Structural and biochemical evaluation of bisubstrate inhibitors of protein arginine N-methyltransferases PRMT1 and CARM1 (PRMT4).

Attenuating the function of protein arginine methyltransferases (PRMTs) is an objective for the investigation and treatment of several diseases including cardiovascular disease and cancer. Bisubstrate inhibitors that simultaneously target binding sites for arginine substrate and the cofactor (S-adenosylmethionine (SAM)) have potential utility, but structural information on their binding is required for their development. Evaluation of bisubstrate inhibitors featuring an isosteric guanidine replacement with two prominent enzymes PRMT1 and CARM1 (PRMT4) by isothermal titration calorimetry (ITC), activity assays and crystallography are reported. Key findings are that 2-aminopyridine is a viable replacement for guanidine, providing an inhibitor that binds more strongly to CARM1 than PRMT1. Moreover, a residue around the active site that differs between CARM1 (Asn-265) and PRMT1 (Tyr-160) is identified that affects the side chain conformation of the catalytically important neighbouring glutamate in the crystal structures. Mutagenesis data supports its contribution to the difference in binding observed for this inhibitor. Structures of CARM1 in complex with a range of seven inhibitors reveal the binding modes and show that inhibitors with an amino acid terminus adopt a single conformation whereas the electron density for equivalent amine-bearing inhibitors is consistent with preferential binding in two conformations. These findings inform the molecular basis of CARM1 ligand binding and identify differences between CARM1 and PRMT1 that can inform drug discovery efforts.

Iron-Catalyzed Indolizine Synthesis from Pyridines, Diazo Compounds, and Alkynes.

The iron(III)-catalyzed synthesis of indolizines from commercially available alkyne, pyridine, and diazo precursors is reported. This mild, expedient method is tolerant of various solvents and proceeds with as little as 0.25 mol % [Fe(TPP)Cl]. Significantly, this multicomponent reaction is compatible with electrophilic alkynes; control experiments demonstrate the importance of the catalyst in promoting pyridinium ylide formation over background reactivity.

Stereoselective Synthesis of Tetrahydroindolizines through the Catalytic Formation of Pyridinium Ylides from Diazo Compounds.

Commercially available iron(III) and copper(I) complexes catalyzed multicomponent cycloaddition reactions between diazo compounds, pyridines, and electrophilic alkenes to give alkaloid-inspired tetrahydroindolizidines in high yield with high diastereoselectivity. Hitherto, the catalytic formation of versatile pyridinium ylides from metal carbenes has been poorly developed; the broad utility demonstrated herein sets the stage for the invention of further multicomponent reactions in future.

Calculating singlet excited states: Comparison with fast time-resolved infrared spectroscopy of coumarins.

In contrast to the ground state, the calculation of the infrared (IR) spectroscopy of molecular singlet excited states represents a substantial challenge. Here, we use the structural IR fingerprint of the singlet excited states of a range of coumarin dyes to assess the accuracy of density functional theory based methods for the calculation of excited state IR spectroscopy. It is shown that excited state Kohn-Sham density functional theory provides a high level of accuracy and represents an alternative approach to time-dependent density functional theory for simulating the IR spectroscopy of singlet excited states.

Alkaloid inspired spirocyclic oxindoles from 1,3-dipolar cycloaddition of pyridinium ylides.

Cycloaddition reactions between pyridinium ylides and 3-alkenyl oxindoles that proceed in high yield and with very good regio- and diastereoselectivity are reported. The resulting cycloadducts have the same stereochemistry of biologically active oxindole alkaloids, such as strychnofoline.

An unusual silicon mediated transannular cyclopropanation.

A silicon mediated intramolecular 1,4-conjugate addition of a homoallylic carbon nucleophile leading to cyclopropanation is reported. Specifically, treatment of 6-trimethylsilyl-5,6-dihydroazocinones with fluoride gives 4-azabicyclo(5.1.0)octenones, presenting an unusual extension to the repertoire of silyl group reactivity.

Small molecule inhibitors that discriminate between protein arginine N-methyltransferases PRMT1 and CARM1.

Protein arginine N-methyltransferases (PRMTs) selectively replace N-H for N-CH(3) at substrate protein guanidines, a post-translational modification important for a range of biological processes, such as epigenetic regulation, signal transduction and cancer progression. Selective chemical probes are required to establish the dynamic function of individual PRMTs. Herein, model inhibitors designed to occupy PRMT binding sites for an arginine substrate and S-adenosylmethionine (AdoMet) co-factor are described. Expedient access to such compounds by modular synthesis is detailed. Remarkably, biological evaluation revealed some compounds to be potent inhibitors of PRMT1, but inactive against CARM1. Docking studies show how prototype compounds may occupy the binding sites for a co-factor and arginine substrate. Overlay of PRMT1 and CARM1 binding sites suggest a difference in a single amino acid that may be responsible for the observed selectivity.

Toward the development of potent and selective bisubstrate inhibitors of protein arginine methyltransferases.

Prototype inhibitors of protein arginine methyltransferases (PRMTs) have been constructed by attaching guanidine functionality via a variable linker to non-reactive amine analogues of the cellular co-factor (S)-adenosyl methionine (AdoMet). Potent inhibition of PRMT1 (IC(50) of approximately 3-6 microM) combined with weak inhibition of the lysine methyltransferase SET7 (approximately 50% of activity at 100 microM) was observed for two such compounds.

NAADP-mediated Ca2+ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist.

The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca(2+) signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca(2+) entry. BZ194 specifically and effectively blocked NAADP-stimulated [(3)H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca(2+) mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca(2+) mobilization, such as nuclear translocation of "nuclear factor of activated T cells" (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4(+) effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases.

Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2+ signalling in intact cells.

NAADP (nicotinic acid adenine dinucleotide phosphate) is a recently discovered second messenger, and as such, we have much yet to learn about its functions in health and disease. A bottleneck in this basic research is due to NAADP, like all second messengers, being charged to prevent it from leaking out of cells. This makes for effective biology, but imposes difficulties in experiments, as it must be injected, loaded via liposomes, or electroporated, techniques that are highly technically demanding and are possible only in certain single cell preparations. For the better understood second messenger inositol 1,4,5-trisphosphate, great success has been obtained with cell-permeant derivatives where the charged groups are masked through esterification. We now report NAADP-AM as a cell-permeant analogue of NAADP that is taken up into cells and induces NAADP-mediated Ca(2+) signalling. NAADP-AM is a powerful chemical tool that will be of enormous biological utility in a wide range of systems and will greatly facilitate research into the role of NAADP in health and disease.

Novel octavalent cross-linker displays efficient trapping of protein-protein interactions.

A novel octavalent, resorcin[4]arene derived, cross-linker designed to overcome some of the limitations of commercially available reagents is significantly more efficient for covalent stabilisation of protein-protein interactions.

Chemo-enzymatic synthesis and biological evaluation of photolabile nicotinic acid adenine dinuclotide phosphate (NAADP+).

A chemo-enzymatic synthesis of novel caged NAADP+ without the formation of multiple cage compounds has been achieved. The biological activity of the caged NAADP+ was demonstrated by its fast uncaging in intact sea-urchin eggs.

Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis.

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.

Cell-permeant small-molecule modulators of NAADP-mediated Ca2+ release.

Nicotinic acid adenine dinucleotide phosphate (NAADP, 1) is the most potent intracellular Ca2+ mobilizing agent in important mammalian cells and tissues, yet the identity of the NAADP receptor is elusive. Significantly, the coenzyme NADP is completely inactive in this respect. Current studies are restricted by the paucity of any chemical probes beyond NAADP itself, and importantly, none is cell permeant. We report simple nicotinic acid-derived pyridinium analogs as low molecular weight compounds that (1) inhibit Ca2+ release via the NAADP receptor (IC50 approximately 15 microM - 1 mM), (2) compete with NAADP binding, (3) cross the cell membrane of sea urchin eggs to inhibit NAADP-evoked Ca2+ release, and (4) selectively ablate NAADP-dependent Ca2+ oscillations induced by the external gastric peptide hormone agonist cholecystokinin (CCK) in murine pancreatic acinar cells.

Caged-iron chelators a novel approach towards protecting skin cells against UVA-induced necrotic cell death.

Exposure of human skin cells to solar UVA radiation leads to an immediate dose-dependent increase of labile iron that subsequently promotes oxidative damage and necrotic cell death. Strong iron chelators have been shown to suppress cell damage and necrotic cell death by moderating the amount of labile iron pool (LIP), but chronic use would cause severe side effects owing to systemic iron depletion. Prodrugs that become activated in skin cells at physiologically relevant doses of UVA, such as "caged-iron chelators", may provide dose- and context-dependent release. Herein, we describe prototypical iron chelator compounds derived from salicylaldehyde isonicotinoyl hydrazone and pyridoxal isonicotinoyl hydrazone and demonstrate that the intracellular LIP and subsequent necrotic cell death of human skin fibroblasts is significantly decreased upon exposure to a combination of the prototypical compounds and physiologically relevant UVA doses. Iron regulatory protein bandshift and calcein fluorescence assays reveal decreased intracellular LIP following irradiation of caged-chelator-treated cells, but not in control samples where either UVA light, or caged-chelator is absent. Furthermore, flow cytometry shows that these compounds have no significant toxicity in the skin fibroblasts. This novel light-activated prodrug strategy may therefore be used to protect skin cells against the deleterious effects of sunlight.

Chemical synthesis of the novel Ca2+ messenger NAADP.

The first total chemical synthesis of nicotinamide adenine dinucleotide phosphate (beta-NADP, 2) as a single isomer was achieved. This was subsequently converted into the important second messenger nicotinic acid adenine dinucleotide phosphate (p-NAADP) 1 and the identity of this material confirmed by biological evaluation. This flexible synthetic route offers new opportunities for the generation of NAADP 1 analogues that cannot be generated directly from NADP 2 or mainly enzymatic methods.

Therapeutic potential of phosphoinositide 3-kinase inhibitors.

At least one Holy Grail for many academic researchers and pharmaceutical research divisions alike has been to identify therapeutically useful selective PI3K inhibitors. There are several different but closely related PI3Ks which are thought to have distinct biological roles. Until now, however, researchers have been frustrated by poor selectivity of the available pharmacological inhibitors, which are unable to distinguish the different isoforms of PI3K adequately. Fortunately, recently published work gives cause for optimism; there are now several patent specifications published that describe new PI3K inhibitors, including some that are more selective for the delta isoform of PI3K. Given the involvement of PI3Ks in a plethora of biological settings, such isoform-selective inhibitors may have immense potential use for the treatment of patients with inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases.

Development of high-affinity ligands and photoaffinity labels for the D-fructose transporter GLUT5.

The GLUT5 transporter catalyses the specific uptake of D-fructose and can accept this hexose in its furanose and pyranose ring forms. The transporter does not accept fructose epimers and has very limited tolerance of bulky groups substituted at the 2-, 3-, 4- and 5-OH positions [Tatibouët, Yang, Morin and Holman (2000) Bioorg. Med. Chem. 8, 1825-1833]. To further explore whether bulky groups can be tolerated at the primary OH positions, a D-fructose analogue with an allylamine group substitution to replace the 1-OH group was synthesized and was found to be quite well tolerated ( K (i)=27.1 mM). However, this analogue occurs in multiple ring forms. By contrast, 2,5-anhydro-D-mannitol is a symmetrical molecule that occurs only in a furanose ring form in which C-1 and C-6 are equivalent. We have therefore synthesized new 2,5-anhydro-D-mannitol analogues (substituted at the equivalent of the 6-OH of D-fructose) and from studies in Chinese hamster ovary cells expressing GLUT5 cells report that (i) the allylamine derivative of 2,5-anhydro-D-mannitol is well tolerated ( K (i)=2.66 mM); (ii) introduction of a di-nitrophenyl-substituted secondary amine group enhances affinity ( K (i)=0.56 mM); (iii) introduction of amide-linked biotinylated photolabel moieties is possible without loss of affinity relative to 2,5-anhydro-D-mannitol but a small secondary amine spacer between the biotinylated photolabelling moiety and the fructofuranose ring increases affinity (fructose photolabel 2; K (i)=1.16 mM); (iv) introduction of a hydrophilic tartarate spacer between biotin and the diazirine photoreactive groups can be accomplished without reduction in affinity and (v) photoactivation of biotinylated fructose photolabels leads to specific biotin tagging of GLUT5. These data suggest that substitution of a secondary amine group (-NH) to replace the C-6 (or C-1) -OH of 2,5-anhydro-D-mannitol results in compounds of high affinity; the affinity is enhanced over 10-fold compared with D-fructose.