Development of a protein palmitoylation assay to monitor treatment of CLN1 Batten Disease

SummaryIn the Specific Aims outlined by the following proposal for an NIH SBIR Phase 1 project, we will use a
multi-faceted approach to identify key substrates for targeted assay development to monitor therapy in for
CLN1 Batten Disease (CLN1). CLN1 is an ultra-rare neurodegenerative disease that affects children with
autosomal recessive mutations in the gene for palmitoyl-protein thioesterase 1 (PPT1). PPT1 cleaves the
thioesterase bond between a 16-carbon lipid chain, palmitate, and many proteins expressed in the brain.
Circumvent Pharmaceuticals is developing a small molecule thioesterase mimetic, CIRC825, for the treatment
of CLN1 as there is currently no available therapeutic for CLN1 patients.We propose a multiplexed Acyl-Resin Assisted Capture (Acyl-RAC) discovery assay coupled to an isobaric
peptide-labeling strategy using tandem mass tags (TMT) to profile the neuronal and blood palmitoylomes of a
Cln1-/- mouse model using LC-MS/MS. TMT assays will be followed up with targeted, parallel reaction
monitoring (PRM) mass spectrometry to validate targets quantitatively. We will select proteins for their relative
expression in brain and blood to enable pharmaceutically-relevant routine blood collection for standard of
practice, clinical examination. We will adapt our assay to the assessment of CLN1 patient blood and cultured
cells for PPT1 substrates by discovery Acyl-RAC-PRM-MS to identify candidates for targeted assay
development. Cultured cells will be treated with CIRC825 and assessed, compared to untreated controls, by
targeted Acyl-RAC-PRM-MS. To allow for further optimization and selection based on reproducibility, stability,
and behavior in method development, we will identify 5-10 candidate palmitoylated protein substrates of PPT1
which are differentially expressed in patient-derived LCLs or patient blood spot samples which are also match
our findings in mouse brain and blood.Through a future NIH SBIR Phase II project, the optimal substrates will be used in the development of a
commercial clinical assay which may be expanded to use in numerous diseases which display aberrant protein
palmitoylation. Finally, we plan to perform proof-of-concept for our final assay during CLN1 Phase I/II clinical
trials before offering our assay for licensing.Narrative
CLN1 Batten Disease (CLN1) is a rare and devastating neurodegenerative disease in which children are in a
vegetative state by age three, and die by age 15. Remarkably, these children share some of the same
underlying disease biology as those who suffer from Alzheimer’s Disease and other brain diseases –
specifically through increased protein palmitoylation. We propose development of a clinical-grade test that will
enable monitoring of aberrant protein palmitoylation in CLN1 and possibly other neurodegenerative disorders.