Cyclin Dependent Kinase 2 Inhibitors in Cancer Therapy: an Update

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Cyclin Dependent Kinase 2 Inhibitors in Cancer Therapy: an Update Solomon Tadesse, Elizabeth Caldon, Wayne Tilley, and Shudong Wang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01469 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 13, 2018

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Journal of Medicinal Chemistry

Cyclin Dependent Kinase 2 Inhibitors in Cancer Therapy: an Update Solomon Tadesse1, Elizabeth C. Caldon,2,3 Wayne Tilley4 and Shudong Wang1* 1Centre

for Drug Discovery and Development, University of South Australia Cancer Research Institute, Adelaide, SA 5000, Australia.

2Garvan

Institute of Medical Research, The Kinghorn Cancer Centre, Darlinghurst, NSW 2010, Australia.

3St

Vincent’s Clinical School, UNSW Medicine, UNSW Sydney, Darlinghurst, NSW 2010, Australia. 4Adelaide

Medical School, University of Adelaide, Adelaide, SA 5000, Australia.

1 ACS Paragon Plus Environment

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ABSTRACT

Cyclin-dependent kinase-2 (CDK2) drives the progression of cells into the S and M phases of the cell cycle. CDK2 activity is largely dispensable for normal development, but it is critically associated with tumor growth in multiple cancer types. Although the role of CDK2 in tumorigenesis has been controversial, emerging evidence proposes that selective CDK2 inhibition may provide therapeutic benefit against certain tumors, and it continues to appeal as a strategy to exploit in anticancer drug development. Several small-molecule CDK2 inhibitors have progressed to the clinical trials. But, a CDK2-selective inhibitor is yet to be discovered. Here, we discuss the latest understandings of the role of CDK2 in normal and cancer cells, review the core pharmacophores used to target CDK2, and outline strategies for the rational design of CDK2 inhibitors. We attempt to provide an outlook on how CDK2-selective inhibitors may open new avenues for cancer therapy.

BACKGROUND The cell division cycle is a fundamental process in life where series of events occur in a cell resulting in the formation of two identical daughter cells.1 It governs the transition from quiescence or cytokinesis to cell proliferation, and through its checkpoints, ensures genome stability.2 Cell division cycle involves four sequential phases (Figure 1).3 S phase, when DNA replication occurs, and M phase, when the cell divides into two daughter cells, are separated by gaps known as G1 and G2. In G1, cells undertake most of their growth and synthesize proteins, RNAs and organelles needed for DNA synthesis, whereas in G2 the microtubules that will be used to mobilize the chromosomes in M phase are assembled. Quiescence (G0) represents exit from the cell cycle either


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due to deprivation of mitogen or full differentiation of the cell (e.g., heart muscle cells and neurons).4 Most adult cells are at G0 and the transcriptional activity of E2F transcription factors (E2Fs) is repressed by the retinoblastoma proteins (hereafter called Rb).4 When needed, these cells can go back into the cell division cycle.5, 6 Briefly, cells at G0 enter G1 due to mitogenic stimuli. This requires CDK3-cyclin C, which phosphorylates Rb at Ser807/811.7 During G1, D-type cyclins bind and activate CDK4 and/or CDK6, also resulting in partial phosphorylation of Rb, leading to the activation of E2Fs. At this stage, E2Fs remain bound to Rb, but are able to transcribe genes such as CCNE1, CCNA2, CCNB1, CDK2 and CDK1. In late G1 (after the restriction point) cyclin E binds to CDK2 to further phosphorylate Rb, releasing and fully activating the E2Fs.4-6 E2Fs then trigger the transcription of S phase proteins such as cyclins A and E.

4-6

CDK2-cyclin A,

CDK1-cyclin A and CDK1-cyclin B then sustain the phosphorylation of Rb ensuring cell cycle progression. CDK2-cyclin A facilitates S/G2 transition, and CDK1-cyclin A and CDK1-cyclin B enable the commencement of mitosis and the progression through M phase, respectively (Figure 1). Finally, cyclin B is degraded, and Rb is dephosphorylated by two phosphatases, PP1 and PP2A, returning the cell to G1 state.5, 6, 8 Intriguingly, animal models have demonstrated that CDK2, CDK4 and CDK6 (interphase CDKs) or their cyclin counterparts are not essential for proliferation of non-transformed cells and development of most tissues.9 On the other hand, deregulation of CDKs has been reported to cause unscheduled proliferation, genomic and chromosomal instability resulting in human cancer, and to contribute to both cancer progression and aggressiveness.10 Additionally, many cancers are uniquely dependent on CDKs and hence are selectively sensitive to their inhibition.11 In this regard, the most successful clinical approach to date has involved



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targeting CDK4 and CDK6 where three CDK4/6-selective inhibitors, namely palbociclib, abemaciclib and ribociclib are approved for treatment of breast cancer.12 There are several excellent reviews on the CDK area that include some aspects of CDK2.13-16 But, an updated review comprising the biology of CDK2 and the medicinal chemistry of its inhibitors in conjunction with approaches for designing of CDK2-selective inhibitors is lacking. Thus, this review focuses on the role of CDK2 in non-transformed and cancer cells, the rationale for developing CDK2-targeted cancer therapy, as well as on the design and future therapeutic potential of CDK2-selective inhibitors in cancer treatment.

Figure 1. An overview of the cell division cycle, and the role of CDKs and checkpoints. In cells, DNA replicates in S phase, and chromosome segregation occurs at M phase. Two gap phases separate S phase and M phase: G1 when cells grow and synthesize proteins, and G2 when cells prepare for mitosis. CDK3-cyclin C stimulates Rb phosphorylation to effect G0/G1 transition. 4 ACS Paragon Plus Environment

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CDK4/6-cyclin D and CDK2-cyclin E mediated sequential phosphorylation of Rb relieves suppression of the activity of the E2Fs allowing G1/S transition through the restriction point. As cells prepare to exit from S phase, CDK2-cyclin A directly phosphorylates E2F to deactivate its function preventing apoptosis that might be triggered by persistent E2F activity. CDK1 in complex with cyclin A or B has defined roles in regulating the G2/M checkpoint and progression through mitosis. The cell cycle is controlled by checkpoints. The integrity of the DNA is assessed at the G1/S checkpoint. Proper chromosome replication is checked at the S and G2/M checkpoints. Attachment of each sister chromatids to a spindle fiber is evaluated at the spindle assembly checkpoint (SAC). STRUCTURE AND REGULATION Constituting a major part of phosphotransferases in the human genome, kinases catalyze the reversible transfer of the γ-phosphate group of ATP onto a target substrate, mediate signal transductions and regulate most aspects of cell life.17 Currently, about 518 human protein and 20 lipid kinases have been identified. Protein kinases are enzymes that play key regulatory roles in nearly every aspect of cell biology, and based upon the nature of the target amino acid in their substrates, they are classified as tyrosine kinases, serine/threonine kinases, dual specificity kinases (act as both tyrosine and serine/threonine kinases), and histidine kinases. The phosphorylation of Ser, Thr, or Tyr residues of proteins by kinases results in conformational change altering the activity of the protein substrates.18 CDKs belong to the serine/threonine protein kinase family and their kinase activity requires binding to a cyclin protein.19 They are involved in various aspects of cell biology notably in cell cycle control (CDKs 1, 2, 3, 4 and 6, see above), transcription (CDKs 7, 8, 9, 12 and 13) regulation through phosphorylation of C-terminal tail of RNA polymerase II, metabolism (CDKs 1, 2, 3, 4 5 ACS Paragon Plus Environment


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and 6),20 and in certain cell types, differentiation (CDKs 1, 2 and 4).21 Although CDKs are commonly grouped into cell-cycle or transcriptional CDKs, these roles are frequently combined in many CDKs.19 CDK7 indirectly regulates the cell cycle by activating CDKs 1, 2, 4 and 6. CDKs 5, 10, 11, 14–18 and 20 have heterogeneous and unique functions that are frequently tissuespecific. For example, CDK5 has a pivotal role in modulating the migration of post-mitotic neurons.22 CDK10 is implicated in regulating gene transcription by steroid hormones by promoting the interaction between heat-shock proteins and the ecdysone receptor EcRB1.23 CDK11-cyclin L regulates RNA splicing.24 Among CDKs, sequence and structure similarity is high (Table 1). For instance, there is 74% sequence identity between CDK2 and CDK3, while root-mean-square deviation of Cα atoms ranges from 1.7 Å for CDK4 to 0.9 Å for CDK5.25 In addition, their convergence to a conserved structure upon activation has presented challenges for the design of selective inhibitors.26 Yet, the available structural diversity and conformational plasticity of the CDK fold have been successfully exploited to fine tune potency and selectivity and to identify the first CDK inhibitors to be registered for clinical use targeting CDK4 and CDK6. However, most inhibitors still exhibit substantial activity for a subset of the family.27 Table 1. Percent (%) sequence similarity between CDK2 and other CDKs* CDK

% sequence identity

CDK3

74

CDK1

65

CDK5

58

CDK6

44

CDK4

43 6 ACS Paragon Plus Environment

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CDK7

38

CDK20

37

CDK10

36

CDK18

33

CDKs9, 14, 15 & 16

32

CDK17

30

CDK8

24

CDK19

23

CDK11

16

CDKs12 & 13

9

*Sequence alignments were performed and % sequence similarity determined by using UniPort database (http://www.uniprot.org/align/). CDK2, similar to other protein kinases, has the classic bilobal architecture, N-terminal lobe (residues 1-82) and the C-terminal domain (residues 83-297) (Figure 2A).18, 19, 28, 29 The smaller N-terminal lobe is mainly made up of β-sheets (five anti-parallel β-strands) with one αC-helix (PSTAIRE). The αC-helix contains the sequence PSTAIRE, and is essential for cyclin binding (Figure 2B). The larger C-terminal lobe is rich in α-helices, and contains the activation segment (also known as the T-loop (residues 145(Asp)-172(Glu)) and the activating phosphorylation site Thr160. The T-loop is the platform for binding of the Ser/Thr (phosphor-acceptor) region of substrates for phosphorylation. The N-terminal and C-terminal lobes are connected by the flexible hinge region (residues 81(Glu)-84(His)), which lines a deep cleft, the ATP-binding site. ATP recognition involves residues from both lobes. CDK2 offers adjacent binding sites for ATP and the phospho-acceptor protein substrate so that the γ-phosphate of ATP faces the hydroxylated side chain of Ser/Thr on the substrate surface.



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Extensive biochemical and structural studies have established a clear picture of the activation and regulatory mechanisms that determine the activity of CDK2.18, 19, 29 In the absence of mitogenic signals, CDK2 is in an inactive form. During late G1 phase, CDK2 activity increases as a result of (1) E2F-mediated transcription of CCNE genes, the protein product of which binds and activates CDK2, (2) CDK4/6-cyclin D-mediated sequestration of the CDK-interacting protein/kinase inhibitory protein (Cip/Kip) class of CDK inhibitors, p21Cip1, p27Kip1 and p57Kip2, which bind to CDK2-cyclin complexes and render them inactive, and (3) due to ubiquitin-mediated proteolysis of Cip/Kip following their phosphorylation by CDK2.18, 19, 29 The Cip/Kip family of inhibitors change the shape of the catalytic cleft of CDK2 to completely deactivate the enzyme by inserting a small helix inside the catalytic unit in away similar to ATP (Figure 2C). Cyclins E and A regulate CDK2 activity by being synthesized and destroyed in cell cycle phase-specific manner.18, 19, 29 The Skp/Cullin/F-box containing complex (SCF) mediates the rapid proteasomal degradation of cyclin E during S phase and CDK2 associates with newly synthesized cyclin A to form active CDK2cyclin A complexes. Cyclin A is stable throughout interphase, and is degraded by the anaphasepromoting complex/cyclosome (APC/C) ubiquitination just before the metaphase to anaphase transition.18, 19, 29 Once cyclin A is disassociated or degraded, dephosphorylation of Thr160 (see below) is executed by a Ser/Thr-directed phosphatase, CDK-interacting phosphatase (KAP).30 Cyclins E and A in concert with phosphorylation by CDK-activating kinase (CAK, CDK7-cyclin H-MAT1 complex) play a critical role in the regulation of CDK2 (Figure 2D).

28, 29

Although

cyclin-binding alone confers enzymatic activity on an intrinsically inert CDK2 monomer, T-loop phosphorylation results in ∼300-fold increase of activity towards a substrate. 31, 32 Upon binding to its cyclin partner, CDK2 changes its conformation (Figure 2B).28, 29 Extensive hydrophobic interactions between CDK2 and its cyclin partner move the αC-helix on the N-lobe towards the 8 ACS Paragon Plus Environment

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catalytic cleft. This conformational change moves the side chain of Glu51 of the αC-helix into an inside position favoring a hydrogen bond between Glu51 and Lys33 allowing Lys33 to bind to the α- and β-phosphates of ATP and align them to enable the phospho-transfer reaction of the γphosphate to substrate proteins.

29

Additionally, cyclin binding relieves the obstruction at the

entrance of the active site by moving the T-loop by 20 Å towards the cyclin and displacing onto the C-terminal lobe, leaving the ATP binding site accessible to substrates. 28, 29 Moreover, cyclin binding was previously thought to be required to expose the buried Thr160 of monomeric CDK2 for phosphorylation by CAK, and this phosphorylation was believed to lead to further conformational changes in the substrate binding site of CDK2 for the full activation of CDK2cyclin complex. 28, 29 However, CAK efficiently phosphorylates monomeric CDK2 (Figure 2E). 31, 32

During the S-phase of the cell cycle, in order to surpass the competition for cyclin A from the

more abundant CDK1, Thr160 phosphorylation of CDK2 precedes cyclin A-binding. This is because of CAK’s inability to phosphorylate monomeric CDK1 contributing to a kinetic barrier preventing CDK1-cyclin A assembly. Phosphorylation of the glycine-rich loop (residues 11(Glu)18(Tyr)) residues Thr14 and Tyr15 by Wee1 and Myt1 kinases, respectively, which can be reversed by the cell division cycle 25 (Cdc25) phosphatases (Cdc25A, Cdc25B and Cdc25C) turns off CDK2 activity (Figure 2D and E). 17, 28, 29



Figure 2. Structural basis of CDK2 activation and inhibition. A. Non-activated monomeric CDK2 (PDB: 4EK3). The N-lobe β-sheets and the αC-helix (PSTAIRE) are shown in pink and blue, respectively; the C-lobe is indicated in purple; the hinge region and the T-loop in green. B. Fully active (phosphorylated CDK2-cyclin A complex, PDB: 1JST). Cyclin A and the activating phosphorylation site Thr160 are depicted in cyan and yellow, respectively; ATP is displayed in orange sticks bound in the deep cleft between the two domains. CDK2 apoenzyme is inactive and 10 ACS Paragon Plus Environment

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full activation entails complex formation with cyclins E or A, and phosphorylation of the Thr160 residue. During the activation, the T-loop moves towards the C-terminal domain where it forms a binding area for the substrate protein, and the αC-helix moves into the binding cleft and is rotated. C. Inhibited p27-Thr160 phosphorylated CDK2-cyclin A complex (PDB: 1JSU). The N-terminal of p27-peptide (shown in yellow) binds with cyclin A, and its C-terminal binds to the N-terminal domain of CDK2 to induce structural changes rendering CDK2-cyclin complex inactive. D. Mechanisms of CDK2 regulation. The activity of CDK2 is regulated by four mechanisms. The first level of regulation involves the binding of CDK2 to cyclin E or A, resulting in partially activated CDK2-cyclin E or CDK2-cyclin A complex. Second, the full activation of CDK2-cyclin E or A complexes necessitates the phosphorylation of Thr160 by CAK. The third mechanism includes inhibitory phosphorylation of Thr14 and Tyr15 by Wee1 and Myt1 kinases, respectively. Dephosphorylation of these residues by members of the Cdc25 family of protein phosphatases reactivates CDK2. Fourth, CDK2 is deactivated by the binding of CDK inhibitory protein families Cip and Kip. Cyclins E and A are destroyed by the Skp/Cullin/F-box containing complex (SCF) and the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligases. E. During S phase, CDK2 can follow a distinct path to activation in which T-loop phosphorylation precedes cyclinbinding. Dephosphorylation of CDK2 Thr160 by the Cyclin-Dependent Kinase-Interacting Phosphatase (KAP) occurs in the absence of cyclin A. BIOLOGICAL ROLES AND SUBSTRATES Most normal tissues have low expression of CDK2.33, Such observation is supported by the fact that, with the exception of few tissues that have a functional need for constant proliferation, the majority of normal cells are found in a state of quiescence.433, Among the exceptions to this is the high CDK2 activity observed in testes where CDK2 is thought to play a unique role in meiotic cell 11 ACS Paragon Plus Environment


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division mirroring the essential meiotic functions of CDK2 in mice.33, 34 CDK2-deficient mice are unable to undergo meiotic division of gametes and are thus sterile. Since kinase-dead CDK2 protein was not capable to drive normal meiotic cell division in vivo, CDK2 is proposed to regulate meiosis by phosphorylating the yet to be determined protein substrates.34 In dividing cells CDK2 is a core cell cycle component that is essentially active from late G1-phase and throughout the Sphase. Amongst the key CDK2 substrates during G1/S progression is the Rb (see above). Rb contains 16 sites for phosphorylation by the CDKs that have been characterized as either specific for CDK4/6, CDK2, or able to be phosphorylated by combinations of these kinases.35 In actively cycling cells, the initial monophosphorylation of Rb in early G1 phase is catalyzed by CDK4/6, and occurs on any of 14 sites. Subsequently, CDK2 activation leads to hyperphosphorylation and complete inactivation of the Rb protein.35 Beyond Rb, CDK2 governs the phosphorylation of a wide range transcription factors including mothers against decapentaplegic homolog 3 (SMAD3),36 forkhead box protein M1 (FOXM1),37 forkhead box protein O1 (FOXO1),38 the helix–loop–helix protein inhibitor of DNA binding 2 (ID2),39 as well as upstream binding factor (UBF),40 nuclear factor Y (NFY)41, Myb-related protein B (B-MYB)42 and Myc proto-oncogene protein (MYC),43 which contribute to cell cycle progression at different levels. Besides these cell cycle targets, it is understood that CDK2 plays a role in mammalian DNA replication,44 adaptive immune response,45 cell differentiation46 and apoptosis.47, 48 RATIONALE FOR TARGETING CDK2 CDK2 is a critical modulator of various oncogenic signaling pathways, and its activity is vital for loss of proliferative control during oncogenesis.49 In addition, the overexpression of CDK2 binding partners cyclin A and/or E is key oncogenic process in several cancers.50, 51 It has also been shown that cyclin E-deficient cells are resistant to oncogenic transformation.52 cyclin E 12 ACS Paragon Plus Environment

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overexpression promotes tumor formation in mice and it correlates with poor prognosis in patients with several tumor types.53,

54

Developing small molecules that can directly target cyclins is

implausible, because these cyclins act as regulatory subunits rather than as an enzyme or receptor. Thus, given the relative specificity of cyclin E’s for CDK2 and its deregulation in certain types of cancer, CDK2 is an attractive target in treating tumors of specific genotypes. Initial interest for CDK2 as a cancer therapeutic target was tempered to some extent by the knowledge that CDK2 inhibition using anti-CDK2 shRNA, antisense oligonucleotides, a dominant-negative CDK2, or overexpression of p27Kip1 failed to arrest the proliferation of colon cancer cells.55 In addition, genetic ablation of CDK2 did not appear to have a negative effect on cellular proliferation during early murine development.9 These methods, however, result in ablation of CDK2 protein expression, possibly allowing for compensation by other CDKs, and they are therefore likely to have different effects than acute inhibition of CDK2 kinase activity using small molecules. Besides, as these studies have been carried out in vitro and in mice, the requirement of CDK2 for humans cannot be completely ruled out. Examination of different kinds of human cancers, with defined molecular features, for their susceptibility to CDK2 inhibition has unveiled the scope in which CDK2 might represent a good therapeutic target. For example, in ovarian cancer with amplified CCNE1 expression,51 in MYCNamplified neuroblastoma cells,56 KRAS-mutant lung cancers57 and several cancers with FBW7 mutation and cyclin E1 overexpression,58 CDK2 is a therapeutic target. In glioblastoma59 and Bcell lymphoma,60 CDK2 is highly expressed and is functionally required for cell proliferation. In prostate cancer, CDK2 is significantly associated with metastasis.61 CDK2 contributes to breast cancer progression by phosphorylating and activating hormone receptors,62-64 and it is a target in hepatocellular carcinoma.65 In acute myeloid leukemia, CDK2 inhibition drives differentiation in 13 ACS Paragon Plus Environment


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cell lines and patient derived-primary samples.46 CDK2 is a transcriptional target of melanocyte lineage transcription factor (MITF) and it is critical for growth of melanoma cells. Expression levels of MITF and CDK2 are tightly correlated in primary melanoma specimens and predict susceptibility to the CDK2 inhibition.66 Taken together, CDK2 plays an essential role in tumor growth and CDK2 inhibitors have the potential to induce growth arrest and apoptosis in cancer cells. In line with these observations, CDK2 knockout mice are viable without apparent abnormalities suggesting that CDK2 inhibitors might preferentially target cancer cells while sparing normal tissues.67 Compelling evidence to support a therapeutic role for pharmacological CDK2 inhibition in cancer has also been presented by several recent findings through combination strategies. A combination of CDK2 and PI3K inhibitors induced apoptosis in glioma and colorectal cancer xenografts.68 Synergistic effect of concurrent inhibition of bromodomain-containing protein 4 (BRD4) and CDK2 in MYC amplified medulloblastoma was observed.69 Enhanced sensitivity of apoptotic-resistant cells was shown by combined inhibition of CDK2 and BCL-2 family proteins.70,

71

Besides, a synergistic role, CDK2 inhibition also attenuates the development of

resistance. Pharmacological or molecular targeting of CDK2 sensitizes BRAF and HSP90 inhibition resistant melanoma cell lines.72 Inhibition of CDK2 gives an opportunity to revert acquired resistance to CDK4/6 inhibitors.73 In Rb-deficient cancer cells, the E2Fs are constitutively active and CDK4/6 signalling is redundant. In Rb-positive cells, overexpression of cyclin E or loss of the CIP/KIP proteins might bypass CDK4/6 inhibition by activating CDK2. 74 Thus, CDK2 inhibition is a potential therapeutic strategy for treatment of tumours that are considered as poor candidates for CDK4/6 inhibitor therapy. One such example is triple-negative breast cancer (TNBC) where tumors often demonstrate loss of expression of the RB protein, or 14 ACS Paragon Plus Environment

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high expression of cyclin E – both of which would be expected to confer resistance to treatment with CDK4/6 inhibitors. In addition, in TNBC, CDK2 inhibition is synergistic with chemotherapeutic agents and radiotherapy, and restores chemo- and radio-sensitivity in resistant cases.75, 76 CDK2 inhibition, either using an inhibitor or through cyclin E knockdown, inhibits the growth of trastuzumab

77and

tamoxifen78 resistant breast cancer cells both in vitro and in vivo.

Beyond oncology, the potential applications of CDK2 inhibitors are also expanding in to other clinical settings including hearing loss,79 neurodegenerative80 and infectious diseases.81, 82 CDK2 INHIBITORS IN DRUG DEVELOPMENT While the main incentive behind the development of CDK2 inhibitors lies in their potential application as anticancer drugs, small molecule CDK2-selective inhibitors would be vital chemical probes to dissect the underpinnings of a cellular process or disease.83 Since the currently available CDK2 inhibitors are not selective, phenotypic responses (both cellular and at organism level) to them are defined by all the on- and off-targets. As such, it is difficult to associate the observed responses to CDK2 inhibition only. Inhibitors in Clinical Trials. A few pharmacologic inhibitors of CDK2 are in clinical development as anticancer agents (Table 2). Some have already been discontinued from clinical development due to promiscuity leading to off-target kinase inhibition and associated side effects (e.g. SNS-032, AZD5438 and R547) as well as failure to achieve an acceptable clinical end point (e.g. AG-024322).13, 84 Alvocidib (flavopiridol),85, 86 the first CDK inhibitor in clinical trials, is a flavone alkaloid ATPantagonistic broad spectrum kinase inhibitor. It induces G1 as well as G2 cell cycle arrest due to inhibition of CDK2/4 and CDK1 activity, respectively. Alvocidib has been studied in numerous clinical trials, as a single agent or in combinations with other drugs, but demonstrated 15 ACS Paragon Plus Environment


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unsatisfactory efficacy and high toxicity. Despite these setbacks, clinical efficacy was confirmed in hematological malignancies, and it received orphan drug designation for the treatment of patients with acute myeloid leukemia. Seliciclib (roscovitine, CYC202),85, 87 a purine analog and the second CDK inhibitor to enter clinical trials, is a pan-CDK inhibitor that exhibited some CDK more selectively when compared with alvocidib. However, despite many successful preclinical studies, results from several clinical trials are not promising. It seems that combination therapies may possibly be more encouraging than monotherapy. Thus, both alvocidib and seliciclib are currently in Phase I and Phase II clinical trials in combination with other anticancer agents. A number of other compounds targeting CDK2 are also in various stages of drug development. Table 2 provides a summary of the preclinical and clinical data of the second-generation CDK inhibitors that have shown potent CDK2 inhibition and are currently in clinical trials.


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Table 2. CDK2 inhibitors under clinical evaluation Inhibitor

Major CDK

Preclinical studies

(synonym; company;

targets: IC50, nM

(in vitro or mouse models)

Clinical trial and disease (www.clinicaltrials.gov, accessed 30

structure)

August 2018)  Increases cells in G0/G1 and G2/M and is

AT751988-93

CDK1B: 210

(AT7519M, Astex

CDK2A: 47

cytotoxic in multiple cancer cells including

 Chronic lymphocytic leukemia (CLL)

Therapeutics Ltd)

CDK3E: 360

multiple myeloma (MM), ovarian and colon

 Mantle cell lymphoma (MCL)

CDK4D1: 100

Cl

 Effective in ovarian, colon and MM xenografts

Phase II:

 As a single agent, has modest

O NH HN

Cl

H N

N O

CDK5p35: 513

clinical activity in MCL and

CDK6D3: 170

CLL

NH

CDK7H: 2400

 Metastatic solid tumors or refractory

CDK9T:

Cyclin Dependent Kinase 2 Inhibitors in Cancer Therapy: an Update

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