Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632

Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability.     

Epoxyeicosatrienoic and dihydroxyeicosatrienoic acids dilate human coronary arterioles via BK(Ca) channels: implications for soluble epoxide hydrolase inhibition

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K+ (BK(Ca)) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BK(Ca) channels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11,12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BK(Ca) channels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously.     

Cytoplasmic domain phosphorylation of heparin-binding EGF-like growth factor

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a transmembrane precursor protein that is anchored to the plasma membrane. The extracellular EGF-like domain acts as a mitogen and motogen upon ectodomain shedding, but the functional roles of the transmembrane and cytoplasmic domains are largely unknown. We demonstrate here that cytoplasmic domain of HB-EGF is phosphorylated by external stimuli, and that the phosphorylation site is involved in HB-EGF-dependent tumorigenesis. Treatment of Vero cells overexpressing human HB-EGF with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused ectodomain shedding of HB-EGF and generated two carboxyl (C)-terminal fragments with distinct electrophoretic mobilities. Mutation analysis showed that Ser207 in the cytoplasmic domain of HB-EGF is phosphorylated upon TPA stimulation, generating two C-terminal fragments with distinct phosphorylation states. Treatment of cells with lysophosphatidic acid, anisomycin, and calcium ionophore, all of which are known to induce ectodomain shedding, also caused phosphorylation of HB-EGF. Although ectodomain shedding and phosphorylation of HB-EGF occurred coordinately, Ala substitution of Ser207 had no effect on TPA-induced or constitutive ectodomain shedding. Injection of cells overexpressing HB-EGF into nude mice showed that Ala substitution of Ser207 reduced the tumorigenic activity of HB-EGF, even though the cell surface level and ectodomain shedding of HB-EGF were not affected by the mutation. Moreover, we found that the cytoplasmic domain of another EGFR ligand, transforming growth factor-alpha, is phosphorylated upon TPA stimulation. Thus, the present results suggest a novel role for the cytoplasmic domain of HB-EGF and other EGF family growth factors that is regulated by phosphorylation.     

Effect of flavonoids on androgen and glucocorticoid receptors based on in vitro reporter gene assay

The effect of 32 flavonoids on androgen (AR) and glucocorticoid receptors (GR) was investigated using an MDA-kb2 human breast cancer cell line to predict potential AR and GR activities. Among them, 5-hydroxyflavone (7) had the highest AR antagonistic activity with an IC₅₀ value of 0.3 μM, whereas 6-methoxyflavone (11) had the highest induced luciferase activity with an EC₁₅₀ value of 0.7 μM. Genistein (2) and daizein (1) showed a sufficient increase of luciferase activities as their concentrations increased with EC₁₅₀ values of 4.4 and 10.1 μM, respectively. These findings provide evidence of a fundamental property of their structure–activity relationship with AR and/or GR.     

Molecular mechanism for the regulation of rho-kinase by dimerization and its inhibition by fasudil

Rho-kinase is a key regulator of cytoskeletal events and a promising drug target in the treatment of vascular diseases and neurological disorders. Unlike other protein kinases, Rho-kinase requires both N- and C-terminal extension segments outside the kinase domain for activity, although the details of this requirement have been elusive. The crystal structure of an active Rho-kinase fragment containing the kinase domain and both the extensions revealed a head-to-head homodimer through the N-terminal extension forming a helix bundle that structurally integrates the C-terminal extension. This structural organization enables binding of the C-terminal hydrophobic motif to the N-terminal lobe, which defines the correct disposition of helix alphaC that is important for the catalytic activity. The bound inhibitor fasudil significantly alters the conformation and, consequently, the mode of interaction with the catalytic cleft that contains local structural changes. Thus, both kinase and drug conformational pliability and stability confer selectivity.     

Effect of essential oils,such as raspberry ketone and its derivatives,on antiandrogenic activity based on in vitro reporter gene assay

The effect of essential oils, such as raspberry ketone, on androgen (AR) receptor was investigated using a MDA-kb2 human breast cancer cell line for predicting potential AR activity. Among them, eugenol had the highest AR antagonistic activity with its IC₅₀ value of 19 μM. Raspberry ketone, which has threefold higher anti-obese activity than that of capsaicin, also had AR antagonist activity with its IC₅₀ value of 252 μM. Based on these findings, a more precise CoMFA model was proposed as follows: pIC₅₀ [log (1/IC₅₀)] = 3.77 + [CoMFA field terms] (n = 39, s = 0.249, r² = 0.834, s_cv = 0.507, q² = 0.311 (three components).