Project 1: Bioorthogonal chemistry

Project leader: Nicolas Winssinger

Participants: Jérôme Waser, Gisou van der Goot, Nicolai Cramer, Karl Gademann, Christian Heinis, Elena Dubikovskaya

A central theme of project 1 is to expand small molecule diversity space beyond the commercial available space and provide the required synthetic chemistry expertise to follow up on identified hits

 

In the first phase of project 1, a strong focus on novel synthetic technologies and their application in diversity-oriented synthesis was initiated. 

Successful projects along these lines will be continued and supplemented with a new subproject on DNA-encoded screening approach that is complimentary to HTS platform (ACCESS) and phage display currently available within the NCCR.   

In addition, the scope of project 1 will be broadened to including novel developments in bioorthogonal reactions.  Reactions that are suitable to modify proteins with chemical reporters have attracted significant attention over the past decade. Early technological breakthroughs such as the expressed protein ligation have enabled the introductions of fluorophore, NMR probes or other reporters into complex proteins providing a window into the function of these proteins through biophysical and mechanistic studies. 

To date the collection of techniques that can be used to attach synthetic groups to a protein has expanded substantially.  Several chemical, enzymatic and genetic methods have now been developed to introduce reporters directly or introduce a functionality that can be targeted chemoselectively.  These developments have not only contributed to our understanding of the targeted biomolecule’s function but are also being applied in protein drug conjugation, medical imaging and hybrid materials.  The scope and limitations of current technologies vary significantly in terms of bioothogonality, compatibility with complex cellular environment, yield and selectivity.

While the repertoire of chemical transformations for native purified proteins is fairly broad, this repertoire is dramatically reduced in more complex settings such as lysates and even more in live cells.  These techniques are well entrenched in ongoing NCCR projects and are being applied to specific research problems. 

 

For example, the SNAP tag technology developed by Kai Johnsson was used in FRET-based sensors for tracking Notch signaling.  Activity-based probes that react specifically with targeted enzymes in order to quantify them are being used in collaborative projects between Alexander Adibekian and Howard Riezman. Reagents to deferentially label metabolites will be developed in collaboration between Alexander Adibekian and Robbie Loewith.  While these examples illustrate the potential of existing methodologies, there are clear opportunities that should arise from broadening the scope of bioorthogonal reactions.   This work package focuses on the development and implementation of novel chemistries to access uncharted diversity space, novel screening methods and methodologies that can be applied to interrogate or manipulate biologically relevant systems. 

It is comprised of 7 subprojects:

  1. Approach Towards the Total Synthesis of Strictamine
  2. Diversity oriented synthesis of bioactive nucleoside analogues
  3. Applying palladium-catalyzed reactions to increase chemical and structural diversity of phage-encoded peptide libraries;
  4. DNA display of small molecule libraries for 2D screening
  5. Stabilizing Chemical Modifications of Native Peptides (disulfide mimetic) with minimal disturbance 
  6. Functionalization of biomolecules using novel hypervalent iodine-mediated reactions 
  7. Developing new techniques for sensitive imaging of biological processes in living cells and animals 

Project 2 : Chemical Systems Biology

Project leader: Robbie Loewith

 

Participants: Alexander Adibekian, Howard Riezman, Jean Gruenberg, Kai Johnsson 

 

NCCR participants have screened, and continue to screen, for small molecules that target various signaling and/or biosynthetic pathways in yeast and in higher eukaryotes.

Past NCCR screens have successfully identified inhibitors of the TOR-pathway, tetrahydrobiopterin biosynthesis, sphingolipid metabolism and compounds that alter the intracellular accumulation of cholesterol and lysobisphosphatidic acid.

In some cases, the direct, physical targets of these compounds remain unknown. Therefore, one of the major goals of project 2 is to provide a technology platform that is used throughout the NCCR to help to identify targets and evaluate the effects on pathways impinged upon by chemical interference and other methods, such as physical manipulations, siRNA, or protein engineering technologies. The availability of pathway inhibitors, orphan or otherwise, now provides us the opportunity to probe how perturbations of these pathways affect cellular physiology, ideally, at a system-wide level and the members of project 2, in addition to providing a technology platform, will apply this technology to their previously identified, as well as to be identified, inhibitors or other perturbations. As detailed below, this effort will be supported by innovative chemistry, exploiting isotopically labeled probes, which will greatly facilitate quantification of proteins, lipids and other metabolites. Lastly, in several cases, NCCR screening efforts have led to the discovery of novel signaling pathways. The third goal of project 2 is thus to support clever, dedicated and novel screening approaches, often based on synthetic biology or genetic interactions, aimed at dissecting the molecular events in these new pathways.  

  1. Generic Approaches for Target Identification
  2. System-level studies including Mass Spectrometry-based Quantitative ‘omics
  3. Novel approaches to screening technology 

Project 3 : Protein-based tools for visualization and manipulation of biochemical activities

Project leader: Kai Johnsson

 

Participants: Oliver Hantschel, Pierre Gönczy, Beat Fierz, Christian Heinis 

This project groups three different subprojects whose common theme is to use protein engineering to generate new tools for studying biological questions. In the first subproject, it is proposed to develop monobodies that bind to proteins such as BCR-ABL1 and the centriolar SAS-6 proteins for subsequent mechanistic studies. This will also lead to the establishment of a platform for the generation of monobodies for other projects in the NCCR and beyond. It is also proposed in this subproject to generate switchable monobodies. In the second subproject, expressed protein ligation will be used to generate tubulin proteins with defined post-translational modifications (PTMs) at their C-terminal tail and characterize the importance of these modifications in asymmetric vesicle trafficking and in centriole formation. In the third subproject it is proposed to generate a fluorescence sensor for the key metabolite acetyl-CoA and utilize it to investigate the mechanism of acetyl-CoA homeostasis.

 

Project 4: Sensors and assays to study cell mechanics and endosomal motility

Project leader: Marcos Gonzalez-Gaitan

 

Participants: Aurélien Roux, Suliana Manley, Howard Riezman, Kai Johnsson 

Much of our understanding of the cell focuses on the (bio)chemistry of the compounds present in living matter: most prominently proteins, lipids, sugars and nucleic acids. Inherently, this realm is a subject of chemical biology: indeed, chemical biology approaches in this NCCR consortium generate both sensors for these compounds and reagents to interfere with their abundance and function.  In Project 4, using chemical sensors and biophysical tools, we plan to address a much neglected issue: the cell as a physical object, which is subject to forces, pressures and tensions. These physical constraints have a major impact on the shape and function of cells and tissues, ultimately feeding back and determining the biochemistry of living matter. 

We will generate assays and reagents to study the physics of three key objects: the plasma membrane, the cytoskeleton and the endosomal pathway. Strategically, we will focus on mitosis as a subject where cell dynamics are most overt, with an emphasis on asymmetric cell divisions. Asymmetric cell division, which generates two daughter cells with different properties, will offer an optimal system to test our tools in a scenario of broken symmetry. 

The three main aims of this project are:

  1. to generate sensors and assays to detect physical properties
  2. to develop techniques to measure those properties
  3. to establish tools to interfere with the physics of the cell  

Project 5: Cellular entry and novel membrane probes

Project leader: Stefan Matile

 

Participants: Howard Riezman, Marcos Gonzalez-Gaitan, Aurélien Roux, Kaori Sugihara, Andreas Zumbühl, Robbie Loewith 

 

Project 5 focuses on methods development with regard to biomembranes. This includes conceptually innovative approaches to cross membrane barriers and enter cells, and to image biologically important properties of biomembranes that are otherwise difficult to detect.

For cellular entry, emphasis will be on cell-penetrating poly(disulfide)s that can grow directly on substrates of free choice for covalent delivery. After uptake, they depolymerize to minimize toxicity and liberate the substrates in unmodified form. The covalent delivery of broad range of substrates from the NCCR network will be explored, including proteins (antibodies, etc), bicyclic peptides, quantum dots, probes such as SNAP-tags, or siRNA.

New methods such as templated side-chain exchange will be developed to find the most powerful poly(disulfide) transporters. This project will be complemented by ongoing, more advanced studies on cell-penetrating dynamic amphiphilic peptide dendrons for the delivery of oligonucleotides (siRNA, plasmids). 


Novel membrane probes will include planarizable push-pull probes, ceramide mimics and protein-based probes. Planarizable push-pull probes explore, for the first time, the molecular principles of the color change of lobsters during cooking or the chemistry of vision.

Emphasis will be on the detection of membrane tension because this is so far difficult to achieve and our chemical and biological approaches have the potential to solve this problem. Namely, the concept of  “fluorescent flippers” will be introduced to maximize mechanosensitivity and be used to study the mechanism of TORC2 activation in vivo.

Other topics of interest include the sensing of membrane phases, microdomains (“rafts”) and, if possible, also membrane potentials. In addition, new methods focusing on bendable supported bilayers (“electrofluorescent imaging”) and compressible Langmuir monolayers will be developed to comprehensively characterize new and old fluorescent membrane probes.

 

 

Project 6: Taking advantage of ACCESS

Project leader: Gerardo Turcatti 

 

We have established in the first four years of the NCCR Chemical Biology the infrastructure and  know-how for efficiently executing chemical screens.

ACCESS, a platform for Academic Chemical Screens in Switzerland, enables to run either complex phenotypic screens or relatively simple in vitro enzymatic assays; it provides diverse chemical libraries; and has competences in chemoinformatics to analyze results and to aid in hit expansion.

 

In the next round of the NCCR, we propose to take advantage of this strategic investment and simply run as many screens as possible. Some of the screens we are proposing come from research groups that are not NCCR members, demonstrating the importance of ACCESS for the Swiss research community.  In addition, we will further improve the performance and infrastructure of ACCESS. A particular focus will be put on the establishment of a web portal for data sharing and analysis. In short, project 6 aims at:

  1. further improvements in the performance of ACCESS
  2. performing chemical screens at ACCESS.

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