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The focal adhesion kinase (FAK) scaffold provides FAK-targeted cancer therapeutics with greater efficacy and specificity than traditional kinase inhibitors. The FAK scaffold function largely involves the interaction between FAK’s focal adhesion targeting (FAT) domain and paxillin, ultimately regulating many hallmarks of cancer. We report the design of paxillin LD-motif mimetics that successfully inhibit the FAT-paxillin interaction. Chemical and biochemical screening identifies stapled peptide 1907, a high affinity binder of the FAT four-helix bundle with ~100-fold greater binding affinity than the native LD2-sequence. The X-ray co-crystal structure of the FAT-1907 complex is solved. Myristoylated 1907-analog, peptide 2012, delocalizes FAK from focal adhesions, induces cancer cell apoptosis, reduces in vitro viability and invasion, and decreases tumor burden in B16F10 melanoma female mice. Enzymatic FAK inhibition produces no comparable effects. Herein, we describe a biologically potent therapeutic strategy to target the FAK-paxillin complex, a previously deemed undruggable protein-protein interaction. 

Sample consumption for serial femtosecond crystallography with X-ray free electron lasers remains a major limitation preventing broader use in macromolecular crystallography. This drawback is exacerbated in time-resolved (TR) experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. To reduce this limitation, we demonstrate a segmented droplet generation strategy coupled to a mix-and-inject approach for TR studies at the European XFEL. The injector produces synchronized droplet trains that enable stable and reproducible injection of protein crystal slurries at significantly reduced flow rates. Using the human flavoenzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a test system, we collected diffraction data after mixing with NADH at 0.3 s and 1.2 s delays. The segmented injection approach achieved up to 97% reduction in sample consumption compared with continuous-flow injection while maintaining data quality suitable for TR crystallography. Reproducible electron density features consistent with low-occupancy NADH binding illustrate both the feasibility and the current limits of studying dynamic redox enzymes using this approach. This work establishes segmented droplet generation as a sample-efficient and XFEL-compatible method for future time-resolved serial crystallography experiments. 

Let \(\Sigma\) be a closed subset of \(\mathbb{R}^{n+1}\) which is parabolic Ahlfors-David regular and assume that \(\Sigma\) satisfies a 2-sided corkscrew condition. Assume, in addition, that \(\Sigma\) is either time-forwards Ahlfors-David regular, time-backwards Ahlfors-David regular, or parabolic uniform rectifiable. We then first prove that \(\Sigma\) satisfies a weak synchronized two cube condition. Based on this we are able to revisit the argument of Nyström and Strömqvist (2009) and prove that \(\Sigma\) contain suniform big pieces of Lip(1,1/2) graphs. When \(\Sigma\) is parabolic uniformly rectifiable the construction can be refined and in this case we prove that \(\Sigma\) contains uniform big pieces of regular parabolic Lip(1,1/2) graphs. Similar results hold if \(\Omega\subset\mathbb{R}^{n+1}\) is a connected component of \(\mathbb{R}^{n+1}\setminus\Sigma\) and in this context we also give a parabolic counterpart of the main result of Azzam et al. (2017) by proving that if \(\Omega\) is a one-sided parabolic chord arc domain, and if \(\Sigma\) is parabolic uniformly rectifiable, then \(\Omega\) is in fact a parabolic chord arc domain. Our results give a flexible parabolic version of the classical (elliptic) result of David and Jerison (1990) concerning the existence of uniform big pieces of Lipschitz graphs for sets satisfying a two disc condition.