The simulation temperature was kept at 298 K by means of the stochastic dynamics integrator with the friction of 0.5 ps -1 . The protein-ligand calculation data collected from used molecular dynamics integrator in combination with the velocity rescaling thermostat with the time constant of 0.1 ps. The pressure was controlled by means of the Parrinello-Rahman barostat with the time constant of 5 ps keeping pressure at 1 bar. Electrostatic interactions were treated by means of the Particle Mesh Ewald (PME) with the direct space cutoff of 1.1 nm, relative strength of interactions at the cutoff of 10 -5 , Fourier grid spacing of 0.12 nm. Van der Waals interactions were switched starting at 1.0 nm distance and were completely turned off for the distances reaching 1.1 nm. Dispersion correction was used to adjust energy and pressure. Non-bonded interactions during the alchemical transitions were softened. The functional form of the softcore potential described in (with the default set of parameters) was used for the transitions in PDE2, GALECTIN, BACE (Hunt), BACE, BACE (P2), CMET, JNK1, TYK2, MCL1, CDK2, THROMBIN, PTP1B and P38 systems. For the alchemical transitions in the other systems the softcore potential described in was used with the parameters α = 0.3 and σ = 0.25 nm. The bonds were constrained by means of LINCS algorithm. From the alchemical transitions, work values were collected and free energy differences were calculated based on the Crooks Fluctuation Theorem 46 using maximum likelihood estimator.