User’s meeting, Brussels, November 2016 - Bruker · User’s meeting, Brussels, November 2016 Fragment-based drug discovery Innovation with Integrity . The principle of Fragment

Dr. Till Kühn

VP Applications Development MRS, Bruker BioSpion

User’s meeting, Brussels, November 2016

Fragment-based drug discovery

Innovation with Integrity

The principle of Fragment Based Screening

from efficient fragments to Drug candidates

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

Candidate high LE, low IC50

0.91mM

0.59

0.07mM

0.55

5.9nM

0.49

3.0nM

0.42

IC50:

LE:

Grow, optimize

Advantages of FBS:

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%)

NMR in Fragment based Screening

More than one answer possible

% respondents using NMR technique

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

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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

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 (13C or 15N)

- 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 crystal structure available

Binding site in crystal contact (often for

PPI targets)

- 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 (13C or 15N)

- Size limitation of target

- Amount of protein required depends on

application

Two NMR Screening principles

Excess of ligand (i.e. 0.25 mM)

small molecule, different NMR properties than protein

NMR Fragment Based Screening

The concept

Low protein concentration (i.e. 0.01mM)

Large molecule with distinct NMR properties

High ligand concentration, low protein concentration

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


The concept

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

norm

alized inte

nsity

time

T2


The concept

Binding ligand keeps properties when back in the pool, other ligands are unchanged

norm

alized inte

nsity

T2

time

Excursion: 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




Spin-Echo pulse program for

relaxation measurement

binding fragments: faster relaxation when compared to non-binders

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


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


Problem: Large Spectral Width

Phasing difficult if hard 180°pulses are used




WHAT WORKS @ 700MHz:

1.5ms Crp90.comp4

Power of 13 us 90-square (19.5kHz)

Excitation profile 70kHz

= ca. 110ppm 19F @ 700MHz

Solution: 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



Solution: broadband adiabatic refocusing pulse


1H decoupling significantly improves signal to noise

With 1H decoupling

Without 1H decoupling

Benefits of 1H decoupling


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

Probe options


Three Basic 1H NMR Experiments in FBS

Saturation Transfer Difference (STD)

Binders show up in difference spectrum, non binders don’t

Water-LOGSY

Binders have opposite phase to non-binders

T2/T1rho

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

STD

STD reference

Not binding

Weakly binding

Strongly binding

NMR FBS in practice

STD

STD reference






NMR FBS in practice

STD

STD reference






1. Before screening against any protein, you run each compound of library

2. Then make cocktails

3. Start screening campaigns

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• Simple batch setup for the complete library

Setup

• Optimized experiments (auto water suppression etc.)

for master stock solutions (DMSO) for ligands in buffer (screening reference)

Automation

• Fully automated analysis on-the-fly or batch:

• integrity, concentration, purity, solubility

Analysis

• 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

Results

Screening preparation & library QC on-the-fly

CMC-q sets-up, runs and analyzes your references

December 5, 2016 24

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%

• QC of DMSO stock solution

• QC and reference in buffer

• The actual protein screening

Tools for the practical work

New temperature control option in SampleJet

Individual temperature control for each of the 5 96 sample racks

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


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


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!

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New FBS tool in Topspin


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Customizable Spectra Types by unique identifier



Customizable display layout



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Customizable report layout



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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 39

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

© 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 discusions

Documents

User’s meeting, Brussels, November 2016 - Bruker · User’s meeting, Brussels, November 2016 Fragment-based drug discovery Innovation with Integrity . The principle of Fragment