Daniel Pilch Associate Professor UMDNJ Dept. of Pharmacology RWJMS. Room 422 Piscataway. NJ 08854 (732) 235-3352 FAX - 4073 pilchds@umdnj.edu

Development of topoisomerase I-directed anticancer drugs. investigations of the structure and energetics of drug-RNA interactions

The research conducted in my laboratory combines biophysical (absorbance. fluorescence. circular dichroism. calorimetry. analytical centrifugation. and viscometry). structural (NMR) biochemical (crosslinking. footprinting. gel-shift. and chemical probing). and computer modeling approaches toward investigating the following programs of research: Development of Topoisomerase I-Directed Anticancer Drugs.

The DNA topoisomerases have been established as effective molecular targets for anticancer drugs. Interest in topoisomerase I (TOP1) as a new molecular target for anticancer drugs has stimulated the search for new TOP1 inhibitors. This search has led to the identification of numerous new TOP1-inhibiting compounds. However. despite the increasing number of TOP1 inhibitors. our current understanding of the molecular mechanism(s) underlying TOP1 inhibition is still quite limited. a deficiency that hinders our ability to design new compounds with desired TOP1 inhibiting and tumor cell killing activities. To address this deficiency. the primary goal of this research program is to understand the molecular mechanism by which DNA-binding drugs stimulate human DNA TOP1-Mediated DNA damage (cleavage).

X-ray crystal structure of reconstituted human DNA topoisomerase I in covalent complex with a DNA duplex. The active-site tyrosine residue that it covalently attached to the DNA is shown in a blue space-filling model.

Investigations of the Structure and Energetics of Drug-RNA Interactions.

RNA can fold into a variety of different structures and/or conformations that can serve as specific recognition elements for drugs. Targeting these structural RNA elements in a site-specific manner offers the potential for modulating the biological function of the targeted RNA. To date. little is known about the thermodynamic driving forces that dictate. control. and stabilize drug-RNA interactions. a deficiency that limits our ability to design new agents with predictable RNA binding affinities and specificities over a range of solution conditions. The primary goal of this research program is to define the rules that govern the affinities and specificities of drugs for their RNA targets. Specifically. we are defining the relative contributions of van der Waals contacts. hydrogen bonding. and electrostatic interactions to the binding affinities and specificities of RNA-directed ligands. We also are evaluating how the presence and sequence of loops and bulges in the host RNA modulate drug recognition. Currently. our studies are focused on targeting various bacterial. viral. and group I intron RNA sequences.

Selected Publications

Nudelman I, Rebibo-Sabbah A, Cherniavsky M, Belakhov V, Hainrichson M, Chen F, Schacht J, Pilch DS, Ben-Yosef T, Baasov T. (2009) Development of novel aminoglycoside (NB54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations. J Med Chem. 52(9):2836-45.

Satyanarayana M, Rzuczek SG, Lavoie EJ, Pilch DS, Liu A, Liu LF, Rice JE. (2008) Ring-closing metathesis for the synthesis of a highly G-quadruplex selective macrocyclic hexaoxazole having enhanced cytotoxic potency. Bioorg Med Chem Lett. 18(13):3802-4.

Pilch DS, Barbieri CM, Rzuczek SG, Lavoie EJ, Rice JE. (2008) Targeting human telomeric G-quadruplex DNA with oxazole-containing macrocyclic compounds. Biochimie. 90(8):1233-49.

Barbieri CM, Kaul M, Pilch DS. (2007) Use of 2-aminopurine as a fluorescent tool for characterizing antibiotic recognition of the bacterial rRNA A-site. Tetrahedron. 63(17):3567-6574.

Rzuczek SG, Pilch DS, LaVoie EJ, Rice JE. (2008) Lysinyl macrocyclic hexaoxazoles: synthesis and selective G-quadruplex stabilizing properties. Bioorg Med Chem Lett. 18(3):913-7.

Kondo J, Hainrichson M, Nudelman I, Shallom-Shezifi D, Barbieri CM, Pilch DS, Westhof E, Baasov T. (2007) Differential selectivity of natural and synthetic aminoglycosides towards the eukaryotic and prokaryotic decoding A sites. Chembiochem. 8(14):1700-9.

Barbieri CM, Srinivasan AR, Rzuczek SG, Rice JE, Lavoie EJ, Pilch DS. (2007) Defining the mode, energetics and specificity with which a macrocyclic hexaoxazole binds to human telomeric G-quadruplex DNA. Nucleic Acids Res. 35(10):3272-86.

Kaul M, Barbieri CM, Srinivasan AR, Pilch DS. (2007) Molecular determinants of antibiotic recognition and resistance by Aminoglycoside Phosphotransferase (3')-IIIa: A calorimetric and mutational analysis. J Mol Biol. 369(1):142-56.

Barbieri CM, Kaul M, Bozza-Hingos M, Zhao F, Tor Y, Hermann T, Pilch DS. (2007) Defining the molecular forces that determine the impact of neomycin on bacterial protein synthesis: importance of the 2'-amino functionality. Antimicrob Agents Chemother. 51(5):1760-9.

Qi H, Lin CP, Fu X, Wood LM, Liu AA, Tsai YC, Chen Y, Barbieri CM, Pilch DS, Liu LF. (2006) G-quadruplexes induce apoptosis in tumor cells. Cancer Res. 66(24):11808-16.

Khan QA, Pilch DS. (2007) Topoisomerase I-mediated DNA cleavage induced by the minor groove-directed binding of bibenzimidazoles to a distal site. J Mol Biol. 365(3):561-9.

Khan QA, Barbieri CM, Srinivasan AR, Wang YH, LaVoie EJ, Pilch DS. (2006) Drug self-association modulates the cellular bioavailability of DNA minor groove-directed terbenzimidazoles. J Med Chem. 49(17):5245-51.

Minhas GS. Pilch DS. Kerrigan JE. Lavoie EJ. Rice JE. (2006) Synthesis and G-quadruplex stabilizing properties of a series of oxazole-containing macrocycles. Bioorg Med Chem Lett. 16(15):3891-5.

Kaul M. Barbieri CM. Pilch DS. (2006) Aminoglycoside-induced reduction in nucleotide mobility at the ribosomal RNA A-site as a potentially key determinant of antibacterial activity. J Am Chem Soc. Feb 1;128(4):1261-71.

Barbieri CM. Pilch DS. (2006) Complete thermodynamic characterization of the multiple protonation equilibria of the aminoglycoside antibiotic paromomycin: a calorimetric and natural abundance 15N NMR study. Biophys J. 90(4):1338-49.

Kaul M. Barbieri CM. Pilch DS. (2005) Defining the basis for the specificity of aminoglycoside-rRNA recognition: a comparative study of drug binding to the A sites of Escherichia coli and human rRNA. J Mol Biol. 346(1):119-34.

Barbieri CM. Srinivasan AR. Pilch DS. (2004) Deciphering the origins of observed heat capacity changes for aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA a-sites: a calorimetric. computational. and osmotic stress study. J Am Chem Soc. 126(44):14380-8.

Li TK. Barbieri CM. Lin HC. Rabson AB. Yang G. Fan Y. Gaffney BL. Jones RA. Pilch DS. (2004) Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication. Biochemistry. 43(30):9732-42.

Kaul M. Barbieri CM. Pilch DS. (2004) Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences. J Am Chem Soc. 126(11):3447-53.

Kerrigan JE. Pilch DS. Ruchelman AL. Zhou N. Liu A. Liu L. LaVoie EJ. (2003) 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones and related compounds as TOP1-targeting agents: influence of structure on the ternary cleavable complex formation. Bioorg Med Chem Lett. 13(20):3395-9.

Pilch DS. Kaul M. Barbieri CM. Kerrigan JE. (2003) Thermodynamics of aminoglycoside-rRNA recognition. Biopolymers. 70(1):58-79.

Barbieri CM. Li TK. Guo S. Wang G. Shallop AJ. Pan W. Yang G. Gaffney BL. Jones RA. Pilch DS. (2003) Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes. J Am Chem Soc. 125(21):6469-77.

Kaul M. Barbieri CM. Kerrigan JE. Pilch DS. (2003) Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities. J Mol Biol. 326(5):1373-87.

Zhang Y. Li Z. Pilch DS. Leibowitz MJ. (2002) Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding. Nucleic Acids Res. 30(13):2961-71.

Kaul M. Pilch DS. (2002) Thermodynamics of aminoglycoside-rRNA recognition: the binding of neomycin-class aminoglycosides to the A site of 16S rRNA. Biochemistry. 41(24):7695-706.