Sybyl x 2

The three-dimensional structures of the designed cruzain inhibitors were constructed using the standard geometric parameters of SYBYL-X 2.1 (Certara, Princeton, NJ). Each compound was energetically minimized using the Tripos force field (Clark et al., 1989 (link)) and Powell conjugate gradient algorithm (Powell, 1977 (link)) with a convergence criterion of 0.05 kcal/mol.Å and Gasteiger-Hückel charges (Gasteiger and Marsili, 1980 (link)). The designed imide derivatives were docked into the cruzain catalytic site using GOLD 5.3 (Cambridge Crystallographic Data Centre, Cambridge, UK) (Jones et al., 1997 (link)). The X-ray structure of cruzain (PDB 3KKU, 1.28 Å) (Ferreira et al., 2010 (link)) was prepared by removing the water molecules and adding hydrogen atoms. The active site residues Cys25 and His162 were maintained as negatively charged and protonated, respectively. A sphere with a 10 Å radius centered on the sulfur atom of Cys25 was settled as the binding site. Compounds were docked by applying the GoldScore scoring function with a search efficiency of 200%. Visual analysis of the molecular docking-derived binding conformations was carried out with PyMOL 1.3 (Schrödinger, New York, NY) (Lill and Danielson, 2011 (link)).

The three-dimensional structures of the cruzain inhibitors were constructed using the standard geometric parameters embedded in SYBYL-X 2.1 (Certara, Princeton, NJ). Each compound was energetically minimized employing the Tripos force field (Clark et al., 1989 (link)) and Powell conjugate gradient method (Powell, 1977 (link)), with a convergence value of 0.05 kcal/mol.Å, and the Gasteiger-Hückel model was used for charge calculation (Gasteiger and Marsili, 1980 (link)). The molecules were docked using GOLD 5.3 (Cambridge Crystallographic Data Centre, Cambridge, United Kingdom) (Jones et al., 1997 (link); Verdonk et al., 2003 (link)) against the X-ray structure of cruzain (PDB ID 3KKU, 1.28 Å) (Ferreira et al., 2010 (link)). The preparation of the cruzain structure consisted of removing all water molecules and inserting hydrogen atoms. The active site Cys25 was kept negatively charged, and His162 was kept protonated. The binding site was defined as a sphere with a 10 Å radius centered on the Cys25 sulfur atom. The default GOLD parameters were applied for the molecular docking runs, except for the search efficiency, which was changed to its maximum value of 200%. The generated poses were evaluated using the GoldScore scoring function, and the analysis of the binding conformations was visualized using PyMOL 3.1 (Schrödinger, New York, NY) (Lill and Danielson, 2011 (link)).

Docking studies were performed to understand the binding interactions of INX-315 in the active site (ATP-binding site) of CDK2/cyclin E1. INX-315 was subjected to minimization using 1500 iterations, applied MMFF94 force field (used through the study), assigned Gasteiger charges, with an energy gradient convergence criterion of 0.0001 kcal/mol Å. The lowest energy conformation was determined. The crystal structure was downloaded from RCSB PDB (ID:1W98). Chain A was retained. Gasteiger charges were assigned. The 3D structure of CDK2 was prepared to fix all the defects and errors in the structures. Protein preparation includes addition of hydrogens, repair side chains, treat termini, fixing of atom type, protonation state, bond order, charges, and amides. It was minimized to remove any strain produced during earlier steps. Protomol was generated with a threshold of 0.5 and a bloat value of 1 Å, to generate an active site. INX-315 was docked into this active site using the Surflex-Dock GeomX (SFXC) method. The estimated binding affinity 10.840 for INX-315, (expressed as total_score which represents −logK_d), was reported. All the simulations were performed using SYBYL-X 2.1, and images were generated using Tripos Benchware 3D Explorer Viewer 2.7 (Certara Inc.).

The HQSAR, AutoQSAR, CoMFA, and CoMSIA models were built as previously described [21 (link),22 (link),23 (link),24 (link)] using SYBYL-X 2.1 (Certara Inc., Princeton, NJ, USA) and Maestro (release 2016-3) (Schrӧdinger LLC, New York, NY, USA). The 3D structures of the compounds were built using Epik at pH 5.5 and minimized using LigPrep and OPLS3 (Schrӧdinger LLC, New York, NY, USA) [25 (link),26 (link),27 (link)]. The X-ray structure of cruzain deposited in the Protein Data Bank (PDB 3KKU, 1.28 Å) [28 (link)] and GOLD 5.3 (Cambridge Crystallographic Data Centre, Cambridge, UK) [29 (link)] were used in the molecular docking studies. The enzyme-inhibitor complexes were visualized using Maestro (release 2016-3) and Chimera (University of California, San Francisco, CA, USA) [30 (link)].

Model heparin—spike complexes were created by manual addition of three 31-mer heparin strands to the cryo-electron microscopy structure of the SARS-CoV-2 S protein PDB code 6ZGE [22 (link)]. A single heparin strand was added manually in SybylX2.1 (Certara, Princeton, NJ, USA) to follow the positively charged surface of one monomer of the spike protein structure, and then the strand was copied using the three-fold symmetry of the S protein to generate the trimeric heparin—spike protein complex. The model was minimized to remove minor clashes introduced during the construction process in SybylX2.1. For SARS-CoV-2 variants, sequence differences were either introduced through simple mutations of the residues in the existing models or, in the case of variants with significant insertions or deletions, through the construction of homology models of the S protein using MODELLER version 10.1 [23 (link)]. Heparin—spike protein complex models were inspected with PyMOL (the PyMOL Molecular Graphics System, Version 2.5 Schrödinger, LLC, New York, NY, USA).

A model of the PfAsnRS-Asn-tRNA complex was generated by combining a modified version of the AlphaFold model for PfAsnRS bound to Asn-AMP with the tRNA from the structure of the E. coli aspartyl-tRNA synthase/tRNA complex, 1C0A^{31 (link)}. The catalytic domain of the PfAsnRS model was aligned to the equivalent region of 1C0A using PyMOL⁷⁵ and visual inspection showed an extremely good match for the local structure, with the tRNA from 1C0A positioned appropriately across both the active site and onto the anticodon domain. The only significant clash was of the acceptor stem with residues of the flipping loop adjacent to the active site, due to the PfAsnRS model having these in the closed conformation seen in the tRNA-free structures of class II tRNA synthase enzymes^{31 (link)}. The conformation of the flipping loop in the PfAsnRS model was manually corrected to the open position using COOT (version 0.9.8.1)^{66 (link)}, and the PfAsnRS/Asn-AMP/tRNA complex model was minimized to remove any minor steric overlaps using SybylX2.1 (Certara, NJ, USA). To generate the PfAsnRS/AMP/Asn-tRNA complex, the bond between the asparagine residue and AMP was manually broken and a new bond to the 3’OH oxygen of the acceptor stem terminal adenine was added using SybylX2.1. The modified complex was minimized to correct any errors in bond lengths or angles.