High-Throughput Covalent Modifier Screening with Acoustic Ejection Mass Spectrometry.

Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered "undruggable". The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC-MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising ∼10,000 compounds against a 50 kDa-sized target protein─Werner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein-ligand adducts. The screening results displayed a high degree of fidelity, with a Z' factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC-MS showcased the AEMS platform's low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.

Structure of the complement C5a receptor bound to the extra-helical antagonist NDT9513727.

The complement system is a crucial component of the host response to infection and tissue damage. Activation of the complement cascade generates anaphylatoxins including C5a and C3a. C5a exerts a pro-inflammatory effect via the G-protein-coupled receptor C5a anaphylatoxin chemotactic receptor 1 (C5aR1, also known as CD88) that is expressed on cells of myeloid origin. Inhibitors of the complement system have long been of interest as potential drugs for the treatment of diseases such as sepsis, rheumatoid arthritis, Crohn's disease and ischaemia-reperfusion injuries. More recently, a role of C5a in neurodegenerative conditions such as Alzheimer's disease has been identified. Peptide antagonists based on the C5a ligand have progressed to phase 2 trials in psoriasis and rheumatoid arthritis; however, these compounds exhibited problems with off-target activity, production costs, potential immunogenicity and poor oral bioavailability. Several small-molecule competitive antagonists for C5aR1, such as W-54011 and NDT9513727, have been identified by C5a radioligand-binding assays. NDT9513727 is a non-peptide inverse agonist of C5aR1, and is highly selective for the primate and gerbil receptors over those of other species. Here, to study the mechanism of action of C5a antagonists, we determine the structure of a thermostabilized C5aR1 (known as C5aR1 StaR) in complex with NDT9513727. We found that the small molecule bound between transmembrane helices 3, 4 and 5, outside the helical bundle. One key interaction between the small molecule and residue Trp2135.49 seems to determine the species selectivity of the compound. The structure demonstrates that NDT9513727 exerts its inverse-agonist activity through an extra-helical mode of action.

Extra-helical binding site of a glucagon receptor antagonist.

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

Tau-tubulin kinase 1 phosphorylates TDP-43 at disease-relevant sites and exacerbates TDP-43 pathology.

TDP-43 pathology is a hallmark of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal lobar degeneration (FTLD). Namely, both diseases feature aggregated and phosphorylated TDP-43 containing inclusions in the cytoplasm and a loss of nuclear TDP-43 in affected neurons. It has been reported that tau tubulin kinase (TTBK)1/2 phosphorylate TDP-43 and TTBK1/2 overexpression induced neuronal loss and behavioral deficits in a C. elegans model of ALS. Here we aimed to elucidate the molecular mechanisms of TTBK1 in TDP-43 pathology. TTBK1 levels were observed to be elevated in ALS patients' post-mortem motor cortex. Also, TTBK1 was found to phosphorylate TDP-43 at disease-relevant sites in vitro directly, and this phosphorylation accelerated TDP-43 formation of high molecular species. Overexpression of TTBK1 in mammalian cells induced TDP-43 phosphorylation and the construction of high molecular species, concurrent with TDP-43 mis-localization and cytoplasmic inclusions. In addition, when TTBK1 was knocked down or pharmacologically inhibited, TDP-43 phosphorylation and aggregation were significantly alleviated. Functionally, TTBK1 knockdown could rescue TDP-43 overexpression-induced neurite and neuronal loss in iPSC-derived GABAergic neurons. These findings suggest that phosphorylation plays a critical role in the pathogenesis of TDP-43 pathology and that TTBK1 inhibition may have therapeutic potential for the treatment of ALS and FTLD.

Preparation of purified GPCRs for structural studies.

Since the publication of the first X-ray structure of a GPCR (G-protein couple receptor) in 2000, the rate at which subsequent ones have appeared has steadily increased. This has required the development of new methodology to overcome the challenges presented by instability of isolated GPCRs, combined with a systematic optimization of existing approaches for protein expression, purification and crystallization. In addition, quality control measures that are predictive of successful outcomes have been identified. Repeated attempts at solving the structures of GPCRs have highlighted experimental approaches that are most likely to lead to success, and have allowed definition of a first-pass protocol for new receptors.

Opportunities for therapeutic antibodies directed at G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are activated by a diverse range of ligands, from large proteins and proteases to small peptides, metabolites, neurotransmitters and ions. They are expressed on all cells in the body and have key roles in physiology and homeostasis. As such, GPCRs are one of the most important target classes for therapeutic drug discovery. The development of drugs targeting GPCRs has therapeutic value across a wide range of diseases, including cancer, immune and inflammatory disorders as well as neurological and metabolic diseases. The progress made by targeting GPCRs with antibody-based therapeutics, as well as technical hurdles to overcome, are presented and discussed in this Review. Antibody therapeutics targeting C-C chemokine receptor type 4 (CCR4), CCR5 and calcitonin gene-related peptide (CGRP) are used as illustrative clinical case studies.

Antibodies targeting G protein-coupled receptors: Recent advances and therapeutic challenges.

Le STUDIUM conference was held November 24-25, 2016 in Tours, France as a satellite workshop of the 5th meeting of the French GDR 3545 on "G Protein-Coupled Receptors (GPCRs) -From Physiology to Drugs," which was held in Tours during November 22-24, 2016. The conference gathered speakers from academia and industry considered to be world leaders in the molecular pharmacology and signaling of GPCRs, with a particular interest in the development of therapeutic GPCR antibodies (Abs). The main topics were new advances and challenges in the development of antibodies targeting GPCRs and their potential applications to the study of the structure and function of GPCRs, as well as their implication in physiology and pathophysiology. The conference included 2 sessions, with the first dedicated to the recent advances in methodological strategies used for GPCR immunization using thermo-stabilized and purified GPCRs, and the development of various formats of Abs such as monoclonal IgG, single-chain variable fragments and nanobodies (Nbs) by in vitro and in silico approaches. The second session focused on GPCR Nbs as a "hot" field of research on GPCRs. This session started with discussion of the pioneering Nbs developed against GPCRs and their application to structural studies, then transitioned to talks on original ex vivo and in vivo studies on GPCR-selective Nbs showing promising therapeutic applications of Nbs in important physiologic systems, such as the central nervous and the immune systems, as well as in cancer. The conference ended with the consensus that Abs and especially Nbs are opening a new era of research on GPCR structure, pharmacology and pathophysiology.

The expression pattern of nitric oxide-sensitive guanylyl cyclase in the rat heart changes during postnatal development.

Nitric oxide (NO)-releasing drugs such as glyceryl trinitrate have been used in the treatment of ischemic heart disease for more than a century. Nevertheless, a detailed analysis of the expression of the NO target enzyme soluble guanylyl cyclase (sGC) in the heart is missing. The aim of the current study was to elucidate the expression, cell distribution, and activity of sGC in the rat heart during postnatal development. Using a novel antibody raised against a C-terminal peptide of the rat beta(1)-subunit of sGC, the enzyme was demonstrated in early postnatal and adult hearts by Western blotting analyses, showing maximal expression in 10-day-old animals. Measurements of basal, NO-, and NO/YC-1-stimulated sGC activity revealed an increase of sGC activity in hearts from neonatal to 10-day-old rats, followed by a subsequent decrease in adult animals. As shown by immunohistochemical analysis, sGC expression was present in vascular endothelium and smooth muscle cells in neonatal heart but expression shifted to endothelial cells in adult animals. In isolated cardiomyocytes, sGC activity was not detectable under basal conditions but significant sGC activity could be detected in the presence of NO. An increase in expression during the perinatal period and changes in the cell types expressing sGC at different phases of development suggest dynamic regulation rather than constitutive expression of the NO receptor in the heart.

Biliverdin IX is an endogenous inhibitor of soluble guanylyl cyclase.

Heme oxygenase (HO) converts heme to carbon monoxide (CO) and biliverdin IX. CO is a weak activator of soluble guanylyl cyclase (SGC), the enzyme that catalyzes the conversion of GTP to the second messenger cGMP. HO overexpression has recently been shown to inhibit production of cGMP by SGC in vivo. The aim of the present study was to investigate a possible influence of biliverdin IX on SGC activity. Using recombinant alpha(1)/beta(1) isoform of SGC, we show an inhibitory effect of biliverdin IX in the micromolar range both on basal and NO stimulated guanylyl cyclase activity. Bilirubin IX which differs from biliverdin IX in two hydrogen atoms had no effect. Biliverdin IX reduced maximal guanylyl cyclase activity (V(max) values) while it had no effect on the K(M) values indicating unchanged affinity towards the substrate GTP. Concentration response experiments using the NO donor, 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO), showed that enzyme activities at maximal DEA/NO concentration were reduced by biliverdin IX. The affinity of the NO-donor, DEA/NO, towards SGC was significantly reduced in the presence of biliverdin IX. Biliverdin IX lowered enzyme activity at maximal activator concentrations of YC-1 and protoporphyrin IX (PPIX) while it had no significant effect on the EC(50) values of these two NO independent activators. The inhibitory effect of biliverdin IX on PPIX activated enzyme activity is not shared by ODQ, which indicates that the inhibitory mechanism of biliverdin IX is different from ODQ.

Native human nitric oxide sensitive guanylyl cyclase: purification and characterization.

The only published report of the purification of native human soluble guanylyl cyclase (sGC) used placenta as starting material. This enzyme preparation showed low fold-activation by NO and a maximal absorption of the prosthetic heme-group at 417nm indicative of a prosthetic heme-group in a hexa-coordinate state. These data are in contrast to what has subsequently been found for the recombinant human enzymes. Apart from this placental enzyme preparation, a native functional human NO-sensitive sGC has not been successfully purified. The aim of the current study was to purify and characterize native human sGC from another source, to see whether the discrepancies between native and recombinant sGC seen for placenta are a general phenomenon. We chose human platelets as starting material since the properties of this enzyme are directly relevant for the development of innovative antiplatelet and antianginal drugs. Our results indicate that the native platelet enzyme exists as a highly NO-sensitive, heterodimeric enzyme with an alpha(1) and beta(1) subunit. In contrast to the native human placental enzyme and in accordance with the human recombinant enzymes, the native human platelet enzyme contains a ferrous, penta-coordinate heme group. To our knowledge this is the first report of the successful purification and characterization of the native human nitric oxide sensitive alpha(1)/beta(1) isoform of sGC which is widely expressed in the cardiovascular system and is an important target of innovative drugs.

Monoclonal anti-ß1-adrenergic receptor antibodies activate G protein signaling in the absence of ß-arrestin recruitment.

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-ß1-adrenergic (ß1AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at ß1AR. Immunization with the ß1AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at ß1AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through ß-arrestin. In vitro characterization also verified different antibody receptor interactions reflecting the different epitopes on the extracellular surface of ß1AR to which the mAbs bind. The anti-ß1AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with ß1AR agonism.

Biophysical fragment screening of the ß1-adrenergic receptor: identification of high affinity arylpiperazine leads using structure-based drug design.

Biophysical fragment screening of a thermostabilized ß1-adrenergic receptor (ß1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein-ligand crystal structures of the ß1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized ß1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively.

The amino-terminus of nitric oxide sensitive guanylyl cyclase α1 does not affect dimerization but influences subcellular localization.

BACKGROUND: Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a ß1-subunit. A splice variant (C-α1) of the α1-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61-128 of the α1-subunit are mandatory for quantitative heterodimerization implying that the C-α1-splice variant should lose its capacity to dimerize quantitatively. METHODOLOGY/PRINCIPAL FINDINGS: In the current study we demonstrate preserved quantitative dimerization of the C-α1-splice by co-purification with the ß1-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the ß1-subunit and the α1-subunit or the C-α1 variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α1/ß1 and C-α1/ß1 than the negative control. In addition we show that lack of the amino-terminus in the α1 splice variant directs it to a more oxidized subcellular compartment. CONCLUSIONS/SIGNIFICANCE: We conclude that the amino-terminus of the α1-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.

Therapeutic antibodies directed at G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. A number of new developments in over-expressing receptors, as well as formulating stable pure protein, are contributing to the growing interest in targeting GPCRs with antibodies. This review discusses the opportunities for targeting GPCRs with antibodies using these approaches and describes the therapeutic antibodies that are currently in clinical development.

Characterization of 16 human G protein-coupled receptors expressed in baculovirus-infected insect cells.

Understanding the three-dimensional structure of G protein-coupled receptors (GPCRs) has been limited by the technical challenges associated with expression, purification, and crystallization of membrane proteins, and their low abundance in native tissue. In the first large-scale comparative study of GPCR protein production using recombinant baculovirus, we report the characterization of 16 human receptors. The GPCRs were produced in three insect cell lines and functional protein levels monitored over 72 h using radioligand binding assays. Different GPCRs exhibited widely different expression levels, ranging from less than 1 pmol receptor/mg protein to more than 250 pmol/mg. No single set of conditions was suitable for all GPCRs, and large differences were seen for the expression of individual GPCRs in different cell lines. Closely related GPCRs did not share similar expression profiles; however, high expression (greater than 20 pmol/mg) was achieved for over half the GPCRs in our study. Overall, the levels of protein production compared favourably to other published systems.

A functional domain of the alpha1 subunit of soluble guanylyl cyclase is necessary for activation of the enzyme by nitric oxide and YC-1 but is not involved in heme binding.

Soluble guanylyl cyclase is a heterodimeric enzyme consisting of an alpha(1) and a beta(1) subunit and is an important target for endogenous nitric oxide and the guanylyl cyclase modulator YC-1. The activation of the enzyme by both substances is dependent on the presence of a prosthetic heme group. It has been unclear whether this prosthetic heme group is sandwiched between the alpha(1) and beta(1) subunits or whether it exclusively binds to the beta(1) subunit. Here we analyze progressive amino-terminal deletion mutants of the human alpha(1) subunit after co-expression with the human beta(1) subunit in the baculovirus/Sf9 system. Spectral, biochemical, and pharmacological analysis shows that the first 259 amino acids of the alpha(1) subunit can be deleted without loss of sensitivity to nitric oxide (NO) or YC-1 or loss of heme binding of the respective enzyme complex with the beta(1) subunit. This is in contrast to previous data indicating that NO sensitivity and a functional heme binding site requires full-length amino termini of bovine alpha(1) and beta(1) subunits. Further deletion of the first 364 amino acids of the alpha(1) subunit leads to an enzyme complex with preserved heme binding but loss of sensitivity to NO or YC-1 despite induction of the typical spectral shift by NO binding to the prosthetic heme group. We conclude that 1) the amino-terminal part of the alpha(1) subunit is not involved in heme binding and 2) amino acids 259-364 of the alpha(1) subunit represent an important functional domain for the transduction of the NO activation signal and likely represent the target for NO-sensitizing substances like YC-1.

BAY 41-2272 activates two isoforms of nitric oxide-sensitive guanylyl cyclase.

Soluble guanylyl cyclase is an important target for endogenous nitric oxide and the guanylyl cyclase modulator, YC-1. Recently BAY 41-2272 was identified as a similar but more potent and more specific substance. While YC-1 also acts as non-specific phosphodiesterase inhibitor, BAY 41-2272 is devoid of an effect on phosphodiesterases. BAY 41-2272 has so far only been tested on the alpha(1)/beta(1) heterodimeric isoform of soluble guanylyl cyclase and its binding site has been mapped to a region in the alpha(1) subunit amino-terminal sequence. Although this region is poorly conserved in the alpha(2) subunit, we show in the current study that the alpha(2)/beta(1) heterodimeric enzyme isoform is activated by BAY 41-2272. Deletion analysis of the alpha(2) subunit and co-expression with the beta(1) subunit in the baculovirus/Sf9 system is consistent with the amino-terminal amino acids 104 to 401 of the alpha(2) subunit as binding site for BAY 41-2272.