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Using chemical probes to study the host-pathogen proteome

Timeline

July 2017 – July 2021

Group and collaboration

prof. dr. Mario van der Stelt and prof. dr. Tom Ottenhoff – Leiden University
PhD student: Alexander Bakker

Project description

Bacterial strains are developing resistance to commonly administered antibiotics at an alarming rate. Antibiotic drugs with novel modes-of-action are now needed more than ever in order to combat these so-called superbugs. A novel and elegant way of combatting bacterial infection of host cells is through host-directed therapy (HDT). In HDT the host machinery is targeted instead of pathogenic biomolecules. The rationale behind this is that in natural conditions bacterial species such as Mycobacterium tuberculosis (Mtb) cannot proliferate and survive without a host, and thus it is not only dependent on normal physiological functioning of its own cell but also of its host. Because of host-dependency it would make sense that host proteins play a key role in the survival of the pathogenic species. Therefore, unravelling these proteins might provide us with greater insight in the molecular mechanisms behind infection, and also can lead to potentially interesting therapeutic targets if inhibition of these enzymes proves to have antibiotic effects.

One method to study the proteome involved in host-pathogen interactions is activity-based protein profiling (ABPP). In ABPP proteins are studied activity-based instead of abundance-based as is the case in whole cell proteomics. Central to ABPP is the activity-based probe, which is generally a small molecule containing an electrophilic warhead, which can covalently bind to a nucleophilic amino acid residue of choice, and a reporter tag, which can be functionalized with either a fluorescent group for imaging purposes, or an enrichment tag for mass spectrometry quantification. Adaptations of the warhead and the core scaffold of the molecule leads to specificity for certain enzyme classes, such as serine hydrolases, lipases and kinases.

Amongst others ABPP can be used to quantitatively analyze differences in protein activity between slightly different proteomes. For instance, human cells that have or have not been treated with a pathogenic species, where you then analyze the host proteome for changes in protein activity.

Currently we are using our ABPP method on tuberculosis infection models in collaboration with the Ottenhoff group. They found that kinase inhibitor 97i has a host-directed antimicrobial effect against Mtb. Which means that it eradicates the pathogen by targeting host machinery. The host-directed effect was concluded after seeing that administration of 97i to pure Mtb culture did not result in any kind of reduction of bacterial load, implying that it has no effect on the pathogen itself, while 97i did reduce bacterial load in host-pathogen systems. In order to see what the targets of 97i are in situ we use a kinase-specific probe, which can show us what the inhibitory potency is of 97i against all enzymes enriched by the kinase-specific probe. The predicted key target enzyme of 97i was confirmed by performing ABPP in Mtb treated cells of the MelJuSo melanoma cell line along with two other targets. Currently the goal is to carry out this experiment in Mtb treated human M2 macrophages to find out the inhibition profile of 97i in a more realistic model.

In the future the aim is to successfully make and implement new chemical probes to study kinases that have not yet been labelled by current kinase probes. These can then be used to study changes in the activity levels of these proteins between proteomes, and also to use these probes for target engagement studies of inhibitors. Furthermore, these probes along with other probes we have already synthesized in house can be used in the future to study the host-pathogen proteome.


Disease Intervention Strategies is the overarching theme for more than 10 PhD tracks in NCOH projects to create new interdisciplinary, inter-thematic, and inter-institutional research collaborations.