Book of Abstracts - PSDI 2019 · [email protected] Thermo Fisher Scientiﬁc is the world leader in serving science. We serve both academic and industrial life sciences

Protein Structure Determinationin Industry Meeting

This 2019 PSDI meeting is organised by AstraZeneca

3–5 November 2019Wellcome Conference Centre, Hinxton, Cambridgeshire, UK

Book of Abstracts

27th Protein Structure Determination in Industry Meeting 3–5 November 2019 1Rigaku Europe SE | Hugenottenallee 167 | 63263 Neu Isenburg | Germany | www.rigaku.com | [email protected]

Density maps from a ten minute thaumatin dataset solved by S-SAD phasing

Electron density from a 0.37 Å quantum crystallography measurement of oxalic acid

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Event Management

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Protein Structure Determinationin Industry Meeting3–5 November 2019Wellcome Conference Centre, Hinxton, Cambridgeshire, UKhttp://www.psdi2019.org/

27th Protein Structure Determination in Industry Meeting 3–5 November 2019 3

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I N G S E R V I C E S . C O M

Contents

Sponsors and Exhibitors 4

Delegate Information 10

Accommodation 10

Internet Access 10

Drinks Reception, Networking and Drinks and Conference Dinner 11

Exhibition Floor Plan 14

History of PSDI Meetings 15

Programme 16

Abstracts 20

Notes 60

ALPXALPX is a new startup based in Grenoble, France.The company is offering access to fully automated,high-throughput crystallographic screening pipelines,based on unique proprietary EMBL technologies: theCrystalDirectTM Technology for automated crystalharvesting and data management via CRIMS (theCrystallization Information Management System).

In the life sciences, researchers use Beckman Coulter’sprecision instruments to study complex biologicalproblems, including causes of disease and potentialnew treatments. Our team of experts dedicates the timeand energy to understand the complexity of researchfor laboratory customers in a wide variety of settings.By innovating new processes and technologies, we’rechallenging conventional wisdom; our goal is to developthe best, most widely trusted laboratory solutionsavailable on the market today.

[email protected]

BIOSAXS was founded in late 2015. The company isa service unit for Small Angle X-Ray Scattering (SAXS)based on the great experience of the SAXS group ofEMBL Hamburg. The SAXS technology reveals solutionstructures of biological macromolecules and syntheticnanoparticles at 1-2 nm resolution.

Bruker AXS enables scientists to explore life at themolecular level and make breakthrough discoveries thatimprove the quality of human life. In close co-operationwith our customers, Bruker is enabling innovation,productivity and customer success in life sciencemolecular research. The D8 VENTURE platform forSC-XRD includes state-of-the-art technologies suchas the PHOTON III CPAD, high-brilliance X-ray sourcesand high-level automation maximise success andproductivity in structure-based drug design.

[email protected]/scd

By bringing together modern label-free technology,application development know-how and sophisticatedsoftware, Creoptix offers a unique optical biosensortool for binding kinetics. Engineered around ourproprietary Grating-Coupled Interferometry (GCI)technology, the Creoptix WAVEsystem delivershigh-quality kinetic data across a broader range ofsamples than traditional SPR equipment.

[email protected]

Douglas Instruments designs and manufactures theOryx range of robots for automatic protein crystallization.The robots can also set up plates for DLS/cryoEM.The simple design and user-friendly software allows avariety of screening and optimization experiments torun including powerful approaches to microseeding.Use as little as 7μl for 96 wells.

https://www.douglas.co.uk/ [email protected]

EMBLEM Technology Transfer GmbH (EMBLEM) isthe commercial subsidiary of the European MolecularBiology Laboratory (EMBL) EMBLEM, established in1999 in Heidelberg, Germany, identifies, protects andcommercialises the intellectual property developed inthe EMBL-world, from EMBL alumni and from thirdparties. EMBLEM facilitates and accelerates the transferof innovative technology from basic research to industryby working closely with industrial partners spanningthe biotech, IT and engineering markets to developnew diagnostics, therapies and devices.

[email protected]

27th Protein Structure Determination in Industry Meeting 3–5 November 2019 527th Protein Structure Determination in Industry Meeting 3–5 November 20194

Sponsors and Exhibitors Sponsors and Exhibitors

This 2019 PSDI meeting is organised by AstraZeneca

76 27th Protein Structure Determination in Industry Meeting 3–5 November 201927th Protein Structure Determination in Industry Meeting 3–5 November 2019

Peak Proteins provide tailor-made protein reagents,generate protein structure information using X-raycrystallography and provide protein mass spectrometryservices. Our staff have many years of drug discoveryexperience through working in large pharmaorganisations as well as in academia and have thein-depth scientific knowledge needed to produce highquality protein reagents for assay development,high-throughput screening, biophysics or structuredetermination.

www.peakproteins.com [email protected]

Proteros is a global market leader for protein X-raycrystallography drug discovery services with a mission todeciphering the mode-of-action analysis of challengingdrug discovery targets on behalf of its clients. Inaddition to the company’s established off-the-shelfresearch tools (Gallery Structures, Proteins and Assays),Proteros has a respected track record of deliveringcustomized Gene-to-Protein/-Structure projects.Proteros has expanded its integrated services byadding fragment/small molecules libraries as well asCryoEM to its service portfolio.

[email protected]

Rigaku has been at the forefront of X-ray instrumentationsince the 1950s, and is a world leaders in the fields ofgeneral X-ray diffraction (XRD), thin film analysis (XRF,XRD and XRR), X-ray fluorescence spectrometry (TXRF,EDXRF and WDXRF), small angle X-ray scattering(SAXS), protein and small molecule X-ray crystallography,Raman spectroscopy, X-ray optics, semiconductormetrology (TXRF, XRF, XRD and XRR), X-ray sources,computed tomography, nondestructive testing andthermal analysis.

[email protected]

Schrödinger is a leading provider of advanced molecularsimulations and enterprise software solutions thataccelerate and increase the efficiency of drug discoveryand materials design. Founded in 1990, Schrödingerhas nearly 400 employees and operations across theworld. For more information, please visitwww.schrodinger.com.

[email protected]

The Difference between Today and the Future in Pharma& Biopharma – Speed With SCIEX, you benefit frominnovative technology that makes complex workflowseasier and more efficient, while delivering the utmost indata quality. Highly reliable instrumentation, with highsensitivity and dynamic range for a variety of analyticalapplications. Advanced software with automation tomake data processing easier. And global service andsupport, so your workflows aren’t interruptedunexpectedly.

www.sciex.com/applications/[email protected]

SWISSCI is a technology company with a focus onplastic injection moulding innovation and excellence.Manufacturing high quality innovative products for thelaboratory. Evolving from our injection moulding rootswe have developed specialty products for specialistresearch applications, including Crystallography,Cryo-EM, Assay plates, Dialysis and Ultrafiltration.

www.swissci.com [email protected]

Understand the machinery of life

With the right tool, protein researchers can change theworld around us.

Our award-winning technologies open doors forscientists to understand proteins and their behaviorbetter than ever before. By characterizing proteins andtheir interactions, scientists can make better decisionsabout how we diagnose diseases, develop treatmentsand maintain our personal well-being. Our revolutionarytechnologies give researchers a fundamentally newway to analyze proteins without compromise, becausewe truly believe the right tools in the right hands willchange the world.

[email protected]

FORMULATRIX® was established in 2002 to provideprotein crystallization automation solutions. Since then,we’ve started developing other laboratory automationsolutions including the next generation of liquid handlersusing microfluidic technology. Headquartered inBedford, Massachusetts, we supply software androbotic automation solutions to leading pharmaceuticalcompanies and academic research institutions aroundthe world. Our team works tirelessly to provide thebest products in the industry with support that issecond to none.

https://formulatrix.com/ [email protected]

MiTeGen engineers, manufactures, and distributes afull range of leading products for crystallography andcryoEM. Our customers include academic, medical,pharmaceutical, and government laboratories in over45 countries. Because our customers’ research isimportant, we provide innovative tools and solutionsthat measurably improve the ease, reproducibility, andquality of experiments. Our second generation cryoEMpuck system advances the handling and organizationof grids and grid boxes to a new level of convenienceand accuracy.

[email protected]

At Molecular Dimensions, our love for all thingscrystallography drives who we are and what we do,every hour of every day. Our vision is to simply provideall our customers with the best products out there forstructural biology. At Anatrace, we develop and supplythe industry’s finest high-purity detergents and lipidsOur standards have made Anatrace an internationally-recognized leader in manufacturing reagents formembrane protein studies.

https://www.anatrace.com/[email protected]

NIS has been providing cryo transmission electronmicroscopy services to the drug discovery anddevelopment market since 2007. Housing aK3-quipped Titan Krio and Glacios microscopes, NISdelivers best-in-class services through fee-for-servicesand partnership models resulting in high resolutionstructures of a broad range of macromolecules andmacromolecular complexes in both apo andligand-bound forms.

[email protected]

NovAliX is a drug discovery-focused CRO with severalunique technologies highly proficient in chemistry andbiophysics. The company has set up one of theworld’s most comprehensive biophysics platforms.Our commitment and reliability are much appreciatedby a worldwide client base over the past 16 years.Our pioneering services include protein crystallographyservices, cryo-electron microscopy, biophysicalcharacterization, target validation, fragment screening& highly cost competitive custom synthesis. Visit us onwww.novalix.com

[email protected]

Sponsors and Exhibitors Sponsors and Exhibitors

27th Protein Structure Determination in Industry Meeting 3–5 November 20198

Syngene International Ltd. (BSE: 539268, NSE:SYNGENE, ISIN: INE398R01022), is an innovationfocused global discovery, development andmanufacturing organization providing integrated scientificservices to the pharmaceutical, biotechnology, nutrition,animal health, consumer goods and specialty chemicalindustries around the world. For more details, visitwww.syngeneintl.com

www.syngeneintl.com [email protected]

Thermo Fisher Scientific is the world leader in servingscience. We serve both academic and industrial lifesciences researchers, providing an unmatchedcombination of complete workflow solutions rangingfrom cryo-EM structural determination of macromolecularcomplexes and protein sociology, in the native state,to reconstruction of 3D architecture of tissues andcells. Our solutions help researchers unlock themysteries of underlying protein function and cellularprocess and bridge the gap between basic scienceand translational therapeutics.

www.pharmadrugdiscovery.com/ [email protected]

TTP Labtech continues develop into a more integratedStructural Biology company. See the new mosquito®

Xtal3, an affordable, application driven system forprotein crystallization drop setting, explore the touchscreen software. Add dragonfly® crystal for fast andreproducible optimization of crystallization hits. Enquireabout our chameleon for cryo-EM grid preparation.

www.ttplabtech.com [email protected]

Xenocs provides complete solutions for characterizingthe nanostructure and morphology of materials. Basedon X-ray scattering techniques, the product portfolio ofthe company includes solutions for biostructuralresearch enabling to characterize the macromolecularshape, interactions and dynamics of proteins insolution with high throughput. Founded in 2000 as aspinoff company from the Institute Laue Langevin, inGrenoble, France, Xenocs supplies its solutions toleading research and development institutions aroundthe world.

www.xenocs.com [email protected]

Xtal Concepts GmbH is a technology company locatedin Hamburg, which focuses on the development,production and commercialization of measuringinstruments for the characterization of nano- andmicroparticles in liquids. Besides the imaging technicssuch as the VIS- and UV-Imaging, the core know-howis the dynamic light scattering, that allows measuring ofthe particle sizes in liquid mediums in the submicroscopicrange of ~1 - 5000 nm.

www.xtal-concepts.de [email protected]

Sponsors and Exhibitors

High-resolution Single Particle Analysis and Micro Electron Diffraction workflows to maximize your productivity“We understand that you are under constant pressure to gain relevant

structural insights quickly, with enough details to decipher biological

mechanisms and develop new drug therapies. At Thermo Fisher

Scientific, we have a long history in delivering the best cryo-EM solutions

to solve more structures at higher resolution and for increasingly smaller

molecules. We believe that maximizing productivity is not only about

performance but also about optimizing connectivity and ease of use of

our workflows to accelerate disease learning.”

—Rob Krueger, General Manager Life Sciences, Thermo Fisher Scientific

Learn how to maximize your productivity at pharmadrugdiscovery.com

For current certifications, visit thermofisher.com/certifications. © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

27th Protein Structure Determination in Industry Meeting 3–5 November 201927th Protein Structure Determination in Industry Meeting 3–5 November 2019 1110

Delegate Information Delegate Information

Accommodation

Wellcome Genome Campus

The Wellcome Genome Campus Conference Centre is a beautifulcomplex within the Hinxton Hall Estate and its 100-acre parklandbordering the river Cam. Blending stunning contemporaryarchitecture with the existing Grade II*-listed country house ofHinxton Hall, the world-class Conference Centre provides anexceptional space in an idyllic setting.

Located alongside – and taking inspiration from – researchinstitutions that are both at the forefront of the biomedicalrevolution and home to so much scientific history, nowhere else isblessed with such a unique atmosphere: on the Wellcome GenomeCampus you are removed from everyday life and experience the luxury of concentrating on discussion andreflection, whilst everything else is taken care of for you.

Ibis Cambridge Central Station

It’s all in the detail at the modern, affordable hotel: fostering awelcoming, happy atmosphere, so you’re always guaranteed awarm welcome. And it’s not just the service that’s spotless: thestaff work around the clock to ensure everything’s immaculatefrom the lobby to the very top floor. And with the on-site Chill#2artisan coffee shop and bar and a hearty, varied breakfast spreadlaid out each morning, you certainly won’t go hungry.

Cambridge is a beautiful city known for its prestigious universityand striking architecture. With everything within walking distance,it’s easy to explore its colleges, historic buildings and beautiful river.

Internet Access

There is free Wi-Fi internet access across the Wellcome Genome Campus. Connect to the network‘ConferenceGuest’ and follow the instructions.

Meeting

If you have any questions during the meeting, please report to the registration desk where someone will behappy to answer any queries you may have. Otherwise, PSDI Organisers will be wearing pink badges and willalso be able to assist you. Please see registration desk opening times and locations below:

Date Opening Times Location

Sunday 3rd November 12:00 – 18:30 Conference Centre

Monday 4th November 08:00 – 17:00 Conference Centre

Tuesday 5th November 08:00 – 16:00 Conference Centre

Drinks ReceptionConference Centre, Sunday 3rd November, 2019, 18:30–20:30

Networking and DrinksConference Centre, Monday 4th November 2019, 17:20–18:30

Conference DinnerUniversity Arms Hotel, Cambridge, 4th November 2019, Pre dinner drinks – 19:00, dinner served 19:30

Coaches due to depart at 18:30 from the Wellcome Genome Campus to the University Arms for theConference Dinner.

University Arms Hotel, Cambridge

The Conference Dinner will take place on Monday 4thNovember, 2019, at the University Arms Hotel, Cambridge.

Following a comprehensive redesign by renowned architectJohn Simpson and interior designer Martin Brudnizki, UniversityArms Hotel, Autograph Collection has re-established itself asone of Cambridge’s most remarkable destinations.

The hotel offers easy access to the University, as well asNewmarket Racecourse, IWM Duxford and the attractions ofCambridge city centre. Elsewhere at the hotel, Chef DirectorTristan Welch presides over Parker’s Tavern, a CambridgeInstitution for 185 years; infused with contemporary energy, therestaurant serves innovative English cuisine in a university-inspired ambiance. Additional hotel amenities include an on-sitefitness centre and a graceful ballroom venue with a fireplace and sweeping views.

Please note that delegates must bring their Conference Dinner ticket with them to the University Arms.

If you have not booked onto the shuttle coach service, the University Arms is a 25 minute drive away from theWellcome Campus and a 6 minute drive from Cambridge Central Station.

Coach Service

Please note this is a pre-booked service as spaces are limited.

For delegates who have pre-booked the coach service, please see the below timetable:

Date Time Depart Arrival

3 November 2019 12:00 Ibis City Centre Wellcome Genome Campus

3 November 2019 20:30 Wellcome Genome Campus Ibis City Centre


4 November 2019 18.30 Welcome Genome Campus University Arms (ConferenceDinner)

4 November 2019 22:30 University Arms (Conference Wellcome Genome CampusDinner)


5 November 2019 16:30 Wellcome Genome Campus Ibis City Centre


Delegate Information Delegate Information

Hinxton, Cambridgeshire

Hinxton is a village in South Cambridgeshire. The River Cam runs through the village as does the Cambridge toLiverpool Street railway, though the village has no station. Hinxton parish’s southern boundaries form the borderbetween Cambridgeshire and Essex. The village is five miles north-west of Saffron Walden and 9 miles south ofCambridge. Hinxton is the home of the Wellcome Trust Genome Campus.

Things to do

• River Cam

• Hinxton Mill and Hall

• Linton Zoo

• Mathematical Bridge, Cambridge CB3 9ET

• Cambridge University Botanic Garden, 1 Brookside, Cambridge CB2 1JE

• The Backs, Queen’s Rd, Cambridge CB3 9AH

• Ely Cathedral, Chapter House, The College, Ely CB7 4DL

• Punting

• Botanic Gardens

Museums

• Fitzwilliam Museum, Trumpington Street, Cambridge, CB2 1RB

• Imperial War Museum Duxford, Imperial War Museum Duxford

• Museum of Archaeology and Anthropology, University of Cambridge, Downing Street, Cambridge, CB2 3DZ

• Sedgwick Museum of Earth Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3DZ

• Whipple Museum of the History of Science, Free School Lane, Cambridge, CB2 3RH

Restaurants

The Red Lion Inn Hinxton32 High StreetCambridge, CB10 1QY

The PloughHigh StreetSaffron Walden, CB10 1PL

The John BarleycornThreshers BushHarlow, CM17 0NS

The Chequers1 Town LaneCambridge, CB22 3ER

Kaz’s Indian & Bangladeshi Restaurant84 High StreetCambridge, CB22 3HJ

The Tickell ArmsNorth RoadCambridge, CB22 4NZ

The Dog & Duck63 High StreetLinton, CB21 4HS

Ask Italian62 High StreetWalden, CB10 1EE

La Maison du Steak125 Hills RoadCambridge, CB2 1PG

Flying Pig106 Hills RoadCambridge, CB2 1LQ

Smokeworks2 Free School LaneCambridge, CB2 3QA

The Oak Bistro6 Lensfield RoadCambridge, CB2 1EG

Cambridge Chop House1 King’s ParadeCambridge, CB2 1SJ

The Olive Grove100 Regent StreetCambridge, CB2 1DP

From ibis to University Arms

From Hinxton to University Arms ‰

‰


History of PSDI Meetings

PSDI 1 1993 – Zeneca, United Kingdom

PSDI 2 1994 – SmithKline Beecham, United Kingdom

PSDI 3 1995 – GlaxoWellcome, United Kingdom

PSDI 4 1996 – Pfizer Sandwich, United Kingdom

PSDI 5 1997 – Zeneca, United Kingdom

PSDI 6 1998 – Roche Welwyn, United Kingdom

PSDI 7 1999 – SmithKline Beecham, United Kingdom

PSDI 8 2000 – Aventis, France

PSDI 9 2001 – GlaxoSmithKline, United Kingdom

PSDI 10 2002 – AstraZeneca, United Kingdom

PSDI 11 2003 – Medivir, Sweden

PSDI 12 2004 – Pfizer, United Kingdom

PSDI 13 2005 – Astex, United Kingdom

PSDI 14 2006 – Novartis, France

PSDI 15 2007 – ESRF, France

PSDI 16 2008 – GlaxoSmithKline, United Kingdom

PSDI 17 2009 – Roche, Basel, Switzerland

PSDI 18 2010 – Diamond Light Source, Oxford, United Kingdom

PSDI 19 2011 – AstraZeneca, Molndal, Sweden

PSDI 20 2012 – Sanofi, France

PSDI 21 2013 – PSI/Expose, Lucerne, Switzerland

PSDI 22 2014 – Merck Serono, Cascais, Portugal

PSDI 23 2015 – Proteros Biostructures, Tegernsee, Germany

PSDI 24 2016 – Novo Nordisk/MAX IV Laboratory/SARomics Biostructures, Malmö, Sweden

PSDI 25 2017 – Astex, Cambridge, United Kingdom

PSDI 26 2018 – Sanofi & Soleil, Versailles, France

PSDI 27 2019 – AstraZeneca, Cambridge, United Kingdom

27th Protein Structure Determination in Industry Meeting 3–5 November 2019 15

Exhibition Floor Plan

List of Exhibitors

P1 AB SCIEX UK Limited

P2 Molecular Dimensions

P3 Syngene International Ltd

P4 Schrodinger GmbH

P5 TTP Labtech

P6 NanoImaging Services

P7 Rigaku Europe SE

P8 ThermoFisher

1 Douglas Instruments Ltd

2 NoVAliX

3 Proteros biostructures GmbH

4 MiTeGen

5 SWISSCI

6 Beckman Coulter United Kingdom

7 Peak Proteins

8 Xenocs

9 Fluidic Analytics

10 Formulatrix

11 Creoptix AG

12 EMBLEM Technology Transfer GmbH

13 XtalConcepts GmbH

14 Bruker


Programme Programme

Day 1: 3 November – Workshops12:00 Registration opens

Workshop 1: Hot and new: emerging techniques (chair: Andrea Gohlke)

13:00–13:30 Protein Structural MS techniquesChris Nortcliffe (Sciex)

13:30–14:00 Micro Electron Diffraction – A powerful method for protein and small molecule structuredeterminationAbhay Kotecha (ThermoFisher)

14:00–14:30 Mechanism of inhibitor-induced PARP1 trapping to damaged DNAMichal Bista (AstraZeneca)

14:30–14:45 TEA/COFFEE BREAK

Workshop 2: Cutting edge synchrotrons (chairs: Gerard Bricogne, Lotte van Beek)

14:45–15:00 A new high-throughput method to prepare large quantities of microcrystals in lipidic cubicphase for serial crystallographyIsabel Moraes (NPL)

15:00–15:15 From protein to high quality X-ray diffraction data at the SLS MX beamlinesJustyna Wojdyla (SLS)

15:15–15:30 Sample and time efficient serial approaches for MX at Diamond and beyondRobin Owen (Diamond)

15:30–15:45 New Developments in Serial Crystallography at Beamline P11, PETRA IIIEva Crosas (DESY)

15:45–16:00 Crystallographic Enzymology: using beamline P14 on PETRA III for high resolution inspace and timeThomas Schneider (EMBL)

16:00–16:15 An outlook on using serial femtosecond crystallography in drug discoveryValentin Borshchevskiy (MIPT)


Workshop 3: Model building into cryoEM maps (chair: Taiana Maia de Oliveira)

16:30–17:00 Cryo-EM model building and validation in CCP-EMColin Palmer (CCPEM)

17:00–17:30 Next generation Coot: exploiting modern PC hardware to improve model-building tools forCryo-EM Model-BuildingPaul Emsley (MRC LMB)

17:30–18:00 Using Coot in CryoEM ReconstructionsAna Casañal (MRC LMB)

18:30–20:30 DRINKS RECEPTION

Day 2: 4 November08:00 Registration opens

09:00–09:10 Welcome addressMike Snowden (AstraZeneca)

Session 1 – Drug discovery case studies (chair: David Hargreaves)

09:10–09:30 Discovery of an orally bioavailable efficacious chemical tool to probe the Heat ShockFactor 1 (HSF1) regulated heat shock responseRob van Montfort (ICR)

09:30–09:50 Mapping the conformational space of small molecule drug targets by crystallographyArmin Ruf (Roche)

09:50–10:10 Targeting Cryptic Pockets to Drug the Master Oncogene RAS ProteinAlexey Rak (Sanofi)

10:10–10:30 Structural and biophysical characterisation of novel protease-activated receptor 2 antagonistsKarl Edman (AstraZeneca)

10:30–10:50 Stabilization of a tetrameric receptor state, an unprecedented mode of action of asmall-molecule inhibitorDennis Fiegen (BI)


Session 2 – Sponsored presentations (chair: Jon Read)

11:30–11:40 Taking out the trash: new tools and reagents for cryo-EM sample preparationEdward Pryor (MDL)

11:40–11:50 chameleon: Next Generation Sample Preparation for Cryo-EMPaul Thaw (TTP)

11:50–12:00 Facilitating CryoEM adoption for high resolution structure determination in Drug DiscoveryMelanie Adams-Cioaba (NanoImaging)

12:00–12:10 Combining Computational Modeling Techniques and EM Map Potentials for AccurateStructural Model GenerationTatjana Braun (Schrodinger)

12:10–12:20 TBCSyngene Intl

12:20–12:30 Exploring the landscape pf biological solutions with SAXSAngela Criswell (Rigaku)

12:30–14:00 LUNCH

Keynote lecture (chair: Maria Flocco)

14:00–14:45 Future prospects of cryoEMRichard Henderson (MRC-LMB)

Session 3 part 1 – cryoEM (chair: Alex Pflug)

14:45–15:10 The Cambridge Pharmaceutical Cryo-EM ConsortiumKasim Sader (ThermoFisher)

15:10–15:35 Cryo-EM at Novartis Institutes for Biomedical ResearchCeline Be (Novartis)


Session 3 part 2 – cryoEM (chair: Alex Pflug)

16:05–16:30 Cryo-EM at Astrazeneca: from molecular mechanisms of drug targets to SBDDTaiana Maia de Oliveira (AstraZeneca)

16:30–16:55 Expanding the scope of GPCR structure-based drug design with cryoEMStacey Southall (Sosei Heptares)

16:55–17:20 Mechanisms of human GABAA receptor modulation revealed by structural pharmacologyRadu Aricescu (MRC-LMB)

17:20–18:30 Networking and Drinks

19:00 PRE-CONFERENCE DINNER DRINKS

19:30 CONFERENCE DINNER


Programme

Day 3: 5 November08:00 Registration opens

Session 4 – Drug discovery case studies (chair: Marianne Schimpl)

09:00–09:20 Discovery of clinical candidate ASTX660: a non-peptidic antagonist of inhibitor ofapoptosis proteinsPhil Day (Astex)

09:20–09:40 Discovery and Structure-Based Optimization of Reversible Methionine Aminopeptidase-2(Met AP-2) InhibitorsDjordje Musil (Merck)

09:40–10:00 Discovery of a Potent, Selective and Orally Active Chymase Inhibitor for the Treatment ofCardiac DiseasesMartina Schaefer (Bayer)

10:00–10:20 Unlocking the structure of G6b-B by engineering of N- and O-linked glycosylationDerek Ogg (Peak Proteins)

10:20–10:40 Tackling protein-protein interactions (PPIs) through stabilisation of desired conformationsof target proteinsTom Ceska (UCB)


Session 5 – Integrative structural biology (chair: Jason Breed)

11:20–11:45 MicroED: conception, practice and future opportunitiesTamir Gonen (UCLA)

11:45–12:10 Site-seeing tour of integrated structural biologyChun-wa Chung (GSK)

12:10–12:35 Native LC-MS analysis of monoclonal antibodies and aggregatesChris Nortcliffe (Sciex)

12:35–13:00 Stabilizing inactive conformations of MALT1 as an effective strategy to inhibit its proteaseactivityPaul Erbel (Novartis)

13:00–14:30 LUNCH

Session 6 – New modalities: degraders (chair: Gavin Collie)

14:30–15:00 BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC designGerd Bader (BI)

15:00–15:30 The chemical ligand space of Cereblon – twists and turns on the route to novelsmall-molecule effectorsMarcus Hartmann (MPI)

15:30–16:00 Structural Complementarity in Small Molecule Mediated Ubiquitin Ligase TargetingEric Fischer (Harvard)

16:00 Concluding remarks

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27th Protein Structure Determination in Industry Meeting 3–5 November 201927th Protein Structure Determination in Industry Meeting 3–5 November 2019

Abstracts Abstracts

2120

Workshop 1: Hot and new: emerging techniques Workshop 1: Hot and new: emerging techniques

Protein Structural MS techniques

Chris Nortcliffe, Sibylle Heidelberger, Esme Candish, Ferran SanchezSCIEX

The three-dimensional structure of proteins is intrinsically linked to their cellular function, interactions andchemistries. However, studying proteins in their naturally folded states can be difficult due to the high amounts ofsalts, lipids, and other proteins present in the cell. These can lead to contamination, loss of signal, or obfuscationof data across many analytical techniques. Mass spectrometry has traditionally been used to study proteins ofvarious sizes both intact and digested for many years through the use of liquid chromatography sample introductionwhich reduces the effects of these other molecules, however this separation is often done under denaturingconditions leading to the loss of these important structural elements.

Native mass spectrometry is a technique which seeks to replicate some of the nature of cellular conditions ofproteins whilst being compatible with MS. New column technology has also allowed hyphenated LC/MStechniques to use Native-like buffers through the use of Size-Exclusion-Chromatography.

Hydrogen Deuterium Exchange (HDX) is an analytical technique used in both NMR and Mass Spectrometry toreplace labile backbone hydrogens to change their magnetic properties or mass. By performing HDX under nativelyfolded conditions only surface accessible hydrogens can exchange providing information on the three-dimensionalsurface of the protein. In addition, by performing exchanges whilst proteins are undergoing dynamic processessuch as unfolding, snapshots of the transitions can be observed. HDX can be a very useful tool for comparingexchange in the presence and absence of a ligand or binding partner, with reductions or increase in exchangebeing relatable back to allosteric changes in structures.

This workshop will outline how these two techniques can be applied in tandem to uncover facets of proteininteractions through which drugability and function can be inferred.

References:

1) G.R. Masson, M.L. Jenkins, J.E. Burke, An overview of hydrogen deuterium exchange mass spectrometry (HDX-MS) indrug discovery, Expert Opin. Drug,Discovery 12 (2017) 981–994.

2) J.R. Engen, D.L. Smith, Peer reviewed: investigating protein structure and dynamics by hydrogen exchange MS, Anal.Chem. 73 (2001).

3) B. Deng, C. Lento, D.J. Wilson, Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery anddevelopment – a review, Anal. Chim. Acta 940 (2016) 8–20.

Micro Electron Diffraction - A powerful method for protein and small moleculestructure determination

Abhay KotechaMaterials and Structural Analysis Division, ThermoFisher Scientific, Eindhoven, The Netherlands.

X-ray crystallography had been the gold standard method for many years that allowed visualizing structures ofproteins and other biomolecules. These structures are an important source of insight into the function andmechanism of biological processes. Despite the great advances in X-ray crystallography, the requirement forlarge, well-ordered crystals remains a bottleneck for this technique. Growing protein crystals is difficult, requires alot of time and effort and it is sometimes even impossible. However, crystallization experiments quite oftenproduce plenty of microcrystals which are useless for conventional X-ray diffraction experiments. Moreover, largeprotein crystals are frequently imperfect and often suffer from different defects like high mosaicity. Small crystalsare usually not affected by such defects and may yield better quality data. These kind of small crystals could beused for high-resolution structure determination by electron microscopy methods.

Since, electrons have a much stronger interaction with matter and are less damaging per scattering eventcompared to X-rays, electron diffraction is capable of producing high-quality diffraction data from crystals that arean order of magnitude smaller than what is necessary for X-ray diffraction. In this talk, I will show how we can useelectron cryo-microscope (CryoEM) to collect diffraction data from nano-crystals, comprising of only ~9000 unitcells, yielding high-resolution protein structure in a quick and easy way with micro-electron diffraction (MicroED)method. Such strong interactions of electron also limits the thickness of crystals that can be used (about 500nm).For bigger crystals, ranging from 1μm to 50μm thickness, a cryo-focused ion beam milling (Cryo-FIB) methodcan be used to prepare the crystals to a suitable thickness (150-600). Using lysozyme and proteinase K as thetest systems, I will show that the crystal lattice can be preserved to near atomic resolution resulting in an electrondiffraction pattern which is maintained to Brag spacings of better than 2Å in cryoFIB milled crystals.

MicroED also gives the opportunity to characterize small molecules and newly synthesized compounds “on thefly,” meaning they can gather important details about a sample throughout its creation, potentially guiding thechemical development process. To this end, I will show diffraction data collected from a paracetamol crystalsobtained from a tablet from supermarket.

This method can bypass the hurdles of crystal size and reduce the radiation damage allowing for the use ofextremely small crystals for protein and small molecule structure determination at atomic resolution. There is stillmuch work to be done improving and optimizing this technique, however, we are currently working on testingand automating the workflow to make microED fast, easy and accessible to all users.


Abstracts Abstracts

2322

Workshop 1: Hot and new: emerging techniques Workshop 2: Cutting edge synchrotrons

Mechanism of inhibitor-induced PARP1 trapping to damaged DNA

Michal Bista1, Lisa McWilliams1, Lizzi Underwood2, Joanna Andrecka3

1Discovery Sciences, R&D, AstraZeneca, Milton Road, CB4 0WG, UK; 2Discovery Sciences, R&D, AstraZeneca, AlderleyPark, Macclesfield, SK10 4TG, UK; 3LUMICKS, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands

PARP1 is versatile DNA-damage sensor that is capable of binding to DNA defects and initiating the DNA-damageresponse (DDR) processes that ultimately lead to the repair of DNA lesions. The protein is involved in the detectionand repair of both single-stranded and double-stranded DNA breaks.

Four PARP1 targeting drugs (PARPi) are already used for the treatment of tumours with BRCA1/2 mutations andthe utility of PARPi in a wider spectrum of genetic backgrounds is under investigation. PARPi bind to the polymerasedomain of PARP1 (HD-ART), but have a non-canonical mechanism of action, leading to trapping of PARPproteins on DNA in addition to blocking their catalytic action(1). This interferes with replication, inducing cell deathpreferentially in cancer cells with pre-existing defects in DNA-damage repair. The relationship between inhibition ofcatalysis and DNA trapping is complex, e.g. the domain targeted by PARPi does not bind to DNA on its own andPARP1-DNA trapping is typically observed at drug concentrations significantly higher than inhibition of catalysis.

Here, we discuss our biophysical research on interactions between PARP1, DNA and inhibitors. Using a combinationof biosensor-based DNA trapping assays, optical tweezers and single-molecule light microscopy (C-Trap), wegained surprising insight into the mechanism of PARP1 activation on DNA and inhibitor-induced trapping. Basedon the data, we propose a model where PARP1 activation involves its transient oligomerisation on DNA andPARP1 trapping on DNA requires inhibition of PARP1 oligomers rather than individual PARP1 molecules. Themodel explains the apparent disconnect between inhibition of PARylation and trapping and is in line with broaderpharmacological context of PARPi, e.g. can be used for modelling of pharmacokinetic effects.

Reference:

(1) Murai J, Huang SY, Das BB, Renaud A, Zhang Y, Doroshow JH, Ji J, Takeda S, Pommier Y. Trapping of PARP1 andPARP2 by Clinical PARP Inhibitors. Cancer Res. 2012 Nov 1;72(21):5588-99.

A new high-throughput method to prepare large quantities of microcrystals inlipidic cubic phase for serial crystallography

Isabel MoraesNational Physical Laboratory (NPL)

Serial crystallography has emerged as promising tool for structural studies of membrane proteins. The possibilityof collecting data from microcrystals at room temperature with minimal radiation damage has opened a range ofnew opportunities, in particular in the field of time-resolved studies. However, the production of large quantities ofmicrocrystals in lipidic cubic phase still is a major bottleneck to the membrane protein community. It usually involvesthe use of a large number of gas-tight syringes not only when screening for the best crystallisation conditions butalso when transporting the microcrystals to the serial crystallography beamlines.

Here, we introduce a simple, fast and efficient method to prepare hundreds of microliters of high-densitymicrocrystals in lipidic cubic phase without the need of large quantities of gas-tight syringes.

We also demonstrate that this new approach is advantageous to fragment drug discovery since it facilitates inmeso crystal soaking.

Finally, the method is easily implemented in any standard crystallisation laboratory.


Abstracts Abstracts

2524

Workshop 2: Cutting edge synchrotrons Workshop 2: Cutting edge synchrotrons

From protein to high quality X-ray diffraction data at the SLS MX beamlines

Justyna A. Wojdyla1 on behalf of the SLS MX groupSwiss Light Source, Paul Scherrer Institute, Forschungsstrasse 111, 5232 Villigen PSI, Switzerland

In this talk I will present an update on the latest SLS MX group hardware and software developments, which aidstandard high throughput experiments, as well as, more advanced methodology.

Recent extension of the crystallization facility (CF) adjacent to the PXIII (X06DA) beamline is the fast fragment-and compound-based screening (FFCS) service. It enables efficient and precise drug discovery utilizing X-raycrystallography as primary screen. The development of the new next-generation reliable sample changer systemis currently underway in the SLS MX group. The new TELL system includes large capacity dewar and reducessample exchange time down to 24 seconds. The SLS MX beamlines PXI (X06SA) and PXII (X10SA) provide acomplete suite for real time data collection and processing of serial synchrotron crystallography (SSX) data. Softwarecomponents allow reliable identification of microcrystals, automated collection and processing of multiple smallwedges of data (so called minisets). SSX includes also automatic data merging (adm), which provides automaticonline scaling and merging of minisets and allows identification of minisets subset resulting in the best quality ofthe final merged data.

Sample and time efficient serial approaches for MX at Diamond and beyond

Robin OwenDiamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE

Synchrotron microfocus beamlines have had a considerable impact on macromolecular crystallography in recentyears, with structure determination from crystals less than 10 microns in size now considered commonplace. Asbeam and sample sizes decrease new challenges arise however, as many aspects of the experiment previouslyconsidered routine, from sample identification and presentation to the X-ray beam to data processing, becomedifficult. In a closely related field, Free Electron Lasers (FELs) offer an exciting new frontier in structural biologycomplementing data collected at synchrotron sources. New approaches are continually being developed to fullyexploit the beam, and samples, available.

Despite the apparent contrast in experimental approach, many similarities between data collection at FELs andmicrofocus synchrotron beamlines exist. I will describe some of our recent developments for serial crystallographyof microcrystals at Diamond and SACLA, with focus on a fixed target approach that permits time and sampleefficient delivery of many 1000’s of crystals at both synchrotron and FEL sources. This will be through reference tothe application of our approach to identification and tracking of low-dose radiation driven effects in metalloproteinsand high-throughput ligand binding studies.


Abstracts Abstracts

2726

Workshop 2: Cutting edge synchrotrons Workshop 2: Cutting edge synchrotrons

New Developments in Serial Crystallography at Beamline P11, PETRA III

Eva Crosas1, Sofiane Saouane1, Johanna Hakanpää1, Jan Meyer1, Jakob Urbschat1, Pontus Fischer2,Bernd Reime1, Tim Pakendorf2, Alke Meents2 and Anja Burkhardt1

1Photon Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany; 2Center for Free Electron Laser Science, DESY,Notkestrasse 85, 22607 Hamburg, Germany

The Bio-imaging and Diffraction Beamline P11 at PETRA III in Hamburg is dedicated to structural investigations ofbiological samples from atomic to micrometer length scales. The beamline provides two experimental endstations:an X-ray scanning microscope which is currently under construction and a crystallography experiment open tousers since 2013 which is optimized for high-throughput, being equipped with a Pilatus 6M detector and a rapidautomatic sample changer(1). The optics concept involves two mirror systems. The first mirror system is used togenerate a secondary source at 65.5 m downstream from the source. The second (KB) system is used forrefocusing the secondary source down to 4 x 9 μm2 (v x h, FWHM) with more than 1013 ph/s at the sample position.

The highly intense microbeam available at P11 makes the beamline ideally suited for serial synchrotroncrystallography (SSX)(2). For this, two dedicated experimental setups are offered to the P11 users. For fixed-targetSSX a new sample holder has been developed, which can carry many thousands of microcrystals(3). The crystalscan be directly grown on these micro-patterned silicon chips or are crystallized off-line via standard vapor diffusiontechniques and then pipetted onto the chips. The chips can be directly mounted on the beamline goniometerand then automatically raster-scanned with the X-rays at room temperature or under cryogenic conditions(4). Withthis setup only a few microliters of microcrystal suspension are required for a high-resolution 3D structuredetermination. A tape-drive system has been designed for liquid sample delivery and allows for micro-diffusionexperiments, such as ligand binding studies, at the millisecond timescale(5). Here, the microcrystal suspension iswritten onto a thin polyimide tape and then transported to the X-ray interaction point. Both SSX setups areoptimized for data collection at low sample consumption and reduced background levels. The background duringdata collection can be further decreased by using our capillary beamstop(6).

For 2020, the implementation of a Roadrunner goniometer is planned, which will allow for conventionalcrystallography as well as SSX on fixed-targets. In addition, the Pilatus 6M will be replaced by a faster EIGER216M detector and a new inline microscope for better sample visualization of micrometer-sized crystals will beinstalled. The implementation of a pink beam option will allow for screening whole ligand libraries via SSX in shorttime. Furthermore, the planned machine upgrade from PETRA III to the diffraction limited light source PETRA IVwill enable smaller beam foci and will further reduce exposure and data collection times.

References:

(1) A. Burkhardt et al., EPJ Plus 131, 56 (2016).

(2) F. Stellato et al., IUCrJ 1, 204 (2014).

(3) J. Lieske et al., IUCrJ 6, 714 (2019).

(4) P. Roedig et al., Sci. Rep. 5, 10451 (2015).

(5) K. R. Beyerlein et al., IUCrJ 4, 769 (2017).

(6) A. Meents et al., Nat. Commun. 8, 1281 (2017).

Crystallographic Enzymology: using beamline P14 on PETRA III for highresolution in space and time

Thomas R. SchneiderEuropean Molecular Biology Laboratory, Hamburg Unit c/o DESY, Notkestr. 85, 22607 Hamburg

The P14 beamline on PETRA III (DESY, Hamburg) offers a wide range of options for diffraction experiments onmacromolecular crystals. A total flux of ~1013 ph/s, full tunability between 6 and 30 keV, and beam dimensionsrapidly adjustable between 500 μm and 5μm in combination with robust automation (accessible remotely) allowcollecting data in high-throughput or experimental modes on a wide variety of samples. While large beams havebeen used for high-resolution(1) and phasing(2) data collections, small beams have enabled structure determinationvia serial crystallography methods(3) including time-resolved studies(4). For the accurate location of micro-crystals,we have recently developed and implemented a protocol based on phase-contrast full-field X-ray imaging(5).

In 2018, funded by a grant from the BMBF (Verbund-Forschungsprojekt 05K16GU1) and in collaboration withArwen Pearson’s group (University of Hamburg), we have installed and commissioned a second endstation“T-REXX” on P14 dedicated to time-resolved pump-probe experiments. The endstation offers a high-fluxmonochromatic beam, pre-installed laser systems, and high flexibility for the introduction of different samplepresentation modes. The physical separation from the first endstation allows setting up complicated experimentswithout disturbing the standard user operation on the first endstation. Once an experiment on the 2nd endstationis ready to start, operation on the 1st endstation is interrupted and the beam is guided to the second hutch via arapidly installable bridging tube (insertion/removal in < 30 min.). For the large amounts of data generated duringserial time-resolved experiments, a dedicated IT infrastructure comprising 1 PByte of fast data storage plus morethan 1000 compute cores is attached to the beamline. First successful experiments in which substrates whereadded to crystals mounted in chips on the fly have been reported(6).

The new endstation will be described, use cases for P14 will be discussed, and ideas for upgrades for the nearfuture and for using a diffraction-limited light source PETRA IV(7) will be presented.

Industrial access to the integrated facilities for macromolecular crystallography at EMBL Hamburg is run viaEMBLEM (https://embl-em.de/), for details see: https://www.embl-hamburg.de/mxind).

References:

(1) Schrader et al. (2016) Science 353:594-598.

(2) Nozawa et al. (2017) Nature 545:248-251.

(3) Gati et al. (2014) IUCrJ 1:87-94.

(4) Schulz et al. (2018) Nat Meth 15:901-904, Mehrabi et al. (2019) Science 365:1167-1170.

(5) Polikarpov et al. (2019) Acta Cryst D in press.

(6) Mehrabi et al. (2019) Nat Meth in press, https://doi.org/10.1038/s41592-019-0553-1.

(7) Schroer et al. (2018) J Synch Rad 25:1277-1290.


Abstracts Abstracts

2928

Workshop 2: Cutting edge synchrotrons Workshop 3: Model building into cryoEM maps

An outlook on using serial femtosecond crystallography in drug discovery

Alexey Mishina1, Anastasiia Gusacha1,Aleksandra Lugininaa1, Egor Marina1, Valentin Borshchevskiy1,2,3,Vadim Cherezov1,4

1Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics andTechnology, Dolgoprudny, Russia; 2Institute of Complex Systems, ICS-6: Structural Biochemistry, Research Center Jülich,Jülich, Germany; 3JuStruct: Jülich Center for Structural Biology, Research Center Jülich, Jülich, Germany; 4Bridge Institute,Departments of Chemistry and Biological Sciences, University of Southern California, Los Angeles, CA, USA

Over the last three decades, X-ray crystallography has contributed profoundly to successes in rational drugdiscovery. However, many important drug targets are not amenable to traditional structure-based drug design(SBDD) approaches. The recent emergence of X-ray free electron lasers (XFELs) together with advancements inserial femtosecond crystallography (SFX) have offered new opportunities to overcome limitations of traditionalcrystallography and carry the strong potential of transforming SBDD. By now, five hard XFEL facilities wereavailable for users and a total of 241 Protein Data Bank entries have been published using SFX data collected atXFELs. Among them there are various important drug targets, such as GPCRs, channels, enzymes, ribosomes,riboswitches, toxins, and viruses. Time-resolved SFX opens the possibility of recording molecular movies ofproteins in action, capturing ultra-fast processes and irreversible conformational transitions. In this talk, we willdiscuss the general principles of X-ray generation at XFELs, outline SFX data collection and processing, summarizethe progress in the development of associated instrumentation for sample delivery and X-ray detection, andconclude with advantages which SFX offers for SBDD over traditional goniometer-based cryo-crystallography.

Reference:

1) Mishin, A., Gusach, A., Luginina, A., Marin, E., Borshchevskiy, V. & Cherezov, V. An outlook on serial femtosecondcrystallography application in drug discovery. Expert Opin. Drug Discov. 14, 1–13 (2019).

Cryo-EM model building and validation in CCP-EM

Colin M. PalmerScience & Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, UK

The CCP-EM software suite [1] provides a range of useful tools for building and validating atomic models incryo-EM maps. These include a number of well-known programs from the CCP4 X-ray crystallography suite,such as REFMAC5 and Buccaneer, that have been adapted for cryo-EM. CCP EM provides easy access to thecryo-EM functionality of these programs, as well as offering a number of tools specifically for cryo-EM, includingLocScale for locally-optimised map sharpening, LAFTER for local map de-noising and the new, advanced versionof Coot for interactive model building, refinement and validation. This presentation will give an overview of thesuite with a focus on tools for working with high-resolution reconstructions.

Reference:

(1) Recent developments in the CCP-EM software suite. Acta Cryst. D73, 469-477, 2017.


Abstracts Abstracts

3130

Workshop 3: Model building into cryoEM maps Workshop 3: Model building into cryoEM maps

Next generation Coot: exploiting modern PC hardware to improve model-building tools for Cryo-EM Model-Building

Paul EmsleyMRC Laboratory of Molecular Biology

Coot has long been used for modelling protein structures in the light of x-ray data. Typical operations were local(usually one or perhaps a few residues) and were largely untaxing for the computer hardware. More recently,Coot has been used for modelling larger fragments, particularly when using cryo-EM reconstructions. Suchoperations are more CPU time-consuming and so effort has been made to use multiple processors for somefunctions (most notably in map contouring and the real space refinement).

The graphics of Coot have hardly been updated since 2003 – you can get a long way with coloured sticks, butit's fair to say that Coot has a reputation for looking flat and ugly. Modern hardware provides the means to shadeobjects to improve the perception of depth and shape with an improved frame rate and new Coot takes advantageof these opportunities.

The updates to the tools and graphics will be previewed – both how they have been achieved and what one can(and will be able) to do with the current and forthcoming versions.

Using Coot in CryoEM Reconstructions

A. Casañal, P. EmsleyMRC Laboratory of Molecular Biology

The recent advances in electron cryo-microscopy (cryoEM) have enabled the production of three-dimensional(3D) structures of biological specimens to near-atomic resolution. As a result, there are an increasing number ofcryoEM structures, with resolution ranging 2.5-4 Å. Model building into these maps is challenging, time consumingand requires experience in both biochemistry and building into low-resolution maps. At those resolutions, automaticde-novo model-building programs usually result in incomplete solutions. Because Coot is extensively used in thefield, improvements for building into typical cryo-EM maps were required. Coot has been extended to this end,and now provides a new set of tools including morphing, Geman-McClure restraints, crankshaft peptide orientationoptimisation, improved chain extension, full chain refinement and Fourier-model based residue-type-specificRamachandran restraints.


Abstracts Abstracts

3332

Drug discovery case studies Drug discovery case studies

Discovery of an orally bioavailable efficacious chemical tool to probe the HeatShock Factor 1 (HSF1) regulated heat shock response

Matthew Cheeseman, Nicola Chessum, Carl Rye, Elisa Pasqua, Michael Tucker, Birgit Wilding, LindsayEvans, Susan Lepri, Meirion Richards, Swee Y. Sharp, Salyha Ali, Martin Rowlands, Lisa O’Fee, Asadh Miah,Angela Hayes, Alan T. Henley, Marissa Powers, Robert te Poele, Emmanuel De Billy, Loredana Pellegrino,Florence Raynaud, Rosemary Burke, Suzanne A. Eccles, Paul Clarke, Keith Jones, Paul Workman, and RobL. M. van Montfort

Cancer Research UK Cancer Therapeutics unit and Division of Structural Biology, The Institute of Cancer Research, London

The heat shock stress response pathway is crucial for normal cell homeostasis, but also heavily implicated inneurodegenerative diseases and cancer. Key components include heat shock proteins 90 (HSP90) and 70(HSP70), which have been explored extensively as therapeutic targets by both industry and academia. Themaster regulator of the heat shock stress response is Heat Shock Factor 1 (HSF1) transcription factor. HSF1 hasalso emerged as an essential player in maintaining the malignant phenotype of cancer cells and is essential fortransformation and tumourigenesis induced by a variety of oncogenes. However, HSF1 is a ligandless transcriptionfactor and deemed undruggable with small molecule inhibitors. In this talk I will describe the discovery of thebisamide (CCT251236), a potent HSF1 pathway inhibitor and chemical probe, identified using an unbiasedphenotypic screen. We subsequently identified pirin, a protein belonging to the bicupin family, as a high affinitymolecular target, which was confirmed by SPR and X-ray crystallography. The chemical probe CCT215236 isorally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model which can be used toexplore the regulation of the stress response in vitro and in vivo.

Mapping the conformational space of small molecule drug targets bycrystallography

Markus G. Rudolph, Jörg Benz, Andreas Ehler, Martine Stihle, David W. Banner, Andreas Kuglstatter,Armin RufRoche Innovation Center Basel, Pharma Research and Early Development

Small molecule inhibitors that bind to another conformation of their target provide sometimes additional chancesto increase selectivity, to obtain chemical novelty, to make properties more drug like or to overcome resistance.However, the crystallization of a protein in a less populated conformation is often a challenge.

I will present an overview of various crystallization approaches we used at the Roche Innovation Center Basel tomap the conformational space available to a number of drug targets from different enzyme classes. We trappedspecific enzyme conformations by co-crystallization with inhibitors(1,2), by crystallizing different protein variants(2,3),or by using protein binders as crystallization helpers followed by soaking(3).

References:

(1) Richter et al. 2018, “DNA-Encoded Library-Derived DDR1 Inhibitor Prevents Fibrosis and Renal Function Loss in aGenetic Mouse Model of Alport Syndrome” ACS Chem. Biol. 2019, 14, 37−49.

(2) Ehler et al. 2014, “Mapping the conformational space accessible to catechol-O-methyltransferase” Acta Cryst. D70,2163–2174.

(3) Banner et al. 2013, “Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals withFab fragments, Fynomers and Xaperones” Acta Cryst. D69, 1124–1137.


Abstracts Abstracts

3534


Targeting Cryptic Pockets to Drug the Master Oncogene RAS Protein

Magali Mathieu, Valerie Steier, Thomas Bertrand, Laure Delarbre, Rosalia Arrebola, Laurent Debussche,Alexey RakBioStructure and Biophysics, Integrated Drug Discovery, Sanofi R&D, 13, Quai Jules Guesde, 94403 Vitry sur Seine, France

kRas is well validated oncology target that is known to be difficult to address by small molecules. We havedeveloped various biophysical applications (TSA, MST, SPR, LO-NMR, MS) for screening and validation of ligandbinding to kRas. We have established X-Ray and NMR structural biology platforms enabling rational approachesin targeting kRas that led to validate known cryptic pockets and to discover new ones. We will present and discussstructural biology and biophysics enabled opportunities in the targeting the kRas cryptic pocket.

Reference:Targeting Cryptic Pockets to Drug the Master Oncogene kRAS Protein, manuscript in preparation.

Structural and biophysical characterisation of novel protease-activatedreceptor 2 antagonists

Karl Edman1, Margareta Ek1, Stefan Geschwindner1, Amanda J. Kennedy2, Linda Sundström2, AnneliNordqvist3, Dean G. Brown4, Niek Dekker5

1Structure, Biophysics and Fragments, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden; 2Mechanistic Biology& Profiling, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden; 3Medicinal Chemistry Cardiovascular, Renal &Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 4Hit Discovery, Discovery Sciences, R&D,AstraZeneca, Boston, USA; 5Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors that are irreversibly activated byproteolytic cleavage of the N-terminus, which unmasks a tethered peptide that binds and activates thetransmembrane receptor domain. The protease-activated receptor 2 (PAR2) is predominantly activated by serineproteases and has been shown to have roles in pain, inflammation, metabolic disease and cancer. Despite asignificant pharmaceutical interest, it has been challenging to identify efficacious antagonists(1). By using bothhigh-through-put screening (HTS) and DNA encoded library (DEL) screening, we discovered two novel compoundseries. In collaboration with Heptares therapeutics, we determined the structures of PAR2 in complex withAZ8838 and AZ34512. The structures revealed that the ligands bind at two distinct sites. The imidazole AZ8838binds in a fully occluded pocket beneath the extracellular loop (ECL)(2). The inhibitory effect of AZ8838 was highlydependent on the compound pre-incubation time which is indicative of slow binding kinetics. These findingswere confirmed by surface plasmon resonance (SPR) experiments. Unbiased ligand entry and exit simulationssuggested that His277 may act as a gate-keeper in the binding event. Since the histidine also packs against thefluorophenyl of AZ8838 it might rationalize the long residence time. In contrast, the benzimidazole AZ3451 bindsin a separate allosteric site on the outside of the transmembrane domain. While AZ3451 is a very potent PARantagonist, it exhibits much faster binding kinetics than AZ8838. To build a better understanding of the inhibitorymechanisms of the different compounds, we combined the output from molecular modelling with data from SPRand functional assays, which characterised AZ8838 as a competitive antagonist and AZ3451 as a negativeallosteric modulator. We further demonstrate that the compounds inhibit both G protein dependant and independentsignalling pathways in vitro and exhibit anti-inflammatory efficacy in a rat model of PAR2-agonist-induced oedema.

References:

(1) Ramachandran et al. Targeting proteinase-activated receptors: therapeutic potential and challenges. Nature ReviewsDrug Discovery 11, 69-86 (2012).

(2) Cheng et al. Structural insights into allosteric modulation of protease-activated receptor 2. Nature 545, 112-115 (2017).

(3) Kennedy et al. Under review.


Abstracts Abstracts

3736

Drug discovery case studies Sponsored presentations

Stabilization of a tetrameric receptor state, an unprecedented mode of actionof a small-molecule inhibitor

Frank Büttner, Dennis Fiegen, Sandra Handschuh, Ralf Heilker, Klaus Klinder, Herbert Nar, Heike Neubauer,Jürgen Prestle, Gisela Schnapp, Rainer Walter, Michael Wolff, Markus ZeebBoehringer Ingelheim Pharma GmbH & Co. KG

The target protein belongs to a large family of proteins that share the same fold. The family member has beenobject of intensive research in the past years, since an increasing number of studies suggest that therapeuticallymodulating this receptor will have a broad spectrum of applications. Although there is a large interest indiscovering inhibitors, so far only a few of limited quality are known. In the talk, we describe the identification andcharacterization of a small molecule inhibitor, initially identified by a HTS screening campaign. Biophysicalcharacterization of this compound showed that its binding leads to formation of dimers of the homodimericreceptor, an unprecedented mechanism of inhibition. The ligand co-structure of the receptor with the compoundshowed that the stabilization of the homomeric protein-protein interaction (PPI) is mediated by inter-ligandinteractions at the PPI interface, explaining receptor inhibition. We demonstrate that the identified compound is aselective in-vitro tool compound, which Boehringer Ingelheim is going to provide to the scientific community viaour open innovation initiative.

Taking out the trash: new tools and reagents for cryo-EM sample preparation

Edward PryorAnatrace and Molecular Dimensions

The past few years have been revolutionary for the field of single-particle electron cryo-microscopy (cryo-EM),with over 75% of the total deposited structures being determined since 2015. Currently, there are nearly 2,000unique (<95% sequence identity) cryo-EM structures deposited in the PDB, over 200 of which are membraneproteins. Although we are witnessing many significant strides in this field, challenges in protein production,purification, and structure determination still persist. These challenges often revolve around the steps needed toprepare the protein sample for cryo-EM studies. For soluble proteins, samples need to be highly pure, homogeneous,active, and stable. For membrane proteins, the choice of a correct detergent for protein solubilization andpurification is of paramount importance, as it is well established that the detergent used can have drastic effectson the homogeneity, stability and activity of the protein. In this brief presentation, we will discuss new reagentsand tools Anatrace and Molecular Dimensions have developed to streamline the sample preparation steps forcryo-EM studies of both soluble and membrane proteins.


Abstracts Abstracts

3938

Sponsored presentations Sponsored presentations

chameleon: Next Generation Sample Preparation for Cryo-EM

Paul Thaw, John P. Moore, R. John Walker, Klaus Doering, Michele C. Darrow, Russell S. KingTTP Labtech LTD., Melbourn Science Park, Hertfordshire, UK

In high-resolution structure determination for drug discovery, cryo-EM has witnessed dramatic improvements inmicroscope stability, direct detectors and image processing which have shifted the bottleneck to samplepreparation. The process of obtaining a film of vitreous ice of an appropriate thickness, with evenly distributedparticles is not straightforward. Many of the current vitrification methods are highly variable, necessitating thecostly step of screening each grid in an electron microscope (EM). Additionally, relatively large sample volumesare required and then lost during the process of blotting, further grid losses are sustained during the manualhandling steps required to transfer frozen grids into storage and there is poor traceability of samples throughoutthe workflow.

chameleon is a blot-free, pico-litre dispense instrument for rapid and robust freezing of samples for use incryo-EM. The chameleon system was developed from Spotiton(1,2) and uses self-wicking copper nanowire gridsto form the thin sample film(3). This process occurs ‘on-the-fly’ as the grid passes in front of the dispenser on itsway to the cryogen bowl, resulting in a stripe of sample across the frozen grid.

This method of grid freezing provides many benefits:

• Blot-free high-speed plunging

• Automated grid handling

• Grid screening based on ice thickness

• Intuitive automated workflows

• Sample tracking and recording

• Cryogen feedback and control

• Potential impact on samples with air-water interface issues (preferred orientation, aggregation, denaturation)

In short, the chameleon is a sample vitrification system with walk-up usability that streamlines the flow of goodgrids to the microscope whilst capturing essential data to improve the repeatability of cryo-EM samples.

References:

(1) I Razinkov, et al. Journal of Structural Biology 195 (2016), p. 190-198.

(2) V Dandey, et al. Journal of Structural Biology 202 (2018), p. 161-169.

(3) H Wei, et al. Journal of Structural Biology 202 (2018), p. 170-174.

(4) A Noble, et al. Nature Methods 15 (2018), p. 793-795.

Facilitating CryoEM adoption for high resolution structure determination inDrug Discovery

Melanie Adams-Cioaba Ph.D. Director of Business Development, NanoImaging Services

Cryo-electron microscopy (cryoEM) has rapidly become an established and viable complement to X-raycrystallography for high resolution protein structure determination. The technique is parsimonious in its materialrequirements and captures specimens in their fully hydrated state, more similar to their native environment.Widespread adoption for Drug Discovery, however, has been hampered by the high cost of required infrastructure,a lack of expertise, and the relatively limited number of drug targets with available high resolution cryoEM structures.Headquartered in San Diego with the first of its new facilities opening outside Boston in 2020, NanoImagingServices offers muitimodal access to in-house cryoEM infrastructure and expertise to support both fully outsourcedor mixed resourcing models. Here, we will overview current service offerings, review success rates for industryprograms since commissioning our first Titan Krios in 2017, and highlight plans for continued expansion.


Abstracts Abstracts

4140

Sponsored presentations Sponsored presentations

Combining Computational Modeling Techniques and EM Map Potentials forAccurate Structural Model Generation

Tatjana Braun1, Gydo van Zundert2, Eric Therrien2, Salma Rafi2, Ken Borrelli2

1Schrödinger GmbH, Thierschstraße 27, 80538 München, Germany; 2Schrödinger Inc., 120 West 45th Street, New York,NY 10036, USA

Producing an accurate atomic model of protein-ligand interactions from the data generated by cryo-electronmicroscopy is often a challenging problem due to a combination of the noise in the experiment and the dynamicnature of protein-ligand binding. In order to address this problem we have developed ways to combine establishedcomputational modeling techniques with EM map potentials to create more accurate and more validated structuralmodels of protein ligand binding. We will present modifications to our Glide ligand docking software to allow it toplace ligands into unmodeled density as well as a method of using the highly advanced OPLS3e protein andligand forcefield in place as a restraint model when refining models against real and reciprocal space data usingthe PHENIX refinement package. Combining these techniques together can produce one or more poses thatare consistent with both the experimental data and computational modeling at a range of resolutions for severalligand types.

We will also demonstrate using alchemical FEP calculations to confirm a prospective pose of an allosteric inhibitorof ATP-citrate lyase in a 4.0Å map with some ambiguities by showing that that pose could be used to accuratelypredict the binding affinities of a series of small molecules congeneric to the allosteric inhibitor. These tools will bevaluable for the robust identification and confirmation of ligand poses to enable structure-based drug discoveryefforts enabled by cryo-EM.

Exploring the landscape of biological solutions with SAXS

A.R. Criswell1, M. Del Campo1, L. Jiang2, K. Lokshin2, C. Breese2

1Rigaku Oxford Diffraction, 9009 New Trails Drive, The Woodlands, TX 77381 USA; 2Rigaku Innovative Technologies, Inc.1900 Taylor Road, Auburn Hills, MI 48326 USA

Small angle X-ray scattering (SAXS) is a useful technique for extracting structural information from biological samplesin solution. Most biological SAXS samples are aqueous solutions with proteins present in low concentration. Inother cases, it may be of interest to study macromolecules at high concentration, at high viscosity or at conditionsin which macromolecules are expected to crystallize. In such cases, SAXS can provide useful information aboutthe inherent structure and phase of macromolecules.

Rigaku’s SAXS systems are well suited for analysis of all types of samples, including pharmaceuticals, food,cosmetics, polymers, R&D and quality control. The BioSAXS-2000nano system uses 2D Kratky collimation withconfocal optics to achieve maximum X-ray flux on the samples, completely eliminated parasitic scattering andhigh angular resolution (Δq ~ 0.002 Å-1). The system includes Rigaku’s hybrid photon counting (HPC) detector,the HyPix-3000. The HyPix-3000 is ideal for measuring scattering from biological solutions because the detectorcombines ultra-low noise, high dynamic range and direct detection of X-ray photons. New features of theBioSAXS-2000nano add further capabilities, such as variable q-range (0.003 – 3.6 Å-1), 360° measurement ofanisotropic samples and grazing incidence (GI) compatibility. These capabilities come in the form of hot-swappableattachments and use auto-detection methods, including support for capillary and flow cells with the automaticsample changer (ASC).

In this study, we describe the versatility offered by the SAXS system for biological SAXS and demonstrate thatcapabilities have been extended for samples types other than dilute protein solutions. In particular, we describeexperiments with non-typical macromolecular samples, whether for the purposes of optimization of biologicalpharmaceuticals, for characterizing crystallization suspensions prior to measurement at an XFEL and forcharacterizing structural changes in response to solution conditions.


Abstracts Abstracts

4342

Keynote lecture CryoEM

Future prospects in cryoEM

Richard HendersonMRC Laboratory of Molecular Biology

Recent progress in single particle electron cryo-microscopy (cryoEM) has allowed determination of the structuresof a range of novel macromolecules or macromolecular assemblies, many of which have proved intractable toother approaches. The method requires only a few tens of micrograms of material; the sample does not have tobe completely pure; nor completely stable. It is also proving very useful for protein structure determination inindustry.

In a collaboration with the group of Chris Russo at MRC-LMB, we have been analysing the remaining technicallimitations that prevent cryoEM from reaching its ultimate potential. We have made quantitative evaluations of thecandidate problems: we have shown that charge build up occurs on a shorter timescale than image acquisition,that microscopic charge fluctuations do not significantly degrade the image quality, and confirmed that correctionsfor Ewald sphere curvature remove one of the arguments for using higher energy electrons. The most importantremaining problem is beam-induced specimen motion, which has been only partly cured by new specimensupports and improved motion correction software. Once better solutions to this problem are obtained, thepower of cryoEM will get very close to its full potential. We have also analysed which aspects of current electronmicroscope technology are really essential for cryoEM, with the goal of making it less expensive and more widelyaccessible. We have concluded that a 100 keV cryoEM equipped with a bright (coherent) electron source and agood detector could be as good as (or even for many purposes better than) the best current technology, yetcould be much less expensive.

The Cambridge Pharmaceutical Cryo-EM Consortium

Kasim Sader, Pablo Castro Hartmann, Pu Qian, Rishi Matadeen, Raymond SchrijverThermo Fisher Scientific

In 2016, Thermo Fisher Scientific (then FEI) partnered with the Medical Research Council Laboratory of MolecularBiology (MRC-LMB), the University of Cambridge Nanoscience Centre and five pharmaceutical companies(Astex Pharmaceuticals, AstraZeneca, GSK, Sosei Heptares and UCB) to form the Cambridge PharmaceuticalCryo-EM Consortium. The Consortium has expanded with a second Krios in 2018 at the University of CambridgeDepartment of Materials Science and Metallurgy.

The companies share use of the Krios in exchange for hosting them (University of Cambridge) and expertise andguidance on the use of cryo-EM (MRC-LMB). The facilities include two vitrification labs, Krios G2 and G3i, 10Gbnetwork, and image processing and data storage infrastructure. Our model started close to the MRC-LMB modeof operation of 24-hour weekday slots for the companies, but with all workflow steps and training on them (up toimage processing) performed by Thermo Fisher facility staff from 9:00-17:00, with the remaining hours for datacollection by automated collection of manually selected holes by EPU. The University and MRC-LMB use theweekend days. As the Consortium members have progressed, longer data collections required, and the additionof the second Krios and company screening microscopes (Astex Glacios), we have built in more flexibility includingmore extensive training and independent use.

In terms of general operation, vitrification with the Vitrobot Mk IV is very reproducible. After an initial calibration ofthe “blot force” (a physical offset of the blot pads) to obtain a good gradient of ice thickness, we have neverchanged our Vitrobot settings over 2½ years. Similar setups have been equally successful for grids produced inseveral company vitrification labs. Krios alignments change very little over time, and the only routine alignmentsdone are beam shift, objective aperture centering, and automated coma and astigmatism correction. Someon-the-fly pre-processing is done, which is useful for monitoring changes in instrument performance, but carefulimage/power spectrum analysis often serves the same purpose. We have strived to optimize uptime and achieved355/365 days for Krios 1 for 2018.

Many aspects of the Cambridge Consortium have been important for its success. The University of Cambridgehas made available two rooms with specifications exceeding those required for a Krios, allowing the installationsto proceed immediately after contracts were finalized. The MRC-LMB has provided expert guidance on settingup and running a cryo-EM lab. The companies share risk by pooling their resources together. There is a monthlymeeting in which there is usually a significant technical presentation from external and internal speakers. A keydistinguishing feature of the Consortium is the willingness of the company members to share the information theyhave been learning between them. Overall the validation of cryoEM as a growing tool for drug discovery is internalimpact on projects, but also the resulting expansion of the Consortium with Krios 2 and on-site companyscreening microscopes.


Abstracts Abstracts

4544

CryoEM CryoEM

Cryo-EM at Novartis Institutes for Biomedical Research

Céline Be, Srinivas Honnappa, Maryam Khoshouei, Christian WiesmannNovartis Institutes for Biomedical Research

Cryo-EM has become more and more prominent in structural biology over the past 5 years and is now accountablefor most of the high-resolution structures in fields like membrane proteins and protein complexes. To implementthis technology, Novartis has built its own electron microscopy centre in 2016 in Basel, Switzerland together withits affiliated research institute the Friedrich Miescher Institute. This facility aims at supporting NIBR research globally,but also benefits regularly to other divisions of Novartis. This particular setup offers a lot of opportunities and ahigh freedom to operate, but also brings up some unique challenges. I will discuss this briefly before highlightinga few in-house examples where cryo-EM could successfully contribute to Novartis drug discovery programs.

References:

1) Jendza K, Kato M, Salcius M, Srinivas H, De Erkenez A, Nguyen A, McLaughlin D, Be C, Wiesmann C, Murphy J, BolducP, Mogi M, Duca J, Namil A, Capparelli M, Darsigny V, Meredith E, Tichkule R, Ferrara L, Heyder J, Liu F, Horton P,Romanowski M, Schirle M, Mainolfi N, Anderson K, Michaud G.A small molecule inhibitor of C5 complement proteinNature Chemical Biology 2019, 15 (7), 666-668.

2) Lindner JM, Cornacchione V, Sathe A, Be C, Srinivas H, Riquet E, Leber XC, Hein A, Wrobel MB, Scharenberg M,Pietzonka T, Wiesmann C, Abend J, Traggiai E.Human Memory B Cells Harbor Diverse Cross-Neutralizing Antibodies against BK and JC Polyomaviruses.Immunity. 2019 Mar 19;50(3):668-676.

3) Bussiere DE, Xie L, Srinivas H, Shu W, Burke A, Be C, Zhao J, Godbole A, King D, Karki RG, Hornak V, Xu F, Cobb J,Carte N, Frank AO, Frommlet A, Graff P, Knapp M, Fazal A, Okram B, Jiang S, Michellys PY, Beckwith R, Voshol H,Wiesmann C, Solomon J, Paulk JStructural basis of Indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complexBiorxiv doi: https://doi.org/10.1101/737510, submitted to Nature Chemical Biology.

Cryo-EM at Astrazeneca: from molecular mechanisms of drug targets to SBDD

Taiana Maia de Oliveira1, Domi Baretic2, Judit Debreczeni1, Roger Williams2, Chris Phillips1

1Structure, Biophysics and FBLG, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Cambridge, UK; 2LMB-MRC,Cambridge UK

Cryo-EM has recently taken the field of Structural Biology by storm: the number of near-atomic resolutionstructures obtained with the technique is growing exponentially and its application led to unprecedentedstructures of drug targets historically refractory to crystallography efforts. Astrazeneca is part of the CambridgePharmaceutical Cryo-EM consortium and our cryo-EM efforts have led to the structures of the triad ofphosphatidylinositol 3-kinase-related kinases (PIKKs) at heart of DNA Damage Response (DDR) at subnanometerresolution. ATM, ATR and DNAPKcs have been linked by numerous studies to tumourigenesis and survival ofcancer cells after therapy, rendering these proteins attractive targets for cancer treatment.

ATM and ATR appear as structurally related open dimers, while DNA-PKcs is predominantly a monomer. Whiledimeric DNA-PKcs could also be observed, their arrangement is distinct from the ones adopted by ATM, ATR andthe previously reported structures of the DNA-PKcs. ATM’s closed dimer in which the PIKK regulatory domainsterically blocks the substrate binding site cannot be observed in the other PIKKs, which suggests that ATMmight display an exclusive mechanism of regulation among the members of this family.

The use of cryo-EM in published drug discovery/SBBD reports remains limited. In addition to determiningstructures that enlight mechanistic aspects of DDR, we have successfully established a cryo-EM system for anepigenetic target. The structures have local resolution of 2-2.4 Å within the binding site and allow unambiguousplacement of complexed ligands. These structures can be taken as strong evidence that the technique willincreasingly impact SBBD in industry.


Abstracts Abstracts

4746

CryoEM CryoEM

Expanding the scope of GPCR structure-based drug design with cryoEM

Stacey Southall, Mathieu Rappas, Gabriella Cseke, Giancarlo Tria, Rob CookeSosei Heptares

G protein-coupled receptors (GPCRs) continue to be an important family of drug targets. Despite many successstories, today there are still a significant number of GPCRs with compelling pre-clinical validation that remainhighly challenging for drug discovery. Over the last 10 years there has been substantial progress in the structuralbiology of GPCRs facilitating Structure-Based Drug Design (SBDD) approaches. Sosei Heptares uses its proprietaryStaR® technology to thermostabilise GPCRs to facilitate biophysical and structural studies.

Using the StaR® approach, Sosei Heptares has solved many high-resolution structures of GPCRs by X-raycrystallography, revealing the diversity in GPCR ligand binding sites. These structural insights have presentedopportunities in rational drug design for GPCR targets at both orthosteric and allosteric binding sites.

At Sosei Heptares we have taken advantage of recent advances in the field of cryo-electron microscopy toincorporate single-particle analysis into our SBDD workflow. Here I describe why StaR® proteins are advantageousin generating optimised samples for GPCR structure determination by single-particle cryo-electron microscopy. Ialso reflect on the type of information that can presently be obtained by CryoEM of GPCRs, the challengesassociated with obtaining them and comment on the future role of this technique in SBDD.

Mechanisms of human GABAA receptor modulation revealed by structuralpharmacology

Radu AricescuMRC Laboratory of Molecular Biology

Type-A γ-aminobutyric receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmissionin the human brain. They are pentameric ligand-gated chloride channels, with a very rich pharmacology, involvedin virtually all brain functions. Some of the GABAAR modulators, including benzodiazepines and anaesthetics, areamong the most successful drugs in clinical use, and often substances of abuse. Without structural data, themolecular basis for pharmacological modulation of GABAARs remained virtually unknown. I will discuss our pathto high resolution structural characterisation of human heteromeric GABAARs and their complexes with multiplesmall molecule ligands. This work provides unprecedented insights into the mechanisms of GABA-ergicsignalling, a reliable structural framework to integrate decades of physiology and pharmacology research and arational basis for the development of novel GABAAR modulators.


Abstracts Abstracts

4948


Discovery of clinical candidate ASTX660: a non-peptidic antagonist ofinhibitor of apoptosis proteins

Phil Day on behalf of Astex Pharmaceuticals IAP teamAstex Pharmaceuticals

The inhibitor of apoptosis proteins (IAPs) are key protein-protein interaction regulators of anti-apoptosis andpro-survival pathways which are associated with tumour progression and resistance to treatment. As a result,IAPs have been proposed as anticancer therapeutic targets and several peptidomimetic IAP antagonists, thatare selective for cellular inhibitor of apoptosis protein 1 (cIAP1), have entered clinical trials.

Astex Pharmaceutical’s fragment-based drug discovery platform, PyramidTM, successfully identified non-peptidicfragments that bind with millimolar affinities to both cIAP1 and X-linked inhibitor of apoptosis protein (XIAP). Usingstructure-based drug design (SBDD) we dramatically increased the binding affinity of the starting hits, whilstreducing off-target activity and improving pharmacokinetic properties. This resulted in the clinical candidateASTX660, a potent non-peptidic antagonist of both cellular and X-linked IAPs, that is structurally distinct from allpreviously reported antagonists. ASTX660 is currently in Phase 1/2 clinical trials (NCT02503423) in cancer patients.

References:

1) Chessari et al., J. Med. Chem., 2015, 58, 6574.

2) Tamanini et al., J. Med. Chem., 2017, 60, 4611.

3) Johnson et al., J. Med. Chem., 2018, 61, 16, 7314.

Discovery and Structure-Based Optimization of Reversible MethionineAminopeptidase-2 (Met AP-2) Inhibitors

Timo Heinrich, Beatrix Blume, Jörg Bomke, Michel Calderini, Uwe Eckert, Manja Friese-Hamim, Rainer Kohl,Martin Lehmann, Birgitta Leuthner, Felix Rohdich, Frank T. Zenke, Djordje MusilMerck Healthcare KGaA, Frankfurterstr. 250, 64293 Darmstadt, Germany

Methionine aminopeptidase-2 (Met AP-2) is an enzyme responsible for cleaving N-terminal methionine residuesof proteins, which is an important step of protein maturation during protein synthesis. Met AP-2 proteolytic activityhas been shown to be important for angiogenesis and tumor growth, suggesting that small-molecule inhibitorsof Met AP-2 may be used for treatment of cancer. Fumagillin analogue TNP-470, a covalent Met-AP-2 inhibitor,was extensively evaluated in clinical trials, which were finally discontinued, probably due to unfavorablepharmacokinetics and neurotoxic side-effects. We have investigated a broad chemical basis of non-covalentMet AP-2 inhibitors. Structure-based compound optimization, resulted in nomination of a clinical developmentcandidate, will be presented.

References:

1) Heinrich et al., Bioorg. Med. Chem. Lett. 27 (2017) 551-556.

2) Heinrich et al., J. Med. Chem. 62 (2019) 5025-5039.


Abstracts Abstracts

5150


Discovery of a Potent, Selective and Orally Active Chymase Inhibitor for theTreatment of Cardiac Diseases

Martina Schaefer Bayer AG

In this talk, the discovery and optimization of a potent, selective and orally available Chymase inhibitor will beshown. Chymase is known to be a relevant target for heart remodeling. We screened our internal HTS library of~2.5 million compounds using a biochemical assay with a fluorogenic substrate resulting in 3 interesting hitclusters. All hits were crystallized and were used to aid Medicinal Chemistry efforts. Our compound reducesdose-dependently cardiac fibrosis in hamster and it also improves the heart function of dogs with left ventriculardysfunction, without reduction of blood pressure.

Unlocking the structure of G6b-B by engineering of N- and O-linkedglycosylation

Tina Howard, Derek OggPeak Proteins Ltd.

Glycosylated proteins are frequently difficult to crystallise. Flexibility of the oligosaccharide side chains andheterogeneity of N-glycosylations can hinder the formation of ordered protein crystals. Protein glycosylation canbe simplified using enzymatic methods, specific cell lines and by the addition of inhibitors (Meuris et al 2014,Chang et al 2007). An alternative approach to generating non-glycosylated proteins is to mutate the glycosylatedresidues.

We set out to look at the interaction between G6b-B (a receptor essential for platelet production and function,Senis et al, 2006), a heparin oligosaccharide and a Fab fragment, using x-ray crystallography.

G6b-B is a type I transmembrane protein with a single transmembrane domain. For crystallisation we focussedthe small extracellular domain (~140 residues). After removing the single N-linked glycosylation site by mutationwe found O-glycosylation was also present. In our attempt to find a suitable protein to crystallise we designedand expressed a range of mutants aimed at removing or reducing the number of O-glycosylation sites. In one ofthese mutants the O-glycosylation was identified as occurring on a single threonine. The identification of this solethreonine was achieved by peptide mapping and mass spectrometry. This construct was successfully used togrow crystals which culminated in the determination of a 3Å resolution structure of G6b-B complexed with aphysiological ligand and in addition the epitope of the Fab being mapped (PDB:6ROX, unreleased structure).


Abstracts Abstracts

5352

Drug discovery case studies Integrative structural biology

Tackling protein-protein interactions (PPIs) through stabilisation of desiredconformations of target proteins

Tom CeskaUCB Pharma

Many proteins are dynamic and readily switch between both signalling and non-signalling conformations. Here wereport the discovery of orally available small molecule inhibitors that stabilise a naturally sampled, non-signallingconformer of a protein by binding to an allosteric, cryptic site. These compounds may provide alternatives toconventional highly successful biologics in common disease. Stabilisation of inactive conformers may prove to bea general method of drugging protein interfaces using traditional non-peptide based medicinal chemistry.

MicroED: conception, practice and future opportunities

Tamir Gonen Howard Hughes Medical Institute, Department of Biological Chemistry and Physiology, University of California, Los Angeles,California, 90095 USA

My laboratory studies the structures of membrane proteins that are important in maintaining homeostasis in thebrain. Understanding structure (and hence function) requires scientists to build an atomic resolution map of everyatom in the protein of interest, that is, an atomic structural model of the protein of interest captured in variousfunctional states. In 2013 we unveiled the method Microcrystal Electron Diffraction (MicroED) and demonstratedthat it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensionalcrystals in an electron cryo-microscope (CryoEM). The CryoEM is used in diffraction mode for structural analysisof proteins of interest using vanishingly small crystals. The crystals are often a billion times smaller in volume thanwhat is normally used for other structural biology methods like x-ray crystallography. In this seminar I will describethe basics of this method, from concept to data collection, analysis and structure determination, and illustratehow samples that were previously unattainable can now be studied by MicroED. I will conclude by highlightinghow this new method is helping us understand major brain diseases like Parkinson’s disease; helping us discoverand design new drugs; shedding new light on chemical synthesis and small molecule chemistry; and showing usunprecedented level of details with sub atomic resolutions.

References:

1) Shi D., Nannenga B., Iadanza GM. and Gonen T*. Three Dimensional electron crystallography of protein microcrystals.eLife. 2:e01345: 1 - 17 (2013).

2) Nannenga BL, Shi D., Leslie AGW. and Gonen T*. High-resolution structure determination by continuous rotation datacollection in MicroED. Nature Methods 11 (9): 927 - 930 (2014).

3) Rodriguez A.J., Ivanova M., Sawaya MR., Cascio D., Reyes F., Shi D., Sangwan S., Guenther EL., Johnson L, Zhang M.,Jiang L., Arbing M., Nannega B., Hattne J., Whitelegge J., Brewster AS., Messerschmidt M., Boutet S., Sauter NK., GonenT* and Eisenberg D* Structure of the toxic core of asynuclein from invisible crystals. Nature 525 (7570): 486 - 490 (2015).

4) Shi D., Nannenga B., de la Cruz J., Jiu L., Guillermo C., Hattne J., Reyes FE., Sawtelle S. and Gonen T*. The collection ofMicroED data for macromolecular crystallography. Nature Protocols 11 (5) : 895 - 904 (2016).

5) Gallagher-Jones G., Glynn C., Hernandez E., Miao J., Boyer D., Zee C., Martynowycz MW., McFarlane HT., Helguera GF.,Sawaya MR., Cascio D., Eisenberg D., Gonen T. and Rodriguez JA. Sub-Ångstrom cryoEM structure of a prion protofibrilfrom nano-scale crystals. Nature Structural and Molecular Biology 25: 131 - 134 (2018).

6) Jones CG, Martynowycz MW, Hattne J, Fulton TJ, Stoltz BM, Rodriguez JA, Nelson HM*, Gonen T*. The CryoEM MethodMicroED as a Powerful Tool for Small Molecule Structure Determination. ACS Central Science 4 (11) : 1587-1592 (2018).

7) Nannenga B* and Gonen T* Microcrystal Electron Diffraction (MicroED). Nature Methods 16 (5): 369 – 379 (2019).


Abstracts Abstracts

5554

Integrative structural biology Integrative structural biology

Site-seeing tour of integrated structural biology

Chun-wa Chung GlaxoSmithKline R&D

Pharmacological action begins with the binding of a molecule to its target. However, for many molecules thisknowledge alone is insufficient to understand how target binding translates to biological function. Molecularmechanism of action (MMoA) was a term coined by David Swinney to describe the link between the mechanismof action of a molecule and the physiological response it elicits. This talk give examples of how integratedstructural biology approaches (structural biology, biophysics, biochemical) can be used to understand the MMoAof molecules with distinct biological profiles.

Native LC-MS analysis of monoclonal antibodies and aggregates

Chris Nortcliffe1, Helen Whitby2, Sibylle Heidelberger1, Ferran Sanchez1

1SCIEX; 2Phenomenex

The biological function of proteins is tied to their three-dimensional structure. The interactions of hydrophobicregions can lead to nonspecific aggregation of these proteins which may be reversible or may lead to furthermisfolding resulting in irreversible aggregates. Aggregation can lead to problems in pharmaceutical proteinsincluding precipitation, immune response, safety and efficacy.

Due to the use of denaturing solvents reverse phase LC-MS leads to the loss of these non-covalent interactionsand therefore certain binding information’s are lost upon analysis. ‘Native’ or ‘Native-like’ analysis of proteins hasbeen utilized to preserve these interactions into the gas phase through the use of volatile buffers. This workoutlines a high-flow method for the detection of multi-domain proteins their dimers and further aggregates.

Size exclusion chromatography is able to separate protein fragments and multimers along with providing a bufferexchange from high salt formulation into MS friendly ammonium acetate. Large species are less able to accesspores in the column leading them to elute earlier, whereas smaller species have more or full access and thereforetravel a longer flow path and elute later. This work shows that dimers and high-order aggregation species can beseparated via native-SEC and then ionized and identified on the SCIEX X500B. Protein reconstruction providesexcellent mass match to expected results from ‘traditional flow’ work. In addition, key mAb proteoforms includingglycoforms and c-terminal fragments can be identified. Smaller fragment species are also clearly separated fromthe main eluting monomer peak and can be reconstructed to determine their identity and origin. It is proven overmultiple experiments that both chromatography and MS are stable during repeat measurements.

References:

1) K. J. Light–Wahl, B. L. Schwartz, R. D. Smith, Observation of the noncovalent quaternary associations of proteins byelectrospray ionization mass spectrometry, J. Am. Chem. Soc. 116 12 (1994) 5271–5278.

2) A. A. Rostom, C. V. Robinson, Detection of the intact GroEL chaperonin assembly by mass spectrometry, J. Am. Chem.Soc. 121 19 (1999) 4718–4719.


Abstracts Abstracts

5756

Integrative structural biology New modalities: degraders

Stabilizing inactive conformations of MALT1 as an effective strategy to inhibitits protease activity

Nicola Hughes, Paul Erbel, Frederic Bornancin, Christian Wiesmann, Nikolaus Schiering, Carsten Spanka,Carole Pissot-Soldermann, Jean Quancard, Achim Schlapbach, Oliver Simic, René Beerli, Marina Tintelnot-Blomley, Arnaud Decock, Bertran Gerrits, Frederic Villard, Riccardo Canova, Mickael Sorge, Jean-BaptisteLanglois and Martin RenatusNovartis Institutes for Biomedical Research (NIBR), Novartis Campus, CH-4002, Basel (Switzerland)

The paracaspase MALT1 (mucosa associated lymphoid tissue lymphoma translocation protein 1) plays animportant role in various immune pathways and has been proposed as a therapeutic target for auto-immunedisorders as well as cancers (i.e. DLBCL). We explored different mechanisms to inhibit the protease activity ofMALT1 and discovered unrelated chemical scaffolds. Biophysical and structural studies revealed that both scaffoldsstabilize the protease in an inactive conformation. While one ligand binds to the allosteric site at the interfacebetween the caspase and the IG3 domain, the other ligand binds to the active site in a so far undescribedmechanism. Iterative structure based drug discovery on one scaffold resulted in the identification of a potent,selective and bioavailable MALT1 inhibitor. In this presentation, we like to highlight the contributions of biophysicalstudies covering NMR, SPR and DSF.

References:

1) Structural determinants of MALT1 protease activity. J Mol Biol. 2012 May 25;419(1-2):4-21.

2) An allosteric MALT1 inhibitor is a molecular corrector rescuing function in an immunodeficient patient. Nat Chem Biol.2019 Mar;15(3):304-313.

BAF complex vulnerabilities in cancer demonstrated via structure-basedPROTAC design

Gerd BaderBoehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploitcancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPasesubunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL.High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficientoptimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancercells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify asuccessful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, andpave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

Reference:

Farnaby, W., et al. (2019). “BAF complex vulnerabilities in cancer demonstrated via structure-based PROTACdesign.” Nature Chemical Biology 15(7): 672-680.


Abstracts Abstracts

5958

New modalities: degraders New modalities: degraders

The chemical ligand space of Cereblon – twists and turns on the route tonovel small-molecule effectors

Marcus D. HartmannDepartment of Protein Evolution, Max Planck Institute for Developmental Biology, Tübingen, Germany

Over the past decade, cereblon has gained attention as an effective target for therapeutic protein degradation.Cereblon is the substrate recognition module of an E3 ubiquitin ligase and a target of classical immunomodulatoryimide drugs (IMiDs) such as thalidomide an its derivatives. Via the binding of IMiDs or IMiD-like small-moleculeeffectors, the substrate spectrum of the ligase is shifted towards different target proteins, which explains theefficacy of several IMiDs in a number of clinical contexts. Cereblon is also increasingly employed for targetedprotein degradation via the PROTAC approach, with PROTAC molecules carrying a thalidomide-derived groupas the E3-ligase recruiting moiety. However, a general downside of cereblon effectors may be their teratogenicpotential. Thalidomide exerts its teratogenic effects via its interaction with cereblon, and it is not yet clear if cerebloneffectors without this potential can be developed. Moreover, while the interaction of IMiDs and PROTACS withcereblon is well understood, little is known about cereblon’s mode of action in absence of pharmaceuticalintervention.

We have found that the thalidomide binding domain of cereblon and also its thalidomide-binding activity is highlyconserved in a wide range of species ranging from bacteria to mammals, suggesting a natural ligand universal inall domains of life. We have focused our research on the delineation of the chemical space of cereblon ligands,with three major objectives: i) identification of natural ligands, ii) understanding the correlation between cereblonbinding and teratogenicity, and iii) identification of novel binding scaffolds. We have probed the chemical space ofcereblon binding far beyond classical IMiDs and characterized the binding of a diverse set of compounds viacrystallography, including commonly used pharmaceuticals. Based on our findings, we designed a first activede-novo cereblon effector that is functional in human cell culture, but we also find that already minimalisticcereblon-binding moieties might exert teratogenic effects in zebrafish. While our results provide a framework forthe circumvention of unintended cereblon binding by negative design, they also may guide the design of apost-thalidomide generation of therapeutic cereblon effectors.

Structural Complementarity in Small Molecule Mediated Ubiquitin LigaseTargeting

Faust T, Yoon H, Nowak RP, Donovan KA, Eleuteri NA, Fischer ESDepartment of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistryand Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

Small molecules that induce protein degradation through ligase-mediated ubiquitination, have shown considerablepromise as a new pharmacological modality. Thalidomide and related IMiDs provided the clinical proof of concept,while significant progress has recently been made towards chemically induced targeted protein degradationusing heterobifunctional small molecule ligands. We will present recent work towards a better understanding ofthe molecular principles that govern neo-substrate recruitment. We will specifically address the structuralprinciples that underly the dimerization of ligase receptors with neo-substrates that have not evolved to interact.


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