Small Molecule CD38 Inhibitors: Synthesis of 8-Amino-N1-inosine 5'-monophosphate, Analogues and Early Structure-Activity Relationship.

Although a monoclonal antibody targeting the multifunctional ectoenzyme CD38 is an FDA-approved drug, few small molecule inhibitors exist for this enzyme that catalyzes inter alia the formation and metabolism of the N1-ribosylated, Ca2+-mobilizing, second messenger cyclic adenosine 5'-diphosphoribose (cADPR). N1-Inosine 5'-monophosphate (N1-IMP) is a fragment directly related to cADPR. 8-Substituted-N1-IMP derivatives, prepared by degradation of cyclic parent compounds, inhibit CD38-mediated cADPR hydrolysis more efficiently than related cyclic analogues, making them attractive for inhibitor development. We report a total synthesis of the N1-IMP scaffold from adenine and a small initial compound series that facilitated early delineation of structure-activity parameters, with analogues evaluated for inhibition of CD38-mediated hydrolysis of cADPR. The 5'-phosphate group proved essential for useful activity, but substitution of this group by a sulfonamide bioisostere was not fruitful. 8-NH2-N1-IMP is the most potent inhibitor (IC50 = 7.6 µM) and importantly HPLC studies showed this ligand to be cleaved at high CD38 concentrations, confirming its access to the CD38 catalytic machinery and demonstrating the potential of our fragment approach.

Inositol Adenophostin: Convergent Synthesis of a Potent Agonist of d-myo-Inositol 1,4,5-Trisphosphate Receptors.

d-myo-Inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ channels activated by the intracellular messenger inositol 1,4,5-trisphosphate (IP3, 1). The glyconucleotide adenophostin A (AdA, 2) is a potent agonist of IP3Rs. A recent synthesis of d-chiro-inositol adenophostin (InsAdA, 5) employed suitably protected chiral building blocks and replaced the d-glucose core by d-chiro-inositol. An alternative approach to fully chiral material is now reported using intrinsic sugar chirality to avoid early isomer resolution, involving the coupling of a protected and activated racemic myo-inositol derivative to a d-ribose derivative. Diastereoisomer separation was achieved after trans-isopropylidene group removal and the absolute ribose-inositol conjugate stereochemistry assigned with reference to the earlier synthesis. Optimization of stannylene-mediated regiospecific benzylation was explored using the model 1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol and conditions successfully transferred to one conjugate diastereoisomer with 3:1 selectivity. However, only roughly 1:1 regiospecificity was achieved on the required diastereoisomer. The conjugate regioisomers of benzyl derivatives 39 and 40 were successfully separated and 39 was transformed subsequently to InsAdA after amination, pan-phosphorylation, and deprotection. InsAdA from this synthetic route bound with greater affinity than AdA to IP3R1 and was more potent in releasing Ca2+ from intracellular stores through IP3Rs. It is the most potent full agonist of IP3R1 known and .equipotent with material from the fully chiral synthetic route.

Synthesis of phosphonoacetate analogues of the second messenger adenosine 5'-diphosphate ribose (ADPR).

Adenosine 5'-diphosphate ribose (ADPR) is an intracellular signalling molecule generated from nicotinamide adenine dinucleotide (NAD+). Synthetic ADPR analogues can shed light on the mechanism of activation of ADPR targets and their downstream effects. Such chemical biology studies, however, are often challenging due to the negatively charged pyrophosphate, also sensitive to cellular pyrophosphatases, and prior work on an initial ADPR target, the transient receptor potential cation channel TRPM2, showed complete pyrophosphate group replacement to be a step too far in maintaining biological activity. Thus, we designed ADPR analogues with just one of the negatively charged phosphate groups removed, by employing a phosphonoacetate linker. Synthesis of two novel phosphonoacetate ADPR analogues is described via tandem N,N'-dicyclohexylcarbodiimide coupling to phosphonoacetic acid. Neither analogue, however, showed significant agonist or antagonist activity towards TRPM2, underlining the importance of a complete pyrophosphate motif in activation of this particular receptor.

Synthesis of Terminal Ribose Analogues of Adenosine 5'-Diphosphate Ribose as Probes for the Transient Receptor Potential Cation Channel TRPM2.

TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a nonselective cation channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide adenosine 5'-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9 H) domain activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of ADPR is known to be essential for activation. To study its role in more detail, we designed synthetic routes to novel analogues of ADPR and 2'-deoxy-ADPR that were modified only by removal of a single hydroxyl group from the terminal ribose. The ADPR analogues were obtained by coupling nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2″-based analogues proved to be unstable. The C1″- and C3″-ADPR analogues were evaluated electrophysiologically by patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups of the terminal ribose blocked as its 1″-ß- O-methyl-2″,3″- O-isopropylidene derivative was evaluated. Removal of either C1″ or C3″ hydroxyl groups from ADPR resulted in loss of agonist activity. Both these modifications and blocking all three hydroxyl groups resulted in TRPM2 antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.

Different substrate specificities of the two ADPR binding sites in TRPM2 channels of Nematostella vectensis and the role of IDPR.

NvTRPM2 (Nematostella vectensis Transient Receptor Potential Melastatin 2), the species variant of the human apoptosis-related cation channel hTRPM2, is gated by ADP-ribose (ADPR) independently of the C-terminal NUDT9H domain that mediates ADPR-directed gating in hTRPM2. The decisive binding site in NvTRPM2 is likely to be identical with the N-terminal ADPR binding pocket in zebra fish DrTRPM2. Our aim was a characterization of this binding site in NvTRPM2 with respect to its substrate specificity, in comparison to the classical ADPR interaction site within NUDT9H that is highly homologous in hTRPM2 and NvTRPM2, although only in NvTRPM2, catalytic (ADPRase) activity is conserved. With various ADPR analogues, key differences of the two sites were identified. Particularly, two reported antagonists on hTRPM2 were agonists on NvTRPM2. Moreover, IDP-ribose (IDPR) induced currents both in hTRPM2 and NvTRPM2 but not in NvTRPM2 mutants in which NUDT9H was absent. Thus, IDPR acts on NUDT9H rather than N-terminally, revealing a regulatory function of NUDT9H in NvTRPM2 opposed to that in hTRPM2. We propose that IDPR competitively inhibits the ADPRase function of NUDT9H and evokes ADPR accumulation. The findings provide important insights into the structure-function relationship of NvTRPM2 and will allow further characterization of the novel ADPR interaction site.

Small Molecule Antagonists of NAADP-Induced Ca2+ Release in T-Lymphocytes Suggest Potential Therapeutic Agents for Autoimmune Disease.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date, but the precise NAADP/Ca2+ signalling mechanisms are still controversial. We report the synthesis of small-molecule inhibitors of NAADP-induced Ca2+ release based upon the nicotinic acid motif. Alkylation of nicotinic acid with a series of bromoacetamides generated a diverse compound library. However, many members were only weakly active or had poor physicochemical properties. Structural optimisation produced the best inhibitors that interact specifically with the NAADP/Ca2+ release mechanism, having no effect on Ca2+ mobilized by the other well-known second messengers D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or cyclic adenosine 5'-diphospho-ribose (cADPR). Lead compound (2) was an efficient antagonist of NAADP-evoked Ca2+ release in vitro in intact T lymphocytes and ameliorated clinical disease in vivo in a rat experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Compound (3) (also known as BZ194) was synthesized as its bromide salt, confirmed by crystallography, and was more membrane permeant than 2. The corresponding zwitterion (3a), was also prepared and studied by crystallography, but 3 had more desirable physicochemical properties. 3 Is potent in vitro and in vivo and has found widespread use as a tool to modulate NAADP effects in autoimmunity and cardiovascular applications. Taken together, data suggest that the NAADP/Ca2+ signalling mechanism may serve as a potential target for T cell- or cardiomyocyte-related diseases such as multiple sclerosis or arrhythmia. Further modification of these lead compounds may potentially result in drug candidates of clinical use.

Synthetic cADPR analogues may form only one of two possible conformational diastereoisomers.

Cyclic adenosine 5'-diphosphate ribose (cADPR) is an emerging Ca2+-mobilising second messenger. cADPR analogues have been generated as chemical biology tools via both chemo-enzymatic and total synthetic routes. Both routes rely on the cyclisation of a linear precursor to close an 18-membered macrocyclic ring. We show here that, after cyclisation, there are two possible macrocyclic product conformers that may be formed, depending on whether cyclisation occurs to the "right" or the "left" of the adenine base (as viewed along the H-8 → C-8 base axis). Molecular modelling demonstrates that these two conformers are distinct and cannot interconvert. The two conformers would present a different spatial layout of binding partners to the cADPR receptor/binding site. For chemo-enzymatically generated analogues Aplysia californica ADP-ribosyl cyclase acts as a template to generate solely the "right-handed" conformer and this corresponds to that of the natural messenger, as originally explored using crystallography. However, for a total synthetic analogue it is theoretically possible to generate either product, or a mixture, from a given linear precursor. Cyclisation on either face of the adenine base is broadly illustrated by the first chemical synthesis of the two enantiomers of a "southern" ribose-simplified cIDPR analogue 8-Br-N9-butyl-cIDPR, a cADPR analogue containing only one chiral sugar in the "northern" ribose, i.e. 8-Br-D- and its mirror image 8-Br-L-N9-butyl-cIDPR. By replacing the D-ribose with the unnatural L-ribose sugar, cyclisation of the linear precursor with pyrophosphate closure generates a cyclised product spectroscopically identical, but displaying equal and opposite specific rotation. These findings have implications for cADPR analogue design, synthesis and activity.

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis.

AIMS: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. RESULTS: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2-/- mice. INNOVATION: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. CONCLUSIONS: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110-130.

Second messenger analogues highlight unexpected substrate sensitivity of CD38: total synthesis of the hybrid "L-cyclic inosine 5'-diphosphate ribose".

The multifunctional, transmembrane glycoprotein human CD38 catalyses the synthesis of three key Ca2+-mobilising messengers, including cyclic adenosine 5'-diphosphate ribose (cADPR), and CD38 knockout studies have revealed the relevance of the related signalling pathways to disease. To generate inhibitors of CD38 by total synthesis, analogues based on the cyclic inosine 5'-diphosphate ribose (cIDPR) template were synthesised. In the first example of a sugar hybrid cIDPR analogue, "L-cIDPR", the natural "northern" N1-linked D-ribose of cADPR was replaced by L-ribose. L-cIDPR is surprisingly still hydrolysed by CD38, whereas 8-Br-L-cIDPR is not cleaved, even at high enzyme concentrations. Thus, the inhibitory activity of L-cIDPR analogues appears to depend upon substitution of the base at C-8; 8-Br-L-cIDPR and 8-NH2-L-cIDPR inhibit CD38-mediated cADPR hydrolysis (IC50 7 µM and 21 µM respectively) with 8-Br-L-cIDPR over 20-fold more potent than 8-Br-cIDPR. In contrast, L-cIDPR displays a comparative 75-fold reduction in activity, but is only ca 2-fold less potent than cIDPR itself. Molecular modelling was used to explore the interaction of the CD38 catalytic residue Glu-226 with the "northern" ribose. We propose that Glu226 still acts as the catalytic residue even for an L-sugar substrate. 8-Br-L-cIDPR potentially binds non-productively in an upside-down fashion. Results highlight the key role of the "northern" ribose in the interaction of cADPR with CD38.

Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy.

In experimental autoimmune encephalitis (EAE), autoimmune T cells are activated in the periphery before they home to the CNS. On their way, the T cells pass through a series of different cellular milieus where they receive signals that instruct them to invade their target tissues. These signals involve interaction with the surrounding stroma cells, in the presence or absence of autoantigens. To portray the serial signaling events, we studied a T-cell-mediated model of EAE combining in vivo two-photon microscopy with two different activation reporters, the FRET-based calcium biosensor Twitch1 and fluorescent NFAT. In vitro activated T cells first settle in secondary (2°) lymphatic tissues (e.g., the spleen) where, in the absence of autoantigen, they establish transient contacts with stroma cells as indicated by sporadic short-lived calcium spikes. The T cells then exit the spleen for the CNS where they first roll and crawl along the luminal surface of leptomeningeal vessels without showing calcium activity. Having crossed the blood-brain barrier, the T cells scan the leptomeningeal space for autoantigen-presenting cells (APCs). Sustained contacts result in long-lasting calcium activity and NFAT translocation, a measure of full T-cell activation. This process is sensitive to anti-MHC class II antibodies. Importantly, the capacity to activate T cells is not a general property of all leptomeningeal phagocytes, but varies between individual APCs. Our results identify distinct checkpoints of T-cell activation, controlling the capacity of myelin-specific T cells to invade and attack the CNS. These processes may be valuable therapeutic targets.

2'-Deoxyadenosine 5'-diphosphoribose is an endogenous TRPM2 superagonist.

Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.

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.