Christopher G. Mayne

Rapid parameterization of small molecules using the force field toolkit

The inability to rapidly generate accurate and robust parameters for novel chemical matter continues to severely limit the application of molecular dynamics simulations to many biological systems of interest, especially in fields such as drug discovery. Although the release of generalized versions of common classical force fields, for example, General Amber Force Field and CHARMM General Force Field, have posited guidelines for parameterization of small molecules, many technical challenges remain that have hampered their wide‐scale extension. The Force Field Toolkit (ffTK), described herein, minimizes common barriers to ligand parameterization through algorithm and method development, automation of tedious and error‐prone tasks, and graphical user interface design. Distributed as a VMD plugin, ffTK facilitates the traversal of a clear and organized workflow resulting in a complete set of CHARMM‐compatible parameters. A variety of tools are provided to generate quantum mechanical target data, setup multidimensional optimization routines, and analyze parameter performance. Parameters developed for a small test set of molecules using ffTK were comparable to existing CGenFF parameters in their ability to reproduce experimentally measured values for pure‐solvent properties (<15% error from experiment) and free energy of solvation (±0.5 kcal/mol from experiment).

1,25-Dihydroxyvitamin D3 acts directly on the T lymphocyte vitamin D receptor to inhibit experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an incurable autoimmune neurodegenerative disease. Environmental factors may be key to MS prevention and treatment. MS prevalence and severity decrease with increasing sunlight exposure and vitamin D3 supplies, supporting our hypothesis that the sunlight‐dependent hormone, 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3), inhibits autoimmune T‐cell responses in MS. Moreover, 1,25‐(OH)2D3 inhibits and reverses experimental autoimmune encephalomyelitis (EAE), an MS model. Here, we investigated whether 1,25‐(OH)2D3 inhibits EAE via the vitamin D receptor (VDR) in T lymphocytes. Using bone marrow chimeric mice with a disrupted VDR only in radio‐sensitive hematopoietic cells or radio‐resistant non‐hematopoietic cells, we found that hematopoietic cell VDR function was necessary for 1,25‐(OH)2D3 to inhibit EAE. Furthermore, conditional targeting experiments showed that VDR function in T cells was necessary. Neither 1,25‐(OH)2D3 nor T‐cell‐specific VDR targeting influenced CD4+Foxp3+ T‐cell proportions in the periphery or the CNS in these studies. These data support a model wherein 1,25‐(OH)2D3 acts directly on pathogenic CD4+ T cells to inhibit EAE.

Minireview: Not Picking Pockets: Nuclear Receptor Alternate-Site Modulators (NRAMs)

Because of their central importance in gene regulation and mediating the actions of many hormones, the nuclear receptors (NRs) have long been recognized as very important biological and pharmaceutical targets. Of all the surfaces available on a given NR, the singular site for regulation of receptor activity has almost invariably been the ligand-binding pocket of the receptor, the site where agonists, antagonists, and selective NR modulators interact. With our increasing understanding of the multiple molecular components involved in NR action, researchers have recently begun to look to additional interaction sites on NRs for regulating their activities by novel mechanisms. The alternate NR-associated interaction sites that have been targeted include the coactivator-binding groove and allosteric sites in the ligand-binding domain, the zinc fingers of the DNA-binding domain, and the NR response element in DNA. The studies thus far have been performed with the estrogen receptors, the androgen receptor (AR), the thyroid hormone receptors, and the pregnane X receptor. Phenotypic and conformation-based screens have also identified small molecule modulators that are believed to function through the NRs but have, as yet, unknown sites and mechanisms of action. The rewards from investigation of these NR alternate-site modulators should be the discovery of new therapeutic approaches and novel agents for regulating the activities of these important NR proteins.

Induced and Natural Regulatory T Cells in the Development of Inflammatory Bowel Disease

Abstract: The mucosal immune system mediates contact between the host and the trillions of microbes that symbiotically colonize the gastrointestinal tract. Failure to tolerate the antigens within this “extended self” can result in inflammatory bowel disease (IBD). Within the adaptive immune system, the most significant cells modulating this interaction are Foxp3+ regulatory T (Treg) cells. Treg cells can be divided into 2 primary subsets: “natural” Treg cells and “adaptive” or “induced” Treg. Recent research suggests that these subsets serve to play both independent and synergistic roles in mucosal tolerance. Studies from both mouse models and human patients suggest that defects in Treg cells can play distinct causative roles in IBD. Numerous genetic, microbial, nutritional, and environmental factors that associate with IBD may also affect Treg cells. In this review, we summarize the development and function of Treg cells and how their regulatory mechanisms may fail, leading to a loss of mucosal tolerance. We discuss both animal models and studies of patients with IBD suggesting Treg cell involvement in IBD and consider how Treg cells may be used in future therapies.

Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation

Somatic mutations in the estrogen receptor alpha (ERα) gene (ESR1), especially Y537S and D538G, have been linked to acquired resistance to endocrine therapies. Cell-based studies demonstrated that these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance. Here, we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα. We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist (estradiol) and antagonist (4-hydroxytamoxifen) are reduced. Further, they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα, which leads to a stabilized agonist state and an altered antagonist state that resists inhibition.

Flexibility Coexists with Shape-Persistence in Cyanostar Macrocycles

Shape-persistent macrocycles are attractive functional targets for synthesis, molecular recognition, and hierarchical self-assembly. Such macrocycles are noncollapsible and geometrically well-defined, and they are traditionally characterized by having repeat units and low conformational flexibility. Here, we find it necessary to refine these ideas in the face of highly flexible yet shape-persistent macrocycles. A molecule is shape-persistent if it has a small change in shape when perturbed by external stimuli (e.g., heat, light, and redox chemistry). In support of this idea, we provide the first examination of the relationships between a macrocycles shape persistence, its conformational space, and the resulting functions. We do this with a star-shaped macrocycle called cyanostar that is flexible as well as being shape-persistent. We employed molecular dynamics (MD), density functional theory (DFT), and NMR experiments. Considering a thermal bath as a stimulus, we found a single macrocycle has 332 accessible conformers with olefins undergoing rapid interconversion by up-down and in-out motions on short time scales (0.2 ns). These many interconverting conformations classify single cyanostars as flexible. To determine and confirm that cyanostars are shape-persistent, we show that they have a high 87% shape similarity across these conformations. To further test the idea, we use the binding of diglyme to the single macrocycle as guest-induced stimulation. This guest has almost no effect on the conformational space. However, formation of a 2:1 sandwich complex involving two macrocycles enhances rigidity and dramatically shifts the conformer distribution toward perfect bowls. Overall, the present study expands the scope of shape-persistent macrocycles to include flexible macrocycles if, and only if, their conformers have similar shapes.

Imaging progesterone receptor in breast tumors: synthesis and receptor binding affinity of fluoroalkyl-substituted analogues of tanaproget.

The progesterone receptor (PR) is estrogen regulated, and PR levels in breast tumors can be used to predict the success of endocrine therapies targeting the estrogen receptor (ER). Tanaproget is a nonsteroidal progestin agonist with very high PR binding affinity and excellent in vivo potency. When appropriately radiolabeled, it might be used to image PR-positive breast tumors noninvasively by positron emission tomography (PET). We describe the synthesis and PR binding affinities of a series of fluoroalkyl-substituted 6-aryl-1,4-dihydrobenzo[d][1,3]oxazine-2-thiones, analogues of Tanaproget. Some of these compounds have subnanomolar binding affinities, higher than that of either Tanaproget itself or the high affinity PR ligand R5020. Structure-binding affinity relationships can be rationalized by molecular modeling of ligand complexes with PR, and the enantioselectivity of binding has been predicted. These compounds are being further evaluated as potential diagnostic PET imaging agents for breast cancer, and enantiomerically pure materials of defined stereochemistry are being prepared.

Design of pathway preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues

Structurally redesigned estrogens improve metabolism and vascular repair without activating cell proliferation. Designing better estrogens Estrogen and synthetic versions can enhance the repair of blood vessels after injury or improve metabolism in the liver and adipose tissue, but they can also cause breast or uterine cancer, because they stimulate cell proliferation in these reproductive tissues. Madak-Erdogan et al. designed estrogen-like molecules that had reduced receptor affinity and that did not enhance ductal mammary gland branching (a sign of mammary gland growth) or increase uterine weight in ovariectomized mice. These estrogens provided vascular and metabolic benefits and thus could be further developed as postmenopausal hormone replacement therapies. There is great medical need for estrogens with favorable pharmacological profiles that support desirable activities for menopausal women, such as metabolic and vascular protection, but that lack stimulatory activities on the breast and uterus. We report the development of structurally novel estrogens that preferentially activate a subset of estrogen receptor (ER) signaling pathways and result in favorable target tissue–selective activity. Through a process of structural alteration of estrogenic ligands that was designed to preserve their essential chemical and physical features but greatly reduced their binding affinity for ERs, we obtained “pathway preferential estrogens” (PaPEs), which interacted with ERs to activate the extranuclear-initiated signaling pathway preferentially over the nuclear-initiated pathway. PaPEs elicited a pattern of gene regulation and cellular and biological processes that did not stimulate reproductive and mammary tissues or breast cancer cells. However, in ovariectomized mice, PaPEs triggered beneficial responses both in metabolic tissues (adipose tissue and liver) that reduced body weight gain and fat accumulation and in the vasculature that accelerated repair of endothelial damage. This process of designed ligand structure alteration represents a novel approach to develop ligands that shift the balance in ER-mediated extranuclear and nuclear pathways to obtain tissue-selective, non-nuclear PaPEs, which may be beneficial for postmenopausal hormone replacement. The approach may also have broad applicability for other members of the nuclear hormone receptor superfamily.

All-Atom Molecular Dynamics of Virus Capsids as Drug Targets

Virus capsids are protein shells that package the viral genome. Although their morphology and biological functions can vary markedly, capsids often play critical roles in regulating viral infection pathways. A detailed knowledge of virus capsids, including their dynamic structure, interactions with cellular factors, and the specific roles that they play in the replication cycle, is imperative for the development of antiviral therapeutics. The following Perspective introduces an emerging area of computational biology that focuses on the dynamics of virus capsids and capsid–protein assemblies, with particular emphasis on the effects of small-molecule drug binding on capsid structure, stability, and allosteric pathways. When performed at chemical detail, molecular dynamics simulations can reveal subtle changes in virus capsids induced by drug molecules a fraction of their size. Here, the current challenges of performing all-atom capsid–drug simulations are discussed, along with an outlook on the applicability of virus capsid simulations to reveal novel drug targets.

Macromolecular Crystallography for Synthetic Abiological Molecules: Combining xMDFF and PHENIX for Structure Determination of Cyanostar Macrocycles

Crystal structure determination has long provided insight into structure and bonding of small molecules. When those same small molecules are designed to come together in multimolecular assemblies, such as in coordination cages, supramolecular architectures and organic-based frameworks, their crystallographic characteristics closely resemble biological macromolecules. This resemblance suggests that biomacromolecular refinement approaches be used for structure determination of abiological molecular complexes that arise in an aggregate state. Following this suggestion we investigated the crystal structure of a pentagonal macrocycle, cyanostar, by means of biological structure analysis methods and compared results to traditional small molecule methods. Cyanostar presents difficulties seen in supramolecular crystallography including whole molecule disorder and highly flexible solvent molecules sitting in macrocyclic and intermolecule void spaces. We used the force-field assisted refinement method, molecular dynamics flexible fitting algorithm for X-ray crystallography (xMDFF), along with tools from the macromolecular structure determination suite PHENIX. We found that a standard implementation of PHENIX, namely one without xMDFF, either fails to produce a solution by molecular replacement alone or produces an inaccurate structure when using generic geometry restraints, even at a very high diffraction data resolution of 0.84 Å. The problems disappear when taking advantage of xMDFF, which applies an optimized force field to realign molecular models during phasing by providing accurate restraints. The structure determination for this model system shows excellent agreement with the small-molecule methods. Therefore, the joint xMDFF-PHENIX refinement protocol provides a new strategy that uses macromolecule methods for structure determination of small molecules and their assemblies.