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In this study, an integrated in silico-in vitro approach was employed to discover natural products (NPs) active against SARS-CoV-2. The two SARS-CoV-2 viral proteases, i.e., main protease (Mpro) and papain-like protease (PLpro), were selected as targets for the in silico study. Virtual hits were obtained by docking more than 140,000 NPs and NP derivatives available in-house and from commercial sources, and 38 virtual hits were experimentally validated in vitro using two enzyme-based assays. Five inhibited the enzyme activity of SARS-CoV-2 Mpro by more than 60% at a concentration of 20 mu M, and four of them with high potency (IC50 < 10 mu M). These hit compounds were further evaluated for their antiviral activity against SARS-CoV-2 in Calu-3 cells. The results from the cell-based assay revealed three mulberry Diels-Alder-type adducts (MDAAs) from Morus alba with pronounced anti-SARS-CoV-2 activities. Sanggenons C (12), O (13), and G (15) showed IC50 values of 4.6, 8.0, and 7.6 mu M and selectivity index values of 5.1, 3.1 and 6.5, respectively. The docking poses of MDAAs in SARS-CoV-2 Mpro proposed a butterfly-shaped binding conformation, which was supported by the results of saturation transfer difference NMR experiments and competitive 1H relaxation dispersion NMR spectroscopy., Journal of Natural Products, 86 (2), ISSN:0163-3864, ISSN:1520-6025

While nuclear magnetic resonance (NMR) is regarded asa referencein fragment-based drug design, its implementation in a high-throughputmanner is limited by its lack of sensitivity resulting in long acquisitiontimes and high micromolar sample concentrations. Several hyperpolarizationapproaches could, in principle, improve the sensitivity of NMR alsoin drug research. However, photochemically induced dynamic nuclearpolarization (photo-CIDNP) is the only method that is directly applicablein aqueous solution and agile for scalable implementation using off-the-shelfhardware. With the use of photo-CIDNP, this work demonstrates thedetection of weak binders in the millimolar affinity range using lowmicromolar concentrations down to 5 mu M of ligand and 2 mu Mof target, thereby exploiting the photo-CIDNP-induced polarizationtwice: (i) increasing the signal-to-noise by one to two orders inmagnitude and (ii) polarization-only of the free non-bound moleculeallowing identification of binding by polarization quenching, yieldinganother factor of hundred in time when compared with standard techniques.The interaction detection was performed with single-scan NMR experimentsof a duration of 2 to 5 s. Taking advantage of the readiness of photo-CIDNPsetup implementation, an automated flow-through platform was designedto screen samples at a screening rate of 1500 samples per day. Furthermore,a 212 compounds photo-CIDNP fragment library is presented, openingan avenue toward a comprehensive fragment-based screening method., Journal of the American Chemical Society, 145 (22), ISSN:0002-7863, ISSN:1520-5126

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules and their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging for MALDI-MS. Herein, we successfully used commercially available purified proteins (β-galactosidase and BSA) as internal standards for high-mass MALDI-MS analysis and achieved absolute quantification of several high-mass analytes. We systematically evaluated four sample deposition methods, and using the sandwich deposition method with saturated sinapinic acid as the top layer, we performed a robust quantitative analysis by high-mass MALDI-MS. Combined with chemical cross-linking, this quantitative strategy was further used to evaluate the affinity of protein–protein interactions (PPIs), specifically of two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners. The measured dissociation constants of the protein complexes formed were between 10 nM and 5 μM. We expect this high-throughput, rapid method, which does not require labeling or immobilization of any of the interaction partners, to become a viable alternative to traditional biophysical methods for studying PPIs. ISSN:1520-6882 ISSN:0003-2700

Prebiotic chemical replication is a commonly assumed precursor to and prerequisite for life and as such is the one of the goals of our research. We have previously reported on the role that short peptide amyloids could have played in a template-based chemical elongation. Here we take a step closer to the goal by reproducing amyloid-templated peptide elongation with carbonyl sulfide (COS) in place of the less-prebiotically relevant carbonyldiimidazole (CDI) used in the earlier study. Our investigation shows that the sequence-selectivity and stereoselectivity of the amyloid-templated reaction is similar for both activation chemistries. Notably, the amyloid protects the peptides from some of the side-reactions that take place with the COS-activation.

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 DEAL