Integrating Microflow NMR into Fragmentbased Drug Discovery

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Integrating Microflow NMR into Fragment-based
Drug Discovery
Daniel S. Sem Chemical Proteomics Facility at
Marquette (CPFM) Department of Chemistry Marquette
University Protasis Webinar 7/15/08
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Integrating Microflow NMR into Fragment-based
Drug Discovery

• Research Focus Drug discovery using NMR
• Background on Fragment-based Drug Discovery
• NMR Equipment in the CPFM
• Flow Probe Applications
• Routine quality control of compound collections
• Protein 1H-15N HSQC screening of labeled proteins
• Fragment-based screening using STD (saturation
  transfer difference) NMR

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New Drug Design Paradigm Fragment-Assembly

• Companies using fragment assembly approaches
• Advanced Medicines / Theravance
• Sunesis (Thiol tethering)
• Structural GenomiX
• Abbott (SAR by NMR)
• Vertex (SHAPES)
• And from a Chemical Proteomics slant Triad
  Therapeutics

• San Diego, California
• Initially funded June, 1999
• Raised 42.5 M in A and B rounds 15 M in C
  round
• 50 employees

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Modular Drug Design / Fragment Assembly1
SAR by NMR2
SHAPES3

• Reviewed by Pellecchia, Sem Wuthrich (2002)
  Nat. Rev. Drug Disc. 1, 211-219.
• Shuker, Hajduk, Meadows Fesik (1996) Science
  274, 1531-1534..
• Fejzo et al. (1999) Chem. Biol. 6, 755-769.

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Combinatorial Library for Dehydrogenases

Chemical inhibitors discovered across a gene
family (dehydrogenases)


scaffold
Common
Variable
scaffold
Oxidoreductase-1 Oxidoreductase-2
Oxidoreductase-N
scaffold
Reviewed by Pellecchia, Sem Wuthrich (2002)
Nat. Rev. Drug Disc 1, 211-219. Proof of concept
Sem et al. (2004) Chemistry and Biology 11,
185.
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Screen NMR-designed Library for Inhibitors
Specific for Target versus Antitarget Assay
results
TB Target
Malaria Target
LDH DHPR DOXPR 55 mM 26 mM
gt50 mM 42 nM gt 50 mM 10 mM 12 mM gt 25
mM 202 nM 620 nM 100 nM 7.9 mM
TB target
Malaria target
proteomic leverage
scaffold
target specificity
(IC50)
Sem et al. (2004) Chemistry and Biology 11,
185.
Sem et al. (2004) Chemistry and Biology 11, 185.
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Triad

• Technology Platform Internal use only
• Proprietary NMR-based drug design platform to
  generate drug leads
• Chemicals, proteins, software, databases, methods
• Proof of Principle completed (Series B, 30M)
• Leads for infectious disease targets
• Dual Business Strategy (Drug Discovery)
• Internal drug discovery licensing early-stage
  leads
• Evolved into late-stage licensing (IND
  candidates)
• Triad ceased operations on 3/19/04
• Drug licensing business model not
  practical

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Drug Discovery in Academics

• Can focus on developing enabling methods rather
  than drug leads. The longer view
• Focus on diseases with smaller markets third
  world diseases.
• The CPFM has resources to aid in drug discovery
  compounds databases software NMR screening
  capability automation labeled probes.

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NMR Equipment in the CPFM

• 600 MHz Varian NMR System
• Cryogenic probe (1H/2H/13C/15N)
• z-axis gradients
• 4 channels

• 300 MHz Varian NMR System
• Broadband probe
• z-axis gradients
• 60 sample change

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NMR Equipment in the CPFM

• 400 MHz Varian NMR System
• x,y,z-axis gradients (imaging capability)
• 2 channels
• BB and inverse probes
• Protasis CapNMR Microflowprobe
• TXI triple resonance, 1H/2H/13C/15N
  detection
• z-gradient, variable temp., 10 uL flowcell
• Automated sample introduction using LEAP
  Technologies
• (CTC Analytics) liquid handler
• Automation managed via Protasis One-Minute NMR
  (OMNMR) software

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NMR-based Drug Discoveryat Marquettes CPFM

• Focus on developing new methods
• Blending chemistry, NMR screening, informatics
• Integrating use of microflow NMR
• Focus on infectious disease

www.marquette.edu/cpfm
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• Could we discover a new version of
• this CR-based biligand more easily?
• Avoiding extensive synthesis (for linking)?
• Using microflow NMR (speed conserve samples)?
• Strategy
• Combine thiol tethering and STD-based screening,
  using a Flow NMR platform

CR catechol rhodanine Privileged scaffold
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STD-based screening(STD saturation transfer
difference)
CF-STD NMR1,2 Cofactor fingerprinting with
saturation-transfer-difference NMR
Cofactor structures
STD NMR3

• Stockman Dalvit (2002) Prog. NMR Spectr. 41,
  187-231.
• Yao Sem (2005) FEBS Lett., 579, 661-666.
• Mayer Meyer (1999) Angew. Chem. Int. Ed. 38,
  1784-1788.

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STD-based screening(STD saturation transfer
difference)
CF-STD NMR1,2 Cofactor fingerprinting with
saturation-transfer-difference NMR
STD PKA cAMP, cCMP, cGMP
STD NMR3
1D cAMP, cCMP, cGMP
STD RSP2 cAMP, cCMP, cGMP

• Stockman Dalvit (2002) Prog. NMR Spectr. 41,
  187-231.
• Yao Sem (2005) FEBS Lett., 579, 661-666.
• Mayer Meyer (1999) Angew. Chem. Int. Ed. 38,
  1784-1788.

600 MHz 25 mM protein 1mM cofactors
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Flow Probe Applications
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• Using Flow Probe for HSQC Experiments
• To screen for folding conditions (ex. structural
  proteomics)
• To screen for fragment binding (ex. SAR by NMR)

Our model protein GB1 (IgG binding domain from
protein G) Well studied (ex. Frank et al. (2002)
NSB 9, 877-885)
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Using Flow Probe for HSQC Experiments

• Our model protein GB1 (IgG binding domain from
  protein G 56 AA)
• 1 mM, pH 7
• Spectra taken on the 400 MHz flow probe (10 uL
  sample volume)
• Acquisition time varied

2 hrs.
5 hrs.
10 hrs.
Main advantage automation and low sample
consumption
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• STD based screening with our drug target DHPR
  (Dihydrodipicolinate reductase)
• Optimizing concentrations for flow-based
  screening
• (generally, we need gt 50 uM protein, and high
  ligand concentration)
• This requires use of reporter ligands to detect
  binding!
• Of course, competitor gt enzyme target

100 uM DHPR ligand (NAD)
80 mM NAD
40 mM NAD
20 mM NAD
10 mM NAD
Acquisition time 47 minutes (96-well plate in lt
4 days this might be a secondary assay) Samples
in D2O, 20 mM K-phosphate, pH 7.6, 298K
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STD based screening with our drug target DHPR
(Dihydrodipicolinate reductase) Optimizing
concentrations for flow-based screening binding
2 ligands (generally, we need gt 50 uM protein,
and high ligand concentration)
100 uM DHPR 10 mM PDC ?
10 mM PDC 10 mM NADH
10 mM PDC 10 mM NAD
10 mM PDC
Acquisition time 47 minutes Samples in D2O, 20
mM K-phosphate, pH 7.6, 298K
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• Whats going on?
• Flow-probe-based STD screening sees NAD but not
  NADH
• NADH binds too tightly
• NADH seemed to increase the PDC STD effect? They
  bind to different sites, so possible synergy?
• Follow-up titration (this one is at 600 MHz
  w/cryoprobe)

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• Next combine STD-based screening w/
  thiol-tethering
• Search for fragments that bind in cofactor (NAD
  or CRAA) and substrate (PDC) sites
• CRAA catechol rhodanine acetic acid
• What is thiol tethering?
• Bring weak binding fragments together (link)
  using disulfide bonds
• Pioneered by Erlanson, Wells and other at Sunesis

Erlanson et al. (2000) PNAS 15,
9367-9372. Erlanson et al. (2003) Nat. Biot. 21,
308-314. Erlanson et al. (2004) Curr. Opin. Chem.
Biol. 11, 730-737.
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• Next combine STD-based screening w/
  thiol-tethering
• Search for fragments that bind in cofactor (NAD
  or CRAA) and substrate (PDC) sites
• Our approach
• Bring weak 2 weak binding thiol-containing
  fragments together in the cofactor and substrate
  sites of DHPR, then link them later
• Start with a first fragment that binds (CRAA)
  and screen for the second
• Detect binding based on competition STD and
  reporter ligands (NAD, PDC)

CRAA
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Use STD Screening to Identify Thiol Fragments
that Fit in the PDC Site
Relative STD for 99 thiols (screened in pools of
5) Flow probe, using 10 mM PDC as a reporter (1
mM thiol 100 uM DHPR 45 min acquisition) Thio
l Fragment Database (99 thiols) www.marquet
te.edu/cpfm
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Why do some thiol fragments cause an increase in
the PDC STD signal? Perhaps binding at other
sites in the tetramer.
PDC
Relative STD for 99 thiols (screened in pools of
5) Flow probe, using 10 mM PDC as a reporter (1
mM thiol 100 uM DHPR 45 min acquisition)
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Discovery that TNB (5-thio-2-nitrobenzoic acid)
binds in the PDC site
20 mM TNB, 200 uM DHPR
20 mM TNB, 200 uM DHPR 4 mM PDC
STD with 400 MHz microflowprobe, 1 hr.
acquisition time gt 25 decrease in TNB STD
signal due to PDC
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Proof that PDC and TNB (a thiol fragment) occupy
the same site gt Titration _at_ 600 MHz
Competition of PDC (varied) against 2 mM TNB
(reporter) (100 uM DHPR)
Competition of TNB (varied) against 2 mM PDC
(reporter) (100 uM DHPR)
Note STD doesnt go to zero - perhaps because
there are 4 active sites that are not equivalent?
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• A New Strategy
• In situ thiol tethering and competition STD
  screening a same time
• The Goal
• Screen various thiols (RS-) to see which can
  form a higher affinity biligand, blocking both
  sites (thereby decreasing STD signals for NAD and
  PDC reporters)
• In this way, we discovered a biligand with TNB
  (5-thio-2-nitrobenzoic acid)

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Results of in situ thiol tethering / competitive
STD
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Synthesis of the Thiol-Tethered Biligand (to
verify in situ hit)
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• Secondary Assay
• In-gel binding to the colored CRAA2-TNB biligand

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• Secondary Assay
• Steady-state inhibition CRAA2-TNB biligand is
  
• competitive vs. NADH (Ki lt 72 mM)

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Acknowledgements
Graduate Students Aurora Costache Huili Yao Xia
Ge NMR Facility Manager Sheng Cai, Ph.D.
Chemistry Dept.

• American Heart Association
• Biomedical Technology Alliance
• NIH (600 MHz spectrometer)
• Marquette University

www.marquette.edu/cpfm Fragment database,
created with SciTegic software