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6:30 - 9:00 pm Dinner Workshop: Emerging Methods for Measuring Target Engagement*

The increase in usage of small molecule probes for the validation of novel therapeutic targets requires effective means of assaying target engagement in cells and living systems. Importantly, measuring the extent to which a target is engaged (or not) provides critical information regarding probe selectivity and attributed efficacy modulating a biological process, while informing of any off-target interactions and potential toxicity. The workshop is designed to discuss emerging techniques and technologies for measuring target engagement.

Instructors: 

 

Erik Hett, Ph.D., Senior Scientist, Chemical Biology, Medicinal Chemistry, Pfizer

 

Andrea M. Zuhl, Ph.D., Fellow, Neuroscience, Pfizer; Former Research Associate, Cravatt Lab, The Scripps Research Institute

 

Thomas Lundbäck, Ph.D., Assay Development & Screening, Chemical Biology Consortium Sweden; Senior Scientist, Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet

 

Michael Dabrowski, Ph.D., CEO & Co-Founder, Pelago Bioscience AB

 

 

Overview: 

In-Cell Target Engagement and Selectivity Assessment Using Chemical Biology

Erik Hett, Ph.D., Senior Scientist, Chemical Biology, Medicinal Chemistry, Pfizer

Selectivity is a critical parameter to understand in drug discovery, yet difficult to quantify in a relevant setting, such as in cells. We have developed a novel probe to enable quantification of target engagement in disease relevant cells as well as a selectivity profiling approach. This work reveals interesting findings in terms of target engagement under different cellular environments and provides an example for how this approach can be used for other drug discovery programs.

Confirming Target Engagement in Living Systems: Examples from the Aspartyl Protease and Serine Hydrolase Enzyme Families

Andrea M. Zuhl, Ph.D., Fellow, Neuroscience, Pfizer; Former Research Associate, Cravatt Lab, The Scripps Research Institute

Chemical probes can be designed to confirm target engagement within a complex biological setting by either a compound centric or enzyme class centric approach. In an example of a compound centric design, a β-secretase inhibitor was modified into a chemical probe that allowed for successful identification and quantification of off-target activity in live cells. In a complementary approach, secondary screening of high-throughput screening (HTS) hits using a general serine hydrolase probe was used to profile a novel chemotype for serine hydrolase inhibition. Techniques to be discussed include activity and affinity-based probes, in situ and in vivo target engagement, activity-based HTS and quantitative mass spectrometry.

Physiological Relevant Target Engagement

Michael Dabrowski, Ph.D., CEO & Co-Founder, Pelago Bioscience AB

With the discovery that it is possible to quantify the thermal stability of individual proteins in complex analytes such as lysate, intact cells, tissue and body fluids, it is now possible to quantify Target Engagement in physiologically relevant settings. The innovative Cellular Thermal Shift Assay (CETSA™) is based on the well-known biophysical phenomenon that the melting point of a protein shifts upon binding of small molecules. CETSA™ allows for direct measurements of molecule and target protein interactions in intact cell systems and tissues from animals and humans. We have validated drug binding in mammalian cancer cell lines for a set of important clinical targets and show that a range of critical factors that are important for drug development can be identified at the target engagement level, including drug transport and activation, off-target effects, drug resistance as well as drug distribution in animal tissues. Preliminary data on drug target engagement in tumor tissue samples from patients will also be presented.

High Throughput Adaptation of the Cellular Thermal Shift Assay (CETSA) Using AlphaScreen® Technology to Monitor Target Engagement in Cells

Thomas Lundbäck, Ph.D., Assay Development & Screening, Chemical Biology Consortium Sweden; Senior Scientist, Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institute

The effective binding of a ligand to a protein can be monitored by following the induced shift in thermal stability of the protein. This shift in stability is a well-known phenomenon and assays measuring such thermal shifts have been used extensively on purified proteins to detect interactions and estimate relative affinities of ligands to proteins. Here we present the implementation of a high throughput screening protocol to a thermal shift assay in a cellular format, referred to as the cellular thermal shift assay (CETSA). The method allows studies of target engagement of drug candidates in a cellular context. We have studied human p38α kinase as an example model system to demonstrate two separate protocols for CETSA. To illustrate the feasibility of applying the homogeneous assay for screening purposes we have also tested how the homogeneous assay protocol responds to the presence of a small set of test compounds taken from a diversity library.

*Separate Registration Required



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