Cristina Visintin

Aurora A and B kinases as targets for cancer: will they be selective for tumors?

Aurora A and B kinases are closely related kinases involved in regulating separate points in the cell cycle. This review highlights the rationale for Aurora kinases as cancer targets and examines the currently known Aurora kinase inhibitors in the patent and scientific literature. The known crystal structures of the Aurora kinases are described with relevance to bound ligand interactions and the prospect of the generation of drug-resistant mutant forms. The potential for selectivity versus primary cells will also be discussed. The status of the inhibitors in clinical development is described.

Small-molecule mimics of an alpha-helix for efficient transport of proteins into cells.

We designed and synthesized small-molecule mimics of an alpha-helical peptide protein transduction domain (PTD). These small-molecule carriers, which we termed SMoCs, are easily coupled to biomolecules, and efficiently deliver dye molecules and recombinant proteins into a variety of cell types. We designed the SMoCs using molecular modeling techniques. As an example of a protein cargo, we applied this new technology to the internalization of the DNA replication licensing repressor geminin, in vitro, providing evidence that extracellularly delivered SMoC-geminin can have an antiproliferative effect on human cancer cells. Uptake of SMoC-geminin was inhibited at 4 °C and by chlorpromazine, a compound that induces misassembly of clathrin-coated pits at the cell surface. Thus the mechanism of uptake is likely to be clathrin-mediated endocytosis.

New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis

We previously reported that the compound O‐2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re‐examined the activity of O‐2093 in vivo and synthesized four structural analogs (O‐2247, O‐2248, O‐3246, and O‐3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB1 and CB2 receptors; (b) assays of transient receptor potential of the vanilloid type‐1 (TRPV1) channel functional activity (measurement of [Ca2+]i); (c) [14C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL‐2H3) cells; (d) the mouse ‘tetrad’ tests (analgesia on a hot plate, immobility on a ‘ring’, rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O‐2093, either synthesized by us or commercially available, was inactive in the ‘tetrad’ up to a 20 mg kg−1 dose (i.v.). Like O‐2093, the other four compounds exhibited low affinity in CB1 (Ki from 1.3 to >10 μM) and CB2 binding assays (1.310 μM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC50>25 μM) and were inactive in the ‘tetrad’ up to a 30 mg kg−1 dose (i.v.). While O‐2247 and O‐2248 were poor inhibitors of [14C]AEA cellular uptake (IC50>40 μM), O‐3246 and O‐3262 were quite potent in this assay. O‐3246, which exhibits only a very subtle structural difference with O‐2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC50=1.4 μM) and is 12‐fold more potent than O‐2093. When injected intravenously O‐3246 and O‐3262, again like O‐2093 and unlike O‐2247 and O‐2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective AEA uptake inhibitors as anti‐spasticity drugs in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake, support further the existence of a specific mechanism for this process.

Synergy between Natriuretic Peptides and Phosphodiesterase 5 Inhibitors Ameliorates Pulmonary Arterial Hypertension

RATIONALE Phosphodiesterase 5 (PDE5) inhibitors (e.g., sildenafil) are selective pulmonary vasodilators in patients with pulmonary arterial hypertension. The mechanism(s) underlying this specificity remains unclear, but studies in genetically modified animals suggest it might be dependent on natriuretic peptide bioactivity. OBJECTIVES We explored the interaction between PDE5 inhibitors and the natriuretic peptide system to elucidate the (patho)physiological relationship between these two cyclic GMP (cGMP)-regulating systems and potential of a combination therapy exploiting these cooperative pathways. METHODS Pharmacological evaluation of vascular reactivity was conducted in rat isolated conduit and resistance vessels from the pulmonary and systemic circulation in vitro, and in anesthetized mice in vivo. Parallel studies were undertaken in an animal model of hypoxia-induced pulmonary hypertension (PH). MEASUREMENTS AND MAIN RESULTS Sildenafil augments vasodilatation to nitric oxide (NO) in pulmonary and systemic conduit and resistance arteries, whereas identical vasorelaxant responses to atrial natriuretic peptide (ANP) are enhanced only in pulmonary vessels. This differential activity is mirrored in vivo where sildenafil increases the hypotensive actions of ANP in the pulmonary, but not systemic, vasculature. In hypoxia-induced PH, combination of sildenafil plus the neutral endopeptidase (NEP) inhibitor ecadotril (which increases endogenous natriuretic peptide levels) acts synergistically, in a cGMP-dependent manner, to reduce many indices of disease severity without significantly affecting systemic blood pressure. CONCLUSIONS These data demonstrate that PDE5 is a key regulator of cGMP-mediated vasodilation by ANP in the pulmonary, but not systemic, vasculature, thereby explaining the pulmonary selectivity of PDE5 inhibitors. Exploitation of this mechanism (i.e., PDE5 and neutral endopeptidase inhibition) represents a novel, orally active combination therapy for pulmonary arterial hypertension.

Oxadiazolylindazole sodium channel modulators are neuroprotective toward hippocampal neurones.

We report the discovery of a new class of neuroprotective voltage-dependent sodium channel modulators exemplified by (5-(1-benzyl-1H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)methanamine 11 (CFM1178). The compounds were inhibitors of [(14)C]guanidinium ion flux in rat forebrain synaptosomes and displaced binding of the sodium channel ligand [(3)H]BW202W92. 11 and the corresponding N(2)-benzyl isomer, 38 (CFM6058), demonstrated neuroprotective activity in hippocampal slices comparable to sipatrigine. CYP450 enzyme inhibition observed with 11 was reduced with 38. In electrophysiological experiments on dissociated hippocampal neurons, these two compounds caused use- and voltage-dependent block of sodium currents. Sodium channel isoform profiling against Na(v)1.1-1.8 demonstrated that the standard sodium channel blocker lamotrigine had modest activity against Na(v)1.1, while sipatrigine was generally more potent and less selective. 11 and 38 showed potent activity against Na(v)1.6, pointing to pharmacological block of this isoform being consistent with the neuroprotective effect. 38 also showed use dependent block of Na(v)1.6 in HEK cells.

Vascular pharmacology of a novel cannabinoid-like compound, 3-(5-dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide (VSN16) in the rat

A putative novel cannabinoid receptor mediates vasorelaxation to anandamide and abnormal‐cannabidiol and is blocked by O‐1918 and by high concentrations of rimonabant. This study investigates VSN16, a novel water‐soluble agonist, as a vasorelaxant potentially acting at non‐CB1, non‐CB2 cannabinoid receptors in the vasculature.

Control of spasticity in a multiple sclerosis model using central nervous system-excluded CB1 cannabinoid receptor agonists

The purpose of this study was the generation of central nervous system (CNS)‐excluded cannabinoid receptor agonists to test the hypothesis that inhibition of spasticity, due to CNS autoimmunity, could be controlled by affecting neurotransmission within the periphery. Procedures included identification of chemicals and modeling to predict the mode of exclusion; induction and control of spasticity in the ABH mouse model of multiple sclerosis; conditional deletion of CB1 receptor in peripheral nerves; side‐effect profiling to demonstrate the mechanism of CNS‐exclusion via drug pumps; genome‐wide association study in N2(129×ABH) backcross to map polymorphic cannabinoid drug pump; and sequencing and detection of cannabinoid drug‐pump activity in human brain endothelial cell lines. Three drugs (CT3, SAB378 and SAD448) were identified that control spasticity via action on the peripheral nerve CB1 receptor. These were peripherally restricted via drug pumps that limit the CNS side effects (hypothermia) of cannabinoids to increase the therapeutic window. A cannabinoid drug pump is polymorphic and functionally lacking in many laboratory (C57BL/6, 129, CD‐1) mice used for transgenesis, pharmacology, and toxicology studies. This phenotype was mapped and controlled by 1–3 geneticloci. ABCC1 within a cluster showing linkage is a cannabinoid CNS‐drug pump. Global and conditional CB1 receptor‐knockout mice were used as controls. In summary, CNS‐excluded CB1 receptor agonists are a novel class of therapeutic agent for spasticity.—Pryce, G., Visintin, C., Ramagopalan, S. V., Al‐Izki, S., De Faveri, L. E., Nuamah, R. A., Mein, C. A., Montpetit, A., Hardcastle, A. J., Kooij, G., de Vries, H. E., Amor, S., Thomas, S. A., Ledent, C., Marsicano, G., Lutz, B., Thompson, A. J., Selwood, D. L., Giovannoni, G., Baker, D. Control of spasticity in a multiple sclerosis model using central nervous system‐excluded CB1 cannabinoid receptor agonists. FASEB J. 28, 117–130 (2014). www.fasebj.org

Modular Assembly Using Sequential Palladium Coupling Gives Easy Access to the SMoC Class of Cellular Transporters

The transducing ability of the third helix of transcription factor homeodomains is effectively mimicked by a biphenyl system displaying guanidine groups. The biphenyl class of small molecule carriers (SMoCs) can carry biomolecules into a wide variety of cell types. A “combinatorial” approach to the synthesis of SMoCs is described using sequential Pd0 coupling chemistry to assemble the molecules from highly functionalized building blocks. SMoCs coupled to the DNA licensing repressor protein geminin can inhibit DNA replication in vitro. We conducted a structure–activity investigation utilizing a range of SMoC–geminin conjugates and demonstrate that both electrostatic and structural features are important for efficient uptake and functional activity. The best analogue was more efficient than either (Arg)4 or (Arg)8 linked to geminin. Effective inhibition of DNA synthesis was achieved in fibroblasts and osteosarcoma cell lines.

Exploring the interaction between siRNA and the SMoC biomolecule transporters: Implications for small molecule-mediated delivery of siRNA

The small molecule carrier class of biomolecule transporters, modeled on the third helix of the Antennapedia homeodomain, has previously been shown to transport active proteins into cells. Here, we show an improved synthetic route to small molecule carriers, including Molander chemistry using trifluoroborate salts to improve the yield of the Suzuki–Miyaura coupling step for the formation of the biphenyl backbone. The required boronic acids could be formed by the reaction of a 2‐(dimethylamino)ethyl ether‐modified aryl Grignard reagent with triisopropyl borate. The potential for the use of small molecule carriers as oligonucleotide‐transporting agents was also explored by characterizing the interactions between small molecule carriers and siRNA. Molecular dynamics and NMR analysis indicated that the small molecule carrier guanidines are stabilized by π‐cation interactions with the biphenyl system, thus not only increasing the basicity or pKa but also shielding the charge. The binding affinities of various small molecule carriers for siRNA were investigated using isothermal calorimetry and gel shift assays. Small molecule carrier‐mediated siRNA delivery to cultured fibroblasts is demonstrated, showing that small molecule carriers possess the ability to transport functional siRNA into cells. Knockdown of Cdc7 kinase, a target for cancer, is achieved.

Big conductance calcium-activated potassium channel openers control spasticity without sedation.

Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti‐metabolite approach to identify drugs that target spasticity.