Exploiting the Cryptic αD Pocket of Casein Kinase 2α (CK2α) to Deliver Highly Potent and Selective Type 1 Inhibitors

Abstract

Casein kinase 2α (CK2α) is an oncology drug target that acts as a positive regulator of many tumorigenic signaling pathways. We previously reported that CK2α has a unique cryptic binding site, the αD pocket, that offers the potential for inhibitors with improved kinase selectivity. The prototype bivalent molecule CAM4066 (6) confirmed that improved selectivity could be achieved while binding in both the ATP-binding site and the αD pocket. A drug discovery project to develop a new series of bivalent CK2α inhibitors with increased cell potency and selectivity identified 61f (APL-5125), a highly potent, ATP-competitive CK2α inhibitor with exquisite kinase selectivity and cellular potency. Compound 61f demonstrates in vivo inhibition of p-AKT S129 in tumors (HCT116) following once-daily oral administration and shows a clear PK-PD relationship with unbound drug exposure. 61f has a superior preclinical profile to existing CK2α inhibitors and is currently under evaluation in patients with advanced solid tumors.

1. Chemical Structures of Selected Type 1 CK2α Inhibitors

2. Chemical Structures of Selected Bivalent CK2α Inhibitors

1. Synthesis of Hit Compound 12

2. Synthesis of Hit Compound 14

3. General Synthetic Routes to Compounds 18–26, Incorporating Initial Changes to the Linker

4. Synthesis of Compounds 30–33, Containing Carboxylic Acid Isosteres

5. Synthesis of Compounds 36 and 38, Incorporating Initial Changes to the Linker

6. Synthesis of Compounds 44–46, Containing an Ether Linker

7. Synthesis of Compounds 48–50, Incorporating Alternatives to the Benzylic Amine

8. Synthesis of Compounds 54a–m and 55a–b, Incorporating Substitution of the αD Fragment

9. Synthesis of Compounds 58a–i, Containing Changes to the Ether Linker

10. Synthesis of Compounds 61a–j, Incorporating Substitution of the αD Fragment

1

Binding mode of 18 to CK2α compared to the binding modes of established CK2α inhibitors. A) The binding mode of 18 (green, PDB: 7I8O) to CK2α. The hydrogen bonding networks of the carboxylic acid and the benzylic nitrogen are shown as dotted lines. B) The binding mode of 18 (green) in the ATP-binding site is compared to the binding modes of 1 (CX-4945, silmitasertib) (orange, PDB: 3PE1), 4 (AZ 7h) (purple, PDB: 5H8E) and 6 (CAM4066) (blue, PDB: 5CU4) with H-bonds to the hinge region shown as dotted lines in their respective colors. C) The binding mode of 18 (green) in the ATP-binding site is compared to the binding modes of 6 (blue), 12 (pink, PDB: 7I8N) and 14 (yellow, PDB: 7I8M) with H-bonds to the hinge region shown as dotted lines in their respective colors.

2

Crystal structures of compounds that show the hydrogen bonding networks of carboxylic acid isosteres. A) and E) The binding mode of 30 (blue, PDB: 7I7Y) compared to 18 (green). B) and F) The binding mode of 31 (pink, PDB: 7I7Z) compared to 18 (green). C) and G) The binding mode of 33 (yellow, PDB: 7I83) compared to 18 (green). D) and H) The binding mode of 32 (salmon pink, PDB: 7I84) compared to 18 (green). Hydrogen bonds are shown as dotted lines.

3

Crystal structures showing compounds with various linker lengths. A) The binding mode of 18 (green) compared to 19 (pink, PDB: 7I80), 20 (yellow, PDB: 7I82) and 21 (blue, PDB: 7I8P). Hydrogen bonds between 18 and Val162 and Pro159 are shown as dotted lines. B) and E) The binding mode of 18 (green) compared to 19 (pink). Hydrogen bonds between 19 and Val162 and Pro159 are shown as dotted lines. C) and F) The binding mode of 18 (green) compared to 20 (yellow). Hydrogen bonds between 20 and Val162 and Pro159 are shown as dotted lines. D) and G) The binding mode of 18 (green) compared to 21 (blue). Hydrogen bonds between 21 and Val162 and Pro159 are shown as dotted lines.

4

Crystal structures of compounds binding in the αD pocket. Cross-section of the binding modes of: A) 18 (green); B) 54f (pink, PDB: 7I84); C) 54e (salmon pink, PDB: 7I89); D) 54b (purple, PDB: 7I8B).

5

Crystal structures of compounds that bind in the αD pocket and grow into the adjacent water channel. αD pocket with a closed water channel: A) The binding mode of 54f (pink, PDB: 7I8A); αD pockets with an open water channel:B) The binding mode of 54l (salmon pink, PDB: 7I8Q); C) The binding mode of 54m (yellow, PDB: 7I8R); D) The binding mode of 54k (dark red, PDB: 9QQX); E) Cross-section of the binding mode of 54f with a closed water channel ( pink, PDB: 7I8A); F) Cross-section of the binding mode of 54l with an open water channel (salmon pink, PDB: 7I8Q).

6

Binding mode of 61f to CK2α. A) The crystal structure of 61f bound to CK2α (PDB: 7I8K). B) The interactions of 61f within the ATP-binding site. C) The hydrogen bonding interactions between 61f and the hinge region. D) The hydrophobic interactions between 61f and the αD pocket. E) The hydrogen bonds between the benzylamine and CK2α. F) The hydrogen bonding network between the carboxylic acid group of 61f, Lys68, Glu81 and a water molecule. G) Two-dimensional map of the interactions between CK2α and 61f (gold) with hydrogen bonds shown as dotted lines, and hydrophobic interactions depicted with spiked arcs (or “eyelashes”).

7

Profile of 61f in the KINOMEscan ^TM scanMAX panel. The human kinome is represented as a phylogenetic tree. Each kinase is shown as a sphere with size and color indicating the level of inhibition of each kinase at 100 nM APL-5125, as per legend on the top left corner.

8

In vivo inhibition of p-AKT S129 in HCT116-tumors in mice following oral administration of 61f. Concentration–response curve for p-AKT S129 inhibition in tumor was generated using the unbound concentration of 61f in plasma from all individual mice in each dose group (10, 30, 100 mg/kg) at either 2 or 8 h postfinal dose, and the corresponding p-AKT S129 measurement from tumor tissue determined by Western blotting.