Biochemical and Biophysical Approaches to Protein Ligand and Enzyme Inhibitor Discovery

Abstract

At any given point in time, a myriad of reactions occurs in a living cell. These reactions often serve a specific purpose and are more often than not, co-ordinated by proteins and enzymes. Proteins and enzymes play pivotal roles in cellular metabolism and therefore they are essential in the sustenance of the living cell. The function of proteins and enzymes are often modulated by their interacting partners, which are termed ligand. The application of ligands to modulate the activity of disease-causing enzymes are fundamental to modern medicinal chemistry. Ligands are also increasing applied in agricultural and food science to regulate enzyme systems in plants and microorganisms. The work in this thesis aims at studying protein-ligand interactions using biophysical assays. Three different enzyme systems were investigated. These are methanogenic seryl-tRNA synthetase (SerRS), which has applications in agricultural and food science, and Mycobacterium tuberculosis isocitrate lyase (ICL) and human molecular chaperone heat shock protein 90 (Hsp-90), which are current inhibition targets for the development of new therapeutical interventions against tuberculosis and cancers respectively. Chapter 2 is a detailed draft review article on the biotechnological advancements that exist in the mitigation of rumen methane production. The implications of rumen methane emissions on the environment were first reviewed. This is followed by a survey of the archaeal communities that are found in farmed ruminant animals in New Zealand. Finally, the various approaches that are currently being developed to reduce methane production was discussed. The discussion focussed on inhibitors against methanogen enzymes. In particularly, two approaches were discussed in detail. These included inhibitors that target the enzymes that are directly involved in methane production, such as methyl-coenzyme M reductase, and enzymes that are important to sustain methanogenic archaea such as hydroxymethylglutarly-coenzyme A reductase. This review set the scene for the work that will be discussed in Chapter 3. Chapter 3 focussed on the biochemical characterization and inhibitor discovery against recombinant Methanosarcina barkeri SerRS. M. barkeri SerRS is a crucial enzyme in the protein synthesis pathways in M. barkeri. It is structurally unique to SerRS that are found in other microorganisms such as Escherichia coli. As such, M. barkeri SerRS could be an ideal inhibition target to reduce methane production by rumen methanogens. The initial part of this chapter focussed on the production and characterization of recombinant M. barkeri SerRS. This is followed by the application of a combined virtual high-throughput screening and biophysical assay approach to identify ligands of M. barkeri SerRS. Initial results yielded eight structurally diverse compounds that bind to the enzyme in micromolar affinity. Finally, the development of kinetic assays to study the activity of M. barkeri SerRS was investigated. However, this was unsuccessful in part due to constraints including protein contamination and the availability of M. barkeri tRNASer. Nonetheless, the results obtained in this chapter are an excellent starting point for the further development and optimisation of novel inhibitors against M. barkeri SerRS. Chapter 4 contained two parts. The first part focussed on the optimisation of the virtual screening strategy to reduce wastage by using human Hsp-90 as a model system. One of the biggest drawbacks of virtual high-throughput screening is that many virtual hits lack aqueous solubility and therefore their ability to interact with the target protein cannot be experimentally verified. In this work, chemical descriptors that are linked to the Lipinski Rule of Five were applied to prefilter compounds with drug-like properties. The rationale is that these compounds may have higher aqueous solubility and more optimum binding properties. Two screens, one using the prefiltered library (focussed library) and the other using the non-optimised library, were conducted. The results showed that the chemical descriptors were not able to improve the aqueous solubility of the virtual hits. However, this method led to compounds that are more specific and bind to Hsp-90 with higher affinity. In the second part of this chapter, experiments were conducted to verify the binding of a range of compounds containing the quinazoline moiety against Hsp-90. Although the tested compounds were limited by aqueous solubility, two compounds were found to be micromolar binders to Hsp-90. However, the mapping of their binding pocket using protein nuclear magnetic resonance spectroscopy were unsuccessful due to their limited solubility and relatively weak affinity. Chapter 5 focussed on a thorough comparison of activity and binding assays that were aimed towards the development of inhibitors against M. tuberculosis ICLs, which are crucial enzymes for the survival of the bacterium during infection. First, optimizations of the various assays were first conducted. These included activity assays using 1H NMR spectroscopy that directly measures the activity of ICLs as well as indirect assay that require auxiliary chemicals or enzymes for readout. In addition, binding studies employing the thermal shift assay and intrinsic tryptophan fluorescence spectroscopy were also conducted. This chapter highlights the advantages and limitations of the assays that were tested; thus, it provides a useful guide for the development of ICL inhibitors.