Sonia Colombo

Design, Synthesis and Biological Evaluation of Sugar-Derived Ras Inhibitors

The design and synthesis of novel Ras inhibitors with a bicyclic scaffold derived from the natural sugar D‐arabinose are presented. Molecular modelling showed that these ligands can bind Ras by accommodating the aromatic moieties and the phenylhydroxylamino group in a cavity near the Switch II region of the protein. All the synthetic compounds were active in inhibiting nucleotide exchange on p21 human Ras in vitro, and two of them selectively inhibited Ras‐dependent cell growth in vivo.

Characterization and Properties of Dominant-negative Mutants of the Ras-specific Guanine Nucleotide Exchange Factor CDC25Mm

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25Mm, are described. In vitro, the mutant GEFW1056E and GEFT1184Eproteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21 ras , and strongly reduce affinity of the nucleotide-free ras·GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras·GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEFW1056Emutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.

Molecular cloning, nucleotide sequence and expression of a Sulfolobus solfataricus gene encoding a class II fumarase

Fumarase catalyzes the interconversion of l‐malate and fumarate. A Sulfolobus solfataricus fumarase gene (fumC) was cloned and sequenced. Typical archaebacterial regulatory sites were identified in the region flanking the fumC open reading frame. The fumC gene encodes a protein of 438 amino acids (47,899 Da) which shows several significant similarities with class II fumarases from both eubacterial and eukariotic sources as well as with aspartases. S. solfataricus fumarase expressed in Escherichia coli retains enzymatic activity and its thermostability is comparable to that of S. solfataricus purified enzyme despite a 11 amino acid C‐terminal deletion.

The role of feedback control mechanisms on the establishment of oscillatory regimes in the Ras/cAMP/PKA pathway in S. cerevisiae

In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of cell growth and proliferation in response to nutritional sensing and stress conditions. The pathway is tightly regulated by multiple feedback loops, exerted by the protein kinase A (PKA) on a few pivotal components of the pathway. In this article, we investigate the dynamics of the second messenger cAMP by performing stochastic simulations and parameter sweep analysis of a mechanistic model of the Ras/cAMP/PKA pathway, to determine the effects that the modulation of these feedback mechanisms has on the establishment of stable oscillatory regimes. In particular, we start by studying the role of phosphodiesterases, the enzymes that catalyze the degradation of cAMP, which represent the major negative feedback in this pathway. Then, we show the results on cAMP oscillations when perturbing the amount of protein Cdc25 coupled with the alteration of the intracellular ratio of the guanine nucleotides (GTP/GDP), which are known to regulate the switch of the GTPase Ras protein. This multi-level regulation of the amplitude and frequency of oscillations in the Ras/cAMP/PKA pathway might act as a fine tuning mechanism for the downstream targets of PKA, as also recently evidenced by some experimental investigations on the nucleocytoplasmic shuttling of the transcription factor Msn2 in yeast cells.

Design, Synthesis, and Biological Evaluation of Levoglucosenone‐Derived Ras Activation Inhibitors

A panel of new potential Ras ligands was generated by decorating a tricyclic levoglucosenone‐derived scaffold with aromatic moieties. Some members of the panel show in vitro inhibitory activity toward the nucleotide exchange process on Ras and are toxic to some human cancer cell lines.

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.

Simulation of the Ras/cAMP/PKA pathway in budding yeast highlights the establishment of stable oscillatory states

In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway plays a major role in the regulation of metabolism, stress resistance and cell cycle progression. We extend here a mechanistic model of the Ras/cAMP/PKA pathway that we previously defined by describing the molecular interactions and post-translational modifications of proteins, and perform a computational analysis to investigate the dynamical behaviors of the components of this pathway, regulated by different control mechanisms. We carry out stochastic simulations to consider, in particular, the effect of the negative feedback loops on the activity of both Ira2 (a Ras-GAP) and Cdc25 (a Ras-GEF) proteins. Our results show that stable oscillatory regimes for the dynamics of cAMP can be obtained only through the activation of these feedback mechanisms, and when the amount of Cdc25 is within a specific range. In addition, we highlight that the levels of guanine nucleotides pools are able to regulate the pathway, by influencing the transition between stable steady states and oscillatory regimes.

Live-cell imaging of endogenous Ras-GTP shows predominant Ras activation at the plasma membrane and in the nucleus in Saccharomyces cerevisiae

Ras proteins function as a point of convergence for different signalling pathways in eukaryotes and are involved in many cellular responses; their different subcellular locations could regulate distinct functions. To investigate the localization of active Ras in vivo in Saccharomyces cerevisiae, we expressed a probe consisting of a GFP fusion with a trimeric Ras binding domain of Raf1 (eGFP-RBD3), which binds Ras-GTP with a much higher affinity than Ras-GDP. Our results show that in wild type cells active Ras accumulates mainly at the plasma membrane and in the nucleus during growth on medium containing glucose, while it accumulates mainly in mitochondria in wild type glucose-starved cells and relocalizes to the plasma membrane and to the nucleus upon addition of this sugar. A similar pattern is observed in a strain deleted in the CYR1 gene indicating that the absence of adenylate cyclase does not impair the localization of Ras-GTP. Remarkably, in a gpa2Δ, but not in a gpr1Δ mutant, active Ras accumulates in internal membranes and mitochondria, both when cells are growing on glucose medium or are starved, indicating that Gpa2, but not Gpr1 is required for the recruitment of Ras-GTP at the plasma membrane and in the nucleus. Moreover, deletion of both HXK1 and HXK2 also causes a mitochondrial localization of the probe, which relocalizes to the plasma membrane and to the nucleus upon expression of HXK2 on a centromeric plasmid, suggesting that this kinase is involved in the proper localization of active Ras.

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.

Evidence for adenylate cyclase as a scaffold protein for Ras2-Ira interaction in Saccharomyces cerevisie.

Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.