Molecular Targets For Drug Development Research Articles

Cooperative cell-growth inhibition by combination treatment with ZD1839 (Iressa) and trastuzumab (Herceptin) in non-small-cell lung cancer

An important recent advance in anticancer therapy was the development of molecular-targeting drugs, such as the epidermal growth-factor receptor (EGFR)-targeting drug ZD1839 (Iressa) and the HER2-trageting anti-HER2 monoclonal antibody trastuzumab (Herceptin). ZD1839 and trastuzumab are reported to improve the therapeutic efficacy of treatment for non-small-cell lung cancer (NSCLC) and breast cancer, respectively, although the effectiveness of either drug alone is not satisfactory. NSCLC cells often express both EGFR and HER2. We therefore investigated whether a combination of ZD1839 and trastuzumab had an additive or synergistic antitumor effect. In culture ZD1839 inhibited the growth of four NSCLC cell lines (A549, NCI-H23, NCI-H727, and NCI-H661) that expressed various levels of EGFR, HER2, HER3, and HER4. A significant cytotoxic effect was observed when ZD1839 was combined with trastuzumab in A549 cells. However, this combination had no apparent effect in NCI-H23 cells. Significant G 1-phase arrest, increased p27 expression and decreased cyclin E or D1 levels were detected in A549 cells treated with ZD1839 and trastuzumab. No significant effects were detected in NCI-H23 cells examined. The combination treatment significantly inhibited the phosphorylation of EGFR, HER2, retinoblastoma, extracellular signal-regulated kinase-1/2, and protein kinase B/Akt in A549 cells, but not in NCI-H23 cells. Our results indicated that increased levels of constitutive EGFR/HER2 heterodimers were formed in A549 cells in the presence of ZD1839, whereas no heterodimer formation was detected in NCI-H23 cells. We therefore suggest that combination treatment with ZD1839 and trastuzumab might have improved therapeutic efficacy against NSCLC cells expressing both EGFR and HER2.

Oral Cancer Prevention and the Evolution of Molecular-Targeted Drug Development

The multifaceted rationale for molecular-targeted prevention of oral cancer is strong. Oral cancer is a major global threat to public health, causing great morbidity and mortality rates that have not improved in decades. Oral cancer development is a tobacco-related multistep and multifocal process involving field carcinogenesis and intraepithelial clonal spread. Biomarkers of genomic instability, such as aneuploidy and allelic imbalance, can accurately measure the cancer risk of oral premalignant lesions, or intraepithelial neoplasia (IEN). Retinoid-oral IEN studies (eg, of retinoic acid receptor-beta, p53, genetic instability, loss of heterozygosity, and cyclin D1) have advanced the overall understanding of the biology of intraepithelial carcinogenesis and of preventive agent molecular mechanisms and targets-important advances for monitoring preventive interventions and assessing cancer risk and pharmacogenomics. Clinical management of oral IEN varies from watchful waiting to complete resection, although complete resection does not prevent oral cancer in high-risk patients. New approaches, such as interventions with molecular-targeted agents and agent combinations in molecularly defined high-risk oral IEN patients, are urgently needed to reduce the devastating worldwide consequences of oral cancer.

A Comparative Proteomic Strategy for Subcellular Proteome Research

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

The host component of cancer is a biological target for the development of radiosensitizing compounds in the treatment of cancer

To determine whether enhanced radiosensitization within a host component (tumor blood vessels or stroma) improves tumor control and to determine whether DNA-PK inhibition is a molecular target for drug development. DNA dependent protein kinase (DNA-PK) is crucial to the rapid repair of DNA double strand breaks induced by ionizing radiation. DNA-PK is a heterotrimer composed of two binding subunits (Ku80, Ku70) and the DNA-PK catalytic subunit (DNA-PKcs). In SCID mice, DNA-PKcs has a truncated C-terminus and was therefore used as a DNA-PK knockout for in vivo studies of tumor response.

Drug-loaded red blood cell-mediated clearance of HIV-1 macrophage reservoir by selective inhibition of STAT1 expression.

Current highly active antiretroviral therapy (HAART) cannot eliminate HIV-1 from infected persons, mainly because of the existence of refractory viral reservoir(s). Beyond latently-infected CD4+-T lymphocytes, macrophages (M/M) are important persistent reservoirs for HIV in vivo, that represent a major obstacle to HIV-1 eradication. Therefore, a rational therapeutic approach directed to the selective elimination of long-living HIV-infected M/M may be relevant in the therapy of HIV infection. Here we report that HIV-1 chronic infection of human macrophages results in the marked increase of expression and phosphorylation of STAT1, a protein involved in the regulation of many functions such as cell growth, differentiation, and maintenance of cellular homeostasis, thereby providing a new molecular target for drug development. A single and brief exposure to 9-(beta-D-arabinofuranosyl)-2-fluoroadenine 5'-monophosphate (FaraAMP, Fludarabine), a potent antileukemic nucleoside analog active against STAT1 expressing cells, selectively kills macrophage cultures infected by HIV-1 without affecting uninfected macrophages. Furthermore, encapsulation of Fludarabine into autologous erythrocytes (RBC) and targeting to macrophages through a single-18 h treatment with drug-loaded RBC, not only abolishes the Fludarabine-mediated toxic effect on non-phagocytic cells, but also enhances the selective killing of HIV-infected macrophages. As a final result, a potent (>98%) and long-lasting (at least 4 weeks without rebound) inhibition of virus release from drug-loaded RBC-treated chronically-infected macrophages was achieved. Taken together, the evidence of HIV-1-induced increase of STAT1, and the availability of a selective drug targeting system, may prove useful in the design of new pharmacological treatments to clear the HIV-1 macrophage reservoir.

TRPV4--The EET Is On

The transient receptor potential V4 (TRPV4) channel is highly expressed in endothelial cells where either heat or a synthetic ligand can activate it. Watanabe et al. have now identified an endogenous chemical activator that stimulates this calcium entry channel. The ligand, an epoxyeicosatrienoic acid called 5,6-EET, is a metabolite of arachidonic acid (AA), which, in turn, is a metabolite of an endocannabinoid called anandamide (AEA). AEA was previously identified as an activator of TRPV1, a related channel. By treating transfected cells that overexpressed TRPV4 with pharmacological agents that inhibited various stages of AEA metabolism, the authors found that TRPV4 facilitated calcium entry and current conductance in response to 5,6-EET. When endothelial cells isolated from mouse aorta were treated with 5,6-EET, intracellular calcium concentration increased in a manner that was inhibited by blocking TRPV4. AEA and EETs are well-known vasorelaxants, which suggests that TRPV4 could be a molecular target for drug development. TRPV4 is also expressed in the hypothalamus and could underlie the psychoactive effects of some endocannabinoids. H. Watanabe, J. Vriens, J. Prenen, G. Droogmans, T. Voets, B. Nilius, Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature 424 , 434-438 (2003). [Online Journal]

Metastasis to bone: causes, consequences and therapeutic opportunities.

The most common human cancers --lung, breast and prostate -- have a great avidity for bone, leading to painful and untreatable consequences. What makes some cancers, but not others, metastasize to bone, and how do they alter its physiology? Some of the molecular mechanisms that are responsible have recently been identified, and provide new molecular targets for drug development.

Molecular characterization of recombinant Pneumocystis carinii topoisomerase I: differential interactions with human topoisomerase I poisons and pentamidine.

The Pneumocystis carinii topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized. The P. carinii topoisomerase I protein contains 763 amino acids and has a molecular mass of ca. 90 kDa. The expressed enzyme relaxes supercoiled DNA to completion and has no Mg2+ requirement. Cleavage assays reveal that both the human and P. carinii enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48. As with the human enzyme, no cleavage is stimulated in the presence of 4',6'-diamidino-2-phenylindole (DAPI) or berenil. A yeast cytotoxicity assay shows that P. carinii topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin. In contrast to the human enzyme, P. carinii topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons. The differences in the sensitivities of P. carinii and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development. Additionally, we have characterized the interaction of pentamidine with P. carinii topoisomerase I. We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.

Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells.

Gene expression profiling has revealed that diffuse large B cell lymphoma (DLBCL) consists of at least two distinct diseases. Patients with one DLBCL subtype, termed activated B cell-like (ABC) DLBCL, have a distinctly inferior prognosis. An untapped potential of gene expression profiling is its ability to identify pathogenic signaling pathways in cancer that are amenable to therapeutic attack. The gene expression profiles of ABC DLBCLs were notable for the high expression of target genes of the nuclear factor (NF)-kappaB transcription factors, raising the possibility that constitutive activity of the NF-kappaB pathway may contribute to the poor prognosis of these patients. Two cell line models of ABC DLBCL had high nuclear NF-kappaB DNA binding activity, constitutive IkappaB kinase (IKK) activity, and rapid IkappaB(alpha) degradation that was not seen in cell lines representing the other DLBCL subtype, germinal center B-like (GCB) DLBCL. Retroviral transduction of a super-repressor form of IkappaBalpha or dominant negative forms of IKKbeta was toxic to ABC DLBCL cells but not GCB DLBCL cells. DNA content analysis showed that NF-kappaB inhibition caused both cell death and G1-phase growth arrest. These findings establish the NF-kappaB pathway as a new molecular target for drug development in the most clinically intractable subtype of DLBCL and demonstrate that the two DLBCL subtypes defined by gene expression profiling utilize distinct pathogenetic mechanisms.

Invited Commentary

Treatment of non–small cell lung cancer (NSCLC) poses a challenge to oncologists as the prognosis for most patients with this disease remains poor. In the last decade, the paradigm of treating NSCLC with chemotherapy has shifted from that of nihilism to that of enthusiasm. Randomized clinical trials have demonstrated improvement of survival and quality of life among patients with advanced NSCLC treated with cisplatin-based chemotherapy versus supportive care alone. A plethora of new chemotherapeutic agents, such as gemcitabine HCl, paclitaxel, docetaxel, and vinorelbine tartrate, have also been introduced for treating NSCLC. However, measurable response rates of NSCLC to the modern regimens, for example, paclitaxel/carboplatin, are still in the range of 20% to 30% as demonstrated in randomized clinical trials. It is clear that NSCLCs are relatively chemoresistant (or chemoinsensitive) and that better systemic therapy must be developed for this devastating disease. One promising approach for systemic treatment of lung cancer is molecularly targeted therapy based on the knowledge of molecular biology of a particular tumor type. This approach is successful in introducing novel treatments other than the “traditional chemotherapy” for other tumor types. For example, trastuzumab, a monoclonal antibody targeting the HER-2/neu receptor is being used for breast cancer, and all-trans-retinoic acid, targeting the defective expression of the PML/RAR-α genes as a result of translocation of chromosomes 15 and 17, is being used for promyelocytic leukemia. It is believed that molecular targeting therapy for lung cancer is on the horizon as we better understand the molecular biology of lung cancer such as that reported in the article by Jones and associates. These authors showed that gemcitabine HCl in NSCLC cell lines upregulated the nuclear translocation of a transcription factor, nuclear factor-Kappa B (NF-κB), which is known for inhibiting programmed cell death or apoptosis. Furthermore, inhibition of the function of NF-κB by a repressor protein, IκBα-SR, led to an increase in chemosensitivity to gemcitabine HCl and in apoptosis. Thus, NF-κB and/or IκBα may be potential molecular targets for drug development. The future paradigm for systemic treatment for lung cancer will rest on the successful development of molecularly targeted therapy. Clinical trials are currently underway to investigate a number of target-specific agents such as inhibitors of protein kinases essential for cell cycle regulation, and inhibitors of metalloproteinases and cell surface receptors essential for angiogenesis and metastasis.

B cell maturation protein is a receptor for the tumor necrosis factor family member TALL-1.

TALL-1 is a recently identified member of the tumor necrosis factor (TNF) family that costimulates B lymphocyte proliferation. Here we show that B cell maturation protein (BCMA), a member of the TNF receptor family that is expressed only by B lymphocytes, specifically binds to TALL-1. A soluble receptor containing the extracellular domain of BCMA blocks the binding of TALL-1 to its receptor on the plasma membrane and inhibits TALL-1-triggered B lymphocyte costimulation. Overexpression of BCMA activates NF-kappaB, and this activation is potentiated by TALL-1. Moreover, BCMA-mediated NF-kappaB activation is inhibited by dominant negative mutants of TNF receptor-associated factor 5 (TRAF5), TRAF6, NF-kappaB-inducing kinase (NIK), and IkappaB kinase (IKK). These data indicate that BCMA is a receptor for TALL-1 and BCMA activates NF-kappaB through a TRAF5-, TRAF6-, NIK-, and IKK-dependent pathway. The identification of BCMA as a NF-kappaB-activating receptor for TALL-1 suggests molecular targets for drug development against certain immunodeficient or autoimmune diseases.

Potential mechanisms of mitochondrial cytochrome‐C release during apoptosis

Cytochrome-c release from the intermembrane space of mitochondria to the cytoplasm is a critical signaling event during many forms of apoptotic cell death. Due to the widespread involvement of apoptosis in pathobiology, this mitochondrial event is a logical target for the development of new therapeutic approaches to numerous diseases. Currently impeding such a development is our lack of understanding of the mechanism by which release of this component of the electron transport chain occurs in response to the wide variety of agents known to induce apoptosis. Release induced by the membrane permeability transition (a permeability change at the inner mitochondrial membrane) likely results from mitochondrial swelling, consequent rupture of the outer mitochondrial membrane, and nonspecific release of intermembrane space contents. An alternative mode of release may include the induction of a specific permeability change at the outer mitochondrial membrane. These two mechanisms present distinct molecular targets for drug development. Drug Dev. Res. 46:18–25, 1999. © 1999 Wiley-Liss, Inc.

Potential mechanisms of mitochondrial cytochrome-C release during apoptosis

Cytochrome-c release from the intermembrane space of mitochondria to the cytoplasm is a critical signaling event during many forms of apoptotic cell death. Due to the widespread involvement of apoptosis in pathobiology, this mitochondrial event is a logical target for the development of new therapeutic approaches to numerous diseases. Currently impeding such a development is our lack of understanding of the mechanism by which release of this component of the electron transport chain occurs in response to the wide variety of agents known to induce apoptosis. Release induced by the membrane permeability transition (a permeability change at the inner mitochondrial membrane) likely results from mitochondrial swelling, consequent rupture of the outer mitochondrial membrane, and nonspecific release of intermembrane space contents. An alternative mode of release may include the induction of a specific permeability change at the outer mitochondrial membrane. These two mechanisms present distinct molecular targets for drug development. Drug Dev. Res. 46:18–25, 1999. © 1999 Wiley-Liss, Inc.

Applying non-destructive testing to welds

Serotonin (5-HT) and brain-derived neurotrophin factor (BDNF) are known to modulate behavioral responses to stress and to mediate the therapeutic efficacy of antidepressant agents through neuroplastic and epigenetic mechanisms. While the two systems interact at several levels, this scenario is complicated by a number of variants including brain region specificity, 5-HT receptor selectivity and timing. Based on recent insights obtained using 5-HT transporter (5-HTT) knockout rats we here set-out and discuss the crucial role of neurodevelopmental mechanisms and the contribution of transcription factors and epigenetic modifications to this interaction and its variants. 5-HTT knockout in rats, as well as the low activity short allelic variant of the serotonin transporter human polymorphism, consistently show reduced BDNF mRNA and protein levels in the hippocampus and in the prefrontal cortex. This starts during the second postnatal week, is preceded by DNA hypermethylation during the first postnatal week, and it is developmentally paralleled by reduced expression of key transcription factors. The reduced BDNF levels, in turn, affect 5-HT1A receptor-mediated intracellular signaling and thereby the serotonergic phenotype of the neurons. We propose that such a negative spiral of modifications may affect brain development and reduce its resiliency to environmental challenges during critical time windows, which may lead to phenotypic alterations that persist for the entire life. The characterization of 5-HT–BDNF interactions will eventually increase the understanding of mental illness etiology and, possibly, lead to the identification of novel molecular targets for drug development.