Sridhar R. Vasudevan

Identification of a chemical probe for NAADP by virtual screening

Research into the biological role of the Ca2+-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 3D-shape and electrostatic similarity. 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 Ca2+ increases in mouse pancreatic beta cells.

A safe lithium mimetic for bipolar disorder

Lithium is the most effective mood stabilizer for the treatment of bipolar disorder, but it is toxic at only twice the therapeutic dosage and has many undesirable side effects. It is likely that a small molecule could be found with lithium-like efficacy but without toxicity through target-based drug discovery; however, lithium’s therapeutic target remains equivocal. Inositol monophosphatase is a possible target but no bioavailable inhibitors exist. Here we report that the antioxidant ebselen inhibits inositol monophosphatase and induces lithium-like effects on mouse behaviour, which are reversed with inositol, consistent with a mechanism involving inhibition of inositol recycling. Ebselen is part of the National Institutes of Health Clinical Collection, a chemical library of bioavailable drugs considered clinically safe but without proven use. Therefore, ebselen represents a lithium mimetic with the potential both to validate inositol monophosphatase inhibition as a treatment for bipolar disorder and to serve as a treatment itself.

The CRTC1-SIK1 Pathway Regulates Entrainment of the Circadian Clock

Summary Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.

Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca2+ release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca2+ release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca2+ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca2+ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.

Effect of the Putative Lithium Mimetic Ebselen on Brain Myo -Inositol, Sleep, and Emotional Processing in Humans

Lithium remains the gold standard in treating bipolar disorder but has unwanted toxicity and side effects. We previously reported that ebselen inhibits inositol monophosphatase (IMPase) and exhibits lithium-like effects in animal models through lowering of inositol. Ebselen has been tested in clinical trials for other disorders, enabling us to determine for the first time the effect of a blood–brain barrier-penetrant IMPase inhibitor on human central nervous system (CNS) function. We now report that in a double-blind, placebo-controlled trial with healthy participants, acute oral ebselen reduced brain myo-inositol in the anterior cingulate cortex, consistent with CNS target engagement. Ebselen decreased slow-wave sleep and affected emotional processing by increasing recognition of some emotions, decreasing latency time in the acoustic startle paradigm, and decreasing the reinforcement of rewarding stimuli. In summary, ebselen affects the phosphoinositide cycle and has CNS effects on surrogate markers that may be relevant to the treatment of bipolar disorder that can be tested in future clinical trials.

The Calcium-mobilizing Messenger Nicotinic Acid Adenine Dinucleotide Phosphate Participates in Sperm Activation by Mediating the Acrosome Reaction

Before a sperm can fertilize an egg it must undergo a final activation step induced by the egg termed the acrosome reaction. During the acrosome reaction a lysosome-related organelle, the acrosome, fuses with the plasma membrane to release hydrolytic enzymes and expose an egg-binding protein. Because NAADP (nicotinic acid adenine dinucleotide phosphate) releases Ca2+ from acidic lysosome-related organelles in other cell types, we investigated a possible role for NAADP in mediating the acrosome reaction. We report that NAADP binds with high affinity to permeabilized sea urchin sperm. Moreover, we used Mn2+ quenching of luminal fura-2 and 45Ca2+ to directly demonstrate NAADP regulation of a cation channel on the acrosome. Additionally, we show that NAADP synthesis occurs through base exchange and is driven by an increase in Ca2+. We propose a new model for acrosome reaction signaling in which Ca2+ influx initiated by egg jelly stimulates NAADP synthesis and that this NAADP acts on its receptor/channel on the acrosome to release Ca2+ to drive acrosomal exocytosis.

Shape-based reprofiling of FDA-approved drugs for the H₁ histamine receptor.

Reprofiling of existing drugs to treat conditions not originally targeted is an attractive means of addressing the problem of a decreasing stream of approved drugs. To determine if 3D shape similarity can be used to rationalize an otherwise serendipitous process, we employed 3D shape-based virtual screening to reprofile existing FDA-approved drugs. The study was conducted in two phases. First, multiple histamine H(1) receptor antagonists were identified to be used as query molecules, and these were compared to a database of approved drugs. Second, the hits were ranked according to 3D similarity and the top drugs evaluated in a cell-based assay. The virtual screening methodology proved highly successful, as 13 of 23 top drugs tested selectively inhibited histamine-induced calcium release with the best being chlorprothixene (IC(50) 1 nM). Finally, we confirmed that the drugs identified using the cell-based assay were all acting at the receptor level by conducting a radioligand-binding assay using rat membrane.

Mining free compound databases to identify candidates selected by virtual screening.

Background: The search for effective ways to discover new and better chemical probes to explore basic biology is never ending. With the advent of genomics and proteomics, data are rapidly accumulating on the role of several proteins and their implications in cellular function. However, the accumulation of knowledge on new proteins is not reciprocated at the level of new chemical probes that would help us understand protein function by modulating it. An emerging solution is virtual screening in which millions of molecules are evaluated with computers rapidly and cheaply relative to high-throughput screening. Objective/method: In this perspective we will use our experience to illustrate how small, modestly funded laboratories can use virtual screening successfully to develop chemical probes. The chief contributors to make this possible are the availability of free databases containing purchasable compounds and powerful and accessible software. Conclusion: That virtual screening can be used to find chemical probes is a reality. Unfortunately, groups that would benefit most from this powerful technique underuse it. Making biologists aware of the existence or capability of virtual screening will greatly speed the identification of new chemical probes.

Characterization of NAADP-mediated calcium signaling in human spermatozoa

Ca(2+) signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca(2+) signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca(2+) and pH. Ca(2+) fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca(2+)] increases in human sperm even in the absence of extracellular Ca(2+). Using LysoTracker, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-l-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker, suggesting that these stores are the targets of NAADP action.

Scaffold Hopping with Virtual Screening from IP3 to a Drug-Like Partial Agonist of the Inositol Trisphosphate Receptor

Inositol 1,4,5‐trisphosphate (IP3) is a universal signalling molecule that releases calcium from stores within cells by activating the IP3 receptor. Although chemical tools that modulate the IP3 receptor exist, none is ideal due to trade offs between potency, selectivity and cell permeability, and their chemical properties make them challenging starting points for optimisation. Therefore, to find new leads, we used virtual screening to scaffold hop from IP3 by using the program ROCS to perform a 3D ligand‐based screen of the ZINC database of purchasable compounds. We then used the program FRED to dock the top‐ranking hits into the IP3 binding pocket of the receptor. We tested the 12 highest‐scoring hits in a calcium‐release bioassay and identified SI‐9 as a partial agonist. SI‐9 competed with [3H]IP3 binding, and reduced histamine‐induced calcium signalling in HeLa cells. SI‐9 has a novel 2D scaffold that represents a tractable lead for designing improved IP3 receptor modulators.