Identifying Modulators of CXC Receptors 3 and 4 with Tailored Selectivity Using Multi-Target Docking

The G protein-coupled receptors of the C–X–C subfamily form a group among the chemokine receptors whose endogenous ligands are peptides with a common Cys–X–Cys motif. The CXC chemokine receptors 3 and 4 (CXCR3, CXCR4), which are investigated in this study, are linked to severe diseases such as cancer, multiple sclerosis, and HIV infections. Of particular interest, this receptor pair potentially forms a target for a polypharmacological drug treatment. Considering known ligands from public databases, such dual binders have not been identified yet. We therefore applied large-scale docking to the structure of CXCR4 and a homology model of CXCR3 with the goal to predict such dual binders, as well as compounds selective for either one of the receptors. Using signaling and biochemical assays, we showed that more than 50% of these predictions were correct in each category, yielding ligands with excellent binding efficiencies. These results highlight that docking is a suitable tool for the identification of ligands with tailored binding profiles to GPCRs, even when using homology models. More importantly, we present novel CXCR3–CXCR4 dual modulators that might pave the road to understanding the mechanisms of polypharmacological inhibition of these receptors

Boronic Acids as Probes for Investigation of Allosteric Modulation of the Chemokine Receptor CXCR3

The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109^2.60 and Lys300^7.35 inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid <b>14</b> as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor–ligand interactions and induction of ligand-biased signaling

Additional file 1 of SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency

Additional file 1: Supplemental Figure 1. Screening of healthy donors 1-93 for WT allele. Supplemental Figure 2. Screening of healthy donors 1-93 for mutant allele. Supplemental Figure 3. Screening of healthy donors 94-186 for WT allele. Supplemental Figure 4. Screening of healthy donors 94-186 for mutant allele. Supplemental Figure 5. Screening of healthy donors 187-279 for WT allele. Supplemental Figure 6. Screening of healthy donors 187-279 for mutant allele. Supplemental Figure 7. Screening of healthy donors 280-360 for WT allele. Supplemental Figure 8. Screening of healthy donors 280-360 for mutant allele. Supplemental Figure 9. Repetition of PCRs with no or positive results in first PCR

Discovery and Characterization of Biased Allosteric Agonists of the Chemokine Receptor CXCR3

In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound <b>11</b> (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, <b>14</b> (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment. Concomitant structure–activity relationship studies indicated very steep structure–activity relationships, which steer the ligand bias between the β-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further development and rational design of strongly biased allosteric agonists of CXCR3