The Impacts of the Molecular Education and Research Consortium in Undergraduate Computational Chemistry on the Careers of Women in Computational Chemistry.

The Molecular Education and Research Consortium in Undergraduate Computational Chemistry (MERCURY) has supported a diverse group of faculty and students for over 20 years by providing computational resources as well as networking opportunities and professional support. The consortium comprises 38 faculty (42% women) at 34 different institutions, who have trained nearly 900 undergraduate students, more than two-thirds of whom identify as women and one-quarter identify as students of color. MERCURY provides a model for the support necessary for faculty to achieve professional advancement and career satisfaction. The range of experiences and expertise of the consortium members provides excellent networking opportunities that allow MERCURY faculty to support each other's teaching, research, and service needs, including generating meaningful scientific advancements and outcomes with undergraduate researchers as well as being leaders at the departmental, institutional, and national levels. While all MERCURY faculty benefit from these supports, the disproportionate number of women in the consortium, relative to their representation in computational sciences generally, produces a sizable impact on advancing women in the computational sciences. In this report, the women of MERCURY share how the consortium has benefited their careers and the careers of their students.

Characterization of Subtype Selective Cannabinoid CB2 Receptor Agonists as Potential Anti-Inflammatory Agents.

Activation of the CB2 receptor has been shown to have anti-inflammatory and antinociceptive effects without causing psychoactive effects. Previously, we reported that the compound ethyl 2(2-(N-(2,3-dimethylphenyl) phenylsulfonamido)acetamido)benzoate (ABK5) is a CB2 subtype selective agonist with anti-inflammatory and antinociceptive effects. In the present study, we tested four ABK5 derivatives, ABK5-1, ABK5-2, ABK5-5, and ABK5-6, to analyze the structure of ABK5 to obtain CB2-selective agonists with higher affinity and efficacy. Affinity, subtype selectivity, and G-protein coupling were determined by radioligand binding assays. Selected compounds were then subjected to evaluation of anti-inflammatory effects using two different cell lines, Jurkat (ABK5-1 and 5-2) and BV-2 cells (ABK5-1), which are models of T cells and microglia, respectively. ABK5-1, ABK5-2, and ABK5-6 had comparable CB2 binding affinity with ABK5 (and stimulated G-protein coupling), while only ABK5-1 and ABK5-2 maintained CB2-subtype selectivity. ABK5-5 did not bind CB2 in the detectable range. RT-PCR and ELISA analysis showed that the two compounds also inhibit IL-2 and TNF-α production, and they were more efficacious than ABK5 in inhibiting TNF-α production. CXCL-12 mediated chemotaxis was also evaluated by the transwell migration assay, and both ABK5-1 and ABK5-2 inhibited chemotaxis with a stronger effect observed in ABK5-1. In the microglia cell line BV-2, ABK5-1 inhibited IL-1ß and IL-6 production, which suggests this compound has anti-inflammatory effects through targeting multiple immune cells, and may be a candidate for treatment of inflammation.

Identification and biochemical analyses of selective CB2 agonists.

Cannabinoid CB1 and CB2 receptors are activated by Δ9-tetrahydrocannabinol, a psychoactive component of marijuana. The cannabinoid CB1 receptor is primarily located in the brain and is responsible for the psychoactive side effects, whereas the cannabinoid CB2 receptor is located in immune cells and is an attractive target for immune-related maladies. We identify small molecules that selectively bind to the cannabinoid CB2 receptor and can be further developed into therapeutics. The affinity of three molecules, ABK5, ABK6, and ABK7, to the cannabinoid CB2 receptor was determined with radioligand competition binding. The potency of G-protein coupling was determined with GTPγS binding. The three compounds bound selectively to the cannabinoid CB2 receptor, and no binding to the cannabinoid CB1 receptor was detected up to 10 µM. Immunoblotting studies show that the amount of ERK1/2 and MEK phosphorylation increased in a Gi/o-dependent manner. Furthermore, an immune cell line (Jurkat cells) was treated with ABK5, and as a result, inhibited cell proliferation. These three compounds are novel cannabinoid CB2 receptor agonists and hold promise to be further developed to treat inflammation and the often-associated pain.

Pyrimidinyl Biphenylureas Act as Allosteric Modulators to Activate Cannabinoid Receptor 1 and Initiate ß-Arrestin-Dependent Responses.

Cannabinoid receptor 1 (CB1) is a G-protein-coupled receptor that is abundant in the central nervous system. It binds several compounds in its orthosteric site, including the endocannabinoids, arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, and the plant-derived Δ9-tetrahydrocannabinol, one of the main psychoactive components of marijuana. It primarily couples to Gi/o proteins to inhibit adenylate cyclase activity and typically induces downstream signaling that is Gi-dependent. Since this receptor is implicated in several maladies, such as obesity, pain, and neurodegenerative disorders, there is interest in developing therapeutics that selectively target this receptor. Allosteric modulators of CB1 offer one new approach that has tremendous therapeutic potential. Here, we reveal receptor- and cellular-level properties consistent with receptor activation by a series of pyrimidinyl biphenylureas (LDK1285, LDK1288, LDK1305, and PSNCBAM1), including promoting binding of the agonist CP55940 with positive cooperativity and inhibiting binding of the inverse agonist SR141716A with negative cooperativity, demonstrated via radioligand binding studies. Consistent with these findings, the allosteric modulators induced cellular internalization of the receptor and recruitment of ß-arrestin 2 in human embryonic kidney cell line 293 cells monitored with confocal and total internal reflective fluorescence microscopy, respectively. These allosteric modulators, however, caused G-protein-independent but ß-arrestin 1-dependent phosphorylation of the downstream kinases extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Src, shown by immunoblotting studies. These results are consistent with the involvement of ß-arrestin and suggest that these allosteric modulators induce biased signaling.

Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes.

Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have been associated with calcium dysregulation. In the heart, accumulating evidence suggests that human amylin oligomers form moderately cation-selective [4,5] channels that embed in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentration-dependent manner [5], which is correlated with elevated cytosolic Ca2+. These findings motivate our core hypothesis that non-selective inward Ca2+ conduction afforded by human amylin oligomers increase cytosolic and sarcoplasmic reticulum (SR) Ca2+ load, which thereby magnifies intracellular Ca2+ transients. Questions remain however regarding the mechanism of amylin-induced Ca2+ dysregulation, including whether enhanced SL Ca2+ influx is sufficient to elevate cytosolic Ca2+ load [6], and if so, how might amplified Ca2+ transients perturb Ca2+-dependent cardiac pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca2+ signaling to reflect experimentally-measured changes in SL membrane permeation and decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from acute and transgenic human amylin peptide exposure. With this model, we confirmed the hypothesis that increasing SL permeation alone was sufficient to enhance Ca2+ transient amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca2+ loading of the SR and that greater fractional release may contribute to the Ca2+-dependent activation of calmodulin, which could prime the activation of myocyte remodeling pathways. Importantly, elevated Ca2+ in the SR and dyadic space collectively drive greater fractional SR Ca2+ release for human amylin expressing rats (HIP) and acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca2+ transients. These findings suggest that increased membrane permeation induced by oligomeratization of amylin peptide in cell sarcolemma contributes to Ca2+ dysregulation in pre-diabetes.

Discovery of Selective Cannabinoid CB2 Receptor Agonists by High-Throughput Screening.

The endocannabinoid system (ECS) plays a diverse role in human physiology ranging from the regulation of mood and appetite to immune modulation and the response to pain. Drug development that targets the cannabinoid receptors (CB1 and CB2) has been explored; however, success in the clinic has been limited by the psychoactive side effects associated with modulation of the neuronally expressed CB1 that are enriched in the CNS. CB2, however, are expressed in peripheral tissues, primarily in immune cells, and thus development of CB2-selective drugs holds the potential to modulate pain among other indications without eliciting anxiety and other undesirable side effects associated with CB1 activation. As part of a collaborative effort among industry and academic laboratories, we performed a high-throughput screen designed to discover selective agonists or positive allosteric modulators (PAMs) of CB2. Although no CB2 PAMs were identified, 167 CB2 agonists were discovered here, and further characterization of four select compounds revealed two with high selectivity for CB2 versus CB1. These results broaden drug discovery efforts aimed at the ECS and may lead to the development of novel therapies for immune modulation and pain management with improved side effect profiles.

Assessing Allosteric Modulation of CB1 at the Receptor and Cellular Levels.

The cannabinoid CB1 receptor is abundant in the central nervous system and regulates neuronal transmission and other key physiological processes including those leading to pain, inflammation, memory, and feeding behavior. CB1 is activated by the endogenous ligands, arachidonoyl ethanolamine and 2-arachidonoyl glycerol, by various synthetic ligands (e.g., CP55940), and by Δ9-tetrahydrocannabinol, the psychoactive component of Cannabis sativa. These CB1 ligands are orthosteric and transduce downstream signals by binding CB1 and primarily inducing Gi coupling, but Gs and ß-arrestin coupling are also possible. Recently, allosteric modulators for CB1 were discovered that bind to topographically distinct sites and can noncompetitively impact the potency and efficacy of orthosteric compounds. These offer the exciting potential for mechanistic analyses and for developing therapeutics. Yet, it is critical to elucidate whether a compound is a positive allosteric modulator or a negative allosteric modulator of orthosteric ligand-induced CB1 profiles to understand pathway specificity and ameliorate diseases. In this chapter, we present equilibrium and kinetic binding analysis to reveal the impact of allosteric modulators on CB1. Also described are activities consistent with CB1 activation (or inactivation) and include cellular internalization of CB1 and downstream signaling patterns. Since many CB1 allosteric modulators do not enhance G protein coupling, it is critical to distinguish CB1 activation and biased signaling patterns via ß-arrestin from CB1 inactivation. These strategies can illuminate pathway specificity and are valuable for the fine-tuning of CB1 function.

Understanding Ion Binding Affinity and Selectivity in ß-Parvalbumin Using Molecular Dynamics and Mean Spherical Approximation Theory.

Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat ß-parvalbumin (ß-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the ß-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound ß-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of PV sequence homologues and EF-hand-containing, Ca(2+) binding proteins.

Quantifying the Influence of the Crowded Cytoplasm on Small Molecule Diffusion.

Cytosolic crowding can influence the thermodynamics and kinetics of in vivo chemical reactions. Most significantly, proteins and nucleic acid crowders reduce the accessible volume fraction, ϕ, available to a diffusing substrate, thereby reducing its effective diffusion rate, Deff, relative to its rate in bulk solution. However, Deff can be further hindered or even enhanced, when long-range crowder/diffuser interactions are significant. To probe these effects, we numerically estimated Deff values for small, charged molecules in representative, cytosolic protein lattices up to 0.1 × 0.1 × 0.1 µm(3) in volume via the homogenized Smoluchowski electro-diffusion equation. We further validated our predictions against Deff estimates from ϕ-dependent analytical relationships, such as the Maxwell-Garnett (MG) bound, as well as explicit solutions of the time-dependent electro-diffusion equation. We find that in typical, moderately crowded cell cytoplasm (ϕ ≈ 0.8), Deff is primarily determined by ϕ; in other words, diverse protein shapes and heterogeneous distributions only modestly impact Deff. However, electrostatic interactions between diffusers and crowders, particularly at low electrolyte ionic strengths, can substantially modulate Deff. These findings help delineate the extent that cytoplasmic crowders influence small molecule diffusion, which ultimately may shape the efficiency and timing of intracellular signaling pathways. More generally, the quantitative agreement between computationally expensive solutions of the time-dependent electro-diffusion equation and its comparatively cheaper homogenized form suggest that the latter is a broadly effective model for diffusion in wide-ranging, crowded biological media.

Molecular Basis of S100A1 Activation at Saturating and Subsaturating Calcium Concentrations.

The S100A1 protein mediates a wide variety of physiological processes through its binding of calcium (Ca(2+)) and endogenous target proteins. S100A1 presents two Ca(2+)-binding domains: a high-affinity "canonical" EF (cEF) hand and a low-affinity "pseudo" EF (pEF) hand. Accumulating evidence suggests that both Ca(2+)-binding sites must be saturated to stabilize an open state conducive to peptide recognition, yet the pEF hand's low affinity limits Ca(2+) binding at normal physiological concentrations. To understand the molecular basis of Ca(2+) binding and open-state stabilization, we performed 100 ns molecular dynamics simulations of S100A1 in the apo/holo (Ca(2+)-free/bound) states and a half-saturated state, for which only the cEF sites are Ca(2+)-bound. Our simulations indicate that the pattern of oxygen coordination about Ca(2+) in the cEF relative to the pEF site contributes to the former's higher affinity, whereas Ca(2+) binding strongly reshapes the protein's conformational dynamics by disrupting ß-sheet coupling between EF hands. Moreover, modeling of the half-saturated configuration suggests that the open state is unstable and reverts toward a closed state in the absence of the pEF Ca(2+) ion. These findings indicate that Ca(2+) binding at the cEF site alone is insufficient to stabilize opening; thus, posttranslational modification of the protein may be required for target peptide binding at subsaturating intracellular Ca(2+) levels.

Computational Prediction and Biochemical Analyses of New Inverse Agonists for the CB1 Receptor.

Human cannabinoid type 1 (CB1) G-protein coupled receptor is a potential therapeutic target for obesity. The previously predicted and experimentally validated ensemble of ligand-free conformations of CB1 [Scott, C. E. et al. Protein Sci. 2013 , 22 , 101 - 113 ; Ahn, K. H. et al. Proteins 2013 , 81 , 1304 - 1317] are used here to predict the binding sites for known CB1-selective inverse agonists including rimonabant and its seven known derivatives. This binding pocket, which differs significantly from previously published models, is used to identify 16 novel compounds expected to be CB1 inverse agonists by exploiting potential new interactions. We show experimentally that two of these compounds exhibit inverse agonist properties including inhibition of basal and agonist-induced G-protein coupling activity, as well as an enhanced level of CB1 cell surface localization. This demonstrates the utility of using the predicted binding sites for an ensemble of CB1 receptor structures for designing new CB1 inverse agonists.

SuperBiHelix method for predicting the pleiotropic ensemble of G-protein-coupled receptor conformations.

There is overwhelming evidence that G-protein-coupled receptors (GPCRs) exhibit several distinct low-energy conformations, each of which might favor binding to different ligands and/or lead to different downstream functions. Understanding the function of such proteins requires knowledge of the ensemble of low-energy configurations that might play a role in this pleiotropic functionality. We earlier reported the BiHelix method for efficiently sampling the (12)(7) = 35 million conformations resulting from 30° rotations about the axis (η) of all seven transmembrane helices (TMHs), showing that the experimental structure is reliably selected as the best conformation from this ensemble. However, various GPCRs differ sufficiently in the tilts of the TMHs that this method need not predict the optimum conformation starting from any other template. In this paper, we introduce the SuperBiHelix method in which the tilt angles (θ, ϕ) are optimized simultaneously with rotations (η) efficiently enough that it is practical and sufficient to sample (5 × 3 × 5)(7) = 13 trillion configurations. This method can correctly identify the optimum structure of a GPCR starting with the template from a different GPCR. We have validated this method by predicting known crystal structure conformations starting from the template of a different protein structure. We find that the SuperBiHelix conformational ensemble includes the higher energy conformations associated with the active protein in addition to those associated with the more stable inactive protein. This methodology was then applied to design and experimentally confirm structures of three mutants of the CB1 cannabinoid receptor associated with different functions.

Computationally-predicted CB1 cannabinoid receptor mutants show distinct patterns of salt-bridges that correlate with their level of constitutive activity reflected in G protein coupling levels, thermal stability, and ligand binding.

The cannabinoid receptor 1 (CB1), a member of the class A G-protein-coupled receptor (GPCR) family, possesses an observable level of constitutive activity. Its activation mechanism, however, has yet to be elucidated. Previously we discovered dramatic changes in CB1 activity due to single mutations; T3.46A, which made the receptor inactive, and T3.46I and L3.43A, which made it essentially fully constitutively active. Our subsequent prediction of the structures of these mutant receptors indicated that these changes in activity are explained in terms of the pattern of salt-bridges in the receptor region involving transmembrane domains 2, 3, 5, and 6. Here we identified key salt-bridges, R2.37 + D6.30 and D2.63 + K3.28, critical for CB1 inactive and active states, respectively, and generated new mutant receptors that we predicted would change CB1 activity by either precluding or promoting these interactions. We find that breaking the R2.37 + D6.30 salt-bridge resulted in substantial increase in G-protein coupling activity and reduced thermal stability relative to the wild-type reflecting the changes in constitutive activity from inactive to active. In contrast, breaking the D2.63 + K3.28 salt-bridge produced the opposite profile suggesting this interaction is critical for the receptor activation. Thus, we demonstrate an excellent correlation with the predicted pattern of key salt-bridges and experimental levels of activity and conformational flexibility. These results are also consistent with the extended ternary complex model with respect to shifts in agonist and inverse agonist affinity and provide a powerful framework for understanding the molecular basis for the multiple stages of CB1 activation and that of other GPCRs in general.

Molecular basis for dramatic changes in cannabinoid CB1 G protein-coupled receptor activation upon single and double point mutations.

There is considerable interest in determining the activation mechanism of G protein-coupled receptors (GPCRs), one of the most important types of proteins for intercellular signaling. Recently, it was demonstrated for the cannabinoid CB1 GPCR, that a single mutation T210A could make CB1 completely inactive whereas T210I makes it essentially constitutively active. To obtain an understanding of this dramatic dependence of activity on mutation, we used first-principles-based methods to predict the ensemble of low-energy seven-helix conformations for the wild-type (WT) and mutants (T210A and T210I). We find that the transmembrane (TM) helix packings depend markedly on these mutations, leading for T210A to both TM3+TM6 and TM2+TM6 salt-bridge couplings in the cytoplasmic face that explains the inactivity of this mutant. In contrast T210I has no such couplings across the receptor explaining the ease in activating this mutant. WT has just the TM3+TM6 coupling, known to be broken upon GPCR activation. To test this hypothesis on activity, we predicted double mutants that would convert the inactive mutant to normal activity and then confirmed this experimentally. This CB1 activation mechanism, or one similar to it, is expected to play a role in other constitutively active GPCRs as well.