Many thermodynamic quantities can be extracted from computer simulations that generate an ensemble of microstates according to the principles of statistical mechanics. Among these quantities is the free energy of binding of a small molecule to a macromolecule, such as a protein. Here, we present an introductory overview of a protocol that allows for the estimation of ligand binding free energies via molecular dynamics simulations. While we focus on the binding of organic molecules to proteins, the approach is in principle transferable to any pair of molecules.

Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t = 39.8 min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC = 166 nM), optimised physicochemical properties (LE = 0.43; LLE = 5.74; SFI = 5.96), and greater metabolic stability (t = 388 min).

The Trypanosoma cruzi (T. cruzi) parasite is the cause of Chagas disease, a neglected disease endemic in South America. The life cycle of the T. cruzi parasite is complex and includes transitions between distinct life stages. This change in phenotype (without a change in genotype) could be controlled by epigenetic regulation, and might involve the bromodomain-containing factors 1−5 (TcBDF1−5). However, little is known about the function of the TcBDF1−5. Here we describe a fragment-based approach to identify ligands for T. cruzi bromodomain-containing factor 3 (TcBDF3). We expressed a soluble construct of TcBDF3 in E. coli, and used this to develop a range of biophysical assays for this protein. Fragment screening identified 12 compounds that bind to the TcBDF3 bromodomain. On the basis of this screen, we developed functional ligands containing a fluorescence or 19 F reporter group, and a photo-crosslinking probe for TcBDF3. These tool compounds will be invaluable in future studies on the function of TcBDF3 and will provide insight into the biology of T. cruzi.

Prioritizing molecules for synthesis is a key role of computational methods within medicinal chemistry. Multiple tools exist for ranking molecules, from the cheap and popular molecular docking methods to more computationally expensive molecular‐dynamics (MD)‐based methods. It is often questioned whether the accuracy of the more rigorous methods justifies the higher computational cost and associated calculation time. Here, we compared the performance on ranking the binding of small molecules for seven scoring functions from five docking programs, one end‐point method (MM/GBSA), and two MD‐based free energy methods (PMX, FEP+). We investigated 16 pharmaceutically relevant targets with a total of 423 known binders. The performance of docking methods for ligand ranking was strongly system dependent. We observed that MD‐based methods predominantly outperformed docking algorithms and MM/GBSA calculations. Based on our results, we recommend the application of MD‐based free energy methods for prioritization of molecules for synthesis in lead optimization, whenever feasible.

The <i>Trypanosoma cruzi</i> (<i>T. cruzi</i>) parasite is the cause of Chagas disease, a neglected disease endemic in South America. The life cycle of the <i>T. cruzi</i> parasite is complex and includes transitions between distinct life stages. This change in phenotype (without a change in genotype) could be controlled by epigenetic regulation, and might involve the bromodomain-containing factors 1-5 (<i>Tc</i>BDF1-5). However, little is known about the function of the <i>Tc</i>BDF1-5. Here we describe a fragment-based approach to identify ligands for <i>T. cruzi</i> bromodomain-containing factor 3 (<i>Tc</i>BDF3). We expressed a soluble construct of <i>Tc</i>BDF3 in <i>E. coli</i>, and used this to develop a range of biophysical assays for this protein. Fragment screening identified twelve compounds that bind to the <i>Tc</i>BDF3 bromodomain. Based on this screen, we developed functional ligands containing a fluorescence or ¹⁹F reporter group, and a photo-crosslinking probe for <i>Tc</i>BDF3. These tools compounds will be invaluable in future studies on the function of <i>Tc</i>BDF3 and will provide insight into the biology of <i>T. cruzi</i>.