USE OF RESEARCH INFRASTRUCTURES TO IDENTIFY THERAPIES FOR COVID-19

Philip Gribbon – Discovery Research, Fraunhofer ITMP Partner site of EU-OPENSCREEN European RI for Chemical Biology and early Drug Discovery

 Long-term funding from 8 member countries: CZ, DE, DK, ES, FI, LV, NO, PL

 Distributed RI with 24 partner sites across Europe

 Services offered:  Screening platforms (17) Open-access  Medicinal Chemistry groups (6) database: FAIRification of data

Collaborative data sharing  FAIR Database hosting (1) environment . maximise re-use of FAIR data . data available without restrictions on use . optional embargo period up to 36 months . data links to other databases (e.g. https://ecbd.eu/ ChEMBL) Why drug repurposing for COVID-19, when vaccines are available?

 Multiple vaccines authorized worldwide  May face challenges in  Safety in certain groups  Efficacy in certain groups  Duration of effectiveness  Production scale up  Clinical roll out  Patient Access  Virus mutation effects leading to vaccine escape / resistance  No high efficacy and safe curative therapy in

place yet https://wellcome.org/news/what-different-types-covid-19-vaccine-are-there Clinical trials in Europe, China, US, Japan, Australia..

Some successes  Approved Therapeutics  Dexamethasone in the United Kingdom and Japan;  Avigan (favilavir) in China, Italy and Russia;  Veklury (remdesivir) in the United States, Japan and Australia.  Emergency use authorizations (EUAs) in the US.  Convalescent plasma, initially authorized by the FDA  Eli Lilly and Company’s monoclonal bamlanivimab,  Regeneron monoclonals casirivimab and imdevimab  Combination of Veklury and the JAK inhibitor Olumiant (Eli Lilly and Company) https://doi.org/10.1038/s41577-021-00542-x  Emerging pre-clinical and clinical stage therapeutics  PF-07321332 Phase 1 clinical start for 3-CL/M Pro inhibitor (NSP-5)  EMA has provided advice given on 57 possible therapies (@Feb ‘21) https://www.ema.europa.eu/en/human-regulatory/overview/public-health-threats/coronavirus-disease-covid-19/treatments- vaccines/treatments-covid-19/covid-19-treatments-research-development Repurposing compounds (> 6000) and screening infrastructure

Indications Target s Screening Infrastructure Anti Cytopathic Effect phenotypic assay with Caco-2 cells

1. Read the images 2. Identify cells 3. Calculate morphology, intensity and distance parameters

(with Ciesek group GuF) Anti Cytopathic effect phenotypic assay with Caco-2 cells

Dissemination (EU H2020 European Open Science Cloud – LIFE) Publication @ Ellinger et al Scientific Data https://doi.org/10.1038/s41597-021-00848-4 Bioactivity data @ ChEMBL data sets https://www.ebi.ac.uk/chembl/document_report_card/CHEMBL4303101/ Bioactivity data @ ECBD (imminently) https://ecbd.eu/ Primary images for re-analysis @ IDR https://idr.openmicroscopy.org/webclient/?show=screen-2603 Remdesivir data @ IDR http://idr.openmicroscopy.org/mapr/compound/?value=Remdesivir Jupyter Notebook linking data to analysis workflows https://github.com/IDR/idr0094-ellinger-sarscov2 E4C Study 1 Anti Cytopathic effect phenotypic assay with Vero_E6 cells (KUL)

Screen in primary assay Dose-response curve analysis SARS-CoV-2 Biological validation VeroE6_GFP SARS-CoV-2 Antiviral activity Broad-spectrum VeroE6_GFP, SARS-CoV-2 Huh7_MTS RT-qPCR Toxicity SARS-CoV-1 VeroE6 Antiviral activity VeroE6_GFP

Air-liquid interface

)

t r

e Raloxifene 3 uM

s 8

n i

/ Raloxifene 10 uM

0 5

D 6 CC

I C

T VC

(

c 4 n

o LOQ

C

2 2

S

R

A S

0

g o

L 0 5 10 15 d.p.i.  Virus control  Virus control  Virus control  Remdesivir control  Cell control  Cell control Anti Cytopathic effect phenotypic assay with VeroE6_GFP Cells

primary screening 1-point at 10 µM 8702 compounds

active molecules >18% inhibition 184 compounds

confirmed actives activity in DRC triplicates 132 compounds

relevant drugs IC50 inhibition <10 uM 75 compounds

selection Data can be found here: safety index > 5 https://www.ebi.ac.uk/chembl/docume 23 compounds nt_report_card/CHEMBL4495565/ Raloxifene active ingredient of Optruma®,

. Raloxifene 60 mg oral tablets.

. Available on the market as branded (Optruma® Eli Lilly; Evista® Daiichi Sankyo) or generic (Raloxifene Teva) medication.

. Registered in EU with CP since 1998 for the treatment and prevention of osteoporosis in postmenopausal women.

. Registered in US since 1997 for treatment and prevention of osteoporosis and reduction in risk of invasive breast cancer in postmenopausal women. * www.ema.europa.eu Achievements in EXSCALATE4COV (E4C) Massive X-ray screening against SARS-CoV-2 main protease

High-throughput X-ray synchrotron crystallography

Cooperation led by Alke Meents and Sebastian Günther, DESY /Hamburg SARS-CoV-2 X-ray Screening Initiative X-Ray studies - more than 100 Contributors … from 25 different institutions CFEL Henry N. Chapman Nanostruktur- und Biology Jozef Stefan Institute Stephan Günther Sebastian Günther  Alke Meents Festkörperphysik Henning Tidow Katarina Karničar  Patrick Y. A. Reinke Susanne Meier NMR Aleksandra Usenik Max Planck Institute for Dominik Oberthuer DESY Henry Gieseler Caroline Schmitt Jure Loboda Biophysical Chemistry Oleksandr Yefanov Sofiane Saouane Diogo Melo Thomas Hackel Dusan Turk Ashwin Chari Thomas J. Lane Frank Schlünzen Jo J. Zaitsev-Doyle Luca Gelisio Johanna Hakanpää Cromarte Rogers Center for Bioinformatics Universität Lübeck, Institute of BMWZ, Leibniz University of Martin Domaracky Jan Meyer Arwen R. Pearson Christiane Ehrt Biochemistry Hannover Philipp Middendorf Heshmat Noei Jonathan Pletzer-Zelgert Linlin Zhang Russell Cox Wolfgang Brehm Department of Chemistry Matthias Rarey Xinyuanyuan Sun Julia Lieske Diamond Light Source Structural Biology of Infection Rolf Hilgenfeld EMBL Hamburg Faisal Koua Helen M. Ginn and Inflammation Max Planck Institute for the Robert Schönherr Stephan Niebling Alexandra Tolstikova Nadine Werner Structure and Dynamics of Juliane Boger Christian Günther Thomas A. White European XFEL Hina Andaleeb Matter Lars Redecke Maria Marta Garcia Alai Michael Groessler Kristina Lorenzen Najeeb Ullah Brenna Norton-Baker Holger Fleckenstein Christina Schmidt Sven Falke Eike-Christian Schulz HZB/BESSY Guillaume Pompidor Fabian Trost Robin Schubert Bruno Alves Franca Pedram Mehrabi Jan Wollenhaupt Gleb Bourenkov Marina Galchenkova Huijong Han Martin Schwinzer Christian Feiler David von Stetten Yaroslav Gevorkov Lea Brings Hévila Brognaro Hauptmann Woodward Medical Manfred Weiss Isabel Bento Chufeng Li Vasundara Srinivasan Research Institute Saravanan Panneerselvam Salah Awel CalTech Christian Betzel Diana C. F. Monteiro Heinrich-Pette-Institut Ivars Karpics P. Lourdu Xavier Ariana Peck Boris Krichel Thomas R. Schneider Miriam Barthelmess Physical Chemistry Fraunhofer ITMP Janine Kopicki Juraj Knoska Max Planck Institute for Brandon Seychell Phil Gribbon Charlotte Uetrecht Gisel Esperanza Molecular Genetics Henrick Böhler Bernhard Ellinger Universität Greifswald Aida Rahmani Mashhour Illona Dunkel Marcel Lach Maria Kuzikov Bernhard-Nocht-Institut für Winfried Hinrichs Filip Guicking Laurin Lang Markus Wolf Tropenmedizin Vincent Hennicke Universität Hamburg Tobias Beck Andrea Zaliani Yaiza Fernández-García Dompé Farmaceutici SpA Pontus Fischer Department of Physics Biochemistry and Molecular Beatriz Escudero-Pérez Andrea R. Beccari | Massive X-ray Screening | Sebastian Günther Page 13 Potential SARS-CoV-2 targets Non-structural essential for virus replication

From: Gordon et al., Nature 2020 gene

protein

function

nsp5/ main protease Mpro From: Zhang et al., Science 2020

Structure solved by X-ray crystallography at BESSY

| Massive X-ray Screening | Sebastian Günther Page 14 Hamburg SARS-CoV-2 screening project Idea: High-throughput screening of a 5700 compound re-purposing library against three SARS-CoV-2 relevant target proteins with X-ray crystallography

Fraunhofer ITMP Compound library > 100 contributing scientists WP2 UHH WP1 UHH in-silico pre- Protein production and selection of compound and co-crystallization compound optimization Further partners:

WP3 DESY High- - HZB throughput X-ray - University of Hannover crystal screening and hit identification - SLAC / Caltech HPI / EUXFEL - EMBL Mass spec analysis WP4 Fraunhofer of hits ITMP / UL Protein activity assays of hits WP5 BNITM Virus cell culture experiments Experimental workflow Feedback from experiment

| Massive X-ray Screening | Sebastian Günther Page 15 Workflow for High-throughput X-ray screening Establishing a screening platform during a pandemic

Crystal harvesting and Protein production Protein crystallization flash-freezing and purification

Structure refinement Synchrotron X-ray and ligand identification Data processing data collection

| Massive X-ray Screening | Sebastian Günther Page 16 Hits in active site and beyond 29 Mpro/ligand structures

| Massive X-ray Screening | Sebastian Günther Page 17 From screening to hits

 7857 crystals, incl. apo/other ligand unique Fraunhofer Dompe

Total compounds 5953 5632 607

Total crystals mounted 6976 4777 2199 Unique compounds 3757 3367 58 w/xtals (63.1%) (59.8%) (96.5%) Dataset in hit finding 3757 2545 1212 Unique compounds in hit 2381 1984 491 finding (40%) (35.2%) (80.9%) 36 25 11 Identified hits (0.6%) (0.44%) (1.8%)

| Massive X-ray Screening | Sebastian Günther Page 18 From compounds to hits With antiviral acitivity – our study Pfizer Clinical candidate

Some promising hits In parallel studies @ Pfizer

| Massive X-ray Screening | Sebastian Günther Page 19 Conclusions and next steps

• Identification of several hits which can serve as • Next steps: progress clinical studies to identify new lead structure for future drug developments optimal treatments • Fast response: Project start to completion of data • EU-wide: Five drugs selected for investment, will be collection took months rather than years chosen from a long list of (57) candidates drawn up by the European Medicines Agency (EMA), • High Scientific impact studies and FAIR data dissemination

| Massive X-ray Screening | Sebastian Günther Page 20 Lessons learnt

 Serendipity has a great part to play, but not coincidence….  Repurposing collection - rare disease  Informed by SARS CoV expertises (Hilgenfeld)  In-silico workflows in E4C from Ebola project  Utilizing Cross Infrastructure workflows was essential to success  Chemical screening + structural biology + data- analytics  Image analysis + bioinformatics databases + large scale ‘omics + systems biology tools  High Biosafety labs + animal models + medicinal chemistry  FAIR data working is essential to reach full understanding of the virus function and roles:

Page 21 Acknowledgements

Phil Gribbon Sandra Ciesek Alke Meents Maria Kuzikov Denisa Bojkova Sebastian Guenther Markus Wolf Jindrich Cinatl Patrick Reincke Jeanette Reinshagen Sandra Westhaus Bernhard Ellinger Kristoff Rieken Philipp Reus (+ Fraunhofer) Carsten Claussen Gerd Geisslinger

Pieter Leyssen Laura Vangeel

Katja Herzog Enzo Tramontano Andrea Beccari Angela Corona Carmine Talarico Francesca Esposito Daniela Jaconis Paola Storici Candida Manelfi Elisa Costanzi Barbara Giabbai Extra slides EU-OPENSCREEN Libraries

European Chemical Biology Library The European Academic Compound (ECBL) Library (EACL) Novel compounds sourced from chemists worldwide Diversity library 96.096 . Target is 40.000 compounds . 99.096 structurally highly diverse compounds . Regulated and confidential access (e.g. MTA) . Average MW=350 g/mol . IP stays with the chemist . 0.0005 % of PAINS . Embargo period up to 3 years Horvath D. et al., ChemMedChem 2014, 9, 2309 . User friendly online submission: http://www.eu-openscreen- cmpds-donation.eu/login.php

Fragment Library NEW! European Chemical Biology Library Set of low MW and ultra-low MW fragments . 968 fragments with HAC > 8 in DMSO-d6 (ECBL) 5.016 1.056 . 88 so called “minifrags” with HAC < 8 in DMSO-d6 Pilot library (O’Reilly M. et al., Drug Discov. Today 2019, 24, 1081) . 2.464 bioactives: active against 1039 different targets, . Derived from the fragment space of the ECBL contain 654 approved drugs and 368 highly selective . Collaboration with INSTRUCT/ iNEXT-Discovery probes European Chemical sites . 2.464 representative compounds of the diversity library Biology Database . 88 assay interference compounds in 4 dilutions EU-OPENSCREEN Fragment Library

 Set of low MW and ultra-low MW fragments (1.056  Available at 6 EU-OPENSCREEN-DRIVE compounds) partners  Derived from the fragment space of the ECBL  Latvian Institute of Organic Synthesis (NMR, SPR, ITC, X-Ray)  Designed by Andrea Zaliani, Fraunhofer ITMP and Jordi Mestres, IMIM (manuscript in preparation)  Príncipe Felipe Research Center/ Health Research Institute Hospital La Fe (NMR, SPR,  Library composition: ITC, biochemical experiments)  968 fragments with HAC > 8 in DMSO-d6 (c = 100  Technical University of Denmark (19F NMR, mM) Thermal Shift)  88 so called “minifrags” with HAC < 8 in DMSO-d6 (c =  University of Bergen (SPR, BLI) 1 M)  Natural Science Research Center of the (O’Reilly M. et al., Drug Discov. Today 2019, 24, 1081) Hungarian Academy of Sciences  131 fluorinated fragments  Fraunhofer ITMP in collaboration with DESY  Consistent with a ‘rule of three’ Hamburg (X-Ray)  QC performed at the EU-OPENSCREEN Central  FBDD projects on COVID-19 targets (e.g. Compound Management Facility (LC-MS) Nsp14-nsp10 complex using NMR), viral enzyme endonucleases of the negative strand RNA viruses (X-Ray) and antibiotic targets (BLI). EU-OPENSCREEN Fragment Library

 Set of low MW and ultra-low MW fragments (1.056  Available at 6 iNEXT-Discovery partners compounds)  EMBL Grenoble - High-Throughput  Derived from the fragment space of the ECBL Crystallisation Laboratory  Designed by Andrea Zaliani, Fraunhofer ITMP and  Diamond Light Source - XChem facility Jordi Mestres, IMIM (manuscript in preparation)  Helmholtz Zentrum Berlin - macromolecular crystallography at the BESSY-II storage  Library composition: ring  968 fragments with HAC > 8 in DMSO-d6 (c = 100  Goethe University Frankfurt- Center for mM) Biomolecular Magnetic Resonance (NMR)  88 so called “minifrags” with HAC < 8 in DMSO-d6 (c =  Netherlands Cancer Institute NKI (biophysical 1 M) methods) (O’Reilly M. et al., Drug Discov. Today 2019, 24, 1081)  FragMAX facility at MAX IV Laboratory (X-ray)  131 fluorinated fragments  X-ray FBDD project on Nsp3 Macrodomain 1  Consistent with a ‘rule of three’ of SARS-CoV-2 at Diamond: Science  QC performed at the EU-OPENSCREEN Central Advances, 2021, 7 (16), eabf8711 Compound Management Facility (LC-MS) EU-OPENSCREEN Screening Campaigns using the ECBL  A total of 25 screening campaigns incl. 13 screening

projects funded by EU-OPENSCREEN-DRIVE and 3 SARS- EU-OPENSCREEN Partner Sites CoV-2 related projects Screening campaigns DRIVE funded screening  Researchers from: projects SARS-CoV-2 projects  France, Germany, Italy, Spain, Switzerland, The Netherlands, United Kingdom  Research fields (among others):  Cancer and neurodegenerative disorders  Infectious diseases  Rare diseases  Spinal muscular atrophy  Prion disease  Publication in focus:  Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen. Kuzikov M. et al, ACS Pharmacol Transl Sci 2021, in press