microbial and natural metabolites that inhibit splicing: a powerful

Review ArticleMicrobial and Natural Metabolites That Inhibit SplicingA Powerful Alternative for Cancer Treatment

Nancy Martiacutenez-Montiel1 Nora Hilda Rosas-Murrieta23 Moacutenica Martiacutenez-Montiel1

Mayra Patricia Gaspariano-Cholula1 and Rebeca D Martiacutenez-Contreras1

1Laboratorio de Ecologıa Molecular Microbiana Centro de Investigaciones en Ciencias Microbiologicas Instituto de CienciasBenemerita Universidad Autonoma de Puebla Edificio IC11 Ciudad Universitaria 72570 Colonia San Manuel PUE Mexico2Laboratorio de Bioquımica y Biologıa Molecular Instituto de Ciencias Benemerita Universidad Autonoma de Puebla Edificio 103HCiudad Universitaria 72550 Colonia San Manuel PUE Mexico3Posgrado en Ciencias Quımicas Benemerita Universidad Autonoma de Puebla Edificio 105 I Ciudad Universitaria72570 Colonia San Manuel PUE Mexico

Correspondence should be addressed to Rebeca D Martınez-Contreras rebecamartinezcorreobuapmx

Received 19 March 2016 Revised 27 June 2016 Accepted 3 July 2016

Academic Editor Yiannis Kourkoutas

Copyright copy 2016 Nancy Martınez-Montiel et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In eukaryotes genes are frequently interrupted with noncoding sequences named introns Alternative splicing is a nuclearmechanism bywhich these introns are removed and flanking coding regions named exons are joined together to generate amessagethat will be translated in the cytoplasm This mechanism is catalyzed by a complex machinery known as the spliceosome whichis conformed by more than 300 proteins and ribonucleoproteins that activate and regulate the precision of gene expression whenassembled It has been proposed that several genetic diseases are related to defects in the splicing process including cancer Forthis reason natural products that show the ability to regulate splicing have attracted enormous attention due to its potential usefor cancer treatment Some microbial metabolites have shown the ability to inhibit gene splicing and the molecular mechanismresponsible for this inhibition is being studied for future applications Here we summarize the main types of natural productsthat have been characterized as splicing inhibitors the recent advances regarding molecular and cellular effects related to thesemolecules and the applications reported so far in cancer therapeutics

1 Introduction

In eukaryotes coding regions of the genome called exonsare interrupted by noncoding sequences known as intronsDuring transcription exons are identified while introns areremoved from the immature mRNA (or pre-mRNA) to gen-erate a mature and functional mRNA molecule The mecha-nism responsible for this process corresponds to splicing andthemachinery that performs this highly regulated event is thespliceosome which is integrated by five small nuclear ribonu-cleoproteic particles (snRNPs) and more than 200 proteinsthat include auxiliary regulatory factors and components ofother co- and posttranscriptional machineries [1] Duringsplicing a series of RNA-RNA RNA-protein and protein-protein interactions are responsible for the decisions that

determine which sequences will be included in the maturetranscript [2]Moreover some sequences can be incorporateddifferentially into separated splicing events leading to anincrease in the coding potential of the genome by a processcalled alternative splicing

2 Alternative Splicing and the Spliceosome

The general splicing mechanism involves the recognition ofexonintron boundaries in a sequence-dependent mannerIn mammals the 51015840 end of the intron (51015840 splice site or51015840ss) contains a characteristic TG which recruits snRNP U1On the opposite side the 31015840 end of the intron (31015840ss) showsan invariant region called the branch point sequence (BPS)followed by a polypyrimidine-rich tract (pY-tract) and a

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3681094 14 pageshttpdxdoiorg10115520163681094

2 BioMed Research International

conserved AG dinucleotide that indicates the end of theintron [3] The recognition of the 31015840ss involves the binding ofSF1 to the BPS and the recruitment of the snRNPU2 auxiliaryfactor (U2AF) to the pY-tract and the AG dinucleotide Afterthe recognition of both exonintron boundaries an earlycomplex is formed that commits pre-mRNA to undergoingsplicing where U2 snRNP is also recruited to the 31015840ss U2snRNP recruitment to the pre-mRNA is one of the key stepsthat triggers additional interactions leading to the formationof catalytic spliceosome complexes due to the incorporationof the tri-snRNP U4U5U6 within which numerous RNArearrangements and modifications in protein compositioncontribute to complete a splicing cycle [2 3]

Like most of the snRNPs U2 is a ribonucleoproteic com-plex formed by 7 Smproteins (which are common for spliceo-somal snRNPs) and 17 specific proteins being the largestsnRNP [3] Among the specific snRNP U2 components twoprotein subcomplexes are found SF3a and SF3b [3ndash5] SF3aincludes 3 subunits of 60 66 and 120 kDa [6] while SF3bshows at least 8 specific subunits of 10 14a 14b 49 125130 140 and 155 kDa [7] Components of the SF3a and SF3bsubcomplexes bind to sequences in the pre-mRNA tetheringU2 snRNP to the BPS and the 31015840ss SF3b 155 is one of themostconserved subunits of U2 snRNP and it has shown the abilityto bind splicing factors U2AF65 and p14 [3 8] Interestinglythis subunit has been related to the antiproliferative effectobserved for some natural products that regulate the splicingmechanism and it results clear in the fact that targeting thespliceosome and modulating splice-site recognition could berelevant for the development of new therapeutic approachesas will be further discussed

3 The Role of Alternative Splicing inHuman Disease

Over the past 10 years the role of alternative splicing inhuman disease has been growing When the human genomeproject was completed in silico analysis predicted that 75 ofthe human genes underwent splicing [26] and that 15 to 50of the genetic diseases were related to aberrant splicing events[27] From this initial observation several studies have linkedsplicing defects with specific genetic disorders However thefull significance of the role in alternative splicing in humandisease remains to be elucidated Some diseases that havebeen linked to defects on splicing include dilated cardiomy-opathy autism spectrum disorder spinal muscular atro-phy schizophrenia cardiac hypertrophy amyotrophic lateralsclerosis and frontotemporal dementia [28] In all thesecases the molecular insights related to the splicing defectthat originates the disease have been dissected The preciseregulation of the splicing event varies for each pre-mRNAand for this reason it is time consuming to demonstrate themolecular mechanism that regulates the alternative splicingfor each gene Moreover this regulation also depends on thecellular context complicating the scene In this regard futureefforts need to be developed in order to dissect the alternativesplicing event that is related to each disease and the possibletherapeutic tools that could be applied

One specific group of diseases that have been related tosplicing corresponds to different types of cancer and onlyrecently the determinant role of splicing in cancer has beenacknowledged [29 30] Several features of splicing eventsrelated to tumor progression have been reported and it iswell documented that the alternative splicing of different pre-mRNAs is altered during oncogenic progression with theconcomitant development of cancer features like an increasein vascularization cell proliferation and invasion [31 32]Themolecular hallmarks documented for several types of cancerhave been recapitulated in an attempt to orientate futureefforts towards cancer treatment through alternative splicingmodulation [33ndash35] Considering all this evidences severalstudies have been oriented to modulate alternative splicingin order to treat cancer

4 Microbial Metabolites ThatRegulate Splicing

Natural products have been traditionally sought from acti-nomycetes filamentous fungi and medicinal plants In thisregard several derivatives of bacterial fermentation as well astheir synthetic equivalents possess the ability to interact withcomponents of the spliceosome In some cases the effect onsplicing associated with these drugs is achieved through thedirect regulation of the expression of genes that are relevantfor cancer progression [36] Dozens of small molecule effec-tors targeting the alternative splicing process have been iden-tified and evaluated as drug candidates including a naturalproduct of Pseudomonas sp number 2663 called FR901464[9] natural products from Streptomyces platensis Mer-11107that originated the group of Pladienolides [37] Herboxidiene[15] and Isoginkgetin [38]Thesemolecules and their deriva-tives have shown activity as splicing inhibitors and manyof them demonstrated potent antiproliferative properties inhuman cancer cell lines being in general less toxic to normalhuman cells [39]

41 FR901464 and Derivatives Spliceostatins are a groupof compounds derived from the natural product FR901464which was identified initially as an antitumor compoundIn the original study FR901463 FR901464 and FR901465were isolated from the fermentation broth of Pseudomonassp number 2663 [9] These 3 compounds are soluble inacetonitrile chloroform and ethyl acetate and poorly solublein water and insoluble in hexane They all show strong UVabsorption at 235 nm distinctive of a conjugated diene whilethe IR spectra indicated the presence of hydroxyl ester and aconjugated amide carbonyl (Figure 1) Initially the three com-pounds from the FR9014 series mentioned before were testedfor their biological activity As a result they all enhancedthe transcriptional activity of SV40 in a CAT assay Besidesthey were all cytotoxic according to the MTT method in thefollowing human adenocarcinomas A549 lung cells MCF-7mammary cells or HCT116 colon cells [9] Moreover the 3compounds extended the life of mice bearing ascetic tumorsFR901464 being the one showing themost potent effect on thetumor systems assayed [9] Furthermore FR901464 induced

BioMed Research International 3

FR series

FR901463 FR901464 FR901465Spliceostatins

Spliceostatin A Spliceostatin B Spliceostatin E

Thailanstatins

Thailanstatin A Thailanstatin B Thailanstatin C

Meayamycin

Meayamycins

Sudemycins

Sudemycin E

O

O O O O

O

O

O O O O

O

O

O O O OOHOH OH

OHHOHOHOHO Cl H

NHN

HN

H

HO

OH OH

HO HO HO

HO HO HO

HO

HO

Cl

OMe

NHN

HN

N

HN

HN

HN

HN

HN

HN H

HN

O

O O O O

O

O OO

O

O

O

OO

O

OOO O

O

OO O O

O

OOOOO

O O

O O O OO

O

OOO O O

O

O

OO

O

O

O OO

O O

OO

O

OOOO

O

Cl

Sudemycin F1Sudemycin C1

Figure 1 FR901464 and derivatives The FR9014 series were isolated from Pseudomonas sp number 2663 and constitute the firstantiproliferativemolecules associated with splicing inhibition Spliceostatin A is amethylated derivative of FR901464 Spliceostatin Bwas alsoisolated from Pseudomonas sp number 2663 Spliceostatin E was isolated from Burkholderia sp FERMBP3421Thailanstatins were recoveredfrom Burkholderia thailandensisMSMB43 Meayamycin and Sudemycins are synthetic derivatives from the natural products depicted

characteristic G1 and G2M phase arrest in the cell cycle andsuppressed the transcription of some inducible endogenousbut not housekeeping genes in M-8 cells In this same cellline internucleosomal degradation of genomic DNA showedthe same kinetics corresponding to the activation of SV40promoter-dependent cellular transcription suggesting that achromatin rearrangement occurs upon the treatmentwith thedrug Despite this effect in inducing viral gene promotersit was observed that FR901464 reduces the mRNA levels ofseveral endogenous genes including c-Myc [9]

Spliceostatin A is a methylated andmore stable derivativeof FR901464 (Figure 1) and they both show similar activ-ity [37 40] the synthesis and activity for both moleculeshave also been reported [41 42] Even when there are fewstudies on the molecular interactions that mediate the effectof Spliceostatin and related molecules the antiproliferativeeffect of Spliceostatin has been associated with splicing

and seems to be equivalent to the one registered afterknocking down SF3b155 [10] Using immunoprecipitationassays further studies demonstrated that FR901464 andits methylated derivative Spliceostatin A inhibit pre-mRNAsplicing both in vivo and in vitro by binding noncovalentlyto the SF3b subcomplex in the U2 snRNP In the same studythe treatment with Spliceostatin A allowed the identificationof immature forms of p27 by RT-PCR suggesting thatpre-mRNA molecules that have not been fully spliced aretransported to the cytoplasm inducing the translation ofaberrant mRNAs [11]

Another analogous compound of FR901464 namedSpliceostatin B was purified from the fermentation brothof Pseudomonas sp number 2663 [43] Spliceostatin B issoluble in DMSO acetonitrile acetone water chloroformand dichloromethane The structure of Spliceostatin B wasdetermined using UV IR HR-MS and NMR spectroscopic


analyses showing that it differs structurally from FR901464at four points the substitution of an epoxide group at C3position with a terminal methylene moiety the presence ofa carboxyl moiety at C17 position and the absence of twohydroxyl groups at C1 and C4 positions respectively Thesestructural features are relevant for the biological functiongiven the fact that it has been reported that loosing the C4hydrogen bond donor decreases the cytotoxicity and thatthe C3 epoxide moiety is necessary for bioactivity [44] Thefunctional analog Spliceostatin B showed cytotoxic effect inthree human cancer cell lines HCT-116 MDA-MB-235 andH232A using the MTT method [45] but its activity wasweaker than the one observed for FR901464 according tothe IC values obtained [43] and in good correlation with thestructural features just mentioned

Other natural products considered Spliceostatin analogswere isolated from the fermentation broth of Burkholderiasp strain FERM BP3421 [46] Among these new moleculesSpliceostatin E exhibited good potency against multiplehuman cancer cell lines with IC

50values ranging from 15

to 41 nM The structure of Spliceostatin E was elucidated byextensive spectroscopic studies and resulted structurally inless complex than Spliceostatins A and B [46] Even whenSpliceostatin E maintains the cytotoxic activity the syntheticmolecule showed no inhibition of splicing and it did not alterthe structure of nuclear speckles [42]

It has been determined that the fr9 gene cluster isresponsible for the biosynthesis of FR901464 in Pseudomonassp number 2663 The biosynthetic fr9 gene cluster spans aDNA region of approximately 81 kb and includes 20 genes(fr9A through fr9T) Using this information a bioinformaticapproach was conducted in order to identify other strainsthat could produce Spliceostatin-like metabolites Using thiscomparative analysis while mining the genome of Burkholde-ria thailandensis MSMB43 elicited the identification of abiosynthetic gene cluster similar to fr9 that was named tstreferring to the Thailanstatin compounds it produces whichare functional analogs of Spliceostatins The tst gene clusterspans a DNA region of 78 kb which contains 15 ORFs des-ignated tstA through tstR The putative functions for the tstgene products were deduced by sequence comparisons withthe FR9 proteins and with other bacterial homologs wherethe most striking difference is the absence of the equivalentfr9S and fr9T genes from the tst gene cluster A detailedanalysis of this cluster suggested a possible biosynthetic routefor Thailanstatins which is similar to the one demonstratedfor FR901464 and corresponds to a hybrid pathway involvinga polyketide synthase and a nonribosomal peptide synthetase[47]

Consistent with the bioinformatic approach Thailan-statins A B and C were isolated from the culture brothof Burkholderia thailandensis MSMB43 and they proved tobe significantly more stable natural analogs of FR901464[47] These molecules are more stable because they lack ahydroxyl group found in FR901464 and they show an extracarboxyl moiety instead as revealed by the HR-MS NMRUV and IR spectrometry Thailanstatins possess the samelinear polyketide-peptide framework observed in FR901464but they lack a hydroxyl group at the C1 position while

Pladienolide

HO OO

OAcOH

OHO

Figure 2 Pladienolide structure Pladienolide is a 12-memberedmacrolide that possesses a long side chain at the carbon bearinglactone oxygen

showing an extra carboxyl moiety at the C17 positionThailanstatins B and C have a chloride substituent at the C3position instead of the epoxide functionality observed forboth Thailanstatin A and FR901464 Finally ThailanstatinB possesses a dimethyl acetyl group at the distal end whileThailanstatin C shows an acetyl group instead [47]

During the biological tests performed all Thailanstatinsexhibited strong antiproliferative activities when tested inthe following human cancer cell lines DU-145 (prostatecancer) NCI-H232A (non-small-cell lung cancer) MDA-MB-231 (triple-negative breast cancer) and SKOV-3 (ovariancancer) Thailanstatin A being the one showing the strongesteffect Moreover the three compounds showed the ability toinhibit in vitro splicing and againThailanstatin A showed thebest result being as strong as FR941464 in inhibiting splicing[47] A summary of themolecular effects depicted for FR9414series and other splicing inhibitors is presented in Table 1

Considering that the molecules just presented are struc-turally quite complex this results in difficulty to accomplishtheir structural modification However synthetic derivativeshave been generated (Figure 1) including Meayamycin andSudemycins [48]

42 Pladienolides Pladienolide B is a macrocyclic lactoneoriginally obtained from Streptomyces platensis Mer-11107strain isolated from a soil sample collected in KanagawaJapan (accession number FERM P-18144 Bioconsortia Pro-gram Laboratory National Institute of Advanced IndustrialScience and Technology Japan)The compound deposited inthe Open Chemistry Database (PubChem ID 52946850) hasa molecular weight of 5387132 gmol (Figure 2)

Pladienolide B was initially identified in 2004 as part ofa work that reported the isolation and structural and func-tional characterization of seven 12-membered macrocycliccompounds named Pladienolides A to G [37] All these com-pounds displayed antiproliferative and tumor suppressiveactivities when assayed in cell culture and xenograft modelsparticularly Pladienolides B and D

The initial extraction to isolate Pladienolides was per-formed with n-butanol from the fermentation broth ofStreptomyces platensis Further chromatography steps overSephadex LH-

20and silica gel column were accomplished


Table 1 Molecular effects of different splicing inhibitors

Splicing inhibitor Cell line Effect Reference

FR9014 series

MCF-7 Induces G1 and G2M arrest of the cell cycle [9]

HeLaInhibits the recognition of the branch point sequence [10]Binding affinity to SAP145 [11]Arrest of SF3b [12]

MDA-MB-468 Interacts with SF3b subunit SAP145 [13]

PladienolideWiDr Interacts with SF3b subunit SAP130 [13]

HeLa Interacts with SF3b Remodeling of U2 snRNP to expose the branchpoint-binding region [14]

Herboxidiene

Normal humanfibroblast cell line

WI-382Induces G1 and G2M arrest of the cell cycle [15]

HeLa Causes arrest in G1 and G2M phases and interacts with SF3b1 subunitSAP145 [16]

Trichostatin WiDr Interacts with SF3B subunit SAP130 [13]

Isoginkgetin

HT1080Inhibition of Cathepsin K and MMP9 [17]Inhibits metalloproteinase MMP9 production and increases thesynthesis of metalloproteinase inhibitor TIMP-1 [18]

HEK293 Stimulates IL-8 expression [19]

Thyroid cancer Increases expression of specific IL-32 isoforms and stimulates theexpression of IL-8 and CXCR1 [19]

The bioactive fractions recovered were subjected to prepar-ative HPLC and each fraction containing pure Pladienolideswas freeze-dried

The chemical properties of Pladienolides were deter-mined using spectroscopic methods According to thephysicochemical characterization of Pladienolides (AndashG)they are soluble in methanol acetone n-butanol ethylacetate and DMSO but not in n-hexane or poorly solublein water In all compounds there is a diene system evidencedby the UV absorption at 240 nm The chemical structure ofPladienolides A (1) B (2) C (3) D (4) E (5) F (6) and G (7)(Figure 3) was determined by the analyses of NMR MS IRand 2DNMRspectraThe carbonyl andhydroxyl groupsweredetected in the IR spectra All Pladienolides are 12-memberedmacrolides possessing a diene unit and one epoxide moietywith a long side chain at the carbon that bears lactone oxygen[37] Regarding their biological activity Pladienolides havehighly potent in vitro and in vivo antitumor activities withpotential for use in anticancer therapy [20]

Pladienolide B has shown strong in vitro and in vivo anti-tumor activity and growth inhibitory effect against variouscell lines some of them being resistant to chemotherapeuticagents routinely used Pladienolide B and some for theiranalogs induce cell cycle arrest at both G1 and G2M [49]

A different study using Pladienolides was oriented toidentify compounds that contribute of the adaptation ofcancer cells to hypoxia using HIF-1 an HLH transcriptionfactor involved in hypoxia adaptation in cancer cells Thisapproach consisted in searching inhibitors of hypoxia adapta-tion involved in the regulation of angiogenesis and anaerobicmetabolism considering that hypoxia-inducible genes arerelevant for growth of cancer cells The screening system

consisted of the placental alkaline phosphatase (PLAP) genereporter under the control of the human VEGF promotercontaining the hypoxia-responsive element (HRE) that bindsHIF-1 The reporter construction was transfected and thehypoxia-induced PLAP expression was analyzed in the U251human glioma cells Using a high throughput screening Pla-dienolides were identified as inhibitors of hypoxia-inducedPLAP expression when the cells were exposed to hypoxicconditions [50 51] The pure compounds were probed intheir anti-VEGF-PLAP and antiproliferative activity but onlyPladienolides B and D showed a strong activity in both testswith IC

50of 18 and 35 for Pladienolide B and of 51 and

6 nM for Pladienolide D In other studies Pladienolide Bshowed a potent tumor regression and inhibition of mousexenograft acting at low-nanomolar concentrations (Table 2)The cell growth inhibition properties of Pladienolide B wereidentified in a study using a 39-cell line drug-screening paneland additional cell cycle analysis indicated that PladienolideB blocks cell growth in both the G1 and the G2M phase [13]

Pladienolide B is themost potentmetabolite of S platensiswith antitumor activity but the chemical synthesis of Pla-dienolide is complicated [52] and only three approaches havebeen reported for the synthesis of these unique macrolides[53ndash55] On the other hand the great amount of compoundrequired for in vivo studies remains a significant challengedue to the synthetic complexity inherent to this class ofcompounds In an attempt to generate simple molecules thatretain the biological activity of Pladienolides several analogshave been developedThemore effective Pladienolide analogsfor antitumor or anticancer application reported to date arePladienolide D E7107 and truncated-Pladienolide versionsPladienolide D (16-hydroxylated pladienolide) is produced


OH

OH

O

O

R2R3

R4R5

OR1

O

Pladienolide

A (1)B (2)C (3)D (4)E (5)F (6)G (7)

R3

HHHH

OHH

OH

R4

HH

H=O

HHH

R1

HAcAcAcAcHH

R5

OHOH

OH=O

OHOHOH

R2

H

OH

HH

OHH

H

(a)

H

O

O

O

O

OO

O

O

OAc

OAc

OMe

OH

OH

OH

OH

OH

OH

OH

OH

NN

X

Pruned analoguesX = H F Me and OMe

E7107

FD-895

O

(b)

Figure 3 Pladienolide analogs (a) General structure of Pladienolides AndashG which was determined by 1H 13C NMR MS IR and 2D NMRanalyses Radicals for each isoform are summarized in the table (b) Different functional analogs have also been reported

to a lesser extent than Pladienolide B on S platensis Mer-11107 In order to facilitate the production of Pladienolide D abiotransformation step of Pladienolide B into Pladienolide Dwas developed In this alternative approach the productionof Pladienolide D was increased by 15-fold in the A-1544strain of S bungoensis by overexpressing the psmA genewhich encodes the Pladienolide B 16-hydroxylase (PsmA)responsible for the production of Pladienolide D [56] Usinga similar approach the modified strain S platensisMer-11107expressing the psmA gene from S bungoensis A-1544 wasobtained and in this case the production level of Pladieno-lide D was 10-fold higher [57] Pladienolides B and D arepromising candidates for further drug development becauseof their high efficacy and low toxicity besides their highlycomplex structure has been directed to the analog synthesison a production scale [58]

E-7107 is a synthetic urethane derivative of PladienolideDwith activity against tumor cell lines and human xenografts[13] E-7107 has a selective and potent antitumor activity inhuman tumor xenograft models such as human lung cancerLC-6-JCK where E7107 caused complete tumor remissionwith poor toxicityMoreover E-7107 shows strong cell growthinhibitory activity against a large variety of human cancercell lines (IC

50values range from 02 nM to 211 nM) Using

an in vivo approach E7107 produced significant tumor

regression in a range of xenograft models In this regardanimals with BSY-1 (breast) MDA-MB-468 (breast) LC-6-JCK (lung) NIHOVCAR-3 (ovary) PC-3 (prostate)and WiDr (colon) xenografts were cured [25] For thisreason E7107 rapidly advanced to Phase I clinical trials andthe tests are currently in progress in Europe and the US(httpsclinicaltrialsgovct2showNCT00459823term=E-7107amprank=1) E7107 was tested in a Phase 1 clinical trial withpatients with different types of solid tumors refractory tostandard therapies such as colorectal esophageal pancreaticgastric renal and uterine and was found to stabilize tumorgrowth [24 59] 40 patients received E7107 at doses from 06to 45mgm2 as a 30-minute intravenous infusion on days 1and 8 every 21 days The MTD for E7107 using this scheduleis 40mgm2 [24]

Finally a different Pladienolide analog called FD-895was isolated from Streptomyces hygroscopicus strain A-9561FD-895 is a 12-membered macrolide antibiotic with a pla-nar structure similar to Pladienolide D but FD-895 has ahydroxyl group at the C-17 position and a methoxy groupsubstituted for the hydroxy group at the C-21 position FD-895 showed a cytotoxic activity against several types of cancercells such as Adriamycin-resistant HL-60 [60] In patientswith chronic lymphocytic leukemia FD-895 and Pladieno-lide B induced intron retention and spliceosomemodulation


Table 2 Antitumor activity of pladienolides

Molecule Cancer type or cell line Effect Reference

Pladienolide B

Breast (BSY-1 MCF-7)Central nervous system (SF-539)Colon (HCT-116)Lung (NCI-H522NCI-H460 A549DMS273 and DMS114)Melanoma (OVCAR-3)Stomach (MKN74)Prostate (DU-145)

Growth inhibitionCell viability was evaluated with MTT and alamarBlue assay Thegrowth inhibitory activity corresponded to the concentration atwhich cell growth was inhibited to 50 of control growth (IC

50)

The strongest effect was observed for lung and breast cancer celllines

[20]

Pladienolide B

Anticancer drug-resistant cell linesP388CPT P388ETP P388CDDPP388VCRHCT-1165-FU andMES-SADx5

Growth inhibitionCell viability was evaluated with MTT and alamarBlue reagent andIC50was determined Pladienolide B showed differential strength

depending on the cell line

[20]

Pladienolide BHuman tumor xenograftsBSY-1 PC-3 OVCAR-3 DU-145WiDr and HCT116

AntitumorCell suspensions of various human cancer cells were implantedsubcutaneously into female or male BALBc nunu miceTumor volume (TV) and relative body weight (RBW) weremeasured for 3 months after the treatment Pladienolide B showedstrong inhibitory or regressive activities against these xenografts

[20]

Pladienolide B WiDr and DLD1 human colorectalcancer cell lines

AntiproliferativeCells were incubated with 10 nM pladienolide B 5-fluorouraciltaxol or vincristine and then stained with propidium iodidePladienolide B caused a cell cycle arrest in both G1 and G2Mphases in a time-dependent manner according to FACS analysis

[21]

Pladienolide B

Gastric cancer cell lines and primarycultured cancer cells fromcarcinomatous ascites of gastric cancerpatients

AntitumorUsing an MTT assay the mean IC

50value was 12ndash11 nM for

gastric lung and breast cancer cell linesThe mean IC

50value for primary cultured cells from the 12 cases

studied was 49ndash47 nMIn xenograft models the tumors completely disappeared within 2weeks

[22]

FD-895 Chronic lymphocytic leukemia

ApoptosisPeripheral blood mononuclear cells from CLL patients wereexposed to 100 nM FD-895 Apoptosis was induced after 2 hexposure in an irreversible manner as measured by flow cytometryusing a PIDiOC

6assay

[23]

E7107 Lung breast and colon tumors

AntineoplasticTumor regression in different xenograft models BSY-1 (breast)MDA-MB-468 (breast) LC-6-JCK (NSCLC) and NIHOVCAR-3(ovary)The efficacy was limited in 26 patients enrolled for a trialCell-cycle arrest at G1 and G2M phases was observed by flowcytometry Highest efficiency against tumors was accompanied byfunctional loss of Rb and an increase of the expression of p16 andcyclin E

[24 25]

The cytotoxic effect of FD-895 involved the apoptosis induc-tion in a caspase-dependent pathway [23]

43 Herboxidiene Herboxidiene (GEXA1) is a polyketide[61] with the structure of a tetrahydrofuran with a residueof acetic acid and a conjugated diene [62] These structuralcharacteristics and its biological properties contributed to itsname [63] This compound was initially identified as a sec-ondary metabolite from Streptomyces chromofuscus A7841This compound was extracted with butanol and purified

usingHPLC Further structural analysis was completed usingHRFAB-MS and spectroscopic studies (RMN-1H y 13C)

Initial applications for Herboxidiene included its her-bicide activity [36] In 2002 six molecules sharing similarstructures were isolated from Streptomyces sp andwere calledGEX1 (Figure 4) These compounds showed antibiotic andantitumor activities GEX 1A being the molecule with thestrongest antiproliferative effect which was later identifiedas Herboxidiene [15 64] This cytotoxic activity seems to berelated to cell cycle arrest in G1 and G2M according to some


O

O

OO

OO

OH

OH

OHNH

N

TrichostatinHerboxidiene (GEXA1)

Figure 4 Herboxidiene structure The characteristic structure of GEXA1 consisting of a tetrahydrofuran with a residue of acetic acid and aconjugated diene is shown in the left This natural product was isolated from Streptomyces sp The derivative Trichostatin is shown at right

in vitro experiments [65] Besides the biological activitiesmentioned before Herboxidiene has also shown activity asanticholesterol agent and as a potent splicing inhibitor

Due to the multiple biological effects demonstrated forHerboxidiene several groups have attempted the chemicalsynthesis of the compound The first total synthesis wasaccomplished in 1999 where the relative and absolute config-urations ofHerboxidienewere confirmed [66] Later attemptsused several routes including a stereochemical synthesis in18 steps [67] an enantioselective synthesis in 16 steps [68]or an alternative synthesis in 16 steps with a global yield of34 [69] An additional chemical synthesis reported wasperformed starting from two chiral ketones derived from lac-tate in 14 steps with a global yield of 8 [70] A total enantios-elective synthesis of Herboxidiene was reported in 2014 andthe obtained product showed amild inhibitory activity on thespliceosome [71] In that same year the alternative synthesisof Herboxidiene and some other analogs like a Pladienolide-Herboxidiene hybrid was reported where alternative splicingwas efficiently modulated [72] These observations sup-ported the potential role of Herboxidiene analogs as drugcandidates for cancer treatment

Some molecules with pharmacological activities similarto those reported for Herboxidiene are Trichostatin andTMC-49A (Figure 4) These compounds have also shownanticholesterol activity However Herboxidiene has showna stronger effect on lowering the amount of cholesterol inplasma by regulating the LDL (Low Density Lipoproteins)receptor [73]

It has been shown that Herboxidiene inhibits splicing dueto its ability to bind SAP155 a component of the SF3b com-plex of the spliceosome altering its functionality [65] Thiswas accomplished using tagged molecules of Herboxidiene[73]

The effect of Herboxidiene on the splicing of cancer-related genes has also been demonstrated for a couple of casesFor example Herboxidiene inhibits the splicing of p27 gener-ating an isoform that is unable to bind the E3 ligase inducingthe accumulation of p27which is in turn free to recognize andblock the E-Cdk2 complex responsible for the E3-mediateddegradation of p27 Some of the evidence supporting therole of Herboxidiene in cancer regulation is summarizedin Table 1

Isoginkgetin

O

OO

OO

O

OH

OH

HO

HO

Figure 5 Structure of Isoginkgetin The structure of the 7-O-120573-d-glucopyranoside isolated from dried leaves of Gingko biloba isdepicted

44 Isoginkgetin Isogingketin (7-O-120573-d-glucopyranoside) isa glycosylated biflavonoid (Figure 5) initially isolated fromdried leaves of Gingko biloba a medicinal plant long utilizedin traditional eastern medicine General extraction usesmethanol and isolation was performed by column chro-matography [38] while further characterization was com-pleted using spectroscopic approaches including IR UV HR-FAB-MS and NMR [74]

Baker and Ollis started to work on the isolation of acompound known as Ginkgetin in 1957 however they werenot able to succeed until later when the molecule was sepa-rated using a potassium salt using an approach developed incollaboration with Nakasawa [75] When applying this tech-nique the recovered compound was an isomer of Ginkgetinand for this reason the molecule was called IsoginkgetinFrom that moment several studies have been performedin order to analyze the properties and applications of thisnatural compound

After the initial isolation Isoginkgetin has been obtainedfrom other plants including Dysoxylum lenticellare Gillespie[76] Chamaecyparis obtusa [77] Cephalotaxus koreana [78]and Cycas circinalis [79] as well as from the fruits of Capparisspinosa [80] Cyperus rotundus [81] Selaginella [82] andPodocarpus henkelii [83]


The biological activity of several biflavonoid compoundshas been analyzed in various studies due to its natural originand its abundance in plants especially in fernsDemonstratedactivities for the extract of Ginkgo biloba are miscellaneousaccording to the following evidence The anti-inflammatoryactivity has been related to the inhibition of arachidonicacid [84] the inhibition of COX-2 [85] SOD [86] cAMPphosphodiesterases [87] and the suppression of lymphocyteproliferation [88] Other activities include the neuroprotectorand cytoprotector effects when cells are exposed either toexternal or intrinsic factors like UV radiation [89] or theaccumulation of 120573-amyloid in neurons [90 91] This extracthas also been shown to increase adiponectin secretion [92]and the activity of AMP-kinases [93] Considering thesebiological activities Isoginkgetin has been a candidate com-pound to treat disorders like diabetes Alzheimerrsquos diseaseand other neurodegenerative diseases [94]

In relation to the effect of Isoginkgetin in splicing it hasbeen shown that the molecule has the ability to inhibit splic-ing both in vitro and in vivo at similar concentrations (30ndash33 120583M) Isoginkgetin was identified as a splicing inhibitorusing a cell-based reporter assay in HEK293 cells [95] In thesame study it was suggested that the inhibitory effect possiblyoccurs due to the prevention of the stable recruitment of theU4U5U6 trismall nuclear ribonucleoprotein Using HeLacells Isoginkgetinwas applied to study in vivomRNAdynam-ics where an accumulation of intron-containingmRNAs wasobserved upon the treatment [96] In this same cell lineIsoginkgetin mimics the effect of RNA exosome inhibitionand causes accumulation of long human Telomerase RNAtranscripts [97] The effect of Isoginkgetin on splicing wasalso evaluated by studying the expression of interleukin32 (IL-32) alternative isoforms where IL-32120574 isoform isoverexpressed upon the treatment (Table 1) correlating withcell death in cell lines derived from thyroid cancer [19]

As observed for other splicing inhibitors Isoginkgetinalso shows antitumor activity which has been related tothe inhibition of metalloprotease MMP-9 production and tothe increase of the inhibitors of metalloproteinase TIMP-1resulting in a decrease of tumor invasion [18]

Further applications involving the use of splicinginhibitors can also expand our knowledge concerning theglobal regulation of gene expression In a recent studyIsoginkgetin was coupled to a modified RNA-Seq method inorder to provide a genome-wide insight into gene expressionand to detect specific defects on splicing and transcription[96]

5 Mechanistic Insights

It has been demonstrated that Spliceostatin A PladienolideB and Herboxidiene show the ability to interact directlywith the spliceosome and that the molecular target for thismolecule is the SF3b spliceosome subunit (Figure 6) a sub-complex of U2 snRNP [13] The particular details observedfor Pladienolide B are presented here

Some studies have demonstrated that drug-treatedhuman tumor xenografts can result in complete loss ofthe full-length mRNA for certain genes such as MDM2 in

rhabdomyosarcoma cells Pladienolide treatment causesan accumulation of unspliced or incompletely spliced pre-mRNAs and gives rise to fewer and larger nuclear specklesthe intranuclear sites where splice factors are stored [98]Recently it was demonstrated that splicing inhibition byPladienolide B decreased phospho-Ser2 level [99] suggestingthat the alteration of gene splicing may represent a suitabletarget for those drugs

To identify the interaction between Pladienolides and asplicing protein Pladienolide-tagged probes were used bymodification of the acetoxy group at position 7 of Pladieno-lide B Chemical tags included 3H-labeled fluorescence-tagged and photoaffinitybiotin- (PB-) tagged ldquochemicalprobesrdquo (BODIPY-FL) The chemical probes were used inthe VEGF-reporter gene expression and cell growth inhibi-tion assays at low-nanomolar to submicromolar IC

50 Pla-

dienolide B blocks splicing and prompts nuclear export ofintron-containing transcripts as observed by fluorescencemicroscopy where the BODIPY-FL probe was concentratedin the nuclei of HeLa cells used as expression systemoverlapping with the signal obtained of splicing factor SC-35 a marker of nuclear speckles where splicing factors arelocated [100] Using immunoprecipitation experiments thesplicing proteins that interact with Pladienolide B were iden-tified such as 227-trimethylguanosine (TMG) Sm BB1015840ampD1protein the U2 snRNP-specific protein U2B10158401015840 spliceosome-associated protein 120 (SAP120 SF3a subunit 1) spliceosome-associated protein 155 (SAP155 SF3b subunit 1) and cyclinE Therefore U2 snRNP that functions at the 31015840 splicing site(Figure 6) seemed to correspond to the target for PladienolideB [21] Further immunoprecipitation assays allowed the iden-tification of SAP145 (SF3b subunit 2) and SAP130 (SF3b sub-unit 3) as Pladienolide B partners In addition it was reportedthat there is a direct interaction between the Pladienolide Bprobe and SAP130 The mechanism by which Pladienolideimpairs in vivo splicing involves SF3b modulation by inter-actingwith SAP130 in the SF3b complex but it is also possiblethat Pladienolide B shows a partial interactionwith SAP155 orSAP145 Besides time- and dose-dependent disturbance of invivo splicing with Pladienolide B results in the formation ofenlarged ldquomegaspecklesrdquo as observed when RNPS1 is overex-pressed [101] In a different approach using the CRISPRCas9genome engineering system it was demonstrated that thespliceosomal target of Pladienolide B is the SF3b1 subunit[102] In the search of themechanismbywhich Pladienolide Bor E-7107 promote the formation of a defective spliceosomeit was found that E7 blocks ATP-dependent remodeling ofU2 snRNP that exposes the branch point-binding regionUnder this scenario U2 snRNP fails to bind tightly to the pre-mRNA without disrupting the U2 particle or its associationwith SF3b [49 103]

The use of the CRISPRCas9 system in HEK293T cellsallowed the observation that some mutations in the subunitsof the SF3b complex I promote higher levels of resistance toPladienolide B It was also found that R1074H mutation inSF3b1 could have a role in the resistance to Pladienolide [104]In some reports it has been proposed thatmutations in SF3b1are associated with numerous types of cancers such asacute myeloid leukemia primary myelofibrosis chronic


Spliceostatin A

Spliceostatin A

HerboxidienePladienolideMeayamycin

Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)

Figure 6 Molecular mechanism depicted for the natural products that inhibit splicing (a) It has been demonstrated that FR901464Spliceostatin A Pladienolide B Herboxidiene and Meayamycin have the ability to block splicing by binding SAP130 SAP145 and SAP155subunits of snRNP U2 (green) Besides these natural products block cell cycle in G1 and G2M transitions (gray arrows) (b) On the otherhand Isoginkgetin blocks splicing by inhibiting the incorporation of the tri-snRNP U4U5U6 complex to the spliceosome

myelomonocytic leukemia breast cancer chronic lympho-cytic leukemia and multiple myeloma [105 106] Thesequence of the SF3b1 gene of 2087 patients with myelodys-plastic syndromes (MDS) showed mutations in 20 of all ofthem where the K700E mutation was the most frequently

found [105] Finally the interaction of spliceosome modu-lators as Pladienolides with the SF3b complex leads to animbalance in the splicing program in susceptible cells whichmay induce apoptosis by changing the levels andor ratios ofessential (and aberrant) proteins in tumor cells [106]


6 Concluding Remarks

Alternative splicing is responsible for increasing the codingpotential of the human genome and the implication of thismechanism in human health is starting to be elucidatedFuture studies could analyze particular splicing events relatedto a specific genetic disease making the discovery of newdrugs for the treatment of particular disorders possible Inthe case of the treatment of cancer available molecules thattarget the spliceosome have effectively reverted proliferationof different types of tumors with very low toxicity suggestingthat splicing modulation is an attractive target for cancertreatment Moreover considering that some of the splicinginhibitors recently discovered are natural microbial metabo-lites it is possible to assume that there are several moleculeswith antitumor activity that remain to be discovered Con-sidering that the molecular mechanism related to particulartypes of cancer is being explored it could be possible todevelop new drugs that could be oriented to modulate aprecise splicing event in order to treat a special geneticdisease

Competing Interests

The authors declare that they have no competing interests

References

[1] M S Jurica and M J Moore ldquoPre-mRNA splicing awash in asea of proteinsrdquoMolecular Cell vol 12 no 1 pp 5ndash14 2003

[2] A J Matlin F Clark and C W J Smith ldquoUnderstandingalternative splicing towards a cellular coderdquo Nature ReviewsMolecular Cell Biology vol 6 no 5 pp 386ndash398 2005

[3] C LWill andR Luhrmann ldquoSpliceosomalUsnRNPbiogenesisstructure and functionrdquo Current Opinion in Cell Biology vol 13no 3 pp 290ndash301 2001

[4] B K Das L Xia L Palandjian O Gozani Y Chyung andR Reed ldquoCharacterization of a protein complex containingspliceosomal proteins SAPs 49 130 145 and 155rdquoMolecular andCellular Biology vol 19 no 10 pp 6796ndash6802 1999

[5] A Kramer P Gruter K Groning and B Kastner ldquoCombinedbiochemical and electronmicroscopic analyses reveal the archi-tecture of the mammalian U2 snRNPrdquo Journal of Cell Biologyvol 145 no 7 pp 1355ndash1368 1999

[6] R Brosi H-P Hauri and A Kramer ldquoSeparation of splicingfactor SF3 into two components and purification of SF3aactivityrdquo The Journal of Biological Chemistry vol 268 no 23pp 17640ndash17646 1993

[7] M C Wahl C L Will and R Luhrmann ldquoThe spliceosomedesign principles of a dynamic RNPmachinerdquo Cell vol 136 no4 pp 701ndash718 2009

[8] K R Thickman M C Swenson J M Kabogo Z Gryczynskiand C L Kielkopf ldquoMultiple U2AF65 binding sites withinSF3b155 thermodynamic and spectroscopic characterizationof protein-protein interactions among pre-mRNA splicing fac-torsrdquo Journal of Molecular Biology vol 356 no 3 pp 664ndash6832006

[9] H Nakajima Y Hori H Terano et al ldquoNew antitumorsubstances FR901463 FR901464 and FR901465 II Activitiesagainst experimental tumors inmice andmechanism of actionrdquoJournal of Antibiotics vol 49 no 12 pp 1204ndash1211 1996

[10] A Corrionero B Minana and J Valcarcel ldquoReduced fidelity ofbranch point recognition and alternative splicing induced bythe anti-tumor drug spliceostatin Ardquo Genes and Developmentvol 25 no 5 pp 445ndash459 2011

[11] DKaidaHMotoyoshi E Tashiro et al ldquoSpliceostatinA targetsSF3b and inhibits both splicing and nuclear retention of pre-mRNArdquo Nature Chemical Biology vol 3 no 9 pp 576ndash5832007

[12] G A Roybal and M S Jurica ldquoSpliceostatin A inhibits spliceo-some assembly subsequent to prespliceosome formationrdquoNucleic Acids Research vol 38 no 19 Article ID gkq494 pp6664ndash6672 2010

[13] Y Kotake K Sagane T Owa et al ldquoSplicing factor SF3b as atarget of the antitumor natural product pladienoliderdquo NatureChemical Biology vol 3 no 9 pp 570ndash575 2007

[14] E G Folco K E Coil and R Reed ldquoThe anti-tumor drug E7107reveals an essential role for SF3b in remodeling U2 snRNP toexpose the branch point-binding regionrdquoGenesampDevelopmentvol 25 no 5 pp 440ndash444 2011

[15] Y Sakai T Tsujita T Akiyama et al ldquoGEX1 compoundsnovel antitumor antibiotics related to herboxidene producedby Streptomyces sp II The effects on cell cycle progression andgene expressionrdquo Journal of Antibiotics vol 55 no 10 pp 863ndash872 2002

[16] M Hasegawa T Miura K Kuzuya et al ldquoIdentification ofSAP155 as the target of GEX1A (Herboxidiene) an antitumornatural productrdquo ACS Chemical Biology vol 6 no 3 pp 229ndash233 2011

[17] G-Z Zeng N-H Tan X-J Hao Q-Z Mu and R-T Li ldquoNat-ural inhibitors targeting osteoclast-mediated bone resorptionrdquoBioorganic and Medicinal Chemistry Letters vol 16 no 24 pp6178ndash6180 2006

[18] S-O Yoon S Shin H-J Lee H-K Chun and A-SChung ldquoIsoginkgetin inhibits tumor cell invasion by regulat-ing phosphatidylinositol 3-kinaseAkt-dependent matrix meta-lloproteinase-9 expressionrdquoMolecular CancerTherapeutics vol5 no 11 pp 2666ndash2675 2006

[19] B Heinhuis T S Plantinga G Semango et al ldquoAlternativelyspliced isoforms of IL-32 differentially influence cell deathpathways in cancer cell linesrdquo Carcinogenesis vol 37 no 2 pp197ndash205 2016

[20] YMizui T SakaiM Iwata et al ldquoPladienolides new substancesfrom culture of Streptomyces platensis Mer-11107 III In vitroand in vivo antitumor activitiesrdquo Journal of Antibiotics vol 57no 3 pp 188ndash196 2004

[21] A Yokoi Y Kotake K Takahashi et al ldquoBiological validationthat SF3b is a target of the antitumor macrolide pladienoliderdquoFEBS Journal vol 278 no 24 pp 4870ndash4880 2011

[22] M Sato N Muguruma T Nakagawa et al ldquoHigh antitumoractivity of pladienolide B and its derivative in gastric cancerrdquoCancer Science vol 105 no 1 pp 110ndash116 2014

[23] M K Kashyap D Kumar R Villa et al ldquoTargeting the spliceo-some in chronic lymphocytic leukemiawith themacrolides FD-895 and pladienolide-BrdquoHaematologica vol 100 no 7 pp 945ndash954 2015

[24] F A L M Eskens F J Ramos H Burger et al ldquoPhase I phar-macokinetic and pharmacodynamic study of the first-in-classspliceosome inhibitor E7107 in patients with advanced solidtumorsrdquoClinical Cancer Research vol 19 no 22 pp 6296ndash63042013

[25] M Iwata I Ozawa T Uenaka et al ldquoE7107 a new 7-urethanederivative of pladienolide D displays curative effect against


several human tumor xenograftsrdquo Proceedings of the AmericanAssociation for Cancer Research vol 64 no 7 supplement p691 2014

[26] J M Johnson J Castle P Garrett-Engele et al ldquoGenome-wide survey of human alternative pre-mRNA splicingwith exonjunctionmicroarraysrdquo Science vol 302 no 5653 pp 2141ndash21442003

[27] J Tazi N Bakkour and S Stamm ldquoAlternative splicing anddiseaserdquo Biochimica et Biophysica Acta (BBA)mdashMolecular Basisof Disease vol 1792 no 1 pp 14ndash26 2009

[28] B Chabot and L Shkreta ldquoDefective control of prendashmessengerRNA splicing in human diseaserdquoThe Journal of Cell Biology vol212 no 1 pp 13ndash27 2016

[29] S Bonomi S Gallo M Catillo D Pignataro G Biamontiand C Ghigna ldquoOncogenic alternative splicing switches rolein cancer progression and prospects for therapyrdquo InternationalJournal of Cell Biology vol 2013 Article ID 962038 17 pages2013

[30] M Ladomery ldquoAberrant alternative splicing is another hallmarkof cancerrdquo International Journal of Cell Biology vol 2013 ArticleID 463786 6 pages 2013

[31] RM Hagen andM R Ladomery ldquoRole of splice variants in themetastatic progression of prostate cancerrdquo Biochemical SocietyTransactions vol 40 no 4 pp 870ndash874 2012

[32] S Oltean and D O Bates ldquoHallmarks of alternative splicing incancerrdquo Oncogene vol 33 pp 5311ndash5318 2013

[33] N Martınez-Montiel N Rosas-Murrieta and R Martınez-Contreras ldquoAlternative splicing regulation implications in can-cer diagnosis and treatmentrdquo Medicina Clinica vol 144 no 7pp 317ndash323 2015

[34] M Contreras Rebeca and M Montiel Nancy ldquoAlternativesplicingmodification as a treatment for genetic disordersrdquoGeneTechnology vol 4 article 126 pp 1ndash4 2015

[35] R D Martınez-Contreras and N Martınez-Montiel ldquoThe roleof splicing factors in cancer prognosis and treatmentrdquo inAlternative Splicing and Disease E Massey Ed Nova ScienceNew York NY USA 2016

[36] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome as atarget of novel antitumour drugsrdquoNature Reviews Drug Discov-ery vol 11 no 11 pp 847ndash859 2012

[37] T Sakai N Asai A Okuda N Kawamura and Y MizuildquoPladienolides new substances from culture of Streptomycesplatensis Mer-11107 II Physico-chemical properties and struc-ture elucidationrdquo The Journal of Antibiotics vol 57 no 3 pp180ndash187 2004

[38] S S Kang J S Kim and W J Kawk ldquoFlavonoids from Ginkgobiloba leavesrdquoKorean Journal of Pharmacognosy vol 21 pp 111ndash120 1990

[39] L Fan C Lagisetti C C Edwards T RWebb and P M PotterldquoSudemycins novel small molecule analogues of FR901464induce alternative gene splicingrdquo ACS Chemical Biology vol 6no 6 pp 582ndash589 2011

[40] H Motoyoshi M Horigome K Ishigami et al ldquoStructure-activity relationship for FR901464 a versatile method for theconversion and preparation of biologically active biotinylatedprobesrdquo Bioscience Biotechnology and Biochemistry vol 68 no10 pp 2178ndash2182 2004

[41] A K Ghosh and Z-H Chen ldquoEnantioselective syntheses ofFR901464 and spliceostatin A potent inhibitors of spliceo-somerdquo Organic Letters vol 15 no 19 pp 5088ndash5091 2013

[42] A K Ghosh Z-H Chen K A Effenberger and M S JuricaldquoEnantioselective total syntheses of fr901464 and spliceostatinA and evaluation of splicing activity of key derivativesrdquo Journalof Organic Chemistry vol 79 no 12 pp 5697ndash5709 2014

[43] X Liu S Biswas G-L Tang and Y-Q Cheng ldquoIsolation andcharacterization of spliceostatin B a new analogue of FR901464from Pseudomonas sp No 2663rdquo Journal of Antibiotics vol 66no 9 pp 555ndash558 2013

[44] S Osman B J Albert Y Wang M Li N L Czaicki and KKoide ldquoStructural requirements for the antiproliferative activityof pre-mRNA splicing inhibitor FR901464rdquo Chemistry vol 17no 3 pp 895ndash904 2011

[45] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[46] HHeA S Ratnayake J E Janso et al ldquoCytotoxic spliceostatinsfrom Burkholderia sp and their semisynthetic analoguesrdquo Jour-nal of Natural Products vol 77 no 8 pp 1864ndash1870 2014

[47] X Liu S BiswasM G Berg et al ldquoGenomics-guided discoveryof thailanstatins A B and C as pre-mRNA splicing inhibitorsand antiproliferative agents from Burkholderia thailandensisMSMB43rdquo Journal of Natural Products vol 76 no 4 pp 685ndash693 2013

[48] T Schneider-Poetsch T Usui D Kaida and M Yoshida ldquoGar-bled messages and corrupted translationsrdquo Nature ChemicalBiology vol 6 no 3 pp 189ndash198 2010

[49] D Pham and K Koide ldquoDiscoveries target identifications andbiological applications of natural products that inhibit splicingfactor 3B subunit 1rdquo Natural Product Reports vol 33 no 5 pp637ndash647 2016

[50] C Blancher and A L Harris ldquoThe molecular basis of thehypoxia response pathway tumour hypoxia as a therapy targetrdquoCancer and Metastasis Reviews vol 17 no 2 pp 187ndash194 1998

[51] B Burke A Giannoudis K P Corke et al ldquoHypoxia-inducedgene expression in human macrophages implications forischemic tissues and hypoxia-regulated gene therapyrdquo TheAmerican Journal of Pathology vol 163 no 4 pp 1233ndash12432003

[52] A L Mandel B D Jones J J La Clair and M D Burkart ldquoAsynthetic entry to pladienolide B and FD-895rdquo Bioorganic andMedicinal Chemistry Letters vol 17 no 18 pp 5159ndash5164 2007

[53] A K Ghosh and D D Anderson ldquoEnantioselective totalsynthesis of pladienolide B a potent spliceosome inhibitorrdquoOrganic Letters vol 14 no 18 pp 4730ndash4733 2012

[54] R M Kanada D Itoh M Nagai et al ldquoTotal synthesis ofthe potent antitumor macrolides pladienolide B and Drdquo Ange-wandte Chemie-International Edition vol 46 no 23 pp 4350ndash4355 2007

[55] V P Kumar and S Chandrasekhar ldquoEnantioselective synthesisof pladienolide B and truncated analogues as new anticanceragentsrdquo Organic Letters vol 15 no 14 pp 3610ndash3613 2013

[56] K Machida Y Aritoku T Nakashima A Arisawa and TTsuchida ldquoIncrease in pladienolide D production rate using aStreptomyces strain overexpressing a cytochrome P450 generdquoJournal of Bioscience and Bioengineering vol 105 no 6 pp 649ndash654 2008

[57] K Machida Y Aritoku and T Tsuchida ldquoOne-pot fermenta-tion of pladienolide D by Streptomyces platensis expressing aheterologous cytochrome P450 generdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 596ndash598 2009


[58] S R Bathula S M Akondi P S Mainkar and S Chan-drasekhar ldquolsquoPruning of biomolecules and natural products(PBNP)rsquo an innovative paradigm in drug discoveryrdquo Organicand Biomolecular Chemistry vol 13 no 23 pp 6432ndash64482015

[59] M Salton and T Misteli ldquoSmall molecule modulators ofpre-mRNA splicing in cancer therapyrdquo Trends in MolecularMedicine vol 22 no 1 pp 28ndash37 2016

[60] M Seki-Asano T Okazaki M Yamagishi et al ldquoIsolation andcharacterization of a new 12-membered macrolide FD-895rdquoJournal of Antibiotics vol 47 no 12 pp 1395ndash1401 1994

[61] B G Isaac S W Ayer R C Elliott and R J Stonard ldquoHer-boxidiene a potent phytotoxic polyketide from Streptomyces spA7847rdquo The Journal of Organic Chemistry vol 57 no 26 pp7220ndash7226 1992

[62] A J F Edmunds W Trueb W Oppolzer and P CowleyldquoHerboxidiene determination of absolute configuration bydegradation and synthetic studiesrdquo Tetrahedron vol 53 no 8pp 2785ndash2802 1997

[63] A R Pokhrel D Dhakal A K Jha and J K Sohng ldquoHerbox-idiene biosynthesis production and structural modificationsprospect for hybrids with related polyketiderdquo Applied Microbi-ology and Biotechnology vol 99 no 20 pp 8351ndash8362 2015

[64] Y Sakai T Yoshida K Ochiai et al ldquoGEX1 compounds novelantitumor antibiotics related to herboxidiene produced byStreptomyces sp I Taxonomy production isolation physico-chemical properties and biological activitiesrdquo The Journal ofAntibiotics vol 55 no 10 pp 855ndash862 2002

[65] Y Gao A Vogt C J Forsyth and K Koide ldquoComparison ofsplicing factor 3b inhibitors in human cellsrdquoChemBioChem vol14 no 1 pp 49ndash52 2013

[66] P R Blakemore P J Kocienski A Morley and K Muir ldquoAsynthesis of herboxidienerdquo Journal of the Chemical SocietyPerkin Transactions 1 vol 8 pp 955ndash968 1999

[67] M Banwell M McLeod R Premraj and G Simpson ldquoTotalsynthesis of herboxidiene a complex polyketide from Strepto-myces species A7847rdquo Pure and Applied Chemistry vol 72 no9 pp 1631ndash1634 2000

[68] Z Yun and J S Panek ldquoTotal synthesis of herboxidieneGEX1Ardquo Organic Letters vol 9 no 16 pp 3141ndash3143 2007

[69] T J Murray and C J Forsyth ldquoTotal synthesis of GEX1ArdquoOrganic Letters vol 10 no 16 pp 3429ndash3431 2008

[70] M Pellicena K Kramer P Romea and F Urpı ldquoTotal synthesisof (+)-Herboxidiene from two chiral lactate-derived ketonesrdquoOrganic Letters vol 13 no 19 pp 5350ndash5353 2011

[71] A K Ghosh N Ma K A Effenberger and M S Jurica ldquoTotalsynthesis of GEX1Q1 assignment of C-5 stereoconfigurationand evaluation of spliceosome inhibitory activityrdquo OrganicLetters vol 16 no 11 pp 3154ndash3157 2014

[72] C LagisettiMV Yermolina L K Sharma G Palacios B J Pri-garo and T R Webb ldquoPre-mRNA splicing-modulatory phar-macophores the total synthesis of herboxidiene a pladienolide-herboxidiene hybrid analog and related derivativesrdquo ACSChemical Biology vol 9 no 3 pp 643ndash648 2014

[73] Y Koguchi M Nishio J Kotera K Omori T Ohnuki andS Komatsubara ldquoTrichostatin A and herboxidiene up-regulatethe gene expression of low density lipoprotein receptorrdquo Journalof Antibiotics vol 50 no 11 pp 970ndash971 1997

[74] S K Hyun S S Kang K H Son H Y Chung and J SChoi ldquoBiflavone glucosides from Ginkgo biloba yellow leavesrdquoChemical and Pharmaceutical Bulletin vol 53 no 9 pp 1200ndash1201 2005

[75] W Baker andW D Ollis Recent Developments in the Chemistryof Natural Phenolic Compounds Pergamon Press London UK1961

[76] K He B N Timmermann A J Aladesanmi and L Zeng ldquoAbiflavonoid from Dysoxylum lenticellare gillespierdquo Phytochem-istry vol 42 no 4 pp 1199ndash1201 1996

[77] M Krauze-Baranowska L Pobłocka and A A El-HelaldquoBiflavones from Chamaecyparis obtusardquo Zeitschrift fur Natur-forschung C vol 60 no 9-10 pp 679ndash685 2005

[78] M K Lee S W Lim H Yang et al ldquoOsteoblast differentiationstimulating activity of biflavonoids fromCephalotaxus koreanardquoBioorganic and Medicinal Chemistry Letters vol 16 no 11 pp2850ndash2854 2006

[79] A Moawad M Hetta J K Zjawiony et al ldquoPhytochemicalinvestigation of Cycas circinalis and Cycas revoluta leafletsmoderately active antibacterial biflavonoidsrdquo Planta Medicavol 76 no 8 pp 796ndash802 2010

[80] H-F Zhou C Xie R Jian et al ldquoBiflavonoids from caper (Cap-paris spinosa L) Fruits and their effects in inhibiting NF-kappaB activationrdquo Journal of Agricultural and Food Chemistry vol59 no 7 pp 3060ndash3065 2011

[81] Z Zhou and C Fu ldquoA new flavanone and other constituentsfrom the rhizomes of Cyperus rotundus and their antioxidantactivitiesrdquo Chemistry of Natural Compounds vol 48 no 6 pp963ndash965 2013

[82] J-K Weng and J P Noel ldquoChemodiversity in Selaginella a ref-erence system for parallel and convergent metabolic evolutionin terrestrial plantsrdquo Frontiers in Plant Science vol 4 article 1192013

[83] V P Bagla L J McGaw E E Elgorashi and J N EloffldquoAntimicrobial activity toxicity and selectivity index of twobiflavonoids and a flavone isolated from Podocarpus henkelii(Podocarpaceae) leavesrdquo BMC Complementary and AlternativeMedicine vol 14 no 1 article 383 2014

[84] S J Lee R H Son H W Chang S S Kang and H P KimldquoInhibition of arachidonate release from rat peritoneal macro-phage by biflavonoidsrdquo Archives of Pharmacal Research vol 20no 6 pp 533ndash538 1997

[85] J K Son M J Son E Lee et al ldquoGinkgetin a biflavonefrom Ginko biloba leaves inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cellsrdquo Bio-logical and Pharmaceutical Bulletin vol 28 no 12 pp 2181ndash2184 2005

[86] P S S Yuan ldquoEffect of isoginkgetin on scavenge of oxygenfree radical in anoxic ratsrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 2 pp 1ndash4 1993

[87] R Saponara and E Bosisio ldquoInhibition of cAMP-phosph-odiesterase by biflavones of Ginkgo biloba in rat adipose tissuerdquoJournal of Natural Products vol 61 no 11 pp 1386ndash1387 1998

[88] S J Lee J H Choi K H Son HW Chang S S kang andH PKim ldquoSuppression of mouse lymphocyte proliferation in vitroby naturally-occurring biflavonoidsrdquo Life Sciences vol 57 no 6pp 551ndash558 1995

[89] S-J Kim ldquoEffect of biflavones of Ginkgo biloba against UVB-induced cytotoxicity in vitrordquo Journal of Dermatology vol 28no 4 pp 193ndash199 2001

[90] S K Sam J Y Lee Y K Choi et al ldquoNeuroprotective effectsof naturally occurring biflavonoidsrdquo Bioorganic and MedicinalChemistry Letters vol 15 no 15 pp 3588ndash3591 2005


[91] H Sasaki Y Kitoh M Tsukada et al ldquoInhibitory activities ofbiflavonoids against amyloid-120573 peptide 42 cytotoxicity in PC-12 cellsrdquo Bioorganic andMedicinal Chemistry Letters vol 25 no14 pp 2831ndash2833 2015

[92] G Liu M Grifman J Macdonald P Moller FWong-Staal andQ-X Li ldquoIsoginkgetin enhances adiponectin secretion fromdifferentiated adiposarcoma cells via a novel pathway involvingAMP-activated protein kinaserdquo Journal of Endocrinology vol194 no 3 pp 569ndash578 2007

[93] C R Cederroth M Vinciguerra A Gjinovci et al ldquoDietaryphytoestrogens activate AMP-Activated protein kinase withimprovement in lipid and glucose metabolismrdquo Diabetes vol57 no 5 pp 1176ndash1185 2008

[94] S Choudhary P Kumar and J Malik ldquoPlants and phytochem-icals for Huntingtonrsquos diseaserdquo Pharmacognosy Reviews vol 7no 14 pp 81ndash91 2013

[95] K OrsquoBrien A J Matlin A M Lowell and M J Moore ldquoThebiflavonoid isoginkgetin is a general inhibitor of pre-mRNAsplicingrdquo Journal of Biological Chemistry vol 283 no 48 pp33147ndash33154 2008

[96] J M Gray D A Harmin S A Boswell et al ldquoSnapShot-Seq amethod for extracting genome-wide in Vivo mRNA dynamicsfrom a single total RNA samplerdquo PLoS ONE vol 9 no 2 ArticleID e89673 2014

[97] C-K Tseng H-F Wang A M Burns M R Schroeder MGaspari and P Baumann ldquoHuman telomerase RNA processingand quality controlrdquo Cell Reports vol 13 pp 2232ndash2243 2015

[98] R J van Alphen E A C Wiemer H Burger and F A L MEskens ldquoThe spliceosome as target for anticancer treatmentrdquoBritish Journal of Cancer vol 100 no 2 pp 228ndash232 2009

[99] M Koga M Hayashi and D Kaida ldquoSplicing inhibitiondecreases phosphorylation level of Ser2 in Pol II CTDrdquo NucleicAcids Research vol 43 no 17 pp 8258ndash8267 2015

[100] L L Hall K P Smith M Byron and J B Lawrence ldquoMolecularanatomy of a specklerdquoThe Anatomical Record Part A Discover-ies in Molecular Cellular and Evolutionary Biology vol 288 no7 pp 664ndash675 2006

[101] P Loyer J H Trembley J M Lahti and V J Kidd ldquoTheRNP protein RNPS1 associates with specific isoforms of thep34(cdc2)-related PITSLRE protein kinase in vivordquo Journal ofCell Science vol 111 no 11 pp 1495ndash1506 1998

[102] M Aouida A Eid and M M Mahfouz ldquoCRISPRCas9-mediated target validation of the splicing inhibitor PladienolideBrdquo Biochimie Open 2016

[103] M Iwata Y Ozawa T Uenaka et al ldquoE7107 a new urethanederivative of pladienolide D displays curative effect againstseveral human tumor xenograftsrdquo Proceedings of AmericanAssociation for Cancer Research vol 45 p 691 2004

[104] E Papaemmanuil M Cazzola J Boultwood et al ldquoSomaticSF3B1 mutation in myelodysplasia with ring sideroblastsrdquo TheNew England Journal ofMedicine vol 365 no 15 pp 1384ndash13952011

[105] F Damm F Thol O Kosmider et al ldquoSF3B1 mutations inmyelodysplastic syndromes clinical associations and prognos-tic implicationsrdquo Leukemia vol 26 no 5 pp 1137ndash1140 2012

[106] T RWebb A S Joyner and PM Potter ldquoThe development andapplication of smallmoleculemodulators of SF3b as therapeuticagents for cancerrdquoDrug Discovery Today vol 18 no 1-2 pp 43ndash49 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


conserved AG dinucleotide that indicates the end of theintron [3] The recognition of the 31015840ss involves the binding ofSF1 to the BPS and the recruitment of the snRNPU2 auxiliaryfactor (U2AF) to the pY-tract and the AG dinucleotide Afterthe recognition of both exonintron boundaries an earlycomplex is formed that commits pre-mRNA to undergoingsplicing where U2 snRNP is also recruited to the 31015840ss U2snRNP recruitment to the pre-mRNA is one of the key stepsthat triggers additional interactions leading to the formationof catalytic spliceosome complexes due to the incorporationof the tri-snRNP U4U5U6 within which numerous RNArearrangements and modifications in protein compositioncontribute to complete a splicing cycle [2 3]

Like most of the snRNPs U2 is a ribonucleoproteic com-plex formed by 7 Smproteins (which are common for spliceo-somal snRNPs) and 17 specific proteins being the largestsnRNP [3] Among the specific snRNP U2 components twoprotein subcomplexes are found SF3a and SF3b [3ndash5] SF3aincludes 3 subunits of 60 66 and 120 kDa [6] while SF3bshows at least 8 specific subunits of 10 14a 14b 49 125130 140 and 155 kDa [7] Components of the SF3a and SF3bsubcomplexes bind to sequences in the pre-mRNA tetheringU2 snRNP to the BPS and the 31015840ss SF3b 155 is one of themostconserved subunits of U2 snRNP and it has shown the abilityto bind splicing factors U2AF65 and p14 [3 8] Interestinglythis subunit has been related to the antiproliferative effectobserved for some natural products that regulate the splicingmechanism and it results clear in the fact that targeting thespliceosome and modulating splice-site recognition could berelevant for the development of new therapeutic approachesas will be further discussed

3 The Role of Alternative Splicing inHuman Disease

Over the past 10 years the role of alternative splicing inhuman disease has been growing When the human genomeproject was completed in silico analysis predicted that 75 ofthe human genes underwent splicing [26] and that 15 to 50of the genetic diseases were related to aberrant splicing events[27] From this initial observation several studies have linkedsplicing defects with specific genetic disorders However thefull significance of the role in alternative splicing in humandisease remains to be elucidated Some diseases that havebeen linked to defects on splicing include dilated cardiomy-opathy autism spectrum disorder spinal muscular atro-phy schizophrenia cardiac hypertrophy amyotrophic lateralsclerosis and frontotemporal dementia [28] In all thesecases the molecular insights related to the splicing defectthat originates the disease have been dissected The preciseregulation of the splicing event varies for each pre-mRNAand for this reason it is time consuming to demonstrate themolecular mechanism that regulates the alternative splicingfor each gene Moreover this regulation also depends on thecellular context complicating the scene In this regard futureefforts need to be developed in order to dissect the alternativesplicing event that is related to each disease and the possibletherapeutic tools that could be applied

One specific group of diseases that have been related tosplicing corresponds to different types of cancer and onlyrecently the determinant role of splicing in cancer has beenacknowledged [29 30] Several features of splicing eventsrelated to tumor progression have been reported and it iswell documented that the alternative splicing of different pre-mRNAs is altered during oncogenic progression with theconcomitant development of cancer features like an increasein vascularization cell proliferation and invasion [31 32]Themolecular hallmarks documented for several types of cancerhave been recapitulated in an attempt to orientate futureefforts towards cancer treatment through alternative splicingmodulation [33ndash35] Considering all this evidences severalstudies have been oriented to modulate alternative splicingin order to treat cancer

4 Microbial Metabolites ThatRegulate Splicing

Natural products have been traditionally sought from acti-nomycetes filamentous fungi and medicinal plants In thisregard several derivatives of bacterial fermentation as well astheir synthetic equivalents possess the ability to interact withcomponents of the spliceosome In some cases the effect onsplicing associated with these drugs is achieved through thedirect regulation of the expression of genes that are relevantfor cancer progression [36] Dozens of small molecule effec-tors targeting the alternative splicing process have been iden-tified and evaluated as drug candidates including a naturalproduct of Pseudomonas sp number 2663 called FR901464[9] natural products from Streptomyces platensis Mer-11107that originated the group of Pladienolides [37] Herboxidiene[15] and Isoginkgetin [38]Thesemolecules and their deriva-tives have shown activity as splicing inhibitors and manyof them demonstrated potent antiproliferative properties inhuman cancer cell lines being in general less toxic to normalhuman cells [39]

41 FR901464 and Derivatives Spliceostatins are a groupof compounds derived from the natural product FR901464which was identified initially as an antitumor compoundIn the original study FR901463 FR901464 and FR901465were isolated from the fermentation broth of Pseudomonassp number 2663 [9] These 3 compounds are soluble inacetonitrile chloroform and ethyl acetate and poorly solublein water and insoluble in hexane They all show strong UVabsorption at 235 nm distinctive of a conjugated diene whilethe IR spectra indicated the presence of hydroxyl ester and aconjugated amide carbonyl (Figure 1) Initially the three com-pounds from the FR9014 series mentioned before were testedfor their biological activity As a result they all enhancedthe transcriptional activity of SV40 in a CAT assay Besidesthey were all cytotoxic according to the MTT method in thefollowing human adenocarcinomas A549 lung cells MCF-7mammary cells or HCT116 colon cells [9] Moreover the 3compounds extended the life of mice bearing ascetic tumorsFR901464 being the one showing themost potent effect on thetumor systems assayed [9] Furthermore FR901464 induced


FR series



Thailanstatins


Meayamycin

Meayamycins

Sudemycins

Sudemycin E

O

O O O O

O

O

O O O O

O

O

O O O OOHOH OH

OHHOHOHOHO Cl H

NHN

HN

H

HO

OH OH

HO HO HO

HO HO HO

HO

HO

Cl

OMe

NHN

HN

N

HN

HN

HN

HN

HN

HN H

HN

O

O O O O

O

O OO

O

O

O

OO

O

OOO O

O

OO O O

O

OOOOO

O O

O O O OO

O

OOO O O

O

O

OO

O

O

O OO

O O

OO

O

OOOO

O

Cl














Pladienolide

HO OO

OAcOH

OHO












FR9014 series






Herboxidiene





Isoginkgetin













OH

OH

O

O

R2R3

R4R5

OR1

O

Pladienolide

A (1)B (2)C (3)D (4)E (5)F (6)G (7)

R3

HHHH

OHH

OH

R4

HH

H=O

HHH

R1

HAcAcAcAcHH

R5

OHOH

OH=O

OHOHOH

R2

H

OH

HH

OHH

H

(a)

H

O

O

O

O

OO

O

O

OAc

OAc

OMe

OH

OH

OH

OH

OH

OH

OH

OH

NN

X


E7107

FD-895

O

(b)











Pladienolide B



50)


[20]

Pladienolide B




[20]



[20]



[21]

Pladienolide B







[22]



6assay

[23]



[24 25]






O

O

OO

OO

OH

OH

OHNH

N








Isoginkgetin

O

OO

OO

O

OH

OH

HO

HO















50 Pla-




Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


FR series



Thailanstatins


Meayamycin

Meayamycins

Sudemycins

Sudemycin E

O

O O O O

O

O

O O O O

O

O

O O O OOHOH OH

OHHOHOHOHO Cl H

NHN

HN

H

HO

OH OH

HO HO HO

HO HO HO

HO

HO

Cl

OMe

NHN

HN

N

HN

HN

HN

HN

HN

HN H

HN

O

O O O O

O

O OO

O

O

O

OO

O

OOO O

O

OO O O

O

OOOOO

O O

O O O OO

O

OOO O O

O

O

OO

O

O

O OO

O O

OO

O

OOOO

O

Cl














Pladienolide

HO OO

OAcOH

OHO












FR9014 series






Herboxidiene





Isoginkgetin













OH

OH

O

O

R2R3

R4R5

OR1

O

Pladienolide

A (1)B (2)C (3)D (4)E (5)F (6)G (7)

R3

HHHH

OHH

OH

R4

HH

H=O

HHH

R1

HAcAcAcAcHH

R5

OHOH

OH=O

OHOHOH

R2

H

OH

HH

OHH

H

(a)

H

O

O

O

O

OO

O

O

OAc

OAc

OMe

OH

OH

OH

OH

OH

OH

OH

OH

NN

X


E7107

FD-895

O

(b)











Pladienolide B



50)


[20]

Pladienolide B




[20]



[20]



[21]

Pladienolide B







[22]



6assay

[23]



[24 25]






O

O

OO

OO

OH

OH

OHNH

N








Isoginkgetin

O

OO

OO

O

OH

OH

HO

HO















50 Pla-




Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Pladienolide

HO OO

OAcOH

OHO












FR9014 series






Herboxidiene





Isoginkgetin













OH

OH

O

O

R2R3

R4R5

OR1

O

Pladienolide

A (1)B (2)C (3)D (4)E (5)F (6)G (7)

R3

HHHH

OHH

OH

R4

HH

H=O

HHH

R1

HAcAcAcAcHH

R5

OHOH

OH=O

OHOHOH

R2

H

OH

HH

OHH

H

(a)

H

O

O

O

O

OO

O

O

OAc

OAc

OMe

OH

OH

OH

OH

OH

OH

OH

OH

NN

X


E7107

FD-895

O

(b)











Pladienolide B



50)


[20]

Pladienolide B




[20]



[20]



[21]

Pladienolide B







[22]



6assay

[23]



[24 25]






O

O

OO

OO

OH

OH

OHNH

N








Isoginkgetin

O

OO

OO

O

OH

OH

HO

HO















50 Pla-




Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




FR9014 series






Herboxidiene





Isoginkgetin













OH

OH

O

O

R2R3

R4R5

OR1

O

Pladienolide

A (1)B (2)C (3)D (4)E (5)F (6)G (7)

R3

HHHH

OHH

OH

R4

HH

H=O

HHH

R1

HAcAcAcAcHH

R5

OHOH

OH=O

OHOHOH

R2

H

OH

HH

OHH

H

(a)

H

O

O

O

O

OO

O

O

OAc

OAc

OMe

OH

OH

OH

OH

OH

OH

OH

OH

NN

X


E7107

FD-895

O

(b)











Pladienolide B



50)


[20]

Pladienolide B




[20]



[20]



[21]

Pladienolide B







[22]



6assay

[23]



[24 25]






O

O

OO

OO

OH

OH

OHNH

N








Isoginkgetin

O

OO

OO

O

OH

OH

HO

HO















50 Pla-




Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


OH

OH

O

O

R2R3

R4R5

OR1

O

Pladienolide

A (1)B (2)C (3)D (4)E (5)F (6)G (7)

R3

HHHH

OHH

OH

R4

HH

H=O

HHH

R1

HAcAcAcAcHH

R5

OHOH

OH=O

OHOHOH

R2

H

OH

HH

OHH

H

(a)

H

O

O

O

O

OO

O

O

OAc

OAc

OMe

OH

OH

OH

OH

OH

OH

OH

OH

NN

X


E7107

FD-895

O

(b)











Pladienolide B



50)


[20]

Pladienolide B




[20]



[20]



[21]

Pladienolide B







[22]



6assay

[23]



[24 25]






O

O

OO

OO

OH

OH

OHNH

N








Isoginkgetin

O

OO

OO

O

OH

OH

HO

HO















50 Pla-




Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Pladienolide B



50)


[20]

Pladienolide B




[20]



[20]



[21]

Pladienolide B







[22]



6assay

[23]



[24 25]






O

O

OO

OO

OH

OH

OHNH

N








Isoginkgetin

O

OO

OO

O

OH

OH

HO

HO















50 Pla-




Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


O

O

OO

OO

OH

OH

OHNH

N








Isoginkgetin

O

OO

OO

O

OH

OH

HO

HO















50 Pla-




Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











50 Pla-




Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Spliceostatin A

Spliceostatin A


Cell cycle

M

S

FR901464

G1

G2

U1

U2

GU5998400ss 3

998400ssA AG

U2AF

SAP155O

O

OO

O

OOMe

O

O

OO

O

OOMe

Y(n)

HOHNN

SAP145 SAP130

(a)

U1

U2

U4

U6

U5

GU AG5998400ss 3

998400ssA

U2AF

Y(n)

IsoginkgetinO

O

OO

O

O

HO

HO

OHOH

(b)







Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Competing Interests


References






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

microbial and natural metabolites that inhibit splicing: a powerful

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