Cawston, Erin E.

Abstract Background Clinicians’ views on clinical readiness of Alzheimer's disease (AD) blood biomarkers (BBM) are not well understood. Method The Alzheimer's Association International Society to Advance Alzheimer's Research and Treatment Biofluid‐Based Biomarkers Professional Interest Area conducted a survey to elicit clinician opinions on AD BBM implementation, including contexts of use, assay selection, reporting and result interpretation. Result Clinician respondents ( n = 212) practiced in Europe (56%), North America (24%), Caribbean and Central or South America (11%) and other continents (9%). Most respondents were medical doctors (80%) practicing in secondary or tertiary care (88%) (Table 1). For 56% of respondents, CSF AD biomarkers or amyloid PET were accessible, but 48% agreed and 52% disagreed with implementation of AD BBM in any clinical context (Table 2). Respondents emphasized the need for data from diverse populations and educational resources to support test interpretation. Conclusion Surveyed clinicians generally agreed with published appropriate use recommendations but were divided on AD BBM readiness for clinical use.

Abstract INTRODUCTION Clinicians’ views on the clinical readiness of Alzheimer's disease (AD) blood biomarkers (BBMs) are not well understood. METHODS The Alzheimer's Association International Society to Advance Alzheimer's Research and Treatment Biofluid‐Based Biomarkers Professional Interest Area conducted a survey to elicit clinician opinions on AD BBM implementation, including contexts of use, assay selection, reporting, and result interpretation. RESULTS Clinician respondents ( n = 212) practiced in Europe (56%), North America (24%), the Caribbean and Central/South America (11%), and other continents (9%). Most respondents were medical doctors (80%) practicing in secondary or tertiary care (88%). For 56%, cerebrospinal fluid AD biomarkers or amyloid positron emission tomography were accessible, but 48% agreed and 52% disagreed with the implementation of AD BBMs in any clinical context. Respondents emphasized the need for data from diverse populations and educational resources to support test interpretation. DISCUSSION Surveyed clinicians generally agreed with published appropriate use recommendations but were divided on AD BBM readiness for clinical use. Highlights A survey of clinicians was conducted regarding clinical readiness of Alzheimer's disease (AD) blood biomarkers (BBMs). Views were split on AD BBM clinical readiness: 48% agreed, 52% disagreed. Most responders supported AD BBM use for treatment decisions. Most responders opposed AD BBM testing in asymptomatic individuals. Test performance data and educational materials to aid interpretation were of high importance.

Receptor ligands, identified as antagonists, based on the absence of stimulation of signaling, can rarely stimulate receptor internalization. d-Tyr-Gly-[(Nle28,31,d-Trp30)CCK-26–32]-2-phenylethyl ester (d-Trp-OPE) is such a ligand that binds to the cholecystokinin (CCK) receptor and stimulates internalization. Here, the molecular basis of this trafficking event is explored, with the assumption that ligand binding initiates conformational change, exposing an epitope to direct endocytosis. Ligand-stimulated internalization was studied morphologically using fluorescent CCK and d-Trp-OPE. d-Trp-OPE occupation of Chinese hamster ovary cell receptors stimulated internalization into the same region as CCK. Arrestin-biased action was ruled out using morphological translocation of fluorescent arrestin 2 and arrestin 3, moving to the membrane in response to CCK, but not d-Trp-OPE. Possible roles of the carboxyl terminus were studied using truncated receptor constructs, eliminating the proline-rich distal tail, the serine/threonine-rich midregion, and the remainder to the vicinal cysteines. None of these constructs disrupted d-Trp-OPE-stimulated internalization. Possible contributions of transmembrane segments were studied using competitive inhibition with peptides that also had no effect. Intracellular regions were studied with a similar strategy using coexpressing cell lines. Peptides corresponding to ends of each loop region were studied, with only the peptide at the carboxyl end of the third loop inhibiting d-Trp-OPE-stimulated internalization but having no effect on CCK-stimulated internalization. The region contributing to this effect was refined to peptide 309–323, located below the recognized G protein-association motif. While a receptor in which this segment was deleted did internalize in response to d-Trp-OPE, it exhibited abnormal ligand binding and did not signal in response to CCK, suggesting an abnormal conformation and possible mechanism of internalization distinct from that being studied. This interpretation was further supported by the inability of peptide 309–323 to inhibit its d-Trp-OPE-stimulated internalization. Thus the 309–323 region of the type 1 CCK receptor affects antagonist-stimulated internalization of this receptor, although its mechanism and interacting partner are not yet clear.

The success in screening for drug candidates is highly dependent on the power of the strategy implemented. In this work, we report and characterize a novel fluorescent benzodiazepine antagonist of the type 1 cholecystokinin receptor (3-(3-(7-fluoro-1-(2-isopropyl(4-methoxyphenyl)amino)-2-oxoethyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[ b ][1,4]-diazepin-3-yl)ureido)benzoic acid) that can be used as a receptor ligand in a fluorescence polarization assay, which is ideally suited for the identification of small molecule allosteric modulators of this physiologically important receptor. By binding directly to the small molecule-docking region within the helical bundle of this receptor, this indicator can be displaced by many small molecule candidate drugs, even those that might not affect the binding of an orthosteric cholecystokinin-like peptide ligand. The biological, pharmacological, and fluorescence properties of this reagent are described, and proof-of-concept is provided in a fluorescence polarization assay utilizing this fluorescent benzodiazepine ligand.