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.

Novel Pathway of Adenosine Generation in the Lungs from NAD+: Relevance to Allergic Airway Disease.

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.

CD38/cADPR Signaling Pathway in Airway Disease: Regulatory Mechanisms.

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.

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.

Tolerance, loss of tolerance and regaining tolerance to self by immune-mediated events.

Autoimmunity has both beneficial and harmful aspects. Beneficial aspects include: (1) removal of released intracytoplasmic antigens (ags) (cells at the end of their life span or damaged by outside agents) by specific nonpathogenic IgM autoantibodies and mononuclear cells and (2) recognition and elimination of cancerous cells. In contrast, harmful aspects include: (1) mounting a pathogenic autoimmune response against a tissue-derived ag, a 'modified self,' resulting in autoimmune disease and (2) inability to recognize and eliminate a cancerous clone. The immune system continuously faces internal and external influences; however, even when it is compromised or overwhelmed, it will still endeavor to regain and maintain tolerance to self. To promote this, we developed a 'modified vaccination technique' (MVT) (described as the third vaccination method after active and passive immunizations). It has two components: purified exogenous/endogenous ag (i.e., target ag) and a high-titer-specific antibody (ab) against the target ag made into an immune complex (IC) with predetermined immune-inducing components. The MVT works by ab information transfer (production of same class of immunoglobulin with the same specificity against the target ag that is present in the vaccine), thereby re-establishing tolerance to self (caused by exogenous/endogenous ags) following repeated administration of appropriate ICs. This vaccination technique can be used both prophylactically and therapeutically, and it mimics the immune system's natural abilities to respond to corrective information specifically, rapidly, safely and with minimal side effects and makes this approach a novel solution for many disorders that are difficult or impossible to cure or manage.

A novel modified vaccination technique produces IgG antibodies that cause complement-mediated lysis of multiple myeloma cells carrying CD38 antigen.

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.

Suppression of tumor growth by a heterologous antibody directed against multiple myeloma dominant CD38 antigen in SCID mice injected with multiple myeloma cells.

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.

Porcine CD38 exhibits prominent secondary NAD(+) cyclase activity.

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.

Inhibition of NAADP signalling on reperfusion protects the heart by preventing lethal calcium oscillations via two-pore channel 1 and opening of the mitochondrial permeability transition pore.

AIMS: In the heart, a period of ischaemia followed by reperfusion evokes powerful cytosolic Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

Cyclic adenosine 5'-diphosphate ribose analogs without a "southern" ribose inhibit ADP-ribosyl cyclase-hydrolase CD38.

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.

Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca(2+) signaling pathway.

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.

'Click cyclic ADP-ribose': a neutral second messenger mimic.

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.

Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

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 Ca(2+)-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.

Determination of ADP-ribosyl cyclase activity, cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate in tissue extracts.

Cyclic ADP-ribose (cADPR) is a novel second messenger that releases calcium from intracellular stores. Although first shown to release calcium in the sea urchin egg, cADPR has been shown since to be active in a variety of cells and tissues, from plant to human. cADPR stimulates calcium release via ryanodine receptors although the mechanism is still not completely understood. cADPR is produced enzymatically from NAD by ADP-ribosyl cyclases; several of these proteins have been identified including one isolated from Aplysia californica, two types found in mammals (CD38 and CD157), and three forms in sea urchin. A cyclase activity has been measured in extracts from Arabidopsis thaliana although the protein is still unidentified. Nicotinic acid adenine dinucleotide phosphate (NAADP) is another novel messenger that releases calcium from internal stores and is produced by these same enzymes by an exchange reaction. NAADP targets lysosomal stores whereas cADPR releases calcium from the endoplasmic reticulum. Due to their importance in cell signaling, cADPR and NAADP have been the focus of numerous investigations over the last 25 years. This chapter describes several assay methods for the measurements of cADPR and NAADP concentration and cyclase activity in extracts from cells.

Increased CD38 expression in T cells and circulating anti-CD38 IgG autoantibodies differentially correlate with distinct cytokine profiles and disease activity in systemic lupus erythematosus patients.

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. In mammals, CD38 also functions as a receptor. In this study CD38 expression in CD4(+), CD8(+), or CD25(+) T cells was significantly higher in systemic lupus erythematosus (SLE) patients than in Normal controls. Increased CD38 expression in SLE T cells correlated with plasma levels of Th2 (IL-4, IL-10, IL-13) and Th1 (IL-1ß, IL-12, IFN-γ, TNF-α) cytokines, and was more prevalent in clinically active SLE patients than in Normal controls. In contrast, elevated anti-CD38 IgG autoantibodies were more frequent in clinically quiescent SLE patients (SLEDAI=0) than in Normal controls, and correlated with moderate increased plasma levels of IL-10 and IFN-γ. However, clinically active SLE patients were mainly discriminated from quiescent SLE patients by increased levels of IL-10 and anti-dsDNA antibodies, with odds ratios (ORs) of 3.7 and 4.8, respectively. Increased frequency of anti-CD38 autoantibodies showed an inverse relationship with clinical activity (OR=0.43), and in particular with the frequency of anti-dsDNA autoantibodies (OR=0.21). Increased cell death occurred in CD38(+) Jurkat T cells treated with anti-CD38(+) SLE plasmas, and not in these cells treated with anti-CD38(-) SLE plasmas, or Normal plasmas. This effect did not occur in CD38-negative Jurkat T cells, suggesting that it could be attributed to anti-CD38 autoantibodies. These results support the hypothesis that anti-CD38 IgG autoantibodies or their associated plasma factors may dampen immune activation by affecting the viability of CD38(+) effector T cells and may provide protection from certain clinical SLE features.

Identification of ADP-ribosylation sites of CD38 mutants by precursor ion scanning mass spectrometry.

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.

NAADP/TPC2/Ca(2+) Signaling Inhibits Autophagy.

Nicotinic adenine acid dinucleotide phosphate (NAADP) is one of the most potent endogenous Ca(2+) mobilizing messengers. NAADP mobilizes Ca(2+) from an acidic lysosome-related store, which can be subsequently amplified into global Ca(2+) waves by calcium-induced calcium release (CICR) from ER/SR via Ins(1,4,5)P 3 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/Ca(2+) 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 Ca(2+) release increases lysosomal pH and discuss the role of TPC2 in neural differentiation of mouse ES cells.

Roles of cADPR and NAADP in pancreatic cells.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are Ca(2+)-mobilizing nucleotides that were discovered in the late 1980s. Two decades of investigations have built up a considerable understanding about these two molecules that are related because both are derived from pyridine nucleotides and known to be generated by CD38/ADP-ribosyl cyclases. cADPR has been shown to target the ryanodine receptors in the endoplasmic reticulum whereas NAADP stimulates the two-pore channels in the endo-lysosomes. Accumulating results indicate that cADPR and NAADP are second messenger molecules mediating Ca(2+) signaling activated by a wide range of agonists. This article reviews what is known about these two molecules, especially regarding their signaling roles in the pancreatic cells.