| dc.description.abstract | Despite advances in technology and understanding of biological systems in the past two decades, modern drug discovery is still a lengthy, expensive, difficult and inefficient process with low rate of new therapeutic discovery. The search for new effective drugs remains a somewhat empirical process. There is compelling need for a more fundamental, mechanistic understanding of human cancers and anticancer drugs to design more appropriate drugs.
Radiotherapy is still the major therapy of cancer. It uses high-energy ionizing radiation such as x-rays and charged particle beams to destroy cancer cells. DNA is well known to be the principal biological target of radiotherapy, but the molecular mechanism of ionizing radiation induced DNA damage was elusive. The conventional thought of the ∙OH radical as the major origin for ionizing radiation induced DNA damage is questionable. Although various strategies and types of compounds have been designed and developed as potential radiosensitizers to enhance the radiosensitizing efficiency of radiotherapy, none of them have been approved for clinical use. The general outcomes of clinical trials have been disappointing.
This thesis presents an innovative molecular-mechanism-based drug discovery project to develop novel drugs for effective radiotherapy of cancer through the emerging femtomedicine approach. Its ultimate goal is to develop more effective radiosensitizers, based on our unique molecular understandings of ionizing radiation induced DNA damage and halopyrimidines as a family of potential radiosensitizers.
Direct, real-time observation of molecular reactions is of significant importance in diverse fields from chemistry and biology, environmental sciences to medicine. Femtosecond time-resolved laser spectroscopy (fs-TRLS) is a very powerful, direct technique for real-time observation of molecular reactions. Its key strength lies in short duration laser flashes of a time scale at which reactions actually happen - femtoseconds (fs) (1fs = 1015 second). Since the late 1980s, its application to study chemical and biological systems led to the births of new subfields of science, called femtochemistry and femtobiology. Recently, femtomedicine has been proposed as a new transdisciplinary frontier to integrate ultrafast laser techniques with biomedical methods for advances in fundamental understandings and treatments of major human diseases. This the remarkable opportunity afforded through real-time observation of biochemical reactions at the molecular level. Femtomedicine holds the promise of advances in the radiotherapy of cancer.
Several important findings were made in this thesis. First, our results of careful and high-quality fs-TRLS measurements have resolved the long existing controversies about the physical nature and lifetimes of a novel ultrashort-lived electron species (epre) generated in radiolysis of water. These results have not only resolved the large discrepancies existing in the literature but provided new insights into electron hydration dynamics in bulk water. Such information is important for quantitative understanding and modeling of the role of non-equilibrium epre in electron-driven reactions in diverse environmental and biological systems, from radiation chemistry and radiation biology to atmospheric ozone depletion.
Second, our fs-TRLS results have unraveled how epre plays a crucial role in ionizing radiation induced DNA damage. We found that among DNA bases, only T and especially G are vulnerable to a dissociative electron transfer (DET) reaction with epre leading to bond breaks, while the electron can be stably trapped at C and especially A to form stable anions. The results not only challenge the conventional notion that damage to the genome by ionizing radiation is mainly induced by the oxidizing •OH radical, but provide a deeper fundamental understanding of the molecular mechanism of the DNA damage caused by a reductive agent (epre). Our findings have led to a new molecular mechanism of reductive DNA damage.
Third, halopyrimidines, especially BrdU and IdU, have passed Phase I to II clinical trials as potential hypoxic radiosensitizers, but the outcome of Phase III clinical trials was disappointing. Our results of fs-TRLS studies have provided a new molecular mechanism of action of halopyrimidines (XdUs, X=F, Cl, Br and I) in liquid water under ionizing radiation. We found that it is the ultrashort-lived epre, rather than the long-lived ehyd, that is responsible for DET reactions of XdUs. This reaction leads to the formation of the reactive dU• radical, which then causes DNA strand breaks and cancer cell death. Our results have challenged a long accepted mechanism that long-lived ehyd would be responsible for the radical formation from halogenated molecules. Furthermore, we found that the DET reaction efficacy leading to the formation of the reactive dU• radical is in the order of FdU << CldU < BrdU < IdU. Thus, only BrdU and IdU could be explored as potential radiosensitizers, in agreement with the results of bioactivity tests and clinical trials.
Fourth, our fs-TRLS studies have provided a molecular mechanism for the DNA sequence selectivity of BrdU and IdU in radiosensitization. We found the DET reactions of BrdU/ IdU with dAMP* and dGMP* formed by attachment of epre generated by radiolysis of water in aqueous BrdU-dAMP/dGMP and IdU-dAMP/dGMP complexes under ionizing radiation. This new mechanistic insight into the interaction of BrdU and IdU with DNA provides clues to improve the halogen familty as potential radiosensitizers and to develop more effective radiosensitizers for clinical applications.
Fifth, based on our molecular mechanistic understandings of DNA damage induced by ionizing radiation and halopyrimidines as potential radiosensitizers, we develop more effective new radisensitizing drug candidates through the femtomedicine approach. We have performed a fs-TRLS study of the DET reaction of a candidate compound (RS-1) with epre, and found that the DET reaction of epre with RS-1 is much stronger than that of IdU (and certainly BrdU and CldU). Moreover, we have tested the radiosensitizing effect of RS-1 against human cervical cancer (HeLa) cells exposed to various doses of x-ray irradiation through DNA damage measurements by gel electrophoresis and cell viability/death assays by MTT. Our results have confirmed that RS-1 can largely enhance the radiosensitivity of treated human cervical cancer (HeLa) cells to x-ray (ionizing) radiation. It is clearly demonstrated that RS-1 has a much better radiosensitizing effect than IdU. Although these are just preliminary results, our results have shown promise of developing more effective radiosensitizers.
In summary, our studies have demonstrated the potential of femtomedicine as an exciting new frontier to bring breakthroughs in understanding fundamental biological processes and to provide an efficient and economical strategy for development of new anticancer drugs. | en |
