Rick A. Finch

Wnk1 kinase deficiency lowers blood pressure in mice: A gene-trap screen to identify potential targets for therapeutic intervention

The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in ≈60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

New insights into the biology and pharmacology of the multidrug resistance protein (MRP) from gene knockout models

Growing interest in the MRP (multidrug resistance protein) gene stems from its importance in multidrug resistance to chemotherapy, its possible use in gene therapy, and its relationship with the glutathione system. The recent generation of mrp gene knockout models in vitro and in vivo is providing information on the mechanism of action and the physiological function(s) of mrp. The importance of mrp in protection of normal tissues from the toxicity of the anticancer agent etoposide has been established. A total block of mrp has been found to be compatible with life, suggesting that MRP inhibitors can be safely used for treating cancer patients. In some sub-classes of leukocytes, mrp contributes to the transport of leukotriene C4, an endogenous glutathione-S-conjugate. However, the baseline expression of mrp does not appear to contribute to the export of glutathione-S-conjugates of alkylating agents, and thus does not exert a protective role against their toxicity. Besides being capable of exporting certain glutathione-S-conjugates, mrp also catalyzes the co-transport of GSH and drug and, presumably, a presently unknown endogenous metabolite(s).

Targeting Oncogenes to Improve Breast Cancer Chemotherapy

Despite recent advances in treatment, breast cancer remains a serious health threat for women. Traditional chemotherapies are limited by a lack of specificity for tumor cells and the cell cycle dependence of many chemotherapeutic agents. Here we report a novel strategy to help overcome these limitations. Using triplex-forming oligonucleotides (TFOs) to direct DNA damage site-specifically to oncogenes overexpressed in human breast cancer cells, we show that the effectiveness of the anticancer nucleoside analogue gemcitabine can be improved significantly. TFOs targeted to the promoter region of c-myc directly inhibited gene expression by approximately 40%. When used in combination, specific TFOs increased the incorporation of gemcitabine at the targeted site approximately 4-fold, presumably due to induction of replication-independent DNA synthesis. Cells treated with TFOs and gemcitabine in combination showed a reduction in both cell survival and capacity for anchorage-independent growth (approximately 19% of untreated cells). This combination affected the tumorigenic potential of these cancer cells to a significantly greater extent than either treatment alone. This novel strategy may be used to increase the range of effectiveness of antitumor nucleosides in any tumor which overexpresses a targetable oncogene. Multifaceted chemotherapeutic approaches such as this, coupled with triplex-directed gene targeting, may lead to more than incremental improvements in nonsurgical treatment of breast tumors.

Antitumor Sulfonylhydrazines: Design, Structure–Activity Relationships, Resistance Mechanisms, and Strategies for Improving Therapeutic Utility

1,2-Bis(sulfonyl)-1-alkylhydrazines (BSHs) were conceived as more specific DNA guanine O-6 methylating and chloroethylating agents lacking many of the undesirable toxicophores contained in antitumor nitrosoureas. O(6)-Alkylguanine-DNA alkyltransferase (MGMT) is the sole repair protein for O(6)-alkylguanine lesions in DNA and has been reported to be absent in 5-20% of most tumor types. Many BSHs exhibit highly selective cytotoxicity toward cells deficient in MGMT activity. The development of clinically useful MGMT assays should permit the identification of tumors with this vulnerability and allow for the preselection of patient subpopulations with a high probability of responding. The BSH system is highly versatile, permitting the synthesis of many prodrug types with the ability to incorporate an additional level of tumor-targeting due to preferential activation by tumor cells. Furthermore, it may be possible to expand the spectrum of activity of these agents to include tumors with MGMT activity by combining them with tumor-targeted MGMT inhibitors.

Lead optimization and structure-based design of potent and bioavailable deoxycytidine kinase inhibitors

A series of deoxycytidine kinase inhibitors was simultaneously optimized for potency and PK properties. A co-crystal structure then allowed merging this series with a high throughput screening hit to afford a highly potent, selective and orally bioavailable inhibitor, compound 10. This compound showed dose dependent inhibition of deoxycytidine kinase in vivo.

5-Fluorocytosine derivatives as inhibitors of deoxycytidine kinase

A series of potent piperidine-linked cytosine derivatives were prepared as inhibitors of deoxycytidine kinase (dCK). Compound 9h was discovered to be a potent inhibitor of dCK and shows a good combination of cellular potency and pharmacokinetic parameters. Compound 9h blocks the incorporation of radiolabeled cytosine into mouse T-cells in vitro, as well as in vivo in mice following a T-cell challenge.

Nucleoside and Nucleotide Modulation of Oncogenic Expression: A New Approach to Cancer Chemotherapy

The last 15 years have seen a virtual explosion in knowledge of cancer at the molecular level. Major efforts in the area of molecular genetics were stimulated by a study of viral oncogenes (1) and have centered around the activation of proto-oncogenes, which code for proteins that are involved in the signal transduction events that modulate normal cellular growth and differentiation. More that one hundred normal cellular proto-oncogenes are now known (2). Mechanisms of activation of proto-oncogenes to cellular oncogenes include point mutation, deletion, insertion, amplification, activation by internal rearrangement, chromosomal translocation, and promoter insertion (3). Cancer would appear to have many causes, but a common element is DNA damage resulting in aberrant gene expression which is a multi-step process (4). Chromosomal analyses of tumor cells have revealed many abnormal karyotypes with metastases as the most aberrant. Most cancers exhibit some cytogenetic defect (5).