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Noncovalent inhibitors reveal BTK gatekeeper and auto-inhibitory residues that control its transforming activity
Shenqiu Wang1, Sayan Mondal2, Chunying Zhao1
1Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York USA.
Abstract:
Inhibition of Bruton tyrosine kinase (BTK) is a breakthrough therapy for certain B cell lymphomas and B cell chronic lymphatic leukemia. Covalent BTK inhibitors (e.g., ibrutinib) bind to cysteine C481, and mutations of this residue confer clinical resistance. This has led to the development of noncovalent BTK inhibitors that do not require binding to cysteine C481. These new compounds are now entering clinical trials. In a systematic BTK mutagenesis screen, we identify residues that are critical for the activity of noncovalent inhibitors. These include a gatekeeper residue (T474) and mutations in the kinase domain. Strikingly, co-occurrence of gatekeeper and kinase domain lesions (L512M, E513G, F517L, L547P) in cis results in a 10- to 15-fold gain of BTK kinase activity and de novo transforming potential in vitro and in vivo. Computational BTK structure analyses reveal how these lesions disrupt an intramolecular mechanism that attenuates BTK activation. Our findings anticipate clinical resistance mechanisms to a new class of noncovalent BTK inhibitors and reveal intramolecular mechanisms that constrain BTK's transforming potential.
Insights
New noncovalent Bruton tyrosine kinase (BTK) inhibitors face resistance from gatekeeper and kinase domain mutations. These mutations can paradoxically increase BTK activity and transforming potential, impacting lymphoma and leukemia therapies.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Bruton tyrosine kinase (BTK) inhibitors are crucial for treating B cell lymphomas and chronic lymphatic leukemia.
- Covalent BTK inhibitors, like ibrutinib, target cysteine C481, but resistance emerges through mutations at this site.
- Noncovalent BTK inhibitors offer an alternative therapeutic strategy, bypassing the need for cysteine C481 binding.
Purpose of the Study:
- To identify key residues in Bruton tyrosine kinase (BTK) critical for the efficacy of noncovalent inhibitors.
- To investigate the impact of specific BTK mutations on kinase activity and cellular transformation.
- To elucidate the structural mechanisms underlying resistance to noncovalent BTK inhibitors.
Main Methods:
- Systematic mutagenesis screen of Bruton tyrosine kinase (BTK) to identify resistance-conferring residues.
- In vitro and in vivo assays to assess kinase activity and transforming potential of mutated BTK.
- Computational structural analysis of BTK to understand the impact of mutations on protein activation.
Main Results:
- Identified a gatekeeper residue (T474) and other kinase domain mutations (L512M, E513G, F517L, L547P) critical for noncovalent inhibitor resistance.
- Observed a 10- to 15-fold increase in BTK kinase activity and de novo transforming potential upon co-occurrence of gatekeeper and kinase domain mutations.
- Computational analysis revealed that these mutations disrupt an intramolecular mechanism that normally attenuates BTK activation.
Conclusions:
- The study anticipates clinical resistance mechanisms against novel noncovalent Bruton tyrosine kinase (BTK) inhibitors.
- Specific co-occurring mutations in BTK can lead to enhanced kinase activity and oncogenic transformation.
- Findings reveal intramolecular regulatory mechanisms that constrain BTK's transforming potential, offering insights for future drug development.
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