Covalent protein-protein interaction inhibitor against srpks

Abstract

Provided is a novel serine-arginine protein kinase (SRPK) inhibitor. More specifically, provided is the novel SRPK inhibitor C-DBS. The novel SRPK inhibitor C-DBS is a covalent protein-protein interaction inhibitor against SRPK and is used in methods oftreating cancers or viral infections.

Description

[0001] DESCRIPTION

[0002] TITLE

[0003] COVALENT PROTEIN-PROTEIN INTERACTION INHIBITOR AGAINST SRPKS

[0004] CROSS-REFERENCE TO RELATED APPLICATION

[0005] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 651,527, filed May 24, 2024, which is hereby incorporated by reference in its entirety including any tables, figures, or drawings.

[0006] TECHNICAL FIELD

[0007] The present invention pertains to methods of inhibiting SRPK comprising a novel SRPK-specific modified inhibitor derived from DBS1. The modified DBS1 is an aryl-sulfonyl fluoride converted DBS1 that targets the docking groove of SRPKs via a proximity-enabled lysine-specific conjugation and inhibits SRSF1 phosphorylation by SRPKs. The modified DBS1 can self-cell penetrate and possesses anti-angiogenic and anti-metastatic activities.

[0008] BACKGROUND OF THE INVENTION

[0009] Serine / arginine-rich proteins (SR proteins) are a family of nonsmall nuclear ribonucleoproteins (snRNP) splicing factors that are essential for both constitutive and alternative splicing. They consist of one or two RNA recognition motifs (RRMs) at their N-terminal regions and stretches of arginine / serine dipeptides at their C- terminal domains (RS domains). Reversible phosphorylation of the RS domains regulates the functions of SR proteins during spliceosome assembly, splice site selection, mRNA export, translation, and maintenance of both mRNA and genome stability1 3. Given their roles in multiple aspects of the gene expression program, dysregulated expression or phosphorylation of SR proteins has been implicated in many human diseases4’5. SR protein kinases (SRPKs), one of the key kinase families that specifically phosphorylates SR proteins, are found in both the cytoplasm and nucleus and preferentially phosphorylate RS dipeptides in a regiospecific manner6>7’8. SRPKs phosphorylate multiple RS dipeptides within SR proteins to regulate their subcellular localization and functions during spliceosome assembly9,1°.

[0010] In particular, SRPK-mediated processive phosphorylation of the SR proteins SRSF1 and SRSF3 facilitates their nuclear import and dissociation from nuclear speckles, respectively11,12. In addition, the multisite phosphorylation of SRSF1 promotes the recruitment of U1-70K to the exonic splicing enhancer during formation of the spliceosome E complex13.

[0011] Given the pivotal roles of SRPKs in the regulation of SR splicing factors, their dysregulated expression can serve as predictive and prognostic indicators for various cancers14'16. High expression of SRPK1 or SRPK2 was observed in clinical samples of multiple carcinomas as well as nonepithelial cancers, such as glioblastomas and several forms of leukemia15, 17'19. In some of these malignancies, increased kinase expression is correlated with higher tumor grade, a more advanced stage, higher metastatic potential, and shorter overall survival15.

[0012] In addition to regulating multiple processes during tumorigenesis, SRPKs have also been implicated in both chemotherapy sensitivity and resistance. Dysregulation of the expressions of SRPKs or their inhibition has shown contradicting effects in different cellular context. For instance, downregulated expression of SRPK1 has been shown to confer resistance to platinum-containing drug like cisplatin in the testicular germ cell tumors and retinoblastoma20,21. In contrast, suppressed expression of SRPK1 enhances apoptosis of pancreatic, breast, and colonic cancer cell lines after treatment by cisplatin or gemcitabine17In addition, a recent finding shows that cisplatin-resistant breast career cells could be resensitized to the drug upon SRPK1 inhibition22.

[0013] The assembly of nucleocapsids is a crucial phase in the Hepatitis B virus (HBV) life cycle. These nucleocapsids are composed of the Cp, the virus-encoded polymerase, and the viral DNA genome. Cp, a fundamental component of nucleocapsids, typically forms a homodimer23. It includes an N-terminal domain (NTD, amino acids 1-149), also referred to as the assembly domain, and a C-terminal domain (CTD, amino acids 150-185) that is rich in arginines. The Cp CTD undergoes dynamic phosphorylation and dephosphorylation, which regulates the electrical homeostasis of Cp24This regulation is crucial for viral pgRNA packaging, reverse transcriptase-directed synthesis of viral DNA, capsid stability, CTD externalization, and nuclear import of the capsid25'30. Seven sites within the CTD have been shown to undergo phosphorylation. Since HBV proteins lack intrinsic protein kinase activity, Cp must be phosphorylated by protein kinases of the host cells. SRPK1 and SRPK2 have been identified as specific kinases that interact with the Cp CTD and phosphorylate the key serines, indicating these splicing kinases could be the major kinases responsible for Cp phosphorylation31. These findings highlight the potential of SRPKs as cellular targets for therapeutic interventions. Several ATP-competitive SRPK-specific inhibitors, including the first reported SRPIN340, and SRPINX31, a potent inhibitor with high selectivity for SRPK1 over SRPK2, have been developed in the past decade32,33. In addition, we reported the development of an irreversible inhibitor known as SRPKIN-1, which forms a covalent bond with a tyrosine residue at the ATP -binding pocket of SRPK134Although these compounds have been proven to be effective against SRPKs, certain concerns exist due to their ATP-competitive nature. For instance, ATP-competitive kinase inhibitors must compete with the high intracellular concentration of ATP. Potential drug polypharmacology will also arise because of the high structural similarity of the kinase ATP -binding pockets35. Moreover, many clinically approved ATP-competitive kinase inhibitors encounter drug-resistance-conferring mutations at ATP- binding pockets36'39. Thus, alternative strategies, are preferred for the development of SRPK- specific inhibitors. Therefore, there remains a need for more effective SRPK-specific inhibitors.

[0014] BRIEF SUMMARY OF THE INVENTION

[0015] The subject invention pertains to a novel SRPK-specific inhibitor derived from the DBS1 (Figure IB, top formula) compound that can be used in methods of inhibiting SRPK. In certain embodiments, the novel compound can target the docking groove of SRPKs via a proximity-enabled lysine-specific conjugation. In certain embodiments, the modified DBS1 is an aryl-sulfonyl fluoride converted DBS1, such as, for example, C-DBS (Figure IB, bottom formula). In certain embodiments, the modified DBS1 can conjugate to a specific lysine within the docking groove of SRPK1 and inhibit SRSF1 phosphorylation by SRPK1 with a significantly improved ICso value compared to DBS1. In certain embodiments, the modified DBS1 can self-cell penetrate and possesses anti -angiogenic and anti -metastatic activities in cellulo. In certain embodiments, the modified DBS1 can be an effective covalent PPI inhibitor that specifically targets SRPKs.

[0016] In certain embodiments, the modified DBS1 can be used to treat human diseases, including, for example, cancer, as phosphorylation of the C-terminal RS domains of serinearginine rich proteins (SR proteins) by the serine-arginine protein kinases (SRPKs) regulates their localization and diverse cellular activities, and dysregulation of phosphorylation of SR proteins and dysregulation of expression of SRPKs have been implicated in many human diseases, including cancers, including, but not limited to solid tumors occurring in breast, pancreatic, liver, colorectal, lung, prostate, and ovarian carcinomas; melanoma, glioblastomas, and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and any combination thereof.

[0017] In certain embodiments, the modified DBS1 can be used to inhibit HBV replication in a subject. In preferred embodiments, the modified DBS1 can inhibit the phosphorylation of Cp.

[0018] In certain embodiments, the modified DBS1 can enhance the sensitivity of cisplatinresistant cells to cisplatin in a co-treatment administered to a subject in need thereof.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] Figures 1A-1G illustrate the development of DBS1 into a covalent inhibitor. Figures 1A and IB show a library of peptides designed with lysine-reactive modifications (Figure 1A: SEQ ID NOs: 6-14; Figure IB: SEQ ID NOs: 5 and 1). The peptides comprise 20 amino acids with reactive chemical warheads installed in different positions. The chosen reactive groups were aryl-sulfonyl fluoride (XI), 4-nitrophenyl (X2), l-fluoro-2,4- dinitrobenzene (FDNB) (X3), 4-fluorophenyl (X4), and phenyl (X5) groups. All the peptides were N-terminus acetylated and C-terminus amidated. Figures 1C-1E illustrate adduct formation assays (left panel) and in vitro kinase activity assays (right panel) of the modified peptides. The adduction assays were performed in a concentration of 1 :4 (protein: peptide) for the indicated time periods. The samples were resolved by SDS-PAGE and visualized by Coomassie blue. Kinase activity assays were performed using radioactive [32P]ATP in the presence of the modified peptides. SRPK1 was preincubated with peptides of indicated concentrations before adding substrate SRSF1, and all reactions were carried out for 2 minutes. Among them, DBSl-lp (C-DBS) showed the best conjugation and inhibition of SRPK1. Figure IF illustrates the determination of half maximal inhibitory concentration (ICso) of C- DBS using kinase activity assay. C-DBS inhibited SRPK1 -mediated SRSF1 phosphorylation with an ICso value of 142 nM. Data represent means ± SEM from three independent experiments. Figure 1G illustrates in vitro GST pull-down assay. SRPK1 was premixed with indicated concentrations of C-DBS before incubating with GST-SRSF1. Samples were resolved by SDS-PAGE and visualized by Coomassie blue. C-DBS disrupted the interaction between SRPK1 and SRSF1 dose-dependently. See also Figures 9-11E. Figures 2A-2D show that C-DBS targets the docking groove of SRPK. Figure 2A shows a schematic representation of the SRPK1ANS3 and SRPK1ANS3 DM constructs. Four residues (D548, D564, E571, and K615) that are essential to substrate binding and phosphorylation were mutated to alanines in SRPK1 ANS3 DM. Figure 2B illustrates the binding affinity between SRPK1ANS3 and C-DBS was measured using fluorescence polarization. A constant concentration (100 nM) of FAM-C-DBS titrated with varying concentrations of SRPK1 ANS3, yielding a Kd value of 402 nM. Data represent means ± SEM from three independent experiments. Figure 2C illustrates adduct formation assays of SRPK1ANS3 and SRPK1ANS3 DM in the absence or presence of C-DBS. SRPK1ANS3 DM failed to conjugate with C-DBS. Figure 2D illustrates time-dependent kinase activity of SRPK1ANS3 DM in the absence or presence of C-DBS. C-DBS did not alter the phosphorylation behavior of SRPK1 ANS3 DM, which phosphorylates substrates in a distributive manner.

[0022] Figures 3A-3B show that C-DBS specifically conjugates to K604. Figure 3A shows MS2 analysis of C-DBS labelled SRPK1 ANS3. The adduct band in SDS-PAGE was cut and digested by trypsin and Glu-C overnight for mass analysis, showing that K604 at the docking groove was covalently conjugated with C-DBS. Figure 3B illustrates adduct formation assay of SRPK1 ANS3 and SRPK1 ANS3 K604A with C-DBS. The amount of the adduct formed with SRPK1 ANS3_K604A was significantly reduced compared to that of SRPK1 ANS3.

[0023] Figures 4A-4D show that C-DBS is a highly selective pan-SRPK inhibitor. Figure 4A shows FAM-C-DBS conjugated with recombinant His-tagged SRPK1 ANS3 in the absence or presence of HeLa cell lysate. The samples were resolved by SDS-PAGE and the adduct bands were visualized using Coomassie blue, in-gel fluorescence, and western blotting. Figure 4B shows C-DBS conjugated with endogenous SRPKE MDA-MB-231 cells were collected when reaching full confluency and lysed with lysis buffer on ice. The cell lysate was incubated with C-DBS, and an upshift band of SRPK1 was observed using an anti-SRPKl antibody. Figure 4C shows C-DBS conjugated with SRPK2AS1. C-DBS was incubated with SRPK2AS1, CLK1 kinase domain, BSA, and GST proteins for the indicated time periods. Only SRPK2, which is an SRPK1 homolog, formed adducts. Figure 4D shows that C-DBS selectively inhibited SRPKs. Kinase activity assays were performed toward SRPK1ANS3, SRPK2AS1, CLK1, and Akt in the presence of indicated concentrations of C-DBS. C-DBS inhibited both SRPK1 and SRPK2 but not CLK1 or Akt. The p-SRSFl levels were quantified using ImageJ. Data represents means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA. ***p < 0.001. See also Figure 13.

[0024] Figures 5A-5C show that C-DBS is self-cell permeable. Figure 5A shows Internalization of C-DBS and a control peptide in A549 cells. Both the peptides were labelled with fluorescent FAM group (green). The cells were treated with 10 pM peptides for 24 hours. Hoechst 33342 (blue) and Wheat Germ Agglutinin (WGA) Alexa Fluor 647 (red) were used to stain the nucleus and cell membrane, respectively. C-DBS, rather than the control peptide, was readily up-taken by the cells. Figures 5B and 5C show fluorescence analysis of FAM-C-DBS and Lysotracker. A549 cells were stained with Hoechst 33342 (blue) and Lysotracker Red (red) after C-DBS incubation for the indicated time. Increasing diffused green signals were observed with prolonged incubation time. The fluorescence intensity profiles of the region of interest (ROI, white box) were analyzed using ImageJ.

[0025] Figure 6 illustrates the down-regulation of phosphor-SR proteins in A549 cells. A549 cells were treated with indicated concentrations of C-DBS and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors. Total cell lysate was subjected to western blotting and the phosphorylated SR proteins were probed with mAblO4. The remaining p-SR levels versus P-tubulin were quantified using ImageJ. Data represent means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA. *p < 0.05; **p < 0.01.

[0026] Figures 7A-7D show that C-DBS suppresses angiogenesis, migration, and invasion. Figure 7A shows that C-DBS inhibited endothelial cell tube formation in a dose-dependent manner. 100 pL conditioned medium collected from C-DBS (top panel) or the control peptide (bottom panel) treated A549 cells was added with 1 * 104HUVECs into a Matrigel-coated 96- well plate. The images of capillary-like tubes formed by HUVECs were captured at three random views in each well. The relative node number and relative total length of the tubes were measured using ImageJ. Data represent means ± SEM from three independent experiments. Scale bar: 250 pm. Statistical analysis was performed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. Figures 7B-7C show that C-DBS inhibited the migration (Figure 7B) and invasion (Figure 7C) of A549 cells in a dose-dependent manner. A549 cells were resuspended in an FBS-free RPMI-1640 medium containing different concentrations of C-DBS (top panel) or the control peptide (bottom panel). Full medium containing 10% FBS was used as attractants in the lower chamber. Migrated or invaded cells were fixed and stained with Hoechst 33342 and photographed at five random views by fluorescent microscope. The number of migrated or invaded cells was counted using ImageJ. Data represent means ± SEM from three independent experiments. Scale bar: 100 pm. Statistical analysis was performed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. Figure 7D shows that C-DBS regulated the expression of EMT markers. Epithelial marker E-cadherin was upregulated, while mesenchymal makers vimentin, twist, and snail were downregulated dose-dependently after C-DBS administration. Band intensity quantification was performed using ImageJ. Data represent means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figures 14- 15C

[0027] Figure 8 shows a schematic illustration of C-DBS combating angiogenesis and invasion. C-DBS permanently blocks the docking groove of SRPK1 via proximity-enabled SuFEx reaction with specific lysine, resulting in the anti-angiogenesis and anti-invasion effect by reversing the EMT of tumor cells.

[0028] Figure 9 illustrates SRPK-specific docking groove. Related to Figures 1A-1G. The crystal structure of SRPK1 in complex with a 7mer peptide (PDB ID: 7DD1). The peptide (orange) binds at the SRPK-specific docking groove. Inset: the lysine residues K602, K604 and K615 within the docking groove that serve as potential sites for proximity-enabled conjugation reaction are shown. The sidechain of E2 is not modeled due to lack of electron density. Based on the location of R3, E2 is expected to locate closely to K604 and K602. The distances between the amino groups of both lysines and the closest Ca of the 7 mer are indicated.

[0029] Figures 10A-10B illustrate the synthesis of modified DBS1. Related to Figures 1A- 1G. Figure 10A illustrates the incorporation of aryl-sulfonyl fluoride and l-fluoro-2,4- dinitrobenzene (FDNB) groups. (a) trifluoroacetic acid (TFA) / deionized water / triisopropylsilane (TIPS) (3 / 94 / 3), rt. Figure 10B illustrates the incorporation of 4- fluorophenyl, 4-nitrophenyl, and phenyl groups, (b) tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] and phenylsilane (PhSiH3), DCM, rt; (c) sodium (diethylcarbamothioyl)sulfanide, DMF (lOmg / mL), rt.

[0030] Figures 11A-11E illustrate the development of C-DBS. Related to Figures 1A-1G. Figure 11A illustrates the comparison of ICso values of DBS1-1, DBS1-2, and DBS1-3. SRSF1 was phosphorylated by SRPK1 ANS3 in the presence of different concentrations of the modified peptides using radioactive kinase activity assays. Radiolabeled phosphor- SRSF1 bands were quantified by ImageJ. DBS 1-1 showed the best inhibitory effect among three reactive peptides with an ICso of 640 nM. Data represent means ± SEM from three independent experiments. Figure 11B shows adduct formation assays of different modified peptides with SRPK1 for extended time periods. The reaction was performed in a concentration of 1 :4 (protein: peptide) at indicated time periods. Samples were resolved by SDS-PAGE and visualized by Coomassie blue. Only DBSl-lp fully consumed SRPK1ANS3. Figure 11C shows the chemical structure of C-DBS incorporated with an aryl-sulfonyl fluoride group in the middle. C-DBS was N-terminal acetylated and C-terminus amidated. Figure 11D and Figure HE show HPLC MALDI-TOF MS analysis of C-DBS, respectively. Fragment peaks of [M+5H]5+, [M+6H]6+, [M+7H]7+were found in the mass spectrum.

[0031] Figure 12 shows that SRPK1 docking groove residues are important for the phosphorylation of substrate. SRPK1ANS3 or SRPK1ANS3 DM was used to phosphorylate GST-SRSF1. The activity assay was initiated by adding ATP and quenched by adding SDS loading buffer. Samples were resolved by Phos-tag SDS-PAGE and probed with anti-GST antibody. Nearly all SRSF1 was phosphorylated by SRPK1ANS3 and resulted in shifted bands. In contrast, most SRSF1 remained unphosphorylated in the presence of SRPK1ANS3 DM.

[0032] Figure 13 shows that C-DBS has no inhibitory effect on other kinases. Related to Figures 4A-4D. Screening of the inhibitory effect of C-DBS (500 nM) against 140 protein kinases was performed by MRC PPU International Centre for Kinases Profiling, University of Dundee. C-DBS showed no obvious inhibition toward the kinases. Experiments were conducted in duplicate, and data were presented as means ± SEM.

[0033] Figure 14 shows that C-DBS is non-cytotoxic. Related to Figures 7A-7D. Cell viability in several cell lines, including A549, MDA-MB-231, HeLa, and HEK-293 after C- DBS administration. Cells were treated with increasing concentrations of C-DBS for 24 hours before adding MTT. The absorbance at 570 nm was determined according to the manufacturer’s instructions. C-DBS showed no cytotoxicity to these cell lines at concentrations up to 100 pM. Data represent means ± SEM from three independent experiments.

[0034] Figures 15A-15C show that C-DBS inhibits the migration and invasion of MDA- MB-231 breast cancer cells. Related to Figures 7A-7D. Figure 15A shows C-DBS down- regulated phosphor-SR proteins in MDA-MB-231 cells in a dose-dependent manner. MDA- MB-231 cells were treated with indicated concentrations of C-DBS and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors. Total cell lysate was subjected to western blotting and phosphorylated SR proteins were probed with mAblO4. The remaining p-SR levels versus P-tubulin were quantified using Image! Data represent means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA. *p < 0.05; **p < 0.01. Figures 15B-15C C-DBS inhibited the migration (Figure 15B) and the invasion (Figure 15C) of MDA-MB-231 cells in a dose-dependent manner. MDA-MB-231 cells were resuspended in an FBS-free RPMI-1640 medium containing different concentrations of C-DBS (top panel) or the control peptide (bottom panel). Full medium containing 10% FBS was used as attractants in the lower chamber. Migrated or invaded cells were fixed and stained with Hoechst 33342 and photographed at five random views by fluorescent microscope. The number of migrated or invaded cells was counted using Image! Data represent means ± SEM from three independent experiments. Scale bar: 100 pm. Statistical analysis was performed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

[0035] Figures 16A-16B illustrate the predicted structure of DBS1. DBS-1 is predicted to adopt alpha helical structure by both PEP-FOLD4 and ColabFold - a user-friendly derivative of AlphaFold2. Direct output of DBS1 as predicted by the PEP-FOLD4 server (bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD4 / ) (Figure 16A). The structure predicted by ColabFold is coloured by the pLDDT scores of AlphaFold2 (Figure 16B).

[0036] Figures 17A-17C show that C-DBS re-sensitizes cisplatin-resistant cells to cisplatin. Cisplatin resistant A549 cells were treated with 5-50 pM of cisplatin alone (Figure 17A) or in the presence with 10 pM of C-DBS (Figure 17B). The viability of the cells was determined by MTT assay. Data represent means ± SEM from three independent experiments. Combination index (CI) to assess the degree of drug combination using the CompuSyn software (Figure 17C). CI values less than 1 indicate a synergistic interaction between cisplatin and C-DBS in the co-treatment of A549 cells

[0037] Figures 18A-18C show that C-DBS inhibits the binding and phosphorylation of Cp by SRPKs. Figures 18A-18B show GST-pulldown assays performed using GST-CpY132 (monomeric Cp mutant) and SRPK2WT (Figure 18A) or SRPK1WT in the presence of different concentrations of C-DBS (Figure 18B). The interaction between Cp and the kinases was attenuated in the presence of C-DBS. Kinase assays with SRPK2WT and CpY132A were performed in the presence of different concentrations of C-DBS (Figure 18C). The samples were separated on Phostag gel, then analyzed by Western blot using anti-GST antibody. 10 pM C-DBS nearly abolished the phosphorylation of Cp. The first lane with no ATP added was used as negative control.

[0038] BRIEF DESCRIPTION OF THE SEQUENCES

[0039] SEQ ID NO: 1: C-DBS

[0040] SEQ ID NO: 2: a peptide fragment of C-DBS conjugated to SRPK1

[0041] SEQ ID NO: 3 : A (6-FAM)-labeled reactive control peptide

[0042] SEQ ID NO: 4 : A peptide derived from the trans-activator of transcription (TAT) of HIV and the RRM2 region of SRSF1

[0043] SEQ ID NO: 5: DBS1

[0044] SEQ ID NOs: 6-14: peptides designed with lysine-reactive modifications

[0045] SEQ ID NO: 15: SRPK1

[0046] SEQ ID NO: 16. SRPK1ANS3 DM

[0047] SEQ ID NO: 17. SRPK1 ANS3_K604A

[0048] SEQ ID NO: 18. SRSF1

[0049] SEQ ID NO: 19. SRSF2

[0050] SEQ ID NO: 20. SRSF4

[0051] SEQ ID NO:21. SRSF6

[0052] SEQ ID NO:22. SRSF10

[0053] DETAILED DISCLOSURE OF THE INVENTION

[0054] Selected Definitions

[0055] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”. The transitional terms / phrases (and any grammatical variations thereof) “comprising”, “comprises”, “comprise”, “consisting essentially of’, “consists essentially of’, “consisting” and “consists” can be used interchangeably.

[0056] The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured, ie., the limitations of the measurement system. In the context of compositions containing amounts of ingredients where the terms “about” are used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X ± 10%). In other contexts, the term “about” is providing a variation (error range) of 0-10% around a given value (X ± 10%). As is apparent, this variation represents a range that is up to 10% above or below a given value, for example, X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0057] In the present disclosure, ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. When ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed range) and specific embodiments therein are explicitly included.

[0058] By “reduces” is meant a negative alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0059] By “increases” is meant as a positive alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0060] As used herein, the term “subject” refers to an animal, needing or desiring delivery of the benefits provided by a therapeutic compound. The animal may be for example, humans, pigs, horses, goats, cats, mice, rats, dogs, apes, fish, chimpanzees, orangutans, guinea pigs, hamsters, cows, sheep, birds, chickens, as well as any other vertebrate or invertebrate. These benefits can include, but are not limited to, the treatment of a health condition, disease or disorder; prevention of a health condition, disease or disorder; immune health; enhancement of the function of an organ, tissue, or system in the body. The preferred subject in the context of this invention is a human. The subject can be of any age or stage of development, including infant, toddler, adolescent, teenager, adult, or senior.

[0061] As used herein, the terms “therapeutically-effective amount,” “therapeutically-effective dose,” “effective amount,” and “effective dose” are used to refer to an amount or dose of a compound or composition that, when administered to a subject, is capable of treating or improving a condition, disease, or disorder in a subject or that is capable of providing enhancement in health or function to an organ, tissue, or body system. In other words, when administered to a subject, the amount is “therapeutically effective.” The actual amount will vary depending on a number of factors including, but not limited to, the particular condition, disease, or disorder being treated or improved; the severity of the condition; the particular organ, tissue, or body system of which enhancement in health or function is desired; the weight, height, age, and health of the patient; and the route of administration.

[0062] As used herein, the term “treatment” refers to eradicating, reducing, ameliorating, or reversing a sign or symptom of a health condition, disease or disorder to any extent, and includes, but does not require, a complete cure of the condition, disease, or disorder. Treating can be curing, improving, or partially ameliorating a disorder. “Treatment” can also include improving or enhancing a condition or characteristic, for example, bringing the function of a particular system in the body to a heightened state of health or homeostasis.

[0063] As used herein, “preventing” a health condition, disease, or disorder refers to avoiding, delaying, forestalling, or minimizing the onset of a particular sign or symptom of the condition, disease, or disorder. Prevention can, but is not required, to be absolute or complete; meaning, the sign or symptom may still develop at a later time. Prevention can include reducing the severity of the onset of such a condition, disease, or disorder, and / or inhibiting the progression of the condition, disease, or disorder to a more severe condition, disease, or disorder.

[0064] In some embodiments of the invention, the method comprises administration of multiple doses of the compounds of the subject invention. The method may comprise administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeutically effective doses of a composition comprising the compounds of the subject invention as described herein. In some embodiments, doses are administered over the course of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days, or more than 30 days. Moreover, treatment of a subject with a therapeutically effective amount of the compounds of the invention can include a single treatment or can include a series of treatments. It will also be appreciated that the effective dosage of a compound used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays or imaging techniques for detecting tumor sizes known in the art. In some embodiments of the invention, the method comprises administration of the compounds at several time per day, including but not limiting to 2 times per day, 3 times per day, and 4 times per day. As used herein, an “isolated” or “purified” compound is substantially free of other compounds. In certain embodiments, purified compounds are at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight of the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis.

[0065] As used herein, a “pharmaceutical” refers to a compound manufactured for use as a medicinal and / or therapeutic drug.

[0066] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0067] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0068] SR Protein Kinases and Methods of Use

[0069] The subject invention pertains to a novel SRPK inhibitor compound comprising a modified DBS1 compound (Figure IB, top formula) or a composition comprising said modified DBS1 compound. In certain embodiments, the modified DBS1 is an aryl-sulfonyl fluoride converted DBS1, such as, for example, C-DBS (Figure IB, bottom formula). C-DBS has an amino acid sequence of RERARTRRXRARTRERARTR (SEQ ID NO: 1), in which amino acid X is a diaminopropionic acid analog with an aryl-sulfonyl fluoride group, such as, for example a compound according to formula (I):

[0070] Formula (I) In preferred embodiments, the compositions and methods according to the subject invention utilize a SRPK inhibitor, such as, for example, C-DBS. SRPK inhibitor compounds may be added to compositions at concentrations of 0.01 to 50% by weight (wt %), preferably 0.1 to 20 wt %, and more preferably 0.1 to 10 wt %. In certain embodiments, the SRPK inhibitor can be administered to a subject at a dosage of about 0.01 mg / kg to about 1000 mg / kg.

[0071] In one embodiment, the peptide inhibitor is administered to the subject weekly.

[0072] In preferred embodiments, the peptide inhibitor is administered to the subject daily.

[0073] In preferred embodiments, the peptide inhibitor is administered to the subject daily for about a year.

[0074] In more preferred embodiments, the peptide inhibitor is administered to the subject daily until needed or alternatively until there is disease progression. Administration of the peptide inhibitor is interrupted in case of acute reaction or severe side effects.

[0075] In one embodiment, the subject compositions are formulated as an orally-consumable product, such as, for example a food item, capsule, pill, or drinkable liquid. An orally deliverable pharmaceutical is any physiologically active substance delivered via initial absorption in the gastrointestinal tract or into the mucus membranes of the mouth. The topic compositions can also be formulated as a solution that can be administered via, for example, injection, which includes intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0076] In other embodiments, the subject compositions are formulated to be administered via the skin through a patch or directly onto the skin for local or systemic effects. The compositions can be administered sublingually, buccally, rectally, or vaginally. Furthermore, the compositions can be sprayed into the nose for absorption through the nasal membrane, nebulized, inhaled via the mouth or nose, or administered in the eye or ear.

[0077] Orally consumable products according to the invention are any preparations or compositions suitable for consumption, for nutrition, for oral hygiene, or for pleasure, and are products intended to be introduced into the human or animal oral cavity, to remain there for a certain period of time, and then either be swallowed (e.g., food ready for consumption or pills) or to be removed from the oral cavity again (e.g., chewing gums or products of oral hygiene or medical mouth washes). While an orally-deliverable pharmaceutical can be formulated into an orally consumable product, and an orally consumable product can comprise an orally deliverable pharmaceutical, the two terms are not meant to be used interchangeably herein. Orally consumable products include all substances or products intended to be ingested by humans or animals in a processed, semi-processed, or unprocessed state. This also includes substances that are added to orally consumable products (particularly food and pharmaceutical products) during their production, treatment, or processing and intended to be introduced into the human or animal oral cavity.

[0078] Orally consumable products can also include substances intended to be swallowed by humans or animals and then digested in an unmodified, prepared, or processed state; the orally consumable products according to the invention therefore also include casings, coatings, or other encapsulations that are intended to be swallowed together with the product or for which swallowing is to be anticipated.

[0079] In one embodiment, the orally consumable product is a capsule, pill, syrup, emulsion, or liquid suspension containing a desired orally deliverable substance. In one embodiment, the orally consumable product can comprise an orally deliverable substance in powder form, which can be mixed with water or another liquid to produce a drinkable orally-consumable product.

[0080] In some embodiments, the orally-consumable product according to the invention can comprise one or more formulations intended for nutrition or pleasure. These particularly include baking products (e.g., bread, dry biscuits, cake, and other pastries), sweets (e.g., chocolates, chocolate bar products, other bar products, fruit gum, coated tablets, hard caramels, toffees and caramels, and chewing gum), alcoholic or non-alcoholic beverages (e.g., cocoa, coffee, green tea, black tea, black or green tea beverages enriched with extracts of green or black tea, Rooibos tea, other herbal teas, fruit-containing lemonades, isotonic beverages, soft drinks, nectars, fruit and vegetable juices, and fruit or vegetable juice preparations), instant beverages (e.g., instant cocoa beverages, instant tea beverages, and instant coffee beverages), meat products (e.g., ham, fresh or raw sausage preparations, and seasoned or marinated fresh meat or salted meat products), eggs or egg products (e.g., dried whole egg, egg white, and egg yolk), cereal products (e.g., breakfast cereals, muesli bars, and pre-cooked instant rice products), dairy products (e.g. , whole fat or fat reduced or fat-free milk beverages, rice pudding, yoghurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, and partly or wholly hydrolyzed products containing milk proteins), products from soy protein or other soy bean fractions (e.g, soy milk and products prepared thereof, beverages containing isolated or enzymatically treated soy protein, soy flour containing beverages, preparations containing soy lecithin, fermented products such as tofu or tempeh products prepared thereof and mixtures with fruit preparations and, optionally, flavoring substances), fruit preparations (e.g., jams, fruit ice cream, fruit sauces, and fruit fillings), vegetable preparations (e.g., ketchup, sauces, dried vegetables, deep-freeze vegetables, pre-cooked vegetables, and boiled vegetables), snack articles (e.g., baked or fried potato chips (crisps) or potato dough products and extrudates on the basis of maize or peanuts), products on the basis of fat and oil or emulsions thereof (e.g., mayonnaise, remoulade, and dressings), other readymade meals and soups (e.g., dry soups, instant soups, and pre-cooked soups), seasonings (e.g., sprinkle-on seasonings), sweetener compositions (e.g., tablets, sachets, and other preparations for sweetening or whitening beverages or other food). The present compositions may also serve as semi-finished products for the production of other compositions intended for nutrition or pleasure.

[0081] The subject composition can further comprise one or more pharmaceutically acceptable carriers, and / or excipients, and can be formulated into preparations, for example, solid, semisolid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0082] The term “pharmaceutically acceptable” as used herein means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0083] Carriers and / or excipients according the subject invention can include any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for, e.g., IV use, solubilizers (e.g., Polysorbate 65, Polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, aromatizers, thickeners (e.g. carbomer, gelatin, or sodium alginate), coatings, preservatives (e.g., Thimerosal, benzyl alcohol, polyquaterium), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tonicity controlling agents, absorption delaying agents, adjuvants, bulking agents (e.g., lactose, mannitol) and the like. The use of carriers and / or excipients in the field of drugs and supplements is well known. Except for any conventional media or agent that is incompatible with the target health-promoting substance or with the composition, carrier or excipient use in the subject compositions may be contemplated.

[0084] In one embodiment, the compositions of the subject invention can be made into aerosol formulations so that, for example, it can be nebulized or inhaled. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, powders, particles, solutions, suspensions or emulsions. Formulations for oral or nasal aerosol or inhalation administration may also be formulated with carriers, including, for example, saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents or fluorocarbons. Aerosol formulations can be placed into pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Illustratively, delivery may be by use of a single-use delivery device, a mist nebulizer, a breath-activated powder inhaler, an aerosol metered-dose inhaler (MDI), or any other of the numerous nebulizer delivery devices available in the art. Additionally, mist tents or direct administration through endotracheal tubes may also be used.

[0085] In one embodiment, the compositions of the subject invention can be formulated for administration via injection, for example, as a solution or suspension. The solution or suspension can comprise suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3 -butanediol, water, Ringer's solution, or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, non-irritant, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. One illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI). Other illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01- 0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion. Water or saline solutions and aqueous dextrose and glycerol solutions may be preferably employed as carriers, particularly for injectable solutions. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.

[0086] In one embodiment, the compositions of the subject invention can be formulated for administration via topical application onto the skin, for example, as topical compositions, which include rinse, spray, or drop, lotion, gel, ointment, cream, foam, powder, solid, sponge, tape, vapor, paste, tincture, or using a transdermal patch. Suitable formulations of topical applications can comprise in addition to any of the pharmaceutically active carriers, for example, emollients such as carnauba wax, cetyl alcohol, cetyl ester wax, emulsifying wax, hydrous lanolin, lanolin, lanolin alcohols, microcrystalline wax, paraffin, petrolatum, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax, or yellow beeswax. Additionally, the compositions may contain humectants such as glycerin, propylene glycol, polyethylene glycol, sorbitol solution, and 1,2,6 hexanetri ol or permeation enhancers such as ethanol, isopropyl alcohol, or oleic acid.

[0087] Methods of Using Compounds of the Subject Invention

[0088] In certain embodiments, the novel compounds can target the docking groove of SRPKs via proximity-enabled lysine-specific reaction. In certain embodiments, the modified DBS1 can conjugate to a specific lysine within the docking groove of SRPK1 and inhibit SRSF1 phosphorylation by SRPK1 with a significantly improved ICso value compared to DBS1. In certain embodiments, the modified DBS1 can self-cell penetrate and possesses anti-angiogenic and anti-metastatic activities in cellulo. In certain embodiments, the modified DBS1 can be an effective covalent PPI inhibitor that specifically targets SRPKs.

[0089] In certain embodiments, the modified DBS1 can be used to treat human diseases, including, for example, cancer, as phosphorylation of their C-terminal RS domains by serinearginine protein kinases (SRPKs) regulates their localization and diverse cellular activities, and dysregulation of phosphorylation has been implicated in many human diseases, including cancers, Alzheimer’s disease, Hepatitis B virus infection, neovascular eye disease, and any combination thereof. In certain embodiments, the subject compounds and methods can be used to treat solid tumors and leukemias containing overexpressed SRPKs. In certain embodiments, the solid tumors are in breast tissue, pancreatic tissue, liver tissue, colorectal tissue, lung tissue, prostate tissue, and / or ovarian tissue. In some embodiments, the compositions of the subject invention can be used to treat carcinomas, melanoma, glioblastoma, and leukemias, including, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, or any combination thereof.

[0090] In certain embodiments, the modified DBS1, specifically C-DBS, binds to the docking groove of SRPK1 (Kd = 5.6 micromolar) and inhibits the phosphorylation of SRSF1 at an IC50 of 10.8 micromolar. In certain embodiments, C-DBS inhibits SRPKl-medated phosphorylation of SRSF 1 with an IC50 of 142 nanomolar, which is 75-fold improvement over DBS 1. In certain embodiments, the subject SRPK inhibitor can regulate the functions of viral proteins, including, for example, hepatitis B virus (HBV) core protein (HBc), herpes simplex virus (HSV) protein ICP27, and the nucleocapsid (N) proteins of both SARS-CoV and SARS-CoV2, and regulate the life cycles of these viruses by controlling the phosphorylation by SRPKs. C-DBS contains N-acetylation and C-amidation modifications to enhance its stability and an aryl-sulfonyl fluoride warhead that can effectively conjugate to SRPK1 both in vitro and in cellulo. In certain embodiments, C-DBS is pan-SRPK-specific as it conjugated to SRPK2 and SRPK1 but not unrelated proteins, including, for example, bovine serum albumin (BSA), glutathione S- transf erase (GST), or an SR protein-specific kinase CDC2-like kinase 1 (CLK1). In certain embodiments, C-DBS can inhibit the phosphorylation of endogenous SR proteins, including, for example, SRSF1 or SRSF2, SRSF4, SRSF6, and SRSF10.

[0091] In certain embodiments, C-DBS can inhibit cell migration and invasion of cancerous cells, such as for example, non-small cell lung cancer cells and breast cancer cells. In certain embodiments, C-DBS can alter the expression of epithelial -mesenchymal transition (EMT) markers, such as, for example, downregulating the expression of vimentin, twist, and snail while upregulating the expression that of E-cadherin. In certain embodiments, the subject SRPK inhibitor can penetrate a cell. In certain embodiments, the subject SRPK inhibitor can covalently inhibit a protein-protein interaction interface in the splicing kinases SRPKs. In certain embodiments, the subject SRPK inhibitor can suppress angiogenesis, cell migration and invasion of cancer cells.

[0092] In certain embodiments, the modified DBS1 can be used to inhibit HBV replication in a subject with an HBV infection. In preferred embodiments, the modified DBS1 can inhibit the phosphorylation of Cp by not less than 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In preferred embodiments, the modified DBS1 can inhibit the phosphorylation of Cp by not less than 50%. In more preferred embodiments, the modified DBS1 can inhibit the phosphorylation of Cp by not less than 70%. In most preferred embodiments, the modified DBS1 can inhibit the phosphorylation of Cp by not less than 90%. In preferred embodiments, the modified DBS1 is C-DBS.

[0093] In certain embodiments, the modified DBS1 can enhance the sensitivity of cisplatinresistant cells to cisplatin in a co-treatment administered to subject affected by a cancer. In certain embodiments, the modified DBS1 enhances the sensitivity of cisplatin-resistant cells to cisplatin by not less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in a cotreatment in the subject. In preferred embodiments, the modified DBS1 enhances the sensitivity of cisplatin-resistant cells to cisplatin by not less than 20%, In more preferred embodiments, the modified DBS1 enhances the sensitivity of cisplatin-resistant cells to cisplatin by not less than 30%, In most preferred embodiments, the modified DBS1 enhances the sensitivity of cisplatin-resistant cells to cisplatin by not less than 40%, In preferred embodiments, the modified DBS1 is C-DBS.

[0094] In some embodiments, the methods of the subject invention can be used to treat cancers. In some embodiments, the methods of the subject invention can be used in addition to known cancer treatments, such as, surgery, chemotherapy, radiation therapy, radiofrequency ablation, cryoablation, immunotherapy, hormone therapy, stem cell transplant, targeted drug therapy, or any combination thereof.

[0095] Several SRPK-specific ATP-competitive inhibitors have been reported. However, this type of inhibitor needs to compete with high concentration of cellular ATP and to overcome the development of resistance mutations at the ATP -binding clefts of the targeted kinases. Our new cell-penetrating covalent protein-protein interaction inhibitor of SRPKs that block their substrate docking grooves could overcome the drawbacks of ATP-competitive inhibitors by targeting a SRPK-specific substrate-binding site that is distal to the ATP-binding site. As a non- ATP competitive inhibitor, C-DBS does not need to compete with high cellular concentration of ATP and will not encounter drug-resistance caused by mutations that often occur at the ATP-binding site.

[0096] MATERIALS AND METHODS

[0097] Cell lines

[0098] A549 (CCL-185, human, male) and MDA-MB-231 (HTB-26, human, female) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) with 10% Fetal Bovine Serum (FBS) (Gibco) and 1% penicillin / streptomycin (Gibco). HeLa (CRM-CCL-2, human, female) and HEK-293 (CRL-1573, human, female) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) with 10% FBS plus 1% penicillin / streptomycin. Cell passage was performed every 2-3 days regarding cell confluency. Human umbilical vein endothelial cells (HUVECs) were cultured in Medium 199 (Gibco) with 20% heat-inactivated FBS and 1% penicillin / streptomycin, supplemented with 50 pg / mL endothelial cell growth supplement (ECGS) (Corning) and 100 pg / mL heparin (Sigma- Aldrich). The medium was replaced every other day. All the cells were cultured in a 37°C incubator with humidified 5% CO2. Cloning and expression of recombinant proteins

[0099] The mutation of SRPK1ANS3 K604A was based on the SRPK1 ANS3 construct in the pET15b vector. The mutation was induced using site-direct mutagenesis. PCR products were purified using a gel extraction kit (Invitrogen), and template DNA was cleaved by Dpnl (New England Biolabs). Products were transformed into DH5a strain Escherichia coli cells via heat shock. Sequencing was performed to verify the presence of the mutation.

[0100] Protein expression and purification of SRPK1 and SRSF1 were performed as described previously13. Briefly, recombinant proteins were expressed in BL21(DE3) pLysS strain Escherichia coli cells via heat shock. Large-scale cultures were grown in Terrific Broth (IBI Scientific) with 200 pg / mL ampicillin (Santa-Cruz) and 50 pg / mL chloramphenicol (Sigma- Aldrich) at 37°C. Protein expression was induced by 0.2 mM isopropylthiogalactoside (IPTG) (GOLD BIO) for 18-20 hours at 16°C when O.D. reached 0.6-0.8. E. coli cells were pelleted and lysed via ultrasonication on ice. Recombinant SRPK1 and its mutant proteins were purified using an anion exchange column (Q-Sepharose) and aNi-NTA affinity column (MACHEREY- NAGEL). Purified proteins were dialyzed into a buffer containing 20 mM MES pH 6.5, 300 mM NaCl, and 5% glycerol supplemented with 1 mM DTT. If necessary, the polyhistidine tag would be removed by thrombin (GE Healthcare) overnight at 25°C, and subsequent gel filtration purification would be performed using the Superdex 75 size exclusion column (GE Healthcare). Recombinant GST-tagged SRSF1 protein in pGEX-4T-2 vector was purified using anion exchange (Q-Sepharose) and GST columns (GenScript) and dialyzed into a buffer containing 20 mM Tris-HCl pH 7.5, 300 mM KC1, 10% glycerol, and 1 mM DTT. All the proteins were concentrated, flash-frozen, and stored at -80°C for further use.

[0101] Peptide synthesis and characterization

[0102] All peptides were synthesized based on manual 9-fluorenylmenthoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) chemistry. Rink Amide-ChemMatrix resins with a loading capacity of 0.46 mmol / g were purchased from Biotage® (Uppsala). All Fmoc- protected amino acids were purchased from GL Biochem Ltd. Generally, in each coupling step, a 5-fold excess of Fmoc-protected amino acid, 2-(lH-benzotriazol-l-yl)-l, 1, 3, 3- tetramethyluronium hexafluorophosphate (HBTU), A-hydroxy benzotriazole (HOBt), and N, A-diisopropylethylamine (DIPEA) with a ratio of 1: 1: 1 : 2 in dimethylformamide (DMF) was added to the resin for peptide elongation. Then, the Fmoc group was removed in 20% piperidine in DMF (v / v) to expose the amine group for the next step of the coupling reaction. After finishing the coupling / deprotection cycles, the N terminus of the peptide was acetylated. To install modifications at a certain position of the peptide sequence, amino acids with specific side-chain protections were used. For DBS 1-1 and DBS 1-3, 4-methyltrityl (Mtt)-protected lysine residue was used at the 9thposition. After acetylation, the Mtt group was removed by trifluoroacetic acid (TFA) / deionized water / triisopropyl silane (TIPS) (3 / 94 / 3) at room temperature and the modifications were introduced by reacting the peptidyl-resin with a 5-fold excess of 4-fluorosulfonylbenzoic acid (Sigma- Aldrich) and l,5-difluoro-2,4-dinitrobenzene (Sigma-Aldrich), respectively. For DBS1-2, DBS1-4, and DBS1-5, allyl (All)-protected glutamic acid residue was used at the 9thposition. After acetylation, the All group was removed by tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] (0.2 equiv.) (TCI) and phenylsilane (PhSiHs) (Macklin) in dichloromethane (DCM). Then, the resin was washed with sodium (diethylcarbamothioyl)sulfanide (10 mg / mL in DMF) (J&K Scientific). 4-fluorophenyl, 4- nitrophenyl, and 4-phenyl groups were introduced by 5-fold excess of 4-fluorophenol, 4- nitrophenol, and phenyl (J&K Scientific). The synthesis of DBS1-1.2, DBS1-1.15, DBSl-lb, and DBS 1- Ip was similar to that of DBS 1-1, except the modification was installed at the 2ndor 15thpositions, the Mtt-protected lysine was replaced by diaminobutyric acid (Dab) or diaminopropionic acid (Dap). Carboxyfluorescein (FAM)-label C-DBS was synthesized by introducing a cysteine to the N-terminus after Fmoc deprotection, allowing the reaction with 6-FAM maleimide in 1 M citric acid solution. After finishing modifications, the peptides were cleaved from the resin in a cleavage cocktail (TFA / DCM / TIPS (95:2.5:2.5)) at room temperature. The crude peptide was obtained by precipitation by adding cold diethyl and purified by RP-HPLC (Prominence LC 20- A, Shimadzu, Kyoto) with a Vydac 218TP Cl 8 LC column (10 pm, 250 mm x 10 mm, 300 A) at a flow rate of 3 mL / min and confirmed by MALDI-TOF MS analysis (Bruker Daltonics).

[0103] Protein-peptide adduction assay

[0104] Peptides (DBS1-1, DBS1-2, DBS1-3, DBS1-4, DBS1-5, DBS1-1.2, DBS1-1.15, DBSl-lb, and DBSl-lp) at a concentration of 8 pM were added to 2 pM SRPK1ANS3 in a conjugation buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, and 1 mM benzamidine) for indicated time periods. The reactions were quenched by adding SDS loading buffer and boiling. Obtained mixtures were then resolved by SDS-PAGE and stained with Coomassie Blue. Conjugation assays with SRPK2AS1, BSA, GST, CLKl(148-484), SRPK1 ANS3 DM, and SRPK1 ANS3 K604A with C-DBS were performed in the same buffer condition.

[0105] GST pull-down assay

[0106] GST-SRSF1 (500 nM) was immobilized by glutathione resin (Genescript) in a pulldown buffer (300 mM NaCl, 20 mM HEPES pH 7.5, 5% glycerol, 0.5% Triton X-100, 1 mM DTT, 1 mM benzamidine) for at least 4 hours and washed three times to remove the unbound proteins. SRPK1 ANS3 (1 pM) was incubated with indicated concentrations of C-DBS at room temperature for one hour, and the mixtures were then added to the resins and allowed to incubate for 20 minutes at 4°C. Afterward, the resins were washed with pull-down buffer (four times, 200 pL each), and proteins were eluted with SDS loading buffer, followed by boiling for 5 minutes. Samples were then resolved by SDS-PAGE and visualized by Coomassie Blue staining.

[0107] Kinase activity assay

[0108] SRPK1ANS3, SRPK2AS1, CLK1, and Akt (Abeam) at a concentration of 10 nM were incubated with different concentrations of C-DBS in a kinase reaction buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM MgCh, and 5 mg / mL BSA) at room temperature for 1 hour prior the addition of 500 nM GST-SRSF 1. Reactions were initiated by adding 50 pM unlabelled ATP mixed with 0.5 pCi of [32P]ATP (PerkinElmer) and quenched with SDS loading buffer and boiling. The mixtures were resolved by 10% SDS-PAGE. Gels were dried using a gel dryer (Savant SG2000 Digital Slab Gel Dryer). The radioactive bands were exposed by autoradiography using X-ray films (Fujifilm) and photographed by BioRad ChemiDoc MP System. Band intensities were quantified by ImageJ software, and ICso was calculated by GraphPad Prism software.

[0109] Fluorescence polarization assay

[0110] FAM-C-DBS was used as the tracer titrated with SRPK1ANS3 or SRPK1ANS3 DM proteins. Briefly, 200 nM FAM-C-DBS was added into a 96-well black plate (GREINER, Cat. No. 655209). Serially diluted proteins were added into each well at an equal volume. The peptide and the protein were incubated at room temperature for 1 hour before processing for the fluorescence reading by a microplate reader (Tecan Spark 10M Microplate Reader). The Kd values for the interaction of C-DBS with SRPK1ANS3 or SRPK1ANS3 DM were calculated using GraphPad Prism software.

[0111] LC-MS / MS analysis of the C-DBS-SRPK1 adduct

[0112] The adduct band on SDS-PAGE gel in the conjugation assay was cut and digested with trypsin (Promega) and Glu-C (Promega) at 37°C overnight. The enzymatic digestion was quenched through the addition of formic acid (10%), and the peptides were collected and vacuum-dried prior to mass analysis. MS data were acquired on an Orbitrap Fusion mass spectrometer (Thermo Scientific) equipped with EASY-nLC 1200 system (Thermo Scientific) and EASY-Spray HPLC column (75 pm I.D. * 150 mm, 3 pm, 100 A) and ion source (Thermo Scientific). The chromatographic separation was performed using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in 80% acetonitrile as mobile phase B operated at the flow rate of 300 nL / min. The MS / MS analyses were carried out with the collision-induced dissociation (CID) mode with the collision energy of 35%. Acquired MS raw data were converted as mgf format by msConvert (version 3.0.18165, ProteoWizard), then analyzed using MassMatrix for MS / MS ion search of cross-linked peptides.

[0113] Cell viability assay

[0114] Cells (6 x io3per well) were seeded into a 96-well plate. After overnight incubation, cells were treated with different concentrations of C-DBS for 24 hours. The viability of the cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Roche). Absorption at 570 nm was measured using a microplate reader (BMG, CLARIOstar) according to the manufacturer’s instructions.

[0115] Cellular uptake and endo / lysosomal escape

[0116] A549 cells (20 * 104per well) were seeded into confocal dishes (SPL Life Sciences) and incubated for adhesion overnight at 37°C in 5% CO2. 6-F AM-labelled C-DSB or control peptide was diluted to Opti-MEM medium (Gibco) and added to the cells for 24-hour incubation. C-DBS was washed with PBS containing 20 U / mL heparin (Sigma- Aldrich) and stained with 40 pM Hoechst 33342 (Life Technologies) and 5 pg / mL Wheat Germ Agglutinin Alexa Fluor 647 Conjugate (Life Technologies) for 20 minutes. After removing excess dye, cell images were captured by the Leica TCS SP8 Confocal Microscope System. To detect the endo / lysosomal escape of C-DBS, 50 nM Lysotracker Red (Life Technologies) was used to stain the endo / lysosomes. Fluorescence intensity profiles of the region of interest (ROI) were analyzed using ImageJ software.

[0117] Preparation for mAb!04 antibody

[0118] Hybridoma cells mAblO4 (ATCC, CRL-2067™) were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (ATCC) plus 20% FBS and 1% penicillin / streptomycin. Cells were passaged every 2-3 days. The culture medium was collected and spun at 5000 RPM for 15 min before the supernatant was directly used for western blotting.

[0119] Inhibitor treatment and western blotting

[0120] A549 cells (10 x io4per well) were seeded into a 12-well plate, starved with Opti- MEM (Gibco) for 24 hours, and treated with C-DBS in RPMI 1640 medium. At the time of harvest, the cells were washed with cold PBS and then lysed on ice with RIPA buffer (Cell Signalling Technology) supplemented with protease inhibitor cocktail (MedChemExpress), phosphatase inhibitor cocktail (Sigma), 10 mM sodium deoxy cholate (Sigma- Aldrich), 10 mM sodium pyrophosphate decahydrate (Sigma- Aldrich) and 10 mM sodium fluoride (Sigma- Aldrich). Cell lysates were centrifuged at 4°C for 15 minutes to remove cell debris. The supernatants were collected, heated, and resolved by SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes (0.22 pm, Bio-Rad) and blocked with 5% BSA (AMRESCO) or 5% non-fat milk in TBST buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20). Membranes were incubated with primary antibody mAblO4, P-tubulin (1 :2000, Genescript), vimentin (1 :500, Abeam), E-cadherin (1 :500, Abeam), GAPDH (1 :1000, Abeam), snail (1 :300, Santa Cruz Biotechnology), twist (1 : 150, Santa Cruz Biotechnology) at 4°C overnight and then washed three times with TBST. Mouse IgGx BP-HRP (1 : 10000, Santa Cruz Biotechnology) and Goat anti-rabbit IgG (H+L) HRP (1 : 10000, Life Technologies) were used as secondary antibodies. Membranes were then washed three times with TBST and visualized using enhanced chemiluminescence (Cytiva) substrate or ultra-high sensitivity ECL kit (MedChemExpress). Tube formation assay

[0121] A549 Cells (10 x io4per well) were seeded into a 12-well plate and starved with Opti- MEM for 24 hours. C-DBS or control peptide was diluted in Opti-MEM and added to the cells. After a 16-hour incubation, the culture medium in each well was collected and spun at 900 RPM. The obtained supernatant was used as the conditioned medium (CM). Before harvesting the HUVECs, 50 pM Matrigel (Corning) was added into the 96-well plate and incubated at 37°C for 1 hour to polymerize. HUVECs were then trypsinized and seeded (1 x 104cells per well) into the Matrigel-coated 96-well plate, with the addition of 100 pL of CM to the corresponding wells. After incubation for 4-6 hours, pictures of the formed tubes were captured using a microscope (NIKON, ECLIPSE TE300) at three different views in each well. The tube nodes and total length were measured with Imaged software.

[0122] Cell migration and invasion assays

[0123] A549 or MDA-MB-231 Cells (4 x io4per well) were seeded into 24-well plates and starved for 24 hours. Cells were then trypsinized, washed twice with PBS, resuspended in an FBS-free RPMI 1640 medium containing different concentrations of C-DBS, and seeded into 8 pm transwell inserts (SPL Life Sciences). RPMI 1640 medium containing 10% FBS was added into the lower chamber as an attractant. After incubation for 16 or 24 hours, migrated cells were fixed with 4% formaldehyde solution (Sigma-Aldrich) and stained with 40 pM Hoechst 33342 for 15 minutes. The cells in the upper chamber were removed with cotton swabs. Inserts were rinsed in PBS to remove excess dye and unfixed cells. Images were taken using Carl Zeiss PALM Inverted Microscope at five random regions. Migrated cells were counted by Imaged software. The invasion assay was performed similarly to the migration assay except that the transwell inserts were pre-coated with 0.5 mg / mL Matrigel matrix (Corning).

[0124] Quantification and statistical analysis

[0125] The intensities of protein bands and radioactive bands were quantified using Imaged software. The number of tube nodes, tube total length, migrated and invaded cells were measured with Imaged software. Data were presented as means ± SEM from three replicates. One-way ANOVA was used to determine the statistical significance of the differences in multiple groups. The differences were considered to be statistically significant when p-values were < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001. All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0126] Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

[0127] EXAMPLE 1 — DEVELOPMENT OF DBS 1 INTO A COVALENT INHIBITOR (C-DBS)

[0128] Structural analysis of the SRPK1 docking groove reveals the presence of three solvent- exposed lysines (K602, K604, and K615) (Figure 9). These lysines come into close proximity with arginines when Arg-rich peptide binds to the docking groove. Therefore, the apparent p / faS of the e-amino groups of these lysines might decrease and serve as better nucleophiles for lysine-reactive covalent conjugation. On the other hand, despite the lack of electron density of Glu2 in our SRPKl :7mer structure (PDB ID:7DD1), its side chain carboxy group is predicted to also locate in proximity to K602 and K604. Since we have previously shown that the midregion of DBS 1 contributes most to its inhibitory activity40, we decided to either modify or replace the sidechain of Glu9 of DBS 1 (RERARTRRE9RARTRERARTR) (SEQ ID NO: 5) with different reactive functional groups. We have first chosen aryl-sulfonyl fluoride, 4- nitrophenol, l-fluoro-2,4-dinitrobenezene (FDNB), 4-fluorophenol, and phenyl groups (XI- X5) as warheads and generated five different derivative peptides (Figure 1A, left panel). In brief, DBS1-1 and DBS 1-3 were generated by replacing Glu9 with lysine followed by the incorporation of aryl-sulfonyl fluoride and FDNB, respectively. DBS1-2, DBS1-4 and DBS1- 5 were generated by modifying the Glu with nitrophenol, fluorophenol and phenyl via the esterification reaction between carboxyl and hydroxyl groups (Figures 10A-10B).

[0129] All of the derivative peptides were N-terminally acetylated and C-terminally amidated for stabilization and to prevent proteolysis. To test their conjugation abilities to SRPK1, the peptides were incubated with recombinant SRPK1ANS3, which is an active construct of SRPK1 where the non-conserved N-terminus and spacer domain are truncated. Only three modified peptides DBS1-1, DBS 1-2, and DBS 1-3, formed adducts with SRPK1ANS3 in a time-dependent manner, where DBS1-1 appeared to be the most efficient (Figure 1C, left panel). We next tested whether the three modified peptides could inhibit the phosphorylation of SRSF1 by SRPK1ANS3 (Figure 1C, right panel). While all three peptides inhibited the phosphorylation reaction, DBS 1-1 exhibited the best inhibitory activity against SRPK1 with an ICso value of 640 nM, which is a 17-fold improvement over DBS1 (IC50 = 11 pM) (Figure 11 A). Our results supported that the aryl-sulfonyl fluoride group is a suitable warhead to modify DBS1 into a reactive peptide that targets SRPK1.

[0130] Despite the improvement observed for DBS1-1, the reaction between excess derivative peptides and SRPK1ANS3 proteins failed to reach completeness after prolonged incubation (Figure 11B, left panel). We speculated if the installation of the warheads at different regions of the derivative peptides would improve the conjugation efficiency since the peptides might bind at the alternate regions of the docking groove. We installed the aryl-sulfonyl fluoride at the positions 2 and 15 to generate DBS1-1.2 and DBS1-1.15, respectively (Figure 1A, right panel). Unexpectedly, both DBS 1-1.2 and DBS 1 -1.15 showed no apparent difference in adduct formation efficiency and inhibitory activity (Figure ID and Figure 11B). We next tried to investigate whether the sidechain length of Lys played any role during the conjugation reaction. We replaced the Lys sidechain at position 9 with the lysine analogs diaminobutyric acid (Dab) and diaminopropionic acid (Dap) to generate DBSl-lb and DBSl-lp, respectively, to shorten the distance between the peptide backbone and the reactive site (Figure 1A, right panel). To our surprise, the shortening of the sidechain length enhanced the adduct formation efficiency. In particular, DBSl-lp, which contains the shortest sidechain, formed covalent adducts with nearly all SRPK1ANS3 after 60 min (Figure IE and Figure 11B). Kinase activity assays demonstrated that DBSl-lp inhibited the phosphorylation of SRSF1 by SRPK1 with an IC50 value of 142 nM, indicating a 75-fold improvement compared to DBS1 (Figure IF). This is equivalent to a Ki value of 104 nM obtained using the ICso-to-W converter developed by Lebeda and colleagues42. We next investigated the inhibition mechanism of DBSl-lp. GST pull-down assay was performed to evaluate whether DBSl-lp inhibited SRSF1 phosphorylation by interrupting the interaction between SRPK1 and SRSF1. The results revealed that DBSl-lp inhibited the interaction of SRPK1 and SRSF1 in a dose-dependent manner (Figure 1G), indicating it functions as a PPI inhibitor. We selected DBSl-lp as our covalent inhibitor candidate and hereafter referred to it as Covalent Docking Blocker of SRPK (C-DBS). All C- DBS described in the following sections was purified by high-performance liquid chromatography and verified by mass spectroscopy (Figures 11C-11E). EXAMPLE 2 — C-DBS SPECIFICALLY CONJUGATES TO K604 AT THE DOCKING

[0131] GROOVE

[0132] Since DBS1 targets the docking groove of SRPK, we wanted to confirm the specificity of C-DBS towards the same site. Fluorescence polarization (FP) assay was performed to measure the direct non-covalent interaction between C-DBS and SRPK1ANS3, as well as the docking groove mutant SRPK1ANS3 DM that contains alanine substitutions at four docking groove residues — D548, D564, E571, and K615 — that are essential for the binding and phosphorylation of the substrate11,12’42. The equilibrium dissociation constant Kd) was 402 ± 19 nM (Figure 2B), indicating that C-DBS bound SRPK1 with improved affinity compared to DBS1 (Kd = 3.2 ±1.5 pM) prior to the covalent interaction. In contrast, SRPK1ANS3 DM failed to interact with C-DBS nor form adducts (Figures 2B-2C). In vitro kinase assays further confirmed that the phosphorylation of SRSF1 by SRPK1ANS3 DM, which was weakened compared to that by SRPK1ANS3 (Figure HA), was not affected by C-DBS because it failed to bind to the docking groove (Figure 2D). These indicated that the inhibitor specifically targeted the docking groove to inhibit SRSF1 phosphorylation.

[0133] Next, we identified the lysine residues to which C-DBS was conjugated. We formed adducts of SRPK1 NS3 and C-DBS, followed by in-gel digestion of the excised adduct bands with trypsin / Glu-C. MALDI-TOF / TOF analyses were then performed on the C-DBS-labelled SRPK1 and SRPK1 alone. The experimental MS2 mass spectra revealed the presence of a peptide fragment LK(X-R)PWGLFE (SEQ ID NO: 2), where X is Dap-aryl-SO2F in the SRPK 1 -C-DBS adducts sample but not in the SRPK1 alone sample. The identified peptide corresponds to residues 603 to 610 of SRPK1 and the labelled lysine was confirmed to be K604 (Figure 3A). C-DBS crosslinked LKPW and KPWGL fragments were also detected using trypsin / chymotrypsin digestion (data not shown), further demonstrating that K604 was the conjugation site. We next site-directly mutated K604 to Ala and repeated the adduction assay using C-DBS. The amount of adducts formed with the SRPK1ANS3 K604A mutant was significantly reduced compared to the reaction with SRPK1ANS3, confirming K604 as the major target site for C-DBS (Figure 3B). EXAMPLE 3 — C-DBS IS A HIGHLY SPECIFIC AND SELECTIVE PAN-SRPK INHIBITOR

[0134] Since the docking groove is unique for SRPKs, we next interrogated the specificity of C-DBS by performing the adduction assay in the presence of endogenous proteins. Recombinant His-tagged SRPK1 NS3 proteins were incubated with 6-carboxyfluorescein (6- FAM)-labeled C-DBS (FAM-C-DBS) in the absence or presence of HeLa cell lysate and resolved by SDS-PAGE. New bands corresponding to the adducts could be clearly visualized after Coomasie blue staining regardless HeLa lysate was present or not (Figure 4A). Fluorescence imaging and western blotting of the samples with anti-His antibody further confirmed the new bands were the SRPK1 -C-DBS adducts. C-DBS was next incubated with MDA-MB-231 cell lysate alone to test if it interacts with endogenous SRPK1. Western blot analysis using anti-SRPKl antibody showed that the band corresponding to endogenous SRPK1 shifted upwards after the addition of C-DBS, suggesting the inhibitor effectively formed adducts with the endogenous kinase (Figure 4B).

[0135] SRPK2 and SRPK1 are highly conserved in primary and tertiary structures33. Both kinases contain lysine residues at the same positions at their docking grooves (K604 and K648 in SRPK1 and SRPK2, respectively) (Figure 13). Therefore, like DBS1, C-DBS might be a pan-SRPK inhibitor that could also inhibit SRPK2 via adduction. We tested the selectivity of C-DBS towards SRPK2 and another SR protein-specific kinase CDC-like kinase 1 (CLK1), as well as two other unrelated proteins bovine serum albumin (BSA) and glutathione S-transferase (GST). Results showed that C-DBS also formed adducts with SRPK2 effectively but failed to react with the CLK1 kinase domain, BSA, or GST (Figure 4C), indicating that C-DBS selectively targets SRPKs. Investigation of the inhibitory activity of C-DBS against SRPK2, CLK1, and Akt, another kinase that also has the capacity to phosphorylate SR proteins, further confirmed that C-DBS selectively targets SRPKs (Figure 4D). The selectivity of C-DBS was further assessed by screening its inhibitory activity against 140 different kinases. While C-DBS was highly selective and did not significantly inhibit all the protein kinases tested, it also did not inhibit SRPK1 in the screen (Figure 13). This was not unexpected because free RS-rich peptide instead of the full SRSF1 substrate was used in the phosphorylation assay. Therefore, C-DBS, which targets the docking groove distal to the active site, could not inhibit the phosphorylation of the free peptide. EXAMPLE 4 — C-DBS IS SELF-CELL PERMEABLE

[0136] The arginine-rich sequence of C-DBS resembles those of cell-penetrating peptides (CPPs) and in fact we have previously shown that DBS1 is self-cell permeable. To determine whether C-DBS exhibits cell-penetrating capability, we treated human non-small cell lung cancer (NSCLC) A549 cells with FAM-C-DBS. Confocal fluorescence microscopic analysis showed that C-DBS was readily internalized by A549 cells (Figure 5A). On the contrary, a (6- FAM)-labeled reactive control peptide consisting of irrelevant amino acids sequence (LNGHEDAQ(Dap-aryl-SO2F)FPTRIVYLSKM: SEQ ID NO: 3) failed to enter the cells. The cellular uptake of C-DBS was further investigated by staining the cells with LysoTracker Red after 1.5 h and 24 h of incubation to probe the colocalization of the inhibitor, if any, with the endocytic vesicles. At 1.5 h, some C-DBS was found to be entrapped inside the endosomal / lysosomal compartments while the rest were in the cytoplasm. On the other hand, the signals of C-DBS became more diffused and were more visible in the cytoplasm after 24 h incubation. Plots of fluorescence intensities of C-DBS and LysoTracker Red revealed that the two did not overlap perfectly and the mismatching became more obvious at the longer time point (Figures 5B and 5C). These findings indicated that C-DBS was not trapped in the endolysosomal compartments after internalization. Together, our data supports the notion that C-DBS can be uptaken by cells without any additional assistance.

[0137] EXAMPLE 5 — C-DBS INHIBITS SR PROTEIN PHOSPHORYLATION, ANGIOGENESIS AND CELL INVASION

[0138] We evaluated the phosphorylation levels of SR proteins in A549 cells after treatment with different dosages of C-DBS. The cell lysates were analyzed using the phosphor-SR proteinspecific mAblO4 antibody 43. The phosphorylation of several SR proteins — including SRSF1 or SRSF2, SRSF4, SRSF6, and SRSF10 — was significantly attenuated by C-DBS dose- dependently (Figure 6), demonstrating that C-DBS effectively inhibited the phosphorylation of endogenous SR proteins in cells. RNAi-mediated knockdown or inhibition of SRPK1 by using small molecule inhibitors or DBS1 has been demonstrated to shift the splicing of VEGF and subsequently inhibit angiogenesis44,41,45. To determine whether C-DBS can also inhibit angiogenesis, we performed an in vitro tube formation assay by using human umbilical vein endothelial cells (HUVECs). MTT assay was previously performed to confirm that C-DBS is non-cytotoxic within the concentration range used (Figure 14). Conditioned medium (CM) collected from A549 cells treated with C-DBS or a reactive control peptide derived from the trans-activator of transcription (TAT) of HIV and the RRM2 region of SRSF1 (GRKKRRQR(K-aryl-SO2F)RPQSWQDLKDH) was added to HUVECs. Our results demonstrated that C-DBS, but not the control peptide, reduced both the number of tube nodes and the total length of the capillary-like tubes in a dose-dependent manner (Figure 7A, upper panel). These results confirmed the potential of C-DBS as an inhibitor of angiogenesis.

[0139] To exclude that the observed anti-angiogenic effect was due to the cell-penetrating property or random reactivity of C-DBS, we repeated the experiment with a cell-penetrating reactive control peptide that is derived from the trans-activator of transcription (TAT) of HIV and the RRM2 region of SRSF 1 (GRKKRRQR(K-aryl-SO2F)RPQSWQDLKDH; SEQ ID NO: 4), CM collected from the control peptide-treated cells failed to exhibit anti -angiogenic effect (Figure 7A, bottom panel). Taken together, these results confirmed C-DBS as an inhibitor of angiogenesis.

[0140] Accumulating evidence has correlated the abnormal expression of SRPKs with the invasiveness and metastasis of cancer46'50. Therefore, C-DBS was evaluated using Transwell assays. Migration of A549 cells was markedly inhibited by 50% and 85% after the administration of 1 pM and 10 pM C-DBS, respectively. Contrarily, no changes in cell migration were observed when the reactive control peptide was administered (Figure 7B). In addition, C-DBS significantly inhibited Matrigel invasion by A549 cells compared to the cells treated with the control peptide (Figure 7C). The effects of C-DBS on the expression of epithelial-mesenchymal transition (EMT) markers were also evaluated. The expression of the mesenchymal markers, vimentin, twist, and snail was downregulated while that of E-cadherin was upregulated (Figure 7D). These results suggested that C-DBS possesses anti-metastatic potential against NSCLC cells. To assess whether C-DBS is also effective in other cancers, we analyzed its effects on the phosphorylation of SR proteins in the triple-negative breast cancer (TNBC) cell line MDA-MB-231 and repeated the Transwell migration and invasion assays as well. Similar to the results in A549 cells, the phosphorylation levels of SR proteins in MDA- MB-231 cells were reduced after treatment with C-DBS (Figure 15A). Moreover, C-DBS significantly inhibited the migration and invasion of TNBC cells dose-dependently (Figures 15B, 15C). Collectively, findings in this study demonstrated that C-DBS is an effective SRPK inhibitor that exhibits anti-angiogenesis and anti-metastasis properties (Figure 8). EXAMPLE 6 — C-DBS re-sensitizes cisplatin-resistant cells to cisplatin

[0141] Drug resistance is a major culprit for the treatment failure in NSCLC51,52. The NSCLC A549 cell line has been shown recently to contain chemotherapy -resistant subpopulation53. Therefore, we tested whether SRPKs inhibition by C-DBS can improve the drug responsiveness of A549 cells to cisplatin. Our results showed that the ICso value of cisplatin decreased from 16 mM to 11 mM when cells were co-treated with 10 mM C-DBS (Figures 17A-17B). The combination index (CI), calculated from the CI equation algorithms using CompuSyn software, indicated a synergistic interaction (CI < 1) between cisplatin and C-DBS in the co-treatment of A549 cells (Figure 17C). These findings suggest that C-DBS may enhance the sensitivity of cisplatin-resistant cells to cisplatin, potentially through a synergistic mechanism.

[0142] EXAMPLE 7 — C-DBS serves as a potential inhibitor for Hepatitis B Virus infection

[0143] Since C-DBS serves as a pan-SRPK inhibitor and inhibits both SRPK1 and SRPK2, we tested whether C-DBS can also block the binding of Cp to SRPKs. We performed GST-pull down assay using GST-Cp and SRPK1 / SRPK2 in the presence of C-DBS. Our results showed that C-DBS inhibited the binding of Cp to both SRPK1 and SRPK2 in a dose-dependent manner (Figures 18A-18B). We next tested whether C-DBS can inhibit the phosphorylation of Cp by SRPK2. Recombinant GST-Cp was phosphorylated by SRPK2 with or without the presence of C-DBS (Figure 18C). The samples were first separated on Phos-tag gel followed by Western blotting using anti-GST antibodies. Our results showed that the phosphorylation of Cp was nearly abolished in the presence of 10pM C-DBS. This suggests C-DBS may serve as a potential inhibitor for HBV replication.

[0144] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto. EXEMPLARY EMBODIMENTS

[0145] Embodiment 1. A peptide inhibitor comprising an amino sequence of RERARTRRXRARTRERARTR (SEQ ID NO: 1), wherein amino acid X at position 9 is a diaminopropionic acid analog with an aryl-sulfonyl fluoride group.

[0146] Embodiment 2. The peptide inhibitor of embodiment 1, wherein the di aminopropionic acid analog with the aryl-sulfonyl fluoride group is Formula (I)

[0147] Formula (I):

[0148] Embodiment 3. A method of inhibiting a serine-arginine protein kinase (SRPK), the method comprising contacting the peptide inhibitor of any preceding embodiment to an SPRK.

[0149] Embodiment 4. The method of any preceding embodiment, wherein the peptide inhibitor blocks a docking groove of the SRPK.

[0150] Embodiment 5. The method of any preceding embodiment, wherein the blocking of the docking groove occurs via conjugation between the peptide inhibitor and a lysine residue of the SRPK.

[0151] Embodiment 6. The method of any preceding embodiment, wherein the peptide inhibitor inhibits SRSF1 phosphorylation by the SRPK, and wherein the SRPK is SRPK1 and / or SRPK2.

[0152] Embodiment 7. The method of any preceding embodiment, wherein the SRPK is within a subject. Embodiment 8. The method of any preceding embodiment, wherein the subject has cancer or a viral infection.

[0153] Embodiment 9. The method of embodiment 8, further comprising administering or performing a cancer treatment to the subject.

[0154] Embodiment 10. The method of any preceding embodiment, wherein the cancer treatment is surgically removing cancerous tissue, chemotherapy, radiation therapy, radiofrequency ablation, cryoablation, immunotherapy, hormone therapy, stem cell transplant, or any combination thereof.

[0155] Embodiment 11. The method of any preceding embodiment, wherein the peptide inhibitor is administered to the subject at a dose of about 0.01 mg / kg to about 1000 mg / kg.

[0156] Embodiment 12. The method of any preceding embodiment, wherein the peptide inhibitor is administered to the subject daily.

[0157] Embodiment 13. The method of any preceding embodiment, wherein the peptide inhibitor is administered to the subject daily for about 1 year, or until no longer needed.

[0158] Embodiment 14. The method of any preceding embodiment, wherein the subject has breast cancer or lung cancer.

[0159] Embodiment 15. The method of any preceding embodiment, wherein the viral infection is caused by a coronavirus, a hepatitis virus, or a herpesvirus.

[0160] Embodiment 16. The method of any preceding embodiment, wherein the peptide inhibitor enhances the sensitivity of cisplatin-resistant cells to cisplatin by not less than 30% in a co-treatment in the subject.

[0161] Embodiment 17. The method of embodiment 16, wherein the peptide inhibitor enhances the sensitivity of cisplatin-resistant cells to cisplatin by not less than 40% in a cotreatment in the subject. Embodiment 18. The method of embodiment 15, wherein the peptide inhibitor inhibits HBV replication by inhibiting the binding of Cp to SRPK1 and / or SRPK2 by not less than 50%.

[0162] Embodiment 19. The method of any preceding embodiment, wherein the peptide inhibitor inhibits HBV replication by inhibiting the phosphorylation of Cp by not less than 70%. Embodiment 20. The method of embodiment 19, wherein the peptide inhibitor inhibits HBV replication by inhibiting the phosphorylation of Cp by not less than 90%.

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Claims

CLAIMSWe claim:

1. A peptide inhibitor comprising an amino sequence of RERARTRRXRARTRERARTR (SEQ ID NO: 1), wherein amino acid X at position 9 is a diaminopropionic acid analog with an aryl-sulfonyl fluoride group.

2. The peptide inhibitor of claim 1, wherein the diaminopropionic acid analog with the aryl-sulfonyl fluoride group is Formula (I)Formula (I):

3. A method of inhibiting a serine-arginine protein kinase (SRPK), the method comprising contacting the peptide inhibitor of claim 1 to an SPRK.

4. The method of claim 3, wherein the peptide inhibitor blocks a docking groove of the SRPK.

5. The method of claim 4, wherein the blocking of the docking groove occurs via conjugation between the peptide inhibitor and a lysine residue of the SRPK.

6. The method of claim 4, wherein the peptide inhibitor inhibits SRSF1 phosphorylation by the SRPK, and wherein the SRPK is SRPK1 and / or SRPK2.

7. The method of claim 3, wherein the SRPK is within a subject.

8. The method of claim 7, wherein the subject has cancer or a viral infection.

9. The method of claim 8, further comprising administering or performing a cancer treatment to the subject.

10. The method of claim 9, wherein the cancer treatment is surgically removing cancerous tissue, chemotherapy, radiation therapy, radiofrequency ablation, cryoablation, immunotherapy, hormone therapy, stem cell transplant, or any combination thereof.

11. The method of claim 7, wherein the peptide inhibitor is administered to the subject at a dose of about 0.01 mg / kg to about 1000 mg / kg.

12. The method of claim 7, wherein the peptide inhibitor is administered to the subject daily.

13. The method of claim 12, wherein the peptide inhibitor is administered to the subject daily for about 1 year, or until no longer needed.

14. The method of claim 7, wherein the subject has breast cancer or lung cancer.

15. The method of claim 8, wherein the viral infection is caused by a coronavirus, a hepatitis virus, or a herpesvirus.

16. The method of claim 9, wherein the peptide inhibitor enhances the sensitivity of cisplatin-resistant cells to cisplatin by not less than 30% in a co-treatment in the subject.

17. The method of claim 16, wherein the peptide inhibitor enhances the sensitivity of cisplatin-resistant cells to cisplatin by not less than 40% in a co-treatment in the subject.

18. The method of claim 15, wherein the peptide inhibitor inhibits HBV replication by inhibiting the binding of Cp to SRPK1 and / or SRPK2 by not less than 50%.

19. The method of claim 15, wherein the peptide inhibitor inhibits HBV replication by inhibiting the phosphorylation of Cp by not less than 70%.

20. The method of claim 19, wherein the peptide inhibitor inhibits HBV replication by inhibiting the phosphorylation of Cp by not less than 90%.