A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos

Abstract

SUMMARYTo uncover the molecular mechanisms of embryonic development, the ideal loss-of-function strategy would be capable of targeting specific regions of the living embryo with both temporal and spatial precision. To this end, we have developed a novel pharmacological agent that can be light activated to achieve spatiotemporally limited inhibition of Rho kinase activity in vivo. A new photolabile caging group, 6-nitropiperonyloxymethyl (NPOM), was installed on a small-molecule inhibitor of Rho kinase, Rockout, to generate a 'caged Rockout' derivative. Complementary biochemical, cellular, molecular and morphogenetic assays in both mammalian cell culture and Xenopus laevis embryos validate that the inhibitory activity of the caged compound is dependent on exposure to light. Conveniently, this unique reagent retains many of the practical advantages of conventional small-molecule inhibitors, including delivery by simple diffusion in the growth medium and concentration-dependent tuneability, but can be locally activated by decaging with standard instrumentation. Application of this novel tool to the spatially heterogeneous problem of embryonic left-right asymmetry revealed a differential requirement for Rho signaling on the left and right sides of the primitive gut tube, yielding new insight into the molecular mechanisms that generate asymmetric organ morphology. As many aromatic/heterocyclic small-molecule inhibitors are amenable to installation of this caging group, our results indicate that photocaging pharmacological inhibitors might be a generalizable technique for engendering convenient loss-of-function reagents with great potential for wide application in developmental biology.

Axitinib: A Photoswitchable Approved Tyrosine Kinase Inhibitor

“…Most of these inhibitors are caged prodrugs, that is, kinase inhibitors modified with ap hotoremovable protecting group that can be irreversibly cleaved by light. [18][19][20] In line with this notion,o ur group has published photoactivatable prodrugs of the approvedk inase inhibitors vemurafenib [21] and imatinib [22] in the past few years. Ad rawback of the caging approachist hat activation remains irreversible andi nvolves the releaseo fabyproduct upon uncaging.…”

“…Regarding the field of protein kinases, only a few photosensitive small‐molecule compounds have been reported. Most of these inhibitors are caged prodrugs, that is, kinase inhibitors modified with a photoremovable protecting group that can be irreversibly cleaved by light . In line with this notion, our group has published photoactivatable prodrugs of the approved kinase inhibitors vemurafenib and imatinib in the past few years.…”

Illuminating developmental biology through photochemistry

“…It includes caged inhibitors such as a Rockout, a nitropiperonyloxymethyl-functionalized Rho kinase inhibitor that was used to uncover left-right differences in Xenopus laevis gut patterning 62 , and caged proteins such as a 4,5-dimethoxy-2-nitrobenzyl-modified Gal4-VP16 transactivator that was applied in Drosophila , Xenopus , and zebrafish embryos 63 . Endogenous photoresponsive molecules such as retinoic acid can be utilized as well.…”

Design, Synthesis, and Characterization of a Photoactivatable Caged Prodrug of Imatinib

“…[18] For this purpose o-nitrobenzylic [19] and coumarylmethyl [20] derivatives have been widely used, among others,a sP PGs in various biological applications. These include ac aged small-molecule Rho kinase inhibitor, [24] equivalents of Src kinase, [21] and peptidic PKAi nhibitors. These include ac aged small-molecule Rho kinase inhibitor, [24] equivalents of Src kinase, [21] and peptidic PKAi nhibitors.…”

“…[21][22][23] There are currently only few reportso np hotoactivatable kinase inhibitors. These include ac aged small-molecule Rho kinase inhibitor, [24] equivalents of Src kinase, [21] and peptidic PKAi nhibitors. [25] Recently,astudy about photo-switchable RET kinase inhibitors was published.…”