The parametrization and validation of the OPLS3 force field for small molecules and proteins are reported. Enhancements with respect to the previous version (OPLS2.1) include the addition of off-atom charge sites to represent halogen bonding and aryl nitrogen lone pairs as well as a complete refit of peptide dihedral parameters to better model the native structure of proteins. To adequately cover medicinal chemical space, OPLS3 employs over an order of magnitude more reference data and associated parameter types relative to other commonly used small molecule force fields (e.g., MMFF and OPLS_2005). As a consequence, OPLS3 achieves a high level of accuracy across performance benchmarks that assess small molecule conformational propensities and solvation. The newly fitted peptide dihedrals lead to significant improvements in the representation of secondary structure elements in simulated peptides and native structure stability over a number of proteins. Together, the improvements made to both the small molecule and protein force field lead to a high level of accuracy in predicting protein-ligand binding measured over a wide range of targets and ligands (less than 1 kcal/mol RMS error) representing a 30% improvement over earlier variants of the OPLS force field.

Designing tight binding ligands is a primary objective of small molecule drug discovery.Over the past few decades, free energy calculations have benefited from improved force fields and sampling algorithms, as well as the advent of low cost parallel computing.However, it has proven to be challenging to reliably achieve the level of accuracy that would be needed to guide lead optimization (~5X in binding affinity) for a wide range of ligands and protein targets. Not surprisingly, widespread commercial application of free energy simulations has been limited due to the lack of large-scale validation coupled with the technical challenges traditionally associated with running these types of calculations.Here, we report an approach that achieves an unprecedented level of accuracy across a broad range of target classes and ligands, with retrospective results encompassing 200 ligands and a wide variety of chemical perturbations, many of which involve significant changes in ligand chemical structures. In addition, we have applied the method in prospective drug discovery projects and found a significant improvement in the quality of the compounds synthesized that have been predicted to be potent. Compounds predicted to be potent by this approach have a substantial reduction in false positives relative to compounds synthesized based on other computational or medicinal chemistry approaches. Furthermore, the results are consistent with those obtained from our retrospective studies, demonstrating the robustness and broad range of applicability of this approach, which can be used to drive decisions in lead optimization.3

ABSTRACT:The mechanism (or mechanisms) of enthalpy− entropy (H/S) compensation in protein−ligand binding remains controversial, and there are still no predictive models (theoretical or experimental) in which hypotheses of ligand binding can be readily tested. Here we describe a particularly well-defined system of protein and ligandshuman carbonic anhydrase (HCA) and a series of benzothiazole sulfonamide ligands with different patterns of fluorinationthat we use to define enthalpy/entropy (H/S) compensation in this system thermodynamically and structurally. The binding affinities of these ligands (with the exception of one ligand, in which the deviation is understood) to HCA are, despite differences in fluorination pattern, indistinguishable; they nonetheless reflect significant and compensating changes in enthalpy and entropy of binding. Analysis reveals that differences in the structure and thermodynamic properties of the waters surrounding the bound ligands are an important contributor to the observed H/S compensation. These results support the hypothesis that the molecules of water filling the active site of a protein, and surrounding the ligand, are as important as the contact interactions between the protein and the ligand for biomolecular recognition, and in determining the thermodynamics of binding.

Predicting protein-ligand binding free energies is a central aim of computational structure-based drug design (SBDD)--improved accuracy in binding free energy predictions could significantly reduce costs and accelerate project timelines in lead discovery and optimization. The recent development and validation of advanced free energy calculation methods represents a major step toward this goal. Accurately predicting the relative binding free energy changes of modifications to ligands is especially valuable in the field of fragment-based drug design, since fragment screens tend to deliver initial hits of low binding affinity that require multiple rounds of synthesis to gain the requisite potency for a project. In this study, we show that a free energy perturbation protocol, FEP+, which was previously validated on drug-like lead compounds, is suitable for the calculation of relative binding strengths of fragment-sized compounds as well. We study several pharmaceutically relevant targets with a total of more than 90 fragments and find that the FEP+ methodology, which uses explicit solvent molecular dynamics and physics-based scoring with no parameters adjusted, can accurately predict relative fragment binding affinities. The calculations afford R(2)-values on average greater than 0.5 compared to experimental data and RMS errors of ca. 1.1 kcal/mol overall, demonstrating significant improvements over the docking and MM-GBSA methods tested in this work and indicating that FEP+ has the requisite predictive power to impact fragment-based affinity optimization projects.

Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is a viable approach to suppress tumor growth in many human cancers and stabilization of the helical structure of synthetic p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of proteinprotein binding (J. Am. Chem. Soc. 129: 5298). However, details of the structure and dynamic stability of the stapled peptides are not well understood. Here, we use extensive all-atom molecular dynamics simulations to study a series of stapled a-helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted a-helical propensities that are in good agreement with the experimental observations. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute a-helical stability. These simulations provide new insights into the design of a-helical stapled peptides and the development of potent inhibitors of a-helical protein-protein interfaces.Key words: circular dichroism, drug design, hDM2, molecular dynamic simulations, p53, protein-protein interfaces, stapled peptide, a-helicity A renaissance of peptide drug discovery has emerged over the past several years. In particular, the identification of synthetically 'stapled' a-helical peptides having promising pharmacokinetic, metabolic stability, and cell-penetrating properties has sparked tremendous interest in their development for a plethora of therapeutic targets that have otherwise been deemed 'undruggable' by more conventional small-molecule strategies (1). To date, numerous examples of stapled peptides for varying therapeutic targets have been described, including p53 (2), BID BH3 (3,4), BAD BH3 (5), NOTCH (6), and HIV-1 capsid (7). Both single-turn (i + 4 fi i ) and double-turn (i + 7 fi i ) stapling chemistries are represented in these studies.Synthetic p53 peptides incorporating double-turn stapling chemistry have been previously described (2) and have provided insight to the evolution of a prototype series of cell-penetrating and in vitro biologically effective lead compounds. This series of peptides (Table 1) serves as the basis for the computational work presented here. These stapled p53 peptides have incorporated C a -methyl-amino acids having terminal olefin alkyl side chains of different lengths and chirality, such as (S)-CH 2 ) 3 -CH=CH 2 and (R)-CH 2 ) 6 -CH=CH 2 , which upon metathesis form an all-carbon macrocycle via an olefin linkage (see compound 8, Scheme 1). The structure-activity relationships of p53-stapled peptides 1-10 involve further amino acid modifications to decrease negative charge (i.e., Asp and Glu replacement by Asn and Gln, respectively) and facilitate cell penetration as well as point mutations to avoid nuclear export and ubiquitination (i.e., Lys replacement by Arg). In contrast,...