Article Figures & Data
Figures
- Fig. 1.
Comparison of the HIF-2α–binding affinities between PT2385 and belzutifan. (A and B) Chemical structures of PT2385 (A) and belzutifan (B). (C and D) Binding of PT2385 (C) and belzutifan (D) to the HIF-2α PAS-B domain as measured by ITC. The calculated KD value of PT2385 was 10 ± 4.9 nM, while the KD value of belzutifan was 16 ± 4.7 nM. This experiment was independently repeated twice.
- Fig. 2.
Crystal structure of the HIF-2α–ARNT-belzutifan complex. (A) Schematic representation showing the domain arrangements of HIF-2α and ARNT. (B) Binding position for belzutifan (brown) within the entire HIF-2α–ARNT crystal structure. (C) FO–FC omit map of belzutifan contoured at 3σ. (D) 2FO–FC map of belzutifan contoured at 1σ. (E–G) Structures of HIF-2α PAS-B domains in complex with belzutifan (E), PT2385 (F), or in apo state (G). (H) Comparison of key residue side-chain conformations in the HIF-2α PAS-B domains among apo, belzutifan-bound, and PT2385-bound proteins.
- Figure. 3
Allosteric effects of belzutifan revealed by crystal structures. (A) The overall arrangement of ARNT (green) and HIF-2α (cyan) PAS-B domains, with the location of dimer interface affected by belzutifan highlighted by an ellipse. (B) Conformational differences between belzutifan-bound and apo (PDB ID: 4ZP4, yellow) HIF-2α–ARNT complexes. (C) Position comparison of M252 side chain in belzutifan-bound and PT2385-bound (PDB ID: 6E3S, blue) HIF-2α–ARNT complexes.
- Fig. 4.
Inhibition of HIF-2α by belzutifan. (A and B) Disruption of HIF-2α–ARNT dimers containing WT HIF-2α (A) or M252A mutant (B) by belzutifan measured using TR-FRET. Error bars, mean ± S.D. (n = 3). The Ki values were derived from a four-parameter logistic equation. Ki = 23 ± 1.5 nM, slope factor = 1.3 ± 0.1 (A) and Ki = 3.5 ± 0.2 μM, slope factor = 1.0 ± 0.1 (B). (C) Dose-dependent inhibition of HRE luciferase reporter activity by belzutifan. Error bars, mean ± S.D. (n = 3). The IC50 value was derived from a four-parameter logistic equation, IC50 = 17 ± 1.6 nM, slope factor = −1.4 ± 0.1. (D and E) Expression of certain HIF-2α target genes in 786-O (D) or HEK293 (E) cells after treatment by belzutifan at various concentrations (0.01, 0.1, 1, or 10 μM). Error bars, mean ± S.D. (n = 3) (distinct replicates for cell cultures). (F) Comparison of the inhibitory effects of 10 μM belzutifan on HIF-2α target genes in Hep3B cells transfected with WT HIF-2α or M252A mutant. Error bars, mean ± S.D. (n = 3) (distinct replicates for cell cultures).
Tables
HIF-2α–ARNT-belzutifan PDB ID 7W80 Data collection Space group P 21 Cell constants
a, b, c (Å)
α, β, γ (°)
49.19, 98.19, 77.74
90, 107.16, 90Resolution (Å) 50.00 - 2.75 Rmerge 0.06 I/σI 1.98 (at 2.75 angstrom) Completeness (%) 99.7 Redundancy 6.8 Refinement Resolution 46.38 - 2.75 Reflections, n 18322 R-work, R-free 0.216, 0.274 Atoms, n Protein 4406 Ligand/ion 26 B-factors Protein 87.71 Ligand/ion 59.51 R.m.s. deviations Bond lengths (Å) 0.01 Bond angles (°) 1.24
