Combined Proteomic and In Silico Target Identification Reveal a Role for 5-Lipoxygenase in Developmental Signaling Pathways.

Identification and validation of the targets of bioactive small molecules identified in cell-based screening is challenging and often meets with failure, calling for the development of new methodology. We demonstrate that a combination of chemical proteomics with in silico target prediction employing the SPiDER method may provide efficient guidance for target candidate selection and prioritization for experimental in-depth evaluation. We identify 5-lipoxygenase (5-LO) as the target of the Wnt pathway inhibitor Lipoxygenin. Lipoxygenin is a non-redox 5-LO inhibitor, modulates the ß-catenin-5-LO complex and induces reduction of both ß-catenin and 5-LO levels in the nucleus. Lipoxygenin and the structurally unrelated 5-LO inhibitor CJ-13,610 promote cardiac differentiation of human induced pluripotent stem cells and inhibit Hedgehog, TGF-ß, BMP, and Activin A signaling, suggesting an unexpected and yet unknown role of 5-LO in these developmental pathways.

Discovery of Inhibitor of Wnt Production 2 (IWP-2) and Related Compounds As Selective ATP-Competitive Inhibitors of Casein Kinase 1 (CK1) δ/ε.

Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazole-based CK1 inhibitors, we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a plausible binding mode of IWP-2 in the ATP binding pocket of CK1δ which was confirmed by X-ray analysis. In vitro kinase assays demonstrated IWPs to be ATP-competitive inhibitors of wtCK1δ. IWPs also strongly inhibited the gatekeeper mutant M82FCK1δ. When profiled in a panel of 320 kinases, IWP-2 specifically inhibited CK1δ. IWP-2 and IWP-4 also inhibited the viability of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWP-derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but also might influence CK1δ/ε-related pathways.

Cell Permeable Stapled Peptide Inhibitor of Wnt Signaling that Targets ß-Catenin Protein-Protein Interactions.

The Wnt signaling pathway plays a critical role in cell proliferation and differentiation, thus it is often associated with diseases such as cancers. Unfortunately, although attractive, developing anti-cancer strategy targeting Wnt signaling has been challenging given that the most attractive targets are involved in protein-protein interactions (PPIs). Here, we develop a stapled peptide inhibitor that targets the interaction between ß-catenin and T cell factor/lymphoid enhancer-binding factor transcription factors, which are crucially involved in Wnt signaling. Our integrative approach combines peptide stapling to optimize proteolytic stability, with lessons learned from cell-penetrating peptide (CPP) design to maximize cellular uptake resulting in NLS-StAx-h, a selective, cell permeable, stapled peptide inhibitor of oncogenic Wnt signaling that efficiently inhibits ß-catenin-transcription factor interactions. We expect that this type of integrative strategy that endows stapled peptides with CPP features will be generally useful for developing inhibitors of intracellular PPIs.

Small molecules targeting in vivo tissue regeneration.

The field of regenerative medicine has boomed in recent years thanks to milestone discoveries in stem cell biology and tissue engineering, which has been driving paradigm shifts in the pharmacotherapy of degenerative and ischemic diseases. Small molecule-mediated replenishment of lost and/or dysfunctional tissue in vivo, however, is still in its infancy due to a limited understanding of mechanisms that control such endogenous processes of tissue homeostasis or regeneration. Here, we discuss current progress using small molecules targeting in vivo aspects of regeneration, including adult stem cells, stem cell niches, and mechanisms of homing, mobilization, and engraftment as well as somatic cell proliferation. Many of these compounds derived from both knowledge-based design and screening campaigns, illustrating the feasibility of translating in vitro discovery to in vivo regeneration. These early examples of drug-mediated in vivo regeneration provide a glimpse of the future directions of in vivo regenerative medicine approaches.