Jihyun Shim

CHARMM general force field: A force field for drug‐like molecules compatible with the CHARMM all‐atom additive biological force fields

The widely used CHARMM additive all‐atom force field includes parameters for proteins, nucleic acids, lipids, and carbohydrates. In the present article, an extension of the CHARMM force field to drug‐like molecules is presented. The resulting CHARMM General Force Field (CGenFF) covers a wide range of chemical groups present in biomolecules and drug‐like molecules, including a large number of heterocyclic scaffolds. The parametrization philosophy behind the force field focuses on quality at the expense of transferability, with the implementation concentrating on an extensible force field. Statistics related to the quality of the parametrization with a focus on experimental validation are presented. Additionally, the parametrization procedure, described fully in the present article in the context of the model systems, pyrrolidine, and 3‐phenoxymethylpyrrolidine will allow users to readily extend the force field to chemical groups that are not explicitly covered in the force field as well as add functional groups to and link together molecules already available in the force field. CGenFF thus makes it possible to perform “all‐CHARMM” simulations on drug‐target interactions thereby extending the utility of CHARMM force fields to medicinally relevant systems.

Synthesis, modeling, and pharmacological evaluation of UMB 425, a mixed μ agonist/δ antagonist opioid analgesic with reduced tolerance liabilities.

Opioid narcotics are used for the treatment of moderate-to-severe pain and primarily exert their analgesic effects through μ receptors. Although traditional μ agonists can cause undesired side effects, including tolerance, addition of δ antagonists can attenuate said side effects. Herein, we report 4a,9-dihydroxy-7a-(hydroxymethyl)-3-methyl-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one (UMB 425) a 5,14-bridged morphinan-based orvinol precursor synthesized from thebaine. Although UMB 425 lacks δ-specific motifs, conformationally sampled pharmacophore models for μ and δ receptors predict it to have efficacy similar to morphine at μ receptors and similar to naltrexone at δ receptors, due to the compound sampling conformations in which the hydroxyl moiety interacts with the receptors similar to orvinols. As predicted, UMB 425 exhibits a mixed μ agonist/δ antagonist profile as determined in receptor binding and [(35)S]GTPγS functional assays in CHO cells. In vivo studies in mice show that UMB 425 displays potent antinociception in the hot plate and tail-flick assays. The antinociceptive effects of UMB 425 are blocked by naloxone, but not by the κ-selective antagonist norbinaltorphimine. During a 6-day tolerance paradigm, UMB 425 maintains significantly greater antinociception compared to morphine. These studies thus indicate that, even in the absence of δ-specific motifs fused to the C-ring, UMB 425 has mixed μ agonist/δ antagonist properties in vitro that translate to reduced tolerance liabilities in vivo.

Molecular Details of the Activation of the μ Opioid Receptor

Molecular details of μ opioid receptor activations were obtained using molecular dynamics simulations of the receptor in the presence of three agonists, three antagonists, and a partial agonist and on the constitutively active T279K mutant. Agonists have a higher probability of direct interactions of their basic nitrogen (N) with Asp147 as compared with antagonists, indicating that direct ligand-Asp147 interactions modulate activation. Medium-size substituents on the basic N of antagonists lead to steric interactions that perturb N-Asp147 interactions, while additional favorable interactions occur with larger basic N substituents, such as in N-phenethylnormorphine, restoring N-Asp147 interactions, leading to agonism. With the orvinols, the increased size of the C19 substituent in buprenorphine over diprenorphine leads to increased interactions with residues adjacent to Asp147, partially overcoming the presence of the cyclopropyl N substituent, such that buprenorphine is a partial agonist. Results also indicate different conformational properties of the intracellular regions of the transmembrane helices in agonists versus antagonists.

Intrinsic energy landscapes of amino acid side chains

Amino acid side-chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side-chain χ(1) and χ(2) torsions in dipeptides in the alphaR, beta, and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side-chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ(1) and χ(2) free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima of the QM surfaces as expected for sp(3) (or sp(2) for χ(2) of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side-chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side-chain energetics can influence backbone preference identified a strong correlation for residues in the alphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the alphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.

Consensus 3D Model of μ-Opioid Receptor Ligand Efficacy Based on a Quantitative Conformationally Sampled Pharmacophore

Despite being studied for over 30 years, a consensus structure-activity relationship (SAR) that encompasses the full range peptidic and nonpeptidic μ-opioid receptor ligands is still not available. To achieve a consensus SAR the Conformationally Sampled Pharmacophore (CSP) method was applied to develop a predictive model of the efficacy of μ-opioid receptor ligands. Emphasis was placed on predicting the efficacy of a wide range of agonists, partial agonists, and antagonists as well as understanding their mode of interaction with the receptor. Inclusion of all accessible conformations of each ligand, a central feature of the CSP method, enabled structural features between diverse μ-opioid receptor ligands that dictate efficacy to be identified. The models were validated against a diverse collection of peptidic and nonpeptidic ligands, including benzomorphans, fentanyl (4-anilinopiperidine), methadone (3,3-diphenylpropylamines), etonitazene (benzimidazole derivatives), funaltrexamine (C6-substituted 4,5-epoxymorphinan), and herkinorin. The model predicts (1) that interactions of ligands with the B site, as with the 19-alkyl substituents of oripavines, modulate the extent of agonism; (2) that agonists with long N-substituents, as with fentanyl and N-phenethylnormorphine, can bind in an orientation such that the N substitutent interacts with the B site that also allows the basic N-receptor Asp interaction essential for agonism; and (3) that the μ agonist herkinorin, that lacks a basic nitrogen, binds to the receptor in a manner similar to the traditional opioids via interactions mediated by water or a ion. Importantly, the proposed CSP model can be reconciled with previously published SAR models for the μ receptor.

(Ala)4-X-(Ala)4 as a model system for the optimization of the χ1 and χ2 amino acid side-chain dihedral empirical force field parameters

Amino acid side‐chain fluctuations play an essential role in the structure and function of proteins. Accordingly, in theoretical studies of proteins, it is important to have an accurate description of their conformational properties. Recently, new side‐chain torsion parameters were introduced into the CHARMM and Amber additive force fields and evaluated based on the conformational properties of the individual side‐chains using protein simulations in explicit solvent. While effective for validation, molecular dynamics simulations of proteins must be extended into the microsecond regime to obtain full convergence of the side‐chain conformations, limiting their use for force field optimization. To address this, we systematically test the utility of explicit solvent simulations of (Ala)4‐X‐(Ala)4 peptides, where X represents the amino acids, as model systems for the optimization of χ1 and χ2 side‐chain parameters. The effect of (Ala)4‐X‐(Ala)4 backbone conformation was tested by constraining the backbone in the α‐helical, C5, C7eq, and PPII conformations and performing exhaustive sampling using Hamiltonian replica exchange simulations. Rotamer distributions from protein and the (Ala)4‐X‐(Ala)4 simulations showed the highest correlation for the C7eq and PPII conformations, although agreement was the best for the α‐helical conformation for Asn. Hydrogen bond analysis indicates the utility of the C7eq and PPII conformations to be due to specific side‐chain‐backbone hydrogen bonds not being oversampled, thereby allowing sampling of a range of side‐chain conformations consistent with the distributions occurring in full proteins. It is anticipated that the (Ala)4‐X‐(Ala)4 model system will allow for iterative force field optimization targeting condensed‐phase conformational distributions of side‐chains.

Identification of an Efficacy Pharmacophore for μ Opioid Receptor Ligands Using the Conformationally Sampled Pharmacophore (CSP) Method

μ opioid receptor agonists and antagonists play a critical role in the treatment of severe pain and as treatments for drug abuse. Their structure activity relationships (SAR) have been extensively investigated followed by lead optimization. However, challenges remain in improving the utility of m ligands with respect to reducing adverse effects such as tolerance, dependence, constipation, etc. while maintaining the potency of current medicines. To facilitate the meeting of these challenges, consensus pharmacophore models for diverse classes of μ opioid receptor ligands were established and we constructed predictive model differentiating agonists and antagonists activities using the conformationally sampled pharmacophore (CSP) method. The predictability of the models was validated against a number of classes of opioids including 4,5-Epoxymorphinans, Diels-Alder adducts of thebaine, Benzomorphans, Methadone, Fentanyl, and, notably, non-nitrogenous opioids; a collection of compounds which have eluded a consensus SAR of opioids for decades. The consensus model was derived by virtue of CSP method incorporating flexibility of molecules through using all-accessible conformers in model development and by eliminating any limitations associated with alignment procedures. The procedures to develop and features of the CSP model will be presented.

Metadynamics-Based Mechanistic Interpretation of Functional Crosstalk Between Serotonin 2A and Metabotropic Glutamate 2 receptors

Serotonin 2A (2AR) and metabotropic glutamate 2 (mGluR2) receptors have been shown to form a functional and specific heteromeric complex in mammalian brain and in tissue culture preparations with possible implications in the psychotic symptoms of schizophrenia. Unlike non-antipsychotic drugs, clinically effective atypical antipsychotics (e.g., clozapine) that bind to 2AR increase the glutamate-mediated Gi signaling through the 2AR/mGluR2 heterodimer. The molecular mechanisms underlying these allosteric effects and functional crosstalk are unknown. Using molecular dynamics (MD) simulations enhanced with metadynamics, we investigated at the molecular level the conformational changes induced by atypical antipsychotic or non-antipsychotic 2AR ligands in atomistic representations of interacting 2AR and mGluR2 embedded in an explicit lipid-water environment. First, we sampled the conformational transitions from inactive to activated (opsin-like) models of the ligand-free transmembrane regions of 2AR or mGluR2 with adiabatic biased MD simulations. We then reconstructed the free-energy landscape of the 2AR/mGluR2 heterodimer along the pre-determined transition trajectories in the presence of ligands, using a path collective variable approach based on metadynamics. The CHARMM force-field with the CMAP backbone energy correction was used to describe the full systems. All calculations were performed using NAMD enhanced with the Plumed plug-in. Our results suggest that the conformational transitions of 2AR and mGluR2 are populated by different inactive and active metastable states of the receptors, which are differentially stabilized by antipsychotic and non-antipsychotic ligands.

Analysis of Amino Acid Sidechain Chi1, Chi2 Conformational Properties Using Quantum Mechanical and Experimental Crystallographic Survey Data

Amino acid side chain flexibility is an important property that influences the side-chain interactions in proteins as well as protein stability. In molecular mechanics, the conformational properties of sidechains can beis modulated, in part, by torsional parameters. In this study, we analyze the conformational properties of sidechains via quantum mechanical calculations. One and two- dimensional chi energy surfaces were performed on dipeptides representative of the amino acids. Analysis was performed for relevant peptide backbone conformations corresponding to the alpha helical (alpha R), beta stranded (extended) and alpha L conformations. Calculated QM energy surfaces are indicative of the conformational properties of the different amino acid sidechains and were used as target data for optimization of the CHARMM additive and polarizable force fields optimization as well as a the basis for explaining experimental observations the sampling of sidechain conformations in protein crystallographic structures.