Structure-Function Relationships of Covalent and Non-Covalent BTK Inhibitors

Abstract

Low-molecular weight chemical compounds have a longstanding history as drugs. Target specificity and binding efficiency represent major obstacles for small molecules to become clinically relevant. Protein kinases are attractive cellular targets; however, they are challenging because they present one of the largest protein families and share structural similarities. Bruton tyrosine kinase (BTK), a cytoplasmic protein tyrosine kinase, has received much attention as a promising target for the treatment of B-cell malignancies and more recently autoimmune and inflammatory diseases. Here we describe the structural properties and binding modes of small-molecule BTK inhibitors, including irreversible and reversible inhibitors. Covalently binding compounds, such as ibrutinib, acalabrutinib and zanubrutinib, are discussed along with non-covalent inhibitors fenebrutinib and RN486. The focus of this review is on structure-function relationships.

Keywords: BTK inhibitors; acalabrutinib; covalent and non-covalent binding; fenebrutinib; ibrutinib; protein-inhibitor interactions; structure-function relationship; zanubrutinib.

Figure 1

(A) Spatial organization of mouse BTK SH3 (yellow), SH2 (pink) and kinase domains (cyan). Linkers are in gray. The SH2 and SH3 domains are indicated by their positions in the dimer (PDB id 4xi2). (B) Human BTK kinase domain with ibrutinib (red) bound covalently (magenta) to C481 (blue) (5p9j). The upper lobe is in cyan, the lower lobe in yellow and inhibitor in red. (C) Superimposition of closed (5p9j) and open BTK kinase conformation (1k2p). Upper lobe is in cyan and lower lobe in yellow. Lower lobe was used for the superimposition.

Figure 2

ADP and inhibitor binding. (A) BTK sequence indicating amino acids within 5 Å radii from ADP or inhibitors. For ATP binding, residues corresponding to interacting amino acids in BTK are shown in red. Crosses indicate the interacting amino acids in inhibitor complexes. (B) Upper panel: Chemical structure of ATP, adenine is highlighted in blue. Lower panel: ADP bound to ITK kinase domain (4m15). Residues within 5 Å from ADP are in green. Atoms in the ADP are colored based on the elements, carbon gray, nitrogen blue, oxygen red, and phosphorus orange. (C) Differences in the binding modes of the covalent inhibitor, ibrutinib (blue, 5p9j), and non-covalent inhibitor, fenebrutinib (orange, 5vfi), to BTK. C481 is in blue.

Figure 3

Chemical structures and binding of covalent BTK inhibitors, (A) ibrutinib (5p9j), and (B) zanubrutinib (6j6m). Residues within 5 Å distance from inhibitor are shown in green. Atoms in the inhibitors are colored based on the elements, carbon gray, nitrogen blue, oxygen red. Covalent bond between C481 and inhibitor is in magenta. The structures have the same pose as in Figure 2C .

Figure 4

Chemical structure of acalabrutinib and molecular model showing binding of the covalent inhibitor in BTK. Atoms in the inhibitor are colored based on the elements, carbon gray, nitrogen blue, and oxygen red. Covalent bond between C481 and inhibitor is in magenta. The structure has the same pose as in Figure 2C .

Figure 5

Chemical structures and BTK binding of non-covalent inhibitors (A) fenebrutinib (5vfi) and (B) RN486 (5p9g). Residues within 5 Å distance from inhibitor are shown in green. Atoms in the inhibitors are colored based on the elements, carbon gray, nitrogen blue, oxygen red, and fluorine pale green. The structures have the same pose as in Figure 2C .

Figure 6

Loss- and gain-of-function variations in BTK kinase domain. (A) XLA-causing variants (cyan) are distributed all along the kinase domain. (B) Sites of ibrutinib resistance conferring amino acids C481 (red) and T474 (green) along with gain-of-function variants at positions 512, 513, 517 and 547 (yellow), note that variant at 474 alone and together with other variants lead to gain-of-function activities. The structures are for ibrutinib (pink) complex with BTK (5p9j).