IPBS Seminar Series - Ligand Screening Symposium

Virginie Nahoum

Head of the Integrated Screening Platform of Toulouse (PICT), IPBS, Toulouse, France

Bioinformatics and structural biology as screening tools on the integrated screening platform of Toulouse (PICT), an academic platform

The Integrated screening platform of Toulouse (PICT) is a multi-site platform whose activities revolve around (1) the identification and design of ligands interacting with all types of targets and (2) the discovery and engineering of enzymes.

These activities are based on expertise and state-of-the-art equipment for high-throughput screening of ligands or enzymes, their structural characterization, the biophysical and structural analysis of target-ligand interactions, and the chemical synthesis and analysis of low molecular weight molecules.

PICT thus occupies a central position in the development process of new drugs, both downstream of the discovery and validation of a therapeutic target and upstream of the ADMET studies and clinical pharmacology.

Two projects currently being developed on the platform demonstrate the contribution of the Fragment Based Ligand Discovery (FBLD) in the search for effective or inhibitory molecules of a target. In one case it is a question of identifying molecules capable of destabilizing a protein-protein complex by interacting at the dimerization interface and in the other case of identifying new molecules inhibiting an enzyme.

Approaches combining in silico screening, biophysical methods and NMR or crystallography screening have allowed to identify new potentially inhibitory molecules.

Selected publication

Gervais V, Muller I, Mari PO, Mourcet A, Movellan KT, Ramos P, Marcoux J, Guillet V, Javaid S, Burlet-Schiltz O, Czaplicki G, Milon A, Giglia-Mari G. (2018) Small molecule-based targeting of TTD-A dimerization to control TFIIH transcriptional activity represents a potential strategy for anticancer therapy. J Biol Chem 293(39):14974-14988

Jonathan Hopper

OMass Therapeutics, Vice President, Platforms and Founder, Oxford, UK

A mass spectrometry engine for drug discovery

Native mass spectrometry allows proteins to be characterized without disrupting the non-covalent interactions responsible for maintaining protein structures and their interactions with native ligands. This has allowed the technique to become a differentiated, high-resolution method for capturing multiple co-existing binding equilibria separated according to mass. We have demonstrated that the native MS technique not only allows hits from compound libraries to be identified, but also defines the pharmacological response to the compound by capturing interactions with signaling proteins. For GPCRs, monitoring G-protein coupling in response to drugs allows direct observation of its functional effect (agonist, partial agonist, antagonist). The high resolution and high content nature of the native MS approach provides an exciting platform to screen complex libraries as well as characterize the functional consequences of compound binding in a biophysical technique.

Selected publications

• Gupta K, Li J, Liko I, Gault J, Bechara C, Wu D, Hopper JTS, Giles K, Benesch JLP, Robinson CV. (2018) Identifying key membrane protein lipid interactions using mass spectrometry. Nat Protoc13(5):1106-1120
• Yen HY, Hopper JTS, Liko I, Allison TM, Zhu Y, Wang D, Stegmann M, Mohammed S, Wu B, Robinson CV. (2017) Ligand binding to a G protein-coupled receptor captured in a mass spectrometer. Sci Adv 3(6):e1701016
• Gault J, Donlan JA, Liko I, Hopper JT, Gupta K, Housden NG, Struwe WB, Marty MT, Mize T, Bechara C, Zhu Y, Wu B, Kleanthous C, Belov M, Damoc E, Makarov A, Robinson CV. (2016) High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat Methods 13(4):333-6
• Hopper JT, Yu YT, Li D, Raymond A, Bostock M, Liko I, Mikhailov V, Laganowsky A, Benesch JL, Caffrey M, Nietlispach D, Robinson CV. (2013) Detergent-free mass spectrometry of membrane protein complexes. Nat Methods 10(12):1206-8

Ghislaine Marchand

Evotec, Biophysic Group Leader, Toulouse, France

Affinity screening mass spectrometry

Affinity screening mass spectrometry enables rapid identification of small molecules binding to a specific protein or RNA target. This technique adopts a multiplexed screening format in which pools of compounds are incubated with the target of interest, and binders are selected according to their affinity to the target. The process consists of a 2D chromatography coupled with a high-resolution mass spectrometry. A first rapid size-exclusion chromatography step is needed to separate unbound ligands from protein-ligands complexes and a second chromatography step is used to dissociate the ligands from the complexes for their identification by MS. In a second step, primary hits are confirmed in singles with the same methodology. The throughput depends of the size of compound mixtures, but classically we evaluate average 15 000 compounds per day in primary screening stage.

Advantages and limitations of this approach will be presented and discussed together with the workflow in place at Evotec in comparison with other techniques.

Selected publication

McManus J, He T, Gavigan JA, Marchand G, Vougier S, Bedel O, Ferrari P, Arrebola R, Gillespy T, Gregory RC, Licht S, Cheng H, Zhang B, Deng G. (2018) A robust multiplex mass spectrometric assay for screening small-molecule inhibitors of CD73 with diverse inhibition modalities. SLAS Discov 23(3):264-273

Romain Talon

Senior user support scientist at Diamond Light Source, Oxford, UK, and Industrial Liaison Manager at the ESRF, Grenoble, France

X-ray fragment screening on XChem: to Hit identification and Beyond!

Fragment-based screening is now well-established as a powerful approach to early drug ("lead compound") discovery.  Amongst the many suitable biophysical techniques, X-ray crystallography was one of the first to be used and is the most directly informative1 but it is only recently it is being used for primary screening2.

In this talk, I will present XChem: the world-wide first X-ray fragment screening facility we built at the Diamond light source synchrotron. Since April 2015, the facility has been available to users and is part of the MX User Program. It helped to identify fragment hits for more than 50 protein targets and now welcome international proposals. XChem3,4 stands for “X-ray structure-accelerated, synthesis-aligned fragment medicinal chemistry”. As the name implies, XChem aims to not only be a fragment screening platform but to also help users to rapidly progress fragment hit into potent follow-up compounds. First, I will present the experiment and how we help users to yield suitable fragment hits for follow-up progression5,6. The process currently covers crystal soaking, harvesting, automatic data collection, and data analysis. However, making good use of the key structural features X-ray hits provide can be challenging, much as getting affinity measurement for X-ray hits or first-round follow-up compounds. In the second part of the talk, I will present the different strategies we are putting in place to tackle the real problem: proceeding rapidly to potent molecules.

Selected publications & references

1. Patel D, Bauman JD, Arnold E. (2014) Advantages of crystallographic fragment screening: functional and mechanistic insights from a powerful platform for efficient drug discovery. Prog Biophys Mol Biol 116(2-3):92-100
2. Collins PM, Douangamath A, Talon R, Dias A, Brandao-Neto J, Krojer T, von Delft F. (2018) Achieving a good crystal system for crystallographic X-ray fragment screening. Methods Enzymol 610:251-264
3. XChem facility webpage: https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening.html
4. von Delft F. (2016) Fragment screening at (almost) the speed of light. The Translational Scientist
5. Collins PM, Ng JT, Talon R, Nekrosiute K, Krojer T, Douangamath A, Brandao-Neto J, Wright N, Pearce NM, von Delft F. (2017) Gentle, fast and effective crystal soaking by acoustic dispensing. Acta Crystallogr D Struct Biol 3(Pt 3):246-255
6. Pearce NM, Krojer T, Bradley AR, Collins P, Nowak RP, Talon R, Marsden BD, Kelm S, Shi J, Deane CM, von Delft F. (2017) A multi-crystal method for extracting obscured crystallographic states from conventionally uninterpretable electron density. Nat Commun 8:15123