Refolding and characterization of a soluble ectodomain complex of the calcitonin gene-related peptide receptor.

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of two membrane proteins: calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a class B G-protein-coupled receptor (GPCR), possessing a characteristic large amino-terminal extracellular domain (ECD) important for ligand recognition and binding. Dimerization of CLR with RAMP1 provides specificity for CGRP versus related agonists. Here we report the expression, purification, and refolding of a soluble form of the CGRP receptor comprising a heterodimer of the CLR and RAMP1 ECDs. The extracellular protein domains corresponding to residues 23-133 of CLR and residues 26-117 of RAMP1 were shown to be sufficient for formation of a stable, monodisperse complex. The binding affinity of the purified ECD complex for the CGRP peptide was significantly lower than that of the native receptor (IC(50) of 12 microM for the purified ECD complex vs 233 pM for membrane-bound CGRP receptor), indicating that other regions of CLR and/or RAMP1 are important for peptide agonist binding. However, high-affinity binding to known potent and specific nonpeptide antagonists of the CGRP receptor, including olcegepant and telcagepant (K(D) < 0.02 muM), as well as N-terminally truncated peptides and peptide analogues (140 nM to 1.62 microM) was observed.

ROCK enzymatic assay.

We describe the protocols for measuring Rho-associated coiled-coil-containing kinase (ROCK) activity in vitro. A His-tagged, constitutively active form of the protein (lacking C-terminal inhibitory domains) is expressed in baculovirus. The protein is purified by a combination of metal affinity, ion exchange, and size exclusion chromatography. Enzymatic activity is measured spectrophotometrically in a coupled assay format wherein a molecule of NADH is oxidized to NAD+ each time a phosphate is transferred by ROCK.

Design and Synthesis of a Novel Series of Orally Bioavailable, CNS-Penetrant, Isoform Selective Phosphoinositide 3-Kinase γ (PI3Kγ) Inhibitors with Potential for the Treatment of Multiple Sclerosis (MS).

The lipid kinase phosphoinositide 3-kinase γ (PI3Kγ) has attracted attention as a potential target to treat a variety of autoimmune disorders, including multiple sclerosis, due to its role in immune modulation and microglial activation. By minimizing the number of hydrogen bond donors while targeting a previously uncovered selectivity pocket adjacent to the ATP binding site of PI3Kγ, we discovered a series of azaisoindolinones as selective, brain penetrant inhibitors of PI3Kγ. This ultimately led to the discovery of 16, an orally bioavailable compound that showed efficacy in murine experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis.

New insights into the structure-function relationships of Rho-associated kinase: a thermodynamic and hydrodynamic study of the dimer-to-monomer transition and its kinetic implications.

The effect of the length of ROCK (Rho-associated kinase) on its oligomerization state has been investigated by analysing full-length protein and four truncated constructs using light-scattering and analytical ultracentrifugation methods. Changes in size correlate with the kinetic properties of the kinase. Sedimentation velocity, sedimentation equilibrium and light-scattering data analyses revealed that protein constructs of size Ser6-Arg415 and larger exist predominantly as dimers, while smaller constructs are predominantly monomeric. The amino acid segments comprising residues 379-415 and 47-78 are shown to be necessary to maintain the dimeric ROCK structure. kcat values ranged from 0.7 to 2.1 s(-1) and from 1.0 to 5.9 s(-1) using ROCK peptide (KKRNRTLSV) and the 20000 Da subunit of myosin light chain respectively as substrate, indicating that the effect of the ROCK oligomerization state on the kcat is minor. Values of ATP K(m) for monomeric constructs were increased by 50-80-fold relative to the dimeric constructs, and K(i) comparisons using the specific competitive ROCK inhibitor Y-27632 also showed increases of at least 120-fold, demonstrating significant perturbations in the ATP binding site. The corresponding K(m) values for the ROCK peptide and myosin light chain substrates increased in the range 1.4-16-fold, demonstrating that substrate binding is less sensitive to the ROCK oligomerization state. These results show that the oligomerization state of ROCK may influence both its kinase activity and its interactions with inhibitors, and suggest that the dimeric structure is essential for normal in vivo function.

Structural basis for isoform selectivity in a class of benzothiazole inhibitors of phosphoinositide 3-kinase γ.

Phosphoinositide 3-kinase γ (PI3Kγ) is an attractive target to potentially treat a range of disease states. Herein, we describe the evolution of a reported phenylthiazole pan-PI3K inhibitor into a family of potent and selective benzothiazole inhibitors. Using X-ray crystallography, we discovered that compound 22 occupies a previously unreported hydrophobic binding cleft adjacent to the ATP binding site of PI3Kγ, and achieves its selectivity by exploiting natural sequence differences among PI3K isoforms in this region.

Discovery of Highly Isoform Selective Thiazolopiperidine Inhibitors of Phosphoinositide 3-Kinase γ.

A series of high affinity second-generation thiazolopiperidine inhibitors of PI3Kγ were designed based on some general observations around lipid kinase structure. Optimization of the alkylimidazole group led to inhibitors with higher levels of PI3Kγ selectivity. Additional insights into PI3K isoform selectivity related to sequence differences in a known distal hydrophobic pocket are also described.

Phosphoryl transfer is not rate-limiting for the ROCK I-catalyzed kinase reaction.

Rho-associated coiled-coil kinase, ROCK, is implicated in Rho-mediated cell adhesion and smooth muscle contraction. Animal models suggest that the inhibition of ROCK can ameliorate conditions, such as vasospasm, hypertension, and inflammation. As part of our effort to design novel inhibitors of ROCK, we investigated the kinetic mechanism of ROCK I. Steady-state bisubstrate kinetics, inhibition kinetics, isotope partition analysis, viscosity effects, and presteady-state kinetics were used to explore the kinetic mechanism. Plots of reciprocals of initial rates obtained in the presence of nonhydrolyzable ATP analogues and the small molecule inhibitor of ROCK, Y-27632, against the reciprocals of the peptide concentrations yielded parallel lines (uncompetitive pattern). This pattern is indicative of an ordered binding mechanism, with the peptide adding first. The staurosporine analogue K252a, however, gave a noncompetitive pattern. When a pulse of (33)P-gamma-ATP mixed with ROCK was chased with excess unlabeled ATP and peptide, 0.66 enzyme equivalent of (33)P-phosphate was incorporated into the product in the first turnover. The presence of ATPase activity coupled with the isotope partition data is a clear evidence for the existence of a viable [E-ATP] complex in the kinase reaction and implicates a random binding mechanism. The k(cat)/K(m) parameters were fully sensitive to viscosity (viscosity effects of 1.4 +/- 0.2 and 0.9 +/- 0.3 for ATP and peptide 5, respectively), and therefore, the barriers to dissociation of either substrate are higher than the barrier for the phosphoryl transfer step. As a consequence, not all the binding steps are at fast equilibrium. The observation of a burst in presteady-state kinetics (k(b) = 10.2 +/- 2.1 s(-)(1)) and the viscosity effect on k(cat) of 1.3 +/- 0.2 characterize the phosphoryl transfer step to be fast and the release of product and/or the enzyme isomerization step accompanying it as rate-limiting at V(max) conditions. From the multiple kinetic studies, most of the rate constants for the individual steps were either evaluated or estimated.