Depalmitoylation Chemical Probes

SUMMARY

• Protein palmitoylation is a post translational modification of cystine protein residues, the dysregulation of which is implicated in diseases such as Alzheimer’s, cancers, and Huntington’s. Despite the importance of palmitoylation, the regulation of the enzymes that control it is poorly understood.
• The inventors designed an enzyme substrate analogue probe that can monitor the activity of protein depalmitoylases, the enzymes that catalyze palmitoylation. The probe fluoresces upon enzyme modification. 
• The invention is a fluorescence assay that can monitor the activity of protein depalmitoylases in vivo. The assay can be read with traditional fluorescent microscopy or flow cytometry.
• The assay was validated in vitro by monitoring the response of both HEK293T cells and human colon organoids to treatment with the non-specific depalmitoylase inhibitor PalmB.

FIGURE

A) Scheme of fluorophore release after cleavage of depalmitoylation probe by a protein depalmitoylase. A color change from green to blue indicates a cleavage event.  B) Imaging in live human colon organoids using the RDP-2 probe. Color scale represents level of intensity, corresponding with level of enzyme activity.

ADVANTAGES

ADVANTAGES

• Live cell imaging and analysis
• Direct quantification of enzyme activity
• Water-soluble (easy cell uptake)
• Performs in heterogeneous tissue samples
• Compatible with standard imaging protocols

APPLICATIONS

• Diagnostics
• Biosensors
• Imaging
• Pro-drug therapeutic

PUBLICATIONS

• Kathayat, RS; Elvira, PD; Dickinson, BC. A fluorescent probe for cystine depalmitoylation reveals dynamic APT signaling. Nat Chem Biol. 2017 Feb;13(2): 150-152.
• Beck, MW; et al. Michael addition-based probes for ratiometric fluorescence imaging of protein S-depalmitoylases in live cells and tissues. Chem Sci. 2017 Nov 1; 8(11):7588-7592. 

July 23, 2019

Ready for use

Patent Issued

Licensing,Co-development

Michael Beck

• US:10,413,583