Identification and Biological Evaluation of a Potent and Selective JAK1 Inhibitor for the Treatment of Pulmonary Fibrosis.

Janus kinase 1 (JAK1) plays a pivotal role in regulating inflammation and fibrosis via the JAK/STAT signaling pathway, making it a promising target for associated diseases. In this study, we explored the modification of an N-methyl 1H-pyrrolo[2,3-b]pyridine-5-carboxylate core, leading to the identification of 4-(((2S,4S)-1-(4-trifluoromethyl)-2-methylpiperidin-4-yl)amino)-N-methyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (36b) as a highly potent and selective JAK1 inhibitor. Compound 36b exhibited an impressive IC50 value of 0.044 nM for JAK1 and demonstrated remarkable selectivity of 382-fold, 210-fold, and 1325-fold specificity over JAK2, JAK3, and TYK2, respectively. The kinase panel assays further confirmed its specificity, and cell-based experiments established its efficacy in inhibiting JAK1-STAT phosphorylation in human L-132 or SK-MES-1 cells. Pharmacokinetic studies revealed that compound 36b boasts an oral bioavailability exceeding 36%. In a bleomycin-induced fibrosis mouse model, compound 36b significantly reduced STAT3 phosphorylation, resulting in improvement in body weight and reduced collagen deposition, all achieved without significant side effects.

Identification of proteomic landscape of drug-binding proteins in live cells by proximity-dependent target ID.

Direct identification of the proteins targeted by small molecules can provide clues for disease diagnosis, prevention, and drug development. Despite concentrated attempts, there are still technical limitations associated with the elucidation of direct interactors. Herein, we report a target-ID system called proximity-based compound-binding protein identification (PROCID), which combines our direct analysis workflow of proximity-labeled proteins (Spot-ID) with the HaloTag system to efficiently identify the dynamic proteomic landscape of drug-binding proteins. We successfully identified well-known dasatinib-binding proteins (ABL1, ABL2) and confirmed the unapproved dasatinib-binding kinases (e.g., BTK and CSK) in a live chronic myeloid leukemia cell line. PROCID also identified the DNA helicase protein SMARCA2 as a dasatinib-binding protein, and the ATPase domain was confirmed to be the binding site of dasatinib using a proximity ligation assay (PLA) and in cellulo biotinylation assay. PROCID thus provides a robust method to identify unknown drug-interacting proteins in live cells that expedites the mode of action of the drug.

Discovery and Biological Evaluation of N-Methyl-pyrrolo[2,3-b]pyridine-5-carboxamide Derivatives as JAK1-Selective Inhibitors.

Janus kinase 1 (JAK1) plays a key role in most cytokine-mediated inflammatory and autoimmune responses through JAK/STAT signaling; thus, JAK1 inhibition is a promising therapeutic strategy for several diseases. Analysis of the binding modes of current JAK inhibitors to JAK isoforms allowed the design of N-alkyl-substituted 1-H-pyrrolo[2,3-b] pyridine carboxamide as a JAK1-selective scaffold, and the synthesis of various methyl amide derivatives provided 4-((cis-1-(4-chlorobenzyl)-2-methylpiperidin-4-yl)amino)-N-methyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (31g) as a potent JAK1-selective inhibitor. In particular, the (S,S)-enantiomer of 31g (38a) exhibited excellent potency for JAK1 and selectivity over JAK2, JAK3, and TYK2. On investigating the effect of 31g on hepatic fibrosis, it was found that it reduces the proliferation and fibrogenic gene expression of TGF-ß-induced hepatic stellate cells (HSCs). Specifically, 31g significantly inhibited TGF-ß-induced migration of HSCs at 0.25 µM in wound-healing assays.

RRM but not the Asp/Glu domain of hnRNP C1/C2 is required for splicing regulation of Ron exon 11 pre-mRNA.

The Ron proto-oncogene is a human receptor for macrophage-stimulating protein (MSP). The exclusion of exon 11 in alternative splicing generates ΔRON protein that is constitutively activated. Heterogenous ribonucleaoprotein (hnRNP) C1/C2 is one of the most abundant proteins in cells. In this manuscript, we showed that both hnRNP C1 and C2 promoted exon 11 inclusion of Ron pre-mRNA and that hnRNP C1 and hnRNP C2 functioned independently but not cooperatively. Moreover, hnRNP C1 stimulated exon 11 splicing through intron 10 activation but not through intron 11 splicing. Furthermore, we showed that, whereas the RRM domain was required for hnRNP C1 function, the Asp/Glu domain was not. In conclusion, hnRNP C1/C2 promoted exon 11 splicing independently by stimulating intron 10 splicing through RRM but not through the Asp/Glu domain. [BMB Reports 2019; 52(11): 641-646].

Activation of Cryptic 3' Splice-Sites by SRSF2 Contributes to Cassette Exon Skipping.

Here we show that the serine/arginine rich splicing factor 2 (SRSF2) promotes cryptic 3' splice-site (3'AG') usage during cassette exon exclusion in survival of motor neuron (SMN2) minigenes. Deletion of the 3'AG' (3'AG'1), its associated branch point (BP') and polypyrimidine tract (PPT') sequences directs SRSF2 to promote a second 3'AG' (3'AG'2) with less conserved associated region for intron splicing. Furthermore, deletion of both 3'AG'1 and 3'AG'2 and their associated sequences triggered usage of a third 3'AG'3 that has very weak associated sequences. Interestingly, when intron splicing was directed to the 3'AG' cryptic splice-sites, intron splicing from the canonical 3'AG splice-site was reduced along with a decrease in cassette exon inclusion. Moreover, multiple SRSF2 binding sites within the intron are responsible for 3'AG' activation. We conclude that SRSF2 facilitates exon exclusion by activating a cryptic 3'AG' and inhibiting downstream intron splicing.

SRSF2 directly inhibits intron splicing to suppresses cassette exon inclusion.

SRSF2, a Serine-Arginine rich (SR) protein, is a splicing activator that mediates exon inclusion and exclusion events equally well. Here we show SRSF2 directly suppresses intron splicing to suppress cassette exon inclusion in SMN premRNA. Through a serial mutagenesis, we demonstrate that a 10 nt RNA sequence surrounding the branch-point (BP), is important for SRSF2-mediated inhibition of cassette exon inclusion through directly interacting with SRSF2. We conclude that SRSF2 inhibits intron splicing to promote exon exclusion. [BMB Reports 2017; 50(8): 423-428].

Effects of PTCs on nonsense-mediated mRNA decay are dependent on PTC location.

The récepteur d'origine nantais (RON) gene is a proto-oncogene that is responsible for encoding the human macrophage-stimulating protein (MSP) 1 receptor. MSP activation induces RON-mediated cell dissociation, migration and matrix invasion. Isoforms of RON that exclude exons 5 and 6 encode the RONΔ160 protein, which promotes cell transformation in vitro and tumor metastasis in vivo. Premature termination codons (PTCs) in exons activate the nonsense-mediated mRNA decay (NMD) signaling pathway. The present study demonstrated that PTCs at various locations in the alternative exons 5 and 6 could induce NMD of the majority of the spliced, or partially spliced, isoforms. However, the isoforms that excluded exon 6 or exons 5 and 6 were markedly increased when produced from mutated minigenes with inserted PTCs. Furthermore, the unspliced isoform of intron 5 was not observed to be decreased by the presence of PTCs. Notably, these effects may be dependent on the location of the PTCs. The current study demonstrated a novel mechanism underlying the regulation of NMD in alternative splicing.

Suppression of 5' splice-sites through multiple exonic motifs by hnRNP L.

Selection of 5' splice-sites (5'SS) in alternative splicing plays an important role in gene regulation. Although regulatory mechanisms of heterogeneous nuclear ribonucleoprotein L (hnRNP L), a well-known splicing regulatory protein, have been studied in a substantial level, its role in 5'SS selection is not thoroughly defined. By using a KLF6 pre-mRNA alternative splicing model, we demonstrate in this report that hnRNP L inhibits proximal 5'SS but promotes two consecutive distal 5'SS splicing, antagonizing SRSF1 roles in KLF6 pre-mRNA splicing. In addition, three consecutive CA-rich sequences in a CA cassette immediately upstream of the proximal 5'SS are all required for hnRNP L functions. Importantly, the CA-cassette locations on the proximal exon do not affect hnRNP L roles. We further show that the proximal 5'SS but not the two distal 5'SSs are essential for hnRNP L activities. Notably, in a Bcl-x pre-mRNA model that contains two alternative 5'SS but includes CA-rich elements at distal exon, we demonstrate that hnRNP L also suppresses nearby 5'SS activation. Taken together, we conclude that hnRNP L suppresses 5'SS selection through multiple exonic motifs.

SR proteins regulate V6 exon splicing of CD44 pre-mRNA.

CD44 pre-mRNA includes 20 exons, of which exons 1-5 (C1-C5) and exons 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. V6-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 V6 splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 V6 minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect V6 splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit V6 splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a V6 specific primer, we identified that reduced SRSF2 expression significantly reduced the V6 isoform, but increased V6-10 and V6,7-10 isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 V6 splicing. [BMB Reports 2016; 49(11): 612-616].

Detecting RNA-Protein Interaction Using End-Labeled Biotinylated RNA Oligonucleotides and Immunoblotting.

RNA-protein interaction can be detected by RNA pull-down and immunoblotting methods. Here, we describe a method to detect RNA-protein interaction using RNA pull down and to identify the proteins that are pulled-down by the RNA using immunoblotting. In this protocol, RNAs with specific sequences are biotinylated and immobilized onto Streptavidin beads, which are then used to pull down interacting proteins from cellular extracts. The presence of a specific protein is subsequently verified by SDS- polyacrylamide gel electrophoresis and immunoblotting with antibodies. Interactions between the SMN RNA and the PSF protein and between the caspase-2 RNA and the SRSF3 protein (SRp20) in nuclear extract prepared from HeLa cells are illustrated as examples.

Identification of Regulatory-RNAs for Alternative Splicing of Ron Proto-Oncogene.

RON receptor tyrosine kinase is a proto-oncogene that induces cell migration and matrix invasion. RONΔ160 protein, which is produced by exclusion of exon 5 and 6, promotes cell migration, matrix invasion and protection from apoptosis. Alternative splicing regulation of exon 5 and 6 is not well understood. In this manuscript, we identified several new RNA regulatory elements for alternative splicing of Ron proto-oncogene. Firstly, we demonstrated that RNA sequences from EcoRI cleavage sites regulate alternative splicing of Ron exon 5 and 6. Secondly, we showed that the ~30 nt RNA at upstream end of exon 4 and the ~33 nt RNA at downstream end of exon 7 also modulate splicing of exon 5 and 6. Thirdly, our results indicate that the RNA sequences of the ends in exon 4 and 7 are required for the regulatory functions of the RNA from restriction enzyme cleavage sites. Our results provide a new insight for regulation of alternative splicing of Ron proto-oncogene.

Splicing inhibition of U2AF65 leads to alternative exon skipping.

U2 snRNP auxiliary factor 65 kDa (U2AF(65)) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF(65) stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5' splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF(65). U2AF(65) overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF(65) inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF(65) effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF(65) even inhibits general splicing of adenovirus major late (Ad ML) or ß-globin pre-mRNA. Thus, we conclude that U2AF(65) possesses a splicing Inhibitory function that leads to alternative exon skipping.

CD44 alternative splicing and hnRNP A1 expression are associated with the metastasis of breast cancer.

CD44 is a transmembrane receptor for hyaluronic acid. CD44 pre-mRNA contains 19 exons, 9 of which are alternatively spliced. Among the CD44 spliced variants, the v4-7 variant, one of the v6 exon-containing isoforms that contains variable exon 4, 5, 6 and 7, confers metastatic potential to non-metastatic cells. Splicing of CD44 and the function of CD44 isoforms are different in breast cancer cells. hnRNP A1 is a ubiquitously expressed protein with an inhibitory function in pre-mRNA splicing. We showed that CD44v6 isoform, which includes all of the v6-containing mRNA isoforms, had the highest expression level in non-metatatic breast cancer cells (MCF7) when compared to the level in metastatic breast cancer cells (MDA-MB-231) and normal breast cells (MCF10A). Furthermore we showed that hnRNP A1 knockdown regulated splicing of CD44 differently in breast cancer cells. We showed here that CD44 isoform expression is completely different in MDA-MB-231 cells than that in MCF7 and MCF10A cells, whereas MCF7 and MCF10A cells had a similar expression pattern of CD44 isoforms. RT-PCR analysis of CD44v6 showed that MCF7 and MCF10A cells predominantly expressed the c5v6v7v8v9v10c6 isoform. However, in addition to this isoform, MDA-MB-231 cells also expressed the c5v6v8v9v10c6 and c5v6c6 isoforms. We also found that knockdown of hnRNP A1 significantly reduced the expression of c5v6v7v8v9v10c6 and c5v6v8v9v10c6, and promoted the expression of c5v6c6. hnRNP A1 knockdown significantly induced cell death. In addition, hnRNP A1 knockdown induced a decrease in cell invasion in the MDA-MB-231 cells. Our results indicate that the knockdown of hnRNP A1 has a specific function on the splicing of CD44 in breast cancer cells.

hnRNP L inhibits CD44 V10 exon splicing through interacting with its upstream intron.

CD44 is a complex cell adhesion molecule that mediates communication and adhesion between adjacent cells as well as between cells and the extracellular matrix. CD44 pre-mRNA produces various mRNA isoforms through alternative splicing of 20 exons, among which exons 1-5 (C1-C5) and 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. CD44 V10 exon has important roles in breast tumor progression and Hodgkin lymphoma. Here we show that increased expression of hnRNP L inhibits V10 exon splicing of CD44 pre-mRNA, whereas reduced expression of hnRNP L promotes V10 exon splicing. In addition, hnRNP L also promotes V10 splicing of endogenous CD44 pre-mRNA. Through mutation analysis, we demonstrate that the effects of hnRNP L on V10 splicing are abolished when the CA-rich sequence on the upstream intron of V10 exon is disrupted. However, hnRNP L effects are stronger if more CA-repeats are provided. Furthermore, we show that hnRNP L directly contacts the CA-rich sequence. Importantly, we provide evidences that hnRNP L inhibits U2AF65 binding on the upstream Py tract of V10 exon. Our results reveal that hnRNP L is a new regulator for CD44 V10 exon splicing.

SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene.

The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.

3' Splice site sequences of spinal muscular atrophy related SMN2 pre-mRNA include enhancers for nearby exons.

Spinal muscular atrophy (SMA) is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5' splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3' splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3' splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites.

PSF contacts exon 7 of SMN2 pre-mRNA to promote exon 7 inclusion.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease and a leading cause of infant mortality. Deletions or mutations of SMN1 cause SMA, a gene that encodes a SMN protein. SMN is important for the assembly of Sm proteins onto UsnRNA to UsnRNP. SMN has also been suggested to direct axonal transport of ß-actin mRNA in neurons. Humans contain a second SMN gene called SMN2 thus SMA patients produce some SMN but not with sufficient levels. The majority of SMN2 mRNA does not include exon 7. Here we show that increased expression of PSF promotes inclusion of exon 7 in the SMN2 whereas reduced expression of PSF promotes exon 7 skipping. In addition, we present evidence showing that PSF interacts with the GAAGGA enhancer in exon 7. We also demonstrate that a mutation in this enhancer abolishes the effects of PSF on exon 7 splicing. Furthermore we show that the RNA target sequences of PSF and tra2ß in exon 7 are partially overlapped. These results lead us to conclude that PSF interacts with an enhancer in exon 7 to promote exon 7 splicing of SMN2 pre-mRNA.

hnRNP M facilitates exon 7 inclusion of SMN2 pre-mRNA in spinal muscular atrophy by targeting an enhancer on exon 7.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, which causes death of motor neurons in the anterior horn of the spinal cord. Genetic cause of SMA is the deletion or mutation of SMN1 gene, which encodes the SMN protein. Although SMA patients include SMN2 gene, a duplicate of SMN1 gene, predominant production of exon 7 skipped isoform from SMN2 pre-mRNA, fails to rescue SMA patients. Here we show that hnRNP M, a member of hnRNP protein family, when knocked down, promotes exon 7 skipping of both SMN2 and SMN1 pre-mRNA. By contrast, overexpression of hnRNP M promotes exon 7 inclusion of both SMN2 and SMN1 pre-mRNA. Significantly, hnRNP M promotes exon 7 inclusion in SMA patient cells. Thus, we conclude that hnRNP M promotes exon 7 inclusion of both SMN1 and SMN2 pre-mRNA. We also demonstrate that hnRNP M contacts an enhancer on exon 7, which was previously shown to provide binding site for tra2ß. We present evidence that hnRNP M and tra2ß contact overlapped sequence on exon 7 but with slightly different RNA sequence requirements. In addition, hnRNP M promotes U2AF65 recruitment on the flanking intron of exon 7. We conclude that hnRNP M promotes exon 7 inclusion of SMN1 and SMN2 pre-mRNA through targeting an enhancer on exon 7 through recruiting U2AF65. Our results provide a clue that hnRNP M is a potential therapeutic target for SMA.

Polypyrimidine tract binding protein inhibits IgM pre-mRNA splicing by diverting U2 snRNA base-pairing away from the branch point.

The mouse immunoglobulin (IgM) pre-mRNA contains a splicing inhibitor that bears multiple binding sites for the splicing repressor polypyrimidine tract binding protein (PTB). Here we show that the inhibitor directs assembly of an ATP-dependent complex that contains PTB and U1 and U2 small nuclear RNAs (snRNAs). Unexpectedly, although U2 snRNA is present in the inhibitor complex, it is not base-paired to the branch point. We present evidence that inhibitor-bound PTB contacts U2 snRNA to promote base-pairing to an adjacent branch point-like sequence within the inhibitor, thereby preventing the U2 snRNA-branch point interaction and resulting in splicing repression. Our studies reveal a novel mechanism by which PTB represses splicing.

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8.

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26nt RNA from 5' end of exon 8, a 33nt RNA from 3' end of exon 10 and a 2225nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.