Snapka, Robert

Robert M. Snapka, Ph.D.

Professor
Department of Radiology

Joint Appointment
Department of Molecular Virology, Immunology and Medical Genetics

Ph.D. Biochemistry and Molecular Biology, University of California

Office: 103 Wiseman Hall, 400 West 12th Avenue, Columbus, OH 43210. Phone: (614)292-9375, Fax: (614)292-7237, E-mail: snapka.1@osu.edu

Research Interests

DNA replication, DNA tumor viruses, Oncolytic viruses, Virus Host Range Determinates, Cytotoxic Mechanisms of Anticancer Drugs, Drug Resistance, Topoisomerases, Deoxyribo-proteomics.

Research Summary

The laboratory uses molecular, cellular and biochemical, and proteomic approaches to understand the mechanisms by which anticancer drugs kill cancer cells. The primary focus is on drugs targeting DNA replication, and the molecular events involved in cell killing by these drugs. The laboratory is also working in the areas of virus host range, oncolytic viruses (tumor targeting viruses) and proteomics.

Selected Publications:

Gao, H, Yamsaki, E.F., Chan, K.K. Shen, L.L. and Snapka, R.M., (2003) DNA Sequence Specificity for Topoisomerase II Poisoning by the Quinoxaline Anticancer Drugs XK469 and CQS. Molecular Pharmacology 63(6):1382-1388.  web link:
http://molpharm.aspetjournals.org/cgi/reprint/63/6/1382.pdf

Topoisomerase II Poisoning by ICRF-193. Kuan-Chun Huang, Hanlin Gao, Edith F. Yamasaki, Dale R. Grabowski, Shujun Liu, Linus L. Shen, Kenneth K. Chan, and Robert M. Snapka. Journal of Biological Chemistry 276:44488-44494, 2001. [Abstract] [Full Text] [PDF] ICRF-193 is one of the most studied members of the bis(2,6-dioxopiperazine) class of anticancer drugs. They were originally made as cell membrane permeant metal chelators (similar to EDTA) on the theory that metal chelation might be important for anticancer activity. ICRF-193 and its analogs do have anticancer activity, but evidence accumulated that metal chelation was not involved. Eventually T. Andoh and co-workers showed that bis(dioxopiperazine)s were potent mammalian topoisomerase II catalytic inhibitors. Work in many laboratories over a number of years confirmed that these compounds strongly inhibited topoisomerase II in vitro and in vivo (see below), but no direct evidence of topoisomerase poisoning was found. We found that ICRF-193 is a strong topoisomerase II poison, both in vivo and in vitro, when chaotropic denaturants are used in the assays. This is similar to the finding for CQS (see below). It seems that topoisomerase II poisoning was missed due to the almost universal use of the detergent, SDS, in topoisomerase poisoning assays. We also find that ICRF-193 shows selectivity for the beta-isozyme of topoisomerase II, although this preference is not as strong as that of XK469 (see below).

Cytotoxic Mechanism of XK469: Resistance of Topoisomerase IIb Knockout Cells and Inhibition of Topoisomerase I. Robert M. Snapka, Hanlin Gao, Dale R. Grabowski, David Brill, Kenneth K. Chan, Ligeng Li, Gloria C. Li, Ram Ganapathi. Biochem. Biophys. Res. Commun. 280:1155-1160, 2001. [Abstract] [PDF] Topoisomerase IIb knockout mouse cells were found to be much more resistant to XK469 than the parental topoisomerase II +/+ cells. The difference in sensitivity to XK469 was shown to parallel the difference in XK469-induced protein-DNA crosslinks (topoisomerase II poisoning).

Chloroquinoxaline Sulfonamide (NSC 33004) is a Topoisomerase IIa/b poison. Hanlin Gao, Edith F. Yamasaki, Kenneth K. Chan, Linus L. Shen and Robert M. Snapka. Cancer Research 60:5937-5940, 2000. [Abstract] [Full Text] [PDF]. CQS was discovered in a clonogenic assay and was developed to the point of Phase II human trials based on low non-specific cytotoxicity and solid tumor activity - yet its cytotoxic mechanism has remained elusive. We report here that CQS is a topoisomerase II a/b poison. This makes it the second quinoxaline to have topoisomerase II activity and, with our report on XK469 (see below), suggests that solid tumor activity may be a characteristic of quinoxaline anticancer drugs. In contrast to XK469, CQS does not show high selectivity for the beta isozyme of topoisomerase II. However, CQS is unique in one respect. Topoisomerase poisoning by CQS can only be shown clearly with strong chaotropic protein denaturants such as guanidinium chloride or urea. The topoisomerase poisoning is almost undetectable with SDS, the most commonly used protein denaturant in topoisomerase poisoning assays.

XK469, A Selective Topoisomerase IIb poison. Hanlin Gao, Kuan-Chun Huang, Edith F. Yamasaki, Kenneth K. Chan, Lubna Chohan and Robert M. Snapka. Proc. Natl. Acad. Sci. U.S.A. 96:12168-12173 (1999). [Abstract] [PDF] [Full Text] XK469 was discovered as a solid tumor-specific anticancer drug with low non-specific cytotoxicity. Although it advanced to the point of clinical trials based on these unusual characteristics, its mechanism was unknown. In this manuscript, we show that XK469 is a highly specific topoisomerase II beta poison. This is the first report of a drug with high specificity for the poorly understood beta isozyme of topoisomerase II and the first report of topoisomerase poisoning for quinoxalines.

Maintenance of Episomal SV40 Genomes in GM637 Human Fibroblasts. Kuan-Chun Huang, Edith F. Yamasaki, and Robert M. Snapka. Virology 262:457-469 (1999). Abstract Article (PDF 459K) The mutant gmSV40 genomes maintained at very high levels in GM637 human fibroblasts are shown to represent a persistent lytic infection. GmSV40 is an SV40 host range mutant with greatly increased infectivity for human cells. This paper includes an infective center assay which can be used to titer any virus for which there is a DNA probe on any permissive or semipermissive cell line. It does not require the production of visible cytopathic plaques. Our results indicate that infectivity assays based only on cytopathic plaques can be inaccurate by orders of magnitude.

Inhibition of Topoisomerase II by Aporphine Alkaloids. Sung Ho Woo, Nan Jun Sun, John M. Cassady and Robert M. Snapka. Biochemical Pharmacology 57:1141-1145, 1999. The aporphine alkaloid, dicentrine is shown to be a topoisomerase II inhibitor, but the very similar compound bulbocapnine is inactive. The evidence suggests that dicentrine can attain a planar conformation and intercalate into DNA while bulbocapnine is prevented from doing this due to steric interaction between the 11-OH group and an oxygen of the methylenedioxy ring. This suggests that dicentrine is an adaptive DNA intercalator and the the activity of such DNA binding drugs can be modulated by substitutions that affect their ability to adopt a planar structure.

DNA Polymerase and Topoisomerase II Inhibitors from Psoralea corylifolia. Nan Jun Sun, Sung Ho Woo, John M. Cassady and Robert M. Snapka. Journal of Natural Products 61:362-366, 1998. 
 
Psoralea corylifolia is an ancient medicinal plant of India and China. It is still cultivated for medicinal purposes in China today. Since P. corylifolia's folkloric uses included cancer treatment, we tested the plant extract using and intact cell assay which can detect inhibitors of specific DNA replication enzymes. Drugs which target enzymes of DNA replication often have anticancer activity. The test indicated the presence of one or more DNA polymerase inhibitors, and the assay was then used to guide their isolation. The activity of the plant was mainly due to bakuchiol and to corylifolin (a new compound). Several weakly active topoisomerase II and DNA polymerase inhibitors were also detected as subfractions of the plant were concentrated and tested. Psoralen, a DNA photocrosslinker, is the compound that P. corylifolia is best known for, but it was inactive in our assay. The psoralen analog, bakuchicin, however was found to be a very weak topoisomerase II inhibitor. After consideration of the structure of the active compounds corylifolin and bakuchiol, resveratrol was obtained from a commercial source and tested for activity. It also had significant activity as a replicative DNA polymerase inhibitor. Resveratrol is more often studied as an antioxidant.

Unbalanced Growth in Mouse Cells With Amplified dhfr Genes. R.M. Snapka, S. Ge, J. Trask and F. Robertson. Cell Proliferation 30:385-399, 1997.

Inhibition of Topoisomerase II by Liriodenine. Sung Ho Woo, Marc C. Reynolds, Nan Jun Sun, J.M. Cassady and R.M. Snapka. Biochemical Pharmacology 54:467-473, 1997.

In a search for new anticancer drugs targeting enzymes of DNA replication, Cananga odorata was found to contain an oxoaporhine alkaloid, liriodenine, which was a strong topoisomerase II inhibitor and a topoisomerase II poison. Liriodenine is not a substrate for the verapamil-sensitive MDR (multi-drug resistance) drug efflux pump in monkey kidney cells. A number of effective anticancer drugs are topoisomerase II poisons, but cancer cells can often become resistant to them by pumping them out through the MDR pump (p-glycoprotein, a membrane protein). One approach to this problem is to use MDR pump blocking drugs such as verapamil in combination with the anticancer drugs. Another approach is to find new anticancer drugs which are not substrates for the MDR pump. This result suggests that the large family of aporphine alkaloids should be explored for anticancer activity and that they may serve as a promising starting point for construction of combinatorial libraries of potential new drugs.

The SV40 Replicon Model for Analysis of Anticancer Drugs. R.M. Snapka, Ed. (1996) Academic Press, Inc. & R.G. Landes Co. ISBN 0-12-65360-9

Inhibition of Topoisomerase II by ICRF-193, the Meso Isomer of 2,3-Bis(2,6-dioxopiperazin-4-yl)butane. Critical Dependence on 2,3-Butanediyl Linker Absolute Configuration. (1996). R.M. Snapka, S.H. Woo, A.V. Blokhin and Donald T. Witiak. Biochemical Pharmacology. Biochemical Pharmacology 52:543-549, 1996.

Inverse Relationship Between Catenation and Superhelicity in Newly Replicated Simian Virus 40 Daughter Chromosomes. (1994) P.A. Permana, C.A. Ferrer and R.M. Snapka. Biochemical and Biophysical Research Communications 201:1510-1517.

SV40 DNA Replication Intermediates: Analysis of Drugs Which Target Mammalian DNA Replication. (1993) R.M. Snapka and P.A. Permana, BioEssays 15:121-127.

Gene Amplification as a Target for Cancer Chemotherapy. (1992) R.M. Snapka, Oncology Research 4:145-150.