Developments in NMR Fragment Screening at UCB. Richard J. Taylor CCPN Conference 13th.July.2017

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1 Developments in NMR Fragment Screening at UCB Richard J. Taylor CCPN Conference 13th.July.2017

2 High Quality, Soluble, Chemically Diverse Fragment Subset

3 NMR Hardware AVIII HD 600 MHz. 5 mm QCI-F He Cryoprobe SampleJet with cooling

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6 Assemble 19 F NMR database Collaboration with ACD/Labs to automate 19 F NMR ligand screening Construct Screening Cocktails Automated Deconvolution 19 F NMR screen is sufficiently rapid to allow ranking in proteins for STD screening High hit rates may be indicative of non-specific binding

7 Results of 19 F CPMG Screens Target A 131 hits (12 %) 94 / 131 confirmed by STD NMR (20 strong) Crystal structure of 2 of the hits Target B isoform 1 30 hits (3 %) 18 / 30 confirmed by STD NMR (6 strong) Target B isoform 2 24 hits (2 %) 18 / 24 confirmed by STD NMR (8 strong) Target B isoform 3 15 hits (1.4 %) 8 / 15 confirmed by STD NMR (8 strong) Target C (tetrameric ion channel) 18 hits (1.6 %) 11 / 18 confirmed by STD NMR (7 strong) HSQC interaction site mapping of 2 of the hits UCB: Christine Prosser, Zara Sands UoL: Philip Renshaw, Sarah Strong, Gareth Hall, Lorna Waters, Vaclav Veverka, Mark Carr

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10 Protein Observe experiments (HSQC, HNCO) are information rich BUT are often dismissed..uses too much protein. is too slow to inform Medicinal Chemistry iteration Ph.D. Studentship Goals: Increase sensitivity Decrease sample consumption Reduce data acquisition time

11 Problem: 2D Experiments are slow Optimise Spectral Width ASCOM Shaped Pulses Ernst-angle excitation cos(θ) = e -(d1+at) / T1 Fast Pulsing Non-Uniform Sampling of t1 Fewer Scans t 1 t 2 BEST-TROSY SOFAST-HMQC band-selective Optimised Flip-Angle Short-Transient (SOFAST) Band-selective Excitation Short Transient (BEST) Tranverse Relaxation Optimised SpectroscopY (TROSY) Automated Spectral COMpression (ASCOM) Dr. Matthew Goodwin

12 BEST-TROSY Reducing D1 Standard TROSY No shaped pulses Slower scans interscan delay 1s 294 μg 9.5 hours 294 μg 3.5 hours BEST-TROSY Shaped pulses Faster scans interscan delay 0.25 s More scans in same time 1.6x more S/N 100 μm 42 kda protein Allows time reduction from 9.5 h to 3.5 h Dr. Matthew Goodwin

13 Buffer Optimisation Buffers: Proteins require salt for solubility and stability, however, salt reduces S/N Low salt buffers result in a ~20 % increase in peak intensity Increase relaxation: Gd 3+ chelate based contrast agents ~ 1mM Gadbutrol, available commercially as Gadovist, used as an MRI contrast agent Phosphate Buffered Saline Arg-Glu Buffer Gd 3+ - Sibille et al. Low concentrations of a Gd-chelate increases the signal to noise ratio in fast pulsing BEST experiments. J. Magn. Reson. 224, (2012) Dr. Matthew Goodwin

14 700 MHz 1.7 mm Microcryoprobe Bruker 700 MHz 1.7 mm microcryoprobe ~ 5x the mass sensitivity of the 600 MHz. 5mm cryoprobe Optimised for sample volumes of 35 µl rather than µl in a 5mm probe Tube based operation SampleJet for automation Gilson 215 robot for sample preparation High Sensitivity - Low volume - Automated Prof. Matthew Crump, Dr. Matthew Goodwin

15 SOFAST 1 H- 15 N HMQC 700 MHz 1.7 mm Microcryoprobe 25 μm 15 N IL6 (~18 μg) 1 mm ligand Arg Glu buffer 1 hour (cf 100 μm 15 N IL6 (~147 μg) 1 mm ligand 30 minutes at 600 MHz in 70mL Schanda et al. SO-FAST HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins in a few seconds. J. Biomol. NMR, 33, (2005) Dr. Matthew Goodwin

16 Ligand Observe MHz 1.7 mm Microcryoprobe WaterLOGSY 25 μm IL6 (~18 μg) 1mM binder 6 PBS, 10% D 2 O 5 minutes Binders (and water) Saturation Transfer Difference 25 μm IL6 (~18 μg) 1mM binder 6 PBS, 63% D 2 O 15 C 8 minutes Non-Binders 63% D 2 O, 15 C, 8 mins experiment time are optimised conditions for acquisition of STD and WaterLOGSY on the same sample with sufficient S/N. Water Ligand Observe via Gradient SpectroscpY (WaterLOGSY) Dr. Matthew Goodwin

17 Conclusion A series of options each offer incremental improvements in sensitivity and acquisition rate 1.7 mm Microcryoprobe 25% NUS with Compressed Sensing reconstruction Arg-Glu Low Conductivity Buffers Chelated PREs (e.g. Gadbutrol) Optimisation of SOFAST-HMQC ( <20k Da) and BEST TROSY (>20 kda) For Example To screen 1000 fragments by STD and waterlogsy on microcryoprobe: To screen 100 compounds by protein detect 15 N- 1 H SOFAST on microcryoprobe: ~20 days, ~18 mg protein Compares to ~7 days ~62 mg on 5 mm probe, running in 5 ligand mixtures, plus deconvolution experiments 4 days, ~180 μg protein Compares to ~1 days ~31 mg on 5 mm probe No ideal single solution still need to compromise Likely to be protein and application specific Dr. Matthew Goodwin

18 Where next? GPRCs and lipodisqs (SMALPS) Antibody Assisted Drug Discovery Selective labelling strategies Explore application of APSY for 3D NHCO fragment screening IDPs by Solid State NMR Automated Projection SpectrocscopY (APSY) Ralph Adams, Rebecca J. Burnley, Chiara R. Valenzano, Omar Qureshi, Carl Doyle, Simon Lumb, Maria del Carmen Lopez, Robert Griffin, David McMillan, Richard D. Taylor, Chris Meier, Prashant Mori, Laura M. Griffin, Ulrich Wernery, Jörg Kinne, Stephen Rapecki, Terry S. Baker, Alastair D. G. Lawson, Michael Wright and Anna Ettorre

19 Acknowledgements Professor Matthew Crump Dr. Matthew Goodwin Erik Landin Professor Mark Carr Dr. Lorna Waters Dr. Vaclav Veverka Dr. Frederick Muskett Dr. Christine Prosser Harry Mackenzie Dr. Alistair Henry

20 Questions? 20