Development of autotaxin inhibitors: A series of tetrazole cinnamides.

A series of inhibitors of Autotaxin (ATX) have been developed from a high throughput screening hit, 1a, which shows an alternative binding mode to known catalytic site inhibitors. Selectivity over the hERG channel and microsomal clearance were dependent on the lipophilicity of the compounds, and this was optimised by reduction of clogD whilst maintaining high affinity ATX inhibition. Compound 15a shows good oral exposure, and concentration dependent inhibition of formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic (PK/PD) experiments.

Development of autotaxin inhibitors: A series of zinc binding triazoles.

A series of inhibitors of Autotaxin (ATX) has been developed using the binding mode of known inhibitor, PF-8380, as a template. Replacement of the benzoxazolone with a triazole zinc-binding motif reduced crystallinity and improved solubility relative to PF-8380. Modification of the linker region removed hERG activity and led to compound 12 - a selective, high affinity, orally-bioavailable inhibitor of ATX. Compound 12 concentration-dependently inhibits autotaxin and formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic experiments.

Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds.

Lipid droplet formation and subsequent steatosis (the abnormal retention of lipids within a cell) has been reported to contribute to hepatotoxicity and is an adverse effect of many pharmacological agents including the antiepileptic drug valproic acid (VPA). In this study, we have developed a simple model system (Dictyostelium discoideum) to investigate the effects of VPA and related compounds in lipid droplet formation. In mammalian hepatocytes, VPA increases lipid droplet accumulation over a 24-hour period, giving rise to liver cell damage, and we show a similar effect in Dictyostelium following 30 minutes of VPA treatment. Using (3)H-labelled polyunsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown that VPA treatment of Dictyostelium gives rise to an increased accumulation of both types of fatty acids in phosphatidylcholine, phosphatidylethanolamine and non-polar lipids in this time period, with a similar trend observed in human hepatocytes (Huh7 cells) labelled with [(3)H]arachidonic acid. In addition, pharmacological inhibition of ß-oxidation in Dictyostelium phenocopies fatty acid accumulation, in agreement with data reported in mammalian systems. Using Dictyostelium, we then screened a range of VPA-related compounds to identify those with high and low lipid-accumulation potential, and validated these activities for effects on lipid droplet formation by using human hepatocytes. Structure-activity relationships for these VPA-related compounds suggest that lipid accumulation is independent of VPA-catalysed teratogenicity and inositol depletion. These results suggest that Dictyostelium could provide both a novel model system for the analysis of lipid droplet formation in human hepatocytes and a rapid method for identifying VPA-related compounds that show liver toxicology.

The chemoselective one-step alkylation and isolation of thiophosphorylated cdk2 substrates in the presence of native cysteine.

The elucidation of signalling pathways relies heavily upon the identification of protein kinase substrates. Recent investigations have demonstrated the efficacy of chemical genetics using ATP analogues and modified protein kinases for specific substrate labelling. Here we combine N(6) -(cyclohexyl)ATPγS with an analogue-sensitive cdk2 variant to thiophosphorylate its substrates and demonstrate a pH-dependent, chemoselective, one-step alkylation to facilitate the detection or isolation of thiophosphorylated peptides.

Cyclin D1 and cyclin D3 show divergent responses to distinct mitogenic stimulation.

D-type cyclins predominantly regulate progression through the cell cycle by their interactions with cyclin-dependent kinases (cdks). Here, we show that stimulating mitogenesis of Swiss 3T3 cells with phorbol esters or forskolin can induce divergent responses in the expression levels, localization and activation state of cyclin D1 and cyclin D3. Phorbol ester-mediated protein kinase C stimulation induces S phase entry which is dependent on MAPK activation and increases the levels and activation of cyclin D1, whereas forskolin-mediated cAMP-dependent protein kinase A stimulation induces mitogenesis that is independent of MAPK, but dependent upon mTor and specifically increases the level and activation of cyclin D3. These findings uncover additional levels of complexity in the regulation of the cell cycle at the level of the D-type cyclins and thus may have important therapeutic implications in cancers where specific D-cyclins are overexpressed.

Synthesis and O-phosphorylation of 3,3,4,4-tetrafluoroaryl-C-nucleoside analogues.

Enantioenriched tetrafluorinated aryl-C-nucleosides were synthesised in four steps from 1-benzyloxy-4-bromo-3,3,4,4-tetrafluorobutan-2-ol. The presence of the tetrafluorinated ethylene group is compatible with O-phosphorylation of the primary alcohol, as demonstrated by the successful preparation of the tetrafluorinated naphthyl-C-nucleotide.

Dose-dependent changes in cyclin D1 in response to 4-nitroquinoline 1-oxide-induced DNA damage.

Cells respond to DNA damage by either repairing the damage or committing to a death or senescence pathway, dependent on the level of damage sustained. In this study, we show that the protein levels of cyclin D1 and the CDK inhibitor, p21(CIP1), respond in a dose-dependent manner to the DNA damaging agent, 4-nitroquinoline 1-oxide (4NQO). Cyclin D1 responses were independent of p53 and resulted in a partial loss of Retinoblastoma protein phosphorylation. The differential responses of cyclin D1 and p21(CIP1) were associated with distinct cellular responses: in low dose treatments the cells recovered after a lag period whilst at medium and high doses, the cells died through seemingly distinct mechanisms. Our data suggest that the balance between cyclin D1 and p21(CIP1) following exposure to DNA damage may play a key role in determining the subsequent cellular responses.

Synthesis and reactivity of novel gamma-phosphate modified ATP analogues.

We hereby present a simple yet novel chemical synthesis of a family of gamma-modified ATPs bearing functional groups on the gamma-phosphate that are amenable to further derivatization by highly selective chemical manipulations (e.g., click chemistry, Staudinger ligations). A preliminary screen of these compounds as phosphate donors with a typical wild type protein kinase (cdk2) and one of its known substrates p27(kip1) is also presented.

A quantitative comparison of wild-type and gatekeeper mutant cdk2 for chemical genetic studies with ATP analogues.

Chemical genetic studies with enlarged ATP binding sites and unnatural ATP analogues have been applied to protein kinases for characterisation and substrate identification. Although this system is becoming widely used, there are limited data available about the kinetic profile of the modified system. Here we describe a detailed comparison of the wild-type cdk2 and the mutant gatekeeper kinase to assess the relative efficiencies of these kinases with ATP and unnatural ATP analogues. Our data demonstrate that mutation of the kinase alters neither the substrate specificity nor the phosphorylation site specificity. We find comparable K(M)/V(max) values for mutant cdk2 and wild-type kinase. Furthermore, F80G cdk2 is efficiently able to compensate for a defective cdk in a biological setting.

Exploring the roles of protein kinases using chemical genetics.

The protein kinase superfamily is one of the most important families of enzymes in molecular biology. Protein kinases typically catalyze the transfer of the γ-phosphate from ATP to a protein substrate (a highly ubiquitous cellular reaction), thereby controlling key areas of cell regulation. Deregulation of protein kinases is known to contribute to many human diseases, and selective inhibitors of protein kinases are a major area of interest in medicinal chemistry. However, a detailed understanding of many kinase pathways is currently lacking. Before we can effectively design medicinally relevant selective kinase inhibitors, it is necessary to understand the role played by a given kinase in specific signal-transduction cascades and to decipher its protein targets. Here, we describe recent advances towards dissecting protein kinase function through the use of chemical genetics.

Opposing roles for caspase and calpain death proteases in L-glutamate-induced oxidative neurotoxicity.

Oxidative glutamate toxicity in HT22 murine hippocampal cells is a model for neuronal death by oxidative stress. We have investigated the role of proteases in HT22 cell oxidative glutamate toxicity. L-glutamate-induced toxicity was characterized by cell and nuclear shrinkage and chromatin condensation, yet occurred in the absence of either DNA fragmentation or mitochondrial cytochrome c release. Pretreatment with the selective caspase inhibitors either benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (pan-caspase), N-acetyl-Leu-Glu-His-Asp-aldehyde (caspase 9) or N-acetyl-Ile-Glu-Thr-Asp-aldehyde (caspase 8), significantly increased L-glutamate-induced cell death with a corresponding increase in observed nuclear shrinkage and chromatin condensation. This enhancement of glutamate toxicity correlated with an increase in L-glutamate-dependent production of reactive oxygen species (ROS) as a result of caspase inhibition. Pretreating the cells with N-acetyl-L-cysteine prevented ROS production, cell shrinkage and cell death from L-glutamate as well as that associated with the presence of the pan-caspase inhibitor. In contrast, the caspase-3/-7 inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde was without significant effect. However, pretreating the cells with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO, but not the cathepsin B inhibitor CA-074, prevented cell death. The cytotoxic role of calpains was confirmed further by: 1) cytotoxic dependency on intracellular Ca(2+) increase, 2) increased cleavage of the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC and 3) immunoblot detection of the calpain-selective 145 kDa alpha-fodrin cleavage fragment. We conclude that oxidative L-glutamate toxicity in HT22 cells is mediated via calpain activation, whereas inhibition of caspases-8 and -9 may exacerbate L-glutamate-induced oxidative neuronal damage through increased oxidative stress.

Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68.

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.

Using chemical genetics and ATP analogues to dissect protein kinase function.

Protein kinases catalyze the transfer of the gamma-phosphate of ATP to a protein substrate and thereby profoundly alter the properties of the phosphorylated protein. The identification of the substrates of protein kinases has proven to be a very difficult task because of the multitude of structurally related protein kinases present in cells, their apparent redundancy of function, and the lack of absolute specificity of small-molecule inhibitors. Here, we review approaches that utilize chemical genetics to determine the functions and substrates of protein kinases, focusing on the design of ATP analogues and protein kinase binding site mutants.

Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct.

A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.