2. Academic Validation
  3. Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist

  • J Med Chem. 2024 May 9;67(9):7245-7259. doi: 10.1021/acs.jmedchem.4c00072.
Esra Balıkçı 1 2,  Anne-Sophie M C Marques 1 2,  Ludwig G Bauer 1 2,  Raina Seupel 1 2,  James Bennett 1 2,  Brigitt Raux 1 2,  Karly Buchan 1 2,  Klemensas Simelis 1 2,  Usha Singh 1 2,  Catherine Rogers 1 2,  Jennifer Ward 1 2,  Carol Cheng 1 2,  Tamas Szommer 1 2,  Kira Schützenhofer 3,  Jonathan M Elkins 1 2,  David L Sloman 4,  Ivan Ahel 3,  Oleg Fedorov 1 2,  Paul E Brennan 1 2 5,  Kilian V M Huber 1 2
Affiliations

Affiliations

  • 1 Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, U.K.
  • 2 Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, U.K.
  • 3 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K.
  • 4 Departments of Discovery Chemistry, Merck & Co. Inc., 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.
  • 5 Alzheimer's Research UK Oxford Drug Discovery Institute, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, U.K.
PMID: 38635563 DOI: 10.1021/acs.jmedchem.4c00072
Abstract

Cofactor mimicry represents an attractive strategy for the development of Enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical Btk inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical Btk Inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

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