Epidermal growth factor receptor inhibitors plus chemotherapy in non-small-cell lung cancer: biologic rationale for combination strategies

Chemotherapy continues to play an essential role in the treatment of most stages of non-small-cell lung cancer (NSCLC). In fact, within the past 5 years, this role has greatly expanded into adjuvant therapy for early-stage resected disease. Likewise, agents targeting the epidermal growth factor receptor (EGFR), particularly the tyrosine kinase inhibitors gefitinib and erlotinib, have proven to be clinically active in patients with advanced-stage NSCLC. Because of these findings, it is logical to expect that combinations of these 2 classes of antineoplastic agents would prove more efficacious than either one alone. Yet 4 large randomized phase III trials of chemotherapy with or without an EGFR tyrosine kinase inhibitor in unselected patients with advanced-stage NSCLC, altogether totaling > 4000 patients, did not demonstrate improvement in clinical outcomes with the combination. Whether these negative results will be reproduced in ongoing combination studies of chemotherapy plus monoclonal antibodies directed against EGFR remain to be determined. Herein, we review recent preclinical and clinical data addressing this topic and explore the biologic rationale for developing new combination strategies based on patient selection by molecular and clinical factors, or by pharmacodynamic parameters.

Aurora kinases as anticancer drug targets

The human aurora family of serine-threonine kinases comprises three members, which act in concert with many other proteins to control chromosome assembly and segregation during mitosis. Aurora dysfunction can cause aneuploidy, mitotic arrest, and cell death. Aurora kinases are strongly expressed in a broad range of cancer types. Aurora A expression in tumors is often associated with gene amplification, genetic instability, poor histologic differentiation, and poor prognosis. Aurora B is frequently expressed at high levels in a variety of tumors, often coincidently with aurora A, and expression level has also been associated with increased genetic instability and clinical outcome. Further, aurora kinase gene polymorphisms are associated with increased risk or early onset of cancer. The expression of aurora C in cancer is less well studied. In recent years, several small-molecule aurora kinase inhibitors have been developed that exhibit preclinical activity against a wide range of solid tumors. Preliminary clinical data from phase I trials have largely been consistent with cytostatic effects, with disease stabilization as the best response achieved in solid tumors. Objective responses have been noted in leukemia patients, although this might conceivably be due to inhibition of the Abl kinase. Current challenges include the optimization of drug administration, the identification of potential biomarkers of tumor sensitivity, and combination studies with cytotoxic drugs. Here, we summarize the most recent preclinical and clinical data and discuss new directions in the development of aurora kinase inhibitors as antineoplastic agents.

Binding of Metallocenes to Short Oligonucleotides

Cancer is one of the most severe and widespread diseases and an ideal treatment has not yet been found. In the last decades, cisplatinum was commonly applied in cancer therapy with very good results. However, serious side effects and resistant tumors necessitated the development of new antineoplastic agents, such as metallocenes dihalides. These are metal-based compounds exhibiting two cyclopentadienyl ligands and a cis-dihalide motif. They resemble the cis-chloro configuration of cisplatinum, which propounds a similar mode of action. Metallocenes comprising one of the transition metals titanium, molybdenum, vanadium, niobium, and zirconium as the metal center have been shown to be effective against several cancer cell lines. Evidence for the accumulation of metallocenes in the nucleus implied that DNA is one of the major targets. Although several studies reported adduct formation of metallocenes with nuclear DNA, as yet substantial information about the general binding pattern and the binding to higher-order structures is lacking. Mass spectrometry can fill this gap as it constitutes a powerful technique to investigate the formation of organometallic adducts. Presented data demonstrate that the two agents titanocene dichloride and molybdenocene dichloride bind to single-stranded DNA and RNA. Distinct fragment ions formed upon collision-induced dissociation help to unravel preferential binding sites within the oligonucleotides. Moreover, adducts with duplexes and quadruplexes shed light on the molecular mechanism of action.

Tandem Mass Spectrometric Analysis of Metallocene Adducts with Short Oligonucleotides

Metallocene dichlorides constitute a remarkable class of antineoplastic agents that are highly effective against several cancer cell lines. They were shown to accumulate in the DNA-rich region, which suggests DNA as the primary target. These compounds exhibit two cyclopentadienyl ligands and two labile halide ligands, resulting in a bent sandwich structure. The cis-dihalide motif is structurally related to the cis-chloro configuration of cisplatin and similar modes of action can thus be assumed. Cisplatin binds to two neighboring guanine nucleobases in DNA and consequently, distorts the double-helix, thereby inhibiting DNA replication and transcription. Platinum is classified as a soft Lewis acid and binds preferentially to the nitrogen atoms within the nucleobases. The metallocene dichlorides investigated in this study comprise the metal centers Ti, V, Nb, Mo, Hf, and W, which are classified as hard or intermediate Lewis acids, and thus, favor binding to the phosphate oxygen. Although several studies reported adduct formation of metallocene dichlorides with nucleic acids, substantial information about the adduct composition, the binding pattern, and the nucleobase selectivity has not been provided yet. ESI-MS analyses gave evidence for the formation of metallocene adducts (M = Ti, V, Mo, and W) with single-stranded DNA homologues at pH 7. No adducts were formed with Nb and Hf at neutral pH, albeit adducts with Nb were observed at a low pH. MS2 data revealed considerable differences of the adduct compositions. The product ion spectra of DNA adducts with hard Lewis acids (Ti, V) gave evidence for the loss of metallocene ligands and only moderate backbone fragmentation was observed. By contrast, adducts with intermediate Lewis acids (Mo, W) retained the hydroxy ligands. Preliminary results are in good agreement with the Pearson concept and DFT calculations. Since the metallodrugs were not lost upon CID, the nucleobase selectivity, stoichiometry, and binding patterns can be elucidated by means of tandem mass spectrometry.

Role of electrostatic interactions for ligand recognition and specificity of peptide transporters.

BACKGROUND Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. RESULTS The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. CONCLUSION This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.