We report on the
feasibility to harness embryonic development in vitro for the identification of small-molecule cytokine
mimetics and signaling activators. Here, a phenotypic, target-agnostic,
high-throughput assay is presented that probes bone morphogenetic
protein (BMP) signaling during mesodermal patterning of embryonic
stem cells. The temporal discrimination of BMP- and transforming growth
factor-β (TGFβ)-driven stages of cardiomyogenesis underpins
a selective, authentic orchestration of BMP cues that can be recapitulated
for the discovery of BMP activator chemotypes. Proof of concept is
shown from a chemical screen of 7000 compounds, provides a robust
hit validation workflow, and afforded 2,3-disubstituted 4H-chromen-4-ones as potent BMP potentiators with osteogenic efficacy.
Mechanistic studies suggest that Chromenone 1 enhances
canonical BMP outputs at the expense of TGFβ-Smads in an unprecedented
manner. Pharmacophoric features were defined, providing a set of novel
chemical probes for various applications in (stem) cell biology, regenerative
medicine, and basic research on the BMP pathway.
Phenotypic drug discovery (PDD) continues to fuel the research and development pipelines with first-in-class therapeutic modalities, but success rates critically depend on the quality of the underlying model system. Here, we employed a stem cell-based approach for the target-agnostic, yet pathway-centric discovery of small-molecule cytokine signaling activators to act as morphogens during development and regeneration. Unbiased screening identified triazolo[1,5-c]quinazolines as a new-in-class in vitro and in vivo active amplifier of the bone morphogenetic protein (BMP) pathway. Cellular BMP outputs were stimulated via enhanced and sustained availability of BMP-Smad proteins, strictly dependent on a minimal BMP input. Holistic target deconvolution unveiled a unique mechanism of dual targeting of casein kinase 1 and phosphatidyl inositol 3-kinase isoforms as key effectors for efficient amplification of osteogenic BMP signaling. This work underscores the asset of PDD to discover unrecognized polypharmacology signatures, in this case significantly expanding the chemical and druggable space of BMP modulators.
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