KH CB19

KH CB19 is a potent and selective inhibitor of CLK1 and CLK4 with IC50 values of 19.7 and 530 nM for CLK1 and CLK3, respectively [1] [2].

The cdc2-like kinases (CLKs) are dual specificity protein kinases that phosphorylate the serine- and arginine-rich (SR) proteins, which are involved in regulating the alternative pre-mRNA splicing process [1].

KH CB19 is a potent and selective inhibitor of CLK1 and CLK4. In endothelial cells, KH CB19 inhibited the phosphorylation of serine- and arginine-rich (SR) proteins stimulated by TNF-α and decreased the mRNA expression of tissue factor splice variants [1]. KH CB19 bound to the ATP binding site in CLK3 and CLK1. In enzyme kinetic assays, KH CB19 inhibited DYRK1A with IC50 value of 55.2 nM. In human microvascular endothelial cells (HMEC-1), KH-CB19 (10 µM) reduced the phosphorylation of SRp75, SRp55 and SRp20. However, in TNF-α stimulated HMEC-1, KH-CB19 (10 µM) inhibited the phosphorylation of SRp75, SRp55, SRp40, SC35, SF2/ASF and SRp20. In HMEC-1, KH CB19 (10 µM) significantly reduced the expression of the membrane bound full-length tissue factor (flTF) as well as the soluble asHTF in TNF-α-induced and TNF-α-non-induced cells [2].

References:
[1].  Grant SK, Lunney EA. Kinase inhibition that hinges on halogen bonds. Chem Biol, 2011, 18(1): 3-4.
[2].  Fedorov O, Huber K, Eisenreich A, et al. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing. Chem Biol, 2011, 18(1): 67-76.

Spatial regulation of the KH domain RNA-binding protein Rnc1 mediated by a Crm1-independent nuclear export system in Schizosaccharomyces pombe.[Pubmed:28142187]

Mol Microbiol. 2017 May;104(3):428-448.

RNA-binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1(+) encodes a K Homology (KH)-type RBP, which binds and stabilizes the Pmp1 MAPK phosphatase mRNA thereby suppressing the Cl(-) hypersensitivity of calcineurin deletion and MAPK signaling mutants. Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES)Rnc1 , which dictates the cytoplasmic localization of Rnc1 in a Crm1-independent manner. Notably, mutations in the NESRnc1 altered nucleocytoplasmic distribution of Rnc1 and abolished its function to suppress calcineurin deletion, although the Rnc1 NES mutant maintains the ability to bind Pmp1 mRNA. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH-domains abolished the mRNA-binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1-dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1.

The interaction of a Trypanosoma brucei KH-domain protein with a ribonuclease is implicated in ribosome processing.[Pubmed:27965085]

Mol Biochem Parasitol. 2017 Jan;211:94-103.

Ribosomal RNA maturation is best understood in yeast. While substantial efforts have been made to explore parts of these essential pathways in animals, the similarities and uniquenesses of rRNA maturation factors in non-Opisthokonts remain largely unexplored. Eukaryotic ribosome synthesis requires the coordinated activities of hundreds of Assembly Factors (AFs) that transiently associate with pre-ribosomes, many of which are essential. Pno1 and Nob1 are two of six AFs that are required for the cytoplasmic maturation of the 20S pre-rRNA to 18S rRNA in yeast where it has been almost exclusively analyzed. Specifically, Nob1 ribonucleolytic activity generates the mature 3'-end of 18S rRNA. We identified putative Pno1 and Nob1 homologues in the protist Trypanosoma brucei, named TbPNO1 and TbNOB1, and set out to explore their rRNA maturation role further as they are both essential for normal growth. TbPNO1 is a nuclear protein with limited cytosolic localization relative to its yeast homologue. Like in yeast, it interacts directly with TbNOB1, with indications of associations with a larger AF-containing complex. Interestingly, in the absence of TbPNO1, TbNOB1 exhibits non-specific degradation activity on RNA substrates, and its cleavage activity becomes specific only in the presence of TbPNO1, suggesting that TbPNO1-TbNOB1 interaction is essential for regulation and site-specificity of TbNOB1 activity. These results highlight a conserved role of the TbPNO1-TbNOB1 complex in 18S rRNA maturation across eukaryotes; yet reveal a novel role of their interaction in regulation of TbNOB1 enzymatic activity.

FUBP/KH domain proteins in transcription: Back to the future.[Pubmed:28301294]

Transcription. 2017 May 27;8(3):185-192.

Drosophila genetic studies demonstrate that cell and tissue growth regulation is a primary developmental function of P-element somatic inhibitor (Psi), the sole ortholog of FUBP family RNA/DNA-binding proteins. Psi achieves growth control through interaction with Mediator, observations that should put to rest controversy surrounding Pol II transcriptional functions for these KH domain proteins.

The herbal formula KH-204 is protective against erectile dysfunction by minimizing oxidative stress and improving lipid profiles in a rat model of erectile dysfunction induced by hypercholesterolaemia.[Pubmed:28235412]

BMC Complement Altern Med. 2017 Feb 24;17(1):129.

BACKGROUND: Hypercholesterolaemia (HC) is a major risk factor for ischemic heart disease and is also known to be a risk factor for erectile dysfunction (ED). ED caused by HC is thought to be related to HC-induced oxidative stress damage in the vascular endothelium and erectile tissue. KH-204 is an herbal formula with a strong antioxidant effect. We evaluated the effects of KH-204 on erectile function in a rat model of HC-induced ED. METHODS: Male Sprague-Dawley rats (6 weeks old) were divided into normal control, high-fat and cholesterol diet (HFC), and HFC with KH-204 treatment (HFC + KH) groups (n = 12 each). Normal control group rats were fed normal chow diet. HFC and HFC + KH group rats were fed high-fat and cholesterol diets and treated with or without daily oral doses of KH-204 for 12 weeks. Subsequently, intracavernous pressure (ICP) and mean arterial pressure (MAP) were measured, and lipid profiles, expression of endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase, oxidative stress (8-hydroxy-2-deoxyguanosine), and ratio of smooth muscle cells and collagen fibres were evaluated in the serum and corpora tissue. RESULTS: Compared to the HFC group, the HFC + KH group showed statistically significant increases in peak ICP and ICP/MAP ratio, expression of eNOS and nNOS, and ratio of smooth muscle cells and collagen fibres (p < 0.05). The HFC + KH group also showed statistically significant decreases in oxidative stress (p < 0.05). Further the lipid profiles of this group were ameliorated compared to those of the HFC group (p < 0.05). CONCLUSIONS: The current study shows that the antioxidant and hypolipidemic effects of KH-204 are effective in ameliorating ED by restoring endothelial dysfunction and suggests that KH-204 may be a potential therapeutic agent for ED by correcting the fundamental cause of ED.