Studies On The Structure-activity Relationships And Action Mechanism Of Third-generation Bisphosphonates

The mevalonate pathway presents in all higher eukaryotes and many viruses, and the intermediates are important for the protein posttranslational modifications in cell signal transduction pathways and play a key role in cell growth, differentiation and expression. Farnesyl pyrophosphate synthase(FPPS) is a key enzyme in the mevalonate pathway and is also one of the important target enzymes of anti-tumor drugs. Currently, biphosphonates as main FPPS inhibitors are used for treatment of various diseases, including bone dieases caused by excessive bone resorption, such as osteoporosis, Paget’s disease, bone metastases of cancers, as well as infectious disease caused by parasites. It also has been demonstrated that bisphosphonates, especially the third-generation bisphosphonates, have direct or indirect antitumor potential, such as multiple myeloma, breast cancer and lung cancer. Zoledronic acid(Zol), a typical third-generation nitrogen-containing bisphosphonates(NBPs), has been widely used in treating osteoporosis and other bone diseases because of its higher biological activity than other bisphosphonates used in the clinical trial. However, the drug tolerance of Zol is still not clear for long-term medication, and Zol exhibits poor cell-membrane permeability and oral bioavailability due to its high polarity and hydrophilicity. When it was injected into the body, a part was absorbed by the skeleton quickly and the rest was excreted by the kidneys, and hence its treatment effect on nonskeletal tissues reduced. Therefore, it is necessary to find new FPPS inhibitors with lower hydrophilicity in order to increase the drug concentration in nonskeletal tissues and bioavailability, which will improve the curative effect for nonskeletal diseases. In this paper, the computer-aided drug design method was used to systematically study the structure-activity relationships and action mechanism of third-generation bisphosphonates in order to looking for new FPPS inhibitors with lower polarity and hydrophilicity, higher anti-tumor activity and lower side effects, which will provide a guide for the design and synthesis of novle FPPS inhibitors.Four different density functional theory(DFT) methods with four basis sets were employed to study the structures and spectral properties of monoclinic(IM) and triclinic(IT) Zol with the unprotonated and protonated conformations. According to the overall average deviation analysis of geometric parameters, IR and Raman spectra, B3LYP/6-31+G* and B3LYP/6-311++G** are relatively more accurate than other methods for predicting the unprotonated and protonated conformations of Zol, respectively. From the calculated total energy, it is inferred that the protonated structures are more stable than the unprotonated ones, and the predicted structures and IR/Raman spectra are in reasonable agreement with the experimental data. The solvent effect on protonated structures of Zol was calculated at B3LYP/6-311++G** level. The presence of intramolecular hydrogen bond was indentified in the solvent, and the geometric parameters were close r to experimental values. The intramolecular hydrogen bonding interactions resulted in the shift of vibrational frequencies of hydroxyl to the low band by 12-22 cm-1 and 24-26 cm-1 for IM and IT conformations, respectively. Furthermore, the analysis of thermodynamic properties, molecular electrostatic potential and frontier molecular orbitals in the gas and aqueous phases reveals the relationship between the physicochemical characteristics and the bioactivity of Zol. And the nature of intramolecular hydrogen bonding was studied by NBO and AIM analyses.The molecular structures and properties of Zol dimers were investigated. The geometry optimization of nine conformations of Zol dimers were performed at different DFT levels with different basis sets, and the best method was found to be B3LYP/6-311++G**. Based on this method, the intermolecular hydrogen bonding interactions, IR spectra, interaction energies and thermodynamic properties were further investigated for all the dimers. The results showed that the stability of IM and IT conformations decreased in the following sequence: IM-Dimer II ≈ IM-Dimer IV > IM-Dimer I > IM-Dimer III and IT-Dimer II > IT-Dimer V > IT-Dimer IV > IT-Dimer I > IT-Dimer III, respectively. And the factors influencing the stability of dimers included the number of hydrogen bond, the intensity of hydrogen bond and the symmetry of monomer. The intra-/inter-molecular hydrogen bonding interactions resulted in the red shift of vibrational frequencies of hydroxyl in Zol dimers, and the corresponding intensities increased. Furthermore, the intensity of intramolecular hydrogen bonding is stronger than the intermolecular hydrogen bonding and the former plays a critical role in the stabilization of Zol.Comparative molecular field analysis(Co MFA) was employed to explore the quantitative structure-activity relationship(QSAR) of 53 NBPs inhibitors of hFPPS. The results showed that the training set yielded a good correlation between the predicted and experimental activities with Y = 0.975 X + 0.179, r2=0.975, and the test set with Y = 0.806 X + 1.245, r2=0.753, which proved that the 3D-QSAR model would have good predication ability. Based on the three-dimensional coefficient equipotential graph and taking into account of the requirement of lipophilicity in vivo, a series of novel nitrogen-containing bisphosphonates inhibitors were designed and their activities were predicted. The results showed that introduction of amino substituent can obviously improve the biological activities of these drugs and 15 novel potent inhibitors have higher bioactivity pIC50 values(pIC50 =-logIC50) than 8.00, among which one inhibitor exceeds Zol and ten inhibitors exceed Ris(Risedronate).The action mechanism of novel bisphosphonates inhibiting hFPPS was studied using molecular docking, molecular dynamics and ONIOM methods, which will provide a guide for the design and synthesis of novle FPPS inhibitors. According to the molecular docking, the binding models of bisphosphonates with hFPPS were determined and found that the bisphosphonates interacted with the active site of hFPPS mainly by hydrogen bonding interactions, hydrophobic interactions and cation-π interactions, in which the amino acids Arg126, Lys214, Thr215, Gln254, Asp257, Lys271 and magnesium ion Mg2+909 possessed important interactions with the nitrogen-containing bisphosphonates. Molecular dynamics simulations showed that all systems were come to dynamic balance after 4 ns. The calculated interaction energies between the ligand and the receptor showed that the amino acids Arg126, Lys214 and Lys271 made the most important contributions to the total interaction energy, followed by the amino acids Thr215 and Gln254. Furthermore, the calculated hydrogen bonding interactions between the ligand and the amino acids also indicated that the number of hydrogen bond and the intensity of hydrogen bond play an important role in the NBPs inhibition activity. ONIOM(B3LYP/6-31+G*:PM6:Amber) calculations showed that the conformations of ligand changed slightly, which was only affected by the formation of hydrogen bonds with surrounding amino acids and waters. The calculated binding energies of the ligand with the receptor FPPS were in correlation with the NBPs inhibition activities. The intramolecular hydrogen bonding interactions and hydrophobic interactions played a vital role in determining the FPPS inhibitor activity, and the amino acids participated in the interaction were in good agreement with the results derived from molecular docking and molecular dynamics simulations.