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Introduction STING (stimulator of interferon genes) is an emerging target for cancer immunotherapy. 2’,3'-cGAMP, a natural cyclic dinucleotide (CDN) STING agonist, and its phosphorothioate analogs, have drawn broad attention as lead molecules for STING targeted drug discovery. These CDNs, however, lack efficacy in some common STING genotypes disproportionally represented in non-Caucasians. Moreover, such CDNs have not fully addressed liability in chemical/metabolic stability. Here we report our chemistry approach to control STING agonist conformation to enhance binding affinity across all common STING genotypes and broaden the therapeutic potential of such compounds. Methods Our SBDD approach started with analysis of the binding pocket and key protein-ligand interactions to prioritize a focused set of analogs for chemical synthesis. Systematic SAR was built upon in vitro assays for STING binding affinity and activation of STING genotypes. X-ray single crystal structures were established for STING and diverse analogs, in free and bound states, to provide structural insight for rational analog design. Results Structural modeling was refined to evaluate different binding modes and dynamic conformational changes in the STING-ligand interface. We observed that STING-bound CDNs had the two ancillary nucleobases specifically oriented in close proximity with parallel pi-pi stacking and discovered that covalently linking the nucleobases advantageously pre-organize the bioactive constrained conformation for enhanced STING affinity. Our discovery established a novel class of macrocycle-bridged STING agonists (MBSAs). E7766, a representative of Eisai MBSA platform, shows superior in vitro activity against all the major human STING genotypes over reference CDNs, most distinctly in STINGREF. E7766 co-crystal structures with STINGWT and STINGREF provide structural basis for the added benefit of the topological novelty. The macrocyclic linker bridging the top of nucleobases perturbs the STING lid loop conformation and create new and specific interactions with both genotypes. In twelve subcutaneous tumor models in immune competent mice, single intra-tumoral injections achieved either complete regression or significant tumor growth delay with no serious adverse effect. E7766 also shows excellent chemical and metabolic stability, presumably conferred by conformational rigidity of the unique macrocycle bridge. More biological characterization of E7766 can be found in abstract #. Conclusion Eisai successfully discovered E7766, a representative of a novel class of macrocycle-bridged STING agonist topologically distinct from conventional STING agonists. E7766 demonstrated pan-genotypic STING activation, potent anti-cancer activities and excellent chemical and metabolic stability for further development. Citation Format: Atsushi ENDO, Dae-Shik Kim, Kuan-Chun Huang, Ming-Hong Hao, Steven Mathieu, Hyeong-wook Choi, Utpal Majumder, Xiaojie Zhu, Yongchun Shen, Kristen Sanders, Thomas Noland, Dinesh Chandra, Yu Chen, Karen TenDyke, Kara Loiacono, Donna Kolber-Simonds, Rongrong Jiang, Vaishali Dixit, Janna Hutz, John Wang, Xingfeng Bao, Francis Fang, Nadeem Sarwar. Discovery of E7766: A representative of a novel class of macrocycle-bridged STING agonists (MBSAs) with superior potency and pan-genotypic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4456.

Chlorotoxin is an established tumor targeting peptide that naturally occurs in scorpion venom. 131I-labelled chlorotoxin assessed in early phase human glioma clinical trials achieved promising results. A peptide drug conjugate (PDC) composed of chlorotoxin peptide linked to a cytotoxic payload (cryptophycin analog) was used as a tool to probe the tumor targeting mechanism of chlorotoxin. The PDC proved efficacious, yet differential sensitivity was observed in multiple human tumor models. Previously described chlorotoxin targets did not align with observed PDC activity; therefore, studies to further elucidate its mechanism were undertaken. PDC treatment of distinct xenograft models led to a wide range of antitumor activity even though similar levels of active metabolite were present in tumor lysates; thus it was hypothesized that a role for chlorotoxin in uptake may be relevant. We identified that the endocytic receptor Neuropilin1 (NRP1) binds to chlorotoxin peptide fragments following proteolytic digestion in vitro. NRP1 binding was selective for peptides with a free C-terminal arginine, while native chlorotoxin which has an amidated C-terminal arginine did not bind. Recovery of chlorotoxin from ex vivo tumor lysate revealed its metabolism to a carboxylated C-terminal arginine version of the peptide, capable of binding to NRP1. These data suggest that chlorotoxin acts as a cryptic peptide incapable of binding to NRP1 systemically and only when metabolized in the tumor microenvironment is NRP1 binding revealed. The expression level of human NRP1 in tumors correlated to the PDC antitumor activity in multiple xenograft models; a wider therapeutic window was observed when NRP1 was highly expressed. Reduction of NRP1 levels in vivo through administration of NRP1 blocking antibodies or by NRP1 knockout in tumor cells blunted PDC antitumor activity while not affecting activity of the cytotoxic payload alone. Reduced PDC antitumor activity correlated with significantly lower levels of active metabolite detected in NRP1-deficient tumors. Together, our findings suggest that chlorotoxin metabolized in the tumor microenvironment binds NRP1 on tumor cells to increase uptake of active metabolite into cells, resulting in enhanced antitumor activity. The identification of NRP1 as an uptake mechanism for chlorotoxin will enable selection of tumors for treatment with chlorotoxin-based therapeutics. Citation Format: Donna Kolber-Simonds, Jiayi Wu, Utpal Majumder, Daniel Custar, Danyang Li, Hong Du, Maarten H. Postema, Thomas Noland, Andrew Hart, George Lai, Sean Eckley, Vaishali Dixit, Karen Tendyke, Kenichi Nomoto, Mary Woodall-Jappe, Sharon McGonigle. Role for neuropilin1 in mode of action of chlorotoxin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3961.