Design, synthesis, and structure-activity relationship of TAK-418 and its derivatives as a novel series of LSD1 inhibitors with lowered risk of hematological side effects.

Lysine-specific demethylase 1 (LSD1) is an enzyme that demethylates methylated histone H3 lysine 4 (H3K4). Inhibition of LSD1 enzyme activity could increase H3K4 methylation levels and treat diseases associated with epigenetic dysregulation. However, known LSD1 inhibitors disrupt the interaction between LSD1 and cofactors such as GFI1B, causing the risk of hematological toxicity, including thrombocytopenia. Starting from a known LSD1 inhibitor (±)1 as a lead compound, a novel series of LSD1 inhibitors that do not induce the expression of GFI1 mRNA, an in vitro surrogate marker of LSD1-GFI1B dissociation, has been designed and synthesized. Initial structure-activity relationship (SAR) studies revealed the structural features key to avoiding GFI1 mRNA induction. Such SAR information enables optimization of LSD1 inhibitors with lowered risk of hematological side effects; TAK-418 ((1R,2R)-2n), the clinical candidate compound found through this optimization, has a hematological safety profile in rodents and humans. We further confirmed that oral administration of TAK-418 at 0.3 and 1 mg/kg for 2 weeks ameliorated memory deficits in mice with NMDA receptor hypofunction, suggesting potential of efficacy in neurodevelopmental disorders. TAK-418 warrants further investigation as a novel class of LSD1 inhibitors with a superior safety profile for the treatment of CNS disorders.

Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays.

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds.

T-448, a specific inhibitor of LSD1 enzyme activity, improves learning function without causing thrombocytopenia in mice.

Dysregulation of histone H3 lysine 4 (H3K4) methylation has been implicated in the pathogenesis of several neurodevelopmental disorders. Targeting lysine-specific demethylase 1 (LSD1), an H3K4 demethylase, is therefore a promising approach to treat these disorders. However, LSD1 forms complexes with cofactors including growth factor independent 1B (GFI1B), a critical regulator of hematopoietic differentiation. Known tranylcypromine-based irreversible LSD1 inhibitors bind to coenzyme flavin adenine dinucleotide (FAD) and disrupt the LSD1-GFI1B complex, which is associated with hematotoxicity such as thrombocytopenia, representing a major hurdle in the development of LSD1 inhibitors as therapeutic agents. To discover LSD1 inhibitors with potent epigenetic modulation and lower risk of hematotoxicity, we screened small molecules that enhance H3K4 methylation by the inhibition of LSD1 enzyme activity in primary cultured rat neurons but have little impact on LSD1-GFI1B complex in human TF-1a erythroblasts. Here we report the discovery of a specific inhibitor of LSD1 enzyme activity, T-448 (3-((1S,2R)-2-(cyclobutylamino)cyclopropyl)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzamide fumarate). T-448 has minimal impact on the LSD1-GFI1B complex and a superior hematological safety profile in mice via the generation of a compact formyl-FAD adduct. T-448 increased brain H3K4 methylation and partially restored learning function in mice with NMDA receptor hypofunction. T-448-type LSD1 inhibitors with improved safety profiles may provide unique therapeutic approaches for central nervous system disorders associated with epigenetic dysregulation.

Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability.

The modulation of pre-mRNA splicing is proposed as an attractive anti-neoplastic strategy, especially for the cancers that exhibit aberrant pre-mRNA splicing. Here, we discovered that T-025 functions as an orally available and potent inhibitor of Cdc2-like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T-025 reduced CLK-dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive-associated biomarker of T-025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T-025 treatment. MYC activation, which altered pre-mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti-tumor efficacy of T-025 in an allograft model of spontaneous, MYC-driven breast cancer, at well-tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC-driven cancer patients.

Discovery of spiro[indole-3,2'-pyrrolidin]-2(1H)-one based inhibitors targeting Brr2, a core component of the U5 snRNP.

Bad response to refrigeration 2 (Brr2) is a member of the Ski2-like RNA helicases, and an essential component of the U5 small nuclear ribonucleoprotein (snRNP). A particularly important role of Brr2 is the ATP-dependent unwinding of the U4/U6 RNA duplex, which is a critical step in spliceosomal activation. Despite its biological importance, selective inhibitor for Brr2 had not been reported until our recent report. Here, we describe novel and structurally distinct spiro[indole-3,2'-pyrrolidin]-2(1H)-one based Brr2 inhibitors with superior activity to the previously reported 4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione series. Using an RNA dependent ATPase assay as a guide, high-throughput screening, hit validation by structure-activity relationship (SAR) study, and subsequent chemical optimization to increase the ATPase inhibitory activity were performed. Thereafter, selectivity and helicase inhibitory activity of optimized compounds were confirmed. In the course of the study, compounds were synthesized using a three-component reaction, which accelerated the optimization process. All these efforts finally culminated in the discovery of the potent and selective Brr2 inhibitors (32a and 33a) exhibiting helicase inhibitory activity at submicromolar concentrations. Thus, compounds 32a and 33a could be valuable molecular probes to study the functions of Brr2 and molecular machinery of RNA splicing.

Discovery of selective ATP-competitive eIF4A3 inhibitors.

Eukaryotic initiation factor 4A3 (eIF4A3), an ATP-dependent RNA helicase, is a core component of exon junction complex (EJC). EJC has a variety of roles in RNA metabolism such as translation, surveillance, and localization of spliced RNA. It is worthwhile to identify selective eIF4A3 inhibitors with a view to investigating the functions of eIF4A3 and EJC further to clarify the roles of the ATPase and helicase activities in cells. Our chemical optimization of hit compound 2 culminated in the discovery of ATP-competitive eIF4A3 inhibitor 18 with submicromolar ATPase inhibitory activity and excellent selectivity over other helicases. Hence, compound 18 could be a valuable chemical probe to elucidate the detailed functions of eIF4A3 and EJC.

CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor.

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.

Prolyl-tRNA synthetase inhibition promotes cell death in SK-MEL-2 cells through GCN2-ATF4 pathway activation.

Protein translation is highly activated in cancer tissues through oncogenic mutations and amplifications, and this can support survival and aberrant proliferation. Therefore, blocking translation could be a promising way to block cancer progression. The process of charging a cognate amino acid to tRNA, a crucial step in protein synthesis, is mediated by tRNA synthetases such as prolyl tRNA synthetase (PRS). Interestingly, unlike pan-translation inhibitors, we demonstrated that a novel small molecule PRS inhibitor (T-3861174) induced cell death in several tumor cell lines including SK-MEL-2 without complete suppression of translation. Additionally, our findings indicated that T-3861174-induced cell death was caused by activation of the GCN2-ATF4 pathway. Furthermore, the PRS inhibitor exhibited significant anti-tumor activity in several xenograft models without severe body weight losses. These results indicate that PRS is a druggable target, and suggest that T-3861174 is a potential therapeutic agent for cancer therapy.

A Novel LSD1 Inhibitor T-3775440 Disrupts GFI1B-Containing Complex Leading to Transdifferentiation and Impaired Growth of AML Cells.

Dysregulation of lysine (K)-specific demethylase 1A (LSD1), also known as KDM1A, has been implicated in the development of various cancers, including leukemia. Here, we describe the antileukemic activity and mechanism of action of T-3775440, a novel irreversible LSD1 inhibitor. Cell growth analysis of leukemia cell lines revealed that acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia cells (AMKL) were highly sensitive to this compound. T-3775440 treatment enforced transdifferentiation of erythroid/megakaryocytic lineages into granulomonocytic-like lineage cells. Mechanistically, T-3775440 disrupted the interaction between LSD1 and growth factor-independent 1B (GFI1B), a transcription factor critical for the differentiation processes of erythroid and megakaryocytic lineage cells. Knockdown of LSD1 and GFI1B recapitulated T-3775440-induced transdifferentiation and cell growth suppression, highlighting the significance of LSD1-GFI1B axis inhibition with regard to the anti-AML effects of T-3775440. Moreover, T-3775440 exhibited significant antitumor efficacy in AEL and AMKL xenograft models. Our findings provide a rationale for evaluating LSD1 inhibitors as potential treatments and indicate a novel mechanism of action against AML, particularly AEL and AMKL. Mol Cancer Ther; 16(2); 273-84. ©2016 AACR.

Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing.

Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

Structure-based discovery of cellular-active allosteric inhibitors of FAK.

In order to develop potent and selective focal adhesion kinase (FAK) inhibitors, synthetic studies on pyrazolo[4,3-c][2,1]benzothiazines targeted for the FAK allosteric site were carried out. Based on the X-ray structural analysis of the co-crystal of the lead compound, 8-(4-ethylphenyl)-5-methyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazine 4,4-dioxide 1 with FAK, we designed and prepared 1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin derivatives which selectively inhibited kinase activity of FAK without affecting seven other kinases. The optimized compound, N-(4-tert-butylbenzyl)-1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin-8-amine 4,4-dioxide 30 possessed significant FAK kinase inhibitory activities both in cell-free (IC50=0.64µM) and in cellular assays (IC50=7.1µM). These results clearly demonstrated a potential of FAK allosteric inhibitors as antitumor agents.

Discovery and characterization of novel allosteric FAK inhibitors.

Focal adhesion kinase (FAK) regulates cell survival and proliferation pathways. Here we report the discovery of a highly selective series of 1,5-dihydropyrazolo[4,3-c][2,1]benzothiazines that demonstrate a novel mode of allosteric inhibition of FAK. These compounds showed slow dissociation from unphosphorylated FAK and were noncompetitive with ATP after long preincubation. Co-crystal structural analysis revealed that the compounds target a novel allosteric site within the C-lobe of the kinase domain, which induces disruption of ATP pocket formation leading to the inhibition of kinase activity. The potency of allosteric inhibition was reduced by phosphorylation of FAK. Coupled SAR analysis revealed that N-substitution of the fused pyrazole is critical to achieve allosteric binding and high selectivity among kinases.

Genome-wide DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) residing in mouse pluripotent stem cells.

DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), has elucidated tissue-specific gene function in mouse tissues. Here, we identified and profiled thousands of T-DMRs in embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPSCs). T-DMRs of ESCs compared with somatic tissues well illustrated gene function of ESCs, by hypomethylation at genes associated with CpG islands and nuclear events including transcriptional regulation network of ESCs, and by hypermethylation at genes for tissue-specific function. These T-DMRs in EGCs and iPSCs showed DNA methylation similar to ESCs. iPSCs, however, showed hypomethylation at a considerable number of T-DMRs that were hypermethylated in ESCs, suggesting existence of traceable progenitor epigenetic information. Thus, DNA methylation profile of T-DMRs contributes to the mechanism of pluripotency, and can be a feasible solution for identification and evaluation of the pluripotent cells.

Resistance to 5-aza-2'-deoxycytidine in genic regions compared to non-genic repetitive sequences.

The DNA methyltransferase (Dnmt) inhibitor and demethylating agent 5-aza-2'-deoxycytidine (5azadC) has been used to induce cellular differentiation and gene activation. It has been approved for treating several kinds of malignancies due to its ability to reactivate silenced tumor suppressor genes. Considering the potential effect of 5azadC on non-targeted genomic regions in normal cells, we investigated its effect on repetitive sequences and selected gene loci, Oct-4, Sall3, Per1, Clu, Dpep1 and Igf2r, including tissue-dependent and differentially methylated regions, by treating mouse NIH/3T3 fibroblast cells with concentrations of 5azadC ranging from 0.001 to 5 microM. Demethylation of minor satellite repeats and endogenous viruses was concentration dependent, and the demethylation was strong at 1 and 5 microM. In genic regions, the methylation level decreased only at 0.1 microM, but was minimally altered at concentrations lower or higher, regardless of the abundance of CpG sites. Thus, repeats are strongly demethylated, but genic regions are only demethylated at effective doses. Genes were activated by 5azadC treatment and were accompanied by a unique combination of histone modifications in genic regions, including an increased level of H3K9me3 and a decreased level of AcH3. Increase of H3K9me3 in genic regions was not observed in Dnmt knock out cells. We identified differential effects of 5azadC on repetitive sequences and genic regions and revealed the importance of choosing appropriate 5azadC doses to achieve targeted gene recovery.

Establishment of trophoblast stem cell lines from somatic cell nuclear-transferred embryos.

Placental abnormalities occur frequently in cloned animals. Here, we attempted to isolate trophoblast stem (TS) cells from mouse blastocysts produced by somatic cell nuclear transfer (NT) at the blastocyst stage (NT blastocysts). Despite the predicted deficiency of the trophoblast cell lineage, we succeeded in isolating cell colonies with typical morphology of TS cells and cell lines from the NT blastocysts (ntTS cell lines) with efficiency as high as that from native blastocysts. The established 10 ntTS cell lines could be maintained in the undifferentiated state and induced to differentiate into several trophoblast subtypes in vitro. A comprehensive analysis of the transcriptional and epigenetic traits demonstrated that ntTS cells were indistinguishable from control TS cells. In addition, ntTS cells contributed exclusively to the placenta and survived until term in chimeras, indicating that ntTS cells have developmental potential as stem cells. Taken together, our data show that NT blastocysts contain cells that can produce TS cells in culture, suggesting that proper commitment to the trophoblast cell lineage in NT embryos occurs by the blastocyst stage.

Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells.

In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a(-/-) embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a(-/-) cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a(-/-) cells. These data indicate that G9a site-selectively contributes to DNA methylation.

Dimethyl sulfoxide has an impact on epigenetic profile in mouse embryoid body.

Dimethyl sulfoxide (DMSO), an amphipathic molecule, is widely used not only as a solvent for water-insoluble substances but also as a cryopreservant for various types of cells. Exposure to DMSO sometimes causes unexpected changes in cell fates. Because mammalian development and cellular differentiation are controlled epigenetically by DNA methylation and histone modifications, DMSO likely affects the epigenetic system. The effects of DMSO on transcription of three major DNA methyltransferases (Dnmts) and five well-studied histone modification enzymes were examined in mouse embryonic stem cells and embryoid bodies (EBs) by reverse transcription-polymerase chain reaction. Addition of DMSO (0.02%-1.0%) to EBs in culture induced an increase in Dnmt3a mRNA levels with increasing dosage. Increased expression of two subtypes of Dnmt3a in protein levels was confirmed by Western blotting. Southern blot analysis revealed that DMSO caused hypermethylation of two kinds of repetitive sequences in EBs. Furthermore, restriction landmark genomic scanning, by which DNA methylation status can be analyzed on thousands of loci in genic regions, revealed that DMSO affected DNA methylation status at multiple loci, inducing hypomethylation as well as hypermethylation depending on the genomic loci. In conclusion, DMSO has an impact on the epigenetic profile: upregulation of Dnmt3a expression and alteration of genome-wide DNA methylation profiles with phenotypic changes in EBs.