This article outlines the benefits of using 'Design of Experiments' (DoE) optimisation during the development of new synthetic methodology. A particularly important factor in the development of new chemical reactions is the choice of solvent which can often drastically alter the efficiency and selectivity of a process. Whilst solvent optimisation is usually done in a non-systematic way based upon a chemist's intuition and previous laboratory experience, we illustrate how optimisation of the solvent for a reaction can be carried out by using a 'map of solvent space' in a DoE optimisation. A new solvent map has been developed specifically for optimisation of new chemical reactions using principle component analysis (PCA) incorporating 136 solvents with a wide range of properties. The new solvent map has been used to identify safer alternatives to toxic/hazardous solvents, and also in the optimisation of an S(N)Ar reaction.
Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition.Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues.
Aurora A is a cell cycle protein kinase implicated in multiple human cancers, and several Aurora Aspecific kinase inhibitors have progressed into clinical trials. In this study, we report structural and cellular analysis of a novel biochemical mode of Aurora A inhibition, which occurs through reversible covalent interaction with the universal metabolic integrator coenzyme A (CoA). Mechanistically, the CoA 3'-phospho ADP moiety interacts with Thr 217, an Aurora A selectivity filter, which permits the formation of an unprecedented covalent bond with Cys 290 in the kinase activation segment, lying some 15 Å away. CoA modification (CoAlation) of endogenous Aurora A is rapidly induced by oxidative stresses at Cys 290 in human cells, and microinjection of CoA into mouse embryos perturbs meitoic spindle formation and chromosome alignment. Aurora A regulation by CoA reveals how targeting of Aurora A might be accomplished in the future by development of a 'doubleanchored' covalent inhibitor.
The Aurora kinases are a family of highly homologous serine/threonine kinases, 1,2,3 which have welldocumented roles in the control of meiosis, mitosis and cell division. 4 There are three distinct subfamilies, Aurora A, Aurora B and Aurora C, which vary in their function and subcellular location.Since the first observation of the overexpression of Aurora kinases in cancer cell lines by Bischoff et al., 5 they have been regarded as promising drug targets for cancer chemotherapy. Aurora A overexpression is frequently detected in leukemia, breast, prostate and colon cancers, 2,5,6 with a lower overall survival rate seen in colorectal cancer patients with increased Aurora A levels. 7 This has seen a number of Aurora kinase inhibitors progressing through different stages of clinical trials. 3,8,9,10,11 The Aurora kinases are highly homologous, with a highly conserved C-terminal catalytic domain, a short N-terminal domain which varies in size, and an activation loop (Asp274-Glu299) which is conserved between the three family members. 3 The kinase activity of Aurora A is dependent on autophosphorylation of Thr288 (and possibly Thr287), found in the activation loop. In addition, binding of Aurora A to microtubule-associated proteins, in particular TPX2, alters the structure of Aurora A by stabilisation of the activation loop, allowing ATP to bind and driving the kinase into the optimal conformation for catalysis. 1,3,12 Inhibition of Aurora A results in inhibition of this phosphorylation, giving delayed entry into mitosis and the failure of the centrosomes to assemble bipolar spindles, resulting in aneuploidy and mitotic arrest. 13 Different conformational states of Aurora A can be induced or stabilised by different small molecule inhibitors, 14,15,16,17 and in particular the conformation of the activation loop modulates the interaction of Aurora A with its binding partners. 18 Coenzyme A (CoA) is an essential and ubiquitous cofactor made from vitamin B5 (pantothenate), ATP, and cysteine. CoA and its thioester derivatives (Acetyl CoA, Malonyl CoA, HMG CoA among others) are involved in diverse anabolic and catabolic pathways, biosynthesis of neurotransmitters and the regulation of gene expression. 19,20 Dysregulation of CoA biosynthesis or CoA thioester homoeostasis is associated with various human pathologies, including neurodegeneration, cancer and metabolic disorders. [21][22][23][24] Recent studies have uncovered a novel function of CoA in redox regulation, involving covalent modification of cellular proteins by disulphide bond formation, termed CoAlation, in cellular response to oxidative and metabolic stress. 25,26 Protein CoAlation is a widespread and reversible posttranslational modification, which occurs in single-cell and multicellular organisms, and modulates catalytic activity, regulatory interactions, subcellular localization and the stability of modified proteins. [27][28][29][30] Recently, we have reported that CoA is a specific ATP-competitive Aurora A inhibitor in vitro. 31 Using a combination of bioc...
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