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Fragment Screening by NMR in Drug Discovery – 19 F Methods Dr. Stefan Jehle, Bruker BioSpin Dr. Pavel Kessler, Bruker BioSpin German Users Meeting, November 9 th , 2016

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Page 1: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Fragment Screening by NMR in Drug Discovery – 19F Methods

Dr. Stefan Jehle, Bruker BioSpin Dr. Pavel Kessler, Bruker BioSpin

German Users Meeting, November 9th, 2016

Page 2: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

The principle of Fragment Based Lead Discovery from efficient fragments to Drug candidates

Fragment high “ligand efficiency” (LE), medium IC50

Candidate high LE, low IC50

0.91µM

0.59

0.07µM

0.55

5.9nM

0.49

3.0nM

0.42

IC50: LE:

Grow, optimize

Advantages of FBLD – start small and grow big: Fragment chemical space (109) << drug like chemical space (1030) • Fragment space easier to explore • Smaller libraries with higher diversity (~ 1’000 – 5’000 compounds) • Higher hit rates (0.1% - 10%) • Cost efficient

Page 3: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

NMR in Fragment based Screening

More than one answer possible

http://practicalfragments.blogspot.ch/2016/10/poll-results-affiliation-metrics-and.html

% respondents using NMR technique

Page 4: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 4

Fragment Based Screening NMR vs. SPR

NMR Fragment Screening SPR Fragment Screening

100 samples, 500-1000 compounds per day (19F 3000 compounds/day) (5-10 compounds per sample)

500 compounds per day, single point measurement (depends on instrument)

Operational costs: ~45k per year (96 well format NMR tubes, cryogens and service contract)

Operational costs: 45-50k per year (chips for target immobilization, consumables, solvents and service)

QC of fragments possible as part of process, inherent concentration information aids hit validation

No QC of fragments possible during process, independent QC required

No issue, one tube per sample: bad sample does not stop the screen

Sticky compounds may dismantle the chip during screening

Clumsy, home-built acquisition automation, data organization, no workflow support

Easy to use interface for operation, data analysis and export

Throughput

Running costs

Data Quality

Bad samples

Data Analysis

New in Topspin: FBS Analysis Tool

Page 5: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

NMR Screening Methods

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) No crystal structure available? Binding site in crystal contact (often for

PPI targets)?

- Binding site on target can be identified - Isotopic protein labeling required (13C or 15N) - Size limitation of target - Amount of protein required depends on

application

Page 6: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Affinity Ranges and NMR Detection Limits

KD = 𝑘𝑜𝑜𝑜𝑘𝑜𝑜

Page 7: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Competition Experiments – Spy Molecules

+ +

Reporter/Spy, eventually with known affinity and binding site

Ligand or cocktail; affinity can be determined

19F 19F

No protein labeling required Minimal amount of protein required (low µM) Binding site on protein is known from spy molecule Affinity of test ligand can be calculated if affinity of spy molecule is known Also high affinity ligands detected (replacement of spy)

Page 8: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Experimental Parameters

Test Samples Mixture of tryptophan and tyrosine (2 mM each) and 0.04 mM human serum albumin

(HSA) (65 kDa) in 100 mM phosphate buffer pH 7.4 Mixture of benzamidine and sucrose (2 mM each) and 0.04 mM trypsin (23.3 kDa) in 100

mM phosphate buffer pH 7.4

The protein concentration and fragment to protein ratio strongly depend on the molecular weight (MW) of the protein

For targets with low sample availability the ratio can be as high as ~1:400

Sample Concentration

Page 9: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Excess of ligand (i.e. 0.050 mM) small molecule, different NMR properties than protein

NMR Fragment Based Screening The concept

Low protein concentration (i.e. 0.005mM) Large molecule with distinct NMR properties

High ligand concentration, low protein concentration: ideal for ligand observed NMR!

Page 10: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Ligand in contact with protein (=binding) adopt it’s physical properties during residence time

NMR Fragment Based Screening The concept

Excess of ligand (i.e. 0.050 mM) small molecule, different NMR properties than protein

Low protein concentration (i.e. 0.005mM) Large molecule with distinct NMR properties

Page 11: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

norm

aliz

ed in

tens

ity

time

T2

Binding ligand keeps properties when back in the pool, non-binding ligands are unchanged

norm

aliz

ed in

tens

ity T2

time

NMR Fragment Based Screening The concept

Page 12: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Fluorine Fragment Screening

5’-19F-Tryptophan, H2O/D2O, phosphate buffer, pH 7.4

5’-19F-Tryptophan + Human Serum Albumin, H2O/D2O, phosphate buffer, pH 7.4

20 ms relaxation delay

200 ms relaxation delay

20 ms relaxation delay

200 ms relaxation delay

Spin-Echo pulse program for relaxation measurement

Two point measurement, binding fragments show faster relaxation when compared to non-binders

Page 13: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Fluorine Fragment Screening Advantages and Opportunities

Usually one peak per fragment

No water suppression No buffer signals Up to 30 fragments

per mixture Increased throughput Low compound and

protein concentration

Page 14: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

typical conditions:

protein = 8 µM

compound = 20 µM

sample volume = 170 µL

(3mm tube)

measuring time = 8 min

600 MHz QCI-F CryoProbe

19F-detected FBS

Courtesy of Dr. M. Blommers, Novartis Pharma, Switzerland

Page 15: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Fluorine Fragment Screening Problem: Large Spectral Width

Phasing of the spectra is difficult if hard 180°pulses or adiabatic pulses with insufficient bandwidth are used

Page 16: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Fluorine Fragment Screening

Spin-Echo pulse program for relaxation measurement

Broadband Adiabatic Refocusing Pulse

WHAT WORKS @ 700MHz: 1.5ms Crp90.comp4 Power of 13 us 90-square (19.5kHz) Excitation profile 70kHz = ca. 110ppm 19F @ 700MHz

Page 17: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Fluorine Fragment Screening Broadband Adiabatic Refocusing Pulse

WHAT WORKS @ 700MHz: 1.5ms Crp90.comp4 Power of 13 us 90-square (19.5kHz) Excitation profile 70kHz = ca. 110ppm 19F @ 700MHz

Spin-Echo pulse program for relaxation measurement

Page 18: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

Fluorine Fragment Screening Benefits of 1H decoupling

1H decoupling significantly improves signal to noise in 19F detected screening spectra

With 1H decoupling

Without 1H decoupling

Page 19: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

CryoProbe QCIF

19F-observe 1H-decouple 19F S/N 6000:1 @ 600MHz

Smart probe BBFO

19F-observe, 1H-decouple 19F S/N 565:1 @ 600MHz

N2 cooled CryoProbe Prodigy TCI

19F-observe without 1H-decoupling 19F S/N 2400:1 @ 600MHz

19F Probe Options for FBS

Page 20: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

How about “druggability assessment”? Determine the “ligandability” of a target

Computational hot-spot mapping is used since > 15 for druggability determination

Hereby a small set of probes is mapped on the surface and favorable binding modes are detected

If a certain number of probes bind and sufficient large hot spots are found, a target is considered druggable

Page 21: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

How about “druggability assessment”? Determine the ligandability of a target

Computational methods

depend on the protein structure that is used Experimental condition like buffer, pH, cofactors etc. are not considered

Experimental druggability assessment

19F library with a diverse set of 900 fragments Cocktails with 30 fragments each makes 30

samples Measurement time 8 min per sample = 4 h total Data analysis 2 hours

One day of 19F NMR fragment screening can save many months ($$$) of work on a target or in buffer conditions under which ligands cannot be found

Immediate ROI for a typical NMR instrument 600 + SampleJet + CP QCIF

Page 22: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

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

Page 23: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

The actual screening campaign Data analysis and interpretation – TS3.5 next pl

With TopSpin now, you

• Point to mixture table

• Indicate parent directory for single compound reference

• Indicate parent blank screening data (if applicable)

Page 24: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

The actual screening campaign Data analysis and interpretation – TS3.5 next pl

Customizable Spectra Types by unique identifier

Page 25: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

The actual screening campaign Data analysis and interpretation – TS3.5 next pl

Customizable display layout

…. you start analyzing!

Page 26: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 27

New FBS tool in Topspin

Data analysis and interpretation – TS3.5 next pl

Page 27: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 28

New FBS tool in Topspin

Data analysis and interpretation – TS3.5 next pl

Page 28: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 29

New FBS tool in Topspin

Data analysis and interpretation – TS3.5 next pl

Page 29: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 30

New FBS tool in Topspin

Data analysis and interpretation – TS3.5 next pl

Page 30: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 31

New FBS tool in Topspin

Data analysis and interpretation – TS3.5 next pl

Page 31: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 32

New FBS tool in Topspin

Data analysis and interpretation – TS3.5 next pl

My comment

Page 32: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 33

New FBS tool in Topspin

Data analysis and interpretation – TS3.5 next pl

Page 33: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 34

New FBS tool in Topspin Data analysis and interpretation – TS3.5 next pl

Page 34: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

The actual screening campaign Data analysis and interpretation – TS3.5 next pl

Customizable report layout

Page 35: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

November 10, 2016 36

Alavar Gossert, Wolfgang Jahnke, Marcel Blommers, Cesar Fernandez, Paul Erbel

Daniel Wyss and Hugh Eaton

Stefan Jehle, Pavel Kessler, Fabrice Moriaud, Matteo Penestri, Anna Codina

Bettina Elshorst

Markus Schade

Page 36: Fragment Screening by NMR in Drug Discovery – F Methods · Fragment Screening by NMR in Drug Discovery – 19F Methods Dr. Stefan Jehle, Bruker BioSpin ... from efficient fragments

© Copyright Bruker Corporation. All rights reserved.

Innovation with Integrity

Copyright © 2011 Bruker Corporation. All rights reserved. www.bruker.com

Thank you for your attention … and come see us for demos and discussions


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