Roswell Park Comprehensive Cancer Center
- Department of Pharmacology and Therapeutics
Special Interests:Molecular targets for drug discovery Tumor-associated methionine auxotrophy Small molecule regulators of the rTS signaling pathway DNA methylation inhibitors Novel Magnetic Resonance Imaging agents
The research interests of this laboratory are centered on the development of antitumor, antimicrobial, and magnetic resonance imaging (MRI) agents that interact with specific molecular targets in the pathways of S-adenosylmethionine (AdoMet) metabolism. Particular emphasis is placed on design and synthesis of molecules that target key AdoMet-associated phenomena: DNA methylation; methionine auxotrophy of human tumors; rTS signaling pathway, discovered by Dr. Bruce Dolnick at RPCI.
Structures of newly synthesized compounds are elucidated by nuclear magnetic resonance NMR) spectroscopy and mass spectroscopy (MS) in RPCI core facilities. Biological properties of newly synthesized compounds are comprehensively studied by a network of collaborators at RPCI as well as outside RPCI. These collaborative studies provide essential information for guiding structural refinements of active lead compounds and are a significant component of our drug discovery and development efforts.
Collaborator: Dr. Bruce Dolnick, Grace Cancer Drug Center, RPCI
AHLs are ubiquitous bacterial signaling molecules that regulate gene expression in a cell density dependent process known as quorum sensing. The Dolnick laboratory at RPCI has discovered that rTSb, a protein encoded by the rTS gene, catalyzes the synthesis of AHLs. rTSb is the first known human homologue of bacterial AHL synthases. Furthermore, rTSb plays an essential role in activating an rTS signaling pathway that regulates levels of the key cancer chemotherapeutic enzyme target, thymidylate synthase (TS). Various analogs of AHLs, synthesized in the Sufrin laboratory, are studied in the Dolnick laboratory for their effects on down regulating TS and inhibiting tumor cell growth. AHL analogs are evaluated in a high throughput screening assay developed in the Dolnick laboratory. Based on thier biological activity profiles, N-3-oxohexanoyl-L-homoserine lactone and N-3-oxododecanoyl-L-homoserine lactone are lead compounds for our design/synthesis of more potent, down regulators of TS function. Our synthetic efforts on this project have been strengthened by the development of a resin-based synthesis of AHLs and by the acquisition of technology for microwave assisted organic synthesis.
Collaborator: Dr. Judith Christman, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE
Our previous studies led to identification of several highly effective 24-mer ODN substrates and inhibitors of mammalian DNA methyltransferases. These ODNs have several novel structural and biochemical features: 1) they are single stranded ODNs; 2) they form a looped structure in the enzyme's active site and 3) they are substrates/inhibitors of mammalian and not bacterial DNA methyltransferases. Building on these earlier structure-activity studies, a new series of modified ODNs was synthesized and first evaluated in a cell free enzyme inhibition assay. Studies of their uptake, cellular localization and effects on DNA methylation in cultured Friend erythroleukemia cells were also done. These studies yielded a 24-mer ODN structure with "stem" and "loop" base modifications that resulted in enhanced enzyme inhibitory activity. This optimized "stem-loop" ODN, designated as a "second generation" lead structure was further modified at its potential methylation site (a cytosine at position 18 of the 24-mer). The inhibitory potency of this third generation ODN structure (3G-ODN), as measured by its Ki value, is four times greater than that of the second generation lead structure. Studies of the biological properties of 3G-ODN in cell culture, are underway. Our evaluations of ODNs in cell free enzyme assays have recently been modified to enable individual characterizations of their activities towards the maintenance DNA methyltransferase, DNMT1 and the de novo DNA methyltransferases, DNMT3a and DNMT3b. DNMT1, the most abundant form of DNA methyltransferase in somatic cells, maintains methylation patterns that are established by de novo DNA methyltransferases during development. Individual evaluations of ODNs against DNMT1, DNMT3a and DNMT3b will assist us in developing selective ODN inhibitors for each of these DNA methyltransferases and in determining the potential of each isozyme as a cancer chemotherapeutic target.
Collaborator: Dr. Richard Mazurchuk, Director, Pre Clinical MR Imaging Facility, RPCI
L-Methionine is uniquely metabolized to the key biochemical intermediate, S-adenosylmethionine (AdoMet). The metabolism of AdoMet includes its precursor roles in polyamine biosynthesis, in methylation of key macromolecular cell constituents such as DNA, RNA, proteins and phospholipids, and in production of the novel class of bacterial signaling molecules, N-acyl homoserine lactones. Methionine metabolism is altered in tumor cells and is characterized by altered DNA methylation patterns leading to aberrant gene expression and by increased utilization of methionine compared to normal cells. To meet these increased demands, some tumor cells turn to the uptake of extracellular methionine and thereby become "methionine-dependent". Tumor-associated methionine auxotrophy, has been attributed to increased demands of cellular methylation pathways rather than the protein machinery. The clinical occurrence of this metabolic defect has been recently demonstrated in surgical specimens of fresh human tumors, suggesting that novel methionine analogs, which are preferentially sequestered within methionine auxotrophic tumors, may be useful for imaging and detection of tumors. Towards this aim, a novel methionine analog (S7-41) has been synthesized in the Sufrin laboratory and is undergoing evaluation in the preclinical MR imaging Facility by Dr. Richard Mazurchuk.