Fragment-based drug discovery

Fragment-based drug discovery Dr. Till Kühn VP Applications Development MRS, Bruker BioSpion User s meeting, Brussels, November 2016 Innovation with Integrity

The principle of Fragment Based Screening from efficient fragments to Drug candidates Fragment high ligand efficiency (LE), medium IC 50 Grow, optimize Candidate high LE, low IC 50 IC 50 : 0.91mM 0.07mM 5.9nM 3.0nM LE: 0.59 0.55 0.49 0.42 Advantages of FBS: Fragment chemical space (10 9 ) << drug like chemical space (10 30 ) Fragment space easier to explore Smaller libraries with higher diversity (~ 1 000 5 000 compounds) Higher hit rates (0.1% - 10%) December 5, 2016 2

NMR in Fragment based Screening % respondents using NMR technique More than one answer possible Summary: FBS is used more and more often NMR is one of the top 2 methods People use more than one technique Ligand and Protein detected NMR used

Fragment Based Screening NMR vs. SPR Throughput Running costs Data Quality Bad samples Data Analysis NMR Fragment Screening 100 samples, 500-1000 compounds per day (19F 3000 compounds/day) (5-10 compounds per sample) Operational costs: ~45k per year (96 well format NMR tubes, cryogens and service contract) QC of fragments possible as part of process, inherent concentration information aids hit validation No issue, one tube per sample: bad sample does not stop the screen Clumsy, home-built acquisition automation, data organization, no workflow support SPR Fragment Screening 500 compounds per day, single point measurement (depends on instrument) Operational costs: 45-50k per year (chips for target immobilization, consumables, solvents and service) No QC of fragments possible during process, independent QC required Sticky compounds may dismantle the chip during screening Easy to use interface for operation, data analysis and export December 5, 2016 4

Two NMR Screening principles Ligand observed methods identify binders from mixtures Protein observed methods identifiy binders and binding site on target - NO isotopic labeling of the protein - NO size limitation of the target - Little amounts of protein needed (4-10 mgs) - Little amounts of ligands needed (0.025 0.250 mm in each sample) - Binding site on target can be identified - Isotopic protein labeling required ( 13 C or 15 N) - Size limitation of target - Amount of protein required depends on application

Two NMR Screening principles Ligand observed methods identify binders from mixtures Protein observed methods identifiy binders and binding site on target - NO isotopic labeling of the protein - NO size limitation of the target - Little amounts of protein needed (4-10 mgs) - Little amounts of ligands needed (0.025 0.250 mm in each sample) - Binding site on target can be identified - Isotopic protein labeling required ( 13 C or 15 N) - Size limitation of target - Amount of protein required depends on application No crystal structure available Binding site in crystal contact (often for PPI targets)

NMR Fragment Based Screening The concept High ligand concentration, low protein concentration Excess of ligand (i.e. 0.25 mm) small molecule, different NMR properties than protein Low protein concentration (i.e. 0.01mM) Large molecule with distinct NMR properties

NMR Fragment Based Screening The concept Ligand in contact with protein (=binding) adopt it s physical properties during residence time Excess of ligand (i.e. 0.25 mm) small molecule, different NMR properties than protein Low protein concentration (i.e. 0.01mM) Large molecule with distinct NMR properties

normalized intensity normalized intensity NMR Fragment Based Screening The concept Binding ligand keeps properties when back in the pool, other ligands are unchanged T 2 T 2 time time

Excursion: Fluorine Fragment Screening Spin-Echo pulse program for relaxation measurement 20 ms relaxation delay 200 ms relaxation delay 20 ms relaxation delay 200 ms relaxation delay 5-19 F-Tryptophan, H 2 O/D 2 O, phosphate buffer, ph 7.4 5-19 F-Tryptophan + Human Serum Albumin, H 2 O/D 2 O, phosphate buffer, ph 7.4 binding fragments: faster relaxation when compared to non-binders

Excursion: Fluorine Fragment Screening Advantages of 19F screening: No water suppression Usually one peak per fragment No buffer signals Up to 30 fragments per mixture Lower compound and protein concentration Increased throughput, higher efficiency

Excursion: Fluorine Fragment Screening typical conditions: protein = 8 µm compound = 20 µm sample volume = 170 µl (3mm tube) measuring time = 8 min 600 MHz QCI-F CryoProbe Courtesy of Dr. M. Blommers, Novartis Pharma, Switzerland

Excursion: Fluorine Fragment Screening Problem: Large Spectral Width Phasing difficult if hard 180 pulses are used

Excursion: Fluorine Fragment Screening Solution: broadband adiabatic refocusing pulse Spin-Echo pulse program for relaxation measurement WHAT WORKS @ 700MHz: 1.5ms Crp90.comp4 Power of 13 us 90-square (19.5kHz) Excitation profile 70kHz = ca. 110ppm 19F @ 700MHz

Excursion: Fluorine Fragment Screening Solution: broadband adiabatic refocusing pulse Spin-Echo pulse program for relaxation measurement WHAT WORKS @ 700MHz: 1.5ms Crp90.comp4 Power of 13 us 90-square (19.5kHz) Excitation profile 70kHz = ca. 110ppm 19F @ 700MHz

Excursion: Fluorine Fragment Screening Benefits of 1H decoupling With 1H decoupling Without 1H decoupling 1 H decoupling significantly improves signal to noise

Excursion: Fluorine Fragment Screening Probe options CryoProbe QCIF 19 F-observe 1 H-decouple 19 F S/N 6000:1 @ 600MHz Smart probe BBFO 19 F-observe, 1 H-decouple 19 F S/N 565:1 @ 600MHz N 2 cooled CryoProbe Prodigy TCI 19 F-observe without 1 H-decoupling 19 F S/N 2400:1 @ 600MHz

Three Basic 1 H NMR Experiments in FBS Water-LOGSY Binders have opposite phase to non-binders Saturation Transfer Difference (STD) Binders show up in difference spectrum, non binders don t T 2 /T 1rho Binders show strong attenuation

NMR FBS in practice You mix several ligands per sample with protein: for higher sample throughput (ligands per sample) AND to identify binding by comparison to non-binder (qualitative aspect) You need a reference spectrum of each individual compound to make the connection to the actual binding molecule Weakly binding Strongly binding Not binding STD reference STD

NMR FBS in practice You mix several ligands per sample with protein: for higher sample throughput (ligands per sample) AND to identify binding by comparison to non-binder (qualitative aspect) You need a reference spectrum of each individual compound to make the connection to the actual binding molecule STD reference STD

NMR FBS in practice You mix several ligands per sample with protein: for higher sample throughput (ligands per sample) AND to identify binding by comparison to non-binder (qualitative aspect) You need a reference spectrum of each individual compound to make the connection to the actual binding molecule STD reference 1. Before screening against any protein, you run each compound of library 2. Then make cocktails 3. Start screening campaigns STD

Screening preparation & library QC on-the-fly CMC-q sets-up, runs and analyzes your references Setup Simple batch setup for the complete library Automation Optimized experiments (auto water suppression etc.) for master stock solutions (DMSO) for ligands in buffer (screening reference) Analysis Fully automated analysis on-the-fly or batch: integrity, concentration, purity, solubility Results All information at a glance Easy cross check and manual editing Spread sheets to sort out bad compounds that don t go into the mixtures December 5, 2016 23

Screening preparation & library QC on-the-fly It actually makes sense to check! Our libraries had ca. 30% bad samples! 20% no compound no compound in stock solution, or not soluble in buffer 10% decayed or wrong compound 50% concentration off by more than +/-30% December 5, 2016 24

Tools for the practical work New temperature control option in SampleJet Individual temperature control for each of the 5 96 sample racks QC of DMSO stock solution QC and reference in buffer The actual protein screening

The actual screening campaign Data acquisition 1. Preparation: run & analyze data for single compound reference (e.g. CMC-q) 2. Mix compound cocktails, document in excel table 3. Run screening campaigns Parameter sets to automatically setup and optimize screening experiments Setup batch run in IconNMR

The actual screening campaign Data analysis and interpretation up to now For each sample, you Open the STD spectrum Open the STD reference Open the Water-LOGSY Open the T2 experiment Open the T2 reference Scale all of them for amplitude Search for, and open all single compound reference spectra for that mixture Before you can start analyzing

The actual screening campaign Data analysis and interpretation TS3.5 next pl With TopSpin 3.5, next pl, you Point to mixture table Indicate directory for single compound reference Indicate parent blank screening data (if applicable). start analyzing!

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl December 5, 2016 29

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl December 5, 2016 30

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl December 5, 2016 31

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl Customizable Spectra Types by unique identifier

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl Customizable display layout

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl December 5, 2016 34

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl December 5, 2016 35

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl December 5, 2016 36

New FBS tool in Topspin Data analysis and interpretation TS3.5 next pl Customizable report layout

Summary New tools & experiments for FBS CMC-q to easily run and analyze single compounds in buffer for reference AND do QC on the fly (in buffer to test solubility!) New Parameter sets in TS3.5 pl6 to run optimized FBS experiments in full automation New SampleJet temperature control option to run DMSO samples in one plate, cooled protein samples in the other New tools in IconNMR to setup batches of screening samples quickly from customized Excel spread sheets Brand new FBS tool in TopSpin to help you quickly organize data, analyze it your way and keep track of your results reporting into configurable excel spread sheets December 5, 2016 38

Alavar Gossert, Wolfgang Jahnke, Marcel Blommers, Cesar Fernandez, Paul Erbel Daniel Wyss and Hugh Eaton Bettina Elshorst Markus Schade Stefan Jehle, Pavel Kessler, Fabrice Moriaud, Matteo Penestri, Anna Codina December 5, 2016 39

Innovation with Integrity Thank you for your attention and come see us for demos and discusions Copyright 2011 Bruker Corporation. Corporation. All rights reserved. All rights www.bruker.com reserved.