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Cis-platin has been one of the most widely used anticancer drug which acts as a DNA alkylating agent for its cytotoxicity. Despite of its profound efficacy, increasing drug resistance and toxicity have limited the use of the drug along with lower survival rate. Drug resistance generally arises through two different ways: Firstly, elevated cellular expression of glutathione that acts as a detoxification agent and secondly, repair of platinated DNA molecules by DNA repair enzymes. In this project, I intend to explore the ability of Pt-Ru bimetallic anticancer drug to overcome drug resistance. The basic concept behind the proposal is that Pt and Ru anticancer drugs are not cross resistance, mainly because of their different mode of binding with DNA and thus, are more difficult to be eliminated by the same type of DNA repair enzymes/mechanism. Also, both the Pt and Ru anticancer drugs tethered with Glutathione S-transferase inhibitors have been reported to be more effective against drug resistant cancer cell line. The specific aims for this proposal are designed to address the issue of drug resistance arising from DNA repair mechanism.
To synthesize and characterize a Pt-Ru bimetallic anticancer drug. A hexacoordinated Pt drug (tetraplatin analogue) will be utilized for the synthesis because of its inertness towards ligand substitution giving a superior survival capability under physiological conditions compared to the parent cisplatin analogues along with reduced cytotoxicity. The employment of covalent attachment is with the intention that the ratio of two drug molecules can be varied directly at the molecular level. Also, it has been reported that Ru-anticancer drugs are accumulated preferentially at cancer cells compared to normal cell by transferrin mediated drug transport. Thus, covalent attachment will ensure the preferential accumulation of Pt-anticancer drug in the same antineoplastic masses along with the Ru-drug.
Schematic representation of bimetallic anticancer drug
Drug molecules will be synthesized using standard inorganic synthetic procedures with functionalized ligands and will be characterized using standard techniques such as NMR, MALDI etc. In the next step, the cytotoxicity of the newly synthesized drug molecules and their ability to overcome drug resistance against different cancer lines will be evaluated in vitro. Uptake and quantification of the drugs in cell will be determined using ICP-AES (Inductively coupled plasma atomic emission spectroscopy).The efficacy of inhibition of cell proliferation by the drug molecules will be determined by exposing the cancer cells at different concentrations of synthesized molecules and measuring cell viability by standard MTT assay, an experimental procedure that quantifies the mitochondrial activity of metabolically active cells colorimetrically.
Background and Introduction:
Since its discovery in 1965, cisplatin has been a harbinger of a new era of anticancer research based on metallopharmaceuticals. To date cisplatin and its analogues have been one of the most effective and most widely used chemotherapeutic agents in use. After treatment with cisplatin, it undergoes an aquation reaction which leads to DNA adduct formation (Fig1) resulting unwinding, bending and finally to cell death.
Fig1: mechanism of action of cisplatin, PDB code: 1AIO
Cisplatin is very effective as an anticancer drug specially in testicular and ovarian cancers because of its DNA metallation capability. However, cisplatin is very toxic to normal healthy cells specially to kidney (nephrotoxicity) and gastrointestinal tract due to its unability to distinguish between normal and cancer cells.
Fig 2: Drug resistance of cisplatin, red arrows indicate drug resistance source.
On the other hand, prolonged use of cisplatin has given rise to drug resistance cancer cell lines. The drug resistance mainly arises from two main reasons: First, Reduced uptake of cisplatin in cell followed by active efflux of the drug from cell and second, Repair of platinated DNA molecules by DNA repair enzymes along with superior tolerance of platinated DNA (Fig 2). Cisplatin is highly polar in nature and enters cell in a very slow manner by passive diffusion or Cu-transporter protein. It has been reported that the uptake process is highly reduced in resistant cell line [ref].Once in the cell, cisplatin undergoes an aquation reaction followed by DNA metallation. In resistant cells, elevated expression of glutathione or cystine rich peptides has been reported that leads to drug detoxification by active efflux of peptide-cisplatin adduct [ref]. Even after DNA adduct formation, resistant cells have managed to evolve for repairing the DNA or high tolerance rate for metallated DNA has been reported [Ref].
Increasing drug resistance along with severe side effects of cisplatin has stimulated the search for novel metalloanticancer drug to overcome the adverse effects imposed by cisplatin. This metallotherapeutics mainly has two branches, one is to synthesize cisplatin analogue whereas the other branch mainly deals with development of non-platinum metalloanticancer drug. One of the
Cisplatin analogue development methodology involves synthesis of hexacoordinated Pt(IV) drug (tetraplatin analogue) utilizing a prodrug approach (Fig.3). There are many advantages of using a tetraplatin analogues. Firstly, hexacoordinated Pt(IV), being coordinatively saturated, are kinetically inert towards ligand substitution which makes them not a target for glutathione mediated detoxification. Secondly, Pt(IV) gets reduced to Pt(II) mainly in cancer cells because of the hypoxic environment and low pH of carcinoma cells compared to normal cell. This makes them extremely superior to their parent cisplatin analogues because not only they get activated in cancer cell but also they have substantially low toxicity towards normal cell. One of the tetraplatin analogue, satraplatin (Fig.3) is under clinical evaluation which also can be administered orally (first of its kind ) validating the design strategy.
Fig 3: a) tetraplatin analogue, a prodrug approach; b) Satraplatin
Ru (II/III) anticancer drug belongs to the later class which shows a very promising result in terms of overcoming drug resistance and toxicity. Although some properties like ligand substitution rate,