Identification of a chemical probe for NAADP by virtual screening

Abstract

Research into the biological role of the Ca(2+)-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca(2+) increases in mouse pancreatic beta cells.

Figure 1

Strategy and results of a ligand-based virtual screen for drug-like molecules with NAADP-like activity. (a) Flow chart outlining the process of ligand-based virtual screening showing the steps (left column), software (yellow boxes), number of compounds and time taken (upper limits on a single computer). (b) Two-dimensional chemical structures of NAADP and Ned-19. (c) Overlay of NAADP (yellow carbons and red surface) and Ned-19 (grey carbons blue surface). (d) Three-dimensional chemical structures of NAADP and Ned-19 with electrostatic surfaces coded by color (red for negative and blue for positive).

Figure 2

Certain virtual screening hits have biological activity. (a) Relative antagonist activity of the hit compounds against NAADP-mediated Ca²⁺ release. Bars with an asterisk indicate inhibition significantly less than the control based on a one-tailed t-test with p ≤ 0.05. Hit compounds were present at 6-125 μM depending on their aqueous solubility (see Supplementary Methods) and NAADP was added at its EC₅₀ (35 nM). (b) The virtual screening hit Ned-19 selectively inhibits NAADP-mediated Ca²⁺ release in a Ca²⁺-mobilizing bioassay (sea urchin egg homogenate; left panel). All three Ca²⁺-releasing second messengers release sequestered Ca²⁺ in the control when added at their half-maximal concentration (right trace). Traces show the fluorescence (Relative Fluorescence Units, RFU) from the Ca²⁺-reporting dye fluo-3. (c) Concentration-inhibition curves for Ned-19 on Ca²⁺ release mediated by the EC₅₀ concentrations of inositol 1,4,5-trisphosphate (IP₃; 1 μM), cyclic ADP-ribose (cADPR; 300 nM) and NAADP (35 nM). (d,e) Concentration-inhibition curves for the compounds Ned-19 (100 μM) (d) and Ned-20 (100 μM) (e) on NAADP-mediated Ca²⁺ release. (f) The compound Ned-19 competes with [³²P]NAADP binding. “Con” is the amount of binding in the control. (g) Dissociation of Ned-19 (10 μM) and NAADP (10 nM) determined by the recovery of [³²P]NAADP binding after 5 days of incubation. A value of 100 percent corresponds to about 1,500 counts per min. (h) Chemical structures of the ‘trans’ and ‘cis’ diastereomers of Ned-19. (i) Concentration-inhibition curves for Ned-19 inhibition of NAADP-medicated Ca²⁺ release. (j) Concentration-inhibition curves for Ned-19 on [³²P]NAADP binding.

Figure 3

The virtual screening hit Ned-19 antagonizes Ca²⁺ signaling in intact cells. (a) Ned-19 (100 μM in the bath) inhibits NAADP-induced Ca²⁺ release in an intact sea urchin egg. Ca²⁺ was monitored with the Ca²⁺-reporting dye Oregon Green 488-BAPTA Dextran (10 μM, microinjected). NAADP was pressure microinjected (100 μM in the pipette) so that each injection delivered approximately 1 μM. (b) Ned-19 inhibits NAADP-induced Ca²⁺ increases in mouse pancreatic beta cells. Ca²⁺-dependent current traces are from beta cells that were voltage-clamped with a patch pipette containing 100 nM NAADP in the presence or absence of 100 μM Ned-19 in the bath.

Figure 4

The virtual screening hit Ned-19 is fluorescent and labels receptors in intact cells. (a) Excitation (368 nm) and emission (425 nm) spectra of Ned-19. Ned-19 was in dimethylsulfoxide at 4 mM. (b) Ned-19 but not Lysotracker Red labeling of pancreatic beta cells is resistant to the V-type proton pump inhibitor bafilomycin A1. Lyostracker red but not Ned-19 labeling is resistant to pre-treatment with NAADP-acetyoxymethyl ester (NAADP-AM). Scale bars, 5 μm.

Figure 5

The virtual screening hit Ned-19 reveals that glucose-induced Ca²⁺ increases require NAADP signaling. (a) Ned-19 inhibits glucose-induced Ca²⁺ oscillations. (b) Ned-20 does not affect glucose-induced Ca²⁺ increases. (c,d) Ned-19 reduces glucose-induced Ca²⁺ increases in a concentration-dependent manner. Islets were pre-incubated with the indicated concentration of Ned-19 for 30 min before the addition of 15 mM glucose. “Con” is the control. (e) Ned-19 does not affect the activation of voltage-gated Ca²⁺ channels. Cells were incubated in glucose (10 mM) and diazoxide (100 μM) and then depolarized with KCl (45 mM) in the presence and absence of Ned-19 (100 μM). (f) Ned-19 does not interfere with mitochondrial production of NAD(P)H. All cells were maintained in 3 mM glucose unless otherwise indicated.

Figure 6