Constrained catecholamines gain ß2AR selectivity through allosteric effects on pocket dynamics.

G protein-coupled receptors (GPCRs) within the same subfamily often share high homology in their orthosteric pocket and therefore pose challenges to drug development. The amino acids that form the orthosteric binding pocket for epinephrine and norepinephrine in the ß1 and ß2 adrenergic receptors (ß1AR and ß2AR) are identical. Here, to examine the effect of conformational restriction on ligand binding kinetics, we synthesized a constrained form of epinephrine. Surprisingly, the constrained epinephrine exhibits over 100-fold selectivity for the ß2AR over the ß1AR. We provide evidence that the selectivity may be due to reduced ligand flexibility that enhances the association rate for the ß2AR, as well as a less stable binding pocket for constrained epinephrine in the ß1AR. The differences in the amino acid sequence of the extracellular vestibule of the ß1AR allosterically alter the shape and stability of the binding pocket, resulting in a marked difference in affinity compared to the ß2AR. These studies suggest that for receptors containing identical binding pocket residues, the binding selectivity may be influenced in an allosteric manner by surrounding residues, like those of the extracellular loops (ECLs) that form the vestibule. Exploiting these allosteric influences may facilitate the development of more subtype-selective ligands for GPCRs.

An allosteric modulator binds to a conformational hub in the ß2 adrenergic receptor.

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the ß2-adrenergic receptor (ß2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for ß2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

Structure-Based Design and Discovery of New M2 Receptor Agonists.

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

Development of molecular tools based on the dopamine D3 receptor ligand FAUC 329 showing inhibiting effects on drug and food maintained behavior.

Dopamine D3 receptor-mediated networks have been associated with a wide range of neuropsychiatric diseases, drug addiction and food maintained behavior, which makes D3 a highly promising biological target. The previously described dopamine D3 receptor ligand FAUC 329 (1) showed protective effects against dopamine depletion in a MPTP mouse model of Parkinson's disease. We used the radioligand [18F]2, a [18F]fluoroethoxy substituted analog of the lead compound 1 as a molecular tool for visualization of D3-rich brain regions including the islands of Calleja. Furthermore, structural modifications are reported leading to the pyrimidylpiperazine derivatives 3 and 9 displaying superior subtype selectivity and preference over serotonergic receptors. Evaluation of the lead compound 1 on cocaine-seeking behavior in non-human primates showed a substantial reduction in cocaine self-administration behavior and food intake.

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αßγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

Dual targeting of adenosine A(2A) receptors and monoamine oxidase B by 4H-3,1-benzothiazin-4-ones.

Blockade of A2A adenosine receptors (A2AARs) and inhibition of monoamine oxidase B (MAO-B) in the brain are considered attractive strategies for the treatment of neurodegenerative diseases such as Parkinson's disease (PD). In the present study, benzothiazinones, e.g., 2-(3-chlorophenoxy)-N-(4-oxo-4H-3,1-benzothiazin-2-yl)acetamide (13), were identified as a novel class of potent MAO-B inhibitors (IC50 human MAO-B: 1.63 nM). Benzothiazinones with large substituents in the 2-position, e.g., methoxycinnamoylamino, phenylbutyrylamino, or chlorobenzylpiperazinylbenzamido residues (14, 17, 27, and 28), showed high affinity and selectivity for A2AARs (Ki human A2AAR: 39.5-69.5 nM). By optimizing benzothiazinones for both targets, the first potent, dual-acting A2AAR/MAO-B inhibitors with a nonxanthine structure were developed. The best derivative was N-(4-oxo-4H-3,1-benzothiazin-2-yl)-4-phenylbutanamide (17, Ki human A2A, 39.5 nM; IC50 human MAO-B, 34.9 nM; selective versus other AR subtypes and MAO-A), which inhibited A2AAR-induced cAMP accumulation and showed competitive, reversible MAO-B inhibition. The new compounds may be useful tools for validating the A2AAR/MAO-B dual target approach in PD.

The soluble guanylyl cyclase inhibitor NS-2028 reduces vascular endothelial growth factor-induced angiogenesis and permeability.

Nitric oxide (NO) is known to promote vascular endothelial growth factor (VEGF)-stimulated permeability and angiogenesis. However, effector molecules that operate downstream of NO in this pathway remain poorly characterized. Herein, we determined the effect of soluble guanylyl cyclase (sGC) inhibition on VEGF responses in vitro and in vivo. Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pretreatment with the sGC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS-2028) blunted cGMP levels without affecting VEGF-receptor phosphorylation. Incubation of cells with NS-2028 blocked the mitogenic effects of VEGF. In addition, cells in which sGC was inhibited exhibited no migration and sprouting in response to VEGF. To study the mechanisms through which NS-2028 inhibits EC migration, we determined the effects of alterations in cGMP levels on p38 MAPK. Initially, we observed that inhibition of sGC attenuated VEGF-stimulated activation of p38. In contrast, the addition of 8-Br-cGMP to EC stimulated p38 phosphorylation. The addition of cGMP elevating agents (BAY 41-2272, DETA NO and YC-1) enhanced EC migration. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally displayed a reduced angiogenic response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 in vascular leakage. Using a modified Miles assay, we observed that NS-2028 attenuated VEGF-induced permeability. Overall, we provide evidence that sGC mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC as a downstream effector of VEGF-triggered responses.

Synthesis and biological evaluation of oxadiazole derivatives as inhibitors of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is an ubiquitously expressed enzyme that generates the second messenger cGMP and hence, leads to a number of physiological responses including vasodilation, inhibition of platelet aggregation and neurotransmission. Whilst many activating and stimulating modulators of sGC were identified and studied in recent years, only two selective inhibitors are known: ODQ and NS 2028. Furthermore, a synthetic approach to these inhibitors has not been reported yet. Herein, we describe a novel and efficient synthesis of these inhibitors, as well as the preparation of three different classes of NS 2028 analogues. Biological evaluation of this library using rat aortic smooth muscle cells revealed four new compounds with good to moderate sGC inhibitory activity. Our experiments underline the major importance of the oxadiazole ring in ODQ and NS 2028 for the efficiency of this class of inhibitors.