Development of small molecule inhibitors of natural killer group 2D receptor (NKG2D).

Natural killer group 2D (NKG2D) is a homodimeric activating immunoreceptor whose function is to detect and eliminate compromised cells upon binding to the NKG2D ligands (NKG2DL) major histocompatibility complex (MHC) molecules class I-related chain A (MICA) and B (MICB) and UL16 binding proteins (ULBP1-6). While typically present at low levels in healthy cells and tissue, NKG2DL expression can be induced by viral infection, cellular stress or transformation. Aberrant activity along the NKG2D/NKG2DL axis has been associated with autoimmune diseases due to the increased expression of NKG2D ligands in human disease tissue, making NKG2D inhibitors an attractive target for immunomodulation. Herein we describe the discovery and optimization of small molecule PPI (protein-protein interaction) inhibitors of NKG2D/NKG2DL. Rapid SAR was guided by structure-based drug design and accomplished by iterative singleton and parallel medicinal chemistry synthesis. These efforts resulted in the identification of several potent analogs (14, 21, 30, 45) with functional activity and improved LLE.

pH Dependence of a GPR4 Selective Antagonist Hampers Its Therapeutic Potential.

Inflammatory bowel disease (IBD) is characterized by chronic mucosal inflammation of the gastrointestinal tract and is associated with extracellular acidification of mucosal tissue. Several extracellular pH-sensing receptors, including G protein-coupled receptor 4 (GPR4), play an important role in the regulation of inflammatory and immune responses, and GPR4 deficiency has been shown to be protective in IBD animal models. To confirm the therapeutic potential of GPR4 antagonism in IBD, we tested Compound 13, a selective GPR4 antagonist, in the interleukin 10-/- mouse model of colitis. Despite good exposures and albeit there was a trend toward improvement for a few readouts, Compound 13 treatment did not improve colitis in this model, and there were no signs of target engagement. Interestingly, Compound 13 behaved as an "orthosteric" antagonist, i.e., its potency was pH dependent and mostly inactive at pH levels lower than 6.8 with preferential binding to the inactive conformation of GPR4. Mutagenesis studies confirmed Compound 13 likely binds to the conserved orthosteric binding site in G protein-coupled receptors, where a histidine sits in GPR4 likely preventing Compound 13 binding when protonated in acidic conditions. While the exact mucosal pH in the human disease and relevant IBD mice models is unknown, it is well established that the degree of acidosis is positively correlated with the degree of inflammation, suggesting Compound 13 is not an ideal tool to study the role of GPR4 in moderate to severe inflammatory conditions. SIGNIFICANCE STATEMENT: Compound 13, a reported selective GPR4 antagonist, has been widely used to assess the therapeutic potential of GPR4, a pH-sensing receptor, for numerous indications. Its pH dependence and mechanism of inhibition identified in this study clearly highlights the limitations of this chemotype for target validation.

Identification of small-molecule protein-protein interaction inhibitors for NKG2D.

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ+, and CD8+ T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein-protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure-activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

Impact of Protein Preparation on Resulting Accuracy of FEP Calculations.

Relative free energy perturbation (FEP) methods have become increasingly popular within the pharmaceutical industry; however, despite time constraints within drug discovery cycles, caution should be applied in the deployment of such methods as protein preparation and system setup can greatly impact the accuracy of free energy predictions.

Breaking the Glass Ceiling in Simulation and Modeling: Women in Pharmaceutical Discovery.

The topic of gender equality within the United States workforce is receiving a great deal of attention. The field of chemistry is no exception and is increasingly focused on taking steps to achieve gender diversity within the chemistry workforce. Over the past several years, many computational chemistry groups within large pharmaceutical companies have realized growth in the number of women, and here we discuss the key factors that we believe have played a role in attracting and retaining the authors of this review as computational chemists in pharma. Furthermore, we combine our professional experiences in the context of how computational methodology and technology have evolved over the past decades and how that evolution has facilitated the inclusion of more women into the field. Our hope is to be a part of a solution and provide insight that will allow the chemistry workforce to continue to make steps forward in attaining gender diversity in the workplace.

Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNav1.7.

Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S-Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 µm, and Nav1.5 is inactive at 10 µm We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.

Predicting the Binding of Fatty Acid Amide Hydrolase Inhibitors by Free Energy Perturbation.

Since a goal of most drug discovery projects in either academia or industry is to design molecules that selectively bind to the desired protein, determination of protein-ligand binding free energies is of utmost importance in computer aided drug design. With the help of significant improvements in computer power, enhanced sampling techniques and accuracy of force fields, FEP (free energy perturbation) is becoming an important tool to estimate binding free energies in many drug discovery projects both retrospectively and prospectively. We have evaluated the ability of Schrödinger's FEP+ to predict relative binding free energies of a congeneric series of noncovalent fatty acid amide hydrolase (FAAH) inhibitors using an in-house crystal structure. This study shows that although an impressively accurate correlation can be obtained with experimental IC50s considering small perturbations on the deeper side of the pocket, the same was not observed with small perturbations on the relatively more open-ended and solvent-accessible side of the pocket. To understand these observations, we thoroughly investigated several key factors including the sampling of asymmetrically substituted rings, different perturbation maps, impact of simultaneous perturbations at two different ends of the ligand, and selecting the perturbations in a "chemically sensible" way.

Dynamic Structural Differences between Human and Mouse STING Lead to Differing Sensitivity to DMXAA.

The stimulator-of-interferon-genes (STING) protein is involved in innate immunity. It has recently been shown that modulation of STING can lead to an aggressive antitumor response. DMXAA is an antitumor agent that had shown great promise in murine models but failed in human clinical trials. The molecular target of DMXAA was subsequently shown to be murine STING (mSTING); however, human STING (hSTING) is insensitive to DMXAA. Molecular dynamics simulations were employed to investigate the differences between hSTING and mSTING that could influence DMXAA binding. An initial set of simulations was performed to investigate a single lid region mutation G230I in hSTING (corresponding residue in mSTING is an Ile), which rendered the protein sensitive to DMXAA. The simulations found that an Ile side chain was enough to form a steric barrier that prevents exit of DMXAA, whereas in WT hSTING, the Gly residue that lacks a side chain formed a porous lid region that allowed DMXAA to exit. A second set of molecular dynamics simulations compared the tendency of STING to be in an open-inactive conformation or a closed-active conformation. The results show that hSTING prefers to be in an open-inactive conformation even with cGAMP, the native ligand, bound. On the other hand, mSTING prefers a closed-active conformation even without a ligand bound. These results highlight the challenges in translating a mouse active STING compound into a human active compound, while also providing avenues to pursue for designing a small-molecule drug targeting human STING.

Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (µ-TRTX-Hh2a).

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.

Identification of structural motifs critical for epstein-barr virus-induced molecule 2 function and homology modeling of the ligand docking site.

Epstein-Barr virus-induced molecule 2 (EBI2) (also known as G-protein-coupled receptor 183) is a G-protein-coupled receptor (GPCR) that is best known for its role in B cell migration and localization. Our recent deorphanization effort led to the discovery of 7α,25-dihydroxycholesterol (7α,25-OHC) as the endogenous ligand for EBI2, which provides a tool for mechanistic studies of EBI2 function. Because EBI2 is the first GPCR known to bind and to be activated by an oxysterol, the goal of this study was to understand the molecular and structural bases for its ligand-dependent activation; this was achieved by identifying structural moieties in EBI2 or in 7α,25-OHC that might affect receptor-ligand interactions. By using a series of chemically related OHC analogs, we demonstrated that all three hydroxyl groups in 7α,25-OHC contributed to ligand-induced activation of the receptor. To determine the location and composition of the ligand binding domain in EBI2, we used a site-directed mutagenesis approach and generated mutant receptors with single amino acid substitutions at selected positions of interest. Biochemical and pharmacological profiling of these mutant receptors allowed for structure-function analyses and revealed critical motifs that likely interact with 7α,25-OHC. By using a hybrid ß(2)-adrenergic receptor-C-X-C chemokine receptor type 4 structure as a template, we created a homology model for EBI2 and optimized the docking of 7α,25-OHC into the putative ligand binding site, so that the hydroxyl groups interact with residues Arg87, Asn114, and Glu183. This model of ligand docking yields important structural insight into the molecular mechanisms mediating EBI2 function and may facilitate future efforts to design novel therapeutic agents that target EBI2.

Lipid-protein correlations in nanoscale phospholipid bilayers determined by solid-state nuclear magnetic resonance.

Nanodiscs are examples of discoidal nanoscale lipid-protein particles that have been extremely useful for the biochemical and biophysical characterization of membrane proteins. They are discoidal lipid bilayer fragments encircled and stabilized by two amphipathic helical proteins named membrane scaffolding protein (MSP), ~10 nm in size. Nanodiscs are homogeneous, easily prepared with reproducible success, amenable to preparations with a variety of lipids, and stable over a range of temperatures. Here we present solid-state nuclear magnetic resonance (SSNMR) studies on lyophilized, rehydrated POPC Nanodiscs prepared with uniformly (13)C-, (15)N-labeled MSP1D1 (Δ1-11 truncated MSP). Under these conditions, by SSNMR we directly determine the gel-to-liquid crystal lipid phase transition to be at 3 ± 2 °C. Above this phase transition, the lipid (1)H signals have slow transverse relaxation, enabling filtering experiments as previously demonstrated for lipid vesicles. We incorporate this approach into two- and three-dimensional heteronuclear SSNMR experiments to examine the MSP1D1 residues interfacing with the lipid bilayer. These (1)H-(13)C and (1)H-(13)C-(13)C correlation spectra are used to identify and quantify the number of lipid-correlated and solvent-exposed residues by amino acid type, which furthermore is compared with molecular dynamics studies of MSP1D1 in Nanodiscs. This study demonstrates the utility of SSNMR experiments with Nanodiscs for examining lipid-protein interfaces and has important applications for future structural studies of membrane proteins in physiologically relevant formulations.

Maturation of high-density lipoproteins.

Human high-density lipoproteins (HDLs) are involved in the transport of cholesterol. The mechanism by which HDL assembles and functions is not well understood owing to a lack of structural information on circulating spherical HDL. Here, we report a series of molecular dynamics simulations that describe the maturation of discoidal HDL into spherical HDL upon incorporation of cholesterol ester as well as the resulting atomic level structure of a mature circulating spherical HDL particle. Sixty cholesterol ester molecules were added in a stepwise fashion to a discoidal HDL particle containing two apolipoproteins wrapped around a 160 dipalmitoylphosphatidylcholine lipid bilayer. The resulting matured particle, captured in a coarse-grained description, was then described in a consistent all-atom representation and analysed in chemical detail. The simulations show that maturation results from the formation of a highly dynamic hydrophobic core comprised of cholesterol ester surrounded by phospholipid and protein; the two apolipoprotein strands remain in a belt-like conformation as seen in the discoidal HDL particle, but with flexible N- and C-terminal helices and a central region stabilized by salt bridges. In the otherwise flexible lipoproteins, a less mobile central region provides an ideal location to bind lecithin cholesterol acyltransferase, the key enzyme that converts cholesterol to cholesterol ester during HDL maturation.

Molecular models need to be tested: the case of a solar flares discoidal HDL model.

In the absence of atomic structures of high-density lipoproteins in their lipid-bound states, many molecular models have been produced based on experimental data. Using molecular dynamics, we show that a recently proposed "solar-flares" model of discoidal high-density lipoprotein is implausible. Our simulations show a collapse of the protruding solar-flare loops and a notable protein rearrangement due to an energetically unfavorable orientation of the hydrophobic protein surface toward the aqueous solvent.

Assembly of lipids and proteins into lipoprotein particles.

The self-assembly of reconstituted discoidal high-density lipoproteins, known as nanodiscs, was studied using coarse-grained molecular dynamics and small-angle X-ray scattering. In humans, high-density lipoprotein particles transport cholesterol in the blood and facilitate the removal of excess cholesterol from the body. Native high-density lipoprotein exhibits a wide variety of shapes and sizes, forming lipid-free/poor, nascent discoidal, and mature spherical particles. Little is known about how these lipoprotein particles assemble and transform from one state to another. Multiple 10 micros coarse-grained simulations reveal the assembly of discoidal high-density lipoprotein particles from disordered protein-lipid complexes. Small-angle X-ray scattering patterns were calculated from the final assembled structures and compared with experimental measurements carried out for this study to verify the accuracy of the coarse-grained simulations. Results show that hydrophobic interactions assemble, within several microseconds, the amphipathic helical proteins and lipids into roughly discoidal particles, while the proteins assume a final approximate double-belt configuration on a slower time scale.

Disassembly of nanodiscs with cholate.

Nanodiscs are protein-lipid particles that furnish a nanometer-sized membrane environment for the investigation of membrane proteins. Nanodiscs assemble spontaneously upon the removal of cholate from an initial mixture of cholate, lipids, and engineered amphipathic proteins. A combined experimental-computational approach is applied here to study the disassembly of nanodiscs through the addition of cholate to preformed particles. For this purpose, small-angle X-ray scattering experiments and coarse-grained molecular dynamics simulations were performed and compared. The study offers a detailed view of nanodisc dynamics.

Assembly of lipoprotein particles revealed by coarse-grained molecular dynamics simulations.

High-density lipoproteins (HDL) function as cholesterol transporters, facilitating the removal of excess cholesterol from the body. Due to the heterogeneity of native HDL particles (both in size and shape), the details on how these protein-lipid particles form and the structure they assume in their lipid-associated states are not well characterized. We report here a study of the self-assembly of discoidal HDL particles using coarse-grained (CG) molecular dynamics. The microsecond simulations reveal the self-assembly of HDL particles from disordered protein-lipid complexes to form structures containing many of the features of the generally accepted double-belt model for discoidal HDL particles. HDL assembly is found to proceed in two broad steps, aggregation of proteins and lipids driven by the hydrophobic effect which occurs on a approximately 1 micros time scale, followed by the optimization of the protein structure driven by increasingly specific protein-protein interactions.

The role of molecular modeling in bionanotechnology.

Molecular modeling is advocated here as a key methodology for research and development in bionanotechnology. Molecular modeling provides nanoscale images at atomic and even electronic resolution, predicts the nanoscale interaction of unfamiliar combinations of biological and inorganic materials, and evaluates strategies for redesigning biopolymers for nanotechnological uses. The methodology is illustrated in this paper through reviewing three case studies. The first one involves the use of single-walled carbon nanotubes as biomedical sensors where a computationally efficient, yet accurate, description of the influence of biomolecules on nanotube electronic properties through nanotube-biomolecule interactions was developed; this development furnishes the ability to test nanotube electronic properties in realistic biological environments. The second case study involves the use of nanopores manufactured into electronic nanodevices based on silicon compounds for single molecule electrical recording, in particular, for DNA sequencing. Here, modeling combining classical molecular dynamics, material science and device physics, described the interaction of biopolymers, e.g., DNA, with silicon nitrate and silicon oxide pores, furnished accurate dynamic images of pore translocation processes, and predicted signals. The third case study involves the development of nanoscale lipid bilayers for the study of embedded membrane proteins and cholesterol. Molecular modeling tested scaffold proteins, redesigned apolipoproteins found in mammalian plasma that hold the discoidal membranes in the proper shape, and predicted the assembly as well as final structure of the nanodiscs. In entirely new technological areas such as bionanotechnology, qualitative concepts, pictures and suggestions are sorely needed; these three case studies document that molecular modeling can serve a critical role in this respect, even though it may still fall short on quantitative precision.

Coarse grained protein-lipid model with application to lipoprotein particles.

A coarse-grained model for molecular dynamics simulations is extended from lipids to proteins. In the framework of such models pioneered by Klein, atoms are described group-wise by beads, with the interactions between beads governed by effective potentials. The extension developed here is based on a coarse-grained lipid model developed previously by Marrink et al., although future versions will reconcile the approach taken with the systematic approach of Klein and other authors. Each amino acid of the protein is represented by two coarse-grained beads, one for the backbone (identical for all residues) and one for the side-chain (which differs depending on the residue type). The coarse-graining reduces the system size about 10-fold and allows integration time steps of 25-50 fs. The model is applied to simulations of discoidal high-density lipoprotein particles involving water, lipids, and two primarily helical proteins. These particles are an ideal test system for the extension of coarse-grained models. Our model proved to be reliable in maintaining the shape of preassembled particles and in reproducing the overall structural features of high-density lipoproteins accurately. Microsecond simulations of lipoprotein assembly revealed the formation of a protein-lipid complex in which two proteins are attached to either side of a discoidal lipid bilayer.

Molecular dynamics simulations of discoidal bilayers assembled from truncated human lipoproteins.

Human apolipoprotein A-1 (apo A-1) is the major protein component of high-density lipoproteins. The apo A-1 lipid-binding domain was used as a template for the synthesis of amphipathic helical proteins termed membrane scaffold proteins, employed to self-assemble soluble monodisperse discoidal particles called Nanodiscs. In these particles, membrane scaffold proteins surround a lipid bilayer in a belt-like fashion forming bilayer disks of discrete size and composition. Here we investigate the structure of Nanodiscs through molecular dynamics simulations in which Nanodiscs were built from scaffold proteins of various lengths. The simulations showed planar or deformed Nanodiscs depending on optimal length and alignment of the scaffold proteins. Based on mean surface area per lipid calculations, comparison of small-angle x-ray scattering curves, and the relatively planar shape of Nanodiscs made from truncated scaffold proteins, one can conclude that the first 17 to 18 residues of the 200-residue apo A-1 lipid-binding domain are not involved in formation of the protein "belts" surrounding the lipid bilayer. To determine whether the addition of an integral membrane protein has an effect on the overall structure of a Nanodisc, bacteriorhodopsin was embedded into a Nanodisc and simulated using molecular dynamics, revealing a planar disk with a slightly rectangular shape.