Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when ...administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.
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•LY-CoV1404 potently neutralizes SARS-CoV-2 Omicron, BA.2 Omicron, and Delta variants•No loss of potency against currently circulating variants•Binding epitope on RBD of SARS-CoV-2 is rarely mutated based on current GISAID data•Breadth of neutralizing activity and potency supports clinical development
Westendorf et al. show that LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and that its epitope is rarely mutated.
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of ...therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour
kg
, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.
A metabotropic glutamate receptor coupled to phospholipase D (PLD‐mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well ...established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch‐evoked firing. We report here the first identification of this PLD‐mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far‐western blotting indicates PLD‐mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD‐mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD‐mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.
What is the central question of this study?
The metabotropic glutamate receptor coupled to phospholipase D (PLD‐mGluR) is a glutamate receptor previously only characterized pharmacologically but essential for maintaining stretch responsiveness in muscle spindle mechanosensory primary endings: what is the PLD‐mGluR protein?
What is the main finding and its importance?
PLD‐mGluR was identified as a homomeric GluK2 receptor signalling metabotropically. This identifies PLD‐mGluR 30 years after its discovery. This is important because: PLD‐mGluR is essential for muscle spindle stretch sensitivity; it is the first native kainate receptor shown to signal solely metabotropically; and, as it is the only GluR expressed in spindle mechanosensory endings, muscle spindles make a good functional assay of the native receptor.
Objective
To characterize the effects of blocking calcitonin gene‐related peptide (CGRP) activity in a mouse model of gastrointestinal transport.
Background
Migraine management using CGRP modulating ...therapies can cause constipation of varying frequency and severity. This variation might be due to the different mechanisms through which therapies block CGRP activity (e.g., blocking CGRP, or the CGRP receptor) with antibodies or receptor antagonists. The charcoal meal gastrointestinal transit assay was used to characterize constipation produced by these modes of therapy in transgenic mice expressing the human receptor activity–modifying protein 1 (hRAMP1) subunit of the CGRP receptor complex.
Methods
Male and female hRAMP1 mice were dosed with compound or vehicle and challenged with a charcoal meal suspension via oral gavage. The mice were then humanely euthanized and the proportion of the length of the large intestine that the charcoal meal had traveled indicated gastrointestinal transit.
Results
Antibody to the CGRP receptor produced % distance traveled (mean ± standard deviation) of 31.8 ± 8.2 (4 mg/kg; p = 0.001) and 33.2 ± 6.0 (30 mg/kg; p < 0.001) compared to 49.7 ± 8.3 (control) in female mice (n = 6–8), and 35.6 ± 13.5 (30 mg/kg, p = 0.019) compared to 50.2 ± 14.0 (control) in male mice (n = 10). Telcagepant (5 mg/kg, n = 8) resulted in % travel of 30.6 ± 14.7 versus 41.2 ± 8.3 (vehicle; p = 0.013) in male mice. Atogepant (3 mg/kg, n = 9) resulted in % travel of 30.6 ± 12.0, versus 41.2 ± 3.7 (control; p = 0.030) in female mice. The CGRP antibody galcanezumab (n = 7–10; p = 0.958 and p = 0.929) did not have a statistically significant effect.
Conclusions
These results are consistent with reported clinical data. Selectively blocking the CGRP receptor may have a greater impact on gastrointestinal transit than attenuating the activity of the ligand CGRP. This differential effect may be related to physiologically opposing mechanisms between the CGRP and AMY1 receptors, as the CGRP ligand antibody could inhibit the effects of CGRP at both the CGRP and AMY1 receptors.
Context:The classic androgen synthesis pathway proceeds via DHEA, androstenedione and testosterone to 5α-dihydrotestosterone (DHT). However, DHT synthesis can also be achieved by an alternative ...pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens and, in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid therapy is unknown.Objective:We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation and their response to conventional glucocorticoid (GC) therapy and modified release hydrocortisone.Methods:We employed urinary steroid metabolome profiling by gas chromatography-mass spectrometry for 24-h steroid excretion analysis, studying the impact of conventional GCs (hydrocortisone, prednisolone, dexamethasone) in 55 adults with CAH and 60 controls. We studied diurnal variation in steroid excreton by comparing 8-hourly collections (23:00-7:00h, 7:00-15:00h, 15:00-23:00h) in 16 CAH patients on conventional glucocorticoids and during six months of treatment with modified release hydrocortisone, Chronocort.Results:CAH patients on conventional GCs showed low excretion of classic pathway androgen metabolites but excess excretion of the alternative pathway signature metabolites 3α,5α-17-hydroxypregnanolone and 11β-hydroxyandrosterone. Chronocort reduced 17OHP and alternative pathway metabolite excretion to near normal levels more consistently than other GC preparations.Conclusions:Alternative pathway mediated androgen synthesis significantly contributes to androgen excess in CAH. Chronocort therapy appears superior to conventional GC therapy in controlling androgen synthesis via alternative pathways through attenuation of their major substrate, 17OHP.
Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic ...transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.
Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) ...as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.
The binding site for DETQ 2-(2,6-dichlorophenyl)-1-((1
,3
)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1
)-yl)ethan-1-one, a positive allosteric modulator (PAM) of ...the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111
-(6-
-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide, a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a
2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state
2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.
Dual orthosteric agonists of metabotropic glutamate 2 (mGlu2) and mGlu3 receptors are being developed as novel antipsychotic agents devoid of the adverse effects of conventional antipsychotics. ...Therefore, these drugs could be helpful for the treatment of psychotic symptoms associated with Alzheimer's disease (AD). In experimental animals, the antipsychotic activity of mGlu2/3 receptor agonists is largely mediated by the activation of mGlu2 receptors and is mimicked by selective positive allosteric modulators (PAMs) of mGlu2 receptors. We investigated the distinct influence of mGlu2 and mGlu3 receptors in mixed and pure neuronal cultures exposed to synthetic β-amyloid protein (Aβ) to model neurodegeneration occurring in AD. The mGlu2 receptor PAM, N-4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), devoid of toxicity per se, amplified Aβ-induced neurodegeneration, and this effect was prevented by the mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). LY566332 potentiated Aβ toxicity regardless of the presence of glial mGlu3 receptors, but it was inactive when neurons lacked mGlu2 receptors. The dual mGlu2/3 receptor agonist, (-)-2-oxa-4-aminobicyclo3.1.0exhane-4,6-dicarboxylic acid (LY379268), was neuroprotective in mixed cultures via a paracrine mechanism mediated by transforming growth factor-β1. LY379268 lost its protective activity in neurons grown with astrocytes lacking mGlu3 receptors, indicating that protection against Aβ neurotoxicity was mediated entirely by glial mGlu3 receptors. The selective noncompetitive mGlu3 receptor antagonist, (3S)-1-(5-bromopyrimidin-2-yl)-N-(2,4-dichlorobenzyl)pyrrolidin-3-amine methanesulfonate hydrate (LY2389575), amplified Aβ toxicity on its own, and, interestingly, unmasked a neurotoxic activity of LY379268, which probably was mediated by the activation of mGlu2 receptors. These data indicate that selective potentiation of mGlu2 receptors enhances neuronal vulnerability to Aβ, whereas dual activation of mGlu2 and mGlu3 receptors is protective against Aβ-induced toxicity.
Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu
) receptors, but small molecule tools are lacking. As part of ...our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu
and mGlu
receptor subtypes, a series of C4
-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo3.1.0hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu
and mGlu
receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-(3-methoxybenzoyl)aminobicyclo3.1.0hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu
receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu
revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu
receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu
receptors in vivo.
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