Structural Basis for Nitrile-Directed Recognition of the Catalytic Lysine in KIT
5 affiliations
- 1Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Straße 4a, Dortmund, Germany, 44227
- 2Drug Discovery Hub Dortmund (DDHD) am Zentrum für Integrierte Wirkstoffforschung (ZIW, Otto-Hahn-Straße 4a, Dortmund, Germany, 44227
- 3Department of Medical Oncology and Sarcoma Center
- 4Medical School
- 5Compound Management and Screening Center (COMAS
Abstract
KIT and PDGFRA are the major oncogenic drivers in gastrointestinal stromal tumors (GIST). Although clinically approved KIT/PDGFRA-directed tyrosine kinase inhibitors (such as imatinib and avapritinib) have improved patient outcomes, long-term efficacy is often limited by additional resistance mutations in these kinases. To explore alternative interaction modes less susceptible to mutational escape, we investigated the catalytic lysine K623 in KIT as an anchoring point for inhibitor design. Starting from avapritinib, we prepared nitrile-containing analogues in which the canonical pyrimidine core was replaced by a nitrile-substituted aromatic scaffold. The compounds were evaluated biochemically against KIT and the closely related GIST driver PDGFRA, including clinically relevant mutant forms, and were further assessed in cellular models of PDGFRA-driven GIST. Co-crystal structures revealed a distinct binding mode in which the nitrile group is directed toward K623, reorienting the ligand within the ATP-binding site while retaining kinase inhibitory activity. Guided by these structural findings, electrophilic analogues bearing squaric ester warheads were developed to probe covalent target engagement. Intact-protein mass spectrometry demonstrated time-, concentration-, and pH-dependent covalent labeling of KIT, with complete mono-labeling of the detectable soluble protein population observed under near-physiological conditions. Pre-occupation of the ATP-binding site with either avapritinib or a covalent ATP-site binder selectively suppressed the earliest and kinetically favored labeling event, supporting an active-site-dependent component of covalent modification. Together, these results establish the catalytic lysine K623 as a structurally accessible interaction site in KIT and show that this binding geometry can provide a basis for proximity-driven covalent kinase targeting.
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BibTeX
@misc{mancino2026structural,
title = {{Structural Basis for Nitrile-Directed Recognition of the Catalytic Lysine in KIT}},
author = {Giorgia Mancino and Andrea Scrima and Janina Sandig and Hala Zohbi and Matthias P. Müller and Thomas Mühlenberg and Sonja Sievers and Sebastian Bauer and Daniel Rauh},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15010974/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010974/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Structural Basis for Nitrile-Directed Recognition of the Catalytic Lysine in KIT
AU - Giorgia Mancino
AU - Andrea Scrima
AU - Janina Sandig
AU - Hala Zohbi
AU - Matthias P. Müller
AU - Thomas Mühlenberg
AU - Sonja Sievers
AU - Sebastian Bauer
AU - Daniel Rauh
PY - 2026
DA - 2026/10/23
PB - ChemRxiv
DO - 10.26434/chemrxiv.15010974/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010974/v1
AB - KIT and PDGFRA are the major oncogenic drivers in gastrointestinal stromal tumors (GIST). Although clinically approved KIT/PDGFRA-directed tyrosine kinase inhibitors (such as imatinib and avapritinib) have improved patient outcomes, long-term efficacy is often limited by additional resistance mutations in these kinases. To explore alternative interaction modes less susceptible to mutational escape, we investigated the catalytic lysine K623 in KIT as an anchoring point for inhibitor design. Starting from avapritinib, we prepared nitrile-containing analogues in which the canonical pyrimidine core was replaced by a nitrile-substituted aromatic scaffold. The compounds were evaluated biochemically against KIT and the closely related GIST driver PDGFRA, including clinically relevant mutant forms, and were further assessed in cellular models of PDGFRA-driven GIST. Co-crystal structures revealed a distinct binding mode in which the nitrile group is directed toward K623, reorienting the ligand within the ATP-binding site while retaining kinase inhibitory activity. Guided by these structural findings, electrophilic analogues bearing squaric ester warheads were developed to probe covalent target engagement. Intact-protein mass spectrometry demonstrated time-, concentration-, and pH-dependent covalent labeling of KIT, with complete mono-labeling of the detectable soluble protein population observed under near-physiological conditions. Pre-occupation of the ATP-binding site with either avapritinib or a covalent ATP-site binder selectively suppressed the earliest and kinetically favored labeling event, supporting an active-site-dependent component of covalent modification. Together, these results establish the catalytic lysine K623 as a structurally accessible interaction site in KIT and show that this binding geometry can provide a basis for proximity-driven covalent kinase targeting.
ER -