Lead compounds G3Ia and G3Ib bind with high affinity to the NTF2L nsP3 binding pocket of G3BP1. (A) Lead compounds FAZ-3532 (G3Ia) and FAZ-3780 (G3Ib) along with respective enantiomer controls FAZ-3861 (G3Ia′) and FAZ-3852 (G3Ib′). (B) Representative double-reference subtracted sensorgrams of compounds binding to sensor-immobilized human G3BP1. Compounds were tested by 1/2 dilution with top concentrations of 50 μM (G3Ia′), 50 μM (G3Ib′), 10 μM (G3Ia), and 2 μM (G3Ib). Marks on each curve indicate the time span at which equilibrium binding was measured to estimate the equilibrium dissociation constant using a 1:1 Langmuir binding model. (C) Percent inhibition calculated using a peptide displacement assay at indicated doses of G3I compounds. Error bars represent mean ± SD, n = 2 replicates per dose. (D) Crystal structure showing the interaction of G3Ia with the nsP3 binding pocket in the NTF2L domain of G3BP1. The NTF2L domain of G3BP1 (light green cartoon model) crystallized in the presence of G3Ia (yellow sticks), with six copies in the asymmetric unit and copy A shown above. All copies were compound bound, although only half had full compound density. The other three were incomplete in either the ether group (1) or terminal phenylalanine (2), highlighting their flexibility. Tert-butyl (3) functions as a space-filling moiety, maximizing the hydrophobicity of the subpocket lined by V11 and F124. An indirect water-mediated backbone interaction with Q18 is present in four of six copies, including copy A above (large red ball). Modified phenylalanine (4) extends the pi-stacking network formed by F15 and F33. (E) Summary characteristics of the four G3I compounds compared with the nsP3 25-mer peptide. MW, molecular weight; PSA, polar surface area; SPR, surface plasmon resonance; Pep-FRET, peptide-fluorescence resonance energy transfer.