Expanding the Druggable Proteome with Chemical Biology – Best Poster Awards

The 2020 conference season at the EMBL Advanced Training Centre kicked off with the EMBL Conference: Expanding the Druggable Proteome with Chemical Biology (5 – 7 February 2020). Meet the three poster prize winners from the conference – Patrick Zanon, Enric Ros and Rens de Vries.

Identification of novel antibiotic targets using covalent inhibitors and residue-specific proteomics

PHOTO: Patrick Zanon

Authors: Patrick Zanon (1), Stephan Hacker (1)

Bacterial resistance towards all marketed antibiotics poses an imminent threat to global health. In order to overcome this antibiotic crisis, drugs with novel mechanisms-of-action are desperately needed. Covalent inhibitors are especially promising in this regard as they are already prevalent as antibiotics (e.g. β-lactams and fosfomycin), allow targeting protein pockets that are hard to address with non-covalent interactions alone and hold the promise to overcome some mechanisms of resistance development.[1] Furthermore, covalent inhibitors are uniquely suited to identify new binding pockets on proteins using residue-specific proteomics and in this way to broaden the scope of targetable protein targets.
The vast majority of covalent inhibitors so far either hijack the enzymatic activity of the protein by modification of catalytic serines and tyrosines or address cysteines through their inherent outstanding nucleophilicity. Nevertheless, the number of potentially addressable proteins in the bacterial proteome is significantly limited by the requirement for these amino acids to be present in target proteins. By developing electrophilic groups that are selective for other amino acids (e.g. lysine), we strive to expand the number of exploitable interaction sites for covalent inhibitors in the bacterial proteome. Furthermore, to assess the reactivity and selectivity of covalent inhibitors and to streamline the discovery of novel antibiotic targets, we develop new methods for residue-specific activity-based protein profiling.[2,3] In this way, we are convinced, that we will be able to make important contributions to overcome the antibiotic crisis.

References:
[1] R. A. Bauer, Drug Discov. Today 2015, 20, 1061–1073.
[2] K. M. Backus et al., Nature 2016, 534, 570.
[3] P. R. A. Zanon, L. Lewald, S. M. Hacker Angew. Chem. Int. Ed., doi: 10.1002/anie.201912075.

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(1) Technical University of Munich, Germany


Incorporating 1,2,4,5-tetrazines into proteins: A method for targeted drug release

PHOTO: Enric Ros

Authors: Enric Ros (1), Antoni Riera (1), Lluís Ribas de Pouplana (1)

Bioorthogonal reactions, namely reactions that can take place under biocompatible conditions, are having a major impact in the development of new research tools and novel therapeutic strategies. In the latter case, the discovery of the reaction commonly referred to as “click-to-release” (CtR), which triggers the liberation of a given cargo (normally a drug or a fluorophore), has led to several applications in drug delivery. This reaction happens between a 1,2,4,5-Tetrazine (Tz) fragment and certain alkenes or alkynes and, in order to achieve drug delivery specifically at the site of action, one of the two reactant counterparts should be conjugated to a biomolecule acting as a carrier, ideally a protein.
We have synthetized the previously unreported 3-bromo-1,2,4,5-tetrazine and used its excellent reactivity to attain chemoselective protein labelling onto lysines. Due to the chemical features of the formed amino-Tz. The resulting labelled lysines can undergo fast CtR reactions with trans-cyclooctenes, thereby releasing a desired cargo under physiological conditions. To showcase the applicability of this approach, we have labelled the monoclonal antibody Trastuzumab (anti-Her2) and demonstrated the specific release of the cytotoxic drug doxorubicin upon reaction in a mammalian cell culture context, resulting in a decrease in cell viability.
Additionally, we have also used 3-bromo-1,2,4,5-tetrazine to synthetize an amino-Tz containing non-natural amino acid and used it to achieve protein labelling through its genetic incorporation by amber codon suppression in Escherichia coli. The resulting site-selectively labelled proteins can also trigger fast, high yielding CtR reactions.
To summarize, we have successfully applied a new compound, 3-bromo-1,2,4,5-tetrazine, as a reagent to achieve either chemoselective or site selective protein labelling. We have applied the bioconjugated proteins to demonstrate their potential use for targeted drug delivery in a relevant cellular model, opening new therapeutically useful methodologies.

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(1) IRB Barcelona, Spain


Modulation of nuclear receptors through ligand architecture

PHOTO: Rens de Vries

Authors: Rens de Vries (1), Femke Meijer (1), Luc Brunsveld (1)

Nuclear receptors (NRs) have been one of the primary drug targets over the last decades for their ability to regulate gene expression. The traditional approach of modulating NRs is to design small synthetic molecules that interact with the ligand-binding domain (LBD) of the NR. Ligands can thereby either enhance or inhibit gene transcription. Apart from the effects on transcription, recent research shows that minor changes in the ligand scaffold can have a significant impact on the behavior of the NR. In this research, we show how small-molecules can change both the dimerization behavior of NRs and the recruitment of allosteric modulators.
The Retinoic X Receptor α (RXRα) is known as a master regulator among NRs through its ability to heterodimerize with, and thereby modulate, other NRs. We show, using a novel NanoBIT complexation assay, that small directed changes in the RXR ligand scaffold can lead to selective formation of specific hetero- and homodimers. Using our structural data and focused compound library, a model was developed to help to understand this effect of the ligand. This information can serve as a blueprint to design small-molecules that selectively target specific NRs via RXR. This makes RXR as an exciting and versatile target for NR modulation, especially when classical modulation of the partner NR is not possible.
Recently, small-molecules have been found to bind to allosteric sites of NRs. Allosteric ligands are of interest since they do not compete with the endogenous ligand of the NR and often shown an increased selectivity towards their target. We show, using X-ray crystallography and biochemical assays, that there is communication between orthosteric and allosteric ligands in the RAR-related orphan receptor γ t (RORγt). We successfully solved eleven new ternary crystal structures of RORγt in the presence of both orthosteric and allosteric ligands. These structures mechanistically show how binding of the orthosteric ligand leads to positive cooperative binding of the allosteric ligand.

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(1) Eindhoven University of Technology, The Netherlands


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Using chemical biology to expand the druggable proteome

Gerard Drewes
Head of Science, GSK Cellzome, Germany

In 2020 the EMBL Resource Development team and  industry partners of the EMBL Corporate Partnership Programme will bring together academic and industrial scientists with interests in chemical biology, chemogenomic libraries, pharmacology, medicinal chemistry and bioinformatics for the EMBL Conference: Expanding the Druggable Proteome with Chemical Biology (5-7 February 2020).

We spoke to co-organiser Gerard Drewes from GSK Cellzome about how chemical biology is helping to expand the druggable proteome.

How would you define the “druggable proteome”?

This is the fraction of our >20,000 human proteins that can be functionally modulated by a drug. Drugs can be small molecules or large molecules such as therapeutic antibodies. Estimates of how many proteins are “tractable” vary widely, I think there may be around 5,000. Only a subset of these 5,000 would be “druggable” which means that modulating them with a drug will also have a therapeutic benefit.

How are advances in chemical biology helping to expand the druggable proteome?

Small molecules are still the main modality for intracellular targets. Deep pockets, typical for enzymes, are more easily tractable than shallow pockets typical for protein-protein interactions. Chemical biology has developed tools to explore different types of pockets. I am excited in particular by the potential of DNA-encoded libraries, and small fragment approaches with covalent modes of action. Some of these compounds will just be “binders” but these can be made into target degraders as PROTACs.

How can these advances help our understanding of disease biology?

If we had more chemical probes, we could use these in a standardised, controlled way to interrogate target function in cell-based models, organoids, and in some cases animal models. Yes, we have gene editing now, but that is not the same as pharmacological modulation.

We also need in vitro models that translate better to in vivo. Our old immortalised cell lines won’t do, we are going to need more work in primary cells, organoids, etc.

What are the main challenges facing scientists in this field?

Lack of standardised probe sets. Bad probe compounds, e.g. with bad selectivity, are still used and wrong conclusions drawn.

Lack of translational in vitro models.

Why is it important to bring industry and academia together to discuss this topic?

Academia brings creativity, agility, fast progress of new ideas and concepts, thinking out of the box.

Industry sometimes lacks these but knows how to develop a compound into a drug, which requires a host of technologies not readily available to academia. Also, industry requires a new generation of drug targets with better validation, and historically targets are often discovered in academia. Once a target hypothesis exists, academics and industry should ideally collaborate to figure out how to drug it.

What will be the main highlight of this conference?

I see many but still hope to be surprised!

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