Atomistic and Systems-level Modeling of Phosphate Catalysis

Phosphate processing enzymes are crucial to the majority of biochemical processes that all living cells depend on, including signal transduction, gene regulation, metabolism and energy transfer. The active sites of these enzymes are remarkably similar, in most cases requiring the presence of a catalytic Mg2+ ion bound to the phosphate groups, as the biological cofactor. However, there are distinct, specific differences in the coordination geometry of these enzymes that contain hidden information about the corresponding function.

To gain insight towards constructing a general, unifying theory of how phosphate processing enzymes modulate the rates of their catalytic reactions, we propose to study two important systems. We will study the RAF kinase catalytic reaction. RAF kinases are part of the ERK signalling pathway, which is aberrantly activated in >30% of all cancers, with RAS and RAF being the main oncogenic factors.

We will also study the catalytic reaction of a bacterial ligase, Ddl, from M. tuberculosis, the causative agent of TBC. Ddl is an essential enzyme in the biosynthesis of bacterial cell wall. It is one of the validated drug targets of the antibiotic drug Seromycine, and a major drug target against TBC. Seromycine is used as a second line of treatment, for multiple drug-resistant and extensively drug-resistant strains of M. tuberculosis.

Using our novel computational methods that can provide accurate free energies and kinetic information about the dynamics of the systems, we will identify coupled proton transfer steps that occur together with the phosphate cleavage reactions. We will use our calculated structures and mechanism to develop and test novel inhibitor design strategies.

We will carry out computational studies of BRAF kinase and Ddl catalytic reactions using novel enhanced sampling algorithms, building on our recently developed accurate free energy calculation method that coupled Hamiltonian replica exchange with the finite temperature string method. We will continue to use MD and QM/MM calculations to study phosphate cleavage and transfer reactions that occur together with rate determining proton transfer processes. We will analyse the results from biased and unbiased dynamical trajectories using our newly developed Markov chain-based kinetic analysis method, DHAM. We will focus on the role of Mg2+ ions in the active site, and identify general features of this essential Mg2+ cofactor - using a systems-level approach - to better understand why magnesium is ubiquitously involved in the catalysis of phosphate cleavage and transfer catalytic reactions. We will use our calculated structures and mechanism to develop and test novel inhibitor design strategies.

Academia
Phosphate processing enzymes are essential in all biological processes, therefore our research is potentially relevant to most biological research fields that study processes related to phosphate cleavage and transfer. For example, research related to regulation and signal transduction by kinases, gene expression and replication by polymerases or topoisomerases, ATPase function in molecular motors such as ATP synthase, myosin, transporters, and other activated mechanical, structural or chemical processes occurring in the cells that requires the energy transfer from ATP hydrolysis.
The proposed project is driven by computational molecular modelling-based data and methods, and it is embedded in an inter-disciplinary research environment. Our research results on applications and developments of molecular modelling methods (e.g., sampling enhanced methods for rare events, statistical analysis of stochastic simulation results to obtain free energies and molecular kinetics) are beneficial for a wide range of research fields related to atomistic computational modelling in general. This includes biomolecular simulations, as well as material science modelling, biotechnology, and synthetic biology modeling.

Public Sector, Business, Industry
On long term, health-related public sectors will benefit from basic research on structure and function of phosphate processing enzymes. Our study may be inspirational to a large number of projects targeting phosphate-processing enzymes that are relevant to many diseases. Phosphate processing enzymes are validated targets of a large number of drugs used in current clinical practices treating a wide range of diseases. These include reverse transcriptase and integrase inhibitors used against HIV and hepatitis B, proton pump inhibitors used in gastric diseases, kinase and topoisomerase inhibitors used in chemotherapy to treat cancers.
In particular, BRAF inhibitor drugs present recent examples for targeted cancer therapy: (http://www.cancer.gov/cancertopics/treatment/types/targeted-therapies/targeted-therapies-fact-sheet).
Another example is related to basic research at the US National Cancer Institute, which supports Federally Funded Research and Development Center (FFRDC) devoted to biomedical research. Within these efforts, the "Ras initiative" is announced as a major initiative and research priority to "improving treatment, diagnosis, and prevention of the many human cancers driven by mutant RAS genes" by studies of mutant Ras and downstream signalling proteins (including BRAF): http://www.cancer.gov/researchandfunding/priorities/ras. Both Ras (GTPases) and downstream kinases are examples of enzymes processing NTP hydrolysis and transfer, the subject of this proposal.
Our second example, Ddl is a validated target in combating antimicrobial resistance, and Ddl inhibitors are used as second line of treatment drugs against tuberculosis. For example, mutation in the chromosomal Ddl gene, encoding a cytoplasm enzyme is directly involved in glycopeptides resistance.
Our basic research results are also relevant for charities linked to these diseases e.g., Cancer Research UK. In addition to drug design, insights related to controlling enzyme activity is also relevant for biotechnology industries, e.g., businesses developing industrial enzymes such as Novozymes.

General Public, Education
The general public, high school and university students will benefit from new basic research developments in general, by public lectures in the UK and world-wide (e.g., via the Alchemy Today seminar series of the Eotvos University, where I've been recently invited as a speaker), or by the Open Days at King's. My lab also hosted 6 high school students to date in the past 2 years, who were introduced to on-going research in my lab via the In2Science and Nuffield Research Placement programs.