NMR in fragment based drug discovery

NMR Studies in Fragment-Based Drug Discovery

Katie Strong

March 20, 2013

Advisor: Dr. Dennis Liotta, Ph.D.

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Bolten, B.M.; DeGregorio, T. Nat. Rev. Drug Discov. 2002, 1, 335.Keseru, G.M.; Makara, G.M. Nat. Rev. Drug Discov. 2009, 8, 203.

Early Stage Research and Discovery

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Hit generation is dominated by high-throughput screening (HTS)

The overall success rate of HTS by measuring progression to lead optimization is 45-55%• Estimated size of drug-like compound library is 1060 compounds, while corporate

chemical library is only 106 compounds• Increasing the size of the screening library does not proportionally yield more hits • Twice as likely to fail for newer targets

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Typical compound hit from HTS screen• Large molecule (MW between 250 – 600) • Broad surface contact with no high quality

interactions in key pockets• May contain functional groups that contribute

poorly to protein binding • Emphasis on potency (30 μM – nM hit activity)

Alternative to HTS: Fragment-Based Drug Discovery (FBDD)

Rees, D.C.; Congreve, M.; Murray, C.W.; Carr, R. Nature 2004, 3, 660.Scott, D.E.; Coyne, A.G.; Hudson, S.A.; Abell, C. Biochemistry 2012, 51, 4990.

Rees, D.C.; Congreve, M.; Murray, C.W.; Carr, R. Nature 2004, 3, 660.Scott, D.E.; Coyne, A.G.; Hudson, S.A.; Abell, C. Biochemistry 2012, 51, 4990.

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Typical compound hit from HTS screen• Large molecule (MW between 250 – 600) • Broad surface contact with no high quality

interactions in key pockets• May contain functional groups that contribute

poorly to protein binding • Emphasis on potency (30 μM – nM hit activity)

The idea that large molecules can be considered combinations of two or more individual fragments is a fundamental principle of fragment-based drug discovery

Typical compound hits from FBDD• Smaller molecule (MW between 150 – 300)• High proportion of the functional groups

involved in binding• Clearly interacts with pockets• Potency in the range of mM to 30 μM• Emphasis on efficiency and design

Alternative to HTS: Fragment-Based Drug Discovery (FBDD)

Rees, D.C.; Congreve, M.; Murray, C.W.; Carr, R. Nature 2004, 3, 660.Erlanson, D.A. Top. Curr. Chem. 2012, 317, 1.

Types of Fragment Elaboration Techniques

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Elaboration of HTS hit

Fragment growing

Fragment linking

Modified compound with higher potency

Every atom that is removed or added in FBDD is modified based on structural reasoning

Lipinski’s Rule of Five for drug-like compounds• Molecular weight < 500 Da• ClogP < 5• Number of hydrogen bond donors < 5• Number of hydrogen bond acceptors < 10

• Number of rotatable bonds < 7 • Polar surface area < 140 Å2

Congreve, M.; Carr, R.; Murray, C.; et al. Drug Discov. Today 2003, 8, 876.Hopkins, A.L.; Groom, C.R.; Alex, A. Drug Discov. Today 2004, 9, 430. Scott, D.E.; Coyne, A.G.; Hudson, S.A.; Abell, C. Biochemistry 2012, 51, 4990

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Fragment Criteria and Characteristics

Ligand efficiency (LE): method to compare different sized fragments• LE = -ΔG/Heavy Atom Content ≈ -RTlnKd/HAC• Fragments are weakly binding, but very “atom efficient” binders • LE > 0.3 kcal/mol per HAC are considered good oral drug candidates

Astex’s Rule of Three for fragments• Molecular weight < 300 Da• ClogP < 3• Number of hydrogen bond donors < 3• Number of hydrogen bond acceptors < 3

• Number of rotatable bonds < 3• Polar surface area < 60 Å2

Scott, D.E.; Coyne, A.G.; Hudson, S.A.; Abell, C. Biochemistry 2012, 51, 4990. 7

Comparison of Fragment and HTS Hits

Pellecchia, M.; Sem, D.S.; Wuthrich, K. Nature 2002, 1, 211.Meyers, B.; Peters, B. Angew. Chem. Int. Ed. 2003, 42, 864.

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NMR Methods for Fragment Discovery and Elaboration

NMR methods for detecting ligand binding are divided into two categories1) Monitor NMR signals from the protein in the presence of ligand

• Chemical-shift mapping and “SAR by NMR”

2) Monitor the ligand bound to target relative to the free ligand• T2 and T1p relaxation • Transferred NOEs • Saturation transfer difference (STD) • Water-ligand Observed via Gradient Spectroscopy (Water-LOGSY)• Diffusion editing

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Use of Relaxation Times to Identify Ligands

Relaxation Time

• Small, rapidly tumbling molecules: high (longer) relaxation times

• Macromolecules that move slowly through solution: low (shorter) relaxation times

Shortening T2 relaxation time leads to peak broadening

Pellecchia, M.; Sem, D.S.; Wuthrich, K. Nature 2002, 1, 211.Meyers, B.; Peters, B. Angew. Chem. Int. Ed. 2003, 42, 864.

Molecular Weight1000 Da

T1

T2

Z

X

Y

After resonance, where v1 = vo, magnetization relaxes back to equilibrium • T1 = relaxation of nuclear spin magnetic

vector parallel to the magnetic field, Bo

• T2 = relaxation of nuclear spin magnetic vector perpendicular to the magnetic field, Bo

Bo

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Pellecchia, M.; Sem, D.S.; Wuthrich, K. Nature 2002, 1, 211.Meyers, B.; Peters, B. Angew. Chem. Int. Ed. 2003, 42, 864. Jahnke, W.; Perez, L.B.; Paris, G.C.; Strauss, A.; Fendrich, G.; Nalin, C.M. J. Am. Chem. Soc. 2000, 122, 7394.

Distance from paramagnetic center

25 Å

T1 and T2

Relaxation Time

1) SLAPSTIC (Spin labeled attached to protein side chains as a tool to identify interacting compounds): Covalently attach a spin label (TEMPO) on the target near the binding site and monitor the enhanced T2 relaxation once a ligand binds

2) Use a spin-labeled ligand to bind to an initial site on the target and then monitor the enhanced T2 relaxation once a ligand binds in the second binding site

The gyromagnetic ratio of an unpaired electron is 658-fold larger than a

proton

γ = μ / Pγ = gyromagnetic ratioμ = magnetic moment

P = spin angular momentum

11Jahnke, W.; Perez, L.B.; Paris, G.C.; Strauss, A.; Fendrich, G.; Nalin, C.M. J. Am. Chem. Soc. 2000, 122, 7394.Meyers, B.; Peters, B. Angew. Chem. Int. Ed. 2003, 42, 864.

Researchers demonstrated the SLAPSTIC method with the FK binding protein by using a mixture of known binders (1) and (2) and 3 non-bonding aromatic ligands.• FKBP has lysine residues 15-20 Å away from the binding site, so all lysine residues

were spin-labeled using N-hydroxysuccinimide ester 3• Monitor the relaxation effect of the ligands in the presence of spin-labeled protein


Kd = 1.1 mM Kd = 9.0 mM

No FKPB FKBP Spin-labeled FKBP

Pellecchia, M.; et al. J. Biomol. NMR 2002, 22, 165.

Pellecchia, M.; Sem, D.S.; Wuthrich, K. Nature 2002, 1, 211.12

Chemical-shift Mapping

Label the target with 15N and/or 13C and observe changes in the chemical environment with the addition of a ligand or mixture of ligands

[13C, 1H]-HMQC of selectively labeled DHPR (13Cε/1H Met, 13Cδ/1H Ile, 13C/1H Thr)

DHPR alone: green

DHPR + : blue

DHPR + : blue

DHPR + : red

Shuker, S.B.; Hajduk, P.J.; Meadows, R.P.; Fesik, S.W. Science 1996, 274, 1531. 13

Chemical-shift Mapping and “SAR by NMR”

Target based screening first reported by Abbott Laboratories in 1996 in an effort to find compounds to

replace FK506, an immunosuppressant that binds to FKBP

FK506

Shuker, S.B.; Hajduk, P.J.; Meadows, R.P.; Fesik, S.W. Science 1996, 274, 1531. 14

Chemical-shift Mapping and “SAR by NMR”

Kd = 2 μM

Kd = 0.8 mM

Kd = 0.1 mM

Kd = 19 nM

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Drugs from FBDD to Reach Clinical Trials

Erlanson, D.A. Top. Curr. Chem. 2012, 317, 1.

Drug Company Target Phase

PLX-4032(Vemurafenib)

Plexxikon B-Raf V600E FDA Approved

ABT 263 Abbott Bcl-2/Bcl-xL Phase 2

ABT869 Abbott VEGF and PDGFR Phase 2

AT9283 Astex Aurora Phase 2

AT5719 Astex CDKs 1,2,4,5 Phase 2

LY-517717 Lilly/Protherics Fxa Phase 2

Indeglitazar Plexxikon PPAR agonist Phase 2

VER-52296 Vernalis/Novartis Hsp90 Phase 2

ABT-518 Abbott MMP-2 and MMp-9 Phase 1

ABT-737 Abbott Bcl-2/Bcl-xL Phase 1

AT13387 Astex Hsp90 Phase 1

LP-261 Locus Tubulin Phase 1

PLX-5568 Plexxikon Kinase Phase 1

Using variety of techniques, a handful of drugs developed by FBDD have entered the clinic

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Bcl-2 (B-cell lymphoma) Family Proteins

Tait, S.W.G.; Green, D.R. Nature Rev. 2010, 11, 621.

Bcl-2 proteins are regulators of programmed cell death, and anti-apoptotic proteins are typically overexpressed in cancer cells

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Bcl-2 Family Proteins and Role in Apoptosis

Youle, R.J.; Strasser, A. Nature Rev. 2008, 9, 47.

• In healthy cells, Bax and Bakpredominately exists in the cytosol, but when under stress will move to the mitochondria and activate apoptosis

• In a mechanism that is not entirely understood, anti-apoptotic proteins can bind and retrotranslocate Baxfrom the mitochondria, inhibiting apoptosis

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Protein-Protein Interactions as a Classically “Difficult” Target

Wells, J.A.; McClendon, C.L. Nature 2007, 450, 1001.Bauer, R.A.; Wurst, J.M.; Tan, D.S. Curr. Opin. Chem. Biol. 2010, 14, 308.Overington, J.P.; Al-Lazikani, B.; Hopkins, A.L. Nat. Rev. Drug Discov. 2006, 5, 993.

It has been proposed that no class of interaction rivals the complexity of protein-protein interactions, and targeting these interactions has been regarded as “difficult.”

• Contact surface area is typically very large at approximately 1500-3000 Å2

• Binding pockets are often flat, featureless, and lack well-defined grooves

• Lack a natural small-molecule partner, so difficult to find a suitable starting lead

• Often a HTS is dominated by compounds that have been used for classic drug targets, and each protein-protein interaction may require a different starting compound

• Protein-protein interactions are key to intracellular signaling pathways

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Binding Site of Bcl-xL and BAX

Sattler, M. et al. Science 1997, 275, 983.Petros, A.M.; et al. J. Med. Chem. 2006, 49, 656.PDB 1G5J

Bcl-xL

α1

α7

α3

α5

α6α4

α8

α2

BH3

BH1

BH2

The Bcl-XL protein consists of 8 α helices with a deep hydrophobic pocket formed by the BH1, BH2, and BH3 domains

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Binding Site of Bcl-xL and BAX

Sattler, M. et al. Science 1997, 275, 983.Petros, A.M.; et al. J. Med. Chem. 2006, 49, 656.PDB 1G5J

Bcl-xL

α1

α7

α3

α5

α6α4

α8

16 residue portion from BH3 domain of BAX

The binding site ofBcl-XL is a deep

hydrophobic pocket and only approximately

500 Å2 of the protein surface is involved

α2

BH3

BH2

BH1

The Bcl-XL protein consists of 8 α helices with a deep hydrophobic pocket formed by the BH1, BH2, and BH3 domains

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“SAR by NMR” to Develop Inhibitor of Bcl-XL: First Fragment

Petros, A.M.; et al. J. Med. Chem. 2006, 49, 656.

R1 R2 R3 NMR Kd (μM)

11 F COOH H 300 + 30

12 H COOH H 1200 + 530

13 F OH H > 5000

14 H COOCH3 H > 5000

15 H CH2COOH H 2000 + 1600

16 H CH2CH2COOH H 1990 + 990

17 OCH3 COOH H 383 + 117

18 Cl COOH H 238 + 110

20 H H COOH > 5000

Initial fragment scaffold that was carried forward

1) Uniformly 15N-label Bcl-XL protein and purify the protein by affinity chromatography 2) [1H-15N]-HSQC NMR screening on 15N-labled protein (100 μM) in presence and

absence of small compounds (average MW of 210)• 9373 compounds were added in increments of 10 • 66 mixtures caused a significant shift in HSQC• The 660 compounds were then retested individually, yielding 49 compounds with

Kd values less than 5 mM.

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“SAR by NMR” to Develop Inhibitor of Bcl-XL: Second Fragment

[1H-15N]-HSQC NMR screening on 15N-labled protein (100 μM) in the presence of biarylderivative (11) and second set of small compounds

• 3472 compounds were added in increments of 5 • 60 mixtures caused a significant shift in HSQC• The 300 compounds were then retested individually, yielding 24 compounds with

Kd values less than 5 mM.R1 NMR Kd (μM)

21 4300 + 1600

22 5000 + 2000

23 2000 + 440

24 9000 + 2000

25 6000 + 2000


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“SAR by NMR” to Develop Inhibitor of Bcl-XL: Chemical-Shift Mapping

Black: 15N-labeled Bcl-XL

Red: Bcl-XL and

Green: Bcl-XL, fragment 11, and


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“SAR by NMR” to Develop Inhibitor of Bcl-XL: Linking the Fragments

The ortho position of fragment 9 was the most direct linker to the second site



2nd site ligand

1st site ligand (11)


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FPA Ki = 1.4 μM

Trans-linker interacts with phenylalanine and prevents fragment from binding deep in the pocket


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“SAR by NMR” to Develop Inhibitor of Bcl-XL: Revisiting First Fragment

The carboxylate of the first fragment was replaced with an acylsulfonamide and 120 analogs were synthesized

using commercially available sulfonamides


NMR Kd = 300 μM NMR Kd = 320 μM

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“SAR by NMR” to Develop Inhibitor of Bcl-XL: Revisiting First Fragment

Bcl-XL FPA Ki = 0.245 μM


Initial most potent sulfonamide

fragment

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“SAR by NMR” to Develop Inhibitor of Bcl-XL: Revisiting the Second Fragment

125 additional compounds that maintained the acylsulfonamide and nitrophenyl moieties were prepared


Bcl-XL FPA Ki = 36 nM

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“SAR by NMR” to Develop Inhibitor of Bcl-XL: Optimizing the Final Compound


Bcl-XL FPA Ki = 0.245 μM Bcl-XL FPA Ki = 36 nM

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Despite the potency, 44 had poor aqueous

solubility and tight binding to human

serum albumin (HSA)


Bcl-XL FPA Ki = 0.245 μM Bcl-XL FPA Ki = 36 nM

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Bruncko, M.; et al. J. Med. Chem. 2007, 50, 641.Petros, A.M.; et al. J. Med. Chem. 2006, 49, 656.Oltersdorf, T.; et al. Nature 2005, 435, 677.

Certain portions of 44 were exposed to lipophilic residues in the complex with HSA, and

these were modified with polar substituents

Basic 2-dimethylaminoethyl group

Basic piperazine and

biphenyl substituents

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Oltersdorf, T.; et al. Nature 2005, 435, 677. Bruncko, M.; et al. J. Med. Chem. 2007, 50, 641.

ABT-737Bcl-XL FPA Ki < 0.5 nMBcl-2 FPA Ki < 1.0 nM

In optimizing the final compound, the first dual inhibitor of Bcl-XL and Bcl-2 was discovered

While no longer binding to HAS, ABT-737 is not orally available and low aqueous solubility makes intravenous delivery challenging

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Park, C-M.; et al. J. Med. Chem. 2008, 51, 6902.

ABT-737Bcl-XL EC50 = 7.7 nMBcl-2 EC50 = 30 nM

10% HS HI46 EC50 = 87 nmAUC = 0.28 μM . h

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50Bcl-XL EC50 = 0.60 nMBcl-2 EC50 = 0.90 nM



10% HS HI46 EC50 = 87 nmAUC = 0.28 μM . h

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5110% HS HI46 EC50 = 40 nm



10% HS HI46 EC50 = 87 nmAUC = 0.28 μM . h

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10% HS HI46 EC50 = 87 nmAUC = 0.28 μM . h


5110% HS HI46 EC50 = 40 nm

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AUC = 1.16 μM . h Park, C-M.; et al. J. Med. Chem. 2008, 51, 6902.

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10% HS HI46 EC50 = 87 nmAUC = 0.28 μM . h

ABT-263Bcl-XL EC50 = 5.9 nMBcl-2 EC50 = 4.2 nM

10% HS HI46 EC50 = 87 nmAUC = 6.26 μM . h

Currently, ABT-263 is in Phase 2 clinical trials for lymphoid malignancies, chronic lymphocytic leukemia, and small cell lung cancer

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“SAR by NMR” to Develop Inhibitor of Bcl-XL: Fragments Leading to Inhibitor

ABT-263Bcl-xL Ki < 0.5 nM

LE = 0.20MW = 975


Kd = 300 μMLE = 0.30

MW = 216

Kd = 6000 μMLE = 0.23

MW = 170Ki = 1.4 μMLE = 0.27

MW = 394

Bcl-xL K i = 36 nMLE = 0.27

MW = 552

ABT-737Bcl-xL Ki = 0.6 nM

LE = 0.22MW = 813

Protein-peptide interactionBcl-XL and 26-residue of BAD

Protein-small molecule interactionBcl-XL and ABT-737

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Bcl-XL Bound to Natural Peptide Partner and Inhibitor

Wells, J.A.; McClendon, C.L. Nature 2007, 450, 1001.

Molecular Mass (Da)

Bcl-xL Ki

(nM)LE

(kcal/mol/HAC)

BAD-derived peptide 3,110 0.6 0.16

ABT-737 813 0.6 0.23

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Protein-Protein Interactions as a Classically “Difficult” Target

Wells, J.A.; McClendon, C.L. Nature 2007, 450, 1001.

It has been proposed that no class of interaction rivals the complexity of protein-protein interactions, and targeting these interactions has been regarded as “difficult.”

1) Binding pockets are often flat, featureless, and lack well-defined grooves• Contact surfaces are adaptable • APT-737 bound to Bcl-XL causes a more puckered conformation

2) Lack a natural small-molecule partner, so difficult to find a suitable starting lead• Small molecule and natural partner possibly have comparable affinities • ABT-737 and BAD both have 0.6 nM affinity, and ABT has higher LE

3) The molecular size of many compounds that interact with protein-protein interfaces are too large • The criteria for defining drug-like characteristics is based on known drugs• HTS may not be successful for more “difficult” targets because libraries are

typically composed of scaffolds for traditional targets.• ABT-263 breaks 3 rules from Lipinkski’s Rule of 5, but is still orally

bioavailable.

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Summary

• FBDD provides an alternative to HTS where small ligands are found and elaborated in a process based on design and efficiency

• NMR techniques can be based on observing changes to the protein or by observing changes to the bound ligand relative to the free ligand• Chemical-shift mapping and “SAR by NMR”• T2 and T1p relaxation

• The first dual inhibitor of Bcl-XL and Bcl-2, developed by “SAR by NMR” is orally available and has entered Phase 2 clinical trials for types of leukemia

• FBDD design is helping to develop modulators for targets that have been classically regarded as difficult and challenging • Protein-protein interactions: Bcl-XL, heat shock protein Hsp90• RNA polymerase: HCV NS5B RNA-dependent polymerase• DNA-binding proteins: E2 transcription factor from human papillomavirus

Coyne, A.G.; Scott, D.E.; Abell, C. Curr. Opin. Chem. Biol. 2010, 14, 299.