Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D (1)H-(15)N correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the Streptococcus protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned ab initio, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D (15)N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.

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Oncogenic Ras plays a key role in cancer initiation but also contributes to malignant phenotypes by stimulating nutrient uptake and promoting invasive migration. Because these latter cellular responses require Rac-mediated remodeling of the actin cytoskeleton, we hypothesized that molecules involved in Rac activation may be valuable targets for cancer therapy. We report that genetic inactivation of the Rac-specific guanine nucleotide exchange factor DOCK1 ablates both macropinocytosis-dependent nutrient uptake and cellular invasion in Ras-transformed cells. By screening chemical libraries, we have identified 1-(2-(3'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-oxoethyl)-5-pyrrolidinylsulfonyl-2(1H)-pyridone (TBOPP) as a selective inhibitor of DOCK1. TBOPP dampened DOCK1-mediated invasion, macropinocytosis, and survival under the condition of glutamine deprivation without impairing the biological functions of the closely related DOCK2 and DOCK5 proteins. Furthermore, TBOPP treatment suppressed cancer metastasis and growth in vivo in mice. Our results demonstrate that selective pharmacological inhibition of DOCK1 could be a therapeutic approach to target cancer cell survival and invasion.

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