Splitomicin

Splitomicin is a selective inhibitor of Sir2p with IC50 value of 60 μM and also a inhibitor of fMLP-induced free radicals with IC50 value of 40.79 ± 9.85 μM [1, 3].
Inhibition of the HDA of Sir2p was the most likely mechanism by which splitomicin caused its phenotypic changes. The direct target of splitomicin is Sir2p deacetylase activity. In addition, splitomicin specific inhibited fMLP-induced superoxide anion release.
In vitro, splitomicin inhibitd NAD+-dependent histone deacetylase activity of the Sir2 protein with an IC50 value of 60μM. By using a [3H]-acetylated histone H4 peptide and measuring the NAD+- dependent release of free [3H]acetate in the presence of whole yeast cell extract from an hst2 strain overexpressing yeast SIR2, a cell extract was obtained from a SIR2-overexpressing hst2 strain. The result established Sir2p deacetylase activity as a direct target of splitomicin. In addition, neutrophils induced by either fMLP (1 μM) or PMA (100 nM) were observed using a flow cytometer and the intracellular production of superoxide anions was investigated at different splitomicin concentrations. Splitomicin inhibited fMLP-induced Mac-1 expressionand increase cAMP levels in human neutrophils [1, 3].
Splitomicin’s naphthoic moiety might be responsible for its inhibitory effects on platelets. By using washed human platelets, the inhibitory effects of splitomicin on platelet aggregation were studied and platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2 μg/ml), arachidonic acid (0.5 mM), U46619 (2 μM) or ADP (10 μM) was monitored. Splitomicin inhibited platelet aggregation in a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 μM) was combined with PGE1 (0.5 μM). The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca ion mobilization, TXB2 formation and ATP release [2].
References:
[1]. Bedalov A, Gatbonton T, Irvine WP, et al. Identification of a small molecule inhibitor of Sir2p. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(26): 15113-15118.
[2]. Liu FC, Liao CH, Chang YW, et al. Splitomicin suppresses human platelet aggregation via inhibition of cyclic AMP phosphodiesterase and intracellular Ca++ release. Thrombosis Research, 2009, 124(2): 199-207.
[3]. Liu FC, Day YJ, Liou JT, et al. Splitomicin inhibits fMLP-induced superoxide anion production in human neutrophils by activate cAMP/PKA signaling inhibition of ERK pathway. European Journal of Pharmacology, 2012, 688: 68-75.

Structure-activity studies on splitomicin derivatives as sirtuin inhibitors and computational prediction of binding mode.[Pubmed:18269226]

J Med Chem. 2008 Mar 13;51(5):1203-13.

NAD (+)-dependent histone deacetylases (sirtuins) are enzymes that cleave acetyl groups from lysines in histones and other proteins. Potent selective sirtuin inhibitors are interesting tools for the investigation of the biological functions of those enzymes and may be future drugs for the treatment of cancer. Splitomicin was among the first two inhibitors that were discovered for yeast sirtuins but showed rather weak inhibition on human enzymes. We present detailed structure-activity relationships on Splitomicin derivatives and their inhibition of recombinant Sirt2. To rationalize our experimental results, ligand docking followed by molecular mechanics Poisson-Boltzmann/surface area (MM-PBSA) calculations were carried out. These analyses suggested a molecular basis for the interaction of the beta-phenylSplitomicins with human Sirt2. Protein-based virtual screening resulted in the identification of a novel Sirt2 inhibitor chemotype. Selected inhibitors showed antiproliferative properties and tubulin hyperacetylation in MCF7 breast cancer cells and are promising candidates for further optimization as potential anticancer drugs.

Splitomicin inhibits fMLP-induced superoxide anion production in human neutrophils by activate cAMP/PKA signaling inhibition of ERK pathway.[Pubmed:22634165]

Eur J Pharmacol. 2012 Aug 5;688(1-3):68-75.

Splitomicin, is a cell-permeable lactone derived from naphthol and known to be a potent selective inhibitor of Sir2 (silent information regulator 2). Previous studies have demonstrated that naphtholic compounds possess an inhibitory effect on neutrophils. Here, we present our investigation on the inhibitory effects of Splitomicin in human neutrophils. The primary goal of our study was to locate a possible candidate on inflammatory reactions and to hopefully develop a novel anti-inflammatory therapy. Neutrophils were prepared following standard procedures. Neutrophils induced by either fMLP (1 muM) or PMA (100 nM) were observed using a flow cytometer and the intracellular production of superoxide anions was investigated at different Splitomicin concentrations. The cytosolic Ca(++) influx concentration was measured using a fluorescence spectrophotometer, and Mac-1 expression was detected with a flow cytometer. The MAP kinases were measured using western blotting. Our results showed that Splitomicin inhibited superoxide anion production by fMLP (1 muM) and NaF (20mM) in a concentration-dependent manner (37.5-450 muM). Splitomicin (300 and 450 muM) also suppressed fMLP-induced intracellular calcium ion mobilization and extracellular-signal regulated kinase (ERK) phosphorylation. Moreover, Splitomicin could inhibit fMLP-induced Mac-1 expression and increase cAMP levels in human neutrophils. Our data demonstrated that Splitomicin exhibits a noticeable inhibitory effect on superoxide anion production in human neutrophils. This negative effect was well-correlated with increased cAMP levels via PKA activity and the subsequent inhibition of ERK (p42/p44) phosphorylation to decrease superoxide anion production.

Splitomicin suppresses human platelet aggregation via inhibition of cyclic AMP phosphodiesterase and intracellular Ca++ release.[Pubmed:19327818]

Thromb Res. 2009 Jun;124(2):199-207.

Splitomicin is derived from beta-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of Splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of Splitomicin on platelet aggregation, we used washed human platelets, and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2 microg/ml), arachidonic acid (AA) (0.5 mM), U46619 (2 microM) or ADP (10 microM). Splitomicin inhibited platelet aggregation induced by thrombin, collagen, AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when Splitomicin (150 microM) was combined with PGE1 (0.5 microM). It did not further increase cAMP when combined with IBMX. This data indicated that Splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition, Splitomicin (300 microM) attenuated intracellular Ca(++) mobilization, and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin, collagen, AA or U46619. The inhibitory mechanism of Splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca(++) mobilization, TXB2 formation and ATP release.

Synthesis and biological activity of splitomicin analogs targeted at human NAD(+)-dependent histone deacetylases (sirtuins).[Pubmed:21315612]

Bioorg Med Chem. 2011 Jun 15;19(12):3669-77.

Small molecules interfering with posttranslational modification of histones are of interest as tools to study epigenetic regulation of gene transcription. Specifically, drugs that interfere with histone deacetylation could be useful to induce differentiation, growth arrest as well as apoptotic cell death in tumor cells. One class of histone deacetylases is known as sirtuins some of which (Saccharomyces cerevisiae Sir2) are for example inhibited by the lactone Splitomicin leading to telomeric silencing in yeast. However, Splitomicin is only a micromolar inhibitor of yeast Sir2 and does not inhibit human subtypes and the lactone is prone to hydrolytic ring opening. In preliminary SAR-studies, Splitomicin analogs lacking this hydrolytically labile ring were described as inactive while the naphthalene moiety could successfully be replaced by smaller aromatic rings in a fragment-like dihydrocoumarin. Here we report the synthesis and biological activity of a series of hydrolytically stable analogs with activity against human SIRT1 and 2. These comparatively small compounds characterized by high ligand efficiency are used as a starting point toward the development of specific inhibitors of histone deacetylases from the class of sirtuins.

Identification of a small molecule inhibitor of Sir2p.[Pubmed:11752457]

Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15113-8.

Sir2p is an NAD(+)-dependent histone deacetylase required for chromatin-dependent silencing in yeast. In a cell-based screen for inhibitors of Sir2p, we identified a compound, Splitomicin, that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at telomeres, silent mating-type loci, and the ribosomal DNA. In addition, whole genome microarray experiments show that Splitomicin selectively inhibits Sir2p. In vitro, Splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein. Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein. By using Splitomicin as a chemical genetic probe, we demonstrate that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells.

Splitomicin (Splitomycin) is a selective Sir2p inhibitor. Splitomicin inhibits NAD+-dependent HDAC activity of Sir2 protein. Splitomicin induces dose-dependent inhibition of HDAC in the yeast extract with an IC50 of 60 μM.

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