Alessandro Di Domizio, Francesco Peri, in, 2013 3.4 Docking analysisAutoDock docking analysis between Ras and compounds 1– 5 ( Fig. 5.5) showed that in high-scoring poses, all compounds bind Ras in a cleft near the Switch 2 region 25. Compounds 1, 2, and 4 share the same pharmacophores, but have different scaffolds. Computed-binding energies for the best poses obtained by docking calculations ( Fig. 5.5) indicate that compound 2 (arabinose scaffold) has a more favorable binding energy than compounds 1 (linear scaffold) and 4 (glucose scaffold).
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The (S)—but not the (R)—stereoisomer of compound 3 (arabinose scaffold) has a similar binding energy as compound 2.Docking analysis shows that the two aromatic pharmacophores are involved in aromatic stacking interactions with the aromatic rings of Y96 and Y64, while a further stabilizing contribution is given by the formation of several hydrogen bonds, where a key role is played by Q99 and some residues of the Switch 2 region, in particular R68 and Q62 25. NMR (STD) and trNOESY experiments in solution also point out for all compounds that aromatic groups are the stronger binding determinants, the phenyloxy or benzyloxy groups of compounds 1– 3 interacting stronger with Ras than the phenylhydroxylamine or 3,4-dihydroxyphenyl groups ( Fig. Consistently with docking analysis, compound 2 was characterized by the strongest STD signals, indicating higher affinity for Ras ( Fig. 5.5, the intensities of STD signals are reported for the different moieties of compounds 1– 5 ranging from low (weak interaction with Ras, light gray square) to high (strong interaction with Ras, black square).The binding region of molecules of type 1 and 2 is localized in the Ras Switch 2 region as described above and overlaps with Ras–Sos binding interface 20. The interface between Ras and Sos is primarily hydrophilic and very extensive, with 3600 A 2 of surface area buried in the complex. At the heart of the interface between Ras and Sos is a cluster of three hydrophobic side chains from the Switch 2 region of Ras (Y64, M67, and Y71) which are buried into the hydrophobic core of Sos at the base of the binding site. Surrounding this hydrophobic anchor is an array of polar and charged interactions between Sos and Ras that results in almost every external side chain of Switch 2 being coordinated by Sos 20.
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The capacity to interfere with Ras–Sos interaction was investigated for type 1 and 2 compounds by surface plasmon resonance (SPR) experiments in collaboration with prof. Marco Vanoni of our Department 27. SPR experiments were performed by using N-terminal His-tagged p21hRas protein immobilized to an NTA sensor chip preactivated with NiCl 2. Sos binding to Ras was detected as an increase in resonance units.
Pretreatment of the NTA-Ras chip with type 1 and 2 compounds (in the 0.5–10 μM range) decreased subsequent binding of Sos in a dose-dependent manner. Type 1 compounds were slightly more potent than type 2 in inhibiting Ras–Sos interaction.The Ras-binding property of compounds type 2, with a glucose scaffold, was investigated in nucleotide exchange experiments on purified Ras 27. These compounds showed higher water solubility than type 1 compound but weaker activity in inhibiting Sos-promoted GDP–GTP exchange in vitro, thus reflecting the less potent capacity to displace Sos from Ras in SPR experiments 27.The NMR experiment (STD, trNOESY) for type 2 compounds confirmed the principal role of the aromatic moieties in the interaction with Ras 27. In the case of AaegOBP1, the docking results demonstrate that AaegOBP1 can bind to MOP at pH 7, with binding energy of − 5.09 kcal mol − 1. On the other hand, it has no affinity to MOP at pH 5, as binding energy is 4.17 kcal mol − 1. In order to further understand the mechanism of pH-dependent binding of AaegOBP1, we select other four ligands: decanal, geranylacetone, nonanal, and octanal, and contrast these four ligands to MOP.
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Compared with MOP, they all have a long lipid tail except a lactone ring head. The four ligands have lower affinity to AaegOBP1 at pH 5, which indicates that the four ligands are less sensitive to pH than MOP. The differences between the binding results of MOP and other four ligands may be derived from the lactone ring head. Figure 5.4A depicts that the lactone ring head of MOP is close to the position of pH-sensing triad of wild-type AaegOBP1 at pH 7. In addition, the binding results of mutation models suggest that the pH-sensing triad is indispensable to MOP binding, but it seems to have no effect on the other four ligands binding, as shown in Fig. This implicates that the lactone ring head of MOP has an important interaction with pH-sensing triad of AaegOBP1. Therefore, AaegOBP1 acts as a selective filter of odor molecule, sensitive to the ligands like MOP with lactone ring head.
And the interaction between ligand head and pH-sensing triad will be disrupted with the decrease in solution pH value. It is probably relevant to the moving of binding pocket made by the C-terminus change at pH 5, which blocks MOP binding to the protein. (A) Docking result of MOP to AaegOBP1 using Autodock at pH 7.0. Substrate MOP is shown in the deep blue ball and stick style, and pH-sensing triad is shown in the yellow stick style. All the oxygen atoms are colored in red and nitrogen atoms are colored in blue. (B) Binding energy plot of the five kinds of ligands binding to five other different proteins.Both CquiOBP1 and AaegOBP1 have high binding affinity with ligand MOP at pH 7, CquiOBP1 has lower binding affinity with MOP at pH 5, but AaegOBP1 loses its binding affinity in the same pH. That seems to have something with the number of H-bonds in pH-sensing triad ( Section 2.3).
At pH 5, all the three H-bonds disappear in AaegOBP1, which may be relative to the MOP binding affinity loss. Himani Tanwar. George Priya Doss, in, 2017 2.5 Molecular Docking AnalysisThe molecular docking analysis was performed using AutoDock tools 4.2 ( Morris et al., 2009). Prior to docking, the flavin adenine dinucleotide (PubChem ID: 643975) was prepared using the Dock prep of the chimera tools ( Pettersen et al., 2004).
Additionally, the native and mutant proteins were energy minimized using Swiss-PdbViewer ( Guex & Peitsch, 1997). To the protein structure, hydrogen bonds and charges were added. Additionally, the protein molecule was assigned the AD4 type. The prepared ligands were then torsion adjusted prior to applying autogrid function with the size of 60 × 60 × 60 along the x-, y-, and z-axes. The grid box was set around the active site of the protein. After the autogrid, autodock was performed using a Lamarckian algorithm.
The docked complex with the best binding energy was then visualized using a Maestro software of the Schrodinger. Ligand 1 has an experimental binding affinity of 0.40 nM to PKCε. Molecular docking using AutoDock suggests 15 pose clusters based on predicted binding energy for this ligand when it is bound to the ATP site of PKCε. Binding energy values of the clusters span between −7.0 and −15 kcal/mol. The main cluster contains 21 poses with predicted binding energy values near −15 kcal/mol ( Fig. 5(A)), which are the most negative among the others.
In this cluster, the strongest docking pose has a binding energy of −14.74 kcal/mol. According to AutoDock result, the docking pose shows an intermolecular energy of −18.02 kcal/mol, a van der Waals and hydrogen bond (H-bond) desolvation energy of −15.90 kcal/mol, electrostatic energy of −2.12 kcal/mol, total internal energy of −1.96 kcal/mol, torsional energy of 3.28 kcal/mol, and unbound energy of −1.96 kcal/mol.
Visual inspection of this docking pose in the ATP site of PKCε suggests that the hydroxyl group on the benzamide moiety (C5′OH) interacts via two H-bonds with Glu489 and Val491 in the adenine subsite ( Fig. The benzamide moiety may interact more tightly by introducing an additional hydroxyl group adjacent to C5′OH. The presence of such additional hydrogen group may strengthen the binding of balanol analog to PKCε. However, introducing such hydroxyl group may be problematic, since the presence of two adjacent hydroxyl groups in the benzamide moiety may lead to the unstable compound. Thus the additional hydroxyl group is incorporated as a hydroxymethyl substituent on the benzamide moiety to form the second ligand. The coordinate file of the second ligand is provided as ligand2.pdb.
H-bond interactions made by ligands (A) 1 and (B) 2 with residues of Glu489 and Val491 in the ATP site of PKCε. H-bonds are indicated by green dashed lines.Molecular docking of the second ligand to PKCε predicted that the incorporation of the hydroxymethyl group results in an improved binding affinity to this protein kinase. Binding-energy-based grouping in ADT categorizes the resulting docking poses into seven clusters ( Fig. The major cluster contains 37 docking poses with predicted binding energy values negative than −15 kcal/mol, which shows stronger interaction than the ligand1. Binding energy components of the second ligand are stronger than those of the first ligand such as intermolecular and desolvation energy which is −18.02 and −15.90 kcal/mol, respectively. Furthermore, visual assessment using Discovery Studio Visualizer shows that the presence of an additional hydroxyl group, as a hydroxymethyl substituent, enhances the H-bond interactions between the benzamide moiety and the ATP site of PKCε ( Fig. This increased interaction resulted in greater binding affinity of the second ligand to PKCε.
The overall virtual screening methodology adopted to find novel drug leads against Plasmodium falciparum Plasmepsin II.Next, compounds with sub-structural features commonly found in Pan Assay Interference Compounds (PAINS) were also filtered out using SMARTS strings inspired from the original Sybyl Line Notation patterns provided in the reference ( Baell and Holloway, 2010). This resulted in the removal of 111,305 compounds. Additionally, the dataset was screened for potentially reactive and promiscuous compounds described by a set of 275 medicinal chemistry rules, developed at Lilly over a period of 18 years ( Bruns and Watson, 2012). Reasons for rejection include reactivity, interference with assay measurements, instability, lack of drug-ability and activities that damage proteins, among others. A total of 1,349,716 compounds were removed and leaving 1,864,623 filtered compounds. To ensure chemical diversity, the approximately 1.8 million remaining molecules were clustered using PubChem fingerprints as implemented in the ChemmineR package ( Cao et al., 2008).
The maximum Tanimoto distance between a compound and the cluster center was set at 0.3. This resulted in the multi-molecule file containing 210,735 molecules which were first protonated for the acidic pH 5 and then minimized using the Ghemical force field. Subsequently, the minimized structures were used as the input to VINA and DOCK for molecular docking analysis. Computational tools for design are easily available online; most of them are free for academic use.
1.Autodock Vina (available from 2.Autodock Tools (available from 3.ChemDraw or comparable software package (available from 4.Rosetta (available from 5.PyMOL or comparable software package for protein structure viewing. PyMOL is available from While different operating systems can be successfully used, Mac OS has provided us with the best overall usability. Installation of the Rosetta package might take up to a day, but the manual provided on the RosettaCommons is very detailed and a person with no extensive computational skills can successfully install it. Familiarity with basic Unix commands is necessary. Minimalist design is inherently rational and it relies on the knowledge of the mechanism of the reaction to be catalyzed. Simplicity of the minimalist approach allows for a stepwise consideration of all of the states along the Michaelis path, such as binding of the substrate by the protein, transition state, and product dissociation.
The overall approach is summarized in Fig. First, an overall idea for potential catalysis is rationally devised, then an appropriate protein scaffold is selected, next docking is performed to determine whether the desired substrate can associate with the protein to any degree, potential placement of the functional groups that are absolutely critical for catalysis is then evaluated, and finally feasibility of the transition state geometry is established.
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