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. Author manuscript; available in PMC: 2018 Nov 21.
Published in final edited form as: Essays Biochem. 2017 Nov 8;61(5):439–452. doi: 10.1042/EBC20170040

Structure-based discovery and development of cyclin-dependent protein kinase inhibitors

Mathew P Martin 1, Jane A Endicott 1, Martin EM Noble 1,1
PMCID: PMC6248306  EMSID: EMS80456  PMID: 29118092

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 illustrates the interactions and properties that confer upon inhibitors affinity and/or selectivity towards 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.

Introduction

Originally identified as regulators of the eukaryotic cell cycle, CDKs also regulate transcription and, in certain cell types, differentiation [1–3]. The activities of CDK family members impact several hallmarks of cancer [4]. In specific cellular settings, inappropriately elevated CDK activity, for example that of the cell cycle regulators CDK2, CDK4 and/or CDK6, has been identified as a cancer driver [5–8]. Expression or mutation of the activating cyclin subunit or decreased expression or mutation of an inhibitor (for example members of the INK family that bind specifically to CDK4 and CDK6) are frequently observed cancer-associated alterations (http://cancer.sanger.ac.uk/cosmic). CDKs 7, 8, 9, 11 and 20 regulate the synthesis of mRNA and therefore, by changing patterns of gene transcription, impact cell survival and differentiation [3, 9]. As a reflection of their differing roles in tissue physiology, aberrant activity of cell cycle or transcription regulatory CDKs can initiate or drive tumor progression in a cell-type specific manner, offering the opportunity to target specific cancers with CDK-selective inhibitors in certain patient populations.

CDK active and inactive states

A conserved feature of protein kinase activation is remodeling of an inactive monomeric kinase fold in response to protein association and /or post-translational modification [10, 11]. The structure of monomeric CDK2 revealed how an inactive state derives from misalignment of residues required for productive substrate binding and catalysis [12]. Association with a cognate cyclin and, for most CDK-cyclin pairs, phosphorylation of a conserved threonine residue within the activation loop, are minimal pre-requisites for full activity [1, 13, 14]. However, the catalytically competent structure is only adopted upon binding of both ATP and peptide substrates (Figure 1a, b [15]).

Figure 1. CDK activation.

Figure 1

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(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.

One challenge for drug development is that 20 members of the CMGC subfamily of the human kinome are sufficiently related to be classified as CDKs [3, 16]. Among these proteins, sequence identity is high within the residues that directly line the CDK active site (Figure 1c, d) and their convergence to a conserved structure upon activation has presented challenges for the design of inhibitors that target individual family members. Nevertheless, the available sequence diversity and conformational plasticity of the CDK fold have together offered opportunities to derive potency and selectivity. However, most inhibitor series exhibit substantial activity for a subset of the family.

The most successful clinical approach to date has involved targeting CDK4 and CDK6 [8, 17, 18]. This strategy exploits structural properties of CDK4 and CDK6 that distinguish them from the rest of the family: CDK4 bound to cyclin D and phosphorylated on the activation loop preserves “inactive” structural features observed in monomeric CDK2, suggesting a mechanism wherein only when both ATP and peptide substrate are engaged is the catalytically competent conformation formed ([19, 20]; Figure 1b).

We will review how CDK structural biology has assisted the design of CDK inhibitors, highlighting structural properties that have been exploited to provide selectivity. Accordingly, we will limit our discussion to inhibitor series for which structures bound to the target CDK have been determined.

The CDK ATP binding site

Conformational change is an essential element of protein kinase function. It has been hypothesized that the conformations that have been observed in protein kinase-inhibitor complexes represent snapshots of states that might exist through the catalytic cycle [21]. Inhibitors that capture the kinase target in particular conformational states have been categorized as type I, II, III or IV depending on whether they mimic the interactions of ATP with a catalytically competent kinase active site (DFG-in, type I); occupy the ATP binding site and stabilize a catalytically inactive DFG-out conformation (type II); are non-competitive with ATP and bind to an hydrophobic pocket adjacent to the ATP binding site (type III) or are allosteric (type IV), binding away from the ATP binding site [22].

The first type I CDK inhibitors whose development was informed by structural biology were targeted against CDK2 (Table S1, [23]). These structures illustrated how inhibitor binding can (i) satisfy the hydrogen bonding potential of the backbone Glu81 carbonyl and Leu83 amide moieties within the hinge sequence (which anchor the adenine N1 and N6 of ATP respectively), (ii) offer a planar moiety that mimics the adenine ring, (iii) fill the binding pocket to contact residues that do not contact ATP, and (iv) probe the ribose and phosphate binding sites (Figure 2).

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

Figure 2

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(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.

Dinaciclib (IC50 values against CDKs 1, 2, 5 and 9 of 3, 1, 1 and 4 nM respectively [8]) has undergone advanced phase III clinical trials and is currently being considered in combination therapies [24, 25]. The structure of dinaciclib bound to CDK2 illustrates the binding mode of this class of type I pan CDK inhibitor (Figure 2b, PDB 4KD1). It also reveals the challenges of achieving selectivity within the highly-conserved ATP binding site: many of the residues that directly contact ATP are identical among CDKs.

There are variations in the ways that adenine mimetics can bind to the hinge that links the N- and C-terminal lobes: 4-(1,3-Benzothiazol-2-yl)thiophene-2-sulfonamide (compound 4a, CDK5 IC50 551 nM) bound to CDK5-p25 makes a water-mediated interaction (PDB 4AU8, [26], Figure 2d), as does a series of 2, 4, 6-tri-substituted quinazolines that targets CDK2 (CDK2-compound 51, PDB 2B53, CDK2 IC50 0.6 ± 0.1 uM, [27]). 6-aza-benzothiophene-containing compounds targeting CDK8 (for example compound 22, IC50 CDK8 5.3 nM) form a single hydrogen bond to the hinge residue Ala100 (equivalent to CDK2 Leu83, PDB 5CEI, [28], Figure 2e), whereas DRB (5,6-dichlorobenzimidazone-1-b-D-ribofuranoside), a CDK9-selective inhibitor (CDK9 IC50 230 nM [29]) exploits halogen bonds (PDBs 3MY1, 4EC8, [29, 30], Figure 2f). The potential to make a third hydrogen bond to the backbone carbonyl equivalent of CDK2 Leu83 may be exploited to anchor and/or orientate the purine mimetic to optimize vectors to exploit sites beyond the adenine binding site (e.g. PDB entries 3DDQ and 1H1S, [31, 32], Figure 2c). Notably, although inhibitors make direct interactions with backbone moieties, sequence differences within the hinge appear to impact inhibitor potency and selectivity, presumably through effects on the relative orientations of the N- and C-terminal lobes and domain flexibility [33–35]. Such effects are difficult to rationalize, but are supported by results derived from CDK2 mutants in which the hinge sequence has been changed to that of CDK4 or CDK6 [36, 37].

The gatekeeper pocket

The CDK active site cleft is larger than is required for cofactor binding, and the additional space within the cleft has been widely exploited. At the back of the cleft, CDKs have a large gatekeeper residue (phenylalanine in all members except CDK10, 11A and 11B where it is a methionine) that can make multiple interactions. For example, edge-to-face aromatic-aromatic interactions are exploited in (i) a series of 4-anilinoquinazolines targeting CDK2 (PDB 1DI8, [38]), (ii) 7-azabenzimidazoles (e.g. CDK6-compound 3, PDB entry 4EZ5, IC50s vs CDK4/6 12 ± 2/300 ± 100 nM, >15 uM vs CDK1/6.2 ± 0.4 uM CDK2, [35], Figure 3a), and 4-(pyrazol-4-yl)-pyrimidines targeting CDK4/6 (e.g. CDK6-compound 37 PDB 3NUP; IC50 CDK4-cyclin D1 12 ± 1 nM; [39]), and (iii) pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidine derivatives characterized as dual FLT3/CDK4 inhibitors (e.g. CDK6-compound 1, PDB 4TTH, FLT3 IC50 14 nM/CDK4-cyclin D1 IC50, 2 nM, [40]).

Figure 3. The gatekeeper pocket.

Figure 3

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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.

Favorable halogen-aromatic interactions are made in a quinazoline series that targets CDK2 (PDB 2B53, [27]), in the 4-(pyrazol-4-yl)-pyrimidines series mentioned above that targets CDK4/CDK6 (CDK6-compound 50 PDB 3NUX, CDK4-cyclin D1 IC50 11 nM; [39]), in CCT251545 that targets CDK8, (PDB entry 5BNJ, [41]) and CCT251921 that targets CDK8 (IC50 2.3 ± 0.8 nM) and CDK19 (IC50 2.6 ± 0.4 nM, PDB 5HBJ [42], Figure 3b). The sulfur atom of the thiadiazole scaffold in a CDK8-compound 6 structure (PDB 5ICP, CDK8 IC50 3.8 ± 1.9 nM [43], Figure 3c) has also been reported to interact with the phenylalanine sidechain. The thio-methylene moiety in a series of 2-amino-5-thioalkyl-substituted thiazoles bound to CDK2 occupies a hydrophobic pocket to which Phe80 contributes (PDB 4LYN, [44].

A preference for type I over type II inhibitors derives in part from the bulky character of the gatekeeper residue in CDKs. Mutation of this residue to a smaller amino acid allows access to the back pocket and has been used in conjunction with modified ATP or inhibitors to respectively identify CDK substrates and as a probe for CDK function [45, 46]. Many inhibitor series build from this region to make either direct or water-mediated interactions with the conserved lysine-glutamate pair (Lys33 and Glu51 in CDK2) that coordinates the ATP alpha-phosphate group. Filling this back part of the cleft with small, branched aromatic or halogen-rich moieties is common feature of a number of inhibitor series. Notably larger moieties can flip the inhibitor binding mode offering alternative options for inhibitor design from a conserved scaffold.

The DFG motif and back pocket remodeling

The character of this region of the active site depends crucially on the DFG conformation [22, 47]. The first observation of a DFG out cyclin-bound CDK structure was that of CDK8 bound to cyclin C and sorafenib (Figure 4a, PDB 3RGF [48]), a type II inhibitor of other protein kinases [49]. Starting from sorafenib, a series of CDK8 inhibitors stabilizing the DFG-out (DMG-out in CDK8) conformation were developed (e.g. CDK8-cyclin C-compound 20, CDK8 IC50 17.4 nM, PDB 5HVY, [50]). CDK8 demonstrates flexibility in this part of the structure as apo-CDK8-cyclin C has a DMG-in structure (PDB 4F7S, [51]). A number of CDK8-cyclin C-type I inhibitor co-complexes have been reported, including CDK8-cyclin C bound to cortistatin A (PDB 4CRL, [52], Figure 4b) and a series of azabenzothiophene derivatives (PDB 5CEI, [28]. Reflecting their close phylogenetic relationship, a number of type I inhibitors have been developed that are selective for CDK8 and CDK19. These include CCT251545 (CDK8-Kd 2 nM, PDB 5BNJ, [41]), 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based ligands [53] and a series of compounds based on a 3-methyl-1H-pyrazolo[3,4-b]-pyridine scaffold (e.g. MSC2530818, CDK8 IC50 2.6 ± 0.1 nM, PDB 5IDN, [43]).

Figure 4. The DFG motif and back pocket remodeling.

Figure 4

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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.

Prolonging the engagement of an inhibitor with its target can lead to improved pharmacokinetic (PK) properties and greater efficacy [54, 55], and has been reported as a characteristic of type II protein kinase inhibitors that target tyrosine kinases [47]. However, within a set of inhibitors that elaborated a pyrazole urea based scaffold (PDBs 4F6S, 4F7J, 4F70, 4F6U, 4F7N, 4F7L and 4F6W, [51]) residency time did not track with DMG conformation, but rather was hypothesized to derive from interactions with the hinge and the “selectivity surface” on the kinase C-terminal lobe. However, some PK optimization of CDK inhibitors has been rationalized by structural insights. Roniciclib (BAY 1000394) is a type I pan CDK inhibitor which exhibits kinetic selectivity for CDK2 and CDK9 [56]. CDK2 showed a DFG loop adaptation as a response to the presence of a 5-(trifluoromethyl) substituent, but not of a hydrogen or bromine atom substitution (Compare PDBs 5IEV and 5IEX). A distinguishing feature of the trifluoromethyl-substituted inhibitor-bound CDK2 structure was the network of water molecules between the inhibitor moiety, the DFG motif and the gatekeeper, Phe80. The DFG conformation was distinct from the DFG-out conformation characteristic of other protein kinases, but had been previously observed in the CDK2-R547 structure (CDK2 Ki 1 nM, PDB 2FVD, [57]).

The ATP ribose phosphate binding pocket

There are fewer reports of rational SAR where interactions offered by the ribose-phosphate binding pocket have been targeted. This part of the active site is composed of a number of flexible loop regions (Figure 5a) making rational design difficult: molecular dynamics simulations conducted on CDKs and CDK-cyclin complexes have illustrated that the glycine-rich lid is highly dynamic in nature [58]. The determination of the structure of CDK5 bound to p25 (PDB 1H4L, [59]) revealed that its active site is very similar to that of CDK2. However, there is local restructuring upon both (R)-roscovitine (PDB 1UNL, [60]) and compound 1.0 binding (PDB 3O0G, [61]) to yield an unusual glycine-rich loop conformation, that presumably reflects the CDK5-specific aspects of the sequence in this region (Figure 5b). Similarly, binding of an aminopurine derivative bearing a bulky biphenyl substituent at the 6-position has been observed to stabilize a glycine-rich loop conformation that is preferred in CDK2 (PDB 5LQE) and that has not been observed in CDK1 (PDB 5LQF) [62]. Though difficult to rationalize, the circa 2000-fold selectivity of this biphenyl derivative for CDK2 over CDK1 must derive from differences in conformational preferences supported by the CDK1 or CDK2 folds.

Figure 5. The ATP ribose phosphate binding pocket.

Figure 5

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(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).

Within a series of 4-(thiazol-5-yl)-2- (phenylamino)pyrimidine-5-carbonitriles, a comparison of inhibitor binding to CDK2 and CDK9 (E.g. compare binding of compound 12u, CDK2 and CDK9 Kis of 568 nM and 7 nM and PDBs 4BCP and 4BCG respectively [63]) illustrates how, despite a broadly conserved binding pose, subdomain movements and local conformational flexibility around the CDK9 active site can drive compound selectivity (Figure 5c, [63, 64]). Comparing the structures of CDK9-cyclin T bound to a more diverse inhibitor set reveals that significant movements of the glycine-rich loop (and also of the beta3-alphaC loop) frequently accompany potent CDK9 inhibitor binding ([29, 65, 66], Figure 5d).

The selectivity surface

To discriminate more effectively between CDKs, the sequence differences immediately outside the active site on the surface of the C-terminal lobe can be probed by extending out from the purine binding site. This surface is quite different in character between CDK1/2 (PDBs 4YC3/1JST), CDK4/6 (PDBs 2W96/1JOW) and CDK8/9 (PDBs 3RGF/3BLQ), (as summarized in Figure 1c).

Various CDK4/6-selective inhibitor series exploit the surface, for example offering substituted piperazine moieties capable of favorable polar interactions with the hydroxyl side chain of CDK4 Thr102 or CDK6 Thr107, but being repulsed by the charge on CDK2 Lys89 [40]. Palbociclib (PDB 2EUF, [34], Figure 6a) and ribociclib (PDB 5L2T, [67], Figure 6b) bound to monomeric CDK6 nicely illustrate how this surface can be exploited. Together with neighboring amino acids, the peptide chain around Lys89 shapes a groove that can be exploited to derive CDK1/2 selectivity. As examples, the 4’-sulfamoylanilino group present in NU6102 sits within this pocket and makes hydrogen bonds to the sidechain carboxylate and peptidic nitrogen of Asp84 (PDB entry 1H1S, [32], Figure 5c). CDK2 Lys89 has also been targeted by a covalent strategy [68]. The flexibility of the CDK9 sequence following the hinge accommodates the bulky, substituted aniline moieties that drives compound selectivity in a series of substituted 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitriles (PDB entries 4BCF, 4BCH, 4BCI, 4BCJ, 4BCM, 4BCN, 4BCK, 4BCO, 4BCQ, [63, 64], Figure 5d). Coupled to groups that probe sequence differences, the most successful compounds have identified a vector from the purine mimetic such that the conformation of the bound inhibitor is relatively strain-free and entropically favored because of pre-organization of the free ligand.

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

Figure 6

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(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).

CDK4/6 lend themselves to selective inhibition and show clear disease linkage

CDK4 and CDK6 are the best validated CDK targets in the largest number of clinical settings [17]. Although no structures have been reported for such inhibitors bound to CDK4/6-cyclin D complexes, drug discovery programs have developed CDK4/6-selective inhibitors of which two, ribociclib [69] and palbociclib [33] are approved for the treatment of hormone receptor positive and human epidermal growth factor receptor-2 negative (HR+/HER2−) breast cancer. This success has been driven in part by the ability to identify patients where enhanced CDK4 or CDK6 activity is a driver of cancer progression (http://cancer.sanger.ac.uk/cosmic). It can also be hypothesized that the unusual plasticity of CDK4, and potentially also that of CDK6, allows the development of potent and selective inhibitors despite the high degree of sequence conservation within CDK active sites. However, it is worth noting that the responses of CDK4 and CDK6 to cognate cyclin binding may differ. CDK4 does not adopt a fully active conformation upon cyclin D and activation segment phosphorylation (PDB 2W96, [20], whereas viral cyclin binding drives CDK6 to adopt an active conformation in which the activation segment is not phosphorylated but would be predicted to accommodate a peptide substrate (PDB 1JOW, [70]).

In the absence of structures for inhibitors bound to active CDK4/6-cyclin D, it is difficult to rationalize inhibitor properties. However, structures were used to guide optimization of a series of 4-(pyrazol-4-yl)-pyrimidines as CDK4/6 inhibitors using monomeric CDK6 as the template (E.g. CDK6-compound 50, CDK4-cyclin D1 IC50 11 nM, [39]). Structures of monomeric CDK6 bound to several clinical candidates have now been determined (PDB entries (5L2S, abemaciclib; 5L2W, dinaciclib; 5L2I, palbociclib; 5L2T, ribociclib, [67]). More detailed rationalization of their inhibitory properties awaits their structure determination bound to a CDK4/6-cyclin D complex.

The C-terminal tail

The transcriptional CDKs are characterized by extended C-terminal sequences beyond the conserved kinase domain. Though few inhibitor co-complex structures have been determined for this transcriptional CDK sub-class, emerging data shows how the C-terminal tail can impact the character of the ATP binding site. The CDK9 tail shapes the catalytic cleft and its conformational cycle results in an ordered binding of substrates and release of products (PDB 4EC8, [30]). It is exploited by the CDK9 inhibitor 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB) that is a more potent inhibitor of full-length CDK9 than C-terminally truncated variants. DRB binding locks the CDK9 N- and C-terminal lobes in an orientation that favors the ordering of the C-terminal sequence (Figure 7a). Similar trapping of AMP-PNP in a closed state assisted by residues located within the C-terminal extension is observed in the structure of a CDK12-cyclin K-AMPPNP complex (PDBs 4NST, [71]; and 4CXA, [72]). Although there are no deposited structures for CDK13-cyclin K bound to ATP-competitive inhibitors, the CDK13 structure reveals a similar C-terminal helix that extends into the active site where it interacts with ATP (PDB 5EFQ, [73]). The CDK8 C-terminal tail also reaches up into the active site and, in the presence of the ATP-competitive inhibitor CCT251545 (a 3,4,5-trisubstituted pyridine) makes a favorable cation-pi interaction between the phenyl ring of the inhibitor and the guanidine moiety of Arg356 (PDB 5BNJ,[41], Figure 7b). Indeed, representatives from diverse chemotypes have now been crystallized with CDK8-cyclin C and, irrespective of the orientation of the DMG sequence, significant interactions are made with the C-terminal tail sequence.

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

Figure 7

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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).

CDK7, CDK12 and CDK13 contain cysteine residues within their extended C-terminal sequences. These residues offer the possibility of targeting by covalent inhibitors and, as they are outside the kinase core fold, achieving greater selectivity. THZ1 is a potent and selective chemical probe targeting CDK7 [74] and covalently interacts with Cys312, a residue that is not built in the CDK7 crystal structure (which terminates at Asn311, PDB 1UA2, [75]). CDK12 and CDK13 have a cysteine residue within 4 residues of CDK7 Cys312 and THZ1 does indeed inhibit CDK12 and CDK13 at higher concentrations [74]. The structure of the THZ531 bound to CDK12-cyclin K reveals the re-arrangements within the CDK12 C-terminal lobe that re-orients the cysteine sidechain (Cys1039) to permit the covalent interaction to form (PDB 5ACB, [76], Figure 7c). Indeed THZ (and derivatives) is proving to be a useful tool compound to delineate transcriptional CDK activity in defined cellular settings [76].

Targeting the monomeric enzyme

Unconstrained by catalytic requirements, CDKs are more diverse in structure when monomeric than cyclin-bound and, correspondingly, show less well conserved patterns of inhibitor binding [77]. Such observations have prompted studies to identify type II and type III inhibitors that can stabilize monomeric CDKs in conformations that are incompatible with catalytic activity and/or cyclin binding.

Most progress towards this aim has been made with CDK2. Although it was originally thought that CDK2 would not be amenable to adopting a DFG-out structure, judicious screening identified an aminopyrimidine-phenyl urea inhibitor (K03861) that stabilized such a conformation [78]. This conformation proved to be competitive with cyclin binding and to have a slow koff, i.e. to have characteristics consistent with a type III inhibitor.

Moving away from the active site, high concentrations of 8-anilino-1-naphthalene sulfonate (ANS) can drive formation of a large pocket that accommodates two adjacent ANS molecules, extending from the DFG region to above the C-helix [79]. The shift in the C-helix position was predicted to be incompatible with cyclin binding, a hypothesis confirmed by competitive binding studies (Figure 8a).

Figure 8. Targeting the monomeric enzyme.

Figure 8

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(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).

In another approach, a high throughput screen has identified compounds that bound to inactive unphosphorylated monomeric CDK2 rather than phosphorylated CDK2-cyclin A. A type I.5 quinolone-based inhibitor (DFG-in, occupying the ATP-binding site and adjacent non-canonical pockets, compound 14) was subsequently developed with a Kd of 5 nM, determined using a temperature-dependent circular dichroism assay [80]. The binding mode of this series is illustrated by a CDK2-compound 2 complex structure (Kd 300 nM). In this structure, the hydroxyphenyl moiety of the inhibitor binds to the hinge, the DFG motif is in the “in” conformation, and a quinolone 3-chlorophenyl group sits in a hydrophobic pocket under the C-helix. Occupation of this pocket translates and rotates the C-helix into a position incompatible with cyclin association (PDB 4NJ3, Figure 8b). Inhibitors that block cyclin binding might be expected to exert different cellular effects from those that sequester cyclins into an inactive complex. Whether such differences in activity translate into novel therapeutic possibilities awaits the identification of cell active type III CDK inhibitors.

Conclusions

With palbociclib and ribociclib now approved for use in therapy, the tractability of this target sub-family is now firmly established, paving the way for further clinical development targeting other CDKs. Results suggest that development of all kinase inhibitor types (I through IV) may be possible for the CDK sub-family, and have identified static and plastic properties of CDKs and their inhibitors which can provide a sufficient degree of selectivity for use in oncology and, potentially other clinical settings.

Supplementary Material

Supplementary Tables

Summary points.

  • Type I, “1.5”, II and III inhibitors have been described for CDKs

  • Type I inhibitors engage the hinge through a range of hydrogen and/or halogen-bonding motifs

  • CDKs have a bulky gatekeeper which can contribute to inhibitor binding or be manipulated to permit selective inhibition/co-factor utilization

  • Plastic rearrangements of the glycine lid may play a role in inhibitor selectivity by allowing read out of sequence differences remote from the active site

  • A “selectivity surface” adjacent to the ATP-binding site differs in character in different CDK subfamilies, and can be predictably targeted to provide a degree of selectivity

  • The C-terminal tail can contribute to inhibitor contacts in transcriptional CDKs

  • A subset of CDKs may be targeted by covalent, thiol and amine-reactive inhibitors

Acknowledgements

We thank Cancer Research UK (grant no. C2115/A21421) and the Medical Research Council (grant no. MR/N009738/1) for financial support.

Abbreviations list

CDK

cyclin-dependent kinase

CBF

cyclin box fold

PK

pharmacokinetic

Footnotes

Declarations of interest

The authors declare that there are no competing interests associated with the manuscript.

Author contribution statement

All authors contributed to the review of the literature and PDB, and to the writing of this article.

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