ProfilE3 is a high throughput assay for detecting target degradation by 150 human E3 ligases. The assay employs native proteins from biological samples and is performed in an ELISA format. The assay can be modified to detect the subcellular compartment of target engagement, or protein modifications necessary for this interaction. Most of the current set of E3 ligases have no known ligand binding capabilities.
Iterating the approach with samples under different physiological conditions can identify the E3 ligase–target pairs which may have changed with treatments, (such as, drug treated vs untreated, diseased vs healthy, and so on). Vistara offers a proteomics service for further analysis of the 'hits', and identify the E3 ligase responsible for degradation of the Target.
Custom assays are available for identifying additional E3 ligases. Using native proteins from biological samples, our proteomics paltform employs mass spectrometry to discover E3 ligases, the targets they engage, accessory proteins collaborating with ubiquitin complexes, glue-like proteins, proteasome subunits, ubiquitins, and some E1 and E2 enzymes.
Besides addressing target engagement, these iterations point to the mechanisms governing cell specificity of the E3L and Target interactions, regulation of ubiquitin proteasome system function, signaling, or drug responses.
The PINTAC platform is a cell-based assay system for observing the in vivo effects of peptide-mediated disruption of a Target or its E3 ligase. PINTAC assays can be performed in 96-well format, or administered to organoid cultures as a peptide or using gene-based approaches. Treatment of cells with PINTACs disruptthe function of the target due to a combination of polyubiquitination and degradation.
Vistara has a current capability of targeting over 5,000 protein with PINTACs. Custom assays are available to identify additional proteins for targeting. Current PINTACs target cytosolic proteins; second generation PINTACs are in development for sub-cellular targeting of specific proteins or their isoforms, motif-based targeting. PINTACs can be used in conjunction with drug treatments for combinatorial targeting of more than one protein(s) or selectively inactivating the drug target in a subcellular compartment.
Vistara also offers proteomics analysis of global changes accompanying disruption of a Target, such as, secretome, gene expression, signaling, sub-cellular localization, etc. The PINTAC assay is a useful adjuct for assessing feasibility of other protein degradation strategies to be applied later in therapeutics in a well defined biological system.
Ligandability of E3 ligases:
Vistara offers discovery services for identifying ligandable E3 ligases and their Targets from biological samples and profile them between different cell types, during the cell cycle, or with drug treatments. By using native samples Vistara processes match E3 ligase-Target combinations for each cell-type.
Cell type specificity of Target engagement:
E3 ligases are known to vary across cell type in their ability to engage a Target for degradation, regardless of expression. Vistara has developed assays for profiling biological samples for Target engagement and mapping the sites of E3 ligase action.
Mutant E3 ligases:
Nearly ∼13% (83 total genes) of the 600 E3 ligases in humans are mutated in 83 neurological disorders representing 70 different types of neurological diseases. Many of these E3 ligases are among the current set of 150 identified by us. These reagents will be used for comparing changes in target engagement between the wild type and mutant E3 ligases.
Glue proteins, Toxic proteins and Motifs:
Vistara has identified a large number of glue proteins, protiens associated with drug toxicity, and motifs among proteins in several gene families which frequently associate with the ubiquitin proteasome system, for use with PINTACs to disrupt multiple proteins in cell cultures, to help with Target identification.
Vistara Bioscience has designed novel tools for extracting proteomic information relating to E3 ligases and the degradation targets engaged by them. Sequence information of E3L-Target interacting regions identified are utilized in the creation of peptide reagents which disrupt E3L-Target interactions in vivo. Termed PINTACs, the peptides exhibit degradation and polyubquitination of the targeted E3 ligase or E3L target. PINTACs have many uses in assay development for drug discovery research, and may also be developed for therapeutic use as peptides, or delivered via gene-based methods.
Vistara seeks partnerships for applying its ubiquitome discovery, profiling, and PINTAC technologies in preclinical drug discovery research.