Our EMBO Workshop ‘Chemical biology’ is the largest and longest standing conference in the field, being a platform for inspiration, collaboration, and networking for researchers in academia and industry. This year we hosted over 200 people on site (and 50 virtually) and are pleased to share with you the four best poster prize winners!

Zinc finger proteins are novel targets of proton pump inhibitors

Presenter: Teresa Marker

Abstract:

Since the approval of Omeprazole in the late 1980s, proton pump inhibitors (PPIs) have become top-selling drugs worldwide. It has long been understood that PPIs are pro-drugs activated by protonation in highly acidic environments. It has also been well established that they inhibit stomach acid secretion by covalently modifying cysteines of gastric H⁺/K⁺-ATPase on the parietal cell surface. However, almost nothing is known about alternative activation mechanisms and intracellular protein targets. To identify previously unknown PPI target proteins, we synthesized azide-functionalized Rabeprazole. Using a chemoproteomic approach in intact cells, we find Rabeprazole forms covalent conjugates with a few dozen intracellular proteins. Remarkably, we find a significant enrichment of zinc-binding proteins, wherein the most highly enriched proteins all harbour the same type of zinc binding motif. We selected one of the main target proteins, density-regulated protein (DENR), for further study, and found that its conjugation to Rabeprazole depends on an intact zinc binding site. We propose that the zinc acts as a Lewis acid, obviating the need for low pH, to promote the activation of Rabeprazole, and possibly of other PPIs as well. Our findings may be relevant for the understanding of secondary drug effects and/or the repurposing of PPIs.

Due to the confidentiality of the unpublished data, we cannot share the poster.

Toward a phenotypic selection platform for the continuous evolution of biocatalysts

Presenter: Suzanne Jansen

Abstract:

Enzymes are impressive catalysts that accelerate reactions with unmatched rates and selectivity under benign conditions. Moreover, enzymatic features that warrant improvement can be tailored to fit a user’s needs through directed evolution, i.e. iterative cycles of diversification, selection, and amplification. Rather than assessing each variant’s activity one-by-one (screening), we have recently developed a phenotypic selection platform that utilizes genetic code expansion to link enzymatic activity to cellular fitness. Here, we describe how this high-throughput, survival-based platform allows for selection on a population level, mimicking nature’s survival of the fittest. Specifically, we challenge this platform to selectively amplify the best catalysts from increasingly complex populations, starting from two to millions of enzyme variants. Lastly, we demonstrate how our platform can be adapted to evolve enzymes in an autonomous and continuous manner, allowing the researcher to explore multiple evolutionary trajectories simultaneously.

View poster

Directed evolution of near-infrared fluorescent chemogenetic reporters for deep tissue imaging in vivo

Presenter: Lina El Hajji

Abstract:

Deep-tissue imaging has largely benefitted from the engineering of far-red and near-infrared fluorescent proteins from bacterial phytochromes, that allow one to circumvent autofluorescence, scattering and absorption encountered in tissues. Alternatively, chemogenetic labels have emerged as a hybrid approach complementing the toolkit of genetically-encoded reporters. 

Here we present our current progress towards the engineering of a near-infrared fluorescent chemogenetic reporter, starting from the far-red Fluorescence Activating and absorption-Shifting Tag (frFAST), a 14 kDa protein tag able to form a bright far-red emitting assembly with a fluorogenic chromophore. Molecular engineering of the fluorogen combined with structure-oriented engineering of frFAST allowed us to red-shift both absorption and emission wavelengths by 50 nm, reaching the near-infrared region. The binding affinity and molecular brightness was further improved by directed protein evolution.  Promising fluorogenic near-infrared emitting variants were isolated from yeast-displayed libraries of frFAST mutants by Fluorescence-Activated Cell Sorting (FACS), and further refined by site-directed mutagenesis. Here, we show how coordinating molecular engineering, rational design and directed evolution can help tailor a chemogenetic system’s spectral properties for specific deep-tissue imaging applications.

Due to the confidentiality of the unpublished data, we cannot share the poster

Conformational nanoswitches for continuous biosensing

Presenter: Anna Swietlikowska

Abstract:

There is an increasing need for biosensors that can measure biomarkers continuously. An accurate diagnostics of certain diseases, like sepsis, depends on it. The technology of such a sensor can be based on an enzymatic reaction (like for glucose monitoring) or a conformational change (e.g. aptamer). Hence, I am developing a molecular switch, made of DNA, that undergoes a structural reorganization when the target biomarker is bound. The system is based on long DNA functionalized in 2 regions.  Upon analyte binding, 2 distant parts of the switch are brought together and form a loop. In this research, a protein G dimer was conjugated to maleimide-modified DNA and used as a general antibody binder. The protein was then successfully photo-crosslinked to infliximab, an immunoglobulin recognizing TNFα. The presence of analytes was detected in various ways. Firstly, when run on an agarose gel, looped and unlooped structures migrated differently in electric field and the target was detected in the concentration as low as 3 nM. Then, split NanoLuc system was fused to protein G to enable bioluminescent readout. Upon addition of TNFα, the bioluminescence level increased seven-fold. Although, the latter is not suitable for continuous sensing, it is a great tool to characterize the switching behavior and the importance of distances between binders. The use of such a modular material as DNA enables easy immobilization of a sensor that in the future, could be studied in flow with single molecule optical detection.

Due to the confidentiality of the unpublished data, we cannot share the poster

The EMBO Workshop ‘Chemical Biology 2022‘ took place from 5 – 8 September 2022 at EMBL Heidelberg and virtually.