This family of small molecules shares a common cyclopenta[species of angiosperms (King et al., 1982). shares a common cyclopenta[species of angiosperms (King et al., 1982). To date, numerous rocaglate analogs have been synthesized with the goals of improving potency and bioavailability (Ebada et al., 2011; Pan et al., 2014; Ribeiro et al., 2012). Studies using silvestrol, a natural product isolated from its eIF4E subunit, remodels adjacent mRNA structure via eIF4A, and recruits 43S pre-initiation complexes (40S subunit and associated factors) through its eIF4G subunit (Pelletier and Sonenberg, 2019). The dependency on eIF4F for ribosome recruitment by different mRNAs varies and scales with the degree of 5 leader secondary structure (Svitkin et al., 2001). Only ~5% Tomatidine of eIF4A is present in the eIF4F complex, suggesting that multiple eIF4A molecules may be used per initiation round and/or eIF4A may have non-eIF4F related activities in translation (Sokabe and Fraser, 2017). Assembly of the eIF4F complex CAB39L is usually under mTOR regulation, ensuring that rheostatic and selective regulation of mRNA translation is usually linked to extra- and intra-cellular cues (Pelletier and Sonenberg, 2019). Structural elucidation of a rocaglate[RocA]:eIF4A1:polypurine RNA complex revealed that rocaglates function as interfacial inhibitors and make crucial contacts with eIF4A1 (F163L, Q195) and two adjacent RNA purine bases (Iwasaki et al., 2019). When present within 5 mRNA leader regions, polypurine sequences serve as nucleation sites for rocaglate:eIF4A1 complexes, leading to the formation of steric barriers that impede 43S PIC scanning (Iwasaki et al., 2016). A rocaglate-resistant eIF4A1 mutant (F163L) has been characterized and introduction of this allele into cells using CRISPR/Cas9-mediated gene editing confers resistance to rocaglate cytotoxicity (Chu et al., 2016; Iwasaki et al., 2019), further demonstrating that this mechanism of action of these compounds is dependent on their ability to interfere with eIF4A1 activity. Mammalian cells also express a second eIF4A paralog (known as eIF4A2) that shares 90% amino acid identity with eIF4A1 and has been shown to participate in translation initiation (Rogers et al., 2002; Yoder-Hill et al., 1993). However, the effects of rocaglates on eIF4A2 have been largely unexplored as eIF4A1 is the predominant paralog in most cell types (Galicia-Vazquez et al., 2012; Nielsen and Trachsel, 1988). Desire for rocaglates as potential anti-neoplastic brokers and the significant efforts made towards development of synthetic strategies have greatly expanded the number of members in this family (Qian et al., 2016). Herein, we characterized the activity of 200 rocaglates from an in-house library (the BU-CMD collection) to induce RNA clamping of eIF4A1 and eIF4A2. During the course of these studies, we uncovered and characterized amidino-rocaglates (ADRs) that rank among the most potent synthetic derivatives recognized to date. Results and Conversation Rocaglates similarly enhance RNA binding of eIF4A1 and eIF4A2. In order to rapidly evaluate the ability of rocaglates to activate binding of eIF4A to RNA, we required advantage of a fluorescence polarization (FP) assay using a FAM (fluorescein amidite)-labelled RNA probe (Fig 1a) (Iwasaki et al., 2016). The ATPase activity of eIF4A1 is usually stimulated by Tomatidine the presence of RNA; previous studies have documented that this homoribopolymers poly r(A) and poly r(U) are more potent than poly r(C), poly r(I), poly r(G), globin mRNA, tRNA, poly r(I-C), or poly r(A)?poly r(U) substrates, suggesting that eIF4A1 has an inherent nucleotide bias for RNA binding (Abramson et al., 1987). Using the FP assay, we revisited the RNA sequence specificity of eIF4A1, and required the opportunity to characterize the RNA binding Tomatidine activity of its paralog, eIF4A2. Studies focusing on the pharmacological targeting of eIF4A2 have been comparatively limited due to the fact that it is the less abundant eIF4A variant in many cell types. However, this remains an area of interest to explore as there is evidence suggesting that eIF4A2 may have tumor promoting effects (Chen et al., 2019) and in certain cancers, eIF4A2 is the predominant paralog (Wolfe et al., 2014)..