Studies On The Interactions Of Active Drug Molecules With Proteins And Nucleic Acids

Proteins and DNA are the important biomacromolecules in life science, biochemistry and medicinalchemistry. Proteins are the carrier of many physiological functions, and are also the direct expresser ofbiological characters. Nucleic acids are the primary materials in showing the heredity and variation of life.Therefore, it is very significant to explore the interaction mechanisms of these biomacromolecules withorganic molecules and metal ions, especially for those targeting-drug molecules, which is useful forexpounding the secrets of life and enhancing the health of human beings. Additionally, these studies are ofthe current interests in life science, biochemistry, medicinal chemistry and so on.As a part of the projects supported by the National Natural Science Foundation of China (No.20673034) and the Research Fund for the Doctoral Program of Higher Education of China (No.20060476001), in this report, we systematically investigated the molecular interaction mechanisms ofproteins and nucleic acids with some active drug molecules. Furthermore, the effects of substituentmodifications and the existence of metal ions on the binding properties of drug molecules with thesebiomacromolecules were also analyzed. The present work consists of the following four sections.1. Under the physiological conditions, the molecular binding mechanisms of CPFX and NRF withtrypsin have been investigated by using fluorescence spectroscopy, UV-vis absorbance spectra andmolecular modeling techniques. At the same time, the effects of alkyl substituent on the interactionbehaviors have been analyzed. It is shown that the fluorescence quenching mechanism of CPFX and NRFwith trypsin is a static process with ground state complex formation. The binding affinity of NRF withtrypsin is greater than that of CPFX, which suggests that the structure of alkyl affects the bindinginteraction to a certain degree. In addition, it is implied that the main acting forces for the bindinginteraction are hydrophobicity and hydrogen interactions according to the results of thermodynamic studiesand the calculation of accessible surface area. The results of synchronous fluorescence and molecularmodeling indicate that the binding sites of CPFX and NRF on trypsin are close to tryptophan residue (Trp208).2. In this part, the molecular binding mechanisms of two dihydropyrimidinones derivatives5-(ethoxycarbonyl)-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (EMPD) and 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (EMMD) withhuman serum albumin (HSA) have been studied with fluorescence spectroscopy, UV-vis absorption spectra,isothermal titration calorimetry (ITC) and molecular modeling methods. The results show that theinteraction of EMPD with HSA is stronger than that of EMMD, which indicates that the substituents inbenzene ring affects the binding interaction to some extent. Moreover, the results of the competitivebinding experiment carried out with fluorescence probe and molecular modeling studies confirm that thespecific binding sites of EMPD and EMMD on HSA are located at subdomain IIA. The binding distancebetween the two drug molecules and the tryptophan residue of HSA (Trp214) were estimated, respectively,by the theory of fluorescence resonance energy transfer. The calculated results demonstrate that thenon-radiation energy transfer can occur between the donor and the receptor with high probability. The mainacting forces for the binding interaction are hydrophobicity and hydrogen interactions as illustrated by theresults of ITC and molecular modeling studies. Furthermore, the interactions of the two drug moleculeswith HSA were investigated in the presence of common metal ions, indicating that the addition of metalions can enhance their binding affinities. Therefore, the effectiveness of drugs in vivo can be enhanced byadding the different metal ions.3. Under the physiological conditions, the specific binding properties, including binding affinities,thermodynamic parameters and specifie binding modes for the binding interactions of a series ofdihydropyrimidinones derivatives with ctDNA were systematically studied by spectroscopic, viscometric,ITC and molecular modeling techniques. The five compounds studied here are, respectively,5-(ethoxycarbonyl)-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (a),5-(ethoxycarbonyl)-6-methyl-4-(4-dimethylaminophenyl)-3,4-dihydropyrimidin-2(1H)-one (b),5-(ethoxycarbonyl)-6-methyl-4-(4-nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one (c),5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (d) and5-(ethoxycarbonyl)-6-methyl-4-(4-chlorophenyl)-3,4-dihydropyrimidin-2(1H)-one (e). The results showthat the different substituents in benzene ring affect greatly the binding affinities and binding modes ofthese compounds with ctDNA. The compounds with electron-donating substituents are more favorable forthe binding with ctDNA, and the binding mode is mainly intercalation. However, the compounds withelectron-withdrawing substituents have the relatively weak binding with ctDNA, and the binding mode seems to be a mixed type of partial intercalation. In addition, it is verified that the favorable bindingsequences of the compounds with electron-donating substituents are G–C base pairs. However, thecompounds with electron-withdrawing substituents are not evident sequense selectivity of G–C bases.4. In order to further investigate the structure activity correlations of dihydropyrimidinones derivatives,antitumor activities of a series of compounds modified with different substituents were evaluated in vitroagainst two cancer cell lines, human hepatocellular carcinoma (BEL-7402) cells and ratpheochromocytoma (PC-12) cells using MTT assay method. The results suggest that compounds modifiedwith electron-donating substituents can efficiently inhibit the growth and proliferation of the two cancercells in a dose-dependent manner, possessing more potent anticancer activities. Whereas, compoundsmodified with electron-withdrawing substituents can hardly affect their biological activities. Therefore, thedihydropyrimidinones drugs can be modified in structures by changing the substituents of benzene ring toenhance their activities and effectiveness.