Chun-Zhi Ai

Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis

MicroRNAs (miRNAs) are endogenously produced ∼21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5′-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg2+) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago.

Anti-androgen-independent prostate cancer effects of ginsenoside metabolites in vitro: mechanism and possible structure-activity relationship investigation.

Treatment of androgen-independent prostate cancer (AIPC) remains unsatisfactory. In our present experiment, natural occurring ginsenosides (NOGs) and intestinal bacterial metabolites (IBMs) were employed to investigate their anti-AIPC cell growth activity using PC-3 cells. Our results showed that the IBMs exerted more portent anti-AIPC activity than NOGs, by decreasing survival rate, inhibiting proliferation, inducing apoptosis, and leading to cell cycle arrest in AIPC PC-3 cells. The increase of LogP and decrease of C-6 steric hindrance, which were caused by deglycosylation by intestinal bacteria, may be the reason for the higher anti-AIPC activity of IBMs.

In Silico Prediction of Estrogen Receptor Subtype Binding Affinity and Selectivity Using Statistical Methods and Molecular Docking with 2-Arylnaphthalenes and 2-Arylquinolines

Over the years development of selective estrogen receptor (ER) ligands has been of great concern to researchers involved in the chemistry and pharmacology of anticancer drugs, resulting in numerous synthesized selective ER subtype inhibitors. In this work, a data set of 82 ER ligands with ERα and ERβ inhibitory activities was built, and quantitative structure-activity relationship (QSAR) methods based on the two linear (multiple linear regression, MLR, partial least squares regression, PLSR) and a nonlinear statistical method (Bayesian regularized neural network, BRNN) were applied to investigate the potential relationship of molecular structural features related to the activity and selectivity of these ligands. For ERα and ERβ, the performances of the MLR and PLSR models are superior to the BRNN model, giving more reasonable statistical properties (ERα: for MLR, Rtr2 = 0.72, Qte2 = 0.63; for PLSR, Rtr2 = 0.92, Qte2 = 0.84. ERβ: for MLR, Rtr2 = 0.75, Qte2 = 0.75; for PLSR, Rtr2 = 0.98, Qte2 = 0.80). The MLR method is also more powerful than other two methods for generating the subtype selectivity models, resulting in Rtr2 = 0.74 and Qte2 = 0.80. In addition, the molecular docking method was also used to explore the possible binding modes of the ligands and a relationship between the 3D-binding modes and the 2D-molecular structural features of ligands was further explored. The results show that the binding affinity strength for both ERα and ERβ is more correlated with the atom fragment type, polarity, electronegativites and hydrophobicity. The substitutent in position 8 of the naphthalene or the quinoline plane and the space orientation of these two planes contribute the most to the subtype selectivity on the basis of similar hydrogen bond interactions between binding ligands and both ER subtypes. The QSAR models built together with the docking procedure should be of great advantage for screening and designing ER ligands with improved affinity and subtype selectivity property.

A model of in vitro UDP-glucuronosyltransferase inhibition by bile acids predicts possible metabolic disorders

Increased levels of bile acids (BAs) due to the various hepatic diseases could interfere with the metabolism of xenobiotics, such as drugs, and endobiotics including steroid hormones. UDP-glucuronosyltransferases (UGTs) are involved in the conjugation and elimination of many xenobiotics and endogenous compounds. The present study sought to investigate the potential for inhibition of UGT enzymes by BAs. The results showed that taurolithocholic acid (TLCA) exhibited the strongest inhibition toward UGTs, followed by lithocholic acid. Structure-UGT inhibition relationships of BAs were examined and in vitro-in vivo extrapolation performed by using in vitro inhibition kinetic parameters (Ki) in combination with calculated in vivo levels of TLCA. Substitution of a hydrogen with a hydroxyl group in the R1, R3, R4, R5 sites of BAs significantly weakens their inhibition ability toward most UGTs. The in vivo inhibition by TLCA toward UGT forms was determined with following orders of potency: UGT1A4 > UGT2B7 > UGT1A3 > UGT1A1 ∼ UGT1A7 ∼ UGT1A10 ∼ UGT2B15. In conclusion, these studies suggest that disrupted homeostasis of BAs, notably taurolithocholic acid, found in various diseases such as cholestasis, could lead to altered metabolism of xenobiotics and endobiotics through inhibition of UGT enzymes.

Dynamic communication between androgen and coactivator: Mutually induced conformational perturbations in androgen receptor ligand‐binding domain

The transcriptional activity of androgen receptor (AR) is regulated by the sequential binding of various ligands (e.g., dihydrotestosterone, DHT) and coactivators (e.g., SRC/p160) to the AR ligand binding domain (LBD) (Askew et al., J Biol Chem 2007;282:25801–25816, Lee and Chang, Cell Mol Life Sci 2003;60:1613–1622). However, the synergism between the recruitments of coactivator (SRC 2–3) and ligand (such as DHT) to AR at atomic level remains unclear. Thus, in this work, extensive explicit‐solvent molecular dynamics (MD) simulations on four independent trajectories, that is, AR‐apo (unbound), DHT·AR, AR·SRC, and DHT·AR·SRC, are performed to investigate the potential communications between the two events in the AR transcriptional process. The MD simulations, analysis of the dynamical cross‐correlation maps, comparisons of the binding energy, and thermodynamic analysis reveal a definite structural and functional link between Activation Function‐2 (AF‐2) surface and the ligand binding site influenced by the binding of ligand and coactivator to the LBD: (I) The DHT binding can increase the LBD volume to 753.0 Å3 from its compact ligand‐free state (372.1 Å3), resulting in a group of helices (1, 2, 8, and loop 2′) to move outward and exert added traction on the ligand binding pathway, which subsequently leads to rearrange the AF‐2 region to well recruit the SRC; (II) Similarly, the SRC recruitment is also found to facilitate the ligand binding through transmitting a concomitant push‐pull effort from the AF‐2 surface to the DHT binding site, leading to the opening of entrance to the LBD formed by Val684, Met745, and Arg752, increase of the volume of binding pocket (896.4 Å3) and stabilization of the dynamic structure of the LBD. These results, in a dynamic form, initially show a bidirectional structural and functional relay between the bound DHT and SRC that establishes AR functional potency. Proteins 2011.

Investigation of binding features: effects on the interaction between CYP2A6 and inhibitors.

A computational investigation has been carried out on CYP2A6 and its naphthalene inhibitors to explore the crucial molecular features contributing to binding specificity. The molecular bioactive orientations were obtained by docking (FlexX) these compounds into the active site of the enzyme. And the density functional theory method was further used to optimize the molecular structures with the subsequent analysis of molecular lipophilic potential (MLP) and molecular electrostatic potential (MEP). The minimal MLPs, minimal MEPs, and the band gap energies (the energy difference between the highest occupied molecular orbital and lowest unoccupied molecular orbital) showed high correlations with the inhibition activities (pIC50s), illustrating their significant roles in driving the inhibitor to adopt an appropriate bioactive conformation oriented in the active site of CYP2A6 enzyme. The differences in MLPs, MEPs, and the orbital energies have been identified as key features in determining the binding specificity of this series of compounds to CYP2A6 and the consequent inhibitory effects. In addition, the combinational use of the docking, MLP and MEP analysis is also demonstrated as a good attempt to gain an insight into the interaction between CYP2A6 and its inhibitors.

Insight into the effects of chiral isomers quinidine and quinine on CYP2D6 inhibition

The distinct inhibitory effects against CYP2D6 enzyme of the stereoisomers quinidine and quinine were investigated in this work by employing various methods, including the comparative molecular field analysis (CoMFA), the comparative molecular similarity indices analysis (CoMSIA), the molecular electrostatic potential (MEP) analysis and the docking method. Several 3D-QSAR models with proper reliability were well established, with a CoMFA model with steric and electrostatic fields exhibiting 0.67, 0.99 and 0.88 of q(2), r(2) and r(pred)(2), respectively, a CoMSIA model with steric, electrostatic and H-bond acceptor fields displaying 0.72, 0.97 and 0.84 of q(2), r(2) and r(pred)(2), respectively. These models and related docking results reveal that quinidine binds to CYP2D6 in an inverse orientation as compared with quinine. Moreover, quinidine blocks the entrance of the active pocket of CYP2D6 more closely than quinine does, which explains well why the inhibitory activity of quinidine is of 2 magnitudes larger than quinine. This investigation provides a better understanding of the stereoisometric effects on the bioactivities of the chiral isomers quinidine and quinine interacting with CYP2D6.

The role of serum albumin in the metabolism of Boc5: Molecular identification, species differences and contribution to plasma metabolism

Boc5, the first nonpeptidic agonist of Glucagon-like peptide-1 receptor, has been recognized as a potential candidate for treatment of diabetes. However, the metabolic behaviors of this novel molecule in both human and experimental animals remain unclear. This study aimed to explore the metabolic behaviors of Boc5 in biological preparations from human, pig and rat. Boc5 was found to be very stable in liver microsomes of human, pig and rat, but it can be degraded to two metabolites in plasma from all three species, via the successive hydrolysis of the C-22 esters. Chemical inhibition studies using selective esterase inhibitors and assays with purified enzymes suggested that Boc5 hydrolysis in human was totally mediated by human serum albumin (HSA) rather than esterases. ESI-TOF-MS/MS analysis revealed that Lys525 of HSA could be modified by treatment with Boc5, strongly suggesting the pseudo-esterase activity of albumin. Studies on species differences in this albumin-mediated metabolism showed large species differences in degradation rate of Boc5, the half lives of Boc5 in plasma from three various species varied from 23.5 h to 83.1h, but they were much closer to the half lives of Boc5 in corresponding serum albumins, implying the predominant role of serum albumin in plasma metabolism of Boc5. Additionally, the effects of various ligands including fatty acids and several drugs with unambiguous binding sites on HSA, on the pseudo-esterase activity of HSA, were also investigated using both experimental and molecular modelling studies. These results showed that the binding of various ligands to HSA could significantly affect the pseudo-esterase activity of HSA towards Boc5, due to the ligand-induced conformation changes of HSA.

Regulation profile of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) components towards UDP-glucuronosyltransferases (UGTs) isoforms

Abstract 1. Endogenous compounds have been reported to be the regulators of UDP-glucuronosyltransferases (UGTs) isoforms. This study aims to investigate the regulatory effects of the activity of UGT isoforms by two important lipid components phosphatidylcholine (PC) and lysophosphatidylcholines (LPC) using in vitro incubation system. 2. UGTs supersomes-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as the probe reaction to evaluate the inhibition of compounds towards UGT isoforms except UGT1A4, and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation reaction was utilized to phenotype the activity of UGT1A4. 3. About 50 μM of LPC15:0, LPC16:0, LPC17:0, LPC18:0, LPC18:1 and PC16:0, 2:0 exhibited inhibition towards more than 90% activity of UGT isoforms, and other LPC and PC components showed negligible inhibitory potential towards all the UGT isoforms. UGT1A6 and UGT1A8 were identified to be the most sensitive UGT isoforms susceptible for the inhibition by LPC15:0, LPC16:0, LPC17:0, LPC18:0, LPC18:1 and PC16:0, 2:0, indicating the strong influence of these LPC and PC components towards UGT1A6 and UGT1A8-catalyzed metabolic reaction when the concentrations of these components increased.

Stereochemical differentiation of C-7 hydroxyltaxane isomers by electrospray ionization mass spectrometry

In this study, different electrospray ionization mass spectrometric (ESI-MS) methods were utilized to analyze several pairs of taxane stereoisomers including paclitaxel and 7-epi-paclitaxel. Both ESI-MS and tandem mass spectrometry (MS/MS) techniques provided stereochemically dependent mass spectra in negative-ion mode, and all studied stereoisomers could be easily distinguished based on their characteristic ions or distinct fragmentation patterns. MS/MS experiments for several taxane analogues at various collision energies were performed to elucidate potential dissociation pathways. The gas-phase deprotonation potentials were also calculated to estimate the most thermodynamically favorable deprotonation site using DFT B3LYP/6-31G(d). The results of the theoretical studies agreed well with the fragmentation patterns of paclitaxel and 7-epi-paclitaxel observed from MS/MS experiments. In addition, it was found that liquid chromatography (LC)/ESI-MS was a useful and sensitive technique for assignment of C-7 taxane stereoisomers from realistic samples.