Conference Agenda

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Session Overview
Session
MS-89: Fragment Screening, LCP, and Automation
Time:
Saturday, 21/Aug/2021:
10:20am - 12:45pm

Session Chair: Alice Douangamath
Session Chair: Lisa J. Keefe
Location: Club B

50 1st floor

Invited: Martin Noble (UK), Kenton Longenecker (USA)


Session Abstract

The increasing speed of detectors and the ever increasing automation and beam intensities at synchrotrons have made it possible to collect several hundred diffraction data sets within 24 hours of beam time. This had made extensive screening-by-crystallography experiments feasible. Fragments can be screened for binding in early-stage drug discovery approaches, metabilites can be screened for functional investigations, etc. Such experiments also require a different way of thinking about individual diffraction data sets. In a screening campaign consisting of several hundred data sets, the individual data sets are essentially meaningless. The power of such an experiments lies in the simultaneous analysis of all data sets at once.


Introduction
Presentations
10:20am - 10:25am

Introduction to session

Alice Douangamath, Lisa J. Keefe



10:25am - 10:55am

Fragment-Based Discovery of Orally Efficacious Allosteric Inhibitors of TNF-alpha

Kenton Longenecker

AbbVie, North Chicago, United States of America

TBD



10:55am - 11:25am

FragLites: a library of small molecules incorporating anomalous scatterers with applications in screening and protein interaction mapping

Martin E M Noble, Jane Endicott, Gemma Davison, Ian Hope, Mathew P Martin, Duncan Miller, Natalie Tatum, Max Temple, Shannon Turberville, James Sanderson, Dan Wood, Mike Waring

Newcastle University, Newcastle upon Tyne, United Kingdom

We have previously described FragLites, a library of small molecules that incorporate an anomalous scatterer for ready detection in X-ray crystallographic fragment screening. Here I will describe our findings as we have assessed their the potential of Fraglites for use in phase determination and to map protein structures for interaction hotspots.



11:25am - 11:45am

Using sound pulses to solve the crystal harvesting bottleneck

Yasmin Samara1, Haley Brennan2, Liam McCarthy3, Mary Bollard4, Denise Laspina3, Jakub Wlodek5, Stefanie Campos6, Ramya Natarajan7, Kazimierz Gofron8, Sean McSweeney8, Alexei Soares8, Ludmila Leroy9

1Universidade Federal de Santa Maria, 97105-900 Santa Maria-RS; 2Department of Biology, College of William and Mary, Williamsburg, VA 23187, USA; 3Department of Biology, Stony Brook University, New York, NY 11794-5215, USA; 4Department of Biology, York College of Pennsylvania, York, PA 17403, USA; 5Department of Computer Science, Stony Brook University, New York, NY 11794-5215, USA; 6Department of Clinical Nutrition, Stony Brook University, New York, NY 11794-5215, USA; 7Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA; 8Energy Sciences Directorate, NSLS II, Brookhaven National Laboratory, Upton, NY 11973-5000 USA; 9Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte-MG, Brazil

Crystal harvesting remains the bottleneck in protein crystallography experiments and is the rate-limiting step for many structure determination, high-throughput screening and femtosecond crystallography studies. Huge progress has been made towards the automation of high-throughput crystallization, even for membrane proteins. Moreover, free electron lasers and fourth generation synchrotrons support extraordinarily rapid rates of data acquisitions and put further pressure on the crystal-harvesting step. Here [1], a simple solution is reported in which crystals can be acoustically harvested from slightly modified MiTeGen In Situ-1 crystallization plates. Acoustic harvesting uses the automated and keyboard driven acoustic droplet ejection (ADE) technology, in which an acoustic pulse ejects each crystal out of its crystallization well, through a short air column and directly onto a micro-mesh. Crystals can be individually harvested or can be serially combined with a chemical library such as a fragment library.

As crystallization plates are used in most automated high-throughput crystallization robots, ejecting crystals directly from their crystallization wells eliminates the laborious and time-consuming manual harvesting of fragile protein crystals. We here made it possible with a very simple modification of the MiTeGen In Situ-1 crystallization plate, that consists in a light sanding of their edge pedestal (Figure 1a). This is enough to make the plate acoustically compatible with the Echo 550 liquid handler (Labcyte Inc.) and would not be needed if the plates were designed with acoustically compatible plastic. An acoustic compatible plate enables multiple acoustic harvests of crystals from different wells, directly to the X-ray diffraction data collection media (micro-meshes), (Figure 1b). Each harvested aliquot can then be combined with distinct chemicals, making combinatorial crystallography an obvious application. Our results demonstrate that acoustic harvesting is not merely a viable and gentle crystal harvesting technique, but it also makes protein crystal harvesting remarkably more efficient.



11:45am - 12:05pm

High throughput approach to prepare high-density microcrystals in lipidic cubic phase for serial crystallography and fragment screening

Isabel Moraes1, Danny Axford2, Agata Butryn2, Pierre Aller2, Tristan Kwan1, Peter J Judge3

1National Physical Laboratory, Teddington, UK; 2Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, UK; 3Biochemistry Department, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

In recent years, serial crystallography has emerged as promising method for structural studies of integral membrane proteins. The possibility of collecting data from very small crystals at room temperature, with reduced radiation damage, has opened new opportunities to the membrane protein structural biology community. In particular in the field of time-resolved studies. However, one of the technical bottlenecks of the method is the production of large amounts of tiny optimized crystals in mesophases. Here, we present a simple and fast method to prepare hundreds of microliters of high-density microcrystals in lipidic cubic phase (LCP) for serial crystallography including time resolved measurements. This approach not only eliminates the need for large quantities of expensive gas-tight syringes, but also may be used as a high-throughput tool when screening conditions for the growth of high density well-diffracting crystals.

We also demonstrate, with practical examples, that this new approach is of great advantage to fragment drug discovery since it facilitates in situ crystal soaking with minimal disturbance to the crystals in LCP.

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



12:05pm - 12:25pm

Fragment-based development of bacterial DsbA inhibitors as novel anti-virulence agents

Geqing Wang1, Wesam Alwan2, Matthew Bentley2, Biswaranjan Mohanty2, Bradley Doak2, Rabeb Dhouib3, Makrina Totsika3, Benvenuto Capuano2, Peter Scammells2, Jennifer Martin4, Martin Scanlon2, Begoña Heras1

1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia; 2Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia; 3Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, 4059; 4Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD, 4111, Australia

Antibiotic resistance is growing to dangerously high levels and poses a serious threat to global public health. The emergence and spread of resistance mechanisms to all antibiotics introduced into the clinic jeopardize the effectiveness of current treatments. Traditionally, antibiotics have been designed to inhibit bacterial viability or impair their growth; these mechanisms induce a strong selection pressure for resistance development. To overcome this problem, an alternate approach is to disarm bacterial virulence without killing them, which potentially reduces selection pressure and delays the emergence of resistance.

In this project, we target the thiol-disulfide oxidoreductase enzyme DsbA which catalyzes disulfide bond formation in the periplasm of Gram-negative bacteria. DsbA facilitates folding of multiple virulent factors and acts as a major regulator of bacterial virulence. Bacteria lacking a functional DsbA display reduced virulence, increased sensitivity to antibiotics and diminished capacity to cause infection in many Gram-negative pathogens [1].

We carried out a fragment screening campaign against Escherichia coli DsbA and identified the first small molecule inhibitors that bind to the catalytic site of DsbA and inhibit DsbA activity in vitro and cell-based assays [2,3,4]. By exploiting an array of biophysical/biochemical tools (NMR, SPR, X-ray crystallography and in vitro assays), we aim to optimize these DsbA inhibitors from fragment hits to high-affinity leads. Herein we report our established drug discovery pipeline and current efforts in developing DsbA inhibitors. The goal of this work is to develop a new generation of antimicrobials with a novel mode of action that could be used alone or in combination with existing drugs to treat multi-drug resistant infections.



12:25pm - 12:45pm

Efficiently from Library to Hit – Crystallographic Fragment Screening in Berlin via Structurally Diverse Compound Libraries

Jan Wollenhaupt1, Tatjana Barthel1, Alexander Metz2, Gustavo M.A. Lima3, Dirk Wallacher4, Elmir Jagudin3, Tobias Krojer3, Christian G. Feiler1, Uwe Mueller1, Gerhard Klebe2, Manfred S. Weiss1

1Helmholtz-Zentrum Berlin, Macromolecular Crystallography; 22 Philipps-Universität Marburg, Institute of Pharmaceutical Chemistry, Drug Design Group; 3MAX IV Laboratory, BioMAX; 4Helmholtz-Zentrum Berlin, Department Sample Environment

Crystallographic fragment screening (CFS) is an established method in academia and the pharmaceutical industry thanks to dedicated workflows established and optimized at several synchrotron sites. Apart from the hit identity, this technique also provides the structural 3D-information of the fragment hits on the protein surface and therefore fosters rational tool compound development and drug discovery.
At Helmholtz-Zentrum Berlin (HZB), a dedicated CFS-workflow is available that enables stream-lined experiments and data analysis [1]. The outcome of such a workflow depends to a large extend on the quality of the fragment library employed. Higher count and chemical diversity of the resulting fragment hits increases the chances for successful design of follow-up compounds. To this end, in collaborative effort with the drug design group at Marburg University, we designed libraries that are highly diverse in terms of their 3D‑pharmacophores and representative for the chemical space of fragments. The resulting F2X‑Universal Library of 1103 compounds and its representative sub-selection of 96 compounds - the F2X‑Entry Screen - are now the libraries of choice for CFS user campaigns at our facility due to their high performance [2]. Validation campaigns and several user campaigns were performed and showed hit rates of usually 15-25%, reaching 30% in exceptional cases. Another advantage of the libraries is their physical presentation as ready-to-use 96-well plates with dried-in compounds which also enables CFS for sensitive crystals without DMSO tolerance.
Apart from the exceptional library, a dedicated tool was developed at HZB to ease handling of large amounts of crystals - the EasyAccess Frame [3]. By supplying the users with such and other tools, as well as the dried-in libraries we can provide CFS campaigns as “fragment screening to go”, i.e., campaigns can be conducted inside our facility or in the user’s home laboratory. Furthermore, the robot assisted, and remotely controllable beamlines BL 14.1 and BL 14.2 are a key part of the HZB workflow and deliver high-quality diffraction data. Subsequently, processing of the data up to the identification of even weakly bound fragments is highly automated and provided via FragMAXapp, a user friendly, web-based tool developed in collaboration with the FragMAX facility at MAX IV [4]. Several CFS campaigns have been conducted successfully using the described workflow at HZB. As part of the EU-funded iNEXT Discovery project, application for access to the facility is straightforward and convenient for users from academia and industry.

[1] Wollenhaupt, J., Barthel, T., Lima, G.M.A., Metz, A., Wallacher, D., Jagudin, E., Huschmann, F.U., Hauß, T., Feiler, C.G., Gerlach, M., Hellmig, H., Förster, R., Steffien, M., Heine, A., Klebe, G., Mueller, U. & Weiss, M.S. (2021). J. Vis. Exp. 169, e62208
[2] Wollenhaupt, J., Metz, A., Barthel, T., Lima, G.M.A., Heine, A., Mueller, U., Klebe, G. & Weiss, M.S. (2020). Structure. 28, 694.
[3] Barthel, T., Huschmann, F.U., Wallacher, D., Feiler, C.G., Klebe, G., Weiss, M.S. & Wollenhaupt, J., (2021). J. Appl. Cryst. 54, 376.
[4] Lima, G.M.A., Jagudin, E., Talibov, V.O., Benz, L.S., Costantino, M., Barthel, T., Wollenhaupt, J., Weiss, M.S. & Mueller, U. (2021). Acta Cryst. D. accepted.