Pikfyve assay

A high-throughput assay using selective proteolysis of PIKfyve generates stable kinase domain fragments for efficient screening of covalent inhibitors, overcoming the inefficiencies and false negatives of previous methods, thereby accelerating inhibitor discovery.

WO2026136219A1 Publication Date: 2026-06-25DUNAD THERAPEUTICS LTD +1
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Patent Information

Application Number
PCT/US2025/059623
Authority / Receiving Office
WO · WO
Patent Type
Applications
Current Assignee / Owner
Priority Date
2024-12-16
Filing Date
2025-12-15
Publication Date
2026-06-25

AI Technical Summary

Technical Problem

Current methods for screening covalent inhibitors of the PIKfyve protein are inefficient and prone to false negatives due to the instability of the kinase domain and the need for large protein amounts, which is exacerbated by the aggregation-prone nature of the C-terminal kinase domain.

Method used

A high-throughput assay is developed using selective proteolysis of recombinant full-length PIKfyve at an endogenous caspase 3 cleavage site to generate stable C-terminal kinase domain fragments, allowing for rapid analysis by mass spectrometry, such as RapidFire-MS, and confirming adduct formation with cysteine-mutant proteins.

Benefits of technology

The assay enables rapid and robust identification of covalent binders, accelerating the discovery and optimization of PIKfyve inhibitors by stabilizing the kinase domain and improving screening efficiency.

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Abstract

The invention provides for a screening assay-using a selective proteolysis of recombinant full-length PIKfyve at an endogenous caspase 3 cleavage site to liberate the 34 kDa C -terminal kinase domain. Despite instability of the kinase domain to isolation, the liberated kinase domain is stable in the presence of the N-terminal PIKfyve protein fragment and can thus be treated with compounds of interest and analyzed by mass spectrometry, such as RapidFire-MS, with each injection taking under one minute. The assay was established to triage small molecules for covalent modification of PIKfyve. The assay was also established using cysteine-mutant proteins, where the reactive cysteine was replaced with an inert amino acid which when run through the same experimental protocol, was used to confirm adduct formation at the target cysteine.
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