Research

Miha Homsak

PLP-dependent chemoproteomics for identification and characterisation

Miha Homsak - 3rd year PhD

Abstract

Background
Tuberculosis, an infectious disease caused by M. tuberculosis, accounted for 10 million new infections, almost 2 million deaths, and 0.5 million infections with multidrug resistant strain of M. tuberculosis in 2015 alone.1 Especially the latter is a clear signal that novel ways of targeting the bacterium are urgently required.
Pyridoxal-5’-phosphate (PLP; Vitamin B6) is the most versatile cofactor in biology, utilised by 145 different enzymes. Many of those are involved in vital cell processes, including protein and polyamide biosynthesis, and glucose metabolism.2 This makes the PLP-dependent proteome an attractive drug target, which is not fully exploited yet.

M. tuberculosis depends on the correct function of the PLP-dependent enzymes to survive. For example, an important PLP-dependent enzyme in the synthesis of the peptidoglycans, needed to create the Mtb’s cell wall, is alanine racemase. Hence, targeting the PLP-dependent proteome could be very beneficial to developing a novel TB therapeutic.

Research plan
Chemical proteomics is the approach of choice for this project, since it is able to selectively target the active (functional) forms of a particular enzyme class and enables one to study post-translational modifications (PTMs), substrate interactions, enzyme dynamics etc.

The method relies on creating a small molecule – a probe – that is designed to selectively bind to the target enzymes. The probe carries a reactive handle (an alkyne), which makes it possible to attach to it labels after selective protein binding has taken place. TAMRA and Biotin are commonly used for this purpose; they enable visualisation of the probe-enzyme complex on gels, as well as isolation and subsequent analysis of the probe-protein complex by LC-MS/MS.

The aims of the project
- Synthesise a chemical proteomic probe to be used to study PLP-dependent enzymes in M. tuberculosis
- Develop a novel PLP-dependent proteome assay and diagnostic tool to perform various studies:
o Activity profiling
o Drug selectivity profiling
o De novo enzyme/substrate discovery
o Biomarker discovery
o Post-translational modification studies
o Substrate/enzyme interactions
o Enzyme dynamics