Reviews pubs.acs.org/acschemicalbiology
Modulation of Alternative Splicing with Chemical Compounds in New Therapeutics for Human Diseases Kenji Ohe†,‡ and Masatoshi Hagiwara*,† †
Department of Anatomy and Developmental Biology and ‡Training Program of Leaders for Integrated Medical System for Fruitful Healthy-Longevity Society (LIMS), Kyoto University Graduate School of Medicine, Kyoto 606-8315, Japan ABSTRACT: Alternative splicing is a critical step where a limited number of human genes generate a complex and diverse proteome. Various diseases, including inherited diseases with abnormalities in the “genome code,” have been found to result in an aberrant misspliced “transcript code” with correlation to the resulting phenotype. Chemical compound-based and nucleic acid-based strategies are trying to target this mis-spliced “transcript code”. We will briefly mention about how to obtain splicing-modifyingcompounds by high-throughput screening and overview of what is known about compounds that modify splicing pathways. The main focus will be on RNA-binding protein kinase inhibitors. In the main text, we will refer to diseases where splicing-modifying-compounds have been intensively investigated, with comparison to nucleic acidbased strategies. The information on their involvement in missplicing as well as nonsplicing events will be helpful in finding better compounds with less off-target effects for future implications in mis-splicing therapy. splicing events using bichromatic fluorescent reporters (Figure 1) in cell-based systems,4,5 in in vivo systems such as C.
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NAs transcribed from the genome are vulnerable to various types of alternative splicing. Exon-skipping, mutually exclusive exons, alternative 5′ splice sites, alternative 3′ splice sites, and intron retention may alter the pre-mRNA into many splice variants of mRNA which are then translated into proteins with different functions.1 Ten years have passed since the completion of the Human Genome Project, and reports have shown that over 95% of genes undergo alternative splicing, which underscores the importance of this process.2,3 Abnormalities in a gene can cause dysfunctions of its proteins through aberrant alternative splicing events, which can be corrected by modulating alternative splicing. The challenge of curing a disease by correcting aberrant splicing events is not an easy one. The spliceosome is a multimegadalton ribonucleoprotein complex where various factors and subcomplexes interact to achieve the splicing reaction. Alternative splicing is regulated by additional factors that interact with this spliceosome, making the situation complicated. Some compounds have been shown to directly target a specific factor of the spliceosome but most have the possibility to function through acting on multiple factors or even indirectly through other steps of gene regulation. Moreover, compounds that target a splicing factor have the potential to regulate alternative splicing of multiple transcripts due to the fact that splicing factors bind to short consensus cis-elements. Logically, this will end up in a multitude of off-target splicing events. Thus, new approaches need to be developed to seek new chemical compounds that correct aberrant alternative splicing of specific transcripts with minimum off-target effects. Powerful screening methods have been developed to recapitulate alternative © XXXX American Chemical Society
Figure 1. Scheme of bichromatic fluorescent reporter. When the exon is included, the reading frame of the reporter is in-frame with green fluorescent protein (GFP) codons. When the exon is skipped, the reading frame of the reporter is in-frame with red fluorescent protein (RFP) codons.
elegans,6,7 and in transgenic mice.8 Evaluating the ratio of differentially spliced mRNA transcribed from a single reporter avoids the influence of differential transcription efficiencies, which is a drawback when using two monochromatic reporters. Applying this system to transgenic mouse embryos9 as well as in transgenic C. elegans,10 a switching mechanism of tissue- or developmental-specific alternative splicing can be uncovered. Regulatory proteins can also be identified with the use of fulllength cDNA libraries. Flipping the two fluorescence exons avoid false positives due to their introduction in these bichromatic fluorescent reporters.11 With this novel bichromatic screening strategy, cardiotonic steroids were unexpectReceived: September 2, 2014 Accepted: January 5, 2015
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ACS Chemical Biology
known to alter post-translational modification of U2 small nuclear ribonucleoprotein auxiliary factor (U2AF),13 which is known to bind to the polypyrimidine tract and is important for exon definition. Iron modulates lysyl-hydroxylation of U2AF65 and corrects aberrant splicing of the ferrochelatase gene, the partial deficiency of which is found in erythropoietic protoporphyria.14 (4) Splicing factor 3b, subunit 1 (SF3B1) is a component of U2 small nuclear ribonucleoprotein (U2 snRNP), which is important for branch-point recognition by the core spliceosome. Compounds targeting SF3B1 can be found in the Myelodysplastic Syndrome and SF3B1 Inhibitors section. (5) A natural product, isoginkgetin, is a biflavonoid that generally inhibits splicing by inhibiting U4/U5/U6 tri-snRNP association to the spliceosome.15 (6) A previously known DNA-binding compound, pyrvinium pamoate,16 was found to bind to structured RNA and change alternative splicing of serotonin receptor 2C pre-mRNA.17 A lead identification strategy named Inforna uses an annotated database of RNA motif and compound interaction.18 Using Inforna, a compound was identified to bind and increase the stability of the mutant RNA of microtubule-associated protein tau (MAPT) and significantly reduce the abnormal inclusion of exon 10.19 (7) Tannic acid was found to increase expression of polyprimidine tract-binding protein (PTB), which improved deleterious inclusion of a mutated exon in congenital myasthenic syndrome.20 Although many pathways have been elucidated, we would like to emphasize that many hnRNPs and RNAbinding proteins (RBPs) have not yet been targeted by compounds (Figure 2). Herein, after an overview of RNA-binding protein kinases, we will categorize splicing-modifying-compounds in the sections of disease where they have been intensively investigated.
edly found to correct mis-splicing of microtubule-associated protein tau (MAPT) exon 10, which causes frontotemporal dementia.12 In order to screen for a compound that alters aberrant splicing with minimum off-target effects, this bichromatic screening strategy is essential. Model systems using induced pluripotent stem (iPS) cells or genomic editing strategies (clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9) will further improve the screening efficiency and may enable to obtain compounds which can correct single splicing events and eventually clear clinical trials. Various chemical compounds have been found to affect various steps of splicing, as summarized below and shown in Figure 2 (red numbers). (1) Several RNA-binding protein
Figure 2. Steps of splicing regulation where chemical compounds are known to target (red numbers). (1) Phosphorylation of SR proteins. (2) 5′ splice site recognition by U1 snRNP. (3) Polypyrimidine tract recognition by U2AF. (4) Branch point recognition by SF3B1. (5) Recruitment of U5, U4/U6 snRNP splicing complex to the spliceosome. (6) RNA structure. (SR: serine/arginine-rich protein. hnRNP: heterogeneous nuclear ribonucleoprotein. U1: U1 snRNP (U1 small nuclear ribonucleoprotein). U2: U2 snRNP. U4: U4 snRNP. U5: U5 snRNP. U6: U6 snRNP. SF1: splicing factor 1. U2AF: U2 small nuclear ribonucleoprotein auxiliary factor. SF3B1: splicing factor 3b, subunit 1. ESE: exonic splicing enhancer. ESS: exonic splicing silencer. ISE: intronic splicing enhancer. ISS: intronic splicing silencer. RBP: RNA-binding protein).
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INHIBITORS OF RNA-BINDING PROTEIN KINASES An elegant approach to modulate alternative splicing is to screen for inhibitors of RNA-binding protein kinases.21 RNAbinding protein kinases mainly target SR proteins, which contain a typical serine (S) and arginine (R)-rich domain, and an RNA-binding motif where they exert their function in exon definition (exon-inclusion).22 Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA−protein complexes formed on newly transcribed pre-mRNA. These SR proteins and hnRNPs are cofactors that bind to regulatory elements and act antagonistically on alternative splicing.23 Phosphorylation of SR proteins is fine-tuned and is differentially regulated by specific kinases such as Cdc2-like kinases (CLKs), dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs), and
kinase inhibitors target the phosphorylation of serine/argininerich (SR) proteins, which mainly bind to exon splicing enhancers (ESEs) and exert their function as major regulators of splicing (see the Inhibitors of RNA-Binding Protein Kinases section). (2) Certain compounds affect the binding of U1 small nuclear ribonucleoprotein (U1 snRNP) to the 5′ splice site, which is an important step of prespliceosome formation in exon definition. These compounds can be found in the Familial Dysautonomia (FD) and Plant Cytokinins section. (3) Iron is
Figure 3. (A) Regulation of alternative splicing by kinases that phosphorylate SR protein via balance of SR proteins and hnRNP proteins. (B) Exonskipping strategy. In order to create a functional protein by exon-skipping, the length of the skipped exon(s) must be a multiple of three. B
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ACS Chemical Biology Table 1. CLK1 Inhibitors
up a new field in regulating alternative splicing. One family of kinases, the CLK1 family, is a member of the dual-specificity kinases and regulates alternative splicing.32 Extensive screening of over 100 000 chemical compounds by in vitro phosphorylation assays using the SR domain of serine/arginine-rich splicing factor 1 (SRSF1) as a substrate, identified TG003 as a potent inhibitor of Clk1 and Clk4.33 TG003 has revealed a novel bypass mechanism of stressresponsive gene expression by promoting Clk1/4 splicing from a nuclear pool of intron-retaining pre-mRNAs and regulating the status of SR phosphorylation.34 TG003 was also used to confirm the involvement of CLK1 in adipogenesis in response to insulin through alternative splicing regulation of protein kinase C beta (PKCβ).35 In a similar way, TG003 was used to confirm Clk1-dependent phosphorylation of splicing factor 45 (SPF45) is involved in exclusion of Fas exon 6, resulting in enhanced cancer cell migration and invasion.36 These are examples where CLK1 inhibitors can be utilized to uncover novel functions of CLK1 in several context-dependent alternative splicing events. Other CLK inhibitors that affect alternative splicing have also been identified (Table 1). Dichloroindolyl enaminonitrile KHBD19 was found to have an effect on alternative splicing of the tissue factor gene.37,38 An analogue of leucettamine B (derived from marine sponge), Leucettine L4139 is capable of inducing autophagy and is implicated in the treatment of neurodegenerative diseases such as Alzheimer’s disease.40 CX-4945, an inhibitor of casein kinase 2 and implicated in cancer treatment,41 inhibited specific SR protein phosphorylation to a greater extent compared with TG003 at the same concentration.42 This differential inhibition of SR protein phosphorylation by various CLK inhibitors may reflect the different patterns of SR repeat regions within SR proteins.43 Another explanation may be that this is a result of “progressive phosphorylation” of SR proteins.44,45 The strength of CLK inhibition may stall this “progressive phosphorylation” at different states and induce various alternative splicing patterns
SR protein-specific kinases (SRPKs) (Figure 3A). Other kinases, such as cAMP-dependent protein kinase A, protein kinase C,24 and topoisomerase I,25 are also known to phosphorylate SR proteins, but there has been no direct evidence yet. Generally, the inhibition of these kinases would inhibit the phosphorylation of SR proteins, and would induce exon-skipping. Since the complete regulatory landscape of SR proteins in regulating context-dependent splicing has yet to be uncovered,26 the detailed mechanism of these RNA-binding protein kinases in regulating specific transcripts compared with their global impact on alternative splicing is under investigation. Recent findings that function of a SR protein can depend on other SR proteins make the situation more complex.27 The following summary will focus on the potential of the inhibitors of RNA-binding protein kinases as therapeutic agents in regulating aberrant splicing related to inherited disorders and malignancies, as well as their potential in uncovering novel context-dependent mechanisms. There is much information unrelated to splicing which may be connected to off-target effects, and thus more specific inhibitors, which specifically correct aberrant splicing are in demand.
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DUCHENNE MUSCULAR DYSTROPHY (DMD) AND CLK INHIBITORS DMD is found in 1 out of 3500 newborn boys and is the most frequent type of life-threatening myopathy. Mutations in the dystrophin gene, which create out-of-frame premature termination codons (PTCs), decrease dystrophin protein expression and cause DMD.28,29 A CLK inhibitor, TG003, was found to induce the skipping of particular mutated exons in patients with Duchenne muscular dystrophy (DMD), converting a nonfunctional dystrophin protein to one that is internally deleted but partially functional.30 This mutated exon-skipping strategy of TG003 develops an in-frame dystrophin protein, which causes a milder type of dystrophy (Figure 3B). Clinical studies have been trying to apply this exon-skipping strategy for the clinical treatment of DMD.31 The search for compounds that inhibit kinases responsible for SR protein phosphorylation has opened C
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ACS Chemical Biology Table 2. DYRK1A Inhibitors and SRPK Inhibitor
deregulated pluripotency and neuronal differentiation.52 In addition to recent findings of epigenetic mechanisms involved in this disease,53 DYRK1A overdosage was reported to enhance specific SR protein phosphorylation, showing the interactions between DYRK1A and specific SR proteins. These interactions induce aberrant splicing of neurotrophic tyrosine kinase, receptor type 2 (NTRK2), and acetylcholinesterase (AChE) genes, which are known to be important for synapses composition in the brains of the patients of this disease.54 DYRK1A has been reported to interact with SRSF155 and SF3B1,56 underscoring the possibility of its involvement in alternative splicing regulation. Next, we will refer to recent interesting findings on DYRK1A inhibitors, whose relationship with splicing regulation is yet to be investigated. Inhibitors of DYRK1A have been developed in the potential treatment of Alzheimer’s disease and Down syndrome.57 The green tea flavonol epigallocatechin-gallate (EGCG) was identified 11 years ago as a DYRK1A inhibitor58 and was shown to rescue defects by DYRK1A on brain morphogenic alterations in animal models,59 and cognitive deficits in mouse models and human.60 Harmine is a plant alkaloid and possessed more potent DYRK1A inhibitory activity than EGCG by binding to the ATP-binding pocket of DYRK1A,61 but it also inhibited monoamine oxidase, limiting its applications.62 INDY (Inhibitor of DYRK) was found as a selective inhibitor without this monoamine oxidase inhibitory activity and was able to reverse aberrant tauphosphorylation and recover Xenopus embryos from head malformation63 (Table 2). Another aspect of DYRK1A is its involvement in the stability of epidermal growth factor receptor (EGFR) in glioblastoma. Inhibition of DYRK1A by harmine or INDY showed a defect in tumor cell self-renewal in glioblastoma primary cells, which implies a promising therapeutic tool where tyrosine kinase inhibitors have partial efficacy.64 Recently, a novel class of kinase inhibitors, 2,4bisheterocyclic substituted thiophenes, were identified as potent DYRK1A inhibitors with reduced cytotoxicity and higher metabolic stability and implicated for the possibility to provide
in a context-dependent manner, but this hypothesis awaits further investigation. The use of steric-blocking oligonucleotides to induce exonskipping by blocking ESEs of the exon should have the advantage of specificity over chemical compounds. Unfortunately, steric-blocking oligonucleotides have encountered various problems such as toxicity (accumulation in the liver, kidney, or spleen), poor intracellular uptake, instability against nucleases, interferon response, complement activation, blood coagulation, off-target effects, and cost problems. Chemical modifications of steric-blocking oligonucleotides such as peptide nucleic acids, deoxynucleotide oligonucleotides, 2′substituted oligonucleotides, and phosphorodiamidate morpholino oligomers have been designed to overcome these problems. Chemical modifications of these steric-blocking oligonucleotides and their use in clinical trials can be found in an excellent review.46 Interestingly, dantrolene was found to enhance the effect of steric-blocking oligonucleotides to improve the efficacy of exon-skipping therapy of DMD.47 This may lead to a new convergence of chemical-based and nucleic acid-based medicine.
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DOWN SYNDROME AND DYRK INHIBITORS Down syndrome is the most frequently found congenital disease caused by chromosomal defect, mostly because of trisomy 21.48 Patients with Down syndrome experience physical growth delays and intellectual disability. Without proper medical care, there is a high risk of early death due to heart problems or infections. The DYRK family of kinases is of the same branch of the kinome as the CLK1 family.49 Twenty years have passed since the identification of the founding member,50 and other members have been found in all eukaryotes with critical functions in developmental processes and homeostasis. A subfamily of the DYRK family contains the pre-mRNA processing protein 4 kinases (PRP4s), a possible checkpoint kinase of splicing that is involved in B complex formation of the spliceosome.51 Analyses of the extra copy of chromosome 21 revealed DYRK1A as an important factor in D
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ACS Chemical Biology Table 3. SF3B1 Inhibitors
inhibitors may also be effective and cause widespread changes in alternative splicing.76 A recent report has shown that differential phosphorylation of adaptor proteins by AKT isoforms recruits a histone methyl transferase to the elongation complex as well as histone methylated-binding protein and polyprimidine tract-binding protein (PTB) to the methylated exon. This process results in alternative splicing of f ibroblast growth factor receptor-2 (FGFR-2), which is linked to lung cancer.77 Thus, further investigation of a possible involvement of SRPK inhibitors as therapeutics in angiogenesis as well as lung cancer is awaited.
higher efficacy for the treatment of tauopathies through modulating alternative splicing.65
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ANGIOGENIC DISEASES, LUNG CANCER, AND SRPK INHIBITORS Angiogenesis is often found as an uninvited state in the progression of various diseases including cancer. The major pro-angiogenic factor is vascular endothelial growth factor (VEGF), isoforms of which are important in controlling antiangiogenic isoforms as well as pro-angiogenic ones. SRPKs are the first kinases found to phosphorylate SR proteins.66,67 Extensive screening by in vitro phosphorylation assays identified SR protein phosphorylation inhibitor (SRPIN) 340 as an inhibitor of SRPK1 and SRPK2.68 This compound was also found to inhibit hepatitis C virus replication.69 Interestingly, a derivative of SRPIN340 had no inhibitory effect on SRPKs but blocked virion assembly of the dengue virus by dislocating the nucleocapsid.70 SRPIN340 did have an impact on inhibiting neovascularization through changing the nucleo-cytoplasmic localization of SRSF171,72 and was reported to attenuate choroidal neovascularization.73 Recent evidence has shown that an mRNA readthrough isoform VEGF-Ax was responsible for the antiangiogenic activity. The enhancer cis-element of this VEGF-Ax contains an hnRNP A2/B1 element and was shown that hnRNP A2/B1 facilitated mRNA readthrough.74 Interestingly, this element is sandwiched by two SRSF1 elements and thus hnRNPA2/B1 may strengthen its function by cytoplasmic relocalization of SRSF1 by SRPIN340. This is also in line with the concept that SRSF1 is a proto-oncogene75 and may block the antiangiogenic VEGF-Ax isoform by preventing readthrough. However, this hypothesis needs to be verified with further experimental evidence. In addition to these findings, SRPK was found to be the major target of v-akt murine thymoma viral oncogene homologue (AKT) in epidermal growth factor (EGF)-induced alternative splicing, where SRPK
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FAMILIAL DYSAUTONOMIA (FD) AND PLANT CYTOKININS FD is a lethal autosomal recessive disease that affects sensory and autosomal nerves by inducing progressive degeneration. Patients are homozygous for a mutation at the sixth nucleotide of the 5′ splice site of intron 20 (IVS20 + 6T > C) in the IKBKAP gene.78,79 Tocotrienols were found to upregulate both full-length and exon 20-lacking transcripts,80 and epigallocatechin gallate (EGCG) reduced the level of hnRNP A2/B1 and corrected aberrant splicing without increasing exon 20-lacking transcripts.81 Using a lymphoblast cell line from an FD patient, 1040 compounds were screened to find the plant cytokinin kinetin to correct splicing of the mutant IKBKAP gene and to increase IKAP protein expression.82 Kinetin is a dietary supplement, and orally administered animal studies have shown that kinetin is distributed to the central nervous system as well as plasma with no clastogenic properties. A 28-day oral administration to FD patients did increase full-length IKBKAP transcripts without adverse effects.83 During a search for food supplements that affect neuronal function, phosphatidylserine was also found to correct splicing and increase IKAP protein levels in lymphoblast cell lines of FD patients84 and FD model mice,85 although these E
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ACS Chemical Biology model mice lacked FD symptoms.86 Cardiac glycosides were also found to correct mutant IKBKAP splicing,87 which we have previously mentioned in correcting MAPT exon 10 missplicing.12 The lack of connection between IKAP production and FD symptoms still needs to be clarified, but recent findings in neural crest-specific ablation of Ikbkap show that IKAP is required for Pax3+ progenitors, which are important for the second wave of neurogenesis after trunk neural crest migration.88 Findings in the brains of FD patients and FD mouse models of down-regulated genes show involvement of genes related to oligodendrocyte differentiation and myelin formation.86 Our novel compound, which rectifies aberrant splicing of IKBKAP, showed direct involvement in correcting aberrant splicing of mutated IKBKAP, recovered the hypomodified levels of uridine at the wobble position in cytoplasmic tRNAs, increased the expression of IKAP protein, and improved cell growth of primary fibroblasts from FD patients (Yoshida et al., accepted).89
nM in tumor cell lines, modulate splicing of the mouse double minute 2 (MDM2) proto-oncogene gene in human tumor xenografts in mice, and can be produced in gram quantities.95 SF3B1 mutations are also found in tumors other than those in MDS,99 leaving the question of how these mutations are connected with tissue-specific tumors, why SF3B1 mutations have opposite effects on the prognosis of MDS and chronic lymphocytic leukemia, and how they are involved in disease progression and tumorigenesis.100,101 The mechanism of these SF3B1 inhibitors, which allow U2 snRNP to recognize upstream, unproductive branch point sites,102 will give us a clue to clarify these questions as well as to determine further, suitable applications of these inhibitors in treating each of these malignant disorders.
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SPINAL MUSCULAR ATROPHY (SMA) AND SPLICING-MODIFYING-COMPOUNDS SMA is found with high prevalence and high infantile mortality rate caused by progressive symmetric weakness and atrophy of the proximal muscle. SMA is caused by deletion, truncation, or mutation of the survival motor neuron 1 (SMN1) gene.103 SMN2 is a nearly identical copy of SMN1 but fails to compensate SMN1 due to a difference at the sixth intronic nucleotide of the 5′ splice site of exon 7, which results in its skipping and unstable SMN protein. The mechanism of abnormal SMN2 exon 7 splicing has been studied104 and has been targeted for compensating the defect of SMN1. A compound that induces exon 7 inclusion of the SMN2 gene would provide tremendous benefits to children suffering from SMA.105 Although it is unclear whether the effect is direct or indirect, the first chemical compounds found to correct aberrant splicing are those of the SMN2 gene found in spinal muscular atrophy (SMA) patients. Sodium butyrate, known as an inhibitor of histone deacetylase (HDAC) activity,106 was found to induce inclusion of SMN2 exon 7.107 Its derivative, phenylbutyrate (PBA), is a compound that has been used clinically to treat patients with sickle cell anemia and thalassemia.108 Although PBA is a well-tolerated FDA approved drug, SMN gene expression varied among individuals and was shown to have short drug half-life in a clinical study.109 Sodium butyrate was shown to induce the expression of two SR proteins and eventually correct SMN2 mis-splicing.107 It was also shown to regulate alternative splicing in HeLa cells correlating with histone H4 acetylation and decreased recruitment of SRSF5.110 Aclarubicin was found to induce exon 7 inclusion in the aberrantly spliced SMN2 gene, but other tetracycline derivatives do not possess this function.111 Interestingly, aclarubicin is known to interact with type II DNA topoisomerase,112 whose defect is critical in motor neuron development.113 In vitro studies could not recapitulate the effect of aclarubicin on the splicing of SMN2 exon 7, but a similar tetracycline derivative, PTK-SMA1, was found to alter SMN2 exon 7 splicing in vitro as well as in fibroblasts in SMA patients and in mouse models.114 While many compounds have been found to induce SMN2 exon 7 splicing in addition to ones that modulate cell signaling, transcription, or translation,115 none have shown significant efficacy in clinical trials. The most successful ones at present are olesoxime, a strong neuroprotective compound with cholesterol-like structure,116 and ISIS-SMNRx, a uniform 2′-O-methoxyethyl modified antisense drug that acts against a strong splicing silencer of SMN2 exon 7. Olesoxime has completed phase II/III pivotal studies and ISISSMNRx is under a phase II trial, both with promising results.117
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MYELODYSPLASTIC SYNDROME AND SF3B1 INHIBITORS MDS is a preleukemic state characterized by deregulated, dysplastic blood cell production. Many gene mutations that were implicated in the pathogenesis of MDS were also found in other myeloid malignancies, making them irrelevant to the disease. Recent whole-exome sequencing of paired tumor/ control DNA revealed unexpected mutations of splicing factors, including SF3B1.90 Recent breakthroughs in the splicing field came from the discovery of splicing inhibitors, the SF3B1 inhibitors (Table 3). These SF3B1 inhibitors, spliceostatin A (SSA),91 pladienolide-B,92 and GEX1A93 were found in a screen of anticancer reagents. Since the SF3B1 mutations found in patients with MDS are likely to be gain-of-function mutations,90 it has been suggested that SF3B1 inhibitors have possible implications in treating this preleukemic state and related myeloid disorders.94 Although inhibition of SF3B1, which plays key roles in the function of the core spliceosome, would be expected to completely inhibit splicing, this is not the case. In addition to the partially inhibiting function of splicing in cultured cells compared with in vitro, the inhibition of splicing by SF3B1 inhibitors generates short transcripts, the truncated proteins of which are less active and show selective action on tumor cells, as found for Spliceostatin A.91 Pladienolide B, an antitumor macrolide was also found to interact with a SF3b subunit and inhibit splicing, implicating SF3b as an antitumor target.92 It is readily synthesized and much more stable other SF3B1 inhibitors.95 E7107 is a urethane derivative of pladienolide D that displayed strong antitumor activity.96 A first-in-human phase I trial of E7107 showed dose-dependent reversible inhibition of pre-mRNA processing target genes in peripheral blood mononuclear cells of patients with solid tumors refractory to standard therapies. Although no response during treatment was observed, one patient had partial response after drug discontinuation.97 Another similar study showed stable disease in eight out of twenty-six patients. 98 E7107 and pladienolide B bind spliceosome-associated protein 130 (SAP130) and pladienolide B regulates splicing of DnaJ (Hsp40) homologue, subfamily B, member 1 (DNAJB1), bromodomain containing 2 (BRD2), and RIO kinase 3 (RIOK3) genes in HeLa cells.92 Compared to E7107, novel analogues called sudemycins do not degrade in human plasma, show much better chemical stability, exhibit half maximal inhibitory concentration (IC50) values of ∼80−500 F
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HUMAN IMMUNODEFICIENCY VIRUS-1 (HIV-1) INFECTION AND SPLICING-MODIFYING-COMPOUNDS HIV-1 infection is the cause of acquired immunodeficiency syndrome (AIDS) and is the result of highly regulated, complex alternative splicing of the HIV-1 genome by host factors. Indole derivatives have been found to interact with specific SR proteins. An in vitro splicing assay using pre-mRNA of adenovirus and β-globin derivatives was used to screen 4000 chemical compounds and found that indole derivatives bind the SR domain of SRSF1 regardless of its phosphorylation state. Specific indole derivatives have differential effects on SRSF1and SRSF6-dependent splicing, implicating its therapeutic potential on aberrant alternative splicing in diseases such as HIV-1.118 In fact, IDC16, an indole derivative, efficiently blocked HIV production of multitherapy-resistant isolates in peripheral blood mononuclear cells or macrophages of patients.119 The aforementioned KH-BD19 (Table 1) is an indole derivative that possesses strong CLK inhibitory activity.38 hnRNPA1 is known as a key regulator of HIV-1 alternative splicing and two models of its mechanism were proposed.120−122 New evidence from a recent phylogenetic and thermodynamic study show the importance of hnRNPA1 binding to RNA structural beacons that may influence HIV-1 splice site usage.123 A novel compound that inhibits hnRNPA1 function may help clarify the mechanism of hnRNPA1 in splicing regulation of HIV-1 in addition to its function in various alternative splicing events.124
possibility of lack of specificity. It is a challenging issue to target specific hnRNPs or RNA-binding proteins (RBPs) to selectively correct mis-splicing with a compound, but recent findings using induced pluripotent stem (iPS) cells for validation show promising results.126 Lastly, in the era of high-throughput sequencing, this technique is essential for drug discovery of splicing-modifying compounds. One of the rate-limiting factors is the precise estimation of alternatively spliced exons from high-throughput sequencing of short cDNA fragments (RNA-Seq). Various strategies have been developed for this purpose including the mixture-of-isoforms (MISO) model127 and others such as the Arabidopsis Tiling-Array-based Detection of Exons 2 model.128 These powerful strategies will become essential not only in evaluating off-target effects but also in elucidating the molecular mechanisms of splicing-modifying compounds.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Grants-in-Aid for Scientific Research (21249013 to M.H. and 24591920 to K.O.), Research Program of Innovative Cell Biology (231006 to MH), and Platform for Drug Discovery (to M.H.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, and by Grants from National Institute of Biomedical Innovation (NIBIO, 241011 to M.H.) and CREST of Japan Science and Technology Agency (JST, 231038 to M.H.). The authors apologize to not properly cited research in this review owing to space limitations.
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CONCLUSIONS AND FUTURE PERSPECTIVES Taken together, we have reviewed the impact of chemical compounds in regulating alternative splicing and their potential in curing disease. We have shown that the targets of most of the compounds listed here have additional roles apart from splicing, which implies the necessity of further structural optimization. Elaborate screening procedures (improving the bichromatic fluorescent splicing reporter system using a closer diseasephenotype strategy or screening compounds with activity below micromolar range) as well as methods to improve selectivity are essential125 for obtaining splicing-specific compounds. The efficacy of a compound is an important factor in target validation, which is of course true in screening compounds that modulate alternative splicing. It is necessary to select the best compound for each inherited disease or malignancy in order to be applicable in clinical studies. The way compounds affect aberrant splicing in a tissuespecific or disease-specific manner is also an issue to be elucidated. Mutations of factors that form the core spliceosome have been found in myelodysplasia syndromes and related disorders.90 Since these are somatic mutations, they may be limited and prone to mutation in the myeloid lineage and other cancerous tissues where these mutations can be found.99 Yet it is puzzling how these diseases can bypass or utilize the splicing machinery for its own sake, and we believe screening for compounds that can correct this phenomenon will give us a deeper insight on the mechanism as well as how to treat these deleterious diseases. We have shown here various compounds that target various pathways involved in splicing. Yet there are many pathways that are to be targeted by compounds. hnRNPA1 is one attractive factor that is involved in numerous splicing events.124 Involvement in numerous splicing events will raise the
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