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Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti-cancer effects and pharmacological properties

Background and purpose:  ABC multidrug transporters (MDR‐ABC proteins) cause multiple drug resistance in cancer and may be involved in the decreased anti‐cancer efficiency and modified pharmacological properties of novel specifically targeted agents. It has been documented that ABCB1 and ABCG2 interact with several first‐generation, small‐molecule, tyrosine kinase inhibitors (TKIs), including the Bcr‐Abl fusion kinase inhibitor imatinib, used for the treatment of chronic myeloid leukaemia. Here, we have investigated the specific interaction of these transporters with nilotinib, dasatinib and bosutinib, three clinically used, second‐generation inhibitors of the Bcr‐Abl tyrosine kinase activity.

Integrating molecular diagnostics into anticancer drug discovery

In the 1990s, the breast cancer drug trastuzumab (Herceptin; Genentech/Roche) — an antibody specific for human epidermal growth factor receptor 2 (HER2; also known as ERBB2) — was approved based on trials in which HER2 expression levels were used to select patients in clinical trials. This provided support for analogous efforts for drugs that target the epidermal growth factor receptor (EGFR). However, the development of these drugs, such as cetuximab (Erbitux; Bristol–Myers Squibb/Lilly) and gefitinib (Iressa; AstraZeneca), has revealed that EGFR expression is an insufficient and unreliable biomarker to select patients for EGFR-targeted therapies in both lung and colon cancer. Indeed, evidence on patient populations that are likely to respond to such therapies, on the basis of specific mutations in proteins of the targeted pathway, has only recently been clinically validated and incorporated into some of the drug labels. This article highlights lessons learned from the development of the first drugs targeting the EGFR family and discusses strategies to decrease the risk of failure in clinical development by more effectively integrating molecular diagnostics into anticancer drug discovery.

Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: Implications for the emergence and reversal of cancer drug resistance

Human ABCG2 is a plasma membrane glycoprotein that provides physiological protection against xenobiotics. ABCG2 also significantly influences biodistribution of drugs through pharmacological tissue barriers and confers multidrug resistance to cancer cells. Moreover, ABCG2 is the molecular determinant of the side population that is characteristically enriched in normal and cancer stem cells. Numerous tumors depend on unregulated EGFR signaling, thus inhibition of this receptor by small molecular weight inhibitors such as gefitinib, and the novel second generation agents vandetanib, pelitinib and neratinib, is a promising therapeutic option. In the present study, we provide detailed biochemical characterization regarding the interaction of these EGFR inhibitors with ABCG2. We show that ABCG2 confers resistance to gefitinib and pelitinib, whereas the intracellular action of vandetanib and neratinib is unaltered by the presence of the transporter. At higher concentrations, however, all these EGFR inhibitors inhibit ABCG2 function, thereby promoting accumulation of ABCG2 substrate drugs. We also report enhanced expression of ABCG2 in gefitinib-resistant non-small cell lung cancer cells, suggesting potential clinical relevance of ABCG2 in acquired drug resistance. Since ABCG2 has important impact on both the pharmacological properties and anti-cancer efficiencies of drugs, our results regarding the novel EGFR inhibitors should provide useful information about their therapeutic applicability against ABCG2-expressing cancer cells depending on EGFR signaling. In addition, the finding that these EGFR inhibitors efficiently block ABCG2 function may help to design novel drug-combination therapeutic strategies.

Anticytolytic Screen Identifies Inhibitors of Mycobacterial Virulence Protein Secretion

Mycobacterium tuberculosis (Mtb) requires protein secretion systems like ESX-1 for intracellular survival and virulence. The major virulence determinant and ESX-1 substrate, EsxA, arrests phagosome maturation and lyses cell membranes, resulting in tissue damage and necrosis that promotes pathogen spread. To identify inhibitors of Mtb protein secretion, we developed a fibroblast survival assay exploiting this phenotype and selected molecules that protect host cells from Mtb-induced lysis without being bactericidal in vitro. Hit compounds blocked EsxA secretion and promoted phagosome maturation in macrophages, thus reducing bacterial loads. Target identification studies led to the discovery of BTP15, a benzothiophene inhibitor of the histidine kinase MprB that indirectly regulates ESX-1, and BBH7, a benzyloxybenzylidene-hydrazine compound. BBH7 affects Mtb metal-ion homeostasis and revealed zinc stress as an activating signal for EsxA secretion. This screening approach extends the target spectrum of small molecule libraries and will help tackle the mounting problem of antibiotic-resistant mycobacteria.

Synthesis and evaluation of phosphorus containing, specific CDK9/CycT1 inhibitors.

Although there is a significant effort in the design of a selective CDK9/CycT1 inhibitor, no compound has been proven to be a specific inhibitor of this kinase so far. The aim of this research was to develop novel and selective phosphorus containing CDK9/CycT1 inhibitors. Molecules bearing phosphonamidate, phosphonate, and phosphinate moieties were synthesized. Prepared compounds were evaluated in an enzymatic CDK9/CycT1 assay. The most potent molecules were tested in cell-based toxicity and HIV proliferation assays. Selectivity of shortlisted compounds against CDKs and other kinases was tested. The best compound was shown to be a highly specific, ATP-competitive inhibitor of CDK9/CycT1 with antiviral activity.

TP53 mutation‐correlated genes predict the risk of tumor relapse and identify MPS1 as a potential therapeutic kinase in TP53‐mutated breast cancers

Breast cancers (BC) carry a complex set of gene mutations that can influence their gene expression and clinical behavior. We aimed to identify genes driven by the TP53 mutation status and assess their clinical relevance in estrogen receptor (ER)‐positive and ER‐negative BC, and their potential as targets for patients with TP53 mutated tumors. Separate ROC analyses of each gene expression according to TP53 mutation status were performed. The prognostic value of genes with the highest AUC were assessed in a large dataset of untreated, and neoadjuvant chemotherapy treated patients. The mitotic checkpoint gene MPS1 was the most significant gene correlated with TP53 status, and the most significant prognostic marker in all ER‐positive BC datasets. MPS1 retained its prognostic value independently from the type of treatment administered. The biological functions of MPS1 were investigated in different BC cell lines. We also assessed the effects of a potent small molecule inhibitor of MPS1, SP600125, alone and in combination with chemotherapy. Consistent with the gene expression profiling and siRNA assays, the inhibition of MPS1 by SP600125 led to a reduction in cell viability and a significant increase in cell death, selectively in TP53‐mutated BC cells. Furthermore, the chemical inhibition of MPS1 sensitized BC cells to conventional chemotherapy, particularly taxanes. Our results collectively demonstrate that TP53‐correlated kinase MPS1, is a potential therapeutic target in BC patients with TP53 mutated tumors, and that SP600125 warrant further development in future clinical trials.

Targeting of a platinum-bound sunitinib analog to renal proximal tubular cells

Background Activated proximal tubular cells play an important role in renal fibrosis. We investigated whether sunitinib and a kidney-targeted conjugate of sunitinib were capable of attenuating fibrogenic events in tubulointerstitial fibrosis. Methods A kidney-targeted conjugate was prepared by linkage of a sunitinib analog (named 17864) via a platinum-based linker to the kidney-specific carrier lysozyme. Pharmacological activity of 17864-lysozyme was evaluated in human kidney proximal tubular cells (HK-2); the capability of the kidney-directed conjugate to accumulate in the kidneys was studied in mice. Potential antifibrotic effects of a single-dose treatment were evaluated in the unilateral ureteral obstruction (UUO) model in mice. Results The 17864-lysozyme conjugate and its metabolites strongly inhibited tyrosine kinase activity. Upon intravenous injection, 17864-lysozyme rapidly accumulated in the kidneys and provided sustained renal drug levels for up to 3 days after a single dose. Renal drug level area under the curve was increased 28-fold versus an equimolar dose of sunitinib malate. Daily treatment of UUO mice with a high dose of sunitinib malate (50 mg/kg) resulted in antifibrotic responses, but also induced drug-related toxicity. A single dose of 17864-lysozyme (equivalent to 1.8 mg/kg sunitinib) was safe but showed no antifibrotic effects. Conclusion Multikinase inhibitors like sunitinib can be of benefit in the treatment of fibrotic diseases, provided that their safety can be improved by strategies as presented in this paper, and sustained renal levels can be achieved.

Dendrimer‐Based Macromolecular Conjugate for the Kidney‐Directed Delivery of a Multitargeted Sunitinib Analogue

The development of a macromolecular conjugate of a multitargeted tyrosine kinase inhibitor is described that can be used for renal-specific delivery into proximal tubular cells. A novel sunitinib analogue, that is, 17864, is conjugated to a NH(2) -PAMAM-G3 dendrimer via the platinum (II)-based Universal Linkage System (ULS™). The activity of 17864 is retained after coordination to the ULS linker alone or when coupled to NH(2) -PAMAM-G3. 17864-UlS-NH(2) -PAMAM-G3 is non-toxic to proximal tubular cells in vitro. After intravenous administration to mice, 17864-UlS-NH(2) -PAMAM-G3 rapidly and efficiently accumulates in the kidneys. These results are encouraging for future studies focusing on the development of novel therapeutics for the treatment of renal diseases.

EVALUATION OF HYDROPHOBICITY AND ANTITUMOR ACTIVITY OF A MOLECULE LIBRARY OF MANNICH KETONES OF CYCLOALKANONES

A series of Mannich ketones of cycloalkanones were synthesized to study the relative importance of structure and specific substitutions in relation to their hydrophobicity and antitumor activity. Substitutions were carried out with morpholinyl, pirrolidinyl, piperidyl, and tetrahydro-isoquinolyl groups in various position on four different base structures. Hydrophobicity of Mannich ketones was characterized by chromatography data (log k′) and by software calculated parameters (c log P). Cell proliferation inhibitory activity of cycloalkanones was evaluated by MTT and apoptosis assays on A431 human adenocarcinoma cells. Our results suggest that the higher the hydrophobicity values (log k′ and c log P) the higher the antitumor- and apoptotic activity of Mannich ketones. Determination of hydrophobicity by measuring the log k′ or by calculating the c log P values of the compounds may help to predict their biological activities.

A NOVEL NORMALIZED RETENTION FACTOR IN MICELLAR ELECTROKINETIC CHROMATOGRAPHY

Characteristic properties of the expression k″ = (tm—to)/(tmc—to) and its applicability in micellar electrokinetic capillary chromatography (MEKC) were compared to the previous expression, k′ = (tm—to)/to(1‐tm/tmc), introduced by Terabe. It was proved with theoretical calculations (curve shape analysis) that the properties of function k″ are in full accordance with the properties of the MEKC system and k″ could be applied advantageously to characterize hydrophobicity of the analytes. This conclusion is now supported by experimental data obtained with homolog series of alkylbenzenes and alkylphenones as well as with hydrophobic protected peptides. Migration times, k′, k″ values, and software‐calculated hydrophobicity data are summarized and analyzed in the present study. Since k″ is a normalized parameter, good relationships between the migration time, the software‐calculated hydrophobicity, and the k″ values were obtained. Differences in hydrophobicity of the analytes could be estimated in a more realistic way with the aid of function k″ than by using function k′. Hydrophobicity data estimated on the basis of the k″ values proved to be in good accordance with the expectations based on the migration times and on the chemical structures of the compounds investigated.