Structure-based discovery of cyclin-dependent protein kinase inhibitors

Abstract

The cell fate-determining roles played by members of the cyclin-dependent protein kinase (CDK) family explain why their dysregulation can promote proliferative diseases, and identify them as potential targets for drug discovery in oncology and beyond. After many years of research, the first efficacious CDK inhibitors have now been registered for clinical use in a defined segment of breast cancer. Research is underway to identify inhibitors with appropriate CDK-inhibitory profiles to recapitulate this success in other disease settings. Here, we review the structural data that illustrate the interactions and properties that confer upon inhibitors affinity and/or selectivity toward different CDK family members. We conclude that where CDK inhibitors display selectivity, that selectivity derives from exploiting active site sequence peculiarities and/or from the capacity of the target CDK(s) to access conformations compatible with optimizing inhibitor-target interactions.

Keywords: CDK; Structure-based drug design; cell cycle; kinase.

Figure 1. CDK activation

(a) Cyclin binding and phosphorylation activate CDKs. A notable exception is CDK8-cyclin C where the residue phosphorylated within the activation segment in most CDKs is replaced by an aspartate (CDK8 Asp191). Monomeric CDK2 (light blue, PDB 1HCK) is superposed on phosphorylated CDK2-cyclin A (colored dark blue and gold respectively, PDB 1JST). The CDK2 activation segment is highlighted in green. (b) Phosphorylated CDK4 bound to cyclin D1 resembles monomeric CDK2. CDK4 and cyclin D1 are colored orange and lime green respectively, and the CDK4 activation segment is brown, PDB 2W96). (c) Sequence alignment over the CDK active site. Zappo color coding is used to distinguish physicochemical properties [81]. (d) Stereo view of key CDK active site residues colored by conservation (green, conserved; red, non-conserved, [82]). Monomeric CDK2 and CDK2 bound to cyclin A are colored light and dark blue respectively throughout.

Figure 2. CDK2 ATP-competitive inhibitors explore the active site

(a) The binding of CDK2 to ATP (PDB 1HCK) is mimicked by inhibitors: (b) CDK2-dinaciclib (PDB 4KD1), (c) CDK2-cyclin A-NU6102 (PDB 1H1S). Within the CDK family, alternative modes of CDK hinge-inhibitor interaction have been observed. (d) CDK5-4a, (PDB 4AU8), (e) CDK8-22, (PDB 5CEI) and (f) CDK9-DRB (PDB 3MY1). The CDK5, CDK8 and CDK9 folds are colored red, dark green and lilac respectively throughout.

Figure 3. The gatekeeper pocket

CDK inhibitors that bind through hinge motif make a number of interactions with the conserved phenylalanine gatekeeper residue. (a) CDK6-compound 3 (PDB 4EZ5, aromatic-aromatic) (b) CDK8- CCT251921 (PDB 5HBJ, aromatic-halogen) (c) CDK8-compound 6 (PDB 5ICP, aromatic-sulfur). The CDK6 fold is colored cyan.

Figure 4. The DFG motif and back pocket remodeling

A comparison of CDK8-cyclin C bound to type II and type I inhibitors (a) CDK8-cyclin C in complex with the type II inhibitor sorafenib (PDB 3RGF). (b) CDK8-cyclin C bound to cortistatin A (PDB 4CRL). Comparing the figures illustrates how sorafenib binding is incompatible with the DMG-in conformation. (c) DFG-out CDK2 in complex with the type II inhibitor K03861 (PDB 5A14). (d) A comparison of CDK2-cyclin A bound to roscovitine in a DFG-in conformation (ice blue, PDB 3DDQ) again illustrates how a type II inhibitor binding is incompatible with a CDK2 DFG-in conformation.

Figure 5. The ATP ribose phosphate binding pocket

(a) An overlay of CDK structures illustrates the dynamic nature of the glycine-rich loop. CDKs are colored as previously. CDKs 4, 7 and 12 are colored orange, magenta and white respectively. (b) The CDK5 glycine-rich loop is restructured upon binding (R)-roscovitine (PDB 1UNL, crimson). The structure of CDK5-(R)-roscovitine is overlaid with CDK2-(R)-roscovitine (PDB 3DDQ, light blue) and CDK2-cyclinA (PDB entry 1FIN, glycine-loop colored green). (c) Comparison of inhibitor 12u binding to CDK2 (PDB 4BCP, CDK2 light blue 12u yellow) and CDK9 (PDB 4BCG, CDK9 lilac and 12u green) illustrates local conformational flexibility around the CDK9 active site can drive compound selectivity. (d) Comparison of CDK9-cyclin T bound to a more diverse inhibitor set reveals significant movements of the glycine-rich loop and also of the beta3-alphaC loop (PDBs 3BLQ, 3BLR, 3LQ5, 3MY1, 3TN8, 4BCG).

Figure 6. CDK-selective inhibitors exploit sequence differences on the surface of the CDK C-terminal lobe

(a) Monomeric CDK6 bound to clinical candidate palbociclib (PDB 5L2I), (b) CDK6 bound to ribociclib (PDB 5L2T). (c) CDK2-cyclin A bound to NU6102 (PDB 1H1S). The structures illustrate selectivity between CDK2 and CDK6 through interactions of Lys89 and Thr107 respectively. (d) CDK9 has a glycine residue (Gly112) at the equivalent position, illustrated in the structure of CDK9-cyclin T bound to compound 4 (PDB 4BCH).

Figure 7. The impact of the C-terminal tail on the catalytic cleft in the transcriptional CDKs.

The transcriptional CDKs are characterized by extended C-terminal sequences beyond the conserved kinase domain, emerging data shows how the C-terminal tail can impact the character of the ATP binding site. (a) CDK9 structure bound to DRB (PDB 3MYC) overlaid with full-length CDK9 (PDB 4EC8). (b) The CDK8 tail also reaches up into the active site as illustrated by the structure of the CDK8-CCT251545 complex. There is a favorable cation-pi interaction between the phenyl ring of the inhibitor and the guanidine moiety of Arg356 (PDB entry 5BNJ). (c) Similar trapping of the inhibitor THZ531 through the formation of an irreversible bond with Cys1039 located within the CDK12 C-terminal extension, as observed in the structure of a CDK12-cyclin K-THZ531 complex (PDB 5ACB).

Figure 8. Targeting the monomeric enzyme

(a) ANS bound to an allosteric site adjacent to the ATP site, the structural rearrangement creates a large pocket that accommodated two ANS molecules. The shift in the C-helix position was predicted to be incompatible with cyclin binding (PDB entry 3PXQ). (b) Structure of CDK2 bound to type I ½ quinolone-based inhibitor (compound 14) in which a phenol hydroxyl binds to the hinge, the DFG motif is in the “in” conformation and the quinolone 3-chlorophenyl group sits in a hydrophobic pocket under the C-helix that displaces it out by a translation and rotation to a position incompatible with cyclin association (PDB entry 4NJ3).