Chang-Guo Zhan

Publications of Chang-Guo Zhan

• Cocaine esterase-cocaine binding process and the free energy profiles by molecular dynamics and potential of mean force simulations.

  Authors: Xiaoqin Huang, Xinyun Zhao, Fang Zheng, Chang-Guo Zhan

  The journal of physical chemistry. B. 03/2012; 116(10):3361-8.

  The combined molecular dynamics (MD) and potential of mean force (PMF) simulations have been performed to determine the free energy profiles for the binding process of (-)-cocaine interacting withThe combined molecular dynamics (MD) and potential of mean force (PMF) simulations have been performed to determine the free energy profiles for the binding process of (-)-cocaine interacting with wild-type cocaine esterase (CocE) and its mutants (T172R/G173Q and L119A/L169K/G173Q). According to the MD simulations, the general protein-(-)-cocaine binding mode is not affected by the mutations; e.g.. the benzoyl group of (-)-cocaine is always bound in a subsite composed of aromatic residues W151, W166, F261, and F408 and hydrophobic residue L407, while the carbonyl oxygen on the benzoyl group of (-)-cocaine is hydrogen-bonded with the oxyanion-hole residues Y44 and Y118. According to the PMF-calculated free energy profiles for the binding process, the binding free energies for (-)-cocaine with the wild-type, T172R/G173Q, and L119A/L169K/G173Q CocEs are predicted to be -6.4, -6.2, and -5.0 kcal/mol, respectively. The computational predictions are supported by experimental kinetic data, as the calculated binding free energies are in good agreement with the experimentally derived binding free energies, i.e., -7.2, -6.7, and -4.8 kcal/mol for the wild-type, T172R/G173Q, and L119A/L169K/G173Q, respectively. The reasonable agreement between the computational and experimental data suggests that the PMF simulations may be used as a valuable tool in new CocE mutant design that aims to decrease the Michaelis-Menten constant of the enzyme for (-)-cocaine.

• New inhibitor of 3-phosphoinositide dependent protein kinase-1 identified from virtual screening.

  Authors: Wenchao Yang, Mohamed Diwan M Abdulhameed, Adel Hamza, Chang-Guo Zhan

  Bioorganic & medicinal chemistry letters. 02/2012; 22(4):1629-32.

  3-Phosphoinositide-dependent protein kinase-1 (PDK1) has been recognized as a promising anticancer target. Thus, it is interesting to identify new inhibitors of PDK1 for anticancer drug discovery.3-Phosphoinositide-dependent protein kinase-1 (PDK1) has been recognized as a promising anticancer target. Thus, it is interesting to identify new inhibitors of PDK1 for anticancer drug discovery. Through a combined use of virtual screening and wet experimental activity assays, we have identified a new PDK1 inhibitor with IC(50)=∼200nM. The anticancer activities of this compound have been confirmed by the anticancer activity assays using 60 cancer cell lines. The obtained new PDK1 inhibitor and its PDK1-inhibitor binding mode should be valuable in future de novo design of novel, more potent and selective PDK1 inhibitors for future development of anticancer therapeutics.

• Reaction pathway and free energy profiles for butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

  Authors: Xi Chen, Lei Fang, Junjun Liu, Chang-Guo Zhan

  Biochemistry. 02/2012; 51(6):1297-305.

  The catalytic mechanism for butyrylcholineserase (BChE)-catalyzed hydrolysis of acetylthiocholine (ATCh) has been studied by performing pseudobond first-principles quantum mechanical/molecularThe catalytic mechanism for butyrylcholineserase (BChE)-catalyzed hydrolysis of acetylthiocholine (ATCh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy (QM/MM-FE) calculations on both acylation and deacylation of BChE. Additional quantum mechanical (QM) calculations have been carried out, along with the QM/MM-FE calculations, to understand the known substrate activation effect on the enzymatic hydrolysis of ATCh. It has been shown that the acylation of BChE with ATCh consists of two reaction steps including the nucleophilic attack on the carbonyl carbon of ATCh and the dissociation of thiocholine ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of substrate and dissociation between the carboxyl carbon of substrate and hydroxyl oxygen of Ser198 side chain. QM/MM-FE calculation results reveal that the acylation of BChE is rate-determining. It has also been demonstrated that an additional substrate molecule binding to the peripheral anionic site (PAS) of BChE is responsible for the substrate activation effect. In the presence of this additional substrate molecule at PAS, the calculated free energy barrier for the acylation stage (rate-determining step) is decreased by ∼1.7 kcal/mol. All of our computational predictions are consistent with available experimental kinetic data. The overall free energy barriers calculated for BChE-catalyzed hydrolysis of ATCh at regular hydrolysis phase and substrate activation phase are ∼13.6 and ∼11.9 kcal/mol, respectively, which are in reasonable agreement with the corresponding experimentally derived activation free energies of 14.0 kcal/mol (for regular hydrolysis phase) and 13.5 kcal/mol (for substrate activation phase).

• Are pharmacokinetic approaches feasible for treatment of cocaine addiction and overdose?

  Authors: Fang Zheng, Chang-Guo Zhan

  Future medicinal chemistry. 02/2012; 4(2):125-8.

• Microscopic binding of M5 muscarinic acetylcholine receptor with antagonists by homology modeling, molecular docking, and molecular dynamics simulation.

  Authors: Xiaoqin Huang, Guangrong Zheng, Chang-Guo Zhan

  The journal of physical chemistry. B. 12/2011; 116(1):532-41.

  By performing homology modeling, molecular docking, and molecular dynamics (MD) simulations, we have developed three-dimensional (3D) structural models of the M5 muscarinic acetylcholine receptorBy performing homology modeling, molecular docking, and molecular dynamics (MD) simulations, we have developed three-dimensional (3D) structural models of the M5 muscarinic acetylcholine receptor (mAChR) and two complexes for M5 mAChR binding with antagonists SVT-40776 and solifenacin in the environment of lipid bilayer and solvent water. According to the simulated results, each of the antagonists is oriented horizontally in the binding pocket formed by transmembrane helices 2, 3, and 5-7. The cationic headgroup of each of the antagonists interacts with a negatively charged residue, Asp110, through electrostatic and hydrogen-bonding interactions. The simulated results also reveal some significant difference between the binding modes of SVT-40776 and solifenacin. In particular, SVT-40776 is persistently hydrogen bonded with the side chain of residue Tyr458, whereas solifenacin cannot form a similar hydrogen bond with residues around its carbonyl group. Such significant difference in the binding structures is consistent with the fact that SVT-40776 has a much higher binding affinity (K(d) = 0.4 nM) to M5 mAChR than that of solifenacin (K(d) = 31 nM) with the same reeptor. The calculated binding free energy change (-2.3 ± 0.3 kcal/mol) from solifenacin to SVT-40776 is in good agreement with the experimentally derived binding free energy change (-2.58 kcal/mol), suggesting that our modeled M5 mAChR structure and its complexes with the antagonists are reliable. The new structural insights obtained from this computational study are expected to stimulate further biochemical and pharmacological studies on the detailed structures of M5 and other subtypes of mAChRs.

• Corneal antifibrotic switch identified in genetic and pharmacological deficiency of vimentin.

  Authors: Paola Bargagna-Mohan, Riya R Paranthan, Adel Hamza, Chang-Guo Zhan, Do-Min Lee, Kyung Bo Kim, Daniel L Lau, Cidambi Srinivasan, Keiko Nakayama, Keiichi I Nakayama, Harald Herrmann, Royce Mohan

  The Journal of biological chemistry. 11/2011; 287(2):989-1006.

  The type III intermediate filaments (IFs) are essential cytoskeletal elements of mechanosignal transduction and serve critical roles in tissue repair. Mice genetically deficient for the IF proteinThe type III intermediate filaments (IFs) are essential cytoskeletal elements of mechanosignal transduction and serve critical roles in tissue repair. Mice genetically deficient for the IF protein vimentin (Vim(-/-)) have impaired wound healing from deficits in myofibroblast development. We report a surprising finding made in Vim(-/-) mice that corneas are protected from fibrosis and instead promote regenerative healing after traumatic alkali injury. This reparative phenotype in Vim(-/-) corneas is strikingly recapitulated by the pharmacological agent withaferin A (WFA), a small molecule that binds to vimentin and down-regulates its injury-induced expression. Attenuation of corneal fibrosis by WFA is mediated by down-regulation of ubiquitin-conjugating E3 ligase Skp2 and up-regulation of cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1). In cell culture models, WFA exerts G(2)/M cell cycle arrest in a p27(Kip1)- and Skp2-dependent manner. Finally, by developing a highly sensitive imaging method to measure corneal opacity, we identify a novel role for desmin overexpression in corneal haze. We demonstrate that desmin down-regulation by WFA via targeting the conserved WFA-ligand binding site shared among type III IFs promotes further improvement of corneal transparency without affecting cyclin-dependent kinase inhibitor levels in Vim(-/-) mice. This dissociates a direct role for desmin in corneal cell proliferation. Taken together, our findings illuminate a previously unappreciated pathogenic role for type III IF overexpression in corneal fibrotic conditions and also validate WFA as a powerful drug lead toward anti-fibrosis therapeutic development.

• Fundamental reaction pathway and free energy profile for hydrolysis of intracellular second messenger adenosine 3',5'-cyclic monophosphate (cAMP) catalyzed by phosphodiesterase-4.

  Authors: Xi Chen, Xinyun Zhao, Ying Xiong, Junjun Liu, Chang-Guo Zhan

  The journal of physical chemistry. B. 10/2011; 115(42):12208-19.

  As important drug targets for a variety of human diseases, cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes sharing a similar catalytic site. We have performed pseudobondAs important drug targets for a variety of human diseases, cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes sharing a similar catalytic site. We have performed pseudobond first-principles quantum mechanical/molecular mechanical-free energy perturbation (QM/MM-FE) and QM/MM-Poisson-Boltzmann surface area (PBSA) calculations to uncover the detailed reaction mechanism for PDE4-catalyzed hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP). This is the first report on QM/MM reaction-coordinate calculations including the protein environment of any PDE-catalyzed reaction system, demonstrating a unique catalytic reaction mechanism. The QM/MM-FE and QM/MM-PBSA calculations revealed that the PDE4-catalyzed hydrolysis of cAMP consists of two reaction stages: cAMP hydrolysis (stage 1) and bridging hydroxide ion regeneration (stage 2). The stage 1 includes the binding of cAMP in the active site, nucleophilic attack of the bridging hydroxide ion on the phosphorus atom of cAMP, cleavage of O3'-P phosphoesteric bond of cAMP, protonation of the departing O3' atom, and dissociation of hydrolysis product (AMP). The stage 2 includes the binding of solvent water molecules with the metal ions in the active site and regeneration of the bridging hydroxide ion. The dissociation of the hydrolysis product is found to be rate-determining for the enzymatic reaction process. The calculated activation Gibbs free energy of ≥16.0 and reaction free energy of -11.1 kcal/mol are in good agreement with the experimentally derived activation free energy of 16.6 kcal/mol and reaction free energy of -11.5 kcal/mol, suggesting that the catalytic mechanism obtained from this study is reliable and provides a solid base for future rational drug design.

• Insight into the binding of the wild type and mutated alginate lyase (AlyVI) with its substrate: A computational and experimental study.

  Authors: Adel Hamza, Yu Lan Piao, Mi-Sun Kim, Cheol Hee Choi, Chang-Guo Zhan, Hoon Cho

  Biochimica et biophysica acta. 09/2011; 1814(12):1739-47.

  The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. ToThe homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure-affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency (k(cat)/K(m)) of the double mutant L224V/D226G increased by two-fold compared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI structures with improved catalytic activity.

• Human butyrylcholinesterase-cocaine binding pathway and free energy profiles by molecular dynamics and potential of mean force simulations.

  Authors: Xiaoqin Huang, Fang Zheng, Chang-Guo Zhan

  The journal of physical chemistry. B. 09/2011; 115(38):11254-60.

  In the present study, we have performed combined molecular dynamics and potential of mean force (PMF) simulations to determine the enzyme-substrate (ES) binding pathway and the corresponding freeIn the present study, we have performed combined molecular dynamics and potential of mean force (PMF) simulations to determine the enzyme-substrate (ES) binding pathway and the corresponding free energy profiles for wild-type butyrylcholinesterase (BChE) binding with (-)/(+)-cocaine and for the A328W/Y332G mutant binding with (-)-cocaine. According to the PMF simulations, for each ES binding system, the substrate first binds with the enzyme at a peripheral anionic site around the entrance of the active-site gorge to form the first ES complex (ES1-like) during the binding process. Further evolution from the ES1-like complex to the nonprereactive ES complex is nearly barrierless, with a free energy barrier lower than 1.0 kcal/mol. So, the nonprereactive ES binding process should be very fast. The rate-determining step of the entire ES binding process is the subsequent evolution from the nonprereactive ES complex to the prereactive ES complex. Further accounting for the entire ES binding process, the PMF-based simulations qualitatively reproduced the relative order of the experimentally derived binding free energies (ΔG(bind)), although the simulations systematically overestimated the magnitude of the binding affinity and systematically underestimated the differences between the ΔG(bind) values. The obtained structural and energetic insights into the entire ES binding process provide a valuable base for future rational design of high-activity mutants of BChE as candidates for an enzyme therapy for cocaine overdose and abuse.

• Novel human mPGES-1 inhibitors identified through structure-based virtual screening.

  Authors: Adel Hamza, Xinyun Zhao, Min Tong, Hsin-Hsiung Tai, Chang-Guo Zhan

  Bioorganic & medicinal chemistry. 08/2011; 19(20):6077-86.

  Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase after exposure to pro-inflammatory stimuli and, therefore, represents a novel target for therapeutic treatmentMicrosomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase after exposure to pro-inflammatory stimuli and, therefore, represents a novel target for therapeutic treatment of acute and chronic inflammatory disorders. It is essential to identify mPGES-1 inhibitors with novel scaffolds as new leads or hits for the purpose of drug design and discovery that aim to develop the next-generation anti-inflammatory drugs. Herein we report novel mPGES-1 inhibitors identified through a combination of large-scale structure-based virtual screening, flexible docking, molecular dynamics simulations, binding free energy calculations, and in vitro assays on the actual inhibitory activity of the computationally selected compounds. The computational studies are based on our recently developed three-dimensional (3D) structural model of mPGES-1 in its open state. The combined computational and experimental studies have led to identification of new mPGES-1 inhibitors with new scaffolds. In particular, (Z)-5-benzylidene-2-iminothiazolidin-4-one is a promising novel scaffold for the further rational design and discovery of new mPGES-1 inhibitors. To our best knowledge, this is the first time a 3D structural model of the open state mPGES-1 is used in structure-based virtual screening of a large library of available compounds for the mPGES-1 inhibitor identification. The positive experimental results suggest that our recently modeled trimeric structure of mPGES-1 in its open state is ready for the structure-based drug design and discovery.

• A bright approach to the immunoproteasome: development of LMP2/β1i-specific imaging probes.

  Authors: Kimberly Cornish Carmony, Do-Min Lee, Ying Wu, Na-Ra Lee, Marie Wehenkel, Jason Lee, Beilei Lei, Chang-Guo Zhan, Kyung-Bo Kim

  Bioorganic & medicinal chemistry. 07/2011; 20(2):607-13.

  While the constitutive, 26S proteasome plays an important role in regulating many important cellular processes, a variant form known as the immunoproteasome is thought to primarily function inWhile the constitutive, 26S proteasome plays an important role in regulating many important cellular processes, a variant form known as the immunoproteasome is thought to primarily function in adaptive immune responses. However, recent studies indicate an association of immunoproteasomes with many physiological disorders such as cancer, neurodegenerative, and inflammatory diseases. Despite this, the detailed functions of the immunoproteasome remain poorly understood. Immunoproteasome-specific probes are essential to gain insight into immunoproteasome function. Here, we describe for the first time the development of cell-permeable activity-based fluorescent probes, UK101-Fluor and UK101-B660, which selectively target the catalytically active LMP2/β1i subunit of the immunoproteasome. These probes facilitate rapid detection of the cellular localization of catalytically active immunoproteasomes in living cells, providing a valuable tool to analyze immunoproteasome functions. Additionally, as LMP2/β1i may serve as a potential tumor biomarker, an LMP2/β1i-targeting fluorescent imaging probe may be applicable to a rapid readout assay to determine tumor LMP2/β1i levels.

• Active site gating and substrate specificity of butyrylcholinesterase and acetylcholinesterase: insights from molecular dynamics simulations.

  Authors: Lei Fang, Yongmei Pan, Jennifer L Muzyka, Chang-Guo Zhan

  The journal of physical chemistry. B. 06/2011; 115(27):8797-805.

  Butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are highly homologous proteins with distinct substrate preferences. In this study we compared the active sites of monomers and tetramersButyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are highly homologous proteins with distinct substrate preferences. In this study we compared the active sites of monomers and tetramers of human BChE and human AChE after performing molecular dynamics (MD) simulations in water-solvated systems. By comparing the conformational dynamics of gating residues of AChE and BChE, we found that the gating mechanisms of the main door of AChE and BChE are responsible for their different substrate specificities. Our simulation of the tetramers of AChE and BChE indicates that both enzymes could have two dysfunctional active sites due to their restricted accessibility to substrates. The further study on catalytic mechanisms of multiple forms of AChE and BChE would benefit from our comparison of the active sites of the monomers and tetramers of both enzymes.

• Catalytic mechanism of cytochrome P450 for 5'-hydroxylation of nicotine: fundamental reaction pathways and stereoselectivity.

  Authors: Dongmei Li, Xiaoqin Huang, Keli Han, Chang-Guo Zhan

  Journal of the American Chemical Society. 05/2011; 133(19):7416-27.

  A series of computational methods were used to study how cytochrome P450 2A6 (CYP2A6) interacts with (S)-(-)-nicotine, demonstrating that the dominant molecular species of (S)-(-)-nicotine in CYP2A6A series of computational methods were used to study how cytochrome P450 2A6 (CYP2A6) interacts with (S)-(-)-nicotine, demonstrating that the dominant molecular species of (S)-(-)-nicotine in CYP2A6 active site exists in the free base state (with two conformations, SR(t) and SR(c)), despite the fact that the protonated state is dominant for the free ligand in solution. The computational results reveal that the dominant pathway of nicotine metabolism in CYP2A6 is through nicotine free base oxidation. Further, first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations were carried out to uncover the detailed reaction pathways for the CYP2A6-catalyzed nicotine 5'-hydroxylation reaction. In the determined CYP2A6-(S)-(-)-nicotine binding structures, the oxygen of Compound I (Cpd I) can abstract a hydrogen from either the trans-5'- or the cis-5'-position of (S)-(-)-nicotine. CYP2A6-catalyzed (S)-(-)-nicotine 5'-hydroxylation consists of two reaction steps, that is, the hydrogen transfer from the 5'-position of (S)-(-)-nicotine to the oxygen of Cpd I (the H-transfer step), followed by the recombination of the (S)-(-)-nicotine moiety with the iron-bound hydroxyl group to generate the 5'-hydroxynicotine product (the O-rebound step). The H-transfer step is rate-determining. The 5'-hydroxylation proceeds mainly with the stereoselective loss of the trans-5'-hydrogen, that is, the 5'-hydrogen trans to the pyridine ring. The calculated overall stereoselectivity of ∼97% favoring the trans-5'-hydroxylation is close to the observed stereoselectivity of 89-94%. This is the first time it has been demonstrated that a CYP substrate exists dominantly in one protonation state (cationic species) in solution, but uses its less-favorable protonation state (neutral free base) to perform the enzymatic reaction.

• Reaction mechanism for cocaine esterase-catalyzed hydrolyses of (+)- and (-)-cocaine: unexpected common rate-determining step.

  Authors: Junjun Liu, Xinyun Zhao, Wenchao Yang, Chang-Guo Zhan

  The journal of physical chemistry. B. 05/2011; 115(17):5017-25.

  First-principles quantum mechanical/molecular mechanical free energy calculations have been performed to examine the catalytic mechanism for cocaine esterase (CocE)-catalyzed hydrolysis ofFirst-principles quantum mechanical/molecular mechanical free energy calculations have been performed to examine the catalytic mechanism for cocaine esterase (CocE)-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. It has been shown that the acylation of (+)-cocaine consists of nucleophilic attack of the hydroxyl group of Ser117 on the carbonyl carbon of (+)-cocaine benzoyl ester and the dissociation of (+)-cocaine benzoyl ester. The first reaction step of deacylation of (+)-cocaine, which is identical to that of (-)-cocaine, is rate-determining, indicating that CocE-catalyzed hydrolyses of (+)- and (-)-cocaine have a common rate-determining step. The computational results predict that the catalytic rate constant of CocE against (+)-cocaine should be the same as that of CocE against (-)-cocaine, in contrast with the remarkable difference between human butyrylcholinesterase-catalyzed hydrolyses of (+)- and (-)-cocaine. The prediction has been confirmed by experimental kinetic analysis on CocE-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. The determined common rate-determining step indicates that rational design of a high-activity mutant of CocE should be focused on the first reaction step of the deacylation. Furthermore, the obtained mechanistic insights into the detailed differences in the acylation between the (+)- and (-)-cocaine hydrolyses provide indirect clues for rational design of amino acid mutations that could more favorably stabilize the rate-determining transition state in the deacylation and, thus, improve the catalytic activity of CocE. This study provides a valuable mechanistic base for rational design of an improved esterase for therapeutic treatment of cocaine abuse.

• Computational design of a thermostable mutant of cocaine esterase via molecular dynamics simulations.

  Authors: Xiaoqin Huang, Daquan Gao, Chang-Guo Zhan

  Organic & biomolecular chemistry. 03/2011; 9(11):4138-43.

  Cocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstabilityCocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only ∼11 min at physiological temperature (37 °C). It is highly desirable to develop a thermostable mutant of CocE for therapeutic treatment of cocaine overdose and addiction. To establish a structure-thermostability relationship, we carried out molecular dynamics (MD) simulations at 400 K on wild-type CocE and previously known thermostable mutants, demonstrating that the thermostability of the active form of the enzyme correlates with the fluctuation (characterized as the root-mean square deviation and root-mean square fluctuation of atomic positions) of the catalytic residues (Y44, S117, Y118, H287, and D259) in the simulated enzyme. In light of the structure-thermostability correlation, further computational modelling including MD simulations at 400 K predicted that the active site structure of the L169K mutant should be more thermostable. The prediction has been confirmed by wet experimental tests showing that the active form of the L169K mutant had a half-life of 570 min at 37 °C, which is significantly longer than those of the wild-type and previously known thermostable mutants. The encouraging outcome suggests that the high-temperature MD simulations and the structure-thermostability relationship may be considered as a valuable tool for the computational design of thermostable mutants of an enzyme.

• Interaction of tyrosine 151 in norepinephrine transporter with the 2β group of cocaine analog RTI-113.

  Authors: Erik R Hill, Xiaoqin Huang, Chang-Guo Zhan, F Ivy Carroll, Howard H Gu

  Neuropharmacology. 03/2011; 61(1-2):112-20.

  Cocaine binds and inhibits dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter. The residues forming cocaine binding sites are unknown. RTI-113, a cocaine analog,Cocaine binds and inhibits dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter. The residues forming cocaine binding sites are unknown. RTI-113, a cocaine analog, is 100× more potent at inhibiting DAT than inhibiting NET. Here we show that removing the hydroxyl group from residue Tyr151 in NET by replacing it with Phe, the corresponding residue in DAT, increased the sensitivity of NET to RTI-113, while the reverse mutation in DAT decreased the sensitivity of DAT to RTI-113. In contrast, RTI-31, another cocaine analog having the same structure as RTI-113 but with the phenyl group at the 2β position replaced by a methyl group, inhibits the transporter mutants equally well whether a hydroxyl group is present at the residue or not. The data suggest that this residue contributes to cocaine binding site and is close to the 2β position of cocaine analogs. These results are consistent with our previously proposed cocaine-DAT binding model where cocaine initially binds to a site that does not overlap with, but is close to, the dopamine-binding site. Computational modeling and molecular docking yielded a binding model that explains the observed changes in RTI-113 inhibition potencies.

• Reaction pathway and free energy profile for butyrylcholinesterase-catalyzed hydrolysis of acetylcholine.

  Authors: Xi Chen, Lei Fang, Junjun Liu, Chang-Guo Zhan

  The journal of physical chemistry. B. 02/2011; 115(5):1315-22.

  A catalytic mechanism for the butyrylcholinesterase (BChE)-catalyzed hydrolysis of acetylcholine (ACh) has been studied by performing pseudobond first-principles quantum mechanical/molecularA catalytic mechanism for the butyrylcholinesterase (BChE)-catalyzed hydrolysis of acetylcholine (ACh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy calculations on both acylation and deacylation of BChE. It has been shown that the acylation with ACh includes two reaction steps, including nucleophilic attack on the carbonyl carbon of ACh and dissociation of choline ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of the substrate and dissociation between the carboxyl carbon of the substrate and the hydroxyl oxygen of the Ser198 side chain. Notably, despite the fact that acetylcholinesterase (AChE) and BChE are very similar enzymes, the acylation of BChE with ACh is rate-determining, which is remarkably different from the AChE-catalyzed hydrolysis of ACh, in which the deacylation is rate-determining. The computational prediction is consistent with available experimental kinetic data. The overall free energy barrier calculated for BChE-catalyzed hydrolysis of ACh is 13.8 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 13.3 kcal/mol.

• Enzyme-therapy approaches for the treatment of drug overdose and addiction.

  Authors: Fang Zheng, Chang-Guo Zhan

  Future medicinal chemistry. 01/2011; 3(1):9-13.

• Computational determination of binding structures and free energies of phosphodiesterase-2 with benzo[1,4]diazepin-2-one derivatives.

  Authors: Bo Yang, Adel Hamza, Guangju Chen, Yan Wang, Chang-Guo Zhan

  The journal of physical chemistry. B. 11/2010; 114(48):16020-8.

  Phosphodiesterase-2 (PDE2) is a key enzyme catalyzing hydrolysis of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) that serve as intracellular second messengers.Phosphodiesterase-2 (PDE2) is a key enzyme catalyzing hydrolysis of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) that serve as intracellular second messengers. PDE2 has been recognized as an attractive drug target, and selective inhibitors of PDE2 are expected to be promising candidates for the memory enhancer, antidepressant, and anxiolytic agent. In the present study, we examined the detailed binding structures and free energies for PDE2 interacting with a promising series of inhibitors, i.e., benzo[1,4]diazepin-2-one derivatives, by carrying out molecular docking, molecular dynamics (MD) simulations, binding free energy calculations, and binding energy decompositions. The computational results provide valuable insights into the detailed enzyme-inhibitor binding modes including important intermolecular interactions, e.g., the π-π stacking interactions with the common benzo[1,4]diazepin-2-one scaffold of the inhibitors, hydrogen bonding and hydrophobic interactions with the substituents on the benzo[1,4]diazepin-2-one scaffold. Future rational design of new, more potent inhibitors of PDE2 should carefully account for all of these favorable intermolecular interactions. By use of the MD-simulated binding structures, the calculated binding free energies are in good agreement with the experimental activity data for all of the examined benzo[1,4]diazepin-2-one derivatives. The enzyme-inhibitor binding modes determined and the agreement between the calculated and experimental results are expected to be valuable for future rational design of more potent inhibitors of PDE2.

• Design of high-activity mutants of human butyrylcholinesterase against (-)-cocaine: structural and energetic factors affecting the catalytic efficiency.

  Authors: Fang Zheng, Wenchao Yang, Liu Xue, Shurong Hou, Junjun Liu, Chang-Guo Zhan

  Biochemistry. 10/2010; 49(42):9113-9.

  The present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (-)-cocaine by modeling theThe present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (-)-cocaine by modeling the rate-determining transition state (TS1), i.e., the transition state for the first step of chemical reaction process, of (-)-cocaine hydrolysis catalyzed by various mutants of human BChE in comparison with the wild type. Molecular modeling of the TS1 structures revealed that mutations on certain nonactive site residues can indirectly affect the catalytic efficiency of the enzyme against (-)-cocaine through enhancing or weakening the overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. Computational insights and predictions were supported by the catalytic activity data obtained from wet experimental tests on the mutants of human BChE, including five new mutants reported for the first time. The BChE mutants with at least ∼1000-fold improved catalytic efficiency against (-)-cocaine compared to the wild-type BChE are all associated with the TS1 structures having stronger overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. The combined computational and experimental data demonstrate a reasonable correlation relationship between the hydrogen-bonding distances in the TS1 structure and the catalytic efficiency of the enzyme against (-)-cocaine.