Authors: Weßeler, Fabian
Title: Stem cell-based phenotypic screening and characterization of novel BMP mimetics
Language (ISO): en
Abstract: The identification of selective small molecule cytokine mimetics and signaling activators holds great promise for numerous applications in biomedicine as they overcome the typical drawbacks of physiological peptide- or protein-based ligands. Yet, the development of such modalities remains a challenging task in drug discovery. In this thesis, a phenotypic, target-agnostic, high-throughput screening assay is presented that probes Bone Morphogenetic Protein (BMP) signaling during mesodermal patterning of murine embryonic stem cells. This approach represents a novelty in BMP activator identification by harnessing embryonic development in vitro, hence potentially expanding the druggable space of BMP signaling activators. During mesoderm specification, BMP signaling can be temporally discriminated from Transforming Growth Factor- (TGF)-driven stages of cardiac differentiation. This selective and authentic orchestration of BMP signaling cues can be recapitulated for the discovery of genuine BMP activator- or potentiator chemotypes. Here, a robust hit validation workflow has been devised, including the orthogonal assessment of BMP activation during osteoblastogenesis as well as BMP-dependent reporter assays. Proof-of-concept is demonstrated from screening of nearly 7,000 chemically diverse compounds, yielding 2,3-disubstituted 4H-chromen-4-ones as a new potent BMP activating chemical modality. Chromenone 1 enhanced osteogenic differentiation and mineralization in vitro. Structure-activity relationship (SAR) studies with 29 different Chromenones revealed pharmacophoric features relevant to BMP activity and provided a valuable toolkit of active and structurally closely related inactive derivatives. Interestingly, mechanistic studies suggested that Chromenone 1 enhances canonical nuclear BMP-Smad signaling outputs through an unparalleled, kinase-independent, negative TGF-Smad feedback loop. This is in sharp contrast to the reported BMP sensitizer PD407824, revealing novel BMP potentiator biology as desired from a conceptionally new morphogenic and phenotypic drug discovery approach. An additional screening of 1,408 known bioactive compounds eventually furnished the triazolo[1,5 c]quinazoline CGS-15943 as a novel and unique BMP activating chemotype that exhibited highest selectivity among all profiled and characterized BMP modulators in the present work. Its capacity to amplify BMP signaling during embryogenesis could be further substantiated in vivo during zebrafish development (i.e., ventralization phenotypes). Target deconvolution studies unraveled novel targets of CGS-15943 with a pronounced potency for CK1- and --isoforms. Mechanistically, CGS-15943 amplifies osteogenic BMP signaling outputs through a dual targeting of CK1/ and PI3K (p110) isoforms by enhancing BMP signaling independent of a TGF-feedback. Together, the presented work underscores proof-of-concept for a novel phenotypic drug discovery strategy to identify unique BMP signaling modulators. Two new chemotypes ultimately expanded the druggable space of osteogenic BMP activators and sensitizers that may serve as valuable tools for various applications in (stem) cell biology and regenerative medicine. Notably, this work might spur future translation to even more complex 3D-gastruloid and organoid systems.
Subject Headings: BMP potentiators
Chemical biology
Stem cell differentiation
Small molecules
Phenotypic screening
Subject Headings (RSWK): Stammzelle
Chemische Biologie
Issue Date: 2021
Appears in Collections:Chemische Biologie

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