MASTER-NAADP: a membrane permeable precursor of the Ca2+ mobilizing second messenger NAADP

Abstract

Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca²⁺ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca²⁺ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca²⁺ signals of low amplitude. Subsequently, also global Ca²⁺ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca²⁺ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca²⁺ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic.

Fig. 1. Synthesis of MASTER-NAADP and derivatives I Reagents and conditions.

[a] (i) 0.10 eq. CuSO₄, 2.8 eq. conc. H₂SO₄, acetone, 3 h, rt, (ii) HCl-solution (0.2 %), 6 h, rt, 99 %; [b] 1.5 eq. Et₃N, 1.1 eq. BzCl, CH₂Cl₂, 1 h, 0 °C, 92 %; [c] 0.2 eq. I₂, MeOH, 4 h, 70 °C, 86 %; [d] 1.5 eq. DAST, dry CH₂Cl₂, 20 h, -78 °C → rt, 43 %; [e] 4.7 eq. Ac₂O, 4.7 eq. conc. H₂SO₄, AcOH, 19 h, rt, 93 %; [f] (i) 1.5 eq. N-benzoyladenine, 1.2 eq. BSA, C₂H₄Cl₂, 1 h, 90 °C, (ii) 1.0 eq. 6, 4.0 eq. TMSOTf, C₂H₄Cl₂, 5.5 h, 90 °C, 61 %; [g] ammonia 7 N in MeOH, 28 h, 70 °C, 84 %; [h] 1.5 eq. TBDMSCl, dry pyridine, 24 h, rt, 71 %; [i] 1.2 eq. 4-hydroxyl benzyl alcohol 10, 1.2 eq. Et₃N, 1.0 eq. heptanoyl chloride, THF, 2 h, 0 °C, 87 %; [j] 1.0 eq. dichloro-N,N-diisopropylaminophosphoramidite, 2.2 eq. 11, 2.3 eq. Et₃N, THF, 0 °C → rt, 16 h, 92 %; [k] (i) 1.3 eq. 12, 1.2 eq. pyridinium trifluoracetate, CH₂Cl₂, 50 min, rt, (ii) 1.5 eq. tert-butylhydroxyperoxide (5.0 M in decane), 0 °C, 1 h, 87 %; [l] 5.2 eq. triethylamine-trihydrofluoride, CH₂Cl₂, 19 h, rt, 81 %; [m] 4.0 eq. POCl₃, TMP, 6.5 h, 0 °C, 31 %.

Fig. 2. Synthesis of MASTER-NAADP and derivatives II A) Reagents and conditions.

[a] Thionylchloride, kat. DMF, 2 h, 80 °C, then 2.0 eq. amino-2-methyl-1-propanol, CH₂Cl₂, 2 h, 0 °C → rt, thionylchloride, 16 h, rt, 87 %; [b] 1.5 eq. 17, 1.6 eq. n-BuLi (1.6 M in hexane), THF, 1 h, –78 °C, then 1.0 eq. 2,3,5-tri-O-benzyl-ribonolactone, THF, 2 h, –78 °C → –30 °C, 3.0 eq. triethylsilane, 2.5 eq. BF₃*Et₂O, CH₂Cl₂, 16 h,–78 °C → rt, 41 %; [c] CH₃NO₂/MeI (2:1) 16 h, reflux, then MeOH/KOH 20 %, 1:1, 16 h, reflux, quantitatively; [d] 0.33 eq. triphosgene, 1.0 eq. pyridine, CH₂Cl₂, 15 h, rt, 93 %; [e] 1.1 eq. 4-hydroxyl benzyl alcohol 10, 1.0 eq. Et₃N, 1.0 eq. 21, CH₂Cl₂, 15 min, 0 °C, then 3 h, rt, 74 %; [f] 1.0 eq. 19, 1.1 eq. Yamaguchi-reagent, 1.2 eq. Et₃N, 1.2 eq. 22, 0.3 eq. DMAP, THF, 3 h 70 °C, 16 h, rt, 58 %; [g] 3.5 eq. BCl₃ (1 M, CH₂Cl₂), 2 h, –78 °C, 67 %; [h] 2.0 eq. POCl₃, 2.5 eq. Bu₃N, trimethylphosphate, 16 h, 0 °C, 42 %. [i] 10.0 eq. TFAA, 16 eq. Et₃N, 1.0 eq. monophosphate 25, CH₃CN, 10 min, 0 °C → Rt, then 6.0 eq. NMI, 10 eq. Et3N, CH₃CN, 10 min, 0 °C → rt, then 1.1 eq. 15, CH₃CN, 3 h, rt, then TEAB-buffer 1 M, 15 min, rt, 57%. B) Release of MASTER-NAADP by porcine liver extract and mass spec characterization of main product. B MASTER-NAADP was analyzed by RP-HPLC for purity (black chromatogram). A representative out of 6 experiments is shown. Then, reaction products obtained upon incubation of MASTER-NAADP with porcine liver extract were separated by RP-HPLC (blue chromatogram). The main product was then identified by ESI-mass spectrum measured in positive ionization mode as benzoic acid C-nucleoside, 2’-phospho-3’F-adenosine-diphosphate. C Molecular modeling: (a) NAADP, (b) overlay of NAADP and benzoic acid C-nucleoside, 2’-phospho-3’F-adenosine-diphosphate, (c) benzoic acid C-nucleoside, 2’-phospho-3’F-adenosine-diphosphate.

Fig. 3. Deprotected MASTER-NAADP evokes Ca²⁺ signals in permeabilized Jurkat T cells and increases the open probability of RYR1.

3.5 × 10⁷ Jurkat T cells were permeabilized (80 µg/mL saponin) and transferred to a fluorimeter in 1 mL cuvettes with gentle magnetic stirring. Free Ca²⁺ concentration was measured every 2 s ratiometrically using emission wavelengths of CalRed (ex 488 nm, em 525/650 nm). Ca²⁺ uptake into the ER was stimulated by the addition of creatine phosphate, creatine kinase and adenosine triphosphate, followed by the addition of 15 µg recombinant human HN1L/JPT2 (except for control experiments) and 10 µM glucose 6-phosphate dehydrogenase inhibitor-1 (g6pdi-1). Deprotected MASTER-compounds or natural nucleotides were diluted in bidestillated water and injected using a Hamilton gas-tight 50μL syringe. A Example tracings showing no response to either biological NAADP or deprotected MASTER-compounds without the re-addition of exogenous HN1L/JPT2. Representative tracings out of 4 experiments are shown. B Example tracings showing increases in the free Ca²⁺ concentration after the injection of NAADP or deprotected MASTER-NAADP, but not of the respective NADP controls. A representative out of at least 5 experiments is shown. C, D The delta Ca²⁺ of the different experiments (mean Ca²⁺ concentration during the first 10 s after injection after subtraction of the mean Ca²⁺ concentration the last 10 s before injection) was plotted against the baseline Ca²⁺ (mean Ca²⁺ concentration the last 10 s before injection) and analyzed by linear regression with two-sided F test of the slope using GraphPad Prism 10. The graph show results from the individual experiments (symbols), linear regression (line) with 95% confidence interval (dotted lines) and significance level of the slope being non-zero, i.e. the amplitude of the responses being correlated with the basal Ca²⁺ concentration (ns = not significant, **p < 0.01, ****p < 0.0001, N = 5 experiments for C and N = 8 experiments for D). E Single channel analysis of deMASTER-NAADP with purified HN1L/JPT2 on RYR1. Representative single-channel current traces of purified RYR1 measured under the following conditions: upper tracing: 1 µM free Ca²⁺, middle tracing: 100 nM deMASTER-NAADP, lower tracing: 100 nM deMASTER-NAADP with 10 µM HN1L/JPT2. F Summarized relative open probability for each condition, presented as mean ± SEM, n = biological replications as indicated above the bars. Two-sided, mixed-effects analysis with Holm-Šídák multiple comparison test, with a single pooled variance *p < 0.05. Each p value is adjusted to account for multiple comparisons. Source data and exact p values are provided as a Source Data file.

Fig. 4. Ca²⁺ microdomains evoked by MASTER-NAADP in different cell types.

Representative high-resolution Ca²⁺ images of Jurkat T cells loaded with both Fluo4-AM and Fura-Red-AM after stimulation with 100 nM MASTER-NAADP (A) or 100 nM MASTER-NADP (B). Number of cells indicated in figure. A, B (upper panel): Pseudocolor images indicate emission ratios between Fluo-4 and Fura-Red ranging from 0.3 -1.3; ratio data were then converted using external calibration corresponding to 8 to 182 nM [Ca²⁺]_i. Scale bars, 5 µm for whole cells. A, B (lower panel): Magnified regions as indicated as 3D surface plots. C Analysis across the first 15 s of stimulation shown as number of Ca²⁺ microdomains. C Data are displayed as mean ± SEM; Jurkat WT MASTER-NAADP, n = 66 cells; Jurkat WT MASTER-NADP, n = 40 cells; Jurkat Hn1l/Jpt2^-/- MASTER-NAADP, n = 43 cells; Jurkat Hn1l/Jpt2^-/- MASTER-NADP, n = 37 cells; Jurkat WT DMSO, n = 17 cells; nonparametric Kruskal-Wallis test and Dunn’s correction for multiple testing *P < 0.05; **P < 0.005. D (upper panel) Representative high-resolution Ca²⁺ images of primary murine T cells loaded with both Fluo4-AM and Fura-Red-AM after stimulation with 100 nM MASTER-NAADP. Pseudocolor images indicate emission ratios between Fluo-4 and Fura-Red ranging from 0.3–1.3; ratio data were then converted using external calibration corresponding to 0 to 347 nM [Ca²⁺]_i. Scale bars, 5 µm for whole cells. D (lower panel) Magnified regions as indicated as 3D surface plots. E Analysis across the first 15 s of stimulation shown as number of Ca²⁺ microdomains. E Data are displayed as mean ± SEM; MASTER-NAADP, n = 89 cells; MASTER-NADP, n = 58 cells; two-sided, nonparametric Mann-Whitney U test: **P < 0.005. F (upper panel) Representative high-resolution Ca²⁺ images of human NK cells (KHYG-1) loaded with both Fluo4-AM and Fura-Red-AM after stimulation with 100 µM MASTER-NAADP. Pseudocolor images indicate emission ratios between Fluo-4 and Fura-Red ranging from 0.3–2.0; ratio data were then converted using external calibration corresponding to 5 to 293 nM [Ca²⁺]_i. Scale bars, 5 µm for whole cells. F (lower panel) Magnified regions as indicated as 3D surface plots. G Analysis across the first 15 s of stimulation shown as number of Ca²⁺ microdomains. G Data are displayed as mean ± SEM; MASTER-NAADP, n = 46 cells; MASTER-NADP, n = 39 cells; two-sided, nonparametric Mann-Whitney U test: *P < 0.05. H (upper panel) Representative high-resolution Ca²⁺ images of Neuro2A cells loaded with both Fluo4-AM and Fura-Red-AM after stimulation with 100 µM MASTER-NAADP. H (lower panel) Magnified regions as indicated as 3D surface plots. Pseudocolor images indicate emission ratios between Fluo-4 and Fura-Red ranging from 0.3–1.3; ratio data were then converted using external calibration corresponding to 0 to 147 nM [Ca²⁺]_i. I Analysis across the first 15 s of stimulation shown as number of Ca²⁺ microdomains. I Data are mean ± SEM; Neuro2A WT MASTER-NAADP, n = 20 cells; Neuro2A WT MASTER-NADP, n = 23 cells; Neuro2A Hn1l/Jpt2^-/- MASTER-NAADP, n = 21 cells; Neuro2A Hn1l/Jpt2^-/- MASTER-NADP, n = 20 cells; nonparametric Kruskal-Wallis test and Dunn’s correction for multiple testing *P < 0.05; **P < 0.005; ***P < 0.001. Source data for (C, E, G, I) and exact p values are provided as a Source Data file.

Fig. 5. Global Ca²⁺ responses evoked by MASTER-NAADP in Jurkat T cells.

Global Ca²⁺ signaling was analyzed in Jurkat WT T cells (A) and Jurkat Hn1l/Jpt2^-/- T cells (B), loaded with Fura2-AM. MASTER-NAADP (100 nM), MASTER-NADP (100 nM), or NAADP-AM (100 nM) were added as indicated. Experiments were carried out at 37 °C and SOCE was blocked by pre-incubation of 50 µM Synta66 for 5 min prior to imaging. Number of cells indicated in figure. Aggregated data are mean amplitude per peak (C), mean number of Ca²⁺ peaks (D), percentage of responding cells (E), and calculation of the mean responsiveness (number of peaks * amplitude; F presented as mean ± SEM; Jurkat WT MASTER-NAADP, n = 240 cells; Jurkat WT MASTER-NADP, n = 120 cells; Jurkat WT NAADP-AM, n = 91 cells; Jurkat Hn1l/Jpt2^-/- MASTER-NAADP, n = 181 cells; Jurkat Hn1l/Jpt2^-/- MASTER-NADP, n = 113 cells; Jurkat Hn1l/Jpt2^-/- NAADP-AM, n = 110 cells. Nonparametric Kruskal-Wallis test and Dunn’s correction for multiple testing *P < 0.05; ****P < 0.0001. Source data and exact p values are provided as a Source Data file.

Fig. 6. Global Ca²⁺ responses evoked by MASTER-NAADP in KHYG-1 NK cells and Neuro2A cells.

Global Ca²⁺ signaling was analyzed in Fura2-loaded KHYG-1 cells (A–E), Neuro2A WT or Neuro2A Hn1l/Jpt2^-/- cells (F–K). A MASTER-NAADP (100 µM) or MASTER-NADP (100 µM) were added at RT as indicated. Number of cells indicated in figure. Aggregated data are mean amplitude per peak (B), mean number of Ca²⁺ peaks (C), responding cells (D), and calculation of the mean responsiveness (number of peaks * amplitude; E; as mean ± SEM; MASTER-NAADP, n = 339 cells; MASTER-NADP, n = 425 cells; two-sided, nonparametric Mann-Whitney U test: ****P < 0.0001. F and G MASTER-NAADP (100 µM) or MASTER-NADP (100 µM) were added at RT as indicated to Neuro2A WT (F) or Neuro2A Hn1l/Jpt2^-/- cells (G). Number of cells is indicated in figure. Aggregated data are mean amplitude per peak (H), mean number of Ca²⁺ peaks (I), responding cells (J), and calculation of the mean responsiveness (number of peaks * amplitude; K; as means ± SEM; Neuro2A WT MASTER-NAADP, n = 71 cells; Neuro2A WT MASTER-NADP, n = 50 cells; Neuro2A Hn1l/Jpt2^-/- clone 1G4 MASTER-NAADP, n = 50 cells; Neuro2A Hn1l/Jpt2^-/- clone 1G4 MASTER-NADP, n = 39 cells; Nonparametric Kruskal-Wallis test and Dunn’s correction for multiple testing ****P < 0.0001. Source data are and exact p values provided as a Source Data file.