have developed synthetic molecules able to inhibit Ras activation. Here we present new, water-soluble Ras inhibitors composed by an aromatic pharmacofore moiety covalently linked to different sugars. New glycosylated compounds bind to Switch 2 region of Ras, also involved in effector binding, inhibit GEF-catalyzed nucleotide exchange on Ras in vitro, and reduce Ras-dependent proliferation of murine fibroblasts. The influence of the sugar unit on Ras binding affinity and on the biological activity of Ras inhibitors has been investigated.

Structure-Activity Studies on Arylamides and Arysulfonamides Ras Inhibitors

This paper reports the synthesis of a panel of small molecules with arylamides and arylsulfonamides groups and their biological activity in inhibiting nucleotide exchange on human Ras. The design of these molecules was guided by experimental and molecular modelling data previously collected on similar compounds. Aim of this work is the validation of the hypothesis that a phenyl hydroxylamine group linked to a second aromatic moiety generates a pharmacophore capable to interact with Ras and to inhibit its activation. In vitro experiments on purified human Ras clearly show that the presence of an aromatic hydroxylamine and a sulfonamide group in the same molecule is a necessary condition for Ras binding and nucleotide exchange inhibition. The inhibitor potency is lower in molecules in which either the hydroxylamine has been replaced by other functional groups or the sulfonamide has been replaced by an amide. In the case both these moieties, the hydroxylamine and sulfonamide are absent, inactive compounds are obtained.

NMR-driven identification of anti-amyloidogenic compounds in green and roasted coffee extracts

To identify food and beverages that provide the regular intake of natural compounds capable of interfering with toxic amyloidogenic aggregates, we developed an experimental protocol that combines NMR spectroscopy and atomic force microscopy, in vitro biochemical and cell assays to detect anti-Aβ molecules in natural edible matrices. We applied this approach to investigate the potential anti-amyloidogenic properties of coffee and its molecular constituents. Our data showed that green and roasted coffee extracts and their main components, 5-O-caffeoylquinic acid and melanoidins, can hinder Aβ on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Coffee extracts and melanoidins also counteract hydrogen peroxide- and rotenone-induced cytotoxicity and modulate some autophagic pathways in the same cell line.

Glycofunctionalization of Poly(lactic-co-glycolic acid) Polymers: Building Blocks for the Generation of Defined Sugar-Coated Nanoparticles

A set of poly(lactic- co-glycolic acid) polymers functionalized with different monosaccharides as well as glycodendrimers and surface-decorated nanoparticles (NPs) were synthesized and characterized. The functionalization of the polymer was carried out through amide bond formation with amino-modified sugar monomers and through a biocompatible chemoselective method exploiting the reducing end of a free sugar. The assemblage of the NPs adopting a nanoprecipitation method was straightforward and allowed the preparation of sugars/sugar dendrimer coated NPs.

bioNMR-based identification of natural anti-Aβ compounds in Peucedanum ostruthium

The growing interest in medicinal plants for the identification of new bioactive compounds and the formulation of new nutraceuticals and drugs prompted us to develop a powerful experimental approach allowing the detailed metabolic profiling of complex plant extracts, the identification of ligands of macromolecular targets of biomedical relevance and a preliminary characterization of their biological activity. To this end, we selected Peucedanum ostruthium, a plant traditionally employed in Austria and Italy for its several potential therapeutic applications, as case study. We combined the use of NMR and UPLC-HR-MS for the identification of the metabolites present in its leaves and rhizome extracts. Due to the significant content of polyphenols, particularly chlorogenic acids, recently identified as anti-amyloidogenic compounds, polyphenols-enriched fractions were prepared and tested for their ability to prevent Aβ1-42 peptide aggregation and neurotoxicity in a neuronal human cell line. STD-NMR experiments allowed the detailed identification of Aβ oligomers ligands responsible for the anti-amyloidogenic activity. These data provide experimental protocols and structural information suitable for the development of innovative molecular tools for prevention, therapy and diagnosis of Alzheimers disease.

Flavonoids in the Treatment of Alzheimer’s and Other Neurodegenerative Diseases

Flavonoids are phytochemicals present in almost all terrestrial plants and, as a consequence, in plant-based foods, and thus consumed by humans through diet. Recent evidences suggest that several flavonoids have positive effects against dementia and Alzheimers disease, reversing age-related declines in neurocognitive performances. In this review, we provide a general classification of natural and synthetic flavonoids, a description of their physico-chemical properties, in particular their redox properties and stability, and an extensive overview about their biological activities and structure-activity relationship in the field of neurodegenerative diseases. In addition, a section will be dedicated to the synthetic strategies for the preparation of bioactive derivatives. This information will be essential for the design and development of new drugs that can improve brain functions.