RESUMEN
Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.
Asunto(s)
Antineoplásicos/farmacología , Materiales Biomiméticos/farmacología , Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/farmacología , Animales , Antígeno B7-H1/metabolismo , Materiales Biomiméticos/química , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Cristalografía por Rayos X , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ratones , Nucleótidos Cíclicos/química , Conformación Proteica/efectos de los fármacosRESUMEN
Characterization of mRNA sequences is a critical aspect of mRNA drug development and regulatory filing. Herein, we developed a novel bottom-up oligonucleotide sequence mapping workflow combining multiple endonucleases that cleave mRNA at different frequencies. RNase T1, colicin E5, and mazF were applied in parallel to provide complementary sequence coverage for large mRNAs. Combined use of multiple endonucleases resulted in significantly improved sequence coverage: greater than 70% sequence coverage was achieved on mRNAs near 3000 nucleotides long. Oligonucleotide mapping simulations with large human RNA databases demonstrate that the proposed workflow can positively identify a single correct sequence from hundreds of similarly sized sequences. In addition, the workflow is sensitive and specific enough to detect minor sequence impurities such as single nucleotide polymorphisms (SNPs) with a sensitivity of less than 1%. LC-MS/MS-based oligonucleotide sequence mapping can serve as an orthogonal sequence characterization method to techniques such as Sanger sequencing or next-generation sequencing (NGS), providing high-throughput sequence identification and sensitive impurity detection.
