Bioorthogonal chemistry | EPFL Graph Search

The term bioorthogonal chemistry refers to any chemical reaction that can occur inside of living systems without interfering with native biochemical processes. The term was coined by Carolyn R. Bertozzi in 2003. Since its introduction, the concept of the bioorthogonal reaction has enabled the study of biomolecules such as glycans, proteins, and lipids in real time in living systems without cellular toxicity. A number of chemical ligation strategies have been developed that fulfill the requirements of bioorthogonality, including the 1,3-dipolar cycloaddition between azides and cyclooctynes (also termed copper-free click chemistry), between nitrones and cyclooctynes, oxime/hydrazone formation from aldehydes and ketones, the tetrazine ligation, the isocyanide-based click reaction, and most recently, the quadricyclane ligation. The use of bioorthogonal chemistry typically proceeds in two steps. First, a cellular substrate is modified with a bioorthogonal functional group (chemical report

Biomolecule gradients on surface initiated graft polymers

Ruben Casanova

Controlling the fate of stem cells in vitro is a key challenge towards using these cells in clinical applications. Adult stem cells (ASC) are known to reside in complex microenvironments called niches in vivo. These niches regulate stem cell fate providing a variety of signals that control in a balanced way self-renewal and differentiation. In vitro ASC culture is challenging using standard culture methods because these are not able to recreate an adequate environment mimicking niche signals. The use of bioengineered polymer surfaces may thus be a solution for controlling stem cell fate in a predictable and scalable manner. Surface initiated polymerisation (SIP) is a method allowing controlled free radical polymerisation. This technique can be used to achieve highly controlled polymer architectures. Combining this versatile technology with a microfluidic gradient maker allowed the patterning of surface gradient arrays of desired tethered biomolecules. We have used monomers carrying functional groups that can be used in highly specific and high-yielding reactions such as biotin methacrylate and alkyne methacrylate to achieve surface gradients of Streptavidin as well as azide-functionalized RGD peptides. Surface characterisation was carried out by X-ray photoelectron spectroscopy (XPS) as well as fluorescence microscopy to verify surface modification steps and gradient patterns. The quantification of immobilized biomolecules was carried out using Europium labelling assays. As a proof of concept gradients of RGD peptides were patterned on polymer brushes expecting dose responding attachment of adherent cells in the presence of these strong attachment motifs. The results showed a dose dependent attachment of fibroblast on gradients of azide-RGD. However this outcome could not be achieved with biotinylated RGD as a result of a failure in binding between the biotinylated peptide and streptavidin. Nevertherless, these experiments demonstrate the suitability of the overall strategy as a mechanism for surface patterning of specific molecules. Improvements of the method established here have the potential to result in a sophisticated platform for the screening of cell-material interactions.

2011

Design and validation of bioorthogonal probes for cellular disease models

Well-validated chemical probes enable testing of biological hypotheses, investigation of target tractability and translatability to the clinical phase. Consequently, these important tool compounds play a key role in the drug discovery process. Moreover, their use for target validation may ultimately help to decrease the attrition rate encountered by new molecular entities in clinical trials. Despite the number of publications describing well-validated chemical probes, for several reasons, it remains a difficult challenge to identify and select the appropriate high quality molecule enabling the desired biological and pharmacological studies in the relevant disease model. Various reports describe sets of principles to be fulfilled by high quality chemical probes, which mainly rely on verifying that chemical probes are potent, engage their intended targets in the relevant cellular model, have sufficient exposure at the desired site of action and express functional pharmacological activities by a selective modulation of their targets. Classical chemical probes include LMW ligands, which inform on the functional consequences of interacting with a particular bio-logical target in a model system. To get information on other parameters (such as sub-cellular distribution of target or compound) different tools may need to be used, often requiring specific chemical functionalization in order to observe the molecule using the currently available technologies. Compared to classical chemical probes, bioorthogonal probes can also be small molecules that elicit a functional response but with the additional advantage of being able to undergo reaction with a variety of chemical reporters (e.g. fluorophores) in situ, in bio-logical model systems such as cells and animals. Therefore, this increases their versatility; for example their visualization using imaging technologies (bioluminescence and fluorescence imaging) can provide important insights on compound permeability, in-tracellular distribution, and potentially co-localization with its protein target in relevant cellular disease models. Hence, the use of a bespoke bioorthogonal probe may answer key biological questions, which are usually only addressed by using multiple classical probes. Herein, we describe the design and the synthesis of bioorthogonal probes to study the inhibition of the p53-Mdm2 protein-protein interaction. Furthermore, we report the development of a set of assays based on fluorescence and bioluminescence techniques enabling the validation of these molecules in a cellular osteosarcoma model. Our approach for chemical probe design and validation in cellular disease models may be applied in principle to a wide range of novel drug discovery projects to provide early mechanistic understanding.

EPFL2019

Development of novel tools for imaging of metabolic fluxes in vivo

Tamara Maric

Bioluminescent imaging is a powerful technique that enables imaging in living organisms with high sensitivity, low background signal, low cost and without the need for radioactivity. The emitted photons are produced in the oxidation reaction of luciferin catalyzed by luciferase. In the caged luciferin approach, the bioluminescent readout is used for measuring the activity of a specific biochemical process. Luciferin is caged in order to prevent catalysis by luciferase, and only when the cage is removed by a specific biochemical event of interest, luciferin is released and becomes a substrate of luciferase. Hence, emitted photons are proportional to the amount of free luciferin, thus allowing to monitor the dynamics of the investigated process. Recent progress in the development of bioorthogonal reactions enabled to answer several fundamental biological questions, improve clinical diagnosis, and to better evaluate therapy efficacy. For example, one of the most robust biorthogonal reactions is the Staudinger ligation, which occurs between a properly tuned azide and a phosphine. In this work, we have used the caged luciferin approach combined with the Staudinger ligation to monitor metabolite uptake in living animals. We have successfully applied this method for the monitoring uptake of two metabolites â glucose and nicotinamide riboside (NR). Glucose is a major source of energy for most living organisms and its aberrant uptake is linked to many pathological conditions. On the other hand, NR is a form of vitamin B3 and represents one of the salvageable NAD+ precursors. Low NAD+ level inside of cells is strongly linked to numerous metabolic and age-related disorders. Despite of their importance, in both cases our understanding of disease-associated glucose or NR flux is limited due to a lack of robust tools. To date, positron emission tomography (PET) imaging remains the gold standard for measuring glucose uptake and no optical tools exist for non-invasive longitudinal imaging of neither of the two metabolites in in vivo settings. Here we report the development of a novel bioluminescent glucose uptake probe (BiGluc) and bioluminescent NR uptake probe (BiNR) for real-time, non-invasive longitudinal imaging of glucose and NR absorption both in vitro and in vivo. The new imaging reagents enable a wide range of applications in the field of metabolism and drug development.

EPFL2020