Structural basis for specificity of TGFβ family receptor small molecule inhibitors

Abstract

Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC(50) (EC(50)=1 μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway.

Figure 1. Crystal structure of TβRI-SB43154 complex

(A) Structure of TβRI Kinase domain (blue) in complex with SB431542. The structure of SB431542 (yellow) is shown as sticks occupying the ATP binding cleft between the kinase N- and C-lobes. (B) Magnified stereo view of SB431542 compound coordination in the binding cleft showing residues contacting SB431542 and the conservation of the ATP binding cleft between TGFβ and ALK2. Hydrogen bonds are represented by dashed lines and a water molecule is depicted as a red sphere.

Figure 2. TβRIca S280T mutant is resistant to SB431542

(A) NIH-3T3 cells were transfected with 3TP-lux. β-gal and constructs expressing TβRI wt, or TβRIca with the indicated ATP binding cleft mutations. After treatment with various doses of SB431542, Luciferase activity was measured and results were normalized to β-gal activity. (B) Worm representation of the N-lobe of TβRI showing residues selected for mutagenesis based on lack of conservation among TGFβ type I receptors (see also sequence alignment in Fig S2). (C) The experiment was conducted as in (A), with the indicated constructs.

Figure 3. Similarities between A8301 and SB431542

(A) Magnified ribbon representation of superimposed co-crystal structures of TβRI- A8301 and TβRI-SB431542 complexes. Dashed lines: Hydrogen bonds; blue sphere: amide nitrogen of His283. (B) NIH-3T3 cells were transfected with 3TP-lux, β-gal and indicated constructs. Dose response was determined by treating cells with varying concentrations of A8301 compound.

Figure 4. ALK2 gatekeeper residue mutant T283S is sensitive to SB431542

(A) NIH-3T3 cells were transfected with BRE-Luc, β-Gal and plasmid expressing ALK2ca or ALK2ca T283S. Dose response was determined by treating cells with varying concentrations of SB431542 compound (A). (B) Superimposition of the ATP binding cleft of TβRI-SB431542 and ALK2-dorsomorphin complexes. (C) The experiment was performed as in (A), but with dorsomorphin.