Using a mammalian cell cycle simulation to interpret differential kinase inhibition in anti-tumour pharmaceutical development.

Systems biology needs to show practical relevance to commercial biological challenges such as those of pharmaceutical development. The aim of this work is to design and validate some applications in anti-cancer therapeutic development. The test system was a group of novel cyclin-dependent kinase (CDK) inhibitors synthesised by Cyclacel Ltd. The measured in vitro IC50s of each compound were used as input data to a proprietary cell cycle model developed by Physiomics plc. The model was able to predict over three orders of magnitude the cytotoxicity of each compound without model adaptation to specific cancer cell types. This pattern matched the experimentally determined data. One class of compounds was predicted to cause an increase of the cell cycle length with a non-linear dose-response curve. Further work will use apoptosis and DNA replication simulations to look at overall cell effects.

Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics.

We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions. The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH. In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH. A stoichiometric conversion of precursors into threonine was observed. We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell. The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions. A response to threonine is only observed in a narrow concentration range from 0.23 to 2 mM.

An integrated study of threonine-pathway enzyme kinetics in Escherichia coli.

We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions.

Control of the threonine-synthesis pathway in Escherichia coli: a theoretical and experimental approach.

A computer simulation of the threonine-synthesis pathway in Escherichia coli Tir-8 has been developed based on our previous measurements of the kinetics of the pathway enzymes under near-physiological conditions. The model successfully simulates the main features of the time courses of threonine synthesis previously observed in a cell-free extract without alteration of the experimentally determined parameters, although improved quantitative fits can be obtained with small parameter adjustments. At the concentrations of enzymes, precursors and products present in cells, the model predicts a threonine-synthesis flux close to that required to support cell growth. Furthermore, the first two enzymes operate close to equilibrium, providing an example of a near-equilibrium feedback-inhibited enzyme. The predicted flux control coefficients of the pathway enzymes under physiological conditions show that the control of flux is shared between the first three enzymes: aspartate kinase, aspartate semialdehyde dehydrogenase and homoserine dehydrogenase, with no single activity dominating the control. The response of the model to the external metabolites shows that the sharing of control between the three enzymes holds across a wide range of conditions, but that the pathway flux is sensitive to the aspartate concentration. When the model was embedded in a larger model to simulate the variable demands for threonine at different growth rates, it showed the accumulation of free threonine that is typical of the Tir-8 strain at low growth rates. At low growth rates, the control of threonine flux remains largely with the pathway enzymes. As an example of the predictive power of the model, we studied the consequences of over-expressing different enzymes in the pathway.

Control of threonine pathway in E. coli. Application to biotechnologies.

Threonine is an essential amino acid for mammals and birds and an adequate supply is necessary for growth and maintenance. Its production has become the aim of metabolic bioengineering and genetic manipulations. We propose in this paper a rational approach for increasing threonine production in an E. coli strain based on metabolic control theory. We have derived a way to measure the control coefficients of threonine pathway in vivo. The method consists in modelling the results of presteady-state experiments. The in vivo concentrations and activities of the enzymes can then be measured and introduced into the model, so that the in vivo steady-state of the pathway can be evaluated. With such a model it is possible to calculate the theoretical values of the control coefficients of the threonine synthesis flux in vivo.