Download DNA Binding Properties of Novel Platinum and Palladium

DNA Binding Properties of Novel Platinum and Palladium Anti­Cancer Drugs by Erica Kennedy In Dr. Granger’s chemistry lab, we have been studying new anti­cancer drugs. Our new compounds belong to a family of compounds related to a common anti­cancer drug called cis­platin (cis­diamminedichloroplatinum). Cis­platin is widely used alone or in combination with other chemotherapeutic drugs in the treatment of several aggressive cancers, including ovarian, lung, testicular, and bladder carcinomas 1, 2 . Unfortunately, resistance to cis­platin often develops and the drug itself is toxic to the patient, with the kidneys, gastrointestinal tract, bone marrow, and nervous system all experiencing distress resulting from treatment. Not all tumors respond to cis­platin, so there is of course the hope that new drugs might be found which would be effective against cis­platin­resistant tumors. In the summer of 2004, I conducted DNA binding studies comparing cis­platin and our new anti­cancer drug. I used the technique of gel electrophoresis to determine whether or not the binding mechanism for our drug, [Pt(dione)Cl4], is in any way similar to the binding mechanism for cis­platin. We speculate that the binding mechanisms will be similar because [Pt(dione)Cl4] is an analog of cis­platin. O H 3 N Cl O Pt H 3 N N Cl N Cl Cl Cl Cl cis­platin [Pt(dione)Cl 4 ] Figure 1. Structures of cis­platin (left) and Pt(dione)Cl4 (right). The technique of gel electrophoresis must first be understood in order to understand the results. The purpose of gel electrophoresis is to separate DNA based on the number of base pairs present in the particular strand. For example, 500 base pairs and 1500 base pairs will appear as different bands on the gel. The change in DNA migration rate can be
­ 1 ­ calculated as percent change relative to a control gel and represents a retardation of electrophoretic mobility in the presence of drug. Gels are made by pouring a heated 1% agarose solution into a mold. Once it cools the gel is just like a small slab of gelatin except with millions of tiny pores unseen to the human eye. While the gel is still warm an apparatus, like a small comb, is inserted into the top of the gel so that when the gel cools, wells, or small holes, will be formed. After cooling, the gel is then carefully moved to a clear, plastic box. Water and salt­based electrolyte is poured into the box until it covers the gel. DNA is then loaded into the wells at the top of the gel. The negative cable is then attached to the end of the gel closest to the wells and the positive cable is attached to the opposite end. When voltage is applied, the DNA moves through the pores in the gel. The high current causes the gel to warm and melt slightly. The buffer is needed, as described earlier, so that the DNA remains a duplex. Since the DNA is negatively charged it will migrate towards the positive end of the gel. When the voltage is turned off, the DNA movement ceases. The gel is stained with ethidium bromide. Ethidium bromide is used because it binds to DNA and fluoresces under ultra­violet (UV) light. When the gel is placed under a UV light source the DNA bands glow and the distance traveled from the edge of the well to the end of the band is measured. The percent change in migration rate of the DNA is calculated compared to a control gel that contains no drug. A change in migration rate is usually the effect of the drug binding to the DNA and as a result the DNA slows down. We directly compared the electrophoretic migration rates of three different DNA sequences * in the presence and absence of our drug and in the presence and absence of cis­platin. This allowed us to ascertain if our drug had any preferential binding tendencies and it allowed us to directly compare our drug to cis­platin. Figures 2 and 3 summarize the results of these experiments.
­ 2 ­ Figure 2: A comparison of the electrophoretic migration rates of poly(dG­dG)­poly(dC­ dC), (Blue); poly(dG­dC)­poly(dG­dC) (Red), and poly(dA­dT)­poly(dA­dT) (Beige) Duplex DNA in the absence of [Pt(dione)Cl4] vs gels containing 10 M [Pt(dione)Cl4]. Data is reported as a % reduction in the migration rate. Data is an average of four replicates and the standard deviation is shown in each bar.
­ 3 ­ Figure 3: A comparison of the electrophoretic migration rates of poly(dG­dG)­poly(dC­ dC) (Blue); poly(dG­dC)­poly(dG­dC) (average zero change), and poly(dA­dT)­poly(dA­ dT) (Beige) Duplex DNA in the absence of cis­platin vs gels containing  M cis­platin. Data is reported as a % reduction in the migration rate. Data is an average of three replicates and the standard deviation is shown in each bar. These studies indicate a DNA binding mechanism for [Pt(dione)Cl4] that is uniquely different than that of cis­platin. An interesting point to notice is that the percent change in migration rate of poly(dG­dG)­poly(dC­dC) in the presence of cis­platin is considerably lower than the percent change in migration rate in the presence of [Pt(dione)Cl4]. It is known that cis­platin will bind to two adjacent guanine base pairs on a piece of DNA (intrastrand) 2,3 . It is also known that cis­platin sheds its two chlorine atoms and binds to the adjacent guanines through those binding sites. Surprisingly, [Pt(dione)Cl4] seemed to bind more effectively as noted by a percent change in migration rate that was five times greater than that of cis­platin. This is surprising because our compound has only two chlorines to shed as does cis­platin. As expected, cis­platin had little effect on the migration rates of poly(dG­dC)­poly(dG­dC) and likewise, [Pt(dione)Cl4] had little effect on the migration rate of this same DNA strand. This suggests that in a manner similar to cis­platin, [Pt(dione)Cl4] has little affinity for binding to adjacent guanines on opposite strands (interstrand) 1, 2 . We also noted that both cis­platin and [Pt(dione)Cl4] had a very similar affinity for poly(dA­dT)­poly(dA­dT). The surprise was in the strong effect relative to cis­platin that [Pt(dione)Cl4] had on poly(dG­dG)­poly(dC­dC). Many quinones have been studied as anti­tumor agents. We speculate that [Pt(dione)Cl4] may first bind to DNA through intrastrand, binding to two adjacent guanines in a manner similar to that of cis­platin, and then the quinone­dione molecule on the adjacent side of the platinum atom interacts further with the DNA strand. We are planning a series of experiments that will compare the effects of cis­platin, [Pt(dipy)Cl2] & [Pt(dione)Cl2] on the electrophoretic migration rates of poly(dG­dG)­poly(dC­dC) and 1 other select DNA duplexes. This will allow us to better understand the effect the dione moiety has on the observed changed in the electrophoretic migration rates of these DNA duplexes. * The three different DNA sequences used were poly(dG­dG)­poly(dC­dC), poly(dG­dC)­poly(dG­dC), and poly(dA­ dT)­poly(dA­dT). (1) Fuertes, M.A.; Alonso, C.; Perez, J.M. Chem Rev. 2003, 103. No.3. (2) (a) Wong, E.; Giandomenico, C.M. Chem. Rev. 1999, 99, 2451—2466. (b) Jamieson, E. R. & Lippard S. J. Chem Rev. 1999, 99, 2467­2498. (3) Full System Reference : Gabe, E.J., Le Page, Y., Charland, J.­P., Lee, F.L. and White, P.S. (1989) J. Appl. Cryst., 22, 384­387.
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