Discovery of 1,8-disubstituted-[1,2,3]triazolo[4,5-c]quinoline derivatives as a new class of Hippo signaling pathway inhibitors.

Inhibitors of the Hippo signaling pathway have been demonstrated to have a potential clinical application in cases such as tissue repair and organ regeneration. However, there is a lack of potent Hippo pathway inhibitors at present. Herein we report the discovery of a series of 1,8-disubstituted-[1,2,3]triazolo[4,5-c]quinoline derivatives as a new class of Hippo pathway inhibitors by utilizing a cell line-based screening model (A549-CTGF). Structure-activity relationship (SAR) of these compounds was also discussed. The most potent compound in the A549-CTGF cell assay, 11g, was then evaluated by real-time PCR and immunofluorescence assays. Overall, this study provides a starting point for later drug discovery targeting the Hippo signaling pathway.

SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK.

The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1-MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.

De novo synthesis and biological evaluation of C6″-substituted C4″-amide analogues of SL0101.

In an effort to improve upon the in vivo half-life of the known ribosomal s6 kinase (RSK) inhibitor SL0101, C4″-amide/C6″-alkyl substituted analogues of SL0101 were synthesized and evaluated in cell-based assays. The analogues were prepared using a de novo asymmetric synthetic approach, which featured Pd-π-allylic catalyzed glycosylation for the introduction of a C4″-azido group. Surprisingly replacement of the C4″-acetate with a C4″-amide resulted in analogues that were no longer specific for RSK in cell-based assays.

Investigation of unanticipated alkylation at the N(π) position of a histidyl residue under Mitsunobu conditions and synthesis of orthogonally protected histidine analogues.

We had previously reported that Mitsunobu-based introduction of alkyl substituents onto the imidazole N(π)-position of a key histidine residue in phosphothreonine-containing peptides can impart high binding affinity against the polo-box domain of polo-like kinase 1. Our current paper investigates the mechanism leading to this N(π)-alkylation and provides synthetic methodologies that permit the facile synthesis of histidine N(π)-modified peptides. These agents represent new and potentially important tools for biological studies.

DNA sequence-specific recognition of peptides incorporating the HPRK and polyamide motifs.

Three peptide amides, HPRK(Py)(4)HPRK-NH(2) (PyH-12), HPRK(Py)(3)HPRK-NH(2) (PyH-11) and HPRK(Py)(2)HPRK-NH(2) (PyH-10), incorporating two HPRK motifs and various 4-amino-1-methylpyrrole-2-carboxylic acid residues (Py) were synthesized by solid-phase peptide methodology. The binding of these three peptides to a 5'-32P-labeled 158-mer DNA duplex (Watson fragment) and to a 5'-32P-labeled 135-mer DNA duplex (complementary Crick fragment) was investigated by quantitative DNase I footprinting. On the 158-mer Watson strand, the most distinctive DNase I blockages seen with all three peptides occur around positions 105-112 and 76-79, corresponding to the sequences 5'-GAGAAAAT-3' and 5'-CGGT-3', respectively. However, on the complementary Crick strand, only PyH-12 strongly discriminates the 5'-TTT-3' site around positions 108-110 whereas both PyH-11 and PyH-10 have moderate binding around positions 102-112 comprising the sequence 5'-ATTTTCTCCTT-3'. Possible bidentate and single interactions of the side-chain functions and alpha-amino protons of the peptides with DNA bases are discussed.

The identification and subcellular localization of human MRK.

A novel human kinase gene, human MRK (hMRK), was cloned by using degenerate RT-PCR. The hMRK encoded a putative 632 amino acids protein and was highly homologous to rat MRK (rMRK) in the entire coding region. The hMRK was located at chromosome 6p12.3 by RH-PCR analysis. The hMRK was generally expressed a single approximately 6.3 kb transcript at a low level in a variety of tissues except at a high level in testis. The full-length hMRK protein was fused to C-terminal of GFP and expressed in Hela cells. The fluorescence microscopy results identified its nuclear localization.

The TGF beta receptor activation process: an inhibitor- to substrate-binding switch.

The type I TGF beta receptor (T beta R-I) is activated by phosphorylation of the GS region, a conserved juxtamembrane segment located just N-terminal to the kinase domain. We have studied the molecular mechanism of receptor activation using a homogeneously tetraphosphorylated form of T beta R-I, prepared using protein semisynthesis. Phosphorylation of the GS region dramatically enhances the specificity of T beta R-I for the critical C-terminal serines of Smad2. In addition, tetraphosphorylated T beta R-I is bound specifically by Smad2 in a phosphorylation-dependent manner and is no longer recognized by the inhibitory protein FKBP12. Thus, phosphorylation activates T beta R-I by switching the GS region from a binding site for an inhibitor into a binding surface for substrate. Our observations suggest that phosphoserine/phosphothreonine-dependent localization is a key feature of the T beta R-I/Smad activation process.

Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity.

In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.