Conformational sensitivity analysis of FKBP–FK506/rapamycin complexes

Sensitivity analysis techniques are applied to the FKBP–FK506 and FKBP–rapamycin complexes to quantify the conformational relationships between FKBP and its ligands. Crystal structures of the two FKBP complexes are energy minimized in the Amber force field using a continuum solvent model, and derived Green's function sensitivity coefficients are developed to describe the relationship between the ?, ψ, and χ₁ torsional angles of the FKBP residues and the bound ligand macrocycle torsional angles. Sensitivity analysis is applied to the entire FKBP structure and reveals that the local conformation of the residues of the 80s and 50s loops and of the active site are sensitive to the ligand conformation. The analysis also reveals that the torsional angles controlling the orientation of the amide and keto carbonyls of FK506 are sensitive to the aromatic side chains in the FKBP carbonyl binding pocket. © 1994 by John Wiley & Sons, Inc.     

Molecular dynamics simulation and free energy calculation studies of the binding mechanism of allosteric inhibitors with p38α MAP kinase

p38 MAP kinase is a promising target for anti-inflammatory treatment. The classical kinase inhibitors imatinib and sorafenib as well as BI-1 and BIRB-796 were reported to bind in the DFG-out form of human p38α, known as type II or allosteric kinase inhibitors. Although DFG-out conformation has attracted great interest in the design of type II kinase inhibitors, the structural requirements for binding and mechanism of stabilization of DFG-out conformation remain unclear. As allosteric inhibition is important to the selectivity of kinase inhibitor, herein the binding modes of imatinib, sorafenib, BI-1 and BIRB-796 to p38α were investigated by molecular dynamics simulation. Binding free energies were calculated by molecular mechanics/Poisson-Boltzmann surface area method. The predicted binding affinities can give a good explanation of the activity difference of the studied inhibitors. Furthermore, binding free energies decomposition analysis and further structural analysis indicate that the dominating effect of van der Waals interaction drives the binding process, and key residues, such as Lys53, Gly71, Leu75, Ile84, Thr106, Met109, Leu167, Asp168, and Phe169, play important roles by forming hydrogen bond, salt bridge, and hydrophobic interactions with the DFG-out conformation of p38α. Finally, we also conducted a detailed analysis of BI-1, imatinib, and sorafenib binding to p38α in comparison with BIRB-796 exploited for gaining potency as well as selectivity of p38 inhibitors. These results are expected to be useful for future rational design of novel type II p38 inhibitors.     

Connectivity and binding‐site recognition: Applications relevant to drug design

Here, we describe a family of methods based on residue–residue connectivity for characterizing binding sites and apply variants of the method to various types of protein–ligand complexes including proteases, allosteric‐binding sites, correctly and incorrectly docked poses, and inhibitors of protein–protein interactions. Residues within ligand‐binding sites have about 25% more contact neighbors than surface residues in general; high‐connectivity residues are found in contact with the ligand in 84% of all complexes studied. In addition, a k‐means algorithm was developed that may be useful for identifying potential binding sites with no obvious geometric or connectivity features. The analysis was primarily carried out on 61 protein–ligand structures from the MEROPS protease database, 250 protein–ligand structures from the PDBSelect (25%), and 30 protein–protein complexes. Analysis of four proteases with crystal structures for multiple bound ligands has shown that residues with high connectivity tend to have less variable side‐chain conformation. The relevance to drug design is discussed in terms of identifying allosteric‐binding sites, distinguishing between alternative docked poses and designing protein interface inhibitors. Taken together, this data indicate that residue–residue connectivity is highly relevant to medicinal chemistry. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Conformational Selection Mechanism Provides Structural Insights into the Optimization of APC-Asef Inhibitors

Metastasis is the major cause of death in colorectal cancer and it has been proven that inhibiting an interaction between adenomatous polyposis coli (APC) and Rho guanine nucleotide exchange factor 4 (Asef) efficaciously restrain metastasis. However, current inhibitors cannot achieve a satisfying effect in vivo and need to be optimized. In the present study, we applied molecular dynamics (MD) simulations and extensive analyses to apo and holo APC systems in order to reveal the inhibitor mechanism in detail and provide insights into optimization. MD simulations suggested that apo APC takes on a broad array of conformations and inhibitors stabilize conformation selectively. Representative structures in trajectories show specific APC-ligand interactions, explaining the different binding process. The stability and dynamic properties of systems elucidate the inherent factors of the conformation selection mechanism. Binding free energy analysis quantitatively confirms key interface residues and guide optimization. This study elucidates the conformation selection mechanism in APC-Asef inhibition and provides insights into peptide-based drug design.     

Rational Design and Asymmetric Synthesis of Potent and Neurotrophic Ligands for FK506‐Binding Proteins (FKBPs)

To create highly efficient inhibitors for FK506‐binding proteins, a new asymmetric synthesis for pro‐(S)‐C⁵‐branched [4.3.1] aza‐amide bicycles was developed. The key step of the synthesis is an HF‐driven N‐acyliminium cyclization. Functionalization of the C⁵ moiety resulted in novel protein contacts with the psychiatric risk factor FKBP51, which led to a more than 280‐fold enhancement in affinity. The most potent ligands facilitated the differentiation of N2a neuroblastoma cells with low nanomolar potency.     

Profiling the structural determinants for the selectivity of representative factor-Xa and thrombin inhibitors using combined ligand-based and structure-based approaches

The current study deciphers the combined ligand- and structure-based computational insights to profile structural determinants for the selectivity of representative diverse classes of FXa-selective and thrombin-selective as well as dual FXa-thrombin high affinity inhibitors. The thrombin-exclusive insertion 60-loop (D-pocket) was observed to be one of the most notable recognition sites for the known thrombin-selective inhibitors. Based on the topological comparison of four common active-site pockets (S1-S4) of FXa and thrombin, the greater structural disparity was observed in the S4-pocket, which was more symmetrical (U-shaped) in FXa as compared to thrombin mainly due to the presence of L99 and I174 residues in latter in place of Y99 and F174 respectively in former protease. The S2 pocket forming partial roof at the entry of 12 ? deep S1-pocket, with two extended β-sheets running antiparallel to each other by undergoing U-turn (～180?), has two conserved glycine residues forming H-bonds with the bound ligand for governing ligand binding affinity. The docking, scoring, and binding pose comparison of the representative high-affinity and selective inhibitors into the active sites of FXa and thrombin revealed critical residues (S214, Y99, W60D) mediating selectivity through direct- and long-range electrostatic interactions. Interestingly, most of the thrombin-selective inhibitors attained S-shaped conformation in thrombin, while FXa-selective inhibitors attained L-shaped conformations in FXa. The role of residue at 99th position of FXa and thrombin toward governing protease selectivity was further substantiated using molecular dynamics simulations on the wild-type and mutated Y99L FXa bound to thrombin-selective inhibitor 2. Furthermore, predictive CoMFA (FXa q2 = 0.814; thrombin q2 = 0.667) and CoMSIA (FXa q2 = 0.807; thrombin q2 = 0.624) models were developed and validated (FXa r2(test) = 0.823; thrombin r(2)(test) = 0.816) to feature molecular determinants of ligand binding affinity using the docking-based conformational alignments (DBCA) of 141 (88(train)+53(test)) and 39 (27(train)+11(test)) nonamidine class of potent FXa (0.004 ≤ K(i) (nM) ≤ 4700) and thrombin (0.001 ≤ K(i) (nM) ≤ 940) inhibitors, respectively. Interestingly, the ligand-based insights well corroborated with the structure-based insights in terms of the role of steric, electrostatic, and hydrophobic parameters for governing the selectivity for the two proteases. The new computational insights presented in this study are expected to be valuable for understanding and designing potent and selective antithrombotic agents.     

Small Molecule Microarray Based Discovery of PARP14 Inhibitors

Poly(ADP‐ribose) polymerases (PARPs) are key enzymes in a variety of cellular processes. Most small‐molecule PARP inhibitors developed to date have been against PARP1, and suffer from poor selectivity. PARP14 has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Herein, we describe a small molecule microarray‐based strategy for high‐throughput synthesis, screening of >1000 potential bidentate inhibitors of PARPs, and the successful discovery of a potent PARP14 inhibitor H10 with >20‐fold selectivity over PARP1. Co‐crystallization of the PARP14/ H10 complex indicated H10 bound to both the nicotinamide and the adenine subsites. Further structure–activity relationship studies identified important binding elements in the adenine subsite. In tumor cells, H10 was able to chemically knockdown endogenous PARP14 activities.     

Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single‐Molecule Spectroscopy

The conformation of the activation loop (T‐loop) of protein kinases underlies enzymatic activity and influences the binding of small‐molecule inhibitors. By using single‐molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora A kinase is in dynamic equilibrium between a DFG‐in‐like active T‐loop conformation and a DFG‐out‐like inactive conformation, and have measured the rate constants of interconversion. Addition of the Aurora A activating protein TPX2 shifts the equilibrium towards an active T‐loop conformation whereas addition of the inhibitors MLN8054 and CD532 favors an inactive T‐loop. We show that Aurora A binds TPX2 and MLN8054 simultaneously and provide a new model for kinase conformational behavior. Our approach will enable conformation‐specific effects to be integrated into inhibitor discovery across the kinome, and we outline some immediate consequences for structure‐based drug discovery.     

Studies of the mechanism of selectivity of protein tyrosine phosphatase 1B (PTP1B) bidentate inhibitors using molecular dynamics simulations and free energy calculations

Bidentate inhibitors of protein tyrosine phosphatase 1B (PTP1B) are considered as a group of ideal inhibitors with high binding potential and high selectivity in treating type II diabetes. In this paper, the binding models of five bidentate inhibitors to PTP1B, TCPTP, and SHP-2 were investigated and compared by using molecular dynamics (MD) simulations and free energy calculations. The binding free energies were computed using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology. The calculation results show that the predicted free energies of the complexes are well consistent with the experimental data. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition analysis indicates that the residues ARG24, ARG254, and GLN262 in the second binding site of PTP1B are essential for the high selectivity of inhibitors. Furthermore, the residue PHE182 close to the active site is also important for the selectivity and the binding affinity of the inhibitors. According to our analysis, it can be concluded that in most cases the polarity of the portion of the inhibitor that binds to the second binding site of the protein is positive to the affinity of the inhibitors while negative to the selectivity of the inhibitors. We expect that the information we obtained here can help to develop potential PTP1B inhibitors with more promising specificity.     

A computational analysis of the binding model of MDM2 with inhibitors

Abstract  

It is a new and promising strategy for anticancer drug design to block the MDM2-p53 interaction using a non-peptide small-molecule inhibitor. We carry out molecular dynamics simulations to study the binding of a set of six non-peptide small-molecule inhibitors with the MDM2. The relative binding free energies calculated using molecular mechanics Poisson–Boltzmann surface area method produce a good correlation with experimentally determined results. The study shows that the van der Waals energies are the largest component of the binding free energy for each complex, which indicates that the affinities of these inhibitors for MDM2 are dominated by shape complementarity. The A-ligands and the B-ligands are the same except for the conformation of 2,2-dimethylbutane group. The quantum mechanics and the binding free energies calculation also show the B-ligands are the more possible conformation of ligands. Detailed binding free energies between inhibitors and individual protein residues are calculated to provide insights into the inhibitor-protein binding model through interpretation of the structural and energetic results from the simulations. The study shows that G1, G2 and G3 group mimic the Phe19, Trp23 and Leu26 residues in p53 and their interactions with MDM2, but the binding model of G4 group differs from the original design strategy to mimic Leu22 residue in p53.     

Theoretical studies on the conformational change of adenosine kinase induced by inhibitors

Adenosine kinase (AK) is a two‐domain protein that catalyzes the phosphorylation of adenosine to adenosine monophosphate. Inhibitors of AK could increase adenosine to levels that activate nearby adenosine receptors and produce a wide variety of therapeutically beneficial activities. To get insight into the interaction mechanism between inhibitors and AK, we chose two kinds of novel inhibitors, alkynylpyrimidine inhibitor (APy) and aryl‐nucleoside inhibitor (AN), and used docking and molecular dynamics simulation methods to study the conformational changes of human AK on binding inhibitors. The calculation results revealed that both APy and AN could induce conformational changes of AK and stabilize AK at different semiopen conformations. On binding APy, the small lid‐domain rotated 14°, and the binding pocket rearranged after MD simulation. But in AK‐AN complex, the rotation of small domain is 22°, and the sugar ring of AN is mobile in the binding pocket. Further docking calculations on APy analogues indicate that the semiopen conformation could well explain the SAR of AK inhibitors. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Small molecule recognition of c-Src via the Imatinib-binding conformation

The cancer drug, Imatinib, is a selective Abl kinase inhibitor that does not inhibit the closely related kinase c-Src. This one drug and its ability to selectively inhibit Abl over c-Src has been a guiding principle in virtually all kinase drug discovery efforts in the last 15 years. A prominent hypothesis explaining the selectivity of Imatinib is that Abl has an intrinsic ability to adopt an inactive conformation (termed DFG-out), whereas c-Src appears to pay a high intrinsic energetic penalty for adopting this conformation, effectively excluding Imatinib from its ATP pocket. This explanation of the difference in binding affinity of Imatinib for Abl versus c-Src makes the striking prediction that it would not be possible to design an inhibitor that binds to the DFG-out conformation of c-Src with high affinity. We report the discovery of a series of such inhibitors. We use structure-activity relationships and X-ray crystallography to confirm our findings. These studies suggest that small molecules are capable of inducing the generally unfavorable DFG-out conformation in c-Src. Structural comparison between c-Src in complex with these inhibitors allows us to speculate on the differential selectivity of Imatinib for c-Src and Abl.     

Novel Dual‐Site‐Binding Neuraminidase Inhibitor from Virtual Screening by Pharmacophore and Molecular Dynamics Methods

Neuraminidase is a significant anti‐influenza target that plays crucial role in virus replication cycle. The discovery of 150‐cavity in Group‐1 neuraminidase provides us a novel mentality of designing inhibitor which can bind with both conserved site and 150‐cavity. In order to discover novel dual‐site‐binding inhibitors, a 3D chemical‐feature‐based pharmacophore model was established to cover dual‐site in neuraminidase. The dual‐site‐binding model was consistent in predicting the binding conformation of Group‐1 neuraminidase inhibitor and applied for virtual screening of Specs database. Compound 4 (ZINC05790048) that aligned well to the model was selected after multiple filtrations for molecular dynamics simulations, indicating improved binding energy with neuraminidase. It can sever as the lead compound for a novel series of inhibitors.     

分子动力学模拟别构抑制剂Efavirenz对HIV-1逆转录酶的作用

为了理解非核苷类逆转录酶抑制剂(NNRTIs)与HIV-1逆转录酶(RT)的相互作用机制,利用新力场ff12SB对未结合和结合Efavirenz (EFV)逆转录酶的三种RT大分子体系分别进行了100 ns的长时间动力学模拟。通过分析EFV对RT结构的影响、不同残基柔性和不同体系构象的动力学行为等,发现EFV的结合会导致RT结构变化,从而影响RT的活性;证实了EFV的“分子楔”作用;还发现EFV的结合不但引起“拇指关节炎”,而且引起轻度“手指关节炎”;整个模拟过程中没有出现不同构象间的跃迁,但是无别构分子时的RT张开构象表现出明显的闭合倾向。这些结果有助于理解NNRTIs的抑制机制和RT构象变化的动力学性质。另外,还比较分析了模拟方法对计算结果的影响,对大分子体系的动力学模拟具有重要借鉴意义。     

Structure‐Based Design of a Macrocyclic PROTAC

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co‐crystal structure of macroPROTAC‐1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12‐fold loss of binary binding affinity for Brd4, macroPROTAC‐1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.     

Deactivation of Mcl‐1 by Dual‐Function Small‐Molecule Inhibitors Targeting the Bcl‐2 Homology 3 Domain and Facilitating Mcl‐1 Ubiquitination

By means of limited proteolysis assay, three‐dimensional NMR, X‐ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl‐2 homology 3 (BH3) domain of Mcl‐1 has been identified as a conformational switch which controls Mcl‐1 ubiquitination. Noxa^BH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl‐1. In contrast, Bim^BH3 binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl‐1 inhibitor, which locates at the BH3 domain of Mcl‐1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro‐apoptotic partners, but also facilitates Mcl‐1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl‐1‐dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.     

Dihydrofolate reductase: structural aspects of mechanisms of enzyme catalysis and inhibition

The mechanism of catalytic reduction of folic and dihydrofolic acids to tetrahydrofolate, which proceeds under the action of dihydrofolate reductase and the coenzyme NADPH, is considered. The roles of the enzyme active site, the coenzyme, individual amino acid residues of the enzyme, and water molecules in the catalytic reaction are discussed. Interactions of the enzyme with competitive inhibitors many of which are widely used in medicine as antitumor and antibacterial drugs are examined. The factors controlling the selectivity of inhibitor binding to bacterial forms of the enzyme are analyzed. The results of X-ray diffraction and NMR spectroscopic studies of the structures of the enzyme and its complexes with the substrate and inhibitors are surveyed. The role of specific interactions and molecular motions of the protein and ligands in the mechanism of catalysis and in the binding of the ligands to the enzyme is discussed.     

3MBA类FtsZ蛋白抑制剂的分子动力学模拟及抗菌作用机制

采用分子动力学模拟、蛋白质二级结构测定(DSSP)、口袋体积测量(POVME)以及MM-PBSA(molecular mechanics Poisson-Boltzmann surface area)方法, 系统研究了金黄色葡萄球菌丝状温度敏感性蛋白Z (SaFtsZ)-二磷酸鸟苷(GDP)二元复合物和SaFtsZ-GDP-3MBA (3-甲氧基苯甲酰胺)类衍生物三元复合物体系的稳定性、蛋白质二级结构、蛋白质构象、关键残基质心距、活性口袋体积以及相对结合自由能的变化规律. 研究表明: 当不含抑制剂存在时SaFtsZ-GDP二元复合物体系稳定性较差, 其T7Loop区域残基(203-209)波动较大, 且蛋白二级结构发生明显变化, 活性口袋体积急剧减小, 底物通道显著变窄且不稳定. 而含有抑制剂PC190723、Compound1 的类衍生物三元复合物体系的表现截然不同, 这主要是由于它们均能和活性口袋T7Loop区周围残基形成关键性的氢键以及疏水作用, 与FtsZ 蛋白紧密结合. 在SaFtsZ-GDP-3MBA三元复合物体系中, 3MBA仅能与活性口袋中部分残基形成疏水作用, 与FtsZ 蛋白亲和力较弱, 使其不能稳定地存在于活性口袋中, 进一步导致它的抗菌活性明显低于PC190723、Compound1. 这些发现深入揭示了3MBA类衍生物对FtsZ 蛋白的作用机制和影响规律, 为该类FtsZ 蛋白抑制剂的结构优化和产品开发应用提供了重要的理论依据.