Discovery of ATX968: An Orally Available Allosteric Inhibitor of DHX9

Abstract

DHX9 is an RNA/DNA helicase integral in the maintenance of genome stability that has emerged as an attractive target for oncology drug discovery. Disclosed herein is the discovery and optimization of a series of DHX9 inhibitors. Compound 1 was identified as a partial inhibitor of DHX9 ATPase activity but a full inhibitor of unwinding activity. Binding of 1 to a pocket distinct from the ATP binding site was confirmed by X-ray crystallography, enabling structure-based drug optimization. During this optimization, a sulfur-halogen bond was identified that increased on-target residence time without impacting equilibrium binding affinity. Analysis shows that cell potency more closely correlates with residence time than with equilibrium measurements of binding affinity or biochemical potency. Further optimization of potency and ADME properties led to the identification of ATX968, a potent and selective DHX9 inhibitor that is efficacious in a tumor xenograft model of microsatellite instability-high (MSI-H) colorectal cancer.

Figure 1

Biochemical and structural properties of 1. (A) Molecular structure of compound 1. (B) Concentration–response curves of 1 in ATPase (blue) and unwinding (red) assays. Partial-inhibition behavior (max inhibition 70%) is seen in the ATPase assay and full enzymatic inhibition is seen in the unwinding assay. (C) Crystal structure of 1 (magenta sticks) and ADP (green sticks) bound to feline DHX9 (PDB 9MFP; PDB 8SZQ used as molecular replacement model). DHX9 is shown in cartoon representation and colored by domain. The binding site of 1 is indicated. (D) Zoomed-in view of the binding site of 1 in feline DHX9. 1 has a robust interaction network with DHX9. Residues within 4 Å distance of 1 shown as sticks, water molecules shown as red spheres, hydrogen bonds shown as black dashes, π-stacking interactions shown as green dashes, and other colors as in C.

Figure 2

Crystal structures of 10 (PDB 9MFQ) and 23 (PDB 9MFR) bound to feline DHX9 (PDB 8SZQ used as a molecular replacement model for both structures). (A) 10 (green sticks) and (B) 23 (orange sticks) bound to feline DHX9 (cartoon representation). Hydrogen bonds are shown in black dashes, and halogen bonds and VDW interactions are shown in yellow dashes with distances shown in Å.

Figure 3

Representative single-cycle SPR sensorgram for 10, 23, and 24 binding to DHX9. Experimental data are in blue, mathematically determined best fit to 1:1 binding model (Biacore Insight Evaluation, Cytiva) in black. The off rate of 23 is decreased relative to 10 or 24, resulting in slower dissociation from the enzyme.

Figure 4

Biochemical, biophysical, and cellular correlations of DHX9 inhibitors. (A) Maximum inhibition (%) vs EC₅₀ (ATPase assay, red) or IC₅₀ (unwinding assay, blue). Dashed line is the mean value (ATPase assay: 70.9%; unwinding assay: 95.3%). (B) circBRIP1 EC₅₀ vs ATPase EC₅₀. Line of best fit drawn (R² = 0.49). (C) circBRIP1 EC₅₀ vs K_D. Line of best fit drawn (R² = 0.55). (D) circBRIP1 EC₅₀ vs SPR residence time. Line of best fit drawn (R² = 0.84).

Figure 5

Crystal structures of ATX968 bound to cat and human DHX9 proteins. A) Crystal structure of ATX968 (cyan sticks) complexed with ADP-bound feline DHX9 (PDB 9MFS; PDB 8SZQ used as a molecular replacement model). Coordinated waters shown as red spheres. B) Structure from A superposed with the human DHX9-ADP-ATX968 complex (gray) (PDB 9MFT; PDB 8SZP used as a molecular replacement model).

Figure 6

Pharmacokinetics of ATX968 at escalating doses in mice. (A) Pharmacokinetic profile of a single oral dose of ATX968 in CD1 mice (10 mg/kg, blue triangles) and Balb/C mice (100 mg/kg, green squares and 300 mg/kg, red circles). The dotted line is the plasma-protein binding corrected circBRIP1 EC₉₀. (B) Plot of dose-normalized C_max (red squares) and AUC_0–24h (blue circles) of ATX968. Line of best fit drawn separately for each end point.

Scheme 1. Synthesis of Compounds 1–9

Reagents and conditions: (a) TCFH, NMI, MeCN, rt. (b) HATU, DIPEA, DMF, rt.

Scheme 2. Synthesis of Arylated Thiophenes

Reagents and conditions: (a) PhB(OH)₂, Pd(dppf)Cl₂, K₃PO₄, 1,4-dioxane, water, 80 °C. (b) LiOH, THF, water, rt. (c) TCFH, NMI, N-(3-anilino)methanesulfonamide, MeCN, rt. (d) B₂pin₂, Pd(dppf)Cl₂, KOAc, 1,4-dioxane, 80 °C. (e) ArBr, Pd(dppf)Cl₂, K₂CO₃, 1,4-dioxane, water, 80 °C. (f) ArB(OH)₂, Pd(PPh₃)₂Cl₂, Na₂CO₃, EtOH, water, 80 °C. (g) ArB(OH)₂, K₂CO₃, Pd(dppf)Cl₂, 1,4-dioxane, water, 80 °C. (h) ArSn(Bu)₃, Pd(PPh₃)₄, toluene, 120 °C.

Scheme 3. Synthesis of ATX968 and Analogs

Reagents and conditions: (a) B₂pin₂, Pd(dppf)Cl₂, potassium acetate, 1,4-dioxane, 80 °C. (b) ArBr, Pd(dppf)Cl₂, K₂CO₃, 1,4-dioxane, water, 90 °C or Pd(PPh₃)Cl₂, Na₂CO₃, EtOH, water, 80 °C. (c) LiOH, EtOH, or MeOH, H₂O, rt. (d) N-(3-Amino-5-chlorophenyl)methanesulfonamide, TCFH, NMI, MeCN, rt. (e) 3-chloro-5-nitroaniline, TCFH, NMI, MeCN. (f) Fe, NH₄Cl, MeOH, H₂O. (g) R₂SO₂Cl, pyridine, rt. (h) 2-(2,6-Difluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, XPhosPdG3, K₃PO₄, 1,4-dioxane, 80 °C or (2-methoxyphenyl)boronic acid, Pd(dppf)Cl₂, K₂CO₃, 1,4-dioxane, 80 °C.