Mutagenicity in a Molecule: Identification of Core Structural Features of Mutagenicity Using a Scaffold Analysis

<div><p>With advances in the development and application of Ames mutagenicity <i>in silico</i> prediction tools, the International Conference on Harmonisation (ICH) has amended its M7 guideline to reflect the use of such prediction models for the detection of mutagenic activity in early drug safety evaluation processes. Since current Ames mutagenicity prediction tools only focus on functional group alerts or side chain modifications of an analog series, these tools are unable to identify mutagenicity derived from core structures or specific scaffolds of a compound. In this study, a large collection of 6512 compounds are used to perform scaffold tree analysis. By relating different scaffolds on constructed scaffold trees with Ames mutagenicity, four major and one minor novel mutagenic groups of scaffold are identified. The recognized mutagenic groups of scaffold can serve as a guide for medicinal chemists to prevent the development of potentially mutagenic therapeutic agents in early drug design or development phases, by modifying the core structures of mutagenic compounds to form non-mutagenic compounds. In addition, five series of substructures are provided as recommendations, for direct modification of potentially mutagenic scaffolds to decrease associated mutagenic activities.</p></div

Cyproheptadine and pizotifen inhibit serotonin-enhanced ADP-induced mouse platelet aggregation <i>ex vivo</i>.

<p>(A) Mouse PRP obtained from vehicle-injected mice (once daily for 5 days) was stimulated with submaximal concentration of ADP (1 µM) in the presence or absence of serotonin (15 µM). (B) Mouse PRP obtained from cyproheptadine-injected mice (1 mg/kg IP once daily for 5 days) was stimulated with submaximal concentration of ADP in the presence or absence of serotonin. (C) Mouse PRP obtained from pizotifen-injected mice (3 mg/kg IP once daily for 5 days) was stimulated with submaximal concentration of ADP in the presence or absence of serotonin. (D) Mouse PRP obtained from EMD 281014 injected mice (5 mg/kg IP once daily for 5 days) was stimulated with submaximal concentration of ADP in the presence or absence of serotonin. Inset shows quantification of the data. Each experiment was repeated at least 3 times, with blood pooled from at least eight mice each time.</p

Cyproheptadine and pizotifen inhibit serotonin-enhanced U46619-induced human platelet aggregation <i>in vitro</i>.

<p>(A) Human PRP was stimulated with submaximal concentration of U46619 (0.125 µM) in the presence or absence of serotonin (15 µM). (B) Human PRP was pre-incubated with increasing doses of cyproheptadine (0.1–250 nM) for 1 min and activated with U46619 (0.125 µM) and serotonin (15 µM). (C) Human PRP was pre-incubated with increasing doses of pizotifen (0.1–30 nM) for 1 min and activated with 0.125 µM U46619 and 15 µM serotonin. (D) Human PRP was pre-incubated with increasing doses of EMD 281014 (5–20 nM) for 1 min and activated with 0.125 µM U46619 and 15 µM serotonin. (E) Human PRP was pre-incubated for 1 min with the highest concentrations of cyproheptadine (250 nM), pizotifen (30 nM), and EMD 281014 (20 nM) used in this set of experiments, and activated with U46619 (0.125 µM). Inset shows quantification of the data. Each experiment was repeated at least 3 times, with blood obtained from three separate donors.</p

Cyproheptadine and pizotifen prolong occlusion times and tail bleeding times in mice.

<p>Each point represents the occlusion time or tail bleeding time of a single animal. Mice were treated IP with vehicle, 6/kg clopidogrel, 1 mg/kg cyproheptadine, 3 mg/kg pizotifen, or 5 mg/kg EMD 281014 once daily for 5 days before experiments. (A) Mean occlusion times for mice treated with: vehicle = 375.3±31.89 sec (n = 11), clopidogrel = 987.6±196.5 sec (n = 8), cyproheptadine = 787.4±84.08 sec (n = 8), pizotifen = 1199±253.1 sec (n = 8), and EMD 281014 = 879.9±270.0 sec (n = 8). **p<0.0022; ****p<0.0001; **p<0.0014; *p<0.0431. (B) Mean bleeding times for mice treated with: vehicle = 213.1±81.03 sec (n = 9), clopidogrel = 793.6±58.17 sec (n = 9), cyproheptadine = 643.1±97.78 sec (n = 9), pizotifen = 714.4±96.78 sec (n = 9), and EMD 281014 = 558.9±67.12 sec (n = 9). ****p<0.0001; **p<0.0038; **p<0.0011; **p<0.0047.</p

The scaffold structures of minor mutagenic scaffold groups.

<p>(A) Naphthalene, (B) Quinoline, and (C) Bezene groups. Below the labels of scaffold names were labelled by the mutagen rates and the numbers of mutagen compounds/the numbers of overall compounds in that scaffolds. In the structures of child scaffolds, the differences from the parent scaffold were colored as red.</p

The scaffold structures of examples of sibling relationships between mutagenic scaffolds and non-mutagenic scaffolds.

<p>(A) Acridine, (B) Phenanthrene, (C) Quinoxaline, (D) Naphthalene, and (E) Quinoline groups. Below the labels of scaffold names were labelled by the mutagen rates and the numbers of mutagen compounds/the numbers of overall compounds in that scaffolds. In the structures of child scaffolds, the differences from the parent scaffold were colored as red.</p

Cyproheptadine and pizotifen inhibit serotonin-enhanced ADP-induced human platelet aggregation <i>in vitro.</i>

<p>(A) Human PRP was stimulated with submaximal concentration of ADP (1 µM) in the presence or absence of serotonin (15 µM). (B) Human PRP was pre-incubated with increasing doses of cyproheptadine (0.1–10 nM) for 1 min and activated with ADP (1 µM) and serotonin (15 µM). (C) Human PRP was pre-incubated with increasing doses of pizotifen (0.01–1 nM) for 1 min and activated with ADP and serotonin. (D) Human PRP was pre-incubated with increasing doses of EMD 281014 (10–40 nM) for 1 min and activated with ADP and serotonin. (E) Human PRP was treated with the highest concentrations of cyproheptadine (10 nM), pizotifen (1 nM), and EMD 281014 (40 nM) used in previous experiments, in the absence of agonists. (F) Human PRP was pre-incubated for 1 min with cyproheptadine (10 nM), pizotifen (1 nM), and EMD 281014 (40 nM) for 1 min and activated with serotonin (15 µM). (G) Human PRP was pre-incubated with cyproheptadine (10 nM), pizotifen (1 nM), and EMD 281014 (40 nM) for 1 min and activated with ADP (1 µM). Inset shows quantification of the data. Each experiment was repeated at least 3 times, with blood obtained from three separate donors.</p

The Toxtree prediction results.

<p>(A) 5-(bromomethyl)-2,3-dimethoxyquinoxaline and (B) acridiine-1,9-diamine. The fitted structural alerts were labelled.</p

The C/S distribution plots according to different mutagenicity cutoff values.

<p>(A) In selecting mutagenic scaffolds, using the mutagens categorized in each selected scaffold as the selection criteria (C₁/S). The detailed scores were listed in <b>Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148900#pone.0148900.s001" target="_blank">S1 File</a></b>. (B) In selecting non-mutagenic scaffolds, using the non-mutagens categorized in each selected scaffold as the selection criteria (C₂/S). The detailed scores were listed in <b>Table B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148900#pone.0148900.s001" target="_blank">S1 File</a>.</b> (C₁: number of mutagenic compounds, C₂: number of non-mutagenic compounds, S: number of mutagenic (for C₁) or non-mutagenic (for C₂) scaffolds).</p

Cyproheptadine, and pizotifen inhibit intracellular calcium elevation and Src activation in human platelets <i>in vitro</i>.

<p>(A) Human platelets were loaded with Fura-2/AM to measure intracellular [Ca2+]_i, in the presence or absence of Cyproheptadine (10 nM), pizotifen (1 nM) or EMD 281014 (40 nM) and activated with ADP (1 µM), serotonin (15 µM) or ADP and serotonin together. (B) Human platelets were incubated in the presence or absence of Cyproheptadine (10 nM), pizotifen (1 nM) or EMD 281014 (40 nM) for 5 minutes and then stimulated with ADP (1 µM), serotonin (15 µM) or ADP and serotonin together for 3 minutes, and subjected to immunoprecipitation followed by immunoblotting with anti-Src and anti-phosphotyrosine antibodies; upper panel shows quantification of the data using densitometry analysis. Each experiment was repeated at least 3 times, with blood obtained from three separate donors.</p