Discovery of new GPCR ligands to illuminate new biology

Although a plurality of drugs target G-protein-coupled receptors (GPCRs), most have emerged from classical medicinal chemistry and pharmacology programs and resemble one another structurally and functionally. Though effective, these drugs are often promiscuous. With the realization that GPCRs signal via multiple pathways, and with the emergence of crystal structures for this family of proteins, there is an opportunity to target GPCRs with new chemotypes and confer new signaling modalities. We consider structure-based and physical screening methods that have led to the discovery of new reagents, focusing particularly on the former. We illustrate their use against previously untargeted or orphan GPCRs, against allosteric sites, and against classical orthosteric sites that selectively activate one downstream pathway over others. The ligands that emerge are often chemically novel, which can lead to new biological effects. © 2017 Nature America, Inc., part of Springer Nature. All rights

Synthetic and Biological Studies on Benzazepine Derivatives as Dopamine Receptor Ligands

Dopamine (DA) receptors, members of the G-protein coupled receptors (GPCRs) family, are divided in two groups based on their transmembrane structural homology domains: D1R-like (D1R, D5R sub-types) and D2R-like DA receptors (D2R, D3R and D4R sub-types). Disturbances in dopaminergic neurotransmission are associated with several CNS disorders. Hence, DA receptor selective ligands have been sought as pharmacological agents to normalize perturbations in the dopaminergic system. Despite several notable efforts, the discovery of highly selective ligands for dopamine receptor sub-types has proved challenging due to close transmembrane structural similarity, especially between DA receptor sub-types within the same group. The 1-phenylbenzazepine scaffold is a well-known template for the discovery of D1R-like ligands with numerous such highly potent and selective compounds possessing a wide range of functional activity (i.e. full agonists, partial agonists, antagonists). Several compounds with 1-phenylbenzazepine framework are used as research tools in pharmacological studies. Fenoldopam (2, Figure 4), a selective D1R-like partial agonist, is currently the only compound from this class in use clinically (as a fast-acting anti-hypertensive drug). In addition to being potential therapeutic agents for Parkinson’s and Alzheimer’s disease, 1-phenylbenzazepine derived D1R partial agonists and antagonists have been extensively studied for their role in attenuating cocaine priming-induced reinstatement of drug-seeking behavior. Aspects related to the chemistry and pharmacology of 1-phenylbenzazepines as well as receptor targets as embodied in the thesis are reviewed in Chapter 1. In our work, we have attempted to address some prominent issues and gaps prevalent in currently available D1R-like selective 1-phenylbenzazepines. Consequently, as detailed in our first aim - Chapter 2, a series of C8, C3’ and C4’ 1-phenylbenzazepines were synthesized and evaluated for their affinity and selectivity towards D1R-like receptors. Our main goal was to replace the pharmacokinetically labile catechol moiety via replacement of the C8 hydroxyl group with various amide, sulfonamide and urea isosteres. In addition, modifications on the 1-phenyl ring were performed. We hypothesized that such modifications to the catechol moiety and 1-phenyl ring would not compromise the D1R affinity, activity and selectivity. An added benefit of these modifications is that on theoretical grounds, the metabolic stability of the analogs would be enhanced. Results from the structure-affinity relationship (SAR) studies revealed that the isosteric replacements did not yield the desired D1R affinity. Thus, we can conclude that amide, sulfonamide and urea groups are not suitable bioisosteres for the C8 hydroxyl group in the 1-phenylbenzazepine scaffold. The results also tend to suggest (in line with historical research on the scaffold) a requirement for the presence of the C8 hydroxyl group for D1R affinity, The second aim of our project (Chapter 3) was to explore halo and methyl substitutions in the 1-phenyl ring of 1-phenylbenzazepines. Extensive SAR studies have been done on the C3’ position on the 1-phenyl ring but the C2’ position has been underexplored in the literature. We hypothesized that substituents at the C2’ position can significantly impact the orientation of the pendant 1-phenyl ring of 1-phenylbenzazepines. The orientation of 1-phenyl group might in turn play a role in affinity and selectivity of these molecules. We substituted the 1-phenyl ring at the C2’ position with larger halo groups such as chloro and bromo and methyl as well as a smaller fluoro group. Moreover, the 1-phenyl ring was disubstituted at the C2’ and C6’ positions with chloro groups that would theoretically disfavour coplanarity of the aryl rings. Results from the SAR studies of these substitutions revealed that C2’ halo (particularly fluoro) and methyl groups are well tolerated for D1R/D5R activity and selectivity, resulting in molecules with single-digit nanomolar affinity. As some molecules exhibited moderate D5R versus D1R selectivity, this study has provided new avenues towards finding novel D5R selective compounds. In another effort to explore the significance of conformational changes in 1-phenylbenzazepines, in our third aim (Chapter 4), we synthesized and evaluated novel conformationally rigid benzazepines (i.e. fluoreno azepines). Via direct arylation chemistry, we achieved rigidification of the benzazepine scaffold in which both aryl rings are directly connected via C9 and C2’, thus forcing coplanarity of the aryl rings. Our goal was to determine the extent to which rigidification of the benzazepine scaffold in this manner would affect affinity and selectivity for D1R/D5R. We hypothesized that rigidification of 1-phenyl ring in this manner would have a significant impact on D1R/D5R activity and selectivity since the pendant 1-phenyl ring is no longer allowed to rotate freely and may lock the molecule in a bioactive conformation. Serendipitously, though lacking significant affinity for dopamine receptors, in this process we discovered a novel class of highly selective and potent 5-HT6 serotonin receptor ligands. These fluoreno azepines are pharmacophorically distinct from currently available 5-HT6 ligands. Molecular docking studies indicate weaker H-bonding interactions between oxygenated groups and binding-pocket amino acid residues of D1R which led to reduced affinity towards D1R. Conversely, docking studies of the most active analogs at the 5-HT6 receptor revealed H-bonding interactions between the hydroxyl groups and Asp106, a protonated nitrogen and Thr196 and pi-pi interactions bewtween the aryl rings and Phe188 and Phe284. Several of these novel compounds displayed \u3e15-fold selectivity for 5-HT6 versus other receptors tested

Dynamics and structural determinants of ligand recognition of the 5-HT6 receptor

In order to identify molecular models of the human 5-HT6 receptor suitable for virtual screening, homology modeling and membrane-embedded molecular dynamics simulations were performed. Structural requirements for robust enrichment were assessed by an unbiased chemometric analysis of enrichments from retrospective virtual screening studies. The two main structural features affecting enrichment are the outward movement of the second extracellular loop and the formation of a hydrophobic cavity deep in the binding site. These features appear transiently in the trajectories and furthermore the stretches of uniformly high enrichment may only last 4-10 ps. The formation of the inner hydrophobic cavity was also linked to the active-like to inactive-like transition of the receptor, especially the so-called connector region. The best structural models provided significant and robust enrichment over three independent ligand sets. © 2015 Springer International Publishing Switzerland