TDP-43 pathology links innate and adaptive immunity in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is the most common fatal motor neuron disease. Approximately 90% of ALS patients exhibit pathology of the master RNA regulator, Transactive Response DNA Binding protein (TDP-43). Despite the prevalence TDP-43 pathology in ALS motor neurons, recent findings suggest immune dysfunction is a determinant of disease progression in patients. Whether TDP-43 pathology elicits disease-modifying immune responses in ALS remains underexplored. In this study, we demonstrate that TDP-43 pathology is internalized by antigen presenting cells, causes vesicle rupture, and leads to innate and adaptive immune cell activation. Using a multiplex imaging platform, we observed interactions between innate and adaptive immune cells near TDP-43 pathological lesions in ALS brain. We used a mass cytometry-based whole-blood stimulation assay to provide evidence that ALS patient peripheral immune cells exhibit responses to TDP-43 aggregates. Taken together, this study provides a novel link between TDP-43 pathology and ALS immune dysfunction, and further highlights the translational and diagnostic implications of monitoring and manipulating the ALS immune response.

The frequency of Treg subsets distinguishes disease activity in ANCA vasculitis.

Objectives: T regulatory cells (Tregs) are a heterogeneous group of immunoregulatory cells that dampen self-harming immune responses and prevent the development of autoimmune diseases. In anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis, Tregs possess diminished suppressive capacity, which has been attributed to the expression of a FOXP3 splice-variant lacking exon 2 in T cells (FOXP3Δ2 CD4+ T cells). However, the suppressive capacity of Tregs varies between subsets. We evaluated the frequency of Treg subsets in ANCA vasculitis as a potential explanation for diminished suppressive capacity. Methods: We developed a custom mass cytometry panel and performed deep immune profiling of Tregs in healthy controls, patients with active disease and in remission. Using these data, we performed multidimensional reduction and discriminant analysis to identify associations between Treg subsets and disease activity. Results: Total Tregs were expanded in ANCA vasculitis, which was associated with remission and the administration of rituximab and/or prednisone. The frequency of FOXP3Δ2 CD4+ T cells did not distinguish disease activity and this population had high expression levels of CD127 and lacked both CD25 and Helios, suggesting that they are not conventional Tregs. The frequency of CXCR3+, CD103+ and CCR7+ Tregs distinguished disease activity, and the combination of the frequency of these three Treg subsets segregated active patients from patients in remission and healthy controls. From these three subsets, the frequency of CXCR3+ Tregs distinguished patients with active disease with renal involvement. Conclusion: Treg heterogeneity can discriminate disease activity and should be explored as a biomarker of disease activity in ANCA vasculitis.

Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor.

Crystallography and computer modeling have been used to exploit a previously unexplored channel in the glucocorticoid receptor (GR). Highly potent, nonsteroidal indazole amides showing excellent complementarity to the channel were designed with the assistance of the computational technique AlleGrow. The accuracy of the design process was demonstrated through crystallographic structural determination of the GR ligand-binding domain-agonist complex of the D-prolinamide derivative 11. The utility of the channel was further exemplified through the design of a potent phenylindazole in which structural motifs, seen to interact with the traditional GR ligand pocket, were abandoned and replaced by interactions within the new channel. Occupation of the channel was confirmed with a second GR crystal structure of this truncated D-alaninamide derivative 13. Compound 11 displays properties compatible with development as an intranasal solution formulation, whereas oral bioavailability has been demonstrated with a related truncated exemplar 14. Data with the pyrrolidinone amide 12 demonstrate the potential for further elaboration within the "meta" channel to deliver compounds with selectivity for the desired transrepressive activity of glucocorticoids. The discovery of these interactions with this important receptor offers significant opportunities for the design of novel GR modulators.

Structure-guided design of N-phenyl tertiary amines as transrepression-selective liver X receptor modulators with anti-inflammatory activity.

A cocrystal structure of T1317 (3) bound to hLXRbeta was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 ( 20) as a high-affinity LXRbeta ligand (IC 50 = 30 nM) that shows separation of anti-inflammatory and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the structurally related analog 19 bound to LXRbeta reveals regions within the receptor that can affect receptor modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with improved therapeutic indexes.

Identification and characterization of a selective peroxisome proliferator-activated receptor beta/delta (NR1C2) antagonist.

The identification of small molecule ligands for the peroxisome proliferator-activated receptors (PPARs) has been instrumental in elucidating their biological roles. In particular, agonists have been the focus of much of the research in the field with relatively few antagonists being described and all of those being selective for PPARalpha or PPARgamma. The comparison of these agonist and antagonist ligands in cellular and animal systems has often led to surprising results and new insights into the biology of the PPARs. The PPARbeta/delta receptor is emerging as an important regulator of energy metabolism, inflammation, and cell growth and differentiation; however, only agonist ligands have been described for this receptor thus far. Here we describe the first report of a PPARbeta/delta small molecule antagonist ligand. This antagonist ligand will be a useful tool for elucidating the biological roles of PPARbeta/delta.

Co-crystal structure guided array synthesis of PPARgamma inverse agonists.

PPARgamma-activating thiazolidinediones and carboxylic acids such as farglitazar exert their anti-diabetic effects in part in PPARgamma rich adipose. Both pro- and anti-adipogenic PPARgamma ligands promote glucose and lipid lowering in animal models of diabetes. Herein, we disclose representatives of an array of 160 farglitazar analogues with atypical inverse agonism of PPARgamma in mature adipocytes.

Effect of microsphere binding site density on the apparent affinity of an interaction partner.

BACKGROUND: Flow cytometric microsphere-based binding assays can be used to measure molecular interactions with high sensitivity. We have used multiplexed microsphere technology to explore the effect that binding site density has on the apparent affinity of a soluble interaction partner. METHODS: The interaction of a nuclear receptor, peroxisome proliferator-activated receptor gamma ligand binding domain (PPARgamma LBD), with a synthetic peptide derived from a nuclear receptor coactivator protein, PPARgamma coactivator-1 alpha (PGC-1alpha), is the interacting system being studied. The density of this peptide coupled to fluorescently unique microsphere populations is varied by co-incubating the biotinylated peptide and avidin-coated microsphere populations with increasing the amounts of free D-biotin. The discrete-density peptide-coupled microsphere populations are combined to conduct a multiplexed binding experiment with Alexa 532-labeled PPARgamma LBD, in the absence or presence of a small molecule ligand. RESULTS: As the immobilized binding site density of PGC-1alpha peptide on fluorescent microspheres is increased the measured apparent affinity for PPARgamma LBD is increased. CONCLUSIONS: The density of binding sites immobilized to a surface has a pronounced effect on the apparent affinity for soluble binding partners. By controlling and varying the binding site density it is possible to increase the sensitivity of an interaction assay. In multiplexed assay formats it should be possible to normalize intrinsically unequal binding interactions by individually optimizing the binding site density of the immobilized interaction partner. However, to quantitatively measure intrinsic affinities of molecular interactions, low binding site densities are required and multivalent reagents must be avoided.

Characterization of nuclear receptor ligands by multiplexed peptide interactions.

This unit describes a method to evaluate the effect that small molecules have on the binding interactions of a nuclear receptor protein with a series of peptides. The multiplexed microsphere-based system employs peptide-coupled microsphere populations that are fluorescently unique and thereby identifiable by flow cytometric analysis. Up to 100 different peptide-nuclear receptor interactions may be analyzed in a single well of a 96-well microtiter plate. This approach allows rapid and sensitive characterization of nuclear receptor ligands based on nuclear receptor protein-peptide interaction profiles. Since nuclear receptor binding interactions are dynamically related to protein conformation, the approach allows rapid evaluation of nuclear receptor ligands that may impart unique protein structure. The no-wash format and the high surface density of the microsphere-coupled interaction partner offer a moderately high-throughput system to examine low- to high-affinity interactions with excellent sensitivity. This approach, although described for nuclear receptors, may also be applied to other types of molecular interactions.

Discovery of novel quinoline-based estrogen receptor ligands using peptide interaction profiling.

Traditional approaches to discovery of selective estrogen receptor modulators (SERMs) have relied on ER binding and cell-based estrogen response element-driven assays to identify compounds that are osteoprotective but nonproliferative in breast and uterine tissues. To discover new classes of potential SERMs, we have employed a cell-free microsphere-based binding assay to rapidly characterize ERalpha interactions with conformation-sensing cofactor or phage display peptides. Peptide profiles of constrained triarenes were compared to known proliferative and nonproliferative ER ligands to discover potent quinoline-based ligands with minimal Ishikawa cell stimulation.

Discovery of novel nuclear receptor modulating ligands: an integral role for peptide interaction profiling.

There is currently a marketed drug for nearly every nuclear receptor for which the natural ligand has been identified. However, because of the complexity of signal transduction by this class of ligand-regulated transcription factors, few of these drugs have been optimized for pharmaceutical effectiveness. Over the past several years, structural and biochemical work has shed light on some of the ligand-induced features of nuclear receptors that enable them to trigger signal transduction cascades. This review will highlight the use of peptide interactions to cluster different classes of ligands and to identify novel nuclear receptor-modulating ligands as potential drug candidates. Phage display and a multiplexed peptide interaction assay are two of the technologies that are key to this approach. When used as part of a drug discovery platform, this type of biochemical characterization can bridge the gap between high-throughput chemical synthesis and disease model testing. Furthermore, the development of these methodologies is timely because there is a significant medical need for new and improved nuclear receptor drugs that retain beneficial effects but do not have undesired side effect activities.

Correlation between in vitro peptide binding profiles and cellular activities for estrogen receptor-modulating compounds.

Numerous biochemical and structural studies have shown that the conformation of the estrogen receptor alpha (ERalpha) can be influenced by ligand binding. In turn, the conformational state of ERalpha affects the ability of the receptor to interact with a wide variety of protein accessory factors. To globally investigate ligand-based cofactor recruitment activities of ERalpha, we have applied a flow cytometric multiplexed binding assay to determine the simultaneous binding of ERalpha to over 50 different peptides derived from both known cofactor proteins and random peptide phage display. Using over 400 ERalpha-binding compounds, we have observed that the multiplexed in vitro peptide-binding profiles are distinct for a number of compounds and that these profiles can predict the effect that ERalpha ligands have on various cellular activities. These cell-based activities include transcriptional regulation at an estrogen response element, MCF-7 cell proliferation, and Ishikawa endometrial cell stimulation. The majority of the compound-induced diversity in the peptide profiling assay is provided by the unique phage display peptides. Importantly, some of these peptides show a sequence relationship with the corepressor motif, suggesting that peptides identified via phage display might represent natural binding partners of ERalpha. These in vitro:cellular correlations may in part explain tissue-specific activities of ERalpha-modulating compounds.

Multiplexed microsphere-based flow cytometric assays.

Flow cytometry has become an indispensable tool for clinical diagnostics and basic research. Although primarily designed for cellular analysis, flow cytometers can detect any particles in the lower micron range, including inert microspheres of different sizes, dyed with various fluorochromes. Over the past 20 years, microspheres have been used as calibrators for flow cytometers and also as a solid support for numerous molecular reactions quantitated by flow cytometry. Proteins, oligonucleotides, polysaccharides, lipids, or small peptides have been adsorbed or chemically coupled to the surface of microspheres to capture analytes that are subsequently measured by a fluorochrome-conjugated detection molecule. More recently, assays for similar analytes have been multiplexed, or analyzed in the same assay volume, by performing each reaction on a set of microspheres that are dyed to different fluorescent intensities and, therefore, are spectrally distinct. Some recent applications with fluorescent microspheres have included cytokine quantitation, single nucleotide polymorphism genotyping, phosphorylated protein detection, and characterization of the molecular interactions of nuclear receptors. The speed, sensitivity, and accuracy of flow cytometric detection of multiple binding events measured in the same small volume have the potential to replace many clinical diagnostic and research methods and deliver data on hundreds of analytes simultaneously.