Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

Prognostic relevance of steroid sulfation in adrenocortical carcinoma revealed by molecular phenotyping using high resolution mass spectrometry imaging.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare tumor with variable prognosis even within the same tumor stage. Cancer-related sex hormones and their sulfated metabolites in body fluids can be used as tumor markers. The role of steroid sulfation in ACC has not yet been studied. MALDI mass spectrometry imaging (MALDI-MSI) is a novel tool for tissue-based chemical phenotyping.METHODS: We performed phenotyping of formalin-fixed, paraffin-embedded tissue samples from 72 ACC by MALDI-MSI at a metabolomics level.RESULTS: Tumoral steroid hormone metabolites-estradiol sulfate [hazard ratio (HR) 0.26; 95% CI, 0.10-0.69; P = 0.005] and estrone 3-sulfate (HR 0.22; 95% CI, 0.07-0.63; P = 0.003)-were significantly associated with prognosis in Kaplan-Meier analyses and after multivariable adjustment for age, tumor stage, and sex (HR 0.29; 95% CI, 0.11-0.79; P= 0.015 and HR 0.30; 95% CI, 0.10-0.91; P = 0.033, respectively). Expression of sulfotransferase SULT2A1 was associated with prognosis to a similar extent and was validated to be a prognostic factor in two published data sets. We discovered the presence of estradiol-17 beta 3,17-disulfate (E2S2) in a subset of tumors with particularly poor overall survival. Electron microscopy revealed novel membrane-delimited organelles in only these tumors. By applying duster analyses of metabolomic data, 3 sulfation-related phenotypes exhibited specific metabolic features unrelated to steroid metabolism.CONCLUSIONS: MALDI-MSI provides novel insights into the pathophysiology of ACC. Steroid hormone sulfation may be used for prognostication and treatment stratification. Sulfation-related metabolic reprogramming may be of relevance also in conditions beyond the rare ACC and can be directly investigated by the use of MALDI-MSI

Landscape of somatic mutations in sporadic GH-secreting pituitary adenomas.

CONTEXT: Alterations in the cAMP signalling pathway are common in hormonally-active endocrine tumors. Somatic mutations at GNAS are causative in 30-40% of GH-secreting adenomas. Recently, mutations affecting the USP8 and PRKACA gene have been reported in ACTH-secreting pituitary adenomas and cortisol-secreting adrenocortical adenomas, respectively. However, the pathogenesis of many GH-secreting adenomas remains unclear. AIM: Comprehensive genetic characterization of sporadic GH-secreting adenomas and identification of new driver mutations. DESIGN: Screening for somatic mutations was performed in 67 GH-secreting adenomas by targeted sequencing for GNAS, PRKACA, and USP8 mutations (n=31) and next-generation exome-sequencing (n=36). RESULTS: By targeted sequencing known activating mutations in GNAS were detected in 5 cases (16.1%), while no somatic mutations were observed in both PRKACA and USP8. Whole exome sequencing identified 134 protein-altering somatic mutations in 31/36 tumors with a median of 3 mutations per sample (range: 1-13). The only recurrent mutations have been observed in GNAS (31.4% of cases). However, 7 genes involved in cAMP signalling pathway were affected in 14 of 36 samples and 8 samples harbored variants in genes involved in the calcium signaling or metabolism. At the enrichment analysis, several altered genes resulted to be associated with developmental processes. No correlation between genetic alterations and the clinical data was observed. CONCLUSIONS: This study provides a comprehensive analysis of somatic mutations in a large series of GH-secreting adenomas. No novel recurrent genetic alterations have been observed, but the data suggest that beside cAMP pathway calcium signalling might be involved in the pathogenesis of these tumors

Genetic landscape of sporadic unilateral adrenocortical adenomas without <em>PRKACA</em> p.Leu206Arg mutation.

CONTEXT: adrenocortical adenomas (ACAs) are among the most frequent human neoplasias. Genetic alterations affecting the cAMP/PKA signaling pathway are common in cortisol-producing ACAs, while activating mutations in the gene encoding &beta;-catenin (CTNNB1) have been reported in a subset of both benign and malignant adrenocortical tumors. However, the molecular pathogenesis of most ACAs is still largely unclear. OBJECTIVE: aim of the study was to define the genetic landscape of sporadic unilateral ACAs. DESIGN AND SETTING: next-generation whole-exome sequencing was performed on fresh-frozen tumor samples and corresponding normal tissue samples. PATIENTS: 99 patients with ACAs (74 cortisol-producing and 25 endocrine inactive) negative for p.Leu206Arg PRKACA mutation. MAIN OUTCOME MEASURES: identification of known and/or new genetic alterations potentially involved in adrenocortical tumorigenesis and autonomous hormone secretion, genotype-phenotype correlation. RESULTS: 706 somatic protein-altering mutations were detected in 88/99 tumors (median: 6 per tumor). We identified several mutations in genes of the cAMP/PKA pathway, including three novel mutations in PRKACA, associated with female sex and Cushing&#39;s syndrome. We also found genetic alterations in different genes involved in the Wnt/&beta;-catenin pathway, associated with larger tumors and endocrine inactivity, and, notably, in many genes of the Ca2+-signaling pathway. Finally, by comparison of our genetic data with those available in the literature, we describe a comprehensive genetic landscape of unilateral ACAs. CONCLUSIONS: This study provides the largest sequencing effort on ACAs up to now. We thereby identified somatic alterations affecting known and novel pathways potentially involved in adrenal tumorigenesis

Genetic landscape of sporadic unilateral adrenocortical adenomas without <em>PRKACA</em> p.Leu206Arg mutation.

CONTEXT: adrenocortical adenomas (ACAs) are among the most frequent human neoplasias. Genetic alterations affecting the cAMP/PKA signaling pathway are common in cortisol-producing ACAs, while activating mutations in the gene encoding &beta;-catenin (CTNNB1) have been reported in a subset of both benign and malignant adrenocortical tumors. However, the molecular pathogenesis of most ACAs is still largely unclear. OBJECTIVE: aim of the study was to define the genetic landscape of sporadic unilateral ACAs. DESIGN AND SETTING: next-generation whole-exome sequencing was performed on fresh-frozen tumor samples and corresponding normal tissue samples. PATIENTS: 99 patients with ACAs (74 cortisol-producing and 25 endocrine inactive) negative for p.Leu206Arg PRKACA mutation. MAIN OUTCOME MEASURES: identification of known and/or new genetic alterations potentially involved in adrenocortical tumorigenesis and autonomous hormone secretion, genotype-phenotype correlation. RESULTS: 706 somatic protein-altering mutations were detected in 88/99 tumors (median: 6 per tumor). We identified several mutations in genes of the cAMP/PKA pathway, including three novel mutations in PRKACA, associated with female sex and Cushing&#39;s syndrome. We also found genetic alterations in different genes involved in the Wnt/&beta;-catenin pathway, associated with larger tumors and endocrine inactivity, and, notably, in many genes of the Ca2+-signaling pathway. Finally, by comparison of our genetic data with those available in the literature, we describe a comprehensive genetic landscape of unilateral ACAs. CONCLUSIONS: This study provides the largest sequencing effort on ACAs up to now. We thereby identified somatic alterations affecting known and novel pathways potentially involved in adrenal tumorigenesis