In cells, myriad membrane-interacting proteins generate and maintain curved membrane domains with radii of curvature around or below 50 nm. To understand how such highly curved membranes modulate specific protein functions, and vice versa, it is imperative to use small liposomes with precisely defined attributes as model membranes. Here, we report a versatile and scalable sorting technique that uses cholesterol-modified DNA "nanobricks" to differentiate hetero-sized

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum (ER) membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit the Sec61 channel, generating cellular effects that are potentially useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of the human Sec61 channel inhibited by a comprehensive panel of structurally distinct small moleculescotransin, decatransin, apratoxin F, ipomoeassin F, mycolactone, cyclotriazadisulfonamide (CADA) and eeyarestatin I (ESI). Remarkably, all inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of the channel. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain of Sec61 in a closed state, thereby preventing the protein-translocation pore from opening. Our study reveals molecular interactions between Sec61 and its inhibitors in atomic detail and offers the structural framework for further pharmacological studies and drug design.

SummaryZika virus (ZIKV) infection causes Guillain-Barré syndrome and severe birth defects. ZIKV envelope (E) protein is the major viral protein involved in cell receptor binding and entry and is therefore considered one of the major determinants in ZIKV pathogenesis. Here we report a gene-wide mapping of functional residues of ZIKV E protein using a mutant library, with changes covering every nucleotide position. By comparing the replication fitness of every viral mutant between mosquito and human cells, we identified that mutations affecting glycosylation display the most divergence. By characterizing individual mutants, we show that ablation of glycosylation selectively benefits ZIKV infection of mosquito cells by enhancing cell entry, whereas it either has little impact on ZIKV infection on certain human cells or leads to decreased infection through the entry factor DC-SIGN. In conclusion, we define the roles of individual residues of ZIKV envelope protein, which contribute to ZIKV replication fitness in human and mosquito cells.

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum (ER) membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit the Sec61 channel, generating cellular effects that are potentially useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of the human Sec61 channel inhibited by a comprehensive panel of structurally distinct small molecules--cotransin, decatransin, apratoxin F, ipomoeassin F, mycolactone, cyclotriazadisulfonamide (CADA) and eeyarestatin I (ESI). Remarkably, all inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of the channel. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain of Sec61 in a closed state, thereby preventing the protein-translocation pore from opening. Our study reveals molecular interactions between Sec61 and its inhibitors in atomic detail and offers the structural framework for further pharmacological studies and drug design.

Sumoylation is a reversible post-translational modification of proteins by small ubiquitin-like modifier (SUMO) that regulates protein function and contributes to cellular response to stress. Sumoylation is an essential pathway for all cells and is involved in diverse processes such as inflammation, DNA damage response, signaling and cell survival/apoptosis. Due to its involvement in such cellular functions, sumoylation intersects with the majority of the hallmarks of cancer. Many cancers display elevated expression of SUMO pathway components, a mechanism co-opted by tumors to survive under stressful conditions. In addition to its tumor intrinsic functions, sumoylation has emerged as a novel target for activating anti-tumor immunity due to its role in regulating type I interferon (IFN) signaling. Targeting sumoylation is a compelling anti-cancer strategy due to the multiple mechanisms of action that may drive anti-tumor activity for cancer patients. We report the discovery of SB-4826, a novel, orally active, covalent small molecule inhibitor of the Sumo Activating Enzyme (SUMO E1), the initiating enzyme of the sumoylation cascade. SB-4826 selectively and irreversibly binds an allosteric pocket of SUMO E1 and drives potent biochemical and cellular activity. SB-4826 inhibits global sumoylation in cells and blocks proliferation across a broad panel (100+) of cancer cell lines. Cysteine-proteome profiling by mass spectrometry indicated that Cys-30 of SUMO E1 was the only cysteine-containing peptide (out of 14,541 measured) that met the criteria for covalent engagement indicating exquisite selectivity across the cellular proteome. In an in vivo pharmacodynamic model measuring IFN signaling SB-4826 treatment led to dose- and time-dependent induction of IFN regulated cytokines. SB-4826 treatment resulted in significant tumor growth inhibition in multiple in vivo models. In a syngeneic A20 lymphoma model SB-4826 led to tumor stasis. In a human RAJI xenograft model, SB-4826 induced near-complete inhibition of tumor growth as a single agent and resulted in tumor regression when combined with rituximab. In a mouse CT-26 colorectal tumor model which is generally refractory to anti-PD-1 treatment, SB-4826 significantly inhibited tumor growth as monotherapy and led to 60% complete tumor responses when combined with anti-PD-1. SB-4826 caused increased CD8+ T cell infiltration in CT-26 tumors supporting the role of SB-4826 in inducing IFN signaling and leading to a tumor microenvironment that is more responsive to immune checkpoint inhibition. SUMO E1 is a clinically validated cancer target as evidenced by recent data showing encouraging responses to TAK-981, an IV-administered, ATP-competitive inhibitor. SB-4826 is a differentiated and optimized SUMO E1 inhibitor that is administered orally and demonstrated improved activity in preclinical models. SB-4826 has favorable properties for development, is a new and promising cancer therapy, and is entering phase 1 clinical studies in 2023. Citation Format: Jude R. Canon, Ashley Callinan, Sarah Bradley, Laurie Wang, Mackenzie Kui, Ariel Wein, Gabriele Sulli, Peter Buchowiecki, Fang-Tsao Hong, Miles Kubota, Mary Walton, Victor J. Cee. SB-4826, a first-in-class oral, covalent inhibitor of SUMO E1 that induces IFN signaling and inhibits tumor growth as monotherapy and in combination with immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB318.