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.

Adenosine and uric acid (UA) play a pivotal role in lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). In the present experiments, we measured adenosine synthesis from nicotinamide adenine dinucleotide (NAD+) in membranes prepared from wild type (WT) and CD38 knockout (CD38KO) mouse lungs, from cultured airway smooth muscle and epithelial cells, and in bronchoalveolar lavage fluid after airway challenge with epidemiologically relevant allergens. Adenosine was determined using an enzymatically coupled assay that produces ATP and is detected by luminescence. Uric acid was determined by ELISA. Exposure of cultured airway epithelial cells to Alternaria alternata extract caused significant nucleotide (NAD+ and ATP) release in the culture media. The addition of NAD+ to membranes prepared from WT mice resulted in faster generation of adenosine compared to membranes from CD38KO mice. Formation of adenosine from NAD+ affected UA and ATP concentrations, its main downstream molecules. Furthermore, NAD+ and adenosine concentrations in the bronchoalveolar lavage fluid decreased significantly following airway challenge with house-dust mite extract in WT but not in CD38KO mice. Thus, NAD+ is a significant source of adenosine and UA in the airways in mouse models of allergic airway disease, and the capacity for their generation from NAD+ is augmented by CD38, a major NADase with high affinity for NAD+. This novel non-canonical NAD+-adenosine-UA pathway that is triggered by allergens has not been previously described in the airways.

Asthma is an inflammatory disease in which proinflammatory cytokines have a role in inducing abnormalities of airway smooth muscle function and in the development of airway hyperresponsiveness. Inflammatory cytokines alter calcium (Ca2+) signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca2+ regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca2+ regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3) and CD38-cyclic ADP-ribose (CD38/cADPR) are two major pathways mediating agonist-induced Ca2+ regulation in airway smooth muscle. Altered CD38 expression or enhanced cyclic ADP-ribosyl cyclase activity associated with CD38 contributes to human pathologies such as asthma, neoplasia, and neuroimmune diseases. This review is focused on investigations on the role of CD38-cyclic ADP-ribose signaling in airway smooth muscle in the context of transcriptional and posttranscriptional regulation of CD38 expression. The specific roles of transcription factors NF-kB and AP-1 in the transcriptional regulation of CD38 expression and of miRNAs miR-140-3p and miR-708 in the posttranscriptional regulation and the underlying mechanisms of such regulation are discussed.

Ying-Ying Lu, Bai-Xia Hao, Richard Graeff, Connie W. M. Wong, Wu-Tian Wu, and Jianbo Yue This article has been withdrawn by the authors. After reviewing the data, the authors found that the first author of the paper made inappropriate modifications to the background of the following figures: Figs. 2C, 2D, 3B, 3E, 4A, 4E, 5E, 6D, 8A, 8C, and supplemental Figs. S1A, S2B, and S5B. The first author admits to making the modifications. The modifications include covering cells not expressing RFP-GFP-LC3, GFP-LC3, or RFP-Lamp, incomplete cells on the edge of the images, or contaminant staining dots outside of the cells in images (Figs. 2C, 2D, 3B, 3E, 4A, 4E, 6D, 8A, 8C, and supplemental Figs. S1A and S2B). Additionally, other modifications include covering dirty dots above the bands in the Western blots (Fig. 5E and supplemental Fig. S5B) and inadvertently inserting a box in LC3 blot of Fig. 4E. Although these modifications did not change the results or interpretations of this work, these figures were not prepared according to the publication policy of the journal. Therefore, the authors wish to withdraw this article and sincerely apologize for these mistakes. WITHDRAWALS/RETRACTIONS

The multifunctional, transmembrane glycoprotein human CD38 catalyses the synthesis of three key Ca 2+ -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” N 1-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-NH 2 -L-cIDPR inhibit CD38-mediated cADPR hydrolysis (IC 50 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.

Employing passive immunization - using a heterologous anti-CD38 IgG antibody containing serum - in SCID mice injected subcutaneously with human multiple myeloma cells, we have shown that treatments with the antiserum - especially in the presence of complement - significantly decreased cancer growth. However, administered antibody and complement was not sufficient in amount to prevent cancer cell multiplication and cancer growth expansion to a satisfactory degree. Larger volumes of the same components more than likely would have further reduced cancer growth and prolonged the life of mice. In control mice, cancer growth progressed faster proving that lytic immune response against multiple myeloma cells is necessary for cancer cell kill.

Objectives were to: 1) induce a lytic IgG antibody (ab) response (via the so called `third vaccination method') against CD38 antigen (ag) residing on the extra-cellular domain of multiple myeloma (MM) cells in recipient rabbits, by combining the CD38 ag with donor-derived anti-CD38 ag lytic IgG ab into an immune complex (IC); and 2) determine whether abs produced would cause complement-mediated lysis (in vitro) of human MM cells containing CD38 ag. The vaccine was created in a two-step process. First, ab (rabbit anti-CD38 ag IgG ab) was raised in donor rabbits by injections of low molecular weight soluble CD38 ag in Freund's complete adjuvant (FCA) and aqueous solution. Second, transfer of pathogenic lytic IgG ab response into recipient rabbits was achieved by injections of ICs composed of CD38 ag and homologous anti-CD38 ag IgG ab. Consequently, recipient rabbits produced the same ab with the same specificity against the target ag as was present in the inoculum, namely agglutinating, precipitating and lytic (as demonstrated in vitro). In an in vitro study, in the presence of complement, donor and recipient rabbits' immune sera caused lysis of CD38 ag associated human MM cells. The most effective lytic ab response causing sera were those from donor rabbits injected with CD38 ag in FCA and those from rabbits injected with ICs, especially when they were administered in adjuvants. These results provided proof of concept that the third vaccination method has good potential as a stand-alone and efficacious method of controlling cancer.

Cyclic ADP‐ribose (cADPR) mobilizes intracellular Ca 2+ stores and activates Ca 2+ influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD + by the multifunctional CD38/ADP‐ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD + can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD + to form linear ADPR while Aplysia ADP‐ribosyl cyclase prefers cyclizing NAD + to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD + cyclase activity producing cADPR. We also determined the X‐ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD + reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively.

Aims In the heart, a period of ischaemia followed by reperfusion evokes powerful cytosolic Ca2+ oscillations that can cause lethal cell injury. These signals represent attractive cardioprotective targets, but the underlying mechanisms of genesis are ill-defined. Here, we investigated the role of the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which is known in several cell types to induce Ca2+ oscillations that initiate from acidic stores such as lysosomes, likely via two-pore channels (TPCs, TPC1 and 2). Methods and results An NAADP antagonist called Ned-K was developed by rational design based on a previously existing scaffold. Ned-K suppressed Ca2+ oscillations and dramatically protected cardiomyocytes from cell death in vitro after ischaemia and reoxygenation, preventing opening of the mitochondrial permeability transition pore. Ned-K profoundly decreased infarct size in mice in vivo. Transgenic mice lacking the endo-lysosomal TPC1 were also protected from injury. Conclusion NAADP signalling plays a major role in reperfusion-induced cell death and represents a potent pathway for protection against reperfusion injury.

Analogues of the potent Ca(2+) releasing second messenger cyclic ADP-ribose (cADPR) with a 1,2,3-triazole pyrophosphate bioisostere were synthesised by click-mediated macrocyclisation. The ability to activate Ca(2+) release was surprisingly retained, and hydrolysis of cADPR by CD38 could also be inhibited, illustrating the potential of this approach to design drug-like signalling pathway modulators.

Cyclic adenosine 5′-diphosphate ribose (cADPR) analogs based on the cyclic inosine 5′-diphosphate ribose (cIDPR) template were synthesized by recently developed stereo- and regioselective N1-ribosylation. Replacing the base N9-ribose with a butyl chain generates inhibitors of cADPR hydrolysis by the human ADP-ribosyl cyclase CD38 catalytic domain (shCD38), illustrating the nonessential nature of the “southern” ribose for binding. Butyl substitution generally improves potency relative to the parent cIDPRs, and 8-amino-N9-butyl-cIDPR is comparable to the best noncovalent CD38 inhibitors to date (IC50 = 3.3 μM). Crystallographic analysis of the shCD38:8-amino-N9-butyl-cIDPR complex to a 2.05 Å resolution unexpectedly reveals an N1-hydrolyzed ligand in the active site, suggesting that it is the N6-imino form of cADPR that is hydrolyzed by CD38. While HPLC studies confirm ligand cleavage at very high protein concentrations, they indicate that hydrolysis does not occur under physiological concentrations. Taken together, these analogs confirm that the “northern” ribose is critical for CD38 activity and inhibition, provide new insight into the mechanism of cADPR hydrolysis by CD38, and may aid future inhibitor design.

Mobilization of intracellular Ca(2+) stores is involved in many diverse cell functions, including: cell proliferation; differentiation; fertilization; muscle contraction; secretion of neurotransmitters, hormones and enzymes; and lymphocyte activation and proliferation. Cyclic adenosine diphosphate ribose (cADPR) is an endogenous Ca(2+) mobilizing nucleotide present in many cell types and species, from plants to animals. cADPR is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide. The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. It has been shown that many extracellular stimuli can induce cADPR production that leads to calcium release or influx, establishing cADPR as a second messenger. cADPR has been linked to a wide variety of cellular processes, but the molecular mechanisms regarding cADPR signaling remain elusive. The aim of this review is to summarize the CD38/cADPR/Ca(2+) signaling pathway, focusing on the recent advances involving the mechanism and physiological functions of cADPR-mediated Ca(2+) mobilization.

Purpose: Myocardial ischaemia-reperfusion injury (IRI) is characterised by excessive intracellular levels of Ca2+ in cardiomyocytes which leads to mitochondrial pore opening and cell death. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent second messenger which mobilises Ca2+ from lysosomes, which in turn can trigger Ca2+ release from the sarcoplasmic reticulum (SR). Lethal Ca2+ oscillations occur in cardiomyocytes during reoxygenation after simulated ischaemia. Ned-19 is a pharmacological inhibitor of NAADP signalling which we found suppresses Ca2+ oscillations during reoxygenation, reducing mitochondrial pore opening and enhancing cell survival. In line with this, we therefore hypothesised that mice with a genetic ablation of the putative NAADP receptor on lysosomes, the two-pore channel (TPC), would be protected against IRI. We also investigated whether the mechanism of protection of Ned-19 involved a direct effect on mitochondria, or on NAADP levels. Methods: Adult TPC1 knockout mice and wild-type littermates were subjected to IRI in vivo by occlusion of the left coronary artery for 30 min followed by 120 min of reperfusion. Infarct size as a proportion of area at risk was measured using triphenyltetrazolium chloride (TTC) staining and Evan's blue. The effect of Ned-19 (10μM) was assessed in cells subject to laser-induced oxidative stress, and in a mitochondrial swelling assay using 500μM free Ca2+ to induce pore opening. NAADP levels were measured in isolated mouse hearts before and after 30 min ischaemia using an enzymatic cycling assay. Results: Mice lacking TPC1 had significantly reduced infarct size compared to wild-type controls (33 ± 5 vs. 51 ± 5%; P<0.05). Ned-19 delayed mPTP opening in cells (100 ± 0 DMSO vs. 155 ± 20% Ned-19; P<0.05), but had no effect on mPTP opening in isolated mitochondria (12 ± 3 vs. 11 ± 2%; P<0.05), supporting an indirect effect involving suppression of Ca2+ oscillations. Myocardial NAADP levels were significantly reduced after a period of 30 min global ischaemia from 8.3 ± 2.0 fmol.mg-1 protein to 2.0 ± 0.3 fmol.mg-1. Upon reperfusion, NAADP levels recovered. Conclusion: These data show that NAADP levels are dynamically regulated during ischaemia and reperfusion supporting a role for NAADP signalling in IRI. NAADP may augment reperfusion injury by stimulating Ca2+ release via TPC1 channels, indirectly augmenting SR Ca2+ oscillations and mitochondrial pore opening. By inhibiting NAADP signalling, Ned-19 may offer a novel approach to preventing IRI.

Nicotinic adenine acid dinucleotide phosphate (NAADP) is one of the most potent endogenous Ca2+ mobilizing messengers. NAADP mobilizes Ca2+ from an acidic lysosome-related store, which can be subsequently amplified into global Ca2+ waves by calcium-induced calcium release (CICR) from ER/SR via Ins(1,4,5)P3 receptors or ryanodine receptors. A body of evidence indicates that 2 pore channel 2 (TPC2), a new member of the superfamily of voltage-gated ion channels containing 12 putative transmembrane segments, is the long sought after NAADP receptor. Activation of NAADP/TPC2/Ca2+ signaling inhibits the fusion between autophagosome and lysosome by alkalizing the lysosomal pH, thereby arresting autophagic flux. In addition, TPC2 is downregulated during neural differentiation of mouse embryonic stem (ES) cells, and TPC2 downregulation actually facilitates the neural lineage entry of ES cells. Here we propose the mechanism underlying how NAADP-induced Ca2+ release increases lysosomal pH and discuss the role of TPC2 in neural differentiation of mouse ES cells.

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 mu M) 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 mu M). Crystallography of a complex of cyclic ADP-carbocyclic ribose (cADPcR, IC50 129 mu M) 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 mu M). The lower molecular weight and relative simplicity of these compounds compared to cADPR make them attractive as a starting point for further inhibitor design.

Protein ADP-ribosylation, including mono- and poly-ADP-ribosylation, is increasingly recognized to play important roles in various biological pathways. Molecular understanding of the functions of ADP-ribosylation requires the identification of the sites of modification. Although tandem mass spectrometry (MS/MS) is widely recognized as an effective means for determining protein modifications, identification of ADP-ribosylation sites has been challenging due to the labile and hydrophilic nature of the modification. Here we applied precursor ion scanning-triggered MS/MS analysis on a hybrid quadrupole linear ion trap mass spectrometer for selectively detecting ADP-ribosylated peptides and determining the auto-ADP-ribosylation sites of CD38 (cluster of differentiation 38) E226D and E226Q mutants. CD38 is an enzyme that catalyzes the hydrolysis of nicotinamide adenine dinucleotide (NAD) to ADP-ribose. Here we show that NAD can covalently label CD38 E226D and E226Q mutants but not wild-type CD38. In this study, we have successfully identified the D226/Q226 and K129 residues of the two CD38 mutants being the ADP-ribosylation sites using precursor ion scanning hybrid quadrupole linear ion trap mass spectrometry. The results offer insights about the CD38 enzymatic reaction mechanism. The precursor ion scanning method should be useful for identifying the modification sites of other ADP-ribosyltransferases such as poly(ADP-ribose) polymerases.