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B(0)AT2, 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 B(0)AT2 with an IC50 of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B(0)AT2 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 B(0)AT2.

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 (CRF(1)R), 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 CRF(1) 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 (EC(50) = 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.

In the sweet spot: Cocrystal structures of engineered neurotransmitter transporters reveal the binding mode of commonly prescribed antidepressants, providing a basis for a rational drug design for this class of proteins. The picture shows the structure of the dopamine transporter of Drosophila melanogaster in complex with the antidepressant nortriptyline.

The cyclic adenosine monophosphate responsive element binding (CREB) protein is a transcription factor involved in different neural processes, such as learning, neuroplasticity and the modulation of stress response. Alterations in the CREB pathway have been observed in the brains and lymphocytes of patients affected by depression and alcohol abuse. Given the lack of information, our study aimed at investigating the levels of total and activated CREB protein in lympho-monocytes of 20 drug-free patients suffering from post-traumatic stress disorders (PTSD), as compared with 20 healthy control subjects.Blood samples were collected from patients and healthy control subjects on the same time and lympho-monocytes were isolated according to standardized methods. CREB protein levels and activation were measured by means of immunoenzymatic techniques.The results showed that PTSD patients had statistically lower levels of total CREB protein in lympho-monocytes than healthy control subjects. On the contrary, no difference in the activated CREB protein was detected.These findings, albeit preliminary, would suggest that the CREB pathway might be involved in the pathophysiology of PTSD. Future studies should clarify if specific PTSD symptom clusters might be related to the CREB pathway.

In the sweet spot: Cocrystal structures of engineered neurotransmitter transporters reveal the binding mode of commonly prescribed antidepressants, providing a basis for a rational drug design for this class of proteins. The picture shows the structure of the dopamine transporter of Drosophila melanogaster in complex with the antidepressant nortriptyline.

In this work we reported the synthesis of new potential ATI antagonists through the replacement of the biphenyltetrazole portion of the losartan with biphenylaldoximic (2) and phenylsalicylaldoximic (3a) moieties. Moreover, also the trifluoromethylpyrazole analogue of 3a (3b) was prepared. The new compounds synthesized were evaluated for their AT1 affinity through binding assay carried out on rat liver membranes using [ 125 I]Sar1,Ile8-angiotensina II as radioligand.

Taking as model compound the amido-derivative 1 described in the literature from Duncia's group as a good AngII antagonist, we have synthesized a new series of compounds (2-7) in which the principal structural variations reside in the inversion of the amidic sequence between the two phenyl ring and/or in the type of heteroaromatic substituent linked to this portion. The new compounds synthesized were evaluated for their AT1 affinity through binding assays carried out on rat liver membranes using ( 125 I)Sar1,Ile8-angiotensin II as radioligand.