LDL delivery of microbial small RNAs drives atherosclerosis through macrophage TLR8

Macrophages present a spectrum of phenotypes that mediate both the pathogenesis and resolution of atherosclerotic lesions. Inflammatory macrophage phenotypes are pro-atherogenic, but the natural factors that instigate this polarization are largely unknown. Here, we demonstrate that microbial small RNAs (msRNA) are enriched on LDL and drive pro-inflammatory macrophage polarization and cytokine secretion via activation of the ribonucleic acid sensor toll-like receptor 8 (TLR8). Removal of msRNA cargo during LDL re-constitution yields particles that readily promote sterol loading but fail to stimulate inflammatory activation. Competitive antagonism of TLR8 with non-targeting locked nucleic acids (nt-LNA) was found to prevent nLDL-induced macrophage polarization in vitro, and re-organize lesion macrophage phenotypes in vivo, as determined by single-cell RNA sequencing. Critically, this was associated with reduced disease burden in distinct mouse models of atherosclerosis. These results identify LDL-msRNA as instigators of atherosclerosis-associated inflammation and support alternative functions of LDL beyond cholesterol transport.

MODL-06. PRECLINICAL EFFICACY OF THE IMIPRIDONE ONC-206 AGAINST MEDULLOBLASTOMA

Treatment for medulloblastoma (MB) is typically ineffective for MYC amplified or metastatic SHH, Group 3 and 4 subgroups. Promising preclinical and clinical results have been obtained in brain cancers treated with ONC-201, a selective antagonist of DRD2, a G-protein coupled receptor that regulates prosurvival pathways. Herein, we report the activity of ONC-201 and ONC-206, which has increased non-competitive antagonism of DRD2, against MB. We treated three different MB cell types representative of SHH- and Group 3-like cells, with varied levels of DRD2 expression, and consistently observed increased cell death in a dose-dependent manner at lower doses of ONC-206 compared to ONC-201. We also evaluated ClpP as an additional drug target in MB. ClpP is a mitochondrial protease that has been shown to directly bind and be activated by ONC 201, and is highly expressed at the protein level across pediatric MB, malignant glioma and ATRT, but not normal brain. We observed that similar to ONC-201, ONC-206 treatment of MB cells induces the restoration of mitochondrial membrane potential to the non-proliferative state, degradation of the mitochondrial substrate SDHB, reduction in survivin and elevation in ATF4 (integrated stress response). Importantly, ONC-206 treatment induced significant cell death of patient-derived SHH, WNT, and Group 3 tumors ex vivo and Group 4 cells in vitro, while having no observable toxicity in normal brain. Efficacy studies of ONC-206 against MB in vivo will be reported in preparation for a planned Phase I study of ONC-206 in children with malignant brain tumors.

0487 Effects of Suvorexant on Sleep Architecture in Patients with Alzheimer’s Disease and Insomnia

Introduction Suvorexant, an orexin receptor antagonist that enables sleep to occur via competitive antagonism of wake-promoting orexins, improved total sleep time (TST) in a sleep laboratory polysomnography (PSG) study of patients with AD and insomnia. Here we report on the effects of suvorexant on sleep architecture in the study. Methods This was a randomized, double-blind, 4-week trial (ClinicalTrials.gov NCT02750306). Participants who met diagnostic criteria for both probable AD dementia (of mild to moderate severity) and insomnia were randomized to suvorexant 10mg (could be increased to 20mg based on clinical response) or matching placebo. Overnight sleep laboratory PSG was performed on 3 nights: screening, baseline, and Night-29 (last night of dosing). Suvorexant differences from placebo in changes-from-baseline at Night-29 for sleep architecture were analyzed as exploratory endpoints. Results A total of 274 participants were included in the analysis (suvorexant N=135, placebo N=139). At Night-29, suvorexant improved TST by 28 minutes versus placebo (p=0.001). There were no significant differences between suvorexant and placebo in the % of TST spent in REM (1.3%, 95% CI: -0.5, 3.0), N1 (0.6%, 95% CI: -1.2, 2.5), N2 (-1.0%, 95% CI: -3.2, 1.2), or N3 (-0.6%, 95% CI: -1.8, 0.6). There was no significant difference between suvorexant and placebo in latency to REM (-5.4 minutes, 95% CI: -23.4, 12.7). Conclusion Suvorexant improves TST without altering the underlying sleep architecture in AD patients with insomnia. Support Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA

EXTH-33. RECEPTOR PHARMACOLOGY OF ONC201: THE FIRST BITOPIC DRD2 ANTAGONIST FOR CLINICAL NEURO-ONCOLOGY

ONC201 has exhibited durable tumor regressions in midline high grade glioma patients. G protein-coupled receptor (GPCR) dopamine receptor D2 (DRD2) is overexpressed in high grade gliomas, controls several oncogenic mechanisms, and its antagonism causes tumor cell apoptosis. Using bioinformatic and GPCR screens, we previously demonstrated selective DRD2/3 antagonism by ONC201 at therapeutic concentrations that translates into a wide therapeutic window, unlike antipsychotics that also target other dopamine receptors and GPCRs (Prabhu et al. CCR 2019 and Madhukar et al. Nat Comm in press). Here, we identify hallmarks of non-competitive DRD2 antagonism by ONC201 in β-arrestin, cAMP and radioligand competition assays. ONC201 caused both a shift in the IC50 and repression of Emax in the dopamine-dose-response curves for DRD2. Schild analysis and fitting these data to an operational model of allostery were consistent with non-competitive antagonism. Alanine scanning mutagenesis of DRD2 identified 6 orthosteric and allosteric residues that are critical for ONC201-mediated antagonism. The residues identified were predominantly conserved in DRD2/3 and were not relevant for antagonism by antipsychotics further explaining the unique selectivity of ONC201. Molecular docking revealed orthosteric interactions at TM-II and an extended binding pocket into an allosteric site. Other critical residues were clustered at an allosteric area at the interface of TM-IV and –V that has been shown to mediate the DRD2 homodimer interface. Point mutations at either site decreased the affinity of ONC201 in competition assays with radiolabeled methyl-spiperone, suggesting cooperativity between these topographically distinct sites. In summary, ONC201 causes a mixed non-competitive and competitive antagonism of DRD2 consistent with a bitopic mechanism of action that may explain its unique selectivity, safety, and anti-cancer activity in clinical trials as the first compound to target this receptor for clinical neuro-oncology. Non-competitive DRD2 antagonism may be critical for ONC201 anti-tumor efficacy in dopamine-rich microenvironments such as midline gliomas.

Phosphate acts directly on the calcium-sensing receptor to stimulate parathyroid hormone secretion

Extracellular phosphate regulates its own renal excretion by eliciting concentration-dependent secretion of parathyroid hormone (PTH). However, the phosphate-sensing mechanism remains unknown and requires elucidation for understanding the aetiology of secondary hyperparathyroidism in chronic kidney disease (CKD). The calcium-sensing receptor (CaSR) is the main controller of PTH secretion and here we show that raising phosphate concentration within the pathophysiologic range for CKD significantly inhibits CaSR activity via non-competitive antagonism. Mutation of residue R62 in anion binding site-1 abolishes phosphate-induced inhibition of CaSR. Further, pathophysiologic phosphate concentrations elicit rapid and reversible increases in PTH secretion from freshly-isolated human parathyroid cells consistent with a receptor-mediated action. The same effect is seen in wild-type murine parathyroid glands, but not in CaSR knockout glands. By sensing moderate changes in extracellular phosphate concentration, the CaSR represents a phosphate sensor in the parathyroid gland, explaining the stimulatory effect of phosphate on PTH secretion.