Table 2 Summary of CLKs and associated therapeutic strategies in human non-cancer diseases

Alzheimer’s disease (AD)

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Human and murine CLK2

The expression and activity of CLK2 led to the misregulation of tau exon 10 splicing which contributed to AD

CLK2 related alternative splicing of tau exon 10 was regulated by phosphorylating SR proteins

Transfection of CLK2 plasmids at 2 μg significantly increased the skipping of exon 10 of tau in HEK293 cells

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^59,60,61,62

Phelan-McDermid syndrome (PMDS)

TG003

CLK2

CLK2 targeting improved autism and neuronal functions in PMDS

TG003 recovered spine density in Shank3-deficient neurons by targeting CLK2

10 μM TG003 rescued spine density in Shank3 deficient neurons

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⁶⁴

Indazole 1 and Indazole 2

CLK2

Indazole 1 and -2 elevated spine density in Shank3-deficient neurons by targeting CLK2

Indazole 1 and -2 significantly improved spine density at 300 nM and 1 µM.

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⁶³

Duchenne muscular dystrophy (DMD)

TG003

CLKs and SRp30c/SRSF9

TG003 promoted exon skipping only in the c.4303 G > T mutant cells to generate partially functional dystrophin protein

Dephosphorylation of SR protein(s) cooperated with hnRNP A1 to act as a co-inhibitor during the recognition of exon 31, resulting in functional dystrophin protein

Up to 50 μM of TG003 promoted exon 31 skipping in mutated Hela cells and showed no cytotoxicity to cells

Up to 100 mg/kg per day per os showed no toxicity to mice

⁶⁸

TG693

CLK1

TG693 promoted the skipping of exon 31 and restored dystrophin expression in patient cells harboring the c.4303 G > T mutation

TG693 targeted CLK1 to reduce downstream SR proteins phosphorylation, resulting in exon-skipping therapy in DMD

TG693 inhibited the phosphorylation of SRSF4 at 5 μM and SRSF6 at 20 μM by targeting CLK1.

Up to 20 μM of TG693 promoted the skipping of exon 31 and upregulated dystrophin expression in patient cells harboring c.4303 G > T mutation

Oral administration of 30 mg/kg TG693 reduced SRs phosphorylation, particularly SRSF4 in mice

No apparent acute toxicity in rats at up to 100 mg/kg per os

⁶⁷

Tendinopathy

SM04755

CLK2 and DYRK1A

SM04755 treatment or knock-down of CLK2 prevented tendon destruction and promoted tendon regeneration in tendinopathy

SM04755 led to intron retention in the mRNA of Wnt pathway genes; decreased tenocyte catabolic enzyme expression; inhibited inflammatory signaling mediators NF-κB and STAT3 by targeting CLK2 and DYRK1A

0.3 μM of SM04755 inhibited inflammatory cytokine production and protected tenocytes from the catabolic breakdown

0.3 mg/cm² of SM04755 inhibited inflammation and promoted tendon healing in a collagenase-induced acute tendinopathy model in rats

⁶⁹

Knee osteoarthritis (OA)

Lorecivivint

CLK2 and DYRK1A

Inhibition or knock-down of CLK2 induced chondrocyte differentiation and suppressed cartilage catabolic enzyme expression

Lorecivivint inhibited CLK2-mediated phosphorylation of SR splicing factors and DYRK1A-mediated phosphorylation of SIRT1 and FOXO1

Lorecivivint inhibited IL-6 and TNF-α production in IL-1β and LPS-stimulated synovial fibroblasts, and LPS-stimulated THP-1 cells in a dose-dependent manner

Intra-articular injection of lorecivivint (0.1 mg, 0.3 mg, 1 mg) decreased phospho-SRSF, phospho-SIRT1, phospho-FOXO1, and phospho-STAT3 in the ACLT + pMMx model and the MIA model of rat knee OA

^71,72

HIV-1 virus

Chlorhexidine

CLK3 and CLK4, slightly activity against CLK2

CLK3 and CLK4 promoted the expression of HIV-1 Gag and viral RNA abundance

Chlorhexidine significantly inhibited HIV-1 Gag synthesis and suppressed HIV-1 regulatory protein Rev accumulation to prevent virus replication

2.5 μM of chlorhexidine significant repressed HIV-1 replication did not significantly impact cell viability in PBMCs

Chlorhexidine was suggested to applicate on mucosal surfaces to establish well toleration to block HIV-1 in human

⁸¹

Influenza A virus (IAV)

NIH39

CLK1

Inhibition or knock-down of CLK1 reduced the replication of influenza A/WSN/33

NIH39 reduced viral NS mRNA splicing ratio and inhibited viral M1, M2, and NS1 protein levels to suppress viral replication

NIH39 showed antiviral activity with an IC₅₀ of 6.6 μM and reduced viral mRNA splicing and related protein expression at 12.5 μM in A549 cells

CLK1^−/− mice showed a reduction of viral replication compared to wild-type C57BL/6 mice

⁸³

J12098 (Corilagin)

CLK1

J12098 exhibited anti-influenza ability by targeting CLK1

J12098 showed better docking to CLK1 and closely interacted with CLK1

J12098 exerted anti-influenza virus effect with EC50 values of 2.0 ± 2.22 μg/mL a CC50 value of 153.54 μg/mL in vitro

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⁸⁶

J14848 (Pinosylvin)

CLK1

J1484 exhibited anti-influenza ability in vitro by targeting CLK1

J14848 showed better docking to CLK1 and closely interacted with CLK1

J14848 exerted an anti-influenza virus effect with EC50 values of 5.28 ± 2.45 μg/mL and a CC50 value of 18.26 μg/mL in vitro

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⁸⁶

J10688 (Clypearin)

CLK1

J10688 exerted anti-influenza virus activity in vivo and in vitro by potently targeting CLK1

J10688 impaired the synthesis of viral proteins NP and M2 and downregulated the phosphorylation of splicing factors SF2/ASF and SC35

J10688 exerted an anti-influenza virus effect with EC50 values of 1.2 ± 0.28 μg/mL and a CC50 value of >200 μg/mL

3, 10, and 30 μM of J10688 remarkably decreased the NP and M2 expression and reduced the copy number of viral RNA synthesis in a dose-dependent manner

Mice intravenous treated with 30, 10, and 3 mg/kg/day J10688 showed 91.67% survival rates, whereas all mice died in the normal saline group within 9 days

J10688 administration reduced lung virus titer, enhanced immunological function, and alleviated influenza-induced acute lung injury

^86,87

Autophagy-associated diseases

Leucettine L41

CLK1

Leucettine L41 triggered the accumulation of LC3 foci and autophagy by targeting CLK1

Leucettine L41 treatment induced a 150-fold increase of the exon 4-containing CLK1 mRNA to inhibit the activity of CLK1 and induce autophagy

Leucettine L41 induced LC3 foci formation and triggered autophagy in U-2 OS cells without modifying the autophagic flux

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¹⁰⁰

Compound 9e

CLK1

Compound 9e was an efficient inducer of autophagy

Compound 9e redistributed the location of SR proteins and induced the formation of autophagosome in SKOV-3 cells by targeting CLK1

Compound 9e treatment converted LC3I to LC3II and formed autophagosomes in vitro

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⁹¹

CLK1-IN-1

CLK1

CLK1-IN-1 treatment increased autophagic flux and induced autophagy

CLK1-IN-1 treatment reduced the phosphorylation and affected the subcellular redistribution of the downstream SR proteins

CLK1-IN-1 elevated the expression level of LC3II protein as well as the ratio of LC3II to LC3I in a dose- and time- dependent manner

CLK1-IN-1 showed hepatoprotective effects on the APAP-induced acute liver injury mouse model.

¹⁰⁴

Pathological cardiac hypertrophy

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CLK4

CLK4 deletion led to pathological cardiac hypertrophy

CLK4 regulated cardiac function through phosphorylation of NEXN at Ser437 to restore heart failure

CLK4 formed a complex with downstream NEXN and phosphorylated it at Ser437

Exogenous overexpression of the NEXN (S437E) phosphorylation-mimic mutant in Clk4-cKO mice via AAV9 injection reversed the pathological phenotype induced by CLK4 knockout

⁵⁶

Hyperglycemia

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CLK2

CLK2 was induced by insulin/AKT and acted as a suppressor of hepatic gluconeogenesis

The activated CLK2 phosphorylated PGC-1α at SR domain to repress PGC-1α transcriptional activity on gluconeogenic genes

Insulin/AKT phosphorylated CLK2 at Ser342/Thr343 to stabilize CLK2.

CLK2 was downregulated in the diabetic and obese db/db mice (Lepr^db/Lepr^db). Re-introduction of CLK2 in the livers of db/db mice dramatically restored the glucose phenotype

⁵⁰

Circadian body-temperature oscillations

TG003

CLK1/4

CLK1/4 acted as molecular thermometers in response to circadian body-temperature oscillations

TG003 administration abolished the re-phosphorylation of SRSF5 and SRSF6 as well as exon inclusion and intron retention events during the shift of temperature from 42 °C to 35 °C by targeting CLKs

HEK293 cells with 35 °C TG003 treatment showed a similar temperature dependent AS as 39 °C DMSO samples

Alligator CLK4 activity decreased with the temperature increasing from 27 °C to 35 °C.

Turtle CLK1 showed full activity below 26 °C but lost 90% activity above 31 °C

TG003 prevented the increased intron retention at a colder temperature

^109,110