Current Pharmaceutical Biotechnology (IN PRESS)

Inhibiting Cyclin-Dependent Kinase/Cyclin Activity for the Treatment of Cancer and Cardiovascular Disease.

C. Ivorra, H. Samyn, M. D. Edo, C. Castro, S. M. Sanz-González, A. Díez-Juan and V. Andrés*

Unit of Vascular Biology, Instituto de Biomedicina de Valencia (IBV-CSIC), Spanish Council for Scientific Research, Valencia, Spain.

*Author for correspondence:

Instituto de Biomedicina de Valencia (IBV-CSIC)

C/ Jaime Roig, 11

46010 Valencia (Spain)

Tel: +34-96-3391752

FAX: +34-96-3690800

E-mail:

ABSTRACT

Excessive cell proliferation contributes to the pathobiology of human diseases with a high health and socio-economic impact, including cancer and vascular occlusive diseases (e. g., atherosclerosis, in-stent restenosis, transplant vasculopathy, and vessel bypass graft failure). Recent advances in the understanding of the molecular networks governing the hyperplastic growth of tumors and vascular obstructive neointimal lesions have provided new perspectives for preventive and therapeutic strategies against these disorders.

Mammalian cell proliferation requires the activation of several cyclin-dependent protein kinases (CDKs). Postranslational activation of CDKs is a complex process that involves their association with regulatory subunits called cyclins. The activity of CDK/cyclin holoenzymes is negatively regulated through their interaction with members of the CDK family of inhibitory proteins (CKIs). Moreover, over fifty low molecular weight pharmacological CDK inhibitors that target the ATP-binding pocket of the catalytic site of CDKs have been identified. In this review, we will discuss the use of pharmacological and gene therapy strategies against CDK/cyclins in animal models and clinical trials of cancer and cardiovascular disease.

1. INTRODUCTION

Tight control of cellular growth is essential to ensure normal tissue patterning during embryonic and postnatal development. Several cell proliferation regulatory networks have been associated with hyperplasia during both cancer progression and vascular occlusive diseases (e. g., atherosclerosis, restenosis after angioplasty, transplant vasculopathy, and graft atherosclerosis after bypass) [1-4]. Cell cycle progression is controlled by several CDKs that associate with regulatory subunits called cyclins [5,6]. Different CDK/cyclin complexes are orderly activated at specific phases of the cell cycle (Fig. 1). Progression through the first gap-phase (G1) requires both cyclin D- and cyclin E-associated CDK activity. Functional CDK2/cyclin A complexes are required for DNA synthesis (S-phase) and, subsequently, CDC2/cyclin A and CDC2/cyclin B pairs are assembled and activated during the second gap-phase (G2) and mitosis (M-phase), respectively. The requirement of CDK2 for entry into mitosis as a positive regulator of CDC2/cyclin B activity as been suggested [7]. Aside from tight control of cyclin gene expression and protein turnover, the activity of CDK/cyclin pairs is regulated via their phosporylation and dephosphorylation at specific residues.

Active CDK/cyclin holoenzymes are presumed to hyperphosphorylate the retinoblastoma protein (pRb) and the related pocket proteins p107 and p130 from mid G1 to mitosis. The interaction among members of the E2F family of transcription factors and individual pocket proteins is a complex regulatory event that determines whether E2F proteins function as transcriptional activators or repressors [8-11]. Phosphorylation of pRb and related pocket proteins contributes to the transactivation of genes with functional E2F-binding sites, including several growth and cell-cycle regulators (i.e., c-myc, pRb, cdc2, cyclin E, cyclin A), and genes encoding proteins that are required for nucleotide and DNA biosynthesis (i. e., DNA polymerase , histone H2A, proliferating cell nuclear antigen, thymidine kinase) [9,11].

CDK/cyclin activity is negatively regulated by the interaction with specific CDK inhibitory proteins (CKIs) [12] (Fig. 1). CKIs of the Cip/Kip family (for CDK interacting protein/Kinase inhibitory protein) (p21Cip1, p27Kip1 and p57Kip2) bind to and inhibit a wide spectrum of CDK/cyclin holoenzymes, while members of the Ink4 family (for inhibitor of CDK4) (p16Ink4a, p15Ink4b, p18Ink4c, p19Ink4d) are specific for cyclin D-associated CDKs (CDK4 and CDK6). Mitogenic and antimitogenic stimuli affect the rates of synthesis and degradation of CKIs, as well as their redistribution among different CDK/cyclin heterodimers.

2. PHARMACOLOGICAL INHIBITORS OF CDKs

2.1. Families of pharmacological CDK inhibitors

In this section, we will review the different families of pharmacological CDK inhibitors (purines, alkaloids, indirubins, flavonoids, paullones, butyrolactone I, hymenialdisine and pyrazolo[3,4-b]quinoxalines), and their specificity against different protein kinases (Table 1). These low molecular weight compounds compete with ATP for its binding to the ATP-binding pocket of the catalytic site of CDKs. Structural information on pharmacological CDK inhibitors was recently reviewed [13,14].

Purines

This family includes olomoucine, roscovitine, CVT-313, isopentenyl-adenine, purvalanols and other purine analogues obtained by combinatorial chemistry. Interaction of the purine portion of these inhibitors with the adenine-binding pocket of CDKs prevents the binding of ATP [15-17].

Purvalanol B is highly specific for CDKs, with an IC50 value of 6 nM for CDK1 (CDC2), CDK2 and CDK5 [16]. In contrast, the IC50 value of purvalanol B when tested against other protein kinases is higher than 10 M [16]. Purvalanol A is a more permeable analog of purvalanol B that potently inhibits CDK2 (IC50 = 35-70 nM) [16].Aminopurvalanol is a potent inhibitor of CDK1, CDK2, and CDK5 [18].

The inhibitory effect of roscovitine is 10 times higher than that of olomoucine, with IC50 values around 600 nM and 7 M, respectively [19-21]. The inhibitory activity of both compounds is high against CDK1, CDK2 and CDK5 as compared with CDK4. Eight new purine analogues show stronger inhibitory activity than olomoucine against CDK1 and CDK2 (IC50 ranging from 0.03 to 3.8 M) [22].

The relative inhibitory potency of CVT-313 varies from very high for CDK2 (IC50 = 500 nM), moderate for CDK1 (IC50 = 4M), and very low for CDK4 (IC50 = 215 M) [23].

Isopentenyl-adenine is the weakest and less specific protein kinase inhibitor of the purine family, with IC50 values for CDKs higher than 40 M [20,21,24].

Recent structure-activity relationship studies have allowed the design of more potent and selective CDK2 inhibitory purine derivates, including 6-benzylamino-2-thiomorpholinyl-9-isopropylpurine (IC50 = 900 nM) [25] and [6-(3-chloroanilino)-2-(2-hydroxypyrrolidyl)-9-isopropylpurine] (IC50 = 300 nM) [26].

Alkaloids

Members of this family (staurosporine, UCN-01 and CGP 41 251) inhibit a broad-spectrum of protein kinases, including protein kinase C (PKC) and CDKs.

Staurosporine is the most potent CDK inhibitory alkaloid, with IC50 values in the nM range for CDK1, CDK2 and CDK5 [20,24,27]. The binding of staurosporine to CDK2 resembles that of the adenine base of ATP [28].

UCN-01 (7-hydroxystaurosporine) shows high affinity for PKC (IC50 = 20-60 nM) and variable affinity for CDKs (IC50 = 1 M for CDK1, and in the nM range for other CDKs) [29-32]. Likewise, CGP 41 251 (4'-N-benzoylstaurosporine)is a potent PKC inhibitor that shows low affinity for CDKs (IC50 values in the M range) [33].

Indirubins

Indirubin is the active ingredient of a mixture of plants used in traditional Chinese medicine [34]. Indirubin and its derivatives inhibit the activity of a broad spectrum of CDKs (high inhibitory activity against CDK1, CDK2 and CDK5, and 10 times lower for CDK4), and the evolutionarily related glycogen synthase kinase-3 (GSK-3) (IC50 = 5-50 nM) [35]. These inhibitors act by competing with ATP for binding to the ATP-binding site of CDK2 [34]. Indirubin is the least active member of the family, with IC50 values in the M range. The most potent inhibitor of this family is indirubin-5-sulphonic acid, with IC50 values between 35 nM and 300 nM. Other indirubin derivatives with strong CDK inhibitory activity are 5-chloro-indirubin and indirubin-3’-monoxime, with IC50 values between 200 nM and 800 nM for CDK1, CDK2 and CDK5, and around 5 M for CDK4 [34].

Flavonoids

Flavopiridol (L86-8275) and its derivative L86-8276 are relatively potent CDK inhibitors (IC50 values in the nM range) [36,37]. Crystallographic studies revealed the binding of the aromatic portion of L86-8276 to the adenine-binding pocket of CDK2 [38]. Moreover, the position of the phenyl group of L86-8276 enables this inhibitor to make exclusive contacts with CDK2 that are not observed in the CDK2-ATP crystals.

Flavopiridol has higher specificity towards CDK4 (IC50 = 65 nM) than towards CDK1 and CDK2 (IC50 values of 500 nM and 100 nM, respectively). The IC50 values of flavopiridol for other protein kinases are all in the M range [36,37].

Casagrande et al [39] demonstrated that flavonoids which induced G1 arrest (i. e., quercetin, daidzein and luteolin) inhibited CDK2 activity by 40-60%. In contrast, other flavonoids which caused G2/M arrest (i.e., kaempherol, apigenin and genistein) inhibited CDK1 activity by 50-70%, without affecting CDK2 activity. All these flavonoids are relatively weak CDK inhibitors (IC50 > 100 M).

Paullones

Molecular modelling studies have suggested that paullones make contacts in the ATP-binding site of CDKs similar to those observed in the crystal structure of several CDK2-bound inhibitors [40]. Alsterpaullone shows high inhibitory activity against CDK1 (IC50 = 35 nM) [41]. Although kenpaullone is a less potent CDK1 inhibitor (IC50 = 400-800 nM), it retains high specificity for this enzyme (its IC50 value for other protein kinases is in the M range) [40]. Paullones also act as very potent inhibitors of GSK-3 and the neuronal CDK5/p25 kinase (IC50 values in the nM range) [42]

Butyrolactone I

Butyrolactone I is a natural compound isolated from Aspergillus which acts as an ATP binding competitor with high specificity for CDK1 (IC50 = 600 nM) and CDK2 (IC50 = 1.5 M) [43,44].

Hymenialdisine

Hymenialdisine is a natural compound isolated from a marine sponge which contains both bromopyrrole and guanidine groups [45]. The crystal structure of the CDK2-hymenialdisine complex shows hydrogen bonds similar to the links observed in other CDK-inhibitor structures. Hymenialdisine is a very potent inhibitor of CDK1, CDK2 and CDK5 (IC50 = 22-70 nM). Interestingly, it shows high inhibitory activity against three protein kinases seemingly involved in Alzheimer’s disease (GSK-3, casein kinase 1 and CDK5, with IC50 values of 10 nM, 35 nM and 28 nM, respectively) [45].

Pyrazolo[3,4-b]quinoxalines

Members of this new family of synthetic CDK inhibitors have been identified by virtue of their structural similarity with some purine derivates. A preliminary structure-activity relationship study suggests that pyrazolo[3,4-b]quinoxalines can be optimized to inhibit CDKs and GSK-3. The most active compound (R3=NH2) shows strong inhibitory activity against the brain kinases CDK1, CDK5 and GSK-3 [46].

2.2. Biological effects of pharmacological CDK inhibitors on cultured cells

Table 2 summarizes representative studies of the biological effects of pharmacological CDK inhibitors on cultured cells, which include cytostatic and cytotoxic effects, and induction of phenotypic differentiation. It is noteworthy that growth arrest by CVT-313 and alkaloids requires slightly higher concentrations than those needed to inhibit CDK activity in vitro. This disparity is even more pronounced for olomoucine, roscovitine, indirubins and butyrolactone I, whose effective in vivo concentrations are 10-100 times higher than those required to inhibit CDK activity in vitro. Poor membrane permeability of these compounds, and/or differences in the ATP concentrations used in the in vitro experiments compared to cellular conditions might account for these differences.

Pharmacological CDK inhibitors generally cause G1 and/or G2/M arrest, consistent with their potent inhibitory activity against both CDK2 and CDK1 (see Table 1). G1 arrest in cells treated with CDK inhibitory agents correlates with decreased pRb protein levels and/or accumulation of hypophosphorylated pRb [31,34,37,47-49]. Interestingly, pRB status influenced the ability of UCN-01 to induce G1 blockade in several cancer cell lines [50]. Likewise, staurosporine failed to induce G1 arrest in the bladder carcinoma cell line 5637 that lacks a functional pRB, and restoration of pRb function in these cells by retroviral infection led to staurosporine-induced G1 arrest [51]. Nishi et al. examined the effect of staurosporine on the growth of several human tumor cell lines and oncogene-transformed NIH3T3 cell lines [52]. While all the cell lines that underwent G1 arrest in response to staurosporine contained functional pRB, this agent induced growth arrest in embryonic fibroblasts derived from pRb-null mice. Interestingly, staurosporine led to p27kip1 accumulation regardless of whether it induced or not growth suppression. Collectively, these results suggest that pRb inactivation alone is not sufficient for the abrogation of staurosporine-induced G1 arrest, and that the antiproliferative potential of staurosporine depends on the integrity of cell cycle regulators which operate downstream of p27kip1.

Some inhibitors, like olomoucine [53,54], roscovitine [54], staurosporine [55,56],indirubin [34], CVT-313 [23]and flavopiridol [37,57], can cause blockade in different cell cycle phases depending on both the concentration of the drug and the cell line analyzed. Cells treated with butyrolactone I accumulate mainly in G2/M, perhaps due to its higher affinity for CDK1 [58-61]. Evidence has been presented indicating that the kinase(s) involved in the regulation of cell exit from G1 and G2, respectively, in normal and leukemic lymphocytes may have different sensitivities to staurosporine, which suggests that the mechanisms controlling exit from G1 in these cells may be different [56].

Consistent with the observation that differentiation and proliferation are mutually exclusive processes in many cell types, treatment of cultured cells with agents that block cell cycle progression can simultaneously induce phenotypic differentiation. For instance, roscovitine and flavopiridol caused mucinous differentiation of non-small cell lung cancer cells [62]. Likewise, PC12 cells [27] and neuro2a cells [63] underwent neuronal differentiation when treated with staurosporine and butyrolactone I, respectively. Staurosporine can induce morphological and biochemical maturation in mouse keratinocyte cell lines [64], and might induce neuronal differentiation of human prostate cancer TSU-Pr1 cells [65]. Distinctive features of neuronal differentiation have been also observed upon exposure of PC12 cells to butyrolactone I or olomoucin, including the appearance of neurite extensions and induction of microtubule-associated protein 2 expression [66].

During development and morphogenesis of multicellular organisms, physiological mechanisms of cell death operate to control cell number, and as a protective strategy to remove infected, mutated or damaged cells [67]. In many instances, programmed cell death (apoptosis) is associated with changes in CDK activity [68,69]. While cell death can be blocked by several pharmacological CDK inhibitors (i. e., butyrolactone I, olomoucine and roscovitine) [70,71], many tumor-derived cells respond to these agents with increased apoptosis (Table 2). Treatment of several bladder cancer cell lines with flavopiridol for 24 hours resulted in marked G2/M arrest [72]. Although modest apoptosis was also observed, it required 72 hours of continuous drug exposure to become evident. Although “conflicting” signals resulting from forced inhibition of CDKs under conditions of deregulated proliferation might be proposed as a general mechanism leading to apoptosis, different CDK inhibitors induce apoptosis via distinct molecular mechanisms [73].

2.3. Therapeutic use of pharmacological CDK inhibitors in animal models of cancer and vascular proliferative disease.

Table 3 summarizes some therapeutic applications of synthetic CDK inhibitors in animal models of cancer and neointimal thickening after balloon angioplastia.

Hyperplastic neointimal lesion growth is thought to contribute to restenosis after angioplasty, the recurrence of arterial narrowing at the site of intervention that occurs in 20-40% of coronary artery disease patients after successful revascularization [74-76]. The rat carotid model of balloon angioplasty has been used to assess the therapeutical efficacy of CVT-313 and flavopiridol. CVT-313 reduced neointimal lesion formation by 80 % when delivered at a dose of 1.25 mg/kg for 15 minutes under pressure at the site of balloon angioplasty [23]. Likewise, flavopiridol at 5 mg/kg administered orally for 5 days beginning at the day of balloon angioplasty reduced neointima formation by 35 % and by 39 % at day 7 and 14 after intervention, respectively [77].

The antitumor activity of pharmacological CDK inhibitors has been extensively investigated in experimental models of cancer. UCN-01 elicited antitumor activity in several murine xenogratft models using different human tumor cells (epidermoid carcinoma A431, fibrosarcoma HT1080, acute myeloid leukemia HL-60, and pancreatic tumor cells) [78,79]. Koh et al. investigated the therapeutic activity of UCN-01 against 3 human breast carcinoma cell lines (MCF-7, Br-10, and MX-1) serially transplanted into nude mice [80]. UCN-01 had antitumor activity against MCF-7 and Br-10, while MX-1 was resistant to UCN-01. Although UCN-01 induced p21Cip1 in the 3 cell lines in culture, little dephosphorylated pRb protein was expressed in MX-1 compared with Br-10 and MCF-7. Pharmacokinetic studies in mice, rats and dogs demonstrated high total clearance values of UCN-01 and higher concentrations of the drug in tumor tissue compared with those found in the plasma [79].

Ikegami et al. have demonstrated broad antitumor activity of CGP 41 251 in various types of murine and human tumor models [81]. When administered at a dose of 75 mg/kg 3 times daily for 9 days, this staurosporine derivative prolonged the life span of mice bearing B16 melanoma. However, oral administration of CGP 41 251 at doses of 25-225 mg/kg once daily for 9 days was ineffective in 4 kinds of murine tumor models. Oral administration of CGP 41 251 at a dose of 200 mg/kg once daily for 4 weeks showed antitumor activity (inhibition rates of 58-80%) in mice subcutaneously inoculated with several human tumor cells (gastric H-55, colorectal H-26, breast H-31, lung H-74 and LC-376 cancer)

Several studies with murine models have shown antitumor activity of flavopiridol, including different human xenografted tumors (head and neck squamous cell carcinoma, colon carcinoma, prostate cancer, and leukemia and lymphoma xenografts) [82-85]. Treatment of mice bearing human head and neck squamous cell carcinoma xenografts with flavopiridol given as a daily intraperitoneal injection for 5 consecutive days at a concentration of 5 mg/kg resulted in a 23% reduction in tumor growth, reaching a 60% reduction 10 weeks after the end of the treatment [84]. In this study, flavopiridol induced apoptosis in tumor cells. In murine models using human tumors of lymphohematopoietic origin, including HL-60 and SUDHL-4 subcutaneous xenografts and Nalm/6 and AS283 disseminated disease models, intravenous injection of flavopiridol at a concentration of 7.5 mg/kg induced tumor regression [82]. Flavopiridol promoted apoptosis in these tumor models and in lymphoid organs (i. e., spleen, thymus and intestinal lymphoid tissues) of healthy mice [82].

Antitumor activity of some pharmacological CDK inhibitors may result from the combination of growth suppression and anti-angiogenic activity. For example, flavopiridol decreased blood vessel formation in a mouse Matrigel model of angiogenesis [83]. Likewise, the anticancer agent UFT, which is widely used in Japan to treat cancer patients requiring a long-term chemotherapy, inhibited angiogenesis induced by RENCA cells (murine renal carcinoma) in a dorsal air sac assay [86]. This inhibitory effect on angiogenesis was also elicited by the butyrolactone analogues -hydroxybutyric acid (GHB) and -butyrolactone (GBL), 2 UFT-specific metabolites. Interestingly, the antiangiogenic effects of GHB and GBL were increased with administration by continuous infusion, providing a suitable pharmacokinetic profile.

3. GENE THERAPY STRATEGIES BASED ON CDK INHIBITION IN ANIMAL MODELS OF CANCER AND CARDIOVASCULAR DISEASE.