Preclinical optimization of MDM2 antagonist scheduling for cancer treatment by using a model-based approach.

PURPOSE: Antitumor clinical activity has been demonstrated for the MDM2 antagonist RG7112, but patient tolerability for the necessary daily dosing was poor. Here, utilizing RG7388, a second-generation nutlin with superior selectivity and potency, we determine the feasibility of intermittent dosing to guide the selection of initial phase I scheduling regimens. EXPERIMENTAL DESIGN: A pharmacokinetic-pharmacodynamic (PKPD) model was developed on the basis of preclinical data to determine alternative dosing schedule requirements for optimal RG7388-induced antitumor activity. This PKPD model was used to investigate the pharmacokinetics of RG7388 linked to the time-course of the antitumor effect in an osteosarcoma xenograft model in mice. These data were used to prospectively predict intermittent and continuous dosing regimens, resulting in tumor stasis in the same model system. RESULTS: RG7388-induced apoptosis was delayed relative to drug exposure with continuous treatment not required. In initial efficacy testing, daily dosing at 30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was equivalent to 10 mg/kg given daily. The implementation of modeling and simulation on these data suggested several possible intermittent clinical dosing schedules. Further preclinical analyses confirmed these schedules as viable options. CONCLUSION: Besides chronic administration, antitumor activity can be achieved with intermittent schedules of RG7388, as predicted through modeling and simulation. These alternative regimens may potentially ameliorate tolerability issues seen with chronic administration of RG7112, while providing clinical benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd) schedules were selected for RG7388 clinical testing.

Discovery of siRNA lipid nanoparticles to transfect suspension leukemia cells and provide in vivo delivery capability.

As a powerful research tool, siRNA's therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment.

Antitumor activity of BRAF inhibitor vemurafenib in preclinical models of BRAF-mutant colorectal cancer.

The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF(V600E) in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF(V600)-expressing cell lines and inhibiting tumor growth in BRAF(V600E) bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF(V600E)-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF(V600E) mutation.

Resistance to selective BRAF inhibition can be mediated by modest upstream pathway activation.

A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.

Identification of the MEK1(F129L) activating mutation as a potential mechanism of acquired resistance to MEK inhibition in human cancers carrying the B-RafV600E mutation.

Although targeting the Ras/Raf/MEK pathway remains a promising anticancer strategy, mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors in clinical development are likely to be limited in their ability to produce durable clinical responses due to the emergence of acquired drug resistance. To identify potential mechanisms of such resistance, we established MEK inhibitor-resistant clones of human HT-29 colon cancer cells (HT-29R cells) that harbor the B-RafV600E mutation. HT-29R cells were specifically resistant to MEK inhibition in vitro and in vivo, with drug-induced elevation of MEK/ERK and their downstream targets primarily accountable for drug resistance. We identified MEK1(F129L) mutation as a molecular mechanism responsible for MEK/ERK pathway activation. In an isogenic cell system that extended these findings into other cancer cell lines, the MEK1(F129L) mutant exhibited higher intrinsic kinase activity than wild-type MEK1 [MEK1(WT)], leading to potent activation of ERK and downstream targets. The MEK1(F129L) mutation also strengthened binding to c-Raf, suggesting an underlying mechanism of higher intrinsic kinase activity. Notably, the combined use of Raf and MEK inhibitors overcame the observed drug resistance and exhibited greater synergy in HT-29R cells than the drug-sensitive HT-29 parental cells. Overall, our findings suggested that mutations in MEK1 can lead to acquired resistance in patients treated with MEK inhibitors and that a combined inhibition of Raf and MEK may be potentially useful as a strategy to bypass or prevent drug resistance in the clinic.

MDM2 antagonists boost antitumor effect of androgen withdrawal: implications for therapy of prostate cancer.

BACKGROUND: Hormone therapy is the standard of care for newly diagnosed or recurrent prostate cancers. It uses anti-androgen agents, castration, or both to eliminate cancer promoting effect of testicular androgen. The p53 tumor suppressor controls a major pathway that can block cell proliferation or induce apoptosis in response to diverse forms of oncogenic stress. Activation of the p53 pathway in cancer cells expressing wild-type p53 has been proposed as a novel therapeutic strategy and recently developed MDM2 antagonists, the nutlins, have validated this in preclinical models of cancer. The crosstalk between p53 and androgen receptor (AR) signaling suggest that p53 activation could augment antitumor outcome of androgen ablation in prostate cancer. Here, we test this hypothesis in vitro and in vivo using the MDM2 antagonist, nutlin-3 and the p53 wild-type prostate cancer cell line, LNCaP. RESULTS: Using charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line. This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53. In vivo, androgen deprivation followed by two weeks of nutlin administration in LNCaP-bearing nude mice led to a greater tumor regression and dramatically increased survival. CONCLUSIONS: Since majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.

High tumor levels of IL6 and IL8 abrogate preclinical efficacy of the γ-secretase inhibitor, RO4929097.

Interest continues to build around the early application of patient selection markers to prospectively identify patients likely to show clinical benefit from cancer therapies. Hypothesis generation and clinical strategies often begin at the preclinical stage where responder and nonresponder tumor cell lines are first identified and characterized. In the present study, we investigate the drivers of in vivo resistance to the γ-secretase inhibitor RO4929097. Beginning at the tissue culture level, we identified apparent IL6 and IL8 expression differences that characterized tumor cell line response to RO4929097. We validated this molecular signature at the preclinical efficacy level identifying additional xenograft models resistant to the in vivo effects of RO4929097. Our data suggest that for IL6 and IL8 overexpressing tumors, RO4929097 no longer impacts angiogenesis or the infiltration of tumor associated fibroblasts. These preclinical data provide a rationale for preselecting patients possessing low levels of IL6 and IL8 prior to RO4929097 dosing. Extending this hypothesis into the clinic, we monitored patient IL6 and IL8 serum levels prior to dosing with RO4929097 during Phase I. Interestingly, the small group of patients deriving some type of clinical benefit from RO4929097 presented with low baseline levels of IL6 and IL8. Our data support the continued investigation of this patient selection marker for RO4929097 and other types of Notch inhibitors undergoing early clinical evaluation.

Small-molecule inducer of cancer cell polyploidy promotes apoptosis or senescence: Implications for therapy.

Polyploidy results from deregulated cell division and has been considered an undesirable event leading to increased mutation rate and cancer development. However, polyploidy may also render cancer cells more vulnerable to chemotherapy. Here, we identify a small-molecule inducer of polyploidy, R1530, which interferes with tubulin polymerization and mitotic checkpoint function in cancer cells, leading to abortive mitosis, endoreduplication and polyploidy. In the presence of R1530, polyploid cancer cells underwent apoptosis or became senescent which translated into potent in vitro and in vivo efficacy. Normal proliferating cells were resistant to R1530-induced polyploidy thus supporting the rationale for cancer therapy by induced polyploidy. Mitotic checkpoint kinase BubR1 was found downregulated during R1530-induced exit from mitosis, a likely consequence of PLK4 inhibition. BubR1 knockdown in the presence of nocodazole induced an R1530-like phenotype, suggesting that BubR1 plays a key role in polyploidy induction by R1530 and could be exploited as a target for designing more specific polyploidy inducers.

RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models.

The BRAF(V600E) mutation is common in several human cancers, especially melanoma. RG7204 (PLX4032) is a small-molecule inhibitor of BRAF(V600E) kinase activity that is in phase II and phase III clinical testing. Here, we report a preclinical characterization of the antitumor activity of RG7204 using established in vitro and in vivo models of malignant melanoma. RG7204 potently inhibited proliferation and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and ERK phosphorylation in a panel of tumor cell lines, including melanoma cell lines expressing BRAF(V600E) or other mutant BRAF proteins altered at codon 600. In contrast, RG7204 lacked activity in cell lines that express wild-type BRAF or non-V600 mutations. In several tumor xenograft models of BRAF(V600E)-expressing melanoma, we found that RG7204 treatment caused partial or complete tumor regressions and improved animal survival, in a dose-dependent manner. There was no toxicity observed in any dose group in any of the in vivo models tested. Our findings offer evidence of the potent antitumor activity of RG7204 against melanomas harboring the mutant BRAF(V600E) gene.

Preclinical in vivo evaluation of efficacy, pharmacokinetics, and pharmacodynamics of a novel MEK1/2 kinase inhibitor RO5068760 in multiple tumor models.

Targeting the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway represents a promising anticancer strategy. Recently, we have reported a novel class of potent and selective non-ATP-competitive MEK1/2 inhibitors with a unique structure and mechanism of action. RO5068760 is a representative of this class showing significant efficacy in a broad spectrum of tumors with aberrant mitogen-activated protein kinase pathway activation. To understand the relationship between systemic exposures and target (MEK1/2) inhibition as well as tumor growth inhibition, the current study presents a detailed in vivo characterization of efficacy, pharmacokinetics, and pharmacodynamics of RO5068760 in multiple xenograft tumor models. For inhibition of MEK1/2 as measured by the phosphorylated ERK levels, the estimated EC(50)s in plasma were 1.36 micromol/L (880 ng/mL) and 3.35 micromol/L (2168 ng/mL) in LOX melanoma and HT-29 colorectal cancer models, respectively. A similar EC(50) (1.41 micromol/L or 915 ng/mL) was observed in monkey peripheral blood lymphocytes. To achieve tumor growth inhibition (>or=90%), an average plasma drug concentration of 0.65 or 5.23 micromol/L was required in B-RafV600E or K-Ras mutant tumor models, respectively, which were remarkably similar to the IC(90) values (0.64 or 4.1 micromol/L) determined in vitro for cellular growth inhibition. With equivalent in vivo systemic exposures, RO5068760 showed superior efficacy in tumors harboring B-RafV600E mutation. The plasma concentration time profiles indicate that constant p-ERK suppression (>50%) may not be required for optimal efficacy, especially in highly responsive tumors. This study may facilitate future clinical trial design in using biochemical markers for early proof of mechanism and in selecting the right patients and optimal dose regimen.

Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties.

Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of gamma-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way.

Novel regimens of capecitabine alone and combined with irinotecan and bevacizumab in colorectal cancer xenografts.

BACKGROUND: Xenograft and mathematical models have shown that the antitumor activity of capecitabine can be increased by modifying the schedule from 14 days on, 7 off (14/7) to 7/7. MATERIALS AND METHODS: Capecitabine at two-thirds maximum tolerated dose (MTD) administered using 14/7 (267 mg/kg) and 7/7 (467 mg/kg) schedules, alone and in doublet and triplet combinations with irinotecan (40 mg/kg intraperitoneally) and bevacizumab (5 mg/kg intraperitoneally) were studied in mice bearing HT29 colorectal xenografts. RESULTS: Tumor growth inhibition was >100% in doublet and triplet regimens with capecitabine 7/7 compared with 70% and 98%, respectively, with 14/7. Increase in lifespan was significantly greater with the 7/7 triplet than the corresponding doublet without bevacizumab (288% versus 225%, respectively). CONCLUSION: Addition of bevacizumab to capecitabine and irinotecan significantly improved tumor growth inhibition and lifespan in the HT29 xenograft model. Modifying the capecitabine schedule from 14/7 to 7/7 improved the efficacy of doublet and triplet combinations without toxicity.

Characterization of a novel mitogen-activated protein kinase kinase 1/2 inhibitor with a unique mechanism of action for cancer therapy.

The mitogen-activated protein kinase (MAPK) signal transduction pathway plays a central role in regulating tumor cell growth, survival, differentiation, and angiogenesis. The key components of the Ras/Raf/MEK/ERK signal module are frequently altered in human cancers. Targeting this pathway represents a promising anticancer strategy. Small molecule inhibitors targeting MEK1/2 have shown promise in the clinic; however, ultimate clinical proof-of-concept remains elusive. Here, we report a potent and highly selective non-ATP-competitive MEK1/2 inhibitor, RO4927350, with a novel chemical structure and unique mechanism of action. It selectively blocks the MAPK pathway signaling both in vitro and in vivo, which results in significant antitumor efficacy in a broad spectrum of tumor models. Compared with previously reported MEK inhibitors, RO4927350 inhibits not only ERK1/2 but also MEK1/2 phosphorylation. In cancer cells, high basal levels of phospho-MEK1/2 rather than phospho-ERK1/2 seem to correlate with greater sensitivity to RO4927350. Furthermore, RO4927350 prevents a feedback increase in MEK phosphorylation, which has been observed with other MEK inhibitors. We show that B-Raf rather than C-Raf plays a critical role in the feedback regulation. The unique MAPK signaling blockade mediated by RO4927350 in cancer may reduce the risk of developing drug resistance. Thus, RO4927350 represents a novel therapeutic modality in cancers with aberrant MAPK pathway activation.

In vivo activity of novel capecitabine regimens alone and with bevacizumab and oxaliplatin in colorectal cancer xenograft models.

Modifying the capecitabine dosing schedule from 14 days on, 7 days off (14/7) to 7 days on, 7 days off (7/7) may enable higher doses and improved antitumor efficacy in colorectal cancer xenografts. Capecitabine 14/7 (267 or 400 mg/kg) and 7/7 (467 or 700 mg/kg) schedules in doublet and triplet combinations with optimally dosed bevacizumab (5 mg/kg) and oxaliplatin (6.7 mg/kg) were studied in female athymic nude mice bearing HT29 colorectal xenografts. Additional studies of suboptimally dosed bevacizumab (2.5 mg/kg) and capecitabine 7/7 (360 mg/kg) were done in a similar Colo205 tumor xenograft model. Monotherapy and combination regimens were administered to groups of 10 animals and compared with vehicle controls. In the HT29 model, tumor growth inhibition and increase in life span (ILS) were significantly greater with capecitabine 7/7 than with 14/7 (P<0.05). The additional benefit of capecitabine 7/7 versus 14/7 was biologically significant according to National Cancer Institute criteria (>25% ILS). Adding bevacizumab to capecitabine 7/7 resulted in significantly greater survival relative to either agent alone (P<0.0001). When oxaliplatin was added, efficacy was significantly better with the triplet combination including capecitabine 7/7 (tumor growth inhibition>100% and ILS 234%) compared with 14/7 (95% and 81%, respectively). In the Colo205 model, combination therapy with capecitabine 7/7 plus bevacizumab resulted in significantly greater survival relative to either agent alone (P<0.0001). In conclusion, in athymic nude mice bearing moderately thymidine phosphorylase-expressing HT29 or Colo205 colorectal xenografts, a capecitabine 7/7 schedule permits increased drug delivery compared with traditional 14/7 regimens, greatly improving monotherapy activity without major toxicity.

Elevated MDM2 boosts the apoptotic activity of p53-MDM2 binding inhibitors by facilitating MDMX degradation.

The p53 tumor suppressor is a powerful growth suppressive and pro-apoptotic molecule frequently inactivated in human cancer. Many tumors overproduce its negative regulator MDM2, a specific p53 ubiquitin ligase and transcriptional inhibitor, to disable p53 function. Therefore, p53 activation by inhibiting MDM2 has been proposed as a novel strategy for cancer therapy in tumors expressing wild-type p53. Recently developed small-molecule p53-MDM2 binding inhibitors, the nutlins, selectively activate p53 function and induce cell cycle arrest and apoptosis in cancer cells. By stabilizing p53, nutlins also elevate the cellular level of its transcriptional target MDM2. Here, we present evidence that nutlin-induced MDM2 retains its ubiquitin ligase activity and contributes to the anti-tumor activity of p53-MDM2 binding inhibitors by facilitating the degradation of another p53 inhibitor, MDMX. MDM2 and MDMX levels were analyzed in a panel of 12 randomly selected solid tumor cell lines. In the presence of nutlin-3, MDM2 increased in all and MDMX decreased in most of the cell lines. MDMX was resistant to nutlin-induced degradation in 2/12 cell lines. In these cells, MDMX appears to be a major suppressor of the apoptotic response to p53 activation although this effect was only partially p53-dependent. Doxorubicin facilitated MDMX degradation through DNA damage response pathways and restored their sensitivity to nutlin, suggesting that combination therapy may be an effective way to overcome nutlin resistance in cancers with MDMX aberrations.

A selective phosphatase of regenerating liver phosphatase inhibitor suppresses tumor cell anchorage-independent growth by a novel mechanism involving p130Cas cleavage.

The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.

Antitumor activity of capecitabine and bevacizumab combination in a human estrogen receptor-negative breast adenocarcinoma xenograft model.

BACKGROUND: Capecitabine and bevacizumab have each been shown to inhibit tumor growth. Their combination failed to improve survival in a phase III trial of metastatic breast cancer (MBC), although it should be noted patients had been heavily pretreated with anthracyclines and taxanes. Our aim was to evaluate whether combination treatment would increase tumor growth inhibition and survival in a breast cancer model. MATERIALS AND METHODS: Mice bearing KPL-4 human estrogen receptor-negative breast adenocarcinoma xenografts were given capecitabine orally daily for 14 days at the maximum tolerated dose (MTD) or half MTD, alone or with 5 mg/kg intraperitoneal bevacizumab twice weekly. RESULTS: Tumor growth inhibition (TGI) and increased life span (ILS) were superior in the combination groups versus monotherapy (p < 0.05). TGI and ILS were significantly improved in the high- versus low-dose capecitabine combination (p < 0.05). CONCLUSION: Capecitabine in combination with bevacizumab provides a basis for pursuing the combination for first-line treatment of MBC.

In vitro and in vivo activity of R547: a potent and selective cyclin-dependent kinase inhibitor currently in phase I clinical trials.

The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (K(i) = 1-3 nmol/L) and was inactive (K(i) > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC(50)s

Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1.

CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G(2)/M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.

Kinesin spindle protein (KSP) inhibitors. Part 3: synthesis and evaluation of phenolic 2,4-diaryl-2,5-dihydropyrroles with reduced hERG binding and employment of a phosphate prodrug strategy for aqueous solubility.

2,4-Diaryl-2,5-dihydropyrroles have been discovered to be novel, potent and water-soluble inhibitors of KSP, an emerging therapeutic target for the treatment of cancer. A potential concern for these basic KSP inhibitors (1 and 2) was hERG binding that can be minimized by incorporation of a potency-enhancing C2 phenol combined with neutral N1 side chains. Aqueous solubility was restored to these, and other, non-basic inhibitors, through a phosphate prodrug strategy.