Biomimetic screening of class B G protein-coupled receptors

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a hallmark of complex and multifactorial psychiatric diseases such as anxiety and mood disorders. The 41-amino acid neuropeptide Corticotropin Releasing Hormone (CRH) is a major regulator of the mammalian stress response. Upon stressful stimuli, it binds to the Corticotropin Releasing Hormone Receptor 1 (CRHR1), a typical member of the class B GPCRs and a potential novel target for the therapeutic intervention in major depressive disorder. A precise understanding of the peptide-receptor interactions is an essential prerequisite towards the development of efficient CRHR1 specific antagonists. To chemically probe the molecular interaction of CRH with its cognate receptor, a high-throughput conjugation approach which mimics the natural activation mechanism for class B GPCRs was developed. Acetylene-tagged peptide libraries were synthesized and conjugated to high-affinity azide-modified carrier peptides using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstitute potent ligands and were tested in situ for modulation of the CRHR1 activity in a cell-based assay. This approach allows to (i) define the sequence motifs which are required for receptor activation or inhibition, (ii) identify the critical functional groups and investigate structure-activity-relationships, and (iii) develop novel optimized, highly potent peptide probes which are specific for the transmembrane domain of the receptor. The membrane recruitment by a high-affinity carrier peptide enhances the potency of tethered peptides and allows the initial testing of weak fragments that otherwise would be inactive. The biomimetic screening led to the discovery of transtressin, a highly modified and potent CRHR1 transmembrane domain-specific optimized agonist (EC50 = 4 nM). Beyond its intrinsic agonistic activity, transtressin is an essential tool for the pharmacological characterization of CRHR1 antagonists in competition assays

Loratadine and analogs: Discovery and preliminary SAR of inhibitors of the amino acid transporter B0AT2

B0AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain but little is otherwise known about its function. In order to identify inhibitors for this transporter we screened a library of different 3133 bioactive compounds. Loratadine, a clinically used histamine H1 receptor antagonist, was identified as a selective inhibitor of B0AT2 with an IC50 of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B0AT2 was confirmed by electrophysiology. A series of loratadine analogs was synthesized to get insight into the structure-activity relationships. Our studies provide the first chemical tool for B0AT2

Biomimetic Screening of Class-B G Protein-Coupled Receptors

The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF1R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF1 receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure–activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC50 = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo

Loratadine and Analogues: Discovery and Preliminary Structure–Activity Relationship of Inhibitors of the Amino Acid Transporter B⁰AT2

B⁰AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain, but little is otherwise known about its function. To identify inhibitors for this transporter, we screened a library of 3133 different bioactive compounds. Loratadine, a clinically used histamine H₁ receptor antagonist, was identified as a selective inhibitor of B⁰AT2 with an IC₅₀ of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B⁰AT2 was confirmed by electrophysiology. A series of loratadine analogues were synthesized to gain insight into the structure–activity relationships. Our studies provide the first chemical tool for B⁰AT2

A Polymorphism in the Crhr1 Gene Determines Stress Vulnerability in Male Mice

Chronic stress is a risk factor for psychiatric disorders but does not necessarily lead to uniform long-term effects on mental health, suggesting modulating factors such as genetic predispositions. Here we address the question whether natural genetic variations in the mouse CRH receptor 1 (Crhr1) locus modulate the effects of adolescent chronic social stress (ACSS) on long-term stress hormone dysregulation in outbred CD1 mice, which allows a better understanding of the currently reported genes x environment interactions of early trauma and CRHR1 in humans. We identified 2 main haplotype variants in the mouse Crhr1 locus that modulate the long-term effects of ACSS on basal hypothalamic-pituitary-adrenal axis activity. This effect is likely mediated by higher levels of CRHR1, because Crhr1 mRNA expression and CRHR1 binding were enhanced in risk haplotype carriers. Furthermore, a CRHR1 receptor antagonist normalized these long-term effects. Deep sequencing of the Crhr1 locus in CD1 mice revealed a large number of linked single-nucleotide polymorphisms with some located in important regulatory regions, similar to the location of human CRHR1 variants implicated in modulating gene x stress exposure interactions. Our data support that the described gene x stress exposure interaction in this animal model is based on naturally occurring genetic variations in the Crhr1 gene associated with enhanced CRHR1-mediated signaling. Our results suggest that patients with a specific genetic predisposition in the CRHR1 gene together with an exposure to chronic stress may benefit from a treatment selectively antagonizing CRHR1 hyperactivity