Christelle Moreau

Rhodium-catalysed aryl transfer to aldehydes: counterion effects with nitrogen containing ligands

Abstract Cationic rhodium complexes of certain nitrogen-containing ligands exhibit excellent catalytic activity in the addition of arylboronic acids to aldehydes. The counterion had a notable effect with the weakly coordinating carborane anion affording a highly active catalyst.

CATALYTIC ASYMMETRIC HYDROSILYLATION OF KETONES USING MIXED-LIGAND RUTHENIUM COMPLEXES

An efficient ruthenium catalysed asymmetric hydrosilylation of ketones has been developed using high-throughput, parallel screening to optimise ligand combinations.

Structure-activity relationship of adenosine 5'-diphosphoribose at the transient receptor potential melastatin 2 (TRPM2) channel: rational design of antagonists.

Adenosine 5′-diphosphoribose (ADPR) activates TRPM2, a Ca2+, Na+, and K+ permeable cation channel. Activation is induced by ADPR binding to the cytosolic C-terminal NudT9-homology domain. To generate the first structure–activity relationship, systematically modified ADPR analogues were designed, synthesized, and evaluated as antagonists using patch-clamp experiments in HEK293 cells overexpressing human TRPM2. Compounds with a purine C8 substituent show antagonist activity, and an 8-phenyl substitution (8-Ph-ADPR, 5) is very effective. Modification of the terminal ribose results in a weak antagonist, whereas its removal abolishes activity. An antagonist based upon a hybrid structure, 8-phenyl-2′-deoxy-ADPR (86, IC50 = 3 μM), is more potent than 8-Ph-ADPR (5). Initial bioisosteric replacement of the pyrophosphate linkage abolishes activity, but replacement of the pyrophosphate and the terminal ribose by a sulfamate-based group leads to a weak antagonist, a lead to more drug-like analogues. 8-Ph-ADPR (5) inhibits Ca2+ signalling and chemotaxis in human neutrophils, illustrating the potential for pharmacological intervention at TRPM2.

Catalysis-associated Conformational Changes Revealed by Human CD38 Complexed with a Non-hydrolyzable Substrate Analog

Cyclic ADP-ribose (cADPR) is a calcium mobilization messenger important for mediating a wide range of physiological functions. The endogenous levels of cADPR in mammalian tissues are primarily controlled by CD38, a multifunctional enzyme capable of both synthesizing and hydrolyzing cADPR. In this study, a novel non-hydrolyzable analog of cADPR, N1-cIDPR (N1-cyclic inosine diphosphate ribose), was utilized to elucidate the structural determinants involved in the hydrolysis of cADPR. N1-cIDPR inhibits CD38-catalyzed cADPR hydrolysis with an IC50 of 0.26 mm. N1-cIDPR forms a complex with CD38 or its inactive mutant in which the catalytic residue Glu-226 is mutated. Both complexes have been determined by x-ray crystallography at 1.7 and 1.76 Å resolution, respectively. The results show that N1-cIDPR forms two hydrogen bonds (2.61 and 2.64Å) with Glu-226, confirming our previously proposed model for cADPR catalysis. Structural analyses reveal that both the enzyme and substrate cADPR undergo catalysis-associated conformational changes. From the enzyme side, residues Glu-146, Asp-147, and Trp-125 work collaboratively to facilitate the formation of the Michaelis complex. From the substrate side, cADPR is found to change its conformation to fit into the active site until it reaches the catalytic residue. The binary CD38-cADPR model described here represents the most detailed description of the CD38-catalyzed hydrolysis of cADPR at atomic resolution. Our structural model should provide insights into the design of effective cADPR analogs.

A concise route to tiazofurin

Abstract Successful oxidation of a key thiazoline intermediate allows an efficient synthesis of tiazofurin in four steps from commercially available 1′-acetoxy-2′,3′,5′-tri-O-benzoyl-β- d -ribofuranose.

8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist.

cADPR (cyclic ADP-ribose) is a universal Ca2+ mobilizing second messenger. In T-cells cADPR is involved in sustained Ca2+ release and also in Ca2+ entry. Potential mechanisms for the latter include either capacitative Ca2+ entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N1-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N1-cIDPR evoked Ca2+ signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca2+ signalling induced by 8-Br-N1-cIDPR consisted of Ca2+ release and Ca2+ entry. Whereas Ca2+ release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca2+ entry was inhibited by the Ca2+ entry blockers Gd3+ (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca2+ entry evoked by 8-Br-N1-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H2O2 was enhanced dramatically in those cells, Ca2+ signalling induced by 8-Br-N1-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N1-cIDPR. In summary, the sensitivity to the Ca2+ entry blockers Gd3+ and SKF-96365 is in favour of the concept of capacitative Ca2+ entry, secondary to store depletion by 8-Br-N1-cIDPR. Taken together, 8-Br-N1-cIDPR appears to be the first cADPR agonist affecting Ca2+ release and secondary Ca2+ entry, but without effect on TRPM2.

CD38 Structure-Based Inhibitor Design Using the N1-Cyclic Inosine 5′-Diphosphate Ribose Template

Few inhibitors exist for CD38, a multifunctional enzyme catalyzing the formation and metabolism of the Ca2+-mobilizing second messenger cyclic adenosine 5′-diphosphoribose (cADPR). Synthetic, non-hydrolyzable ligands can facilitate structure-based inhibitor design. Molecular docking was used to reproduce the crystallographic binding mode of cyclic inosine 5′-diphosphoribose (N1-cIDPR) with CD38, revealing an exploitable pocket and predicting the potential to introduce an extra hydrogen bond interaction with Asp-155. The purine C-8 position of N1-cIDPR (IC50 276 µM) was extended with an amino or diaminobutane group and the 8-modified compounds were evaluated against CD38-catalyzed cADPR hydrolysis. Crystallography of an 8-amino N1-cIDPR:CD38 complex confirmed the predicted interaction with Asp-155, together with a second H-bond from a realigned Glu-146, rationalizing the improved inhibition (IC50 56 µM). Crystallography of a complex of cyclic ADP-carbocyclic ribose (cADPcR, IC50 129 µM) with CD38 illustrated that Glu-146 hydrogen bonds with the ligand N6-amino group. Both 8-amino N1-cIDPR and cADPcR bind deep in the active site reaching the catalytic residue Glu-226, and mimicking the likely location of cADPR during catalysis. Substantial overlap of the N1-cIDPR “northern” ribose monophosphate and the cADPcR carbocyclic ribose monophosphate regions suggests that this area is crucial for inhibitor design, leading to a new compound series of N1-inosine 5′-monophosphates (N1-IMPs). These small fragments inhibit hydrolysis of cADPR more efficiently than the parent cyclic compounds, with the best in the series demonstrating potent inhibition (IC50 = 7.6 µM). The lower molecular weight and relative simplicity of these compounds compared to cADPR make them attractive as a starting point for further inhibitor design.

Aberrant cyclization affords a C-6 modified cyclic adenosine 5'-diphosphoribose analogue with biological activity in Jurkat T cells.

Two nicotinamide adenine dinucleotide (NAD+) analogues modified at the 6 position of the purine ring were synthesized, and their substrate properties toward Aplysia californica ADP-ribosyl cyclase were investigated. 6-N-Methyl NAD+ (6-N-methyl nicotinamide adenosine 5′-dinucleotide 10) hydrolyzes to give the linear 6-N-methyl ADPR (adenosine 5′-diphosphoribose, 11), whereas 6-thio NHD+ (nicotinamide 6-mercaptopurine 5′-dinucleotide, 17) generates a cyclic dinucleotide. Surprisingly, NMR correlation spectra confirm this compound to be the N1 cyclic product 6-thio N1-cIDPR (6-thio cyclic inosine 5′-diphosphoribose, 3), although the corresponding 6-oxo analogue is well-known to cyclize at N7. In Jurkat T cells, unlike the parent cyclic inosine 5′-diphosphoribose N1-cIDPR 2, 6-thio N1-cIDPR antagonizes both cADPR- and N1-cIDPR-induced Ca2+ release but possesses weak agonist activity at higher concentration. 3 is thus identified as the first C-6 modified cADPR (cyclic adenosine 5′-diphosphoribose) analogue antagonist; it represents the first example of a fluorescent N1-cyclized cADPR analogue and is a new pharmacological tool for intervention in the cADPR pathway of cellular signaling.

Chemical synthesis of the novel Ca2+ messenger NAADP

The first total chemical synthesis of nicotinamide adenine dinucleotide phosphate (β-NADP, 2) as a single isomer was achieved. This was subsequently converted into the important second messenger nicotinic acid adenine dinucleotide phosphate (β-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.

Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349

TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5′-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5′-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.