In the context of drug−receptor binding affinity calculations using molecular dynamics techniques, we implemented a combination of Hamiltonian replica exchange (HREM) and a novel nonequilibrium alchemical methodology, called virtual double-system single-box, with increased accuracy, precision, and efficiency with respect to the standard nonequilibrium approaches. The method has been applied for the determination of absolute binding free energies of 16 newly designed noncovalent ligands of the main protease (3CL pro ) of SARS-CoV-2. The core structures of 3CL pro ligands were previously identified using a multimodal structure-based ligand design in combination with docking techniques. The calculated binding free energies for four additional ligands with known activity (either for SARS-CoV or SARS-CoV-2 main protease) are also reported. The nature of binding in the 3CL pro active site and the involved residues besides the CYS−HYS catalytic dyad have been thoroughly characterized by enhanced sampling simulations of the bound state. We have identified several noncongeneric compounds with predicted low micromolar activity for 3CL pro inhibition, which may constitute possible lead compounds for the development of antiviral agents in Covid-19 treatment.

We describe a step-by-step protocol for the computation of absolute dissociation free energy with GROMACS code and PLUMED library, which exploits a combination of advanced sampling techniques and nonequilibrium alchemical methodologies. The computational protocol has been automated through an open source Python middleware (HPC_Drug) which allows one to set up the GROMACS/PLUMED input files for execution on high performing computing facilities. The proposed protocol, by exploiting its inherent parallelism and the power of the GROMACS code on graphical processing units, has the potential to afford accurate and precise estimates of the dissociation constants in drug-receptor systems described at the atomistic level. The procedure has been applied to the calculation of the absolute dissociation free energy of PF-07321332, an oral antiviral proposed by Pfizer, with the main protease (3CLpro) of SARS-CoV-2.

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In this paper we further develop a combined Hamiltonian replica exchange / non-equilibrium alchemical method for absolute binding free energies and test its performance on 11 ligands of the BRD4 bromodomain protein. We compare the results obtained with our approach with those obtained with other equilibrium and non-equilibrium alchemical methods showing their relative strengths, weaknesses, and limits. We show how using an enhanced sampling technique, before the alchemical transformations, allows to get accurate estimates of the binding free energies even when starting with sub-optimal initial binding poses (e.g. from docking). We also study the effect of different restraining mechanisms on the final result, and introduce a new "Loose-Tight" restraining algorithm. The method proves to strike a good balance between ease of use, automation, speed, and accuracy for absolute ligand binding free energy calculations and our scripts make it easy to integrate it into pre-existing computational drug discovery pipelines.

A simple and efficient method for C‐2 deuterium labeling of 3,4‐di‐tert‐butoxypyrroline N‐oxide, a useful chiral building block in azaheterocycles syntheses, is presented. Selective and quantitative deuterium incorporation (> 99 %) was achieved by base‐catalyzed H/D exchange in D2O under mild reaction conditions. A mechanistic pathway based on kinetic and computational data was proposed. The labeled nitrone was used in the synthesis of C‐8a deuterated (1R,2R,8aR)‐lentiginosine.