Showing papers by "Robert H. Shoemaker published in 2006"

The NCI60 human tumour cell line anticancer drug screen

Abstract: The US National Cancer Institute (NCI) 60 human tumour cell line anticancer drug screen (NCI60) was developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumours in anticancer drug screening. This screening model was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumour-cell kill. Recently, its role has changed to that of a service screen supporting the cancer research community. Here I review the development, use and productivity of the screen, highlighting several outcomes that have contributed to advances in cancer chemotherapy.

Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides

Abstract: The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule The strengths of these additional binding reactions are quantitated The implications of this multivalency for the functions of NC in virus replication are discussed

Targeting the PAS-A domain of HIF-1alpha for development of small molecule inhibitors of HIF-1.

Abstract: Hypoxia inducible factor-1 (HIF-1) is a master regulator of cellular adaptation to oxygen deprivation and activates transcription of genes involved in tumor metabolism, angiogenesis, invasion and metastasis, all of which are implicated in cancer progression. Several domains of HIF-1alpha mediate protein-protein interaction, which is essential for the formation of the active heterodimer with HIF-1beta. Targeting specific domains of HIF-1alpha might lead to the identification of more selective inhibitors. HIF-1alpha and HIF-1beta contain two Per-Arnt-Sim (PAS) domains, A and B, both of which appear to be important for heterodimer formation. In an attempt to identify small molecule inhibitors of the PAS-A domain of HIF-1 we expressed proteins containing amino acids 86-165 of HIF-1alpha and amino acids 159-240 of HIF-1beta fused to a His or FLAG tag, respectively. Expressed proteins retained functional activity as indicated by in vitro immunoprecipitation experiments and activation of luciferase expression in a mammalian two-hybrid system. Interestingly, over-expression of HIF-1alpha-PAS-A domain was sufficient to abrogate hypoxic induction of HIF-1-dependent luciferase expression, supporting its potential role as drug target. An ELISA based on the interaction between FLAG-HIF-1beta-PAS-A and HIF-1alpha-PAS-A-His was developed and used to screen libraries of synthetic compounds. NSC 50352 specifically inhibited PAS-A-dependent interaction between HIF-1alpha and HIF-1beta, but not the interaction mediated by unrelated domains. However, NSC 50352 was devoid of activity in cell-based assays. Our results provide proof-of-principle that the PAS-A domain of HIF-1alpha is a valid target for development of small molecule inhibitors.

Identification of a new natural camptothecin analogue in targeted screening for HIF-1α inhibitors

Abstract: Screening to detect compounds that inhibit the HIF-1alpha transcriptional activation pathway identified an extract of Ophiorrhiza trichocarpon for investigation. A high throughput dereplication strategy was employed, involving chromatography with spectral data acquisition supported by bioactivity testing and literature referencing, which led to rapid identification of camptothecin (1) and three analogues (2 - 4) as the active compounds. 9,10-Methylenedioxy-(20S)-camptothecin (4) was found for the first time from a plant.

A Three-Dimensional Quantitative Structure-Activity Relationship Study of the Inhibition of the ATPase Activity and the Strand Passing Catalytic Activity of Topoisomerase IIα by Substituted Purine Analogs

Abstract: Based on the topoisomerase IIα catalytic inhibitory activity of a previous hit compound, NSC35866, we screened 40 substituted purines or purine-like compounds from the National Cancer Institute repository for their ability to inhibit the ATPase activity of human topoisomerase IIα. Several compounds, including NSC348400, NSC348401 and NSC348402, were inhibitory at submicromolar concentrations. Three-dimensional quantitative structure-activity relationship models using comparative molecular field and comparative molecular similarity indices analyses were constructed using 24 of these compounds. The ability of 10 selected compounds to inhibit the complete DNA strand passage reaction of topoisomerase IIα correlated well with their potency as ATPase inhibitors. None of the 40 compounds significantly increased levels of the topoisomerase IIα-DNA covalent complex, suggesting that they functioned as catalytic topoisomerase II inhibitors and not as topoisomerase II poisons. Although some of these compounds could antagonize the effect of etoposide on the level of topoisomerase IIα-DNA covalent complex formation in vitro, in contrast to NSC35866, they were not capable of antagonizing etoposide-induced cytotoxicity and DNA strand breaks in cells. Two independently selected human SCLC cell lines with reduced topoisomerase IIα expression displayed cross-resistance to NSC348400, NBSC348401, and NSC348402, whereas an MDR1 line was fully sensitive. These results suggest that topoisomerase IIα is a functional cellular target for most of these substituted purine compounds and that these compounds do not display MDR1 liability.

In vivo and in vitro growth of alveolar soft part sarcoma (ASPS)

Abstract: 269 Alveolar soft part sarcoma (ASPS), a rare malignant neoplasm found predominantly in adolescents and young children, is characterized by a non-reciprocal chromosomal translocation in which the C-terminal region of the transcription factor TFE3 (X chromosome) is fused to the N-terminal region of the gene ASPL (Chromosome 17). Clinically, ASPS is characterized by periods of latency, extremely slow growth and a predilection for metastasis to the lung and brain. The fragility and slow growing nature of ASPS tumor cells in a milieu of rapidly proliferating stromal cells has made it extremely difficult to establish in-vivo and in-vitro models of the disease. We report here the development of an ASPS culture system, permitting the first in-vitro experiments intended to explain the phenotypic effects of the ASPS translocation. In-vivo growth of ASPS from several patients was achieved as a subcutaneous xenograft in sex-matched NOD.SCID\NCr mice. To confirm tumor origin, a representative xenograft was harvested 200 days folllowing implantation. Expression of ASPL-TFE3 fusion transcript was confirmed using RT-PCR and histological analysis revealed PAS-Diastase resistant crystals and intense nuclear staining with antibodies to both TFE3 and to a 25aa peptide homologous to the ASPL-TFE3 junction. Multiple lung metastases were present in this mouse and the tumor exhibited characteristic histopathology and immunohistochemistry. Non-enzymatic disaggregation of the tumor enabled in-vitro culture of cells growing with an estimated doubling time of 180 days, similar to that observed in-vivo. A cytokine/growth factor secretory profile of these in-vitro cultured xenograft cells was performed to better understand their signaling pathways. The results demonstrated the secretion of numerous cytokines/growth factors whose genes were localized to chromosome 17. This suggests that the chromosomal translocation t(X;17)(p11;q25) results in disregulation of multiple genes on chromosome 17. This observation was supported and extended by microarray analysis of 12 additional ASPS clinical samples of ASPS, constituting both primary tumors and metastases. Relative levels of the fusion transcript in each tumor were determined by quantitative RT-PCR, and these values were used to standardize microarray data and illuminate ASPS specific gene expression in a background of contaminating stromal cells. This novel technique revealed that a statistically significant (p