Design and Discovery of a High Affinity, Selective and β-Arrestin Biased 5-HT7 Receptor Agonist

2021 ◽  
Author(s):  
Edem K. Onyameh ◽  
Edward Ofori ◽  
Barbara A. Bricker ◽  
Uma M. Gonela ◽  
Suresh V. K. Eyunni ◽  
...  
Keyword(s):  
Receptor Agonist ◽  
Enantiomeric Separation ◽  
Affinity Binding ◽  
Binding Affinities ◽  
Functional Selectivity ◽  
High Affinity Binding ◽  
High Affinity

Abstract Compound 1c, 2-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl)-5-fluoro-2,3-dihydro-1H-inden-1-one was previously reported from our laboratory showing high affinity binding to the 5-HT7 receptor (Ki = 0.5 nM). However, compound 1c racemizes readily upon enantiomeric separation. To prevent racemization, we have redesigned and synthesized a methyl and carboxyethyl analogs, compounds 2 and 3 respectively, whose binding affinities were similar to those of compound 1c. Compound 2 and 3 cannot undergo racemization since tautomerism was no longer possible and thus, compound 2 was selected for enantiomeric separation and further evaluation. Upon enantiomeric separation, the levorotatory enantiomer, (-)2 or 2a demonstrated a higher affinity and a β-arrestin biased functional selectivity for the 5-HT7 receptor. Although 2a showed 22 times less activity than 5-HT in the Gs pathway, it showed 28 times higher activity than 5-HT in the β-arrestin pathway. This constitutes a significant β-arrestin pathway preference and shows 2a to be more potent and more efficacious than the recently published β-arrestin biased 3-(4-chlorophenyl)-1,4,5,6,7,8-hexahydropyrazolo[3,4-d]azepine, the N-debenzylated analog of JNJ18038683 (Compound 7).

scholarly journals Prototypic GABAA Receptor Agonist Muscimol Acts Preferentially Through Forebrain High-Affinity Binding Sites

2009 ◽  
Vol 35 (4) ◽  
pp. 999-1007 ◽  
Author(s):  
Dev Chandra ◽  
Lauri M Halonen ◽  
Anni-Maija Linden ◽  
Chiara Procaccini ◽  
Kati Hellsten ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
Binding Sites ◽  
Receptor Agonist ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity

scholarly journals Comparison of Target Recognition by TRAF1 and TRAF2

2020 ◽  
Vol 21 (8) ◽  
pp. 2895
Author(s):  
Chang Min Kim ◽  
Hyun Ho Park
Keyword(s):  
Receptor Binding ◽  
Target Recognition ◽  
Affinity Binding ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
Critical Determinant ◽  
High Affinity Binding ◽  
High Affinity ◽  
Caspase 2

Although TRAF1 and TRAF2 share common receptors and have extremely conserved amino acid residues, recent studies have shown that key differences in receptor binding preferences with different affinities exist, which might be important for their different functions in TRAF-mediated signal transduction. To better understand TRAF1 and TRAF2 signaling, we analyzed and compared their receptor binding-affinities. Our study revealed that TRADD, TANK, and caspase-2 bind to both TRAF1 and TRAF2 with different affinities in vitro. Sequence and structural analyses revealed that S454 on TRAF2 (corresponding to A369 of TRAF1) is critical for the binding of TRADD, and F347 on TRAF1 (corresponding to L432 of TRAF2) is a critical determinant for high affinity binding of TANK and caspase-2.

scholarly journals Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis

2020 ◽  
Vol 117 (39) ◽  
pp. 24557-24566 ◽  
Author(s):  
Alejandra Freire-Rios ◽  
Keita Tanaka ◽  
Isidro Crespo ◽  
Elmar van der Wijk ◽  
Yana Sizentsova ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Regulatory Elements ◽  
Affinity Binding ◽  
Binding Affinities ◽  
Auxin Response ◽  
High Affinity Binding ◽  
High Affinity ◽  
Dna Elements ◽  
Differential Binding

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF–DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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