Date of Award

5-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Wei Li, Ph.D.

Committee

Ji-seon Kim, Ph.D. Bernd Meibohm, Ph.D. Duane D. Miller, Ph.D. Andrzej Slominski, M.D., Ph.D.

Keywords

Melanoma, tubulin, inhibitor, multi-drug resistance, imidazole, thiazole

Abstract

Malignant melanoma is the most dangerous form of skin cancer and accounts for about 75% of skin cancer deaths. Once diagnosed at the metastatic stage, it has a very poor prognosis with a median survival rate of 6 months and a 5-year survival rate of less than 5%. In addition, melanoma has become an important public health hazard owing to its rising incidence, which has been well documented over the past 50 years. Currently there is no effective way to treat melanoma. It is highly resistant to existing chemotherapy, radiotherapy, and immunotherapy. Over the past 30 years, only two drugs have been approved by the Food and Drug Administration (FDA) for metastatic melanoma: dacarbazine (DTIC) and interleukin-2 (IL-2). But even with these two drugs, fewer than 15% of patients have a favorable response and fewer than 5% of patients reach complete remission. On the other hand, the toxicity associated with DTIC and IL-2 is often significant, resulting in serious or life threatening side effects in many patients. In recent years, great efforts have been made in fighting metastatic melanoma. But neither combinations of DTIC with other chemotherapy drugs (e.g., cisplatin, vinblastine, and carmustine) nor adding interferon-α2b to DTIC have shown a survival advantage over DTIC treatment alone. Extensive clinical trials with a lot of antibodies and vaccines to treat metastatic melanoma have also failed to demonstrate satisfactory efficacy. Therefore, developing more effective drugs for melanoma is urgently needed.

We started our efforts in finding new drugs for melanoma by screening a large compound library. The in vitro cytotoxicity data on several melanoma cell lines led us to the discovery of three active structure scaffolds: serine amides, serine amino alcohols, and arylthiazolidine-4-carboxylic acid amides (ATCAAs). Because ATCAAs showed better selectivity between cancer cells and normal fibroblast cells, the chemists in our group focused on this scaffold and performed extensive structure modifications for structure-activity-relationship (SAR) studies. The SAR results were then used to guide further synthesis in an effort to maximize activity and selectivity. Two active compounds identified during the process were sent to the U.S. National Cancer Institute for anticancer screening using 60 human tumor cell lines. Results showed that these two compounds have extensive cytotoxic activity against all nine types of cancer cells with IC50values ranging from 120 nM (leukemia, CCRF-CEM cell line) to 11 μM (colon cancer, HCC-15 cell line). One compound showed particularly good activity against melanoma cells (IC50=130 nM~1 mM against all eight melanoma cell lines). I then evaluated ATCAAs inhibitory effect on melanoma colony formation and in vivo melanoma tumor growth. The in vivo data were very encouraging. One tested compound significantly inhibited melanoma tumor growth at a dose of 10 mg/kg and showed higher efficacy than did DTIC at a dose of 60 mg/kg. These findings built up a strong basis for the development of novel chemotherapeutic drugs for advanced melanoma.

Furthermore, the chemists in our group also synthesized some new imidazole and imidazoline analogs by focusing on the SAR studies of the central five-member ring. Although the current compounds displayed lower potency when compared with our lead thiazolidine analogs, they may have the distinct advantage of being more stable in vivo with the reduced necessity of chiral separations. Some of these new compounds have activity similar to Sorafenib, an FDA-approved drug that has been tested clinically in melanoma patients.

To further expand our understanding of SARs and to potentially identify new platforms for active compounds, Dr. Li and Dr. Seibel explored a compound library from the University of Cincinnati’s Drug Discovery Center. This library contains 342,910 small molecules. Based on the structure of our lead molecule, two ligand-based virtual screening approaches were used: 1) similarity search based on atom connectivity by using Scitegic Pipeline Pilot software and 2) similarity search based on molecular shape by using Schrodinger software. Results showed that these two approaches are highly complementary and lead to different active molecular structures. These structures are quite suitable for further structural modification and provide new platforms for our anticancer drug discovery efforts.

Subsequently, further lead structure optimization led to the discovery of substituted methoxylbenzoyl-aryl-thiazole (SMART) compounds. To improve solubility and to circumvent the metabolic instability brought by the thiazole ring, our team designed and synthesized a new series of analogs: 2-aryl-4-benzoyl-imidazoles (ABIs). These two classes of compounds showed great in vitro cytotoxicity against melanoma, and the IC50 of the most active compound was below 10 nM. They also showed equal potency against multi-drug resistant melanoma cells and the sensitive parent cells, indicating that these compounds can effectively overcome multi-drug resistance, which is a major cause of cancer chemotherapy failure. In vivo testing on C57BL/6 mice bearing B16-F1 melanoma allograft and on double homozygous SCID (severe combined immunodeficiency) hairless outbred (SHO) mice or athymic nude mice bearing A375 human melanoma xenograft showed these two classes of compounds significantly inhibited melanoma tumor growth. Some compounds even showed substantially better activity than did DTIC, the gold standard anti-melanoma drug. Meanwhile, preliminary toxicity studies suggested that mice can tolerate tested compounds well at effective dose levels. No sign of acute toxicity was observed from the experiments. More importantly, I identified the cellular target for ABI and SMART compounds through a series of biotechniques and molecular modeling studies. Strong experimental evidence has shown that these compounds bind to tubulin at the colchicine binding site in the α/β-tubulin heterodimers to disrupt functional microtubule formation. In the meantime, I also tested the pharmacokinetic properties of some active compounds in mice together with Mr. Chien-ming Li. With their good in vivo anti-melanoma activity and their ability to overcome multi-drug resistance, these new classes of compounds have great potential for melanoma therapy.

DOI

10.21007/etd.cghs.2010.0343

Comments

Two year embargo expired May 2010

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