Two mutations (S157A and Y159F) in the Rieske iron-sulfur subunit of the ubihydroquinone-cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and [2Fe-2S] cluster stability, the enzyme activity and the redox potential of the [2Fe-2S] cluster.In the structure of the water-soluble fragment of the Rieske iron-sulfur protein of the bovine heart mitochondrial bc 1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe-2S] cluster: Ser163 Oγ-S-1 and Tyr165 Oη-Cys139 Sγ [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567Ϫ579]. They are conserved in all known Rieske iron-sulfur proteins from bc 1 complexes including that from P. denitrificans.Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three-subunit complex in P. denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain.The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster. Whereas the turnover number of the bc 1 complex isolated from the parental strain was 145 s Ϫ1 , the turnover numbers for the mutants S157A and Y159F were 10 s Ϫ1 and 52 s Ϫ1 , respectively, corresponding to 7% and 36% activity, respectively. The midpoint potential E m of the Rieske cluster at pH 6 and 5°C was ϩ360 mV for the bc 1 complex from the parental strain ; the values in the mutant complexes were ϩ316 mV (Y159F) and ϩ265 mV (S157A). Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild-type protein.Keywords : Rieske protein; cytochrome bc 1 complex ; Paracoccus denitrificans ; redox potential ; sitedirected mutagenesis.The ubihydroquinone-cytochrome c oxidoreductase is a key and c1 and the Rieske iron-sulfur protein (Yang and Trumpower, 1986; Payne and Trumpower, 1987). The iron-sulfur protein was component of photosynthetic and respiratory electron-transport chains in many organisms. The complex is embedded in the in-first found in bovine heart mitochondrial membranes by Rieske et al. (1964) and has later been identified as the oxidation factor ner mitochondrial membrane of eukaryotes or in the plasma membrane of many bacteria. It catalyzes the reduction of cyto-of the cytochrome bc 1 complex (Trumpower and Edwards, 1979). In contrast to [2Fe-2S] clusters, e.g. of plant type ferrechrome c by ubihydroquinone and translocates protons across the membrane by the Q-cycle mechanism.doxins, Rieske [2Fe-2S] clusters are coordinated by two cysteine and two histidine residues (Gurbiel et al., 1989(Gurbiel et al., , 1991; Britt et In contrast to mitochondrial cytochrome bc 1 complexes containing 10 or 11 subunits, bacterial cytochrome bc 1 complexes al., 1991). In the recently solved X-ray structure of the watersoluble fragment (ISF) of the Rieske iron-sulfur protein f...

The identification of a new class of potent and selective ROCK-II inhibitors is presented. Compound 5 (SR-3677) had an IC50 of ~3 nM in enzyme and cell based assays and had an off-target hit rate of 1.4% against 353 kinases, and inhibited only 3 out of 70 nonkinase enzymes and receptors. Pharmacology studies showed that 5 was efficacious in both, increasing ex vivo aqueous humor outflow in porcine eyes and inhibiting myosin light chain phosphorylation.

AAK1, the adaptor-associated kinase 1, phosphorylates the m2 subunit of AP2 and regulates the recruitment of AP2 to tyrosine-based internalization motifs found on membrane-bound receptors. AAK1 overexpression specifically inhibits the AP2-dependent internalization of transferrin receptor and LDL-receptor related protein by functionally sequestering AP2 (Conner and Schmid. J Cell Biol 2003; 162: 773). However, while AAK1 stably associates with AP2 and specifically targets the m2 subunit in vitro, m2 phosphorylation in vivo was not altered by overexpression of either wild-type or kinase-inactive AAK1. These results suggested that AAK1 might be tightly regulated in the cell. Here, we report that AAK1 is an atypical kinase that is rate limited by its stable association with AP2 and that clathrin stimulates m2 phosphorylation by AAK1. Efficient stimulation of AAK1 by clathrin involves multiple interactions between several domains on AAK1 and both heavy and light chains on clathrin. Importantly, incubation of AAK1 with clathrin cages resulted in even greater stimulation when compared to that of unassembled clathrin triskelia. Collectively, our observations indicate that clathrin function is not limited to structural and/or mechanical roles in endocytic vesicle formation: the stimulatory effects of clathrin on AAK1 activity argue that it also plays a regulatory role by modulating the activity of AP2 complexes through activation of AAK1. We suggest a model in which AAK1 is specifically activated in coated pits to enhance cargo recruitment and efficient internalization. Clathrin-dependent endocytosis is a robust and ubiquitous internalization pathway in eukaryotic cells that is mediated by the action of two oligomeric protein complexes, clathrin and the adaptor protein complex, AP2 (1). Clathrin is a heterohexameric structure composed of three heavy chains of $180 kDa and three tightly associated light chains of $30 kDa that together form three-legged structures called triskelia. While clathrin self-assembly into cages occurs under nonphysiologic conditions (2,3), the specific recruitment and assembly of clathrin into a polyhedral lattice at the plasma membrane, as well as its coupling to endocytic cargo, are coordinated by adaptor proteins, such as AP2. AP2 consists of four subunits: two large (a and b2, $105-115 kDa), one medium (m2, $50 kDa) and one small (s2, $17 kDa). Plasma membrane targeting (4,5), interaction with clathrin (6), and the recruitment of several accessory proteins important for endocytosis are supported by the large AP2 subunits (7,8). A critical step in endocytosis is the coupling of the endocytic machinery to cargo. This is coordinated through the m2 subunit of AP2 that directly interacts with the cytoplasmic tail of membrane-bound receptors marked for internalization by tyrosine-based sorting signals (9). The function of the small subunit has not been specifically elucidated, but structural studies implicate its role in stabilizing the AP2 core (10).Accumulating evidence suggests that the spatial...

A series of urea-based Rho kinase (ROCK) inhibitors were designed and evaluated. The discovered compounds had excellent enzyme and cellular potency, high kinase selectivity, high aqueous solubility, good porcine corneal penetration, and appropriate DMPK profiles for topical applications as antiglaucoma therapeutics.

Rho kinase (ROCK) is a promising drug target for the treatment of many diseases including hypertension, multiple sclerosis, cancer, and glaucoma. The structure-activity relationships (SAR) around a series of tetrahydroisoquinolines were evaluated utilizing biochemical and cell-based assays to measure ROCK inhibition. These novel ROCK inhibitors possess high potency, high selectivity, and appropriate pharmacokinetic properties for glaucoma applications. The lead compound, 35, had subnanomolar potency in enzyme ROCK-II assays as well as excellent cell-based potency (IC(50) = 51 nM). In a kinase panel profiling, 35 had an off-target hit rate of only 1.6% against 442 kinases. Pharmacology studies showed that compound 35 was efficacious in reducing intraocular pressure (IOP) in rats with reasonably long duration of action. These results suggest that compound 35 may serve as a promising agent for further development in the treatment of glaucoma.

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RhoA and its downstream effector ROCK mediate stress fiber formation and cell contraction through their effects on the phosphorylation of myosin light chain (MLC). Inhibition of the RhoA/ROCK pathway has proven to be a promising strategy for several indications such as cardiovascular disease, glaucoma, and inflammatory disease. In 2010, our group reported urea-based ROCK inhibitors as potential antiglaucoma agents. These compounds showed potent IC50 values in enzymatic and cell-based assays and significant intraocular pressure (IOP)-lowering effects in rats (∼7 mmHg). (22) To develop more advanced ROCK inhibitors targeting various potential applications (such as myocardial infarction, erectile dysfunction, multiple sclerosis, etc.) in addition to glaucoma, a thorough SAR for this urea-based scaffold was studied. The detailed optimization process, counter-screening, and in vitro and in vivo DMPK studies are discussed. Potent and selective ROCK inhibitors with various in vivo pharmacokinetic properties were discovered.

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