Deregulation of the cell cycle has long been recognized as an essential driver of tumorigenesis, and agents that selectively target key cell cycle components continue to hold promise as potential therapeutics. We have developed AZD5438, a 4-(1-isopropyl-2-methylimidazol-5-yl)-2-(4-methylsulphonylanilino) pyrimidine, as a potent inhibitor of cyclin-dependent kinase (cdk) 1, 2, and 9 (IC 50 , 16, 6, and 20 nmol/L, respectively). In vitro, AZD5438 showed significant antiproliferative activity in human tumor cell lines (IC 50 range, 0.2-1.7 μmol/L), causing inhibition of the phosphorylation of cdk substrates pRb, nucleolin, protein phosphatase 1a, and RNA polymerase II COOH-terminal domain and blocking cell cycling at G 2 -M, S, and G 1 phases. In vivo, when orally administered at either 50 mg/kg twice daily or 75 mg/kg once daily, AZD5438 inhibited human tumor xenograft growth (maximum percentage tumor growth inhibition, range, 38-153; P < 0.05). In vivo, AZD5438 reduced the proportion of actively cycling cells. Further pharmacodynamic analysis of AZD5438-treated SW620 xenografts showed that efficacious doses of AZD5438 (>40% tumor growth inhibition) maintained suppression of biomarkers, such as phospho-pRbSer 249 /Thr 252 , for up to 16 hours following a single oral dose. A comparison of different schedules indicated that chronic daily oral dosing provided optimal cover to ensure antitumor efficacy. These data indicate that broad cdk inhibition may provide an effective method to impair the dysregulated cell cycle that drives tumorigenesis and AZD5438 has the pharmacologic profile that provides an ideal probe to test this premise.
Ghrelin plays a major physiological role in the control of food intake, and inverse agonists of the ghrelin receptor (GHS-R1a) are widely considered to offer utility as antiobesity agents by lowering the set-point for hunger between meals. We identified an acylurea series of ghrelin modulators from high throughput screening and optimized binding affinity through structure-activity relationship studies. Furthermore, we identified specific substructural changes, which switched partial agonist activity to inverse agonist activity, and optimized physicochemical and DMPK properties to afford the non-CNS penetrant inverse agonist 22 (AZ-GHS-22) and the CNS penetrant inverse agonist 38 (AZ-GHS-38). Free feeding efficacy experiments showed that CNS exposure was necessary to obtain reduced food intake in mice, and it was demonstrated using GHS-R1a null and wild-type mice that this effect operates through a mechanism involving GHS-R1a.
An environment-sensitive probe, Nile Red, tagged to a designed model peptide, is used to probe the structural
changes and dynamics of single calmodulin (CaM):peptide complexes. When the Ca2+ concentration changes
from 0 M to 5 mM, the labeled Nile Red dye undergoes an order of magnitude increase in fluorescence
intensity. By encapsulation of single CaM:peptide−Nile Red complexes into a hydrogel, single-molecule
fluorescence detection has been achieved using confocal microcopy. The fluorescence polarization distribution
obtained for these single complexes shows a mean of 0 and a width of 0.17, for a binning time of 1 ms. This
finding shows that on the 10−100-μs time scale the CaM:peptide complex is tumbling within the hydrogel
matrix. The single-molecule spectral distribution provides the scale of the heterogeneity of the polarity sensed
by the probe. The calcium concentration dependency of the single-molecule-fluorescence lifetime distributions
and photon-arrival-time (PAT) trajectories of the CaM:peptide−Nile Red complexes were also obtained. The
mean and variance of the Nile Red fluorescence decay rate increase 40% and 180%, respectively, as the Ca2+
concentration approaches the titration midpoint of 2 μM. These changes are considerably greater than would
be expected if the chromophore was in a homogeneous static environment, or in a heterogeneous environment
that was exchanging faster than a time scale of 1 s to 10's of seconds. Thus, PAT analysis appears to be
uniquely well suited to study the dynamics of the peptide dissociation from the CaM:peptide complex in the
presence of [Ca2+] and transitions within heterogeneous populations on the microsecond−second time scale.
For the discovery of new candidate molecules in the pharmaceutical industry, library synthesis is a critical step, in which library size, diversity, and time to synthesise are fundamental. In this...
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