Protocol Development for the Discovery of Angiogenesis Inhibitors via Automated Methods Using Zebrafish and the Discovery and Validation of PD 81723 as a Novel Angiogenesis Inhibitor
Bibliographic record
Abstract
Their optical clarity as larvae and embryos, small size, and high fecundity make zebrafish ideal for whole animal high-throughput screening. A high-throughput drug discovery platform (HTP) has been built to perform fully automated screens of compound libraries with zebrafish embryos. A Tg(kdrl:EGFP) line, marking endothelial cell cytoplasm, was used in this work to help develop protocols and functional algorithms for the system, with the intent of screening for angiogenesis inhibitors. Indirubin 3’ Monoxime (I3M), a known angiogenesis inhibitor, was used at various concentrations to validate the protocols. Consistent with previous studies, a dose-dependent inhibitory effect of I3M on angiogenesis was confirmed. The National Institutes of Health Clinical Collections Libraries I II (NIHCCLI) were screened for angiogenesis inhibitors with these methods as a pilot project for the HTP. The 727 compounds were screened in triplicate and 14 compounds were considered hits. The 5 compounds that corresponded to the most diminished endothelial network with a relatively normal overall morphology were selected for further analysis. A dose response experiment of each of the top 5 compounds was conducted and only PD 81723 and raclopride yielded a significantly smaller endothelial network. Evaluation of the chemical phenotypes of PD 81723 and raclopride led to the exclusion of raclopride as a possible candidate. PD 81723 appeared to be working in a dose-dependent manner and was selected as the main compound of interest. Zebrafish exposed to PD 81723 exhibited several signs of a diminished endothelial network due to the inhibition of angiogenesis. Immunochemistry experiments did not reveal any significant apoptotic or mitotic activity in the zebrafish. Assays with cultured human umbilical vein endothelial cells (HUVECs) elucidated the ability of PD 81723 to inhibit capillary tube formation, wound healing/migration, and proliferation of endothelial cells. An annexin V/PI flow cytometry assay did not reveal significant levels of apoptotic events in PD 81723 treated cells. These results indicate that PD 81723 may be driving endothelial cells into a quiescent state. In addition, immunoblotting revealed that PD 81723 significantly down regulated p21, AKT, VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS, with no notable change in endogenous VEGF-A.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.005 | 0.003 |
| Meta-epidemiology (narrow) | 0.002 | 0.002 |
| Meta-epidemiology (broad) | 0.002 | 0.002 |
| Bibliometrics | 0.003 | 0.002 |
| Science and technology studies | 0.002 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.016 | 0.010 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".