PAS domains sense oxygen, redox potential and light, and are implicated in behaviour, circadian rhythmicity, development and metabolic regulation. Although PAS domains are widespread in archaea, bacteria and eukaryota, the mechanism of signal transduction has been elucidated only for the bacterial photo sensor PYP and oxygen sensor FixL. We investigated the signalling mechanism in the PAS domain of Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Forty‐two residues in Aer were substituted using cysteine‐replacement mutagenesis. Eight mutations resulted in a null phenotype for aerotaxis, the behavioural response to oxygen. Four of them also led to the loss of the non‐covalently bound FAD cofactor. Three mutant Aer proteins, N34C, F66C and N85C, transmitted a constant signal‐on bias. One mutation, Y111C, inverted signalling by the transducer so that positive stimuli produced negative signals and vice versa. Residues critical for signalling were mapped onto a three‐dimensional model of the Aer PAS domain, and an FAD‐binding site and ‘active site’ for signal transduction are proposed.

T he phosphoinositide-3-kinase (PI3K) family of lipid kinases is involved in a diverse set of cellular functions, including cell growth, proliferation, motility, differentiation, glucose transport, survival, intracellular trafficking, and membrane ruffling. 1 PI3K's can be categorized into class I, II, or III, depending on their subunit structure, regulation, and substrate selectivity. 2 Class IA PI3K's are activated by receptor tyrosine kinases and consist of a regulatory subunit (p85) and a catalytic subunit (p110). There are three catalytic isoforms: p110R, β, and δ. A single class IB PI3K, activated by GPCRs, consists of only one member: a p110γ catalytic subunit and a p101 regulatory subunit. The primary in vivo substrate of the class I PI3K's is phosphatidylinositol (4,5) diphosphate (PtdIns(4,5)P2), which upon phosphorylation at the 3-position of the inositol ring to form phosphatidylinositol triphosphate (3,4,5)P3 (PIP3) serves as a second messenger by activating a series of downstream effectors that mediate the cellular functions mentioned above. The PI3K isoforms have different distributions and share similar cellular functions, which are context dependent. In particular, p110R pathway deregulation has been demonstrated in ovarian, breast, colon, and brain cancers. 3,4 Inhibitors of PI3KR represent an intriguing therapeutic modality for these indications, and as such, there is much interest in generating suitable molecules to test this hypothesis in the clinic. 5À10 We have previously reported on a series of 6-hydroxyphenyl-2-morpholino pyrimidines, 11 as potent pan class I PI3K inhibitors that exhibit high selectivity toward protein kinases (serine/threonine and tyrosine kinases). We have further reported on non-phenol containing heterocyclic, morpholino pyrimidines 12 such as compound 1 which demonstrate in vivo PI3K pathway modulation and modest tumor growth inhibition. Described herein are our efforts to identify potent morpholino pyrimidinyl inhibitors of class I PI3Ks that exhibit potency and pharmacokinetic properties which allow for maximal pathway modulation in vivo and have druglike properties suitable for clinical development. These efforts culminated in the identification of 15, NVP-BKM120.Aminopyrimidine 1 and analogues such as 3 (Figure 1) exhibit low or sub-nanomolar biochemical potency and sub-micromolar cellular potency against PI3KR. Even with high rodent CL values, such analogues can demonstrate PI3K pathway modulation in mouse xenograft models. 12 During our exploration of the C 6 position, it was noted that C 6 aminopyridine analogue 4, while being less potent than 3 against PI3KR (>10Â potency loss), exhibited a markedly reduced (>9Â) rat CL value, increased %F, and increased oral AUC. Thus, superior pharmacokinetic properties were achievable within this scaffold and the challenge remaining was to retain this kind of pharmacokinetic profile while optimizing all the other attributes (potency, solubility, permeability, safety) necessary for advancement. To address this challenge, ...

BackgroundAlzheimer’s disease (AD) is the leading cause of dementia affecting greater than 26 million people worldwide. Although cerebrospinal fluid (CSF) levels of Aβ42, tau, and p-tau181 are well established as diagnostic biomarkers of AD, there is a need for additional CSF biomarkers of neuronal function that continue to change during disease progression and could be used as pharmacodynamic measures in clinical trials. Multiple proteomic discovery experiments have reported a range of CSF biomarkers that differ between AD and control subjects. These potential biomarkers represent multiple aspects of the disease pathology. The performance of these markers has not been compared with each other, and their performance has not been evaluated longitudinally.ResultsWe developed a targeted-proteomic, multiple reaction monitoring (MRM) assay for the absolute quantitation of 39 peptides corresponding to 30 proteins. We evaluated the candidate biomarkers in longitudinal CSF samples collected from aged, cognitively-normal control (n = 10), MCI (n = 5), and AD (n = 45) individuals (age > 60 years). We evaluated each biomarker for diagnostic sensitivity, longitudinal consistency, and compared with CSF Aβ42, tau, and p-tau181. Four of 28 quantifiable CSF proteins were significantly different between aged, cognitively-normal controls and AD subjects including chitinase-3-like protein 1, reproducing published results. Four CSF markers demonstrated significant longitudinal change in AD: Amyloid precursor protein, Neuronal pentraxin receptor, NrCAM and Chromogranin A. Robust correlations were observed within some subgroups of proteins including the potential disease progression markers.ConclusionUsing a targeted proteomics approach, we confirmed previous findings for a subset of markers, defined longitudinal performance of our panel of markers, and established a flexible proteomics method for robust multiplexed analyses.

Receptor tyrosine kinases (RTK), such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (KIT), and fms-like tyrosine kinase 3 (FLT3), are expressed in malignant tissues and act in concert, playing diverse and major roles in angiogenesis, tumor growth, and metastasis. With

Sunitinib exhibited advantageous pharmacokinetic and dispositional properties in non-clinical species, translating into favorable properties in humans.