A bioorthogonal organometallic reaction is a biocompatible transformation undergone by a synthetic material exclusively through the mediation of a non-biotic metal source; a selective process used to label biomolecules and activate probes in biological environs. Here we report the in vitro bioorthogonal generation of 5-fluorouracil from a biologically inert precursor by heterogeneous Pd0 catalysis. Although independently harmless, combined treatment of 5-fluoro-1-propargyl-uracil and Pd0-functionalized resins exhibits comparable antiproliferative properties to the unmodified drug in colorectal and pancreatic cancer cells. Live-cell imaging and immunoassay studies demonstrate that the cytotoxic activity of the prodrug/Pd0-resin combination is due to the in situ generation of 5-fluorouracil. Pd0-resins can be carefully implanted in the yolk sac of zebrafish embryos and display excellent biocompatibility and local catalytic activity. The in vitro efficacy shown by this masking/activation strategy underlines its potential to develop a bioorthogonally activated prodrug approach and supports further in vivo investigations.
Fascin is an actin-bundling protein involved in filopodia assembly and cancer invasion and metastasis of multiple epithelial cancer types. Fascin forms stable actin bundles with slow dissociation kinetics in vitro and is regulated by phosphorylation of serine 39 by protein kinase C (PKC). Cancer cells use invasive finger-like protrusions termed invadopodia to invade into and degrade extracellular matrix. Invadopodia have highly dynamic actin that is assembled by both Arp2/3 complex and formins; they also contain components of membrane trafficking machinery such as dynamin and cortactin and have been compared with focal adhesions and podosomes. We show that fascin is an integral component of invadopodia and that it is important for the stability of actin in invadopodia. The phosphorylation state of fascin at S39, a PKC site, contributes to its regulation at invadopodia. We further implicate fascin in invasive migration into collagen I-Matrigel gels and particularly in cell types that use an elongated mesenchymal type of motility in 3D. We provide a potential molecular mechanism for how fascin increases the invasiveness of cancer cells, and we compare invadopodia with invasive filopod-like structures in 3D.
SummaryDynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches.
Bioorthogonal
chemistry has become one of the main driving forces
in current chemical biology, inspiring the search for novel biocompatible
chemospecific reactions for the past decade. Alongside the well-established
labeling strategies that originated the bioorthogonal paradigm, we
have recently proposed the use of heterogeneous palladium chemistry
and bioorthogonal Pd0-labile prodrugs to develop spatially
targeted therapies. Herein, we report the generation of biologically
inert precursors of cytotoxic gemcitabine by introducing Pd0-cleavable groups in positions that are mechanistically relevant
for gemcitabine’s pharmacological activity. Cell viability
studies in pancreatic cancer cells showed that carbamate functionalization
of the 4-amino group of gemcitabine significantly reduced (>23-fold)
the prodrugs’ cytotoxicity. The N-propargyloxycarbonyl
(N-Poc) promoiety displayed the highest sensitivity
to heterogeneous palladium catalysis under biocompatible conditions,
with a reaction half-life of less than 6 h. Zebrafish studies with
allyl, propargyl, and benzyl carbamate-protected rhodamines confirmed N-Poc as the most suitable masking group for implementing in vivo bioorthogonal organometallic chemistry.
IRSp53 is a scaffold protein that contains an IRSp53/MIM homology domain (IMD) that bundles actin filaments and interacts with the small GTPase Rac. IRSp53 also binds to the small GTPase Cdc42 and to Scar/WAVE and Mena/VASP proteins to regulate the actin cytoskeleton. We have characterised a novel IMD-containing protein, insulin receptor tyrosine kinase substrate (IRTKS), which has widespread tissue distribution, is a substrate for the insulin receptor and binds Rac. Unlike IRSp53, IRTKS does not interact with Cdc42. Expression of IRTKS induces clusters of short actin bundles rather than filopodia-like protrusions. This difference may be attributable to a short carboxyl-terminal (Ct) extension present on IRTKS, which resembles a WASP-homology 2 (WH2) motif. Addition of the Ct extension to IRSp53 causes an apparent shortening of bundles induced by the IMD in vitro, and in cultured cells, suggesting that the Ct extension of IRTKS modulates the organising activity of the IMD. Lastly, we could not detect actin monomer sequestration by the Ct extension of IRTKS as would be expected with a conventional WH2 motif, but it did interact with actin filaments.
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