Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics.

Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non-ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non-ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer.

Constitutive Cdk2 activity promotes aneuploidy while altering the spindle assembly and tetraploidy checkpoints.

The cell has many mechanisms for protecting the integrity of its genome. These mechanisms are often weakened or absent in many cancers, leading to high rates of chromosomal instability in tumors. Control of the cell cycle is crucial for the function of these checkpoints, and is frequently lost in cancers as well. Overexpression of Cyclin D1 in a large number of breast cancers causes overactivation of the cyclin-dependent kinases, including Cdk2. Constitutive Cdk2 activation through Cyclin D1 generates tumors in mice that are aneuploid and have many characteristics indicative of chromosomal instability. Expression of these complexes in the MCF10A cell line leads to retinoblastoma protein (Rb) hyperphosphorylation, a subsequent increase in proliferation rate, and increased expression of the spindle assembly checkpoint protein Mad2. This results in a strengthening of the spindle assembly checkpoint and renders cells more sensitive to the spindle poison paclitaxel. Constitutive Rb phosphorylation also causes a weakening of the p53-dependent tetraploidy checkpoint. Cells with overactive Cdk2 fail to arrest after mitotic slippage in the presence of paclitaxel or cytokinesis failure during treatment with cytochalasin-B, generating 8N populations. This additional increase in DNA content appears to further intensify the tetraploidy checkpoint in a step-wise manner. These polyploid cells are not viable long-term, either failing to undergo division or creating daughter cells that are unable to undergo subsequent division. This study raises intriguing questions about the treatment of tumors with overactive Cdk2.

Assembly, activation, and substrate specificity of cyclin D1/Cdk2 complexes.

Previous studies have shown conflicting data regarding cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes, and considering the widespread overexpression of cyclin D1 in cancer, it is important to fully understand their relevance. While many have shown that cyclin D1 and Cdk2 form active complexes, others have failed to show activity or association. Here, using a novel p21-PCNA fusion protein as well as p21 mutant proteins, we show that p21 is a required scaffolding protein, with cyclin D1 and Cdk2 failing to complex in its absence. These p21/cyclin D1/Cdk2 complexes are active and also bind the trimeric PCNA complex, with each trimer capable of independently binding distinct cyclin/Cdk complexes. We also show that increased p21 levels due to treatment with chemotherapeutic agents result in increased formation and kinase activity of cyclin D1/Cdk2 complexes, and that cyclin D1/Cdk2 complexes are able to phosphorylate a number of substrates in addition to Rb. Nucleophosmin and Cdh1, two proteins important for centrosome replication and implicated in the chromosomal instability of cancer, are shown to be phosphorylated by cyclin D1/Cdk2 complexes. Additionally, polypyrimidine tract binding protein-associated splicing factor (PSF) is identified as a novel Cdk2 substrate, being phosphorylated by Cdk2 complexed with either cyclin E or cyclin D1, and given the many functions of PSF, it could have important implications on cellular activity.

Amino acid Asp181 of 5'-flap endonuclease 1 is a useful target for chemotherapeutic development.

DNA alkylation-induced damage is one of the most efficacious anticancer therapeutic strategies. Enhanced DNA alkylation and weakened DNA repair capacity in cancer cells are responsible for the effectiveness of DNA-alkylating therapies. 5'-Flap endonuclease 1 (Fen1) is an important enzyme involved in base excision repair (BER), specifically in long-patch BER (LP-BER). Using the site-directed mutagenesis approach, we have identified an important role for amino acid Asp181 of Fen1 in its endonuclease activity. Asp181 is thought to be involved in Mg(2+) binding in the active site. Using structure-based molecular docking of Fen1 targeted to its metal binding pocket M2 (Mg(2+) site), we have identified a potent low-molecular weight inhibitor (LMI, NSC-281680) that efficiently blocks LP-BER. In this study, we have demonstrated that the interaction of this LMI with Fen1 blocked its endonuclease activity, thereby blocking LP-BER and enhancing the cytotoxic effect of DNA-alkylating agent Temozolomide (TMZ) in mismatch repair (MMR)-deficient and MMR-proficient colon cancer cells. The results further suggest that blockade of LP-BER by NSC-281680 may bypass other drug resistance mechanisms such as mismatch repair (MMR) defects. Therefore, our findings provide groundwork for the development of highly specific and safer structure-based small molecular inhibitors targeting the BER pathway, which can be used along with existing chemotherapeutic agents, like TMZ, as combination therapy for the treatment of colorectal cancer.

A Novel, Duodenal-Release Formulation of a Combination of Caraway Oil and L-Menthol for the Treatment of Functional Dyspepsia: A Randomized Controlled Trial.

OBJECTIVES: We conducted a randomized, placebo-controlled trial, which evaluated a novel formulation of caraway oil and L-menthol using microsphere-based site-specific targeting (COLM-SST) vs placebo in patients with functional dyspepsia (FD). METHODS: Adult men and women with FD defined by Rome III criteria were recruited. Patients were randomized to COLM-SST (25 mg of caraway oil and 20.75 mg of L-menthol per capsule, at 2 capsules per dose, twice per day) or placebo. Efficacy was measured at 24 hours, 2 weeks, and 4 weeks. Patients were allowed to take concomitant medications for their FD throughout the trial, and rescue medicines were allowed, 48 hours after start of dosing. RESULTS: Ninety-five patients were enrolled (mean age = 43.4 years; 75.8% women). At 24 hours, the active arm reported a statistically significant reduction in postprandial distress syndrome symptoms (P = 0.039), and a nonsignificant trend toward benefit of epigastric pain syndrome symptoms (P = 0.074). In patients with more severe symptoms, approximately 3 quarters of patients showed substantial global improvement (i.e., clinical global impressions), after 4 weeks of treatment, vs half in the control arm. These differences were statistically significant for patients with epigastric pain syndrome (P = 0.046), and trending toward significance for patients with postprandial distress syndrome (P = 0.091). There was no statistically significant difference between groups for Global Overall Symptom scores for the overall population at 2 and 4 weeks. Treatment emergent adverse events were mild to moderate, and no serious adverse events were reported. DISCUSSION: In patients taking their usual medications for FD, COLM-SST provided rapid relief (within 24 hours) and relief of severe FD symptoms. It was safe and well tolerated.

Crystal structure of the TetR/CamR family repressor Mycobacterium tuberculosis EthR implicated in ethionamide resistance.

Ethionamide has been used for more than 30 years as a second-line chemotherapeutic to treat tuberculosis patients who have developed resistance to first-line drugs, such as isoniazid (INH) and rifampicin. Activation of the pro-drug ethionamide is regulated by the Baeyer-Villiger monooxygenase EthA and the TetR/CamR family repressor EthR, whose open reading frames are separated by 75 bp on the Mycobacterium tuberculosis genome. EthR has been shown to repress transcription of the activator gene ethA by binding to this intergenic region, thus contributing to ethionamide resistance. We have determined the crystal structure of EthR, to 1.7A resolution, revealing a dimeric two-domain molecule with an overall architecture typical for TetR/CamR repressor proteins. A 20A long hydrophobic tunnel-like cavity in the "drug-binding" domain of EthR is occupied by two 1,4-dioxane molecules, a component of the crystallisation buffer. Comparing the present structure to those of the homologues Staphylococcus aureus QacR and Escherichia coli TetR leads to the hypothesis that the hydrophobic cavity constitutes a binding site for an as yet unknown ligand that might regulate DNA-binding of EthR.

Signaling mechanisms that suppress the cytostatic actions of rapamycin.

While rapamycin and the "rapalogs" Everolimus and Temsirolimus have been approved for clinical use in the treatment of a number of forms of cancer, they have not met overarching success. Some tumors are largely refractory to rapamycin treatment, with some even undergoing an increase in growth rates. However the mechanisms by which this occurs are largely unknown. The results presented here reveal novel cell-signaling mechanisms that may lead to this resistance. The absence of TGFß signaling results in resistance to rapamycin. Additionally, we observed that treatment of some cancer cell lines with rapamycin and its analogs not only potentiates mitogenic signaling and proliferation induced by HGF, but also stimulates the pro-survival kinase Akt. Together, the data show that the effectiveness of rapamycin treatment can be influenced by a number of factors and bring to light potential biomarkers for the prediction of responsiveness to treatment, and suggest combination therapies to optimize rapalog anticancer efficacy.

Small-molecule Bax agonists for cancer therapy.

Bax, a central death regulator, is required at the decisional stage of apoptosis. We recently identified serine 184 (S184) of Bax as a critical functional switch controlling its proapoptotic activity. Here we used the structural pocket around S184 as a docking site to screen the NCI library of small molecules using the UCSF-DOCK programme suite. Three compounds, small-molecule Bax agonists SMBA1, SMBA2 and SMBA3, induce conformational changes in Bax by blocking S184 phosphorylation, facilitating Bax insertion into mitochondrial membranes and forming Bax oligomers. The latter leads to cytochrome c release and apoptosis in human lung cancer cells, which occurs in a Bax- but not Bak-dependent fashion. SMBA1 potently suppresses lung tumour growth via apoptosis by selectively activating Bax in vivo without significant normal tissue toxicity. Development of Bax agonists as a new class of anticancer drugs offers a strategy for the treatment of lung cancer and other Bax-expressing malignancies.

An in vivo model of epithelial to mesenchymal transition reveals a mitogenic switch.

The epithelial to mesenchymal transition (EMT) is a process by which differentiated epithelial cells transition to a mesenchymal phenotype. EMT enables the escape of epithelial cells from the rigid structural constraints of the tissue architecture to a phenotype more amenable to cell migration and, therefore, invasion and metastasis. We characterized an in vivo model of EMT and discovered that marked changes in mitogenic signaling occurred during this process. DNA microarray analysis revealed that the expression of a number of genes varied significantly between post-EMT and pre-EMT breast cancer cells. Post-EMT cancer cells upregulated mRNA encoding c-Met and the PDGF and LPA receptors, and acquired increased responsiveness to HGF, PDGF, and LPA. This rendered the post-EMT cells responsive to the growth inhibitory effects of HGF, PDGF, and LPA receptor inhibitors/antagonists. Furthermore, post-EMT cells exhibited decreased basal Raf and Erk phosphorylation, and in comparison to pre-EMT cells, their proliferation was poorly inhibited by a MEK inhibitor. These studies suggest that therapies need to be designed to target both pre-EMT and post-EMT cancer cells and that signaling changes in post-EMT cells may allow them to take advantage of paracrine signaling from the stroma in vivo.

Mammary tumors initiated by constitutive Cdk2 activation contain an invasive basal-like component.

The basal-like subtype of breast cancer is associated with invasiveness, high rates of postsurgical recurrence, and poor prognosis. Aside from inactivation of the BRCA1 tumor-suppressor gene, little is known concerning the mechanisms that cause basal breast cancer or the mechanisms responsible for its invasiveness. Here, we show that the heterogeneous mouse mammary tumor virus-cyclin D1-Cdk2 (MMTV-D1K2) transgenic mouse mammary tumors contain regions of spindle-shaped cells expressing both luminal and myoepithelial markers. Cell lines cultured from these tumors exhibit the same luminal/myoepithelial mixed-lineage phenotype that is associated with human basal-like breast cancer and express a number of myoepithelial markers including cytokeratin 14, P-cadherin, alpha smooth muscle actin, and nestin. The MMTV-D1K2 tumor-derived cell lines form highly invasive tumors when injected into mouse mammary glands. Invasion is associated with E-cadherin localization to the cytoplasm or loss of E-cadherin expression. Cytoplasmic E-cadherin correlates with lack of colony formation in vitro and beta-catenin and p120(ctn) localization to the cytoplasm. The data suggest that the invasiveness of these cell lines results from a combination of factors including mislocalization of E-cadherin, beta-catenin, and p120(ctn) to the cytoplasm. Nestin expression and E-cadherin mislocalization were also observed in human basal-like breast cancer cell lines, suggesting that these results are relevant to human tumors. Together, these results suggest that abnormal Cdk2 activation may contribute to the formation of basal-like breast cancers.

Discovery of novel DNA gyrase inhibitors by high-throughput virtual screening.

The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.