Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.     

The development of benzimidazoles as selective rho kinase inhibitors

Rho Kinase (ROCK) is a serine/threonine kinase whose inhibition could prove beneficial in numerous therapeutic areas. We have developed a promising class of ATP-competitive inhibitors based upon a benzimidazole scaffold, which show excellent potency toward ROCK (IC₅₀ <10 nM). This report details the optimization of selectivity for ROCK over other related kinases such as Protein kinase A (PKA).     

Aromatase inhibitors--gene discovery

Microarray analysis of tumour RNA is an extremely powerful tool which allows global gene expression to be measured. When used in combination with neoadjuvant treatment protocols in which therapy is given with the primary tumour within the breast, sequential biopsies may be analysed and results correlated with clinical and pathological response. In the present study, a neoadjuvant protocol has been used, administering the third generation inhibitor, letrozole, for 3 months and subjecting RNA extracted from biopsies taken before and after 10–14 days of treatment to microarray analysis. The objectives were to discover: (i) genes that change with estrogen deprivation (the only known biological effect of letrozole is to inhibit aromatase activity and reduce endogenous estrogens in postmenopausal women) and (ii) genes whose basal, on treatment or change in expression differ between tumours which are either responsive or resistant to treatment (so that predictive indices of response/resistance may be developed).

Early changes in gene expression were identified by comparing paired tumour core biopsies taken before and after 14 days treatment in 58 patients using three different approaches based on frequency of changes, magnitude of changes and SAM analysis. All three approaches showed a greater number of genes were down-regulated than up-regulated. Merging of the data produced a total of 143 genes which were subject to gene ontology and cluster analysis. The ontology of the 91 down-regulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, up-regulated genes were associated with organ development and extra-cellular matrix turnover and regulation.

Clinical response was assessable in 52 patients; 37 (71%) tumours were classified as clinical responders (>50% reduction in volume at 3 months). Microarray analysis of pre- and 14-day biopsies identified 291 covariates (84 baselines, 72 14-day and 135 changes) highly predictive of response status. A similarity matrix using the covariates showed responding tumours have a similar genetic profile which was dissimilar to non-responding cancers whereas non-responsive cases were distinctive from each other. Changed genes predicting for response showed no concordance with those changed significantly by treatment in the overall group.     

Myeloperoxidase-dependent Lipid Peroxidation Promotes the Oxidative Modification of Cytosolic Proteins in Phagocytic Neutrophils

Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.     

Discovery and optimization of indole and 7-azaindoles as Rho kinase (ROCK) inhibitors (part-II)

Therapeutic interventions with Rho kinase (ROCK) inhibitors may effectively treat several disorders such as hypertension, stroke, cancer, and glaucoma. Herein we disclose the optimization and biological evaluation of potent novel ROCK inhibitors based on substituted indole and 7-azaindole core scaffolds. Substitutions on the indole C3 position and on the indole NH and/or amide NH positions all yielded potent and selective ROCK inhibitors (25, 42, and 50). Improvement of aqueous solubility and tailoring of in vitro and in vivo DMPK properties could be achieved through these substitutions.     

Synthesis and biological evaluation of 4-quinazolinones as Rho kinase inhibitors

Rho kinase (ROCK) is an attractive therapeutic target for various diseases including glaucoma, hypertension, and spinal cord injury. Herein, we report the development of a series of ROCK-II inhibitors based on 4-quinazolinone and quinazoline scaffolds. SAR studies at three positions of the quinazoline core led to the identification of analogs with high potency against ROCK-II and good selectivity over protein kinase A (PKA).