Antimicrobial resistance in bacteria often arises from their ability to actively identify and expel toxic compounds. The bacterium strain Pseudomonas putida DOT-T1E utilizes its TtgABC efflux pump to confer robust resistance against antibiotics, flavonoids, and organic solvents. This resistance mechanism is intricately regulated at the transcriptional level by the TtgR protein. Through molecular dynamics and alchemical free energy simulations, we systematically examine the binding of seven flavonoids and their derivatives with the TtgR transcriptional regulator. Our simulations reveal distinct binding geometries and free energies for the flavonoids in the active site of the protein, which are driven by a range of noncovalent forces encompassing van der Waals, electrostatic, and hydrogen bonding interactions. The interplay of molecular structures, substituent patterns, and intermolecular interactions effectively stabilizes the bound flavonoids, confining their movements within the TtgR binding pocket. These findings yield valuable insights into the molecular determinants that govern ligand recognition in TtgR and shed light on the mechanism of antimicrobial resistance in P. putida DOT-T1E.

An alchemical enhanced sampling (ACES) method has recently been introduced to facilitate importance sampling in free energy simulations. The method achieves enhanced sampling from Hamiltonian replica exchange within a dual topology framework while utilizing new smoothstep softcore potentials. A common sampling problem encountered in lead optimization is the functionalization of aromatic rings that exhibit distinct conformational preferences when interacting with the protein. It is difficult to converge the distribution of ring conformations due to the long time scale of ring flipping events; however, the ACES method addresses this issue by modeling the syn and anti ring conformations within a dual topology. ACES thereby samples the conformer distributions by alchemically tunneling between states, as opposed to traversing a physical pathway with a high rotational barrier. We demonstrate the use of ACES to overcome conformational sampling issues involving ring flipping in ML300-derived noncovalent inhibitors of SARS-CoV-2 Main Protease (Mpro). The demonstrations explore how the use of replica exchange and the choice of softcore selection affects the convergence of the ring conformation distributions. Furthermore, we examine how the accuracy of the calculated free energies is affected by the degree of phase space overlap (PSO) between adjacent states (i.e., between neighboring λ-windows) and the Hamiltonian replica exchange acceptance ratios. Both of these factors are sensitive to the spacing between the intermediate states. We introduce a new method for choosing a schedule of λ values. The method analyzes short “burn-in” simulations to construct a 2D map of the nonlocal PSO. The schedule is obtained by optimizing an alchemical pathway on the 2D map that equalizes the PSO between the λ intervals. The optimized phase space overlap λ-spacing method (Opt-PSO) leads to more numerous end-to-end single passes and round trips due to the correlation between PSO and Hamiltonian replica exchange acceptance ratios. The improved exchange statistics enhance the efficiency of ACES method. The method has been implemented into the FE-ToolKit software package, which is freely available.

AmberTools is a free and open-source collection of programs used to set up, run, and analyze molecular simulations. The newer features contained within AmberTools23 are briefly described in this Application note.