Common and distinctive pathogenetic features of arteriovenous malformations in hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 animal models--brief report.

OBJECTIVE: Hereditary hemorrhagic telangiectasia is a genetic disorder characterized by visceral and mucocutaneous arteriovenous malformations (AVMs). Clinically indistinguishable hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 are caused by mutations in ENG and ALK1, respectively. In this study, we have compared the development of visceral and mucocutaneous AVMs in adult stages between Eng- and Alk1-inducible knockout (iKO) models. APPROACH AND RESULTS: Eng or Alk1 were deleted from either vascular endothelial cells (ECs) or smooth muscle cells in adult stages using Scl-CreER and Myh11-CreER lines, respectively. Latex perfusion and intravital spectral imaging in a dorsal skinfold window chamber system were used to visualize remodeling vasculature during AVM formation. Global Eng deletion resulted in lethality with visceral AVMs and wound-induced skin AVMs. Deletion of Alk1 or Eng in ECs, but not in smooth muscle cells, resulted in wound-induced skin AVMs. Visceral AVMs were observed in EC-specific Alk1-iKO but not in Eng-iKO. Intravital spectral imaging revealed that Eng-iKO model exhibited more dynamic processes for AVM development when compared with Alk1-iKO model. CONCLUSIONS: Both Alk1- and Eng-deficient models require a secondary insult, such as wounding, and ECs are the primary cell type responsible for the pathogenesis. However, Alk1 but not Eng deletion in ECs results in visceral AVMs.

MATRIX platform to analyze translation machinery remodeling in glioblastoma cells.

Here, we present a protocol using MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) platform to investigate changes of the protein synthesis machinery in U87MG glioblastoma cells in response to the rocaglate silvestrol. This protocol describes steps to perform SILAC (stable isotope labeling by amino acids in cell culture), ribosome density fractionation, protein isolation, and mass spectrometry analysis. This approach can be applied to study any adaptive remodeling of protein synthesis machineries. For complete details on the use and execution of this protocol, please refer to Ho et al. (2021).1.

Molecular Dynamics Simulations Identify Tractable Lead-like Phenyl-Piperazine Scaffolds as eIF4A1 ATP-competitive Inhibitors.

eIF4A1 is an ATP-dependent RNA helicase whose overexpression and activity have been tightly linked to oncogenesis in a number of malignancies. An understanding of the complex kinetics and conformational changes of this translational enzyme is necessary to map out all targetable binding sites and develop novel, chemically tractable inhibitors. We herein present a comprehensive quantitative analysis of eIF4A1 conformational changes using protein-ligand docking, homology modeling, and extended molecular dynamics simulations. Through this, we report the discovery of a novel, biochemically active phenyl-piperazine pharmacophore, which is predicted to target the ATP-binding site and may serve as the starting point for medicinal chemistry optimization efforts. This is the first such report of an ATP-competitive inhibitor for eiF4A1, which is predicted to bind in the nucleotide cleft. Our novel interdisciplinary pipeline serves as a framework for future drug discovery efforts for targeting eiF4A1 and other proteins with complex kinetics.

Proteomics reveal cap-dependent translation inhibitors remodel the translation machinery and translatome.

Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling.

Discovery of an eIF4A Inhibitor with a Novel Mechanism of Action.

Increased protein synthesis is a requirement for malignant growth, and as a result, translation has become a pharmaceutical target for cancer. The initiation of cap-dependent translation is enzymatically driven by the eukaryotic initiation factor (eIF)4A, an ATP-powered DEAD-box RNA-helicase that unwinds the messenger RNA secondary structure upstream of the start codon, enabling translation of downstream genes. A screen for inhibitors of eIF4A ATPase activity produced an intriguing hit that, surprisingly, was not ATP-competitive. A medicinal chemistry campaign produced the novel eIF4A inhibitor 28, which decreased BJAB Burkitt lymphoma cell viability. Biochemical and cellular studies, molecular docking, and functional assays uncovered that 28 is an RNA-competitive, ATP-uncompetitive inhibitor that engages a novel pocket in the RNA groove of eIF4A and inhibits unwinding activity by interfering with proper RNA binding and suppressing ATP hydrolysis. Inhibition of eIF4A through this unique mechanism may offer new strategies for targeting this promising intersection point of many oncogenic pathways.