Luigi Aurelio

Allosteric modulators of the adenosine A1 receptor: synthesis and pharmacological evaluation of 4-substituted 2-amino-3-benzoylthiophenes.

A series of 4-substituted 2-amino-3-benzoylthiophenes was screened using a functional assay of A(1)AR-mediated phosphorylation of ERK1/2 in intact CHO cells to identify both potential agonistic effects as well the ability to allosterically modulate the activity of the orthosteric agonist, R-PIA. More detailed concentration-response experiments were subsequently performed on two compounds (9a and 9o) utilizing both the ERK1/2 assay as well as a second assay of [(35)S]GTPgammaS binding to activated G proteins.

Separation of on-target efficacy from adverse effects through rational design of a bitopic adenosine receptor agonist

Significance The adenosine A1 receptor (A1AR) is an important target for cardioprotection, but current A1AR drugs are limited for this indication because of the occurrence of bradycardia as a major adverse effect mediated by the same receptor. To address this problem, we designed a ligand that simultaneously bridges two different sites on the A1AR; the hypothesis is that this bitopic mode would result in unique receptor conformations that will signal to desirable pathways while sparing those pathways mediating undesirable effects. This mechanism was validated in native rodent cells and isolated rat atria, providing proof of concept that the design of bitopic ligands may be a path forward to separating beneficial from harmful effects mediated by the same drug target. The concepts of allosteric modulation and biased agonism are revolutionizing modern approaches to drug discovery, particularly in the field of G protein-coupled receptors (GPCRs). Both phenomena exploit topographically distinct binding sites to promote unique GPCR conformations that can lead to different patterns of cellular responsiveness. The adenosine A1 GPCR (A1AR) is a major therapeutic target for cardioprotection, but current agents acting on the receptor are clinically limited for this indication because of on-target bradycardia as a serious adverse effect. In the current study, we have rationally designed a novel A1AR ligand (VCP746)—a hybrid molecule comprising adenosine linked to a positive allosteric modulator—specifically to engender biased signaling at the A1AR. We validate that the interaction of VCP746 with the A1AR is consistent with a bitopic mode of receptor engagement (i.e., concomitant association with orthosteric and allosteric sites) and that the compound displays biased agonism relative to prototypical A1AR ligands. Importantly, we also show that the unique pharmacology of VCP746 is (patho)physiologically relevant, because the compound protects against ischemic insult in native A1AR-expressing cardiomyoblasts and cardiomyocytes but does not affect rat atrial heart rate. Thus, this study provides proof of concept that bitopic ligands can be designed as biased agonists to promote on-target efficacy without on-target side effects.

Delineating the Mode of Action of Adenosine A1 Receptor Allosteric Modulators

Despite the identification of 2-amino-3-benzoylthiophenes (2A3BTs) as the first example of small-molecule allosteric potentiators of agonist function at a G protein-coupled receptor (GPCR)—the adenosine A1 receptor—their mechanism of action is still not fully understood. We now report the mechanistic basis for the complex behaviors noted for 2A3BTs at A1 receptors. Using a combination of membrane-based and intact-cell radioligand binding, multiple signaling assays, and a native tissue bioassay, we found that the allosteric interaction between 2A3BTs and the agonists 2-chloro-N6-[3H]cyclopentyladenosine or (−)-N6-(2-phenylisopropyl)adenosine (R-PIA) or the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine is consistent with a ternary complex model involving recognition of a single extracellular allosteric site. However, when allowed access to the intracellular milieu, 2A3BTs have a secondary action as direct G protein inhibitors; this latter property is receptor-independent as it is observed in nontransfected cells and also after stimulation of another GPCR. In addition, we found that 2A3BTs can signal as allosteric agonists in their own right but show bias toward certain pathways relative to the orthosteric agonist, R-PIA. These results indicate that 2A3BTs have a dual mode of action when interacting with the A1 receptor and that they can engender novel functional selectivity in A1 signaling. These mechanisms need to be factored into allosteric ligand structure-activity studies.

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief

Therapeutic targeting of the neurokinin 1 receptor in endosomes provides efficacious and prolonged pain relief. Targeting the enemy within endosomes With opioid addiction on the rise, there is a great need for effective nonopioid approaches to treat pain. Jensen et al. examined the function of substance P neurokinin 1 receptor, which plays a role in the transmission of pain signals in the central nervous system. The authors demonstrated that endocytosis of this receptor is required for the transmission of pain signals. Although systemic inhibition of endocytosis would not be feasible in a living organism, the authors discovered another way to take advantage of this information. They conjugated neurokinin 1 receptor antagonists to cholestanol, promoting their incorporation into endosomes, where they successfully inhibited their target to block pain transmission. Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)–coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane–targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal–regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.

Synthesis and characterization of novel 2-amino-3-benzoylthiophene derivatives as biased allosteric agonists and modulators of the adenosine A(1) receptor

A series of novel 2-amino-3-benzoylthiophenes (2A3BTs) were screened using a functional assay of A(1)R mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in intact CHO cells to identify potential agonistic effects as well as the ability to allosterically modulate the activity of the orthosteric agonist, R-PIA. Two derivatives, 8h and 8i, differing only in terms of the absence or presence of an electron-withdrawing group on the benzoyl moiety of the 2A3BT scaffold, were identified as biased allosteric agonists and positive allosteric modulators of agonist function at the adenosine A(1) receptor (A(1)R) in two different functional assays. Our findings indicate that subtle structural variations can promote functionally distinct receptor conformational states.

Scaffold-Divergent Synthesis of Ring-Fused Indoles, Quinolines, and Quinolones via Iodonium-Induced Reaction Cascades

N-(2-Iodophenyl)imines A are readily formed from Schiffs base condensation of 2-iodoanilines with carbonyls and ketals. These imines provide useful substrates in scaffold-divergent synthesis through the attachment of an alkyne (Songashira coupling or acyl substitution of a Weinreb amide) followed by an iodonium-induced reaction cascade to give ring-fused indoles B, quinolines C, or quinolones D depending on the reaction conditions employed.

3- and 6-Substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as A1 adenosine receptor allosteric modulators and antagonists

A series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were prepared and evaluated as potential allosteric modulators at the A(1) adenosine receptor. The structure-activity relationships of the 3- and 6-positions of a series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were explored. Despite finding that 3- and 6-substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines possess the ability to recognize an allosteric site on the agonist-occupied A(1)AR at relatively high concentrations, the structural modifications we have performed on this scaffold favor the expression of orthosteric antagonist properties over allosteric properties. This research has identified 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as novel class of orthosteric antagonist of the A(1)AR and highlighted the close relationship between structural elements governing allosteric modulation and orthosteric antagonism of agonist function at the A(1)AR.

Protein conformation by EPR spectroscopy using gadolinium tags clicked to genetically encoded p-azido-L-phenylalanine

Quantitative cysteine-independent ligation of a Gd(3+) tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd(3+)-Gd(3+) distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd(3+) ion relative to the protein can be predicted with high accuracy.

The synthesis and biological evaluation of 2-amino-4,5,6,7,8,9-hexahydrocycloocta[b]thiophenes as allosteric modulators of the A1 adenosine receptor.

A series of 2-amino-4,5,6,7,8,9-hexahydrocycloocta[b]thiophenes were prepared and evaluated as potential allosteric modulators of the A(1) adenosine receptor (AR). The structure-activity relationships of the 3-position were explored along with varying the size of the cycloalkyl ring. 2-Aminothiophenes with amide and hydrazide groups in the 3-position were completely inactive in an A(1)-AR-mediated ERK1/2 phosphorylation assay, yet most of the 3-benzoyl substituted compounds exhibited allosteric effects on responses mediated by the orthosteric agonist, R-PIA. Despite finding an increase in both agonistic and allosteric activities by going from a cyclopentyl ring to a cyclohexyl ring in the 3-benzoyl series, decreases were observed when further increasing the ring size. Varying the substituents on the phenyl ring of the 3-benzoyl group also affected the activity of these compounds.

Effects of Conformational Restriction of 2-Amino-3-benzoylthiophenes on A1 Adenosine Receptor Modulation

2-Amino-3-benzoylthiophenes (2A3BTs) have been widely reported to act as allosteric enhancers (AEs) at the A(1) adenosine receptor (A(1)AR). Herein we describe the synthesis of a series of 1-aminoindeno[1,2-c]thiophen-8-ones and a series of (2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl)methanones as conformationally rigid analogues of the 2A3BTs. These compounds were screened using a functional assay of A(1)AR-mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in intact Chinese hamster ovary (CHO) cells to identify both potential agonistic effects as well as the ability to allosterically modulate the activity of the orthosteric agonist, N(6)-(R-phenylisopropyl)adenosine (R-PIA). All of the 1-aminoindeno[1,2-c]thiophen-8-ones (14a-c and 17a-f) proved either to be inactive or behaved as antagonists in the functional assay. However, the (2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl)methanones with para-chloro substitution (compounds 25b, 25d, and 25f) did significantly augment the R-PIA response, indicating a positive allosteric effect.