Ken Ishigami

Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA

The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 (1) and its methylated derivative, spliceostatin A (2), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.

Radicicol Binds and Inhibits Mammalian ATP Citrate Lyase

Six different biotinylated radicicol derivatives were synthesized as affinity probes for identification of cellular radicicol-binding proteins. Derivatives biotinylated at the C-17 (BR-1) and C-11 (BR-6) positions retained the activity of morphological reversion in v-src-transformed 3Y1 fibroblasts. Two radicicol-binding proteins, 120 and 90-kDa in size, were detected in HeLa cell extracts by employing BR-1 and BR-6, respectively. The 90-kDa protein bound to BR-6 was identified to be Hsp90 by immunoblotting. The 120-kDa protein bound to BR-1 was purified from rabbit reticulocyte lysate, and its internal amino acid sequence was identical to that of human and rat ATP citrate lyase. The identity of the 120-kDa protein as ATP citrate lyase was confirmed by immunoblotting. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. These results suggest that radicicol binds the two proteins through different molecular portions of its structure. BR-1-bound ATP citrate lyase isolated from rabbit reticulocyte lysate showed no enzymatic activity. The activity of rat liver ATP citrate lyase was inhibited by radicicol and BR-1 but not by BR-6. Kinetic analysis demonstrated that radicicol was a non-competitive inhibitor of ATP citrate lyase withK i values for citrate and ATP of 13 and 7 μm, respectively.

Spliceostatin A blocks angiogenesis by inhibiting global gene expression including VEGF.

Spliceostatin A (SSA) is a methylated derivative of an antitumor natural product FR901464, which specifically binds and inhibits the SF3b spliceosome sub‐complex. To investigate the selective antitumor activity of SSA, we focused on the regulation of vascular endothelial growth factor (VEGF) mRNA, since VEGF is a key regulatory component in tumor angiogenesis and known for the intricate regulation of mRNA processing, such as alternative splicing. We found that in HeLa cells SSA reduced the amount of both mRNA and protein of VEGF. Spliceostatin A not only inhibited the splicing reaction of VEGF pre‐mRNA but also reduced the total amount of VEGF’s transcripts, while SSA affected GAPDH mRNA to a lesser extent. Given a significant reduction in VEGF gene expression, SSA was expected to possess anti‐angiogenic activity in vivo. Indeed, SSA inhibited cancer cell‐derived angiogenesis in vivo in a chicken chorioallantoic membrane (CAM) assay. The inhibition of angiogenesis with SSA was abolished by addition of exogenous VEGF. We also performed global gene expression analyses of HeLa cells and found that the expression levels of 38% of total genes including VEGF decreased to <50% of the basal levels following 16 h of SSA treatment. These results suggest that the global interference of gene expression including VEGF in tumor cells is at least one of the mechanisms by which SSA (or FR901464) exhibits its strong antitumor activity. (Cancer Sci 2010; 101: 2483–2489)

Structure-activity Relationship for FR901464 : A Versatile Method for the Conversion and Preparation of Biologically Active Biotinylated Probes

The structure-activity relationship for FR901464, a potent cell-cycle inhibitor, was examined by synthesizing its analogs. A versatile method for converting FR901464 was devised. This method made it possible to synthesize biologically active FR901464-biotin conjugates which could be used to isolate the binding proteins.

First total synthesis and determination of the absolute configuration of mueggelone

Abstract All the four possible stereoisomers of mueggelone, an inhibitor of fish development, were efficiently synthesized in a stereoselective manner starting from d -arabinose, and the absolute configuration was determined to be 9 R , 12 S , 13 S .

Synthesis of all the four possible stereoisomers of acaterin, naturally occurring ACAT inhibitor, and the determination of its absolute configuration☆

Abstract Enantioselective synthesis of all the possible stereoisomers of acaterin 1 , naturally occurring ACAT inhibitor with acetogenin-type skeleton, was accomplished starting from both the enantiomers of ethyl 3-hydroxy butanoate 3 . Stereochemistry of synthetic samples 1 and pseudo- 1 was unambiguously assigned by converting to the authentic compound. The absolute configuration of natural acaterin was determined as (4 R , 1′ R ) by careful comparison of TLC behavior and spectral and optical data.

Synthetic Studies of Natural 10-Membered Lactones, Mueggelone, Microcarpalide, and Sch 642305, Which Have Interesting Bioactivities

A number of 10-membered lactones have been isolated as secondary metabolites, and many of them have interesting and potent activities. Because of synthetic and biological interests, many synthetic studies of these compounds have been made. This review summarizes synthetic approaches to three natural 10-membered lactones, mueggelone, microcarpalide, and Sch 642305. Mueggelone is an inhibitor of fish development, microcarpalide is a microfilament disrupting agent, and Sch 642305 is an inhibitor of bacterial DNA primase.

Synthesis of microcarpalide, a microfilament disrupting agent

Microcarpalide is a strong microfilament disrupting agent. The convergent and stereoselective synthesis of microcarpalide was succeeded via Julia olefination and macrolactonization.

Short-step Synthesis of Chenodiol from Stigmasterol

Chenodiol is an important bile acid widely used for gallstone dissolution and cholestatic liver diseases. We succeeded in a short-step synthesis of chenodiol, starting from the safer phytosterol, stigmasterol.

First total synthesis of mueggelone

Abstract All the four possible stereoisomers of mueggelone, an inhibitor of fish development, were efficiently synthesized in a stereoselective manner starting from d -arabinose, and the absolute configuration was determined to be 9 R ,12 S ,13 S .