The discovery of AZD5597, a potent imidazole pyrimidine amide CDK inhibitor suitable for intravenous dosing

The development of a novel series of imidazole pyrimidine amides as cyclin-dependent kinase (CDK) inhibitors is described. Optimisation of inhibitory potency against multiple CDK's (1, 2 and 9) resulted in imidazole pyrimidine amides with potent in vitro anti-proliferative effects against a range of cancer cell lines. Excellent physiochemical properties and large margins against inhibition of CYP isoforms and the hERG ion channel were achieved by modification of lipophilicity and amine basicity. A candidate with disease model activity in human cancer cell line xenografts and with suitable physiochemical and pharmacokinetic profiles for intravenous (i.v.) dosing was selected for further development as AZD5597.     

The second Conceptual Assessment in the Biological Sciences workshop

A second National Science Foundation-sponsored workshop on Conceptual Assessment in Biology was held in January 2008. Reports prepared for the workshop revealed that research groups working in a variety of biological sciences are continuing to develop conceptual assessment instruments for use in the classroom. Discussions at this meeting largely focused on two issues: 1) the utility of the backwards design approach of Wiggins and McTighe (11), in which identification of learning outcomes (determining what to assess) lies at the beginning of course design; and 2) the utility of defining expected learning outcomes as the building of runable mental models (and designing conceptual assessments that would test the correctness of these mental models). A third meeting is being planned that will focus on the processes involved in writing and validating conceptual assessment instruments.     

Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions

Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.     

Frankenswine,or bringing home the bacon: How close are we to clinical trials in xenotransplantation?

Xenotransplantation—specifically from pig into human—could resolve the critical shortage of organs, tissues and cells for clinical transplantation. Genetic engineering techniques in pigs are relatively well-developed and to date have largely been aimed at producing pigs that either (1) express high levels of one or more human complement-regulatory protein(s), such as decay-accelerating factor or membrane cofactor protein, or (2) have deletion of the gene responsible for the expression of the oligosaccharide, Galα1,3Gal (Gal), the major target for human anti-pig antibodies, or (3) have both manipulations. Currently the transplantation of pig organs in adequately-immunosuppressed baboons results in graft function for periods of 2–6 months (auxiliary hearts) and 2–3 months (life-supporting kidneys). Pig islets have maintained normoglycemia in diabetic monkeys for >6 months. The remaining immunologic barriers to successful xenotransplantation are discussed, and brief reviews made of (1) the potential risk of the transmission of an infectious microorganism from pig to patient and possibly to the public at large, (2) the potential physiologic incompatibilities between a pig organ and its human counterpart, (3) the major ethical considerations of clinical xenotransplantation, and (4) the possible alternatives that compete with xenotransplantation in the field of organ or cell replacement, such as mechanical devices, tissue engineering, stem cell biology and organogenesis. Finally, the proximity of clinical trials is discussed. Islet xenotransplantation is already at the stage where clinical trials are actively being considered, but the transplantation of pig organs will probably require further genetic modifications to be made to the organ-source pigs to protect their tissues from the coagulation/anticoagulation dysfunction that plays a significant role in pig graft failure after transplantation in primates.Key words: islets, pancreatic, genetic engineering, organogenesis, pig, xenotransplantation     

Phospho-MAPK as a marker of myogenic satellite cell responsiveness to growth factors

To determine if differential response to growth factor stimuli between subpopulations of satellite cells was due to variation in the levels of activated intracellular signaling proteins, the levels of phospho-MAPK (phospho-ERK 1/2) were determined in clonal populations of turkey (Meleagris gallopavo) satellite cells. Relative levels of phospho-ERK 1/2 between clones were determined by Western blotting of extracts from satellite cells exposed to growth factor stimuli. Initial measurements using serum mitogenic stimuli showed differences in phospho-MAPK levels between the clonal subpopulations, but the responses did not correlate with proliferation rates of the individual clones (P>0.05). IGF-I alone did not increase phospho-MAPK levels compared to unstimulated controls (P>0.05), whereas FGF-2 did increase levels (P<0.05). A synergistic response was seen in satellite cells as well as embryonic myoblasts administered both IGF-I and FGF-2. When administered FGF-2 and IGF-I, 2 of the slow growing satellite cell clones exhibited lowest levels of phospho-MAPK (P<0.05). One of the slow growing clones had levels of phospho-MAPK similar to the three fast growing clones (P>0.05). The results suggest that variation in responsiveness to growth factor stimuli among satellite cell populations within muscles may be due to several different reasons. Some differences in cell responsiveness appear to be due to variation in phospho-MAPK generation.     

Energetics and cooperativity of the hydrogen bonding and anchor interactions that bind peptides to MHC class II protein

The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A(d)-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A(d) MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (<5A).     

Whole-genome sequencing reveals Listeria monocytogenes diversity and allows identification of long-term persistent strains in Brazil

Advances in whole-genome sequencing (WGS) technologies have documented genetic diversity and epidemiology of the major foodborne pathogen Listeria monocytogenes (Lm) in Europe and North America, but data concerning South America are scarce. Here, we examined the population structure and genetic diversity of this major foodborne pathogen collected in Brazil. Based on core genome multilocus sequence typing (cgMLST), isolates from lineages I (n = 22; 63%) and II (n = 13; 37%) were distributed into 10 different sublineages (SLs) and represented 31 new cgMLST types (CTs). The most prevalent SLs were SL9 (n = 9; 26%), SL3 (n = 6; 17%) and SL2 and SL218 (n = 5; 14%). Isolates belonging to CTs L2-SL9-ST9-CT4420 and L1-SL315-ST520-CT4429 were collected 3 and 9 years apart, respectively, revealing long-term persistence of Lm in Brazil. Genetic elements associated with stress survival were present in 60% of isolates (57% SSI-1 and 3% SSI-2). Pathogenic islands were present in 100% (LIPI-1), 43% (LIPI-3) and 6% (LIPI-4) of the isolates. Mutations leading to premature stop codons were detected in the prfA and inlA virulence genes. This study is an important contribution to understanding the genomic diversity and epidemiology of Lm in South America. In addition, the results highlight the importance of using WGS to reveal Lm long-term persistence.     

Migration of turkey muscle satellite cells is enhanced by the syndecan-4 cytoplasmic domain through the activation of RhoA

Syndecan-4 (S4) is a cell membrane-associated heparan sulfate proteoglycan that forms oligomers in muscle satellite cells. The S4 oligomers activate protein kinase Cα (PKCα) through the S4 cytoplasmic domain and may regulate the activation of ras homolog gene family member A (RhoA), a signal transduction molecule down-stream of PKCα which is thought to influence cell migration. However, little is known about the function of the S4 cytoplasmic domain in satellite cell migration and RhoA activation. The objective of the current study was to determine the function of S4 and its cytoplasmic domain in cell migration and RhoA activation. To study the objective, clones of S4 and S4 without the cytoplasmic domain (S4C) were used in overexpression studies, and small interference RNAs targeting S4 or RhoA were used in knockdown studies. Satellite cell migration was increased by S4 overexpression, but decreased by the knockdown or deletion of the S4 cytoplasmic domain. The RhoA protein was activated by the overexpression of S4, but not with the deletion of the S4 cytoplasmic domain. The treatment of Rho activator II or the knockdown of RhoA also modulated satellite cell migration. Finally, co-transfection (S4 overexpression and RhoA knockdown) and rescue (the knockdown of S4 and the treatment with Rho activator II) studies demonstrated that S4-mediated satellite cell migration was regulated through the activation of RhoA. The cytoplasmic domain of S4 is required for cell migration and RhoA activation which will affect muscle fiber formation.