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The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular stress can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily post-mitotic and aids regeneration in mammals. In the present study we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of its target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.Significance StatementIn humans and other mammals, many organs regenerate through the proliferation of cells that replace those that have succumbed to aging or injury. However, proliferation is largely absent in certain critical organs, including the heart, the central nervous system, and sensory organs such as the inner ear and retina. The Hippo-Yap biochemical signaling pathway, a cascade of proteins that—when active—inhibits cell division, constitutes one impediment to proliferation. We earlier identified a small molecule that interrupts Hippo-Yap signaling and thus relieves this block for some non-proliferating cells in vitro. In the present investigation, we have chemically modified the original substance to yield a more potent analog that is effective for several hours in mammalian tissues in vivo and initiates the proliferation of heart-muscle cells after cryolesioning. After further refinements, compounds of this family might prove useful in regenerative therapies.