In silico molecular comparisons of C. elegans and mammalian pharmacology identify distinct targets that regulate feeding

Abstract

Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.

Figure 1. Overview of the ligand-target predictions for C. elegans screen actives.

(A) Distribution of ligand predictions per compound expressed as a histogram. (B) Target classes more frequently (positive %) or less frequently (negative %) predicted for C. elegans screen actives, using predictions on ChEMBL's ligands as a baseline. Data are calculated based on ligand–target interactions at a minimum significance threshold of E<0.00001.

Figure 2. SEA predictions that were confirmed by in vitro testing.

Figure 3. Several compounds with predicted and confirmed human targets increase pharyngeal pumping.

(A) Wild-type C. elegans were cultured on media supplemented with either 0.1% DMSO (vehicle control) or 10 µM of each compound. (B) The effects of the compounds on the pharyngeal pumping rate when exposed for differing developmental periods was evaluated for C. elegans exposed to each 10 uM of each compound during different times: L1 to L4 (2 d at 20°C), L1 to gravid adult (3 d at 20°C, and naïve day 1 gravid adults exposed to B16, F15, and D20 for 1 h, H6 for 16 h). The pharyngeal pumping rate of 10–13 animals per condition was quantified. Error bars represent the standard deviation. *p<0.01: ANOVA, Dunnett's multiple comparisons test. In (B) gravid adults exposed to H6 for 16 h was compared to DMSO 16 h (t test: two tailed *p<0.01).

Figure 4. D20 antagonizes a VER-3–dependent pathway to induce pharyngeal pumping.

(A) Animals were cultured on different RNAi clones in media supplemented with either 0.05% DMSO or 5 µM D20. Sequences are arranged based on their BLASTp similarity to the human FLT-3 receptor from most similar (left) to least similar (right). Error bars represent the s.e.m. **p<0.001, *p<0.01 (D20 versus DMSO): two-tailed t test. (B) Pharyngeal pumping rates of wild-type C. elegans treated with serial 3-fold dilutions of 5-flurox or D20. (C) The pharyngeal pumping rates of wild-type, egl-15(n484), ver-1(ok1738), ver-2(ok897), ver-3(ok891), and ver-4(ok1079) mutant animals cultured on 0.1% DMSO, 10 µM D20, 10 µM K9, or 2 µM 5-flurox. Error bars represent the standard deviation. Significance levels: **p<0.001 were determined by one-way ANOVA using Bonferroni's multiple comparison test. (D) C. elegans were treated with serial 3-fold dilutions of K9 and the pharyngeal pumping rate was quantified at each dose. Error bars represent the s.e.m. In (A–D) 10–20 animals were evaluated per condition.

Figure 5. Identification of C. elegans GPCR-regulated feeding pathways that are pharmacologically orthologous to their human targets.

(A) Wild-type C. elegans treated with serial 3-fold dilutions of either F15 or L-371257. Error bars represent the standard error of the mean. (B) Wild-type, ntr-1(ok2780), gnrr-1(ok238), gnrr-2(tm4867), or gnrr-3(tm4152) mutant animals cultured on either 0.1% DMSO, 10 µM F15, 100 nM L-371257, or 200 nM SB222200. (C) Wild-type C. elegans cultured on E. coli expressing either tkr-1 RNAi or vector control, then treated with either 0.1% DMSO,10 µM H6, 10 µM F15, or 200 nM SB222200. (D) Wild-type, mgl-1(tm1811), and mgl-2(tm355) mutant C. elegans cultured on media containing either 0.1% DMSO, 10 µM B16, 10 µM F15, or 2 µM MMPIP. Error bars represent the standard deviation. (A–D) The mean pharyngeal pumping rate of 10–20 C. elegans per condition are shown. Significance levels: **p<0.001, *p<0.05 were determined by one-way ANOVA using Bonferroni's multiple comparison test.

Figure 6. Interaction matrix of all binary combinations of compounds and gene knockdowns that individually increase pharyngeal pumping.

(A) The differences in the pumping rates of compound-treated versus vehicle (0.1% DMSO) treatment on each genetic background for all pair-wise combinations of compounds and mutants were evaluated. Compound concentrations used were 10 µM for B16, D20, K9, F15, and H6; 200 nM for L-371257 and SB 222200; and 2 µM for MMPIP, 5-flurox. The predicted compound–target interactions are outlined in yellow. Red- and blue-labeled interactions indicate pumping rates significantly different (ANOVA, p<0.05 Dunnett's multiple comparison test) from the corresponding vehicle control-treated mutant. (B) The implied genetic interactions on pharyngeal pumping of mgl-2, ver-2, ver-3, and gnrr-1 mutants assayed by mutant–RNAi combinations. Twelve animals per condition were analyzed. Error bars represent 1 standard deviation. * p<0.001 one-way ANOVA using Bonferroni's multiple comparison test.