In silico identification of a ß2-adrenoceptor allosteric site that selectively augments canonical ß2AR-Gs signaling and function.

Activation of ß2-adrenoceptors (ß2ARs) causes airway smooth muscle (ASM) relaxation and bronchodilation, and ß2AR agonists (ß-agonists) are front-line treatments for asthma and other obstructive lung diseases. However, the therapeutic efficacy of ß-agonists is limited by agonist-induced ß2AR desensitization and noncanonical ß2AR signaling involving ß-arrestin that is shown to promote asthma pathophysiology. Accordingly, we undertook the identification of an allosteric site on ß2AR that could modulate the activity of ß-agonists to overcome these limitations. We employed the site identification by ligand competitive saturation (SILCS) computational method to comprehensively map the entire 3D structure of in silico-generated ß2AR intermediate conformations and identified a putative allosteric binding site. Subsequent database screening using SILCS identified drug-like molecules with the potential to bind to the site. Experimental assays in HEK293 cells (expressing recombinant wild-type human ß2AR) and human ASM cells (expressing endogenous ß2AR) identified positive and negative allosteric modulators (PAMs and NAMs) of ß2AR as assessed by regulation of ß-agonist-stimulation of cyclic AMP generation. PAMs/NAMs had no effect on ß-agonist-induced recruitment of ß-arrestin to ß2AR- or ß-agonist-induced loss of cell surface expression in HEK293 cells expressing ß2AR. Mutagenesis analysis of ß2AR confirmed the SILCS identified site based on mutants of amino acids R131, Y219, and F282. Finally, functional studies revealed augmentation of ß-agonist-induced relaxation of contracted human ASM cells and bronchodilation of contracted airways. These findings identify a allosteric binding site on the ß2AR, whose activation selectively augments ß-agonist-induced Gs signaling, and increases relaxation of ASM cells, the principal therapeutic effect of ß-agonists.

GPU-specific algorithms for improved solute sampling in grand canonical Monte Carlo simulations.

The Grand Canonical Monte Carlo (GCMC) ensemble defined by the excess chemical potential, µex , volume, and temperature, in the context of molecular simulations allows for variations in the number of particles in the system. In practice, GCMC simulations have been widely applied for the sampling of rare gasses and water, but limited in the context of larger molecules. To overcome this limitation, the oscillating µex GCMC method was introduced and shown to be of utility for sampling small solutes, such as formamide, propane, and benzene, as well as for ionic species such as monocations, acetate, and methylammonium. However, the acceptance of GCMC insertions is low, and the method is computationally demanding. In the present study, we improved the sampling efficiency of the GCMC method using known cavity-bias and configurational-bias algorithms in the context of GPU architecture. Specifically, for GCMC simulations of aqueous solution systems, the configurational-bias algorithm was extended by applying system partitioning in conjunction with a random interval extraction algorithm, thereby improving the efficiency in a highly parallel computing environment. The method is parallelized on the GPU using CUDA and OpenCL, allowing for the code to run on both Nvidia and AMD GPUs, respectively. Notably, the method is particularly well suited for GPU computing as the large number of threads allows for simultaneous sampling of a large number of configurations during insertion attempts without additional computational overhead. In addition, the partitioning scheme allows for simultaneous insertion attempts for large systems, offering considerable efficiency. Calculations on the BK Channel, a transporter, including a lipid bilayer with over 760,000 atoms, show a speed up of ~53-fold through the use of system partitioning. The improved algorithm is then combined with an enhanced µex oscillation protocol and shown to be of utility in the context of the site-identification by ligand competitive saturation (SILCS) co-solvent sampling approach as illustrated through application to the protein CDK2.

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism.

Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO3-, CO32-, and Cl- necessary for pH regulation, transepithelial H+/base transport, and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl-/HCO3- exchange versus Na+-CO32- symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein-ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32- binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.

Specificity of Molecular Fragments Binding to S100B versus S100A1 as Identified by NMR and Site Identification by Ligand Competitive Saturation (SILCS).

S100B, a biomarker of malignant melanoma, interacts with the p53 protein and diminishes its tumor suppressor function, which makes this S100 family member a promising therapeutic target for treating malignant melanoma. However, it is a challenge to design inhibitors that are specific for S100B in melanoma versus other S100-family members that are important for normal cellular activities. For example, S100A1 is most similar in sequence and structure to S100B, and this S100 protein is important for normal skeletal and cardiac muscle function. Therefore, a combination of NMR and computer aided drug design (CADD) was used to initiate the design of specific S100B inhibitors. Fragment-based screening by NMR, also termed "SAR by NMR," is a well-established method, and was used to examine spectral perturbations in 2D [1H, 15N]-HSQC spectra of Ca2+-bound S100B and Ca2+-bound S100A1, side-by-side, and under identical conditions for comparison. Of the 1000 compounds screened, two were found to be specific for binding Ca2+-bound S100A1 and four were found to be specific for Ca2+-bound S100B, respectively. The NMR spectral perturbations observed in these six data sets were then used to model how each of these small molecule fragments showed specificity for one S100 versus the other using a CADD approach termed Site Identification by Ligand Competitive Saturation (SILCS). In summary, the combination of NMR and computational approaches provided insight into how S100A1 versus S100B bind small molecules specifically, which will enable improved drug design efforts to inhibit elevated S100B in melanoma. Such a fragment-based approach can be used generally to initiate the design of specific inhibitors for other highly homologous drug targets.

Estimation of relative free energies of binding using pre-computed ensembles based on the single-step free energy perturbation and the site-identification by Ligand competitive saturation approaches.

Accurate and rapid estimation of relative binding affinities of ligand-protein complexes is a requirement of computational methods for their effective use in rational ligand design. Of the approaches commonly used, free energy perturbation (FEP) methods are considered one of the most accurate, although they require significant computational resources. Accordingly, it is desirable to have alternative methods of similar accuracy but greater computational efficiency to facilitate ligand design. In the present study relative free energies of binding are estimated for one or two non-hydrogen atom changes in compounds targeting the proteins ACK1 and p38 MAP kinase using three methods. The methods include standard FEP, single-step free energy perturbation (SSFEP) and the site-identification by ligand competitive saturation (SILCS) ligand grid free energy (LGFE) approach. Results show the SSFEP and SILCS LGFE methods to be competitive with or better than the FEP results for the studied systems, with SILCS LGFE giving the best agreement with experimental results. This is supported by additional comparisons with published FEP data on p38 MAP kinase inhibitors. While both the SSFEP and SILCS LGFE approaches require a significant upfront computational investment, they offer a 1000-fold computational savings over FEP for calculating the relative affinities of ligand modifications once those pre-computations are complete. An illustrative example of the potential application of these methods in the context of screening large numbers of transformations is presented. Thus, the SSFEP and SILCS LGFE approaches represent viable alternatives for actively driving ligand design during drug discovery and development. © 2016 Wiley Periodicals, Inc.

Nitro-benzylideneoxymorphone, a bifunctional mu and delta opioid receptor ligand with high mu opioid receptor efficacy.

Introduction: There is a major societal need for analgesics with less tolerance, dependence, and abuse liability. Preclinical rodent studies suggest that bifunctional ligands with both mu (MOPr) and delta (DOPr) opioid peptide receptor activity may produce analgesia with reduced tolerance and other side effects. This study explores the structure-activity relationships (SAR) of our previously reported MOPr/DOPr lead, benzylideneoxymorphone (BOM) with C7-methylene-substituted analogs. Methods: Analogs were synthesized and tested in vitro for opioid receptor binding and efficacy. One compound, nitro-BOM (NBOM, 12) was evaluated for antinociceptive effects in the warm water tail withdrawal assay in C57BL/6 mice. Acute and chronic antinociception was determined, as was toxicologic effects on chronic administration. Molecular modeling experiments were performed using the Site Identification by Ligand Competitive Saturation (SILCS) method. Results: NBOM was found to be a potent MOPr agonist/DOPr partial agonist that produces high-efficacy antinociception. Antinociceptive tolerance was observed, as was weight loss; this toxicity was only observed with NBOM and not with BOM. Modeling supports the hypothesis that the increased MOPr efficacy of NBOM is due to the substituted benzylidene ring occupying a nonpolar region within the MOPr agonist state. Discussion: Though antinociceptive tolerance and non-specific toxicity was observed on repeated administration, NBOM provides an important new tool for understanding MOPr/DOPr pharmacology.

Identification of a novel transport system in Borrelia burgdorferi that links the inner and outer membranes.

Borrelia burgdorferi, the spirochete that causes Lyme disease, is a diderm organism that is similar to Gram-negative organisms in that it contains both an inner and outer membrane. Unlike typical Gram-negative organisms, however, B. burgdorferi lacks lipopolysaccharide (LPS). Using computational genome analyses and structural modeling, we identified a transport system containing six proteins in B. burgdorferi that are all orthologs to proteins found in the lipopolysaccharide transport (LPT) system that links the inner and outer membranes of Gram-negative organisms and is responsible for placing LPS on the surface of these organisms. While B. burgdorferi does not contain LPS, it does encode over 100 different surface-exposed lipoproteins and several major glycolipids, which like LPS are also highly amphiphilic molecules, though no system to transport these molecules to the borrelial surface is known. Accordingly, experiments supplemented by molecular modeling were undertaken to determine whether the orthologous LPT system identified in B. burgdorferi could transport lipoproteins and/or glycolipids to the borrelial outer membrane. Our combined observations strongly suggest that the LPT transport system does not transport lipoproteins to the surface. Molecular dynamic modeling, however, suggests that the borrelial LPT system could transport borrelial glycolipids to the outer membrane.

Computer-Aided Drug Design: An Update.

Computer-aided drug design (CADD) approaches are playing an increasingly important role in understanding the fundamentals of ligand-receptor interactions and helping medicinal chemists design therapeutics. About 5 years ago, we presented a chapter devoted to an overview of CADD methods and covered typical CADD protocols including structure-based drug design (SBDD) and ligand-based drug design (LBDD) approaches that were frequently used in the antibiotic drug design process. Advances in computational hardware and algorithms and emerging CADD methods are enhancing the accuracy and ability of CADD in drug design and development. In this chapter, an update to our previous chapter is provided with a focus on new CADD approaches from our laboratory and other peers that can be employed to facilitate the development of antibiotic therapeutics.

hERG Blockade Prediction by Combining Site Identification by Ligand Competitive Saturation and Physicochemical Properties.

Human ether-a-go-go-related gene (hERG) potassium channel is well-known contributor to drug-induced cardiotoxicity and therefore an extremely important target when performing safety assessments of drug candidates. Ligand-based approaches in connection with quantitative structure active relationships (QSAR) analyses have been developed to predict hERG toxicity. Availability of the recent published cryogenic electron microscopy (cryo-EM) structure for the hERG channel opened the prospect for using structure-based simulation and docking approaches for hERG drug liability predictions. In recent time, the idea of combining structure- and ligand-based approaches for modeling hERG drug liability has gained momentum offering improvements in predictability when compared to ligand-based QSAR practices alone. The present article demonstrates uniting the structure-based SILCS (site-identification by ligand competitive saturation) approach in conjunction with physicochemical properties to develop predictive models for hERG blockade. This combination leads to improved model predictability based on Pearson's R and percent correct (represents rank-ordering of ligands) metric for different validation sets of hERG blockers involving diverse chemical scaffold and wide range of pIC50 values. The inclusion of the SILCS structure-based approach allows determination of the hERG region to which compounds bind and the contribution of different chemical moieties in the compounds to blockade, thereby facilitating the rational ligand design to minimize hERG liability.

Spatial requirements for ITAM signaling in an intracellular natural killer cell model membrane.

FcγRIIIa-FcεRIγ complexes, upon stimulation by antibodies, cluster to initiate intracellular signaling and activate natural killer (NK) cells. Intracellular signaling involves Lck phosphorylation of ITAMs of each monomer of a FcεRIγ homodimer in a FcγRIIIa-FcεRIγ complex and subsequent binding of two phosphotyrosines (pY) in tandem by a Syk family kinase. However, how FcR clustering triggers ITAM signaling is not resolved. Molecular modeling and dynamics (MD) simulations are applied to generate ensembles of structures of the FcγRIIIa and FcεRIγ homodimeric cytoplasmic tails of FcγRIIIa-FcεRIγ complexes based on the transmembrane helices and cytoplasmic tails spaced 120, 80, and 50 Å apart to model different extents of clustering. Site-identification by ligand competitive saturation method with Monte Carlo sampling (SILCS-MC) is used to model how Lck could phosphorylate a diversity of ITAM conformations. At 80 Å separation between FcγRIIIa-FcεRIγ complexes, Lck can perform multiple phosphorylations on individual and multiple ITAMs across complexes, including potential sequential phosphorylation events. Syk may then potentially bind the two pYs within a single ITAM in tandem in isolated FcγRIIIa-FcεRIγ complexes, as observed in CD3ε and ζ chains of T cell receptors by the Syk family kinase ZAP-70. In addition, at 50 Å separation between complexes, unique to natural killer cells over T cells, Syk could potentially bind in tandem to pYs in different ITAMs across FcγRIIIa-FcεRIγ complexes. Thus, we predict that an ensemble of spatial orientations of the ITAMS of FcγRIIIa-FcεRIγ complexes that occur upon clustering lead to ITAM phosphorylation by Lck and subsequent Syk activity thereby facilitating downstream signaling.

Species-Specific Endotoxin Stimulus Determines Toll-Like Receptor 4- and Caspase 11-Mediated Pathway Activation Characteristics.

The innate immune system is the body's first line of defense against pathogens and its protection against infectious diseases. On the surface of host myeloid cells, Toll-like receptor 4 (TLR4) senses lipopolysaccharide (LPS), the major outer membrane component of Gram-negative bacteria. Intracellularly, LPS is recognized by caspase 11 through the noncanonical inflammasome to induce pyroptosis-an inflammatory form of lytic cell death. While TLR4-mediated signaling perturbations result in secretion of cytokines and chemokines that help clear infection and facilitate adaptive immunity, caspase 11-mediated pyroptosis leads to the release of damage-associated molecular patterns and inflammatory mediators. Although the core signaling events and many associated proteins in the TLR4 signaling pathway are known, the complex signaling events and protein networks within the noncanonical inflammasome pathway remain obscure. Moreover, there is mounting evidence for pathogen-specific innate immune tuning. We characterized the major LPS structures from two different pathogens, modeled their binding to the surface receptors, systematically examined macrophage inflammatory responses to these LPS molecules, and surveyed the temporal differences in global protein secretion resulting from TLR4 and caspase 11 activation in macrophages using mass spectrometry (MS)-based quantitative proteomics. This integrated strategy, spanning functional activity assays, top-down structural elucidation of endotoxins, and secretome analysis of stimulated macrophages, allowed us to identify crucial differences in TLR4- and caspase 11-mediated protein secretion in response to two Gram-negative bacterial endotoxins. IMPORTANCE Macrophages and monocytes are innate immune cells playing an important role in orchestrating the initial innate immune response to bacterial infection and the tissue damage. This response is facilitated by specific receptors on the cell surface and intracellularly. One of the bacterial molecules recognized is a Gram-negative bacteria cell wall component, lipopolysaccharide (LPS). The structure of LPS differs between different species. We have characterized the innate immune responses to the LPS molecules from two bacteria, Escherichia coli and Bordetella pertussis, administered either extracellularly or intracellularly, whose structures we first determined. We observed marked differences in the temporal dynamics and amounts of proteins secreted by the innate immune cells stimulated by any of these molecules and routes. This suggests that there is specificity in the first line of response to different Gram-negative bacteria that can be explored to tailor specific therapeutic interventions.

A phase I randomized safety study of a single-size silicone rubber diaphragm used with or without a lactic-acid-containing diaphragm gel.

OBJECTIVES: To evaluate a lactic-acid-containing diaphragm gel (Contragel®) approved outside the United States for use with a silicone rubber diaphragm (Caya®). The study gel is being evaluated as a safer alternative to nonoxynol-9 (N-9) gel, which has been associated with risk of increasing susceptibility to human immunodeficiency virus (HIV). STUDY DESIGN: This was a Phase I randomized, parallel study evaluating the safety of the novel diaphragm gel versus hydroxyethylcellulose (HEC) universal placebo gel delivered by the study diaphragm for two 7-day test cycles of daily use, without and with intercourse. The primary clinical safety endpoint was treatment emergent adverse events. Mucosal safety endpoints included colposcopic findings, anti-Escherichia coli activity of endocervical and vaginal fluid, immune mediators, Nugent score and ectocervical immune cell density. Endpoints were assessed prior to each test cycle and at day 7 of each test cycle. We compared the two independent groups and also evaluated paired changes from baseline in each gel cohort. RESULTS: Twenty-three participants used the study diaphragm with the novel gel (n=11) or with HEC (n=12). Use of either gel resulted in few genital AEs and no colposcopic findings. There were no differences in ectocervical histology and lymphocyte density or phenotype between the two cohorts at baseline or after each test cycle. We found no clinically important differences in the anti-microbial (anti Escherichia coli) activity of endocervical or vaginal fluid or concentrations of genital immune mediators (e.g. anti-inflammatory secretory leukocyte protease inhibitor (SLPI) or pro-inflammatory mediator RANTES) between the two gel cohorts at any visit. There were no important paired changes from baseline among participants using either gel in Nugent score, ectocervical histology or anti-microbial activity of genital secretions. CONCLUSIONS: We found no clinically significant differences in clinical and mucosal safety endpoints between the two cohorts. The mucosal safety profiles of the study gel and HEC placebo gel were similar. IMPLICATIONS: Our data demonstrate no clinically important differences between the safety profiles of the lactic-acid-containing diaphragm gel versus HEC placebo gel when used with the study diaphragm. N-9 can no longer be used with contraceptive diaphragms in high HIV prevalence regions. Although larger studies are needed, the novel gel appears safe for use with the study diaphragm, which is the first over-the-counter, non-hormonal, diaphragm.

A phase I randomized postcoital testing and safety study of the Caya diaphragm used with 3% Nonoxynol-9 gel, ContraGel or no gel.

OBJECTIVES: The Caya® Diaphragm is a newly approved single-size, nonlatex diaphragm. Contragel® is a personal lubricant containing lactic acid approved in Europe and other countries for use with vaginal barrier devices. This study assessed the effectiveness in preventing sperm from penetrating midcycle cervical mucus of Caya with Contragel, Caya with 3% nonoxynol-9 (N-9) and Caya alone. STUDY DESIGN: Phase I multicenter, single-blind, randomized, crossover, nonsignificant risk study at two sites: Eastern Virginia Medical School, Norfolk, VA, USA, and Profamilia, Santo Domingo, Dominican Republic. Healthy, sexually active women 18-45years old, not at risk for pregnancy due to tubal occlusion, were eligible. Each participant was seen in nine visits, completing a baseline cycle (without product use) followed by three test cycles (sequence determined by randomization), each consisting of a cervical mucus check visit and a postcoital test visit. To proceed to test cycles, the baseline postcoital test had to show adequate cervical mucus and >5 progressively motile sperm per high power field (PMS/HPF). RESULTS: All women had an average of <5 PMS/HPF during the test cycle of each study arm, the primary endpoint. Caya with ContraGel and Caya with N-9 reduced the average number of PMS/HPF from 22.5 to 0. Caya alone reduced the average number of PMS/HPF from 22.5 to 0.4. There were two possibly product-related mild adverse events. CONCLUSION: This study supports that Caya with ContraGel is safe and functions as well as Caya with N-9 in preventing PMS from reaching midcycle cervical mucus. IMPLICATIONS: A single-size diaphragm used with a personal lubricant gel containing lactic acid appears to be safe and to function as well as the same diaphragm used with N-9 in preventing PMS from reaching midcycle cervical mucus.

Computer-Aided Drug Design Methods.

Computational approaches are useful tools to interpret and guide experiments to expedite the antibiotic drug design process. Structure-based drug design (SBDD) and ligand-based drug design (LBDD) are the two general types of computer-aided drug design (CADD) approaches in existence. SBDD methods analyze macromolecular target 3-dimensional structural information, typically of proteins or RNA, to identify key sites and interactions that are important for their respective biological functions. Such information can then be utilized to design antibiotic drugs that can compete with essential interactions involving the target and thus interrupt the biological pathways essential for survival of the microorganism(s). LBDD methods focus on known antibiotic ligands for a target to establish a relationship between their physiochemical properties and antibiotic activities, referred to as a structure-activity relationship (SAR), information that can be used for optimization of known drugs or guide the design of new drugs with improved activity. In this chapter, standard CADD protocols for both SBDD and LBDD will be presented with a special focus on methodologies and targets routinely studied in our laboratory for antibiotic drug discoveries.

Vaginal rings for delivery of HIV microbicides.

Following the successful development of long-acting steroid-releasing vaginal ring devices for the treatment of menopausal symptoms and contraception, there is now considerable interest in applying similar devices to the controlled release of microbicides against HIV. In this review article, the vaginal ring concept is first considered within the wider context of the early advances in controlled-release technology, before describing the various types of ring device available today. The remainder of the article highlights the key developments in HIV microbicide-releasing vaginal rings, with a particular focus on the dapivirine ring that is presently in late-stage clinical testing.