Structural Probing of Off-Target G Protein-Coupled Receptor Activities within a Series of Adenosine/Adenine Congeners

<div><p>We studied patterns of off-target receptor interactions, mostly at G protein-coupled receptors (GPCRs) in the µM range, of nucleoside derivatives that are highly engineered for nM interaction with adenosine receptors (ARs). Because of the considerable interest of using AR ligands for treating diseases of the CNS, we used the Psychoactive Drug Screening Program (PDSP) for probing promiscuity of these adenosine/adenine congeners at 41 diverse receptors, channels and a transporter. The step-wise truncation of rigidified, trisubstituted (at N⁶, C2, and 5′ positions) nucleosides revealed unanticipated interactions mainly with biogenic amine receptors, such as adrenergic receptors and serotonergic receptors, with affinities as high as 61 nM. The unmasking of consistent sets of structure activity relationship (SAR) at novel sites suggested similarities between receptor families in molecular recognition. Extensive molecular modeling of the GPCRs affected suggested binding modes of the ligands that supported the patterns of SAR at individual receptors. In some cases, the ligand docking mode closely resembled AR binding and in other cases the ligand assumed different orientations. The recognition patterns for different GPCRs were clustered according to which substituent groups were tolerated and explained in light of the complementarity with the receptor binding site. Thus, some likely off-target interactions, a concern for secondary drug effects, can be predicted for analogues of this set of substructures, aiding the design of additional structural analogues that either eliminate or accentuate certain off-target activities. Moreover, similar analyses could be performed for unrelated structural families for other GPCRs.</p></div

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

25 mars 19121912/03/25 (A13,N4179)

Docking at 5HT₇ serotonergic receptor.

<p>Hypothetical binding mode of compounds <b>4</b> (green carbons) and <b>7</b> (pale pink carbons) at a homology model of the h5HT₇ serotonergic receptor based on the h5HT_1B receptor structure. Ligands are shown in ball and stick, and some residues important for ligand recognition are shown in stick (gray carbons). Hydrogen atoms are not displayed. H-bonds are shown as black dashed lines. The Connolly surface of the amino acids surrounding the binding site is displayed. Surface color indicates the lipophilic potential: lipophilic regions (green), neutral regions (white) and hydrophilic regions (magenta).</p

Points of truncation to generate 10 adenosine/adenine derivatives.

<p>When present, the ribose-like moiety contains a [3.1.0]bicyclohexane ((N)-methanocarba) ring system designed to maintain an A₃ and A₁ ARs preferred conformation, and other substituents are associated with potent activity at these receptors. Using these truncation points, a family of 10 congeners to be evaluated at off-target (non-AR) sites was generated. In one case (compound <b>10</b>) an alternate substitution at the N⁶ position was included.</p

Docking at 5HT_2B serotonergic receptor.

<p>Hypothetical alternative binding modes of selected compounds at the h5HT_2B receptor crystal structure. (A) First proposed binding mode for compounds <b>4</b> (green carbons), <b>7</b> (pale pink carbons) and <b>10</b> (magenta carbons) at the 5HT_2B receptor. (B) Second proposed binding mode for compounds <b>1</b> (orange carbons), <b>4</b> (green carbons) and <b>9</b> (cyan carbons) at the 5HT_2B receptor. Ligands are shown in ball and stick and some residues important for ligand recognition are shown in stick (gray carbons). Hydrogen atoms are not displayed. H-bonds are shown as black dashed lines. The Connolly surface of the amino acids surrounding the binding site is displayed. Surface color indicates the lipophilic potential: lipophilic regions (green), neutral regions (white) and hydrophilic regions (magenta).</p

Docking at α adrenergic receptors.

<p>Hypothetical binding modes of selected compounds at homology models of the hα_2B and hα_2C adrenergic receptors based on the h5HT_1B receptor structure. (A) Compounds <b>8</b> (yellow carbons) and <b>9</b> (cyan carbons) at the α_2B receptor. (B) Compounds <b>1</b> (orange carbons), <b>8</b> (yellow carbons) and <b>9</b> (cyan carbons) at the α_2C receptor. Ligands are show in ball and stick and some residues important for ligand recognition are shown in stick (gray carbons). Hydrogen atoms are not displayed. H-bonds are shown as black dashed lines. The Connolly surface of the amino acids surrounding the binding site is displayed. Surface color indicates the lipophilic potential: lipophilic regions (green), neutral regions (white) and hydrophilic regions (magenta).</p

Definition of pharmacophore structures for individual off-target receptor sites.

<p>(A) Colors code the degree of tolerance of appended groups: pharmacophores (minimum structural requirement for binding, shown on <b>1</b> as template) are shown in black, favorable or tolerated substituents are shown in green and not tolerated substituents are shown in red. Some residues predicted to be in contact with the adenosine derivatives at the off-target receptors are highlighted according to the explanations provided in the text (corresponding to poses shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097858#pone-0097858-g004" target="_blank">Figure 4B</a> for the h5HT₂ receptors and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097858#pone-0097858-g007" target="_blank">Figure 7B</a> for the hβ₃ receptor. This is an approximation based on a limited set of compounds. Pharmacophores for other targets were not well defined with the current data set, and weak hits correspond to individual compounds as noted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097858#pone-0097858-t001" target="_blank">Table 1</a>. (B) A comparison with the residues in contact with compound <b>1</b> at the A₃AR, as previously predicted by docking studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097858#pone.0097858-Tosh1" target="_blank">[16]</a>.</p

Similarity of binding between 5HT_2B serotonergic receptor and adenosine receptors.

<p>Comparison between the docking pose of compound <b>4</b> (green carbons) at the 5HT_2B serotonergic receptor structure (silver ribbon) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097858#pone-0097858-g004" target="_blank">Figure 4A</a> and the crystallographic pose of the AR agonist UK-432097 (yellow carbons) at the hA_2AAR (gold ribbon). Ligands are shown in ball and stick, and some residues important for ligand recognition are shown in stick (silver or gold carbons). Hydrogen atoms are not displayed.</p

Truncated Nucleosides as A₃ Adenosine Receptor Ligands: Combined 2-Arylethynyl and Bicyclohexane Substitutions

C2-Arylethynyladenosine-5′-<i>N</i>-methyluronamides containing a bicyclo[3.1.0]­hexane [(N)-methanocarba] ring are selective A₃ adenosine receptor (AR) agonists. Similar 4′-truncated C2-arylethynyl-(N)-methanocarba nucleosides containing alkyl or alkylaryl groups at the <i>N</i>⁶ position were low-efficacy agonists or antagonists of the human A₃AR with high selectivity. Higher hA₃AR affinity was associated with <i>N</i>⁶-methyl and ethyl (<i>K</i>_i = 3–6 nM) than with <i>N</i>⁶-arylalkyl groups. However, combined C2-phenylethynyl and <i>N</i>⁶-2-phenylethyl substitutions in selective antagonist <b>15</b> provided a <i>K</i>_i of 20 nM. Differences between 4′-truncated and nontruncated analogues of extended C2-<i>p</i>-biphenylethynyl substitution suggested a ligand reorientation in AR binding, dominated by bulky <i>N</i>⁶ groups in analogues lacking a stabilizing 5′-uronamide moiety. Thus, 4′-truncation of C2-arylethynyl-(N)-methanocarba adenosine derivatives is compatible with general preservation of A₃AR selectivity, especially with small <i>N</i>⁶ groups, but reduced efficacy in A₃AR-induced inhibition of adenylate cyclase

Structure-Based Design of Reactive Nucleosides for Site-Specific Modification of the A_2A Adenosine Receptor

Adenosine receptors (ARs) are members of the G protein-coupled receptor (GPCR) superfamily and have shown much promise as therapeutic targets. We have used an agonist-bound A_2AAR X-ray crystallographic structure to design a chemically reactive agonist for site-specific chemical modification of the receptor. To further explore and chemically engineer its binding cavity, a 2-nitrophenyl active ester was attached through an elongated chain at adenine C2 position. This general structure was designed for irreversible transfer of a terminal acyl group to a nucleophilic amino group on the A_2AAR. Preincubation with several <i>O</i>-acyl derivatives prevented radioligand binding that was not regenerated upon extensive washing. In silico receptor docking suggested two lysine residues (second extracellular loop) as potential target sites for an <i>O</i>-acetyl derivative (MRS5854, <b>3a</b>), and site-directed mutagenesis indicated that K153 but not K150 is essential. Similarly, a butyl azide for click reaction was incorporated in the active ester moiety (<b>3b</b>). These promising results indicate a stable, covalent modification of the receptor by several reactive adenosine derivatives, which could be chemical tools for future imaging, structural probing, and drug discovery. Thus, structure-based ligand design has guided the site-specific modification of a GPCR