Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease

There are currently no treatments that can slow the progression of neurodegenerative diseases, such as Alzheimer’s disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein–biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimized by ensuring activating ligands maintain receptor phosphorylation/ arrestin-dependent signaling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signaling delivers neuroprotection that both extends normal animal behavior and prolongs the life span of prion-diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signaling would potentially show low adverse responses while delivering neuroprotection that will slow disease progression

Development and initial evaluation of a novel 11C-labeled PET tracer to image GPR84 expressing-myeloid cells during neuroinflammation

Purpose/Background: Neuroinflammation is a hallmark of many central nervous system (CNS) diseases including Alzheimer’s disease, multiple sclerosis, and stroke. Unfortunately, most previously investigated biomarkers for quantifying neuroinflammation in vivo using positron emission tomography (PET) imaging have several limitations. For example, the most widely evaluated PET tracers for neuroinflammation target the translocator protein 18 kDa (TSPO)— expressed indiscriminately on activated myeloid lineage cells, reactive astrocytes, and endothelial cells. In contrast, GPR84 is a G-protein coupled receptor (GPCR) expressed predominately on myeloid cells.1,2 Importantly, GPR84 is significantly upregulated specifically on pro-inflammatory myeloid cells following CNS injury or insult.3,4 Here we report the synthesis of a new PET tracer for GPR84 (11C-MGX-10S), based on a dihydropyrimido isoquinolinone inhibitor scaffold5, and its initial assessment in cells and mice. Methods: First, we synthesized the cold version of compound MGX-10S, an allosteric inhibitor of GPR84 and determined its kinetic inhibition constant (Ki) via a competition binding assay with 3H-G9543 (known GPR84 inhibitor). We then generated 11C-MGX-10S by alkylating the phenolic precursor with 11C-methyl iodide in DMF (500 L) for 3 min at 65 °C, using 1M NaOH as a base (Fig 1A). Subsequently we evaluated the binding specificity of 11C-MGX-10S using stable human-GPR84-expressing human endothelial kidney (hGPR84-HEK293) cells versus parental control cells in quadruplicate. Lastly, we assessed the in vivo kinetics, distribution, and blood-brain-barrier (BBB) permeability of 11C-MGX-10S in healthy C57BL/6 mice (n=4) using dynamic PET/CT imaging. Results: The 1,4-dioxane ring linked compound (MGX-10S) was shown to have a Ki of 8.22 nM in the competitive binding assay. Automated module synthesis of 11C-MGX-10S was completed in 60 minutes, with a radiochemical yield of 5.07 1.42% (non-decay corrected), radiochemical purity >99%, and molar activity of 6328 396 mCi/mol. Cell binding studies revealed 14.5 fold higher binding of 11C-MGX-10S to hGPR84-HEK293 cells compared to parental HEK293 cells after 40 minutes of incubation (P <0.0001, n= 4). Blocking with GPR84 antagonist (GLPG1205, 35 M), significantly reduced tracer binding to hGPR84-HEK293 cells by 91.7%, demonstrating high specificity of 11C-MGX-10S (P <0.0001, n= 4). A similar pattern of binding was observed for tracer binding after 20 minutes incubation (Fig 1B). Whole brain time-activity curves (Fig 1C), generated by analyzing PET/CT images co-registered with a mouse brain atlas, demonstrate rapid entry of 11C-MGX-10S into the brain with an average peak uptake of 5.90 0.89 %ID/g (n=4) within 22.5 seconds, decreasing to 1.89 0.35 %ID/g by the end of the 60 minute scan. Whole body 3D maximum intensity projection PET/CT images (Fig 1D) illustrate robust brain signal at 0-5 min with increasing renal/hepatic clearance, in addition to low background signal in all other areas of the body, by the later time point (30-60 min). Conclusion: We have successfully synthesized and are currently evaluating a novel PET tracer for the measurement of GPR84 expression. Our data demonstrate the promise of 11C-MGX-10S as a highly specific tracer for detecting GPR84-expressing myeloid cells in vitro and in vivo. Further studies are currently underway to explore the utility of 11C-MGX-10S for imaging GPR84-associated innate immune activation in rodent models of CNS diseases

Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease

Significance The M1 muscarinic acetylcholine receptor (M1-receptor) plays a crucial role in learning and memory and is a validated drug target for the treatment of Alzheimer’s disease (AD). Furthermore, M1-receptor ligands have been demonstrated to display disease-modifying effects in preclinical models of neurodegenerative disease. By employing a genetic mouse model expressing a G protein–biased M1-receptor in combination with a mouse model of terminal neurodegenerative disease, we demonstrate here that the M1-receptor exerts an inherent neuroprotective activity that is dependent on its phosphorylation status. Thus, in AD drug development programs, M1-receptor ligands that maintain the receptor phosphorylation status will be more likely to lead to beneficial neuroprotective outcomes.</jats:p

PET Imaging of Innate Immune Activation Using 11C Radiotracers Targeting GPR84

Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11C-MGX-10S and 11C-MGX-11Svia carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by &gt;90%, demonstrating their high specificity for GPR84 in vitro. In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11C-MGX-10S compared to 11C-MGX-11S. Subsequent use of 11C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11C-DPA-713-an existing radiotracer used to image innate immune activation in clinical research studies-11C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology

The use of spatial intensity distribution analysis to examine G protein-coupled receptor oligomerization

Spatial Intensity Distribution Analysis (SpIDA) is a new approach for detecting protein oligomerization states that can be applied not only to live cells but also fixed cells and native tissue. This approach is based on the generation of pixel-integrated fluorescence intensity histograms from laser scanning fluorescence microscopy images. These histograms are then fit with super-Poissonian distribution functions to obtain density maps and quantal brightness values of the fluorophore that are used to determine the proportions of monomer and dimers/oligomers of the fluorophore-tagged protein. In this chapter we describe SpIDA and highlight its advantages compared to other biochemical or biophysical approaches. We provide guidelines that should be useful to readers who wish to perform SpIDA measurements and describe the application of SpIDA as a post-acquisition imaging histogram analysis software tool to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at the surface of mammalian cells in order to define the steady-state proportion of monomeric and dimeric/oligomeric forms and how this may be regulated by cellular challenges such as ligand treatment

PET Imaging of Innate Immune Activation Using ¹¹C Radiotracers Targeting GPR84

Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11C-MGX-10S and 11C-MGX-11S via carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by >90%, demonstrating their high specificity for GPR84 in vitro. In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11C-MGX-10S compared to 11C-MGX-11S. Subsequent use of 11C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11C-DPA-713an existing radiotracer used to image innate immune activation in clinical research studies11C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology

PET Imaging of Innate Immune Activation Using ¹¹C Radiotracers Targeting GPR84

Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11C-MGX-10S and 11C-MGX-11S via carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by >90%, demonstrating their high specificity for GPR84 in vitro. In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11C-MGX-10S compared to 11C-MGX-11S. Subsequent use of 11C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11C-DPA-713an existing radiotracer used to image innate immune activation in clinical research studies11C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology

PET Imaging of Innate Immune Activation Using ¹¹C Radiotracers Targeting GPR84

Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11C-MGX-10S and 11C-MGX-11S via carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by >90%, demonstrating their high specificity for GPR84 in vitro. In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11C-MGX-10S compared to 11C-MGX-11S. Subsequent use of 11C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11C-DPA-713an existing radiotracer used to image innate immune activation in clinical research studies11C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology