Biology subjects

Windhorst, A. D.

Publications and source records attributed to Windhorst, A. D..

3 recordsLinked to original sources

bioRxiv2024

Characterization of AZ12464237 as a high affinity, non-nucleotide antagonist radioligand for the P2Y12 receptor

The purinergic receptor P2Y12 (P2Y12R) is a well-recognized target for anti-thrombotic agents. This receptor is also expressed in microglia, where it plays a key role in neuroinflammation and microglia activation. To investigate P2Y12R-mediated actions in the central nervous system (CNS), the development of novel brain-penetrant ligands is essential, along with further in vitro studies. A radiolabeled, easily accessible tool compound would significantly advance such drug discovery efforts. Herein, we describe the 3H-labeling of a non-nucleotide P2Y12R antagonist AZ12464237, and its in vitro binding properties to the receptor in membrane preparations form transfected cells, as well as on mouse brain tissues. The radioligand shows high affinity toward both the human and rat P2Y12R in transfected cells, with Kd values of 3.12 {+/-} 0.70 nM (human) and 16.6 {+/-} 3.4 nM (rat), as determined by saturation binding studies. The binding kinetics of [3H]AZ12464237 are rapid with a short target residence time ([~]1 min). We further confirmed the selectivity of the radioligand by performing competitive displacement studies, in which reported P2Y12R ligands and other P2Y receptors ligands were tested for binding against [3H]AZ12464237. Additionally, the radioligand proved useful for in vitro autoradiography studies on mouse brain tissues, although a small amount of off-target binding was observed in P2Y12R knock-out mice. This could be traced to glycogen synthase kinase 3 . Considering the growing interest in P2Y12R as a biomarker for microglia activation, [3H]AZ12464237 represents a promising tool for in vitro studies, including screening assays aimed at identifying novel P2Y12R ligands for CNS applications.

pharmacology and toxicology↗

bioRxiv2023

Pharmacological characterization and radiolabeling ofVUF15485, a high-affinity small-molecule agonist for theatypical chemokine receptor ACKR3

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this {beta}-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a BRET-based {beta}-arrestin2 recruitment assay VUF15485 acts as an ACKR3 agonist with high potency (pEC50 = 7.6) and shows a similar extent of receptor activation compared to CXCL12 when using a newly developed, FRET-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485 was tested against a (atypical) chemokine receptor panel (agonist and antagonist mode) and proves to be selective for ACKR3. VUF15485 was subsequently labeled with tritium at one of its methoxy groups affording [3H]VUF15485. The small-molecule agonist radioligand binds saturably and with high affinity to human ACKR3 (Kd = 8.2 nM). [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (RT < 2 min) for its binding to ACKR3. Displacement of [3H]VUF15485 binding to membranes of HEK293T cells, transiently expressing ACKR3, with a number of CXCR3, CXCR4 or ACKR3 small-molecule ligands confirmed the ACKR3 profile of the [3H]VUF15485 binding site. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose VUF15485 to bind in the major and the minor binding pocket of ACKR3.

pharmacology and toxicology↗

bioRxiv2019

PET and CSF amyloid-β status are differently predicted by patient features: Information from discordant cases

BackgroundAmyloid-{beta} PET and CSF A{beta}42 yield discordant results in 10-20% of patients, possibly providing unique information. Although the predictive power of demographic, clinical, genetic and imaging features for amyloid-positivity has previously been investigated, it is unknown whether these features differentially predict amyloid-{beta} status based on PET or CSF, or whether this differs by disease stage.\n\nMethodsWe included 768 patients (subjective cognitive decline (SCD, n=194), mild cognitive impairment (MCI, n=127), dementia (AD and non-AD, n=447) with amyloid-{beta} PET and CSF A{beta}42 measurement within one year. 97(13%) patients had discordant PET/CSF amyloid-{beta} status. We performed parallel random forest models predicting separately PET and CSF status using 17 patient features (demographics, APOE4 positivity, CSF (p)tau, cognitive performance, and MRI visual ratings) in the total patient group and stratified by syndrome diagnosis. Thereafter, we selected features with the highest variable importance measure (VIM) as input for logistic regression models, where amyloid status on either PET or CSF was predicted by (i) the selected patient feature, and (ii) the patient feature adjusted for the status of the other amyloid modality.\n\nResultsAPOE4, CSF tau and p-tau had highest VIM for PET and CSF in all groups. In the amyloid-adjusted logistic regression models, p-tau was a significant predictor for PET-amyloid in SCD (OR=1.02[1.01-1.04], pFDR=0.03), MCI (OR=1.05[1.02-1.07], pFDR<0.01) and dementia (OR=1.04[1.03-1.05], pFDR<0.001), but not for CSF-amyloid. APOE4 (OR=3.07[1.33-7.07], punc<0.01) was associated with CSF-amyloid in SCD, while it was only predictive for PET-amyloid in MCI (OR=9.44[2.93,30.39], pFDR<0.01). Worse MMSE scores (OR=1.21[1.03-1.41], punc=0.02) were associated to CSF-amyloid status in SCD, whereas worse memory (OR=1.17[1.05-1.31], pFDR=0.02) only predicted PET positivity in dementia.\n\nConclusionAmyloid status based on either PET or CSF was predicted by different patient features and this varied by disease stage, suggesting that PET-CSF discordance yields unique information. The stronger associations of both APOE4 carriership and worse memory z-scores with CSF-amyloid in SCD suggests that CSF-amyloid is more sensitive early in the disease course. The higher predictive value of CSF p-tau for a positive PET scan suggests that PET is more specific to AD pathology. These findings can influence the choice between amyloid biomarkers in future studies or trials.

neuroscience↗