Exhaustively Exploring the Prevalent Interaction Pathways of Ligands Targeting the Ligand-Binding Pocket of Farnesoid X Receptor via Combined Enhanced Sampling.

It is well-known that the potency of a drug is heavily associated with its kinetic and thermodynamic properties with the target. Nuclear receptors (NRs), as an important target family, play important roles in regulating a variety of physiological processes in vivo. However, it is hard to understand the drug-NR interaction process because of the closed structure of the ligand-binding domain (LBD) of the NR proteins, which apparently hinders the rational design of drugs with controllable kinetic properties. Therefore, understanding the underlying mechanism of the ligand-NR interaction process seems necessary to help NR drug design. However, it is usually difficult for experimental approaches to interpret the kinetic process of drug-target interactions. Therefore, in silico methods were utilized to explore the optimal binding/dissociation pathways of the NR ligands. Specifically, farnesoid X receptor (FXR) is considered here as the target system since it has been an important target for the treatment of bile acid metabolism-associated diseases, and a series of structures cocrystallized with diverse scaffold ligands were resolved. By using random acceleration molecular dynamics (RAMD) simulation and umbrella sampling (US), 5 main dissociation pathways (pathways I-V) were identified in 11 representative FXR ligands, with most of them (9/11) preferring to go through Pathway III and the remaining two favoring escaping from Pathway I and IV. Furthermore, key residues functioning in the three main dissociation pathways were revealed by the kinetic residue energy analysis (KREA) based on the US trajectories, which may serve as road-marker residues for rapid identification of the (un)binding pathways of FXR ligands. Moreover, the preferred pathways explored by RAMD simulations are in good agreement with the minimum free energy path identified by the US simulations with the Pearson R = 0.76 between the predicted binding affinity and the experimental data, suggesting that RAMD is suitable for applying in large-scale (un)binding-pathway exploration in the case of ligands with obscure binding tunnels to the target.

Uncovering the Kinetic Characteristics and Degradation Preference of PROTAC Systems with Advanced Theoretical Analyses.

Proteolysis-targeting chimeras (PROTACs), which can selectively induce the degradation of target proteins, represent an attractive technology in drug discovery. A large number of PROTACs have been reported, but due to the complicated structural and kinetic characteristics of the target-PROTAC-E3 ligase ternary interaction process, the rational design of PROTACs is still quite challenging. Here, we characterized and analyzed the kinetic mechanism of MZ1, a PROTAC that targets the bromodomain (BD) of the bromodomain and extra terminal (BET) protein (Brd2, Brd3, or Brd4) and von Hippel-Lindau E3 ligase (VHL), from the kinetic and thermodynamic perspectives of view by using enhanced sampling simulations and free energy calculations. The simulations yielded satisfactory predictions on the relative residence time and standard binding free energy (rp > 0.9) for MZ1 in different BrdBD-MZ1-VHL ternary complexes. Interestingly, the simulation of the PROTAC ternary complex disintegration illustrates that MZ1 tends to remain on the surface of VHL with the BD proteins dissociating alone without a specific dissociation direction, indicating that the PROTAC prefers more to bind with E3 ligase at the first step in the formation of the target-PROTAC-E3 ligase ternary complex. Further exploration of the degradation difference of MZ1 in different Brd systems shows that the PROTAC with higher degradation efficiency tends to leave more lysine exposed on the target protein, which is guaranteed by the stability (binding affinity) and durability (residence time) of the target-PROTAC-E3 ligase ternary complex. It is quite possible that the underlying binding characteristics of the BrdBD-MZ1-VHL systems revealed by this study may be shared by different PROTAC systems as a general rule, which may accelerate rational PROTAC design with higher degradation efficiency.

Deciphering the Shared and Specific Drug Resistance Mechanisms of Anaplastic Lymphoma Kinase via Binding Free Energy Computation.

Anaplastic lymphoma kinase (ALK), a tyrosine receptor kinase, has been proven to be associated with the occurrence of numerous malignancies. Although there have been already at least 3 generations of ALK inhibitors approved by FDA or in clinical trials, the occurrence of various mutations seriously attenuates the effectiveness of the drugs. Unfortunately, most of the drug resistance mechanisms still remain obscure. Therefore, it is necessary to reveal the bottom reasons of the drug resistance mechanisms caused by the mutations. In this work, on the basis of verifying the accuracy of 2 main kinds of binding free energy calculation methodologies [end-point method of Molecular Mechanics with Poisson-Boltzmann/Generalized Born and Surface Area (MM/PB(GB)SA) and alchemical method of Thermodynamic Integration (TI)], we performed a systematic analysis on the ALK systems to explore the underlying shared and specific drug resistance mechanisms, covering the one-drug-multiple-mutation and multiple-drug-one-mutation cases. Through conventional molecular dynamics (cMD) simulation in conjunction with MM/PB(GB)SA and umbrella sampling (US) in conjunction with contact network analysis (CNA), the resistance mechanisms of the in-pocket, out-pocket, and multiple-site mutations were revealed. Especially for the out-pocket mutation, a possible transfer chain of the mutation effect was revealed, and the reason why different drugs exhibited various sensitivities to the same mutation was also uncovered. The proposed mechanisms may be prevalent in various drug resistance cases.