A small-molecule inhibitor of lectin-like oxidized LDL receptor-1 acts by stabilizing an inactive receptor tetramer state.

The C-type lectin family member lectin-like oxidized LDL receptor-1 (LOX-1) has been object of intensive research. Its modulation may offer a broad spectrum of therapeutic interventions ranging from cardiovascular diseases to cancer. LOX-1 mediates uptake of oxLDL by vascular cells and plays an important role in the initiation of endothelial dysfunction and its progression to atherosclerosis. So far only a few compounds targeting oxLDL-LOX-1 interaction are reported with a limited level of characterization. Here we describe the identification and characterization of BI-0115, a selective small molecule inhibitor of LOX-1 that blocks cellular uptake of oxLDL. Identified by a high throughput screening campaign, biophysical analysis shows that BI-0115 binding triggers receptor inhibition by formation of dimers of the homodimeric ligand binding domain. The structure of LOX-1 bound to BI-0115 shows that inter-ligand interactions at the receptor interfaces are key to the formation of the receptor tetramer thereby blocking oxLDL binding.

Muscle to Brain Partitioning as Measure of Transporter-Mediated Efflux at the Rat Blood-Brain Barrier and Its Implementation into Compound Optimization in Drug Discovery.

Movement of xenobiotic substances across the blood-brain barrier (BBB) is tightly regulated by various transporter proteins, especially the efflux transporters P-glycoprotein (P-gp/MDR1) and breast cancer resistance protein (BCRP). Avoiding drug efflux at the BBB is a unique challenge for the development of new central nervous system (CNS) drugs. Drug efflux at the BBB is described by the partition coefficient of unbound drug between brain and plasma (Kp,uu,brain) which is typically obtained from in vivo and often additionally in vitro measurements. Here, we describe a new method for the rapid estimation of the in vivo drug efflux at the BBB of rats: the measurement of the partition coefficient of a drug between brain and skeletal muscle (Kp,brain/muscle). Assuming a closely similar distribution of drugs into the brain and muscle and that the efflux transporters are only expressed in the brain, Kp,brain/muscle, similar to Kp,uu,brain, reflects the efflux at the BBB. The new method requires a single in vivo experiment. For 64 compounds from different research programs, we show the comparability to other approaches used to obtain Kp,uu,brain. P-gp- and BCRP-overexpressing cell systems are valuable in vitro tools for prescreening. Drug efflux at the BBB can be most accurately predicted based on a simple algorithm incorporating data from both in vitro assays. In conclusion, the combined use of our new in vivo method and the in vitro tools allows an efficient screening method in drug discovery with respect to efflux at the BBB.

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. Kp,uu,brain and Kp,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, Kp,uu,brain for the BCRP substrates was low. In contrast, Kp,uu,CSF for both BCRP substrates was close to unity, resulting in Kp,uu,CSF/Kp,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

Ultrafast and Predictive Mass Spectrometry-Based Autotaxin Assays for Label-Free Potency Screening.

Autotaxin (ATX) is a promising drug target for the treatment of several diseases, such as cancer and fibrosis. ATX hydrolyzes lysophosphatidyl choline (LPC) into bioactive lysophosphatidic acid (LPA). The potency of ATX inhibitors can be readily determined by using fluorescence-based LPC derivatives. While such assays are ultra-high throughput, they are prone to false positives compared to assays based on natural LPC. Here we report the development of ultrafast mass spectrometry-based ATX assays enabling the measurement of data points within 13 s, which is 10 times faster than classic liquid chromatography-mass spectrometry. To this end, we set up a novel in vitro and whole-blood assay. We demonstrate that the potencies determined with these assays are in good agreement with the in vivo efficacy and that the whole-blood assay has the best predictive power. This high-throughput label-free approach paired with the translatable data quality is highly attractive for appropriate guidance of medicinal chemists for constructing strong structure-activity relationships.