Probes, Inhibitors and PROTACs

The Probes, Inhibitors and PROTACs Core is a new facility that will provide validated sophisticated small molecules and mass spectrometry services.

These services will  map interaction sites, characterize proteome-wide binding profiles, delineate impact on protein interaction and signaling networks, and identify the rise of resistance mechanisms. 

The rapid discovery and development of high-yielding chemical transformations in biological environments, called bio-orthogonal chemistry, have had far reaching implications in a variety of applications, including genetic code expansion and drug target identification. This, coupled with allele-specific chemical genetics, wherein a small molecule is designed to specifically bind to mutated proteins to overcome the slow kinetics associated with genetic experiments, has provided scientists with complementary tools to generate a molecular view of the associated biology.

These technologies rely on the design and synthesis of chemical  probes that are required to conduct the bio-orthogonal chemistry or chemical genetics experiments and sophisticated mass spectrometry to validate the molecular target and characterize the targeted and untargeted effects of these probes, inhibitors and PROTACs.

The scientific community exclusively relied on genetic methods to alter protein levels until the discovery of proteolysis-targeting chimeras, also known as PROTACs, which are heterobifunctional small molecules that facilitate the formation of ternary complexes between the protein of interest and the E3-ligase. This allows the E3-ligase to polyubiquitinate a proximal lysine on the protein of interest, thus enabling the degradation of the protein of interest by the cellular ubiquitin proteasome system.

This core will provide mass spectrometry services to characterize the effect of inhibitors and PROTACs in cells, organoids, and tissues. These include altered post-translational modifications regulated by signaling molecules, such as phosphorylation by kinases, that can be used for the purposes of deconvoluting signaling pathways or identifying new targets for probes, inhibitors and PROTACs development.

This core uniquely weds two complementary services that will provide the research community not only with cutting-edge chemical tools but also a map of their effects on the proteome.

The PIP Core will pursue these aims:

• Aim 1: Provide validated probes, inhibitors, and PROTACs for the discovery and
  development of therapeutic targets. The services include:

1. Focused chemical libraries for identification of PROTACs that selectively degrade the target of interest or for structure guided hit-to-lead optimization studies.
2. Access to small molecules that are not commercially available (including compounds found in patents), as well as photoactivatable, fluorescent and biotinylated chemical compounds, for assay development and biological target identification.
3. Scale-up services (mg to grams) for additional in vivo studies to demonstrate proof of concept.

• Aim 2: Provide sophisticated mass spectrometry analysis to characterize probes, inhibitors and PROTACs, and support the discovery of novel targets for development. Mass spectrometry services will be provided by the core to support:

1. Validation of probe/inhibitor/PROTAC targets.
2. Quantitative proteome network analysis of differentially expressed proteins following PIP treatment.
3. Target discovery using quantitative mass spectrometry of posttranslational modifications.

• Aim 3: Continue to develop new methods and acquire instrumentation for long-term sustainability of the core. The core will continue to develop and implement state-of-the-art chemistry and mass spectrometry methods to address future needs of center members. Examples include developing methods for single-cell proteomics and photoactivatable PROTACs for controlled (spatio-temporal) target modulation.