The majority of secreted and membrane proteins in eukaryotic cells are either translocated across or integrated into the ER membrane after the ribosome has docked at the start of the secretory pathway. This process can be selectively and reversibly blocked by a small group of macrocyclic natural products (NPs) produced by fungi, cyanobacteria and human pathogenic bacteria. These specialized NP metabolites bind directly to the Sec61 translocon channel to inhibit co-translational translocation of nascent proteins at the ER resulting in a loss of cellular proteostasis both in the ER and cytosol. Exactly how this group of NP compounds induces either selective or broad inhibition of protein biosynthesis with varying degrees of protein substrate selectivity is not clear. Maintenance of proteostasis is a highly regulated process in mammalian cells and the loss of homeostasis in the cellular secretory pathway is implicated in major human diseases such as cancer, neurodegeneration and diabetes. Thus, our discovery of new potent NP Sec61 inhibitors presents an opportunity to both understand the mechanistic basis of protein import into the ER secretory pathway and provide a reservoir of new Sec61 ligands and potential drug leads based on these complex chemical entities from Nature. We plan to utilize a multidisciplinary approach that includes NP discovery, solid-phase peptide synthesis of macrocycles, pharmacology, chemical and structural biology to pursue the following two aims: 1) Expand and define the class of nonpolar NP-derived macrocycles that target cotranslational translocation; 2) Elucidate the mechanistic basis of Sec61 inhibition using analysis of structure activity relationships (SAR) and cryogenic electron microscopy (cryoEM). In Aim 1, existing NP libraries likely to be rich in non-polar medium-sized macrocycles will be screened for new proteostasis modulators using phenotypic and target-based assays for Sec61-dependent inhibition of ER translocation. Early determination of structural motifs using LCMS2-based metabolomics, NMR spectroscopy and cheminformatics will guide the solid phase peptide synthesis of divergent representatives of NP molecular families to provide a platform for discovery of Sec61 ligands for structural biology studies. In Aim 2 we propose to use cryo-EM to study the specific Sec61 binding of two different NP molecular families by analyzing the Sec61 binding interface of suites of closely related synthetic compounds that have different Sec61 substrate specificity. This multidimensional approach will reveal the feasibility of targeting cellular proteostasis for therapeutic needs, while avoiding toxicities due to non-specific inhibition of secretory protein biosynthesis.