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Application of NMR in the Design of Peptide Tools for Chemical Biology and Drug Discovery Dr Andrew Jamieson School of Chemistry University of Glasgow andrew.jamieson.2@glasgow.ac.uk @jamiesonlab Research Programme Peptides/peptidomimetics

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SLIDE 1

Application of NMR in the Design

  • f Peptide Tools for Chemical

Biology and Drug Discovery

Dr Andrew Jamieson

School of Chemistry University of Glasgow andrew.jamieson.2@glasgow.ac.uk

@jamiesonlab

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SLIDE 2

Research Programme

Peptides/peptidomimetics

AcHN N H H N HN O O O NH2 O R2 R1 ZBG R1, R2 = amino acid side chain

A" B" C"

His187"

ACS Chem. Bio., 2016, 11, 3383-3390.

  • Rep. Org Chem., 2015, 5, 65 – 74.
  • Org. Bio. Chem., 2014, 12, 8775-8782.
  • Nat. Commun., 2016, 7, 11262
  • Chem. Comm., 2012, 48, 3709-3711.

β-Strand Mimetic Zinc Dependent Enzyme Inhibitors

HDAC/DUB

Stapled α-Helix Peptides

Aurora-A/TPX2

N N N O O N O O N N O O BocHN R1 R2 R3 H N N H H N N H H N O O O O O R R R R R i i + 1 i + 2 i + 3 i + 4

slide-3
SLIDE 3
  • Highly selective
  • Hormones, neurotransmitters, growth factors, ion channel ligands.
  • Efficacious
  • Relatively safe and well tolerated
  • Lower production complexity compared with protein-based

biopharmaceuticals

  • Enfuvirtide (36 residue peptide HIV therapy)

Ø 60 peptide drugs in clinic Ø 140 peptide drugs in clinical trials Ø 500 therapeutic peptides in preclinical development (2015)

Do Peptides Make Good Drugs?

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SLIDE 4
  • Limited orally bioavailability
  • Low membrane permeability (dissociation of water)
  • Approximately 75% of peptide drugs

are administered intravenously

  • Short circulating plasma half-life - Proteases

O N H O OH H2N

H3N N N O O R3 H O R2 H O R1 O H H H O H H O H

Problems with Peptide Drugs

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SLIDE 5

Peptidomimetic Design

Designed Peptidomimetics for the disruption of protein-protein interactions High-throughput screen to identify small molecule inhibitors

N N O N N OH O O Br Br

Design synthetic mimic of important side-chain residues

R R R

Conformationally constrain native peptide

Science, 2004, 303, 844.

  • J. Am. Chem. Soc., 1997, 119, 455.
  • J. Am. Chem. Soc., 2001, 123, 5382
  • Binding affinity
  • Specificity
  • Protease resistant
  • Cell permeable?
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SLIDE 6

Stapled Helices

NH3 O O

  • R. L. Baldwin

Biochemistry, 1993, 32, 9668 Salt Bridge

O NH O NH

Lactam

  • J. C. Phelan
  • J. Am. Chem. Soc., 1997, 119, 455

L L S S

Disulfide

  • P. G. Schultz
  • J. Am. Chem. Soc., 1991, 113, 9391

N H O NO2 O2N H N O NO2 O2N

Hydrophobic interactions

  • A. D. Hamilton

Biochemistry, 1995, 34, 984 Metal ligation

M

  • P. B. Hopkins
  • J. Am. Chem. Soc., 1990, 112, 9403
  • M. R. Ghadiri
  • J. Am. Chem. Soc., 1990, 112, 9633

Hydrocarbon

  • R. H. Grubbs
  • Angew. Chem. Int. Ed., 1998, 37, 3281
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SLIDE 7

Si, i+3R(8) G.L. Verdine

  • Org. Lett., 2010, 12, 3046

Si, i+4S(8) Y.-W. Kim & G.L. Verdine

  • Bioorg. Med. Chem. Lett.,

2009, 19, 2533 Ri, i+7S(11) G.L. Verdine JACS, 2000, 122, 5891 Stitched staple G.L. Verdine JACS, 2014, 136, 12314 Double staple L.D. Walensky

  • Proc. Natl. Acad. Sci. USA, 2010,

107, 14093

All-Hydrocarbon Stapled Peptides

  • N. S. Robertson, A. G. Jamieson, Rep. Org Chem., 2015, 5, 65 - 74.
  • Hydrocarbon length
  • Stereochemistry
  • α-methyl-α-AA
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SLIDE 8

Conotoxin Proteomimetic

  • Conotoxins are a family mini-proteins
  • Isolated from marine cone snails
  • Predatory sea animal
  • Produces 100s of neurotoxic peptides
  • Conotoxin µ-KIIIA
  • Voltage-gated sodium channels, NaV 1.1-1.9
  • Potential as analgesic
  • Knottin or cystine knot scaffold

www.coneshell.net

Conus Kinoshitai

  • Chem. Rev., 2014, 114, 5815–5847.
slide-9
SLIDE 9

µ-KIIIA Structure Determination

  • K. K. Khoo, K. Gupta, B. R. Green, M.-M. Zhang, M. Watkins, B. M. Olivera, P. Balaram, D. Yoshikami, G. Bulaj, R. S.

Norton, Biochemistry, 2012, 51, 9826–9835.

  • 15 possible foldamers of µ-KIIIA
  • Structural initially assigned as wrongly (Biochemistry, 2009, 48, 1210–1219 )
slide-10
SLIDE 10

Amide and aromatic region of 1D 1H-NMR spectra at 5 °C intervals from 5-25 °C, acquired on a Bruker DRX-600 spectrometer for a 2.6 mM solution of µ-KIIIA (pH 4.8)

µ-KIIIA Structure Determination

  • K. K. Khoo, K. Gupta, B. R. Green, M.-M. Zhang, M. Watkins, B. M. Olivera, P. Balaram, D. Yoshikami, G. Bulaj, R. S.

Norton, Biochemistry, 2012, 51, 9826–9835.

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SLIDE 11

Amide and aromatic region of NOESY spectra (blue) overlayed with TOCSY spectra (red) at 5 °C for µ-KIIIA (pH 4.8).

µ-KIIIA Structure Determination

  • K. K. Khoo, K. Gupta, B. R. Green, M.-M. Zhang, M. Watkins, B. M. Olivera, P. Balaram, D. Yoshikami, G. Bulaj, R. S.

Norton, Biochemistry, 2012, 51, 9826–9835.

slide-12
SLIDE 12

Parameters characterizing the final 20 structures of µ-KIIIA plotted as a function of residue number. Top left panel indicates number of long range (i-j ≥6), short range (2≤i-j≤5), sequential and intra NOE restraints used in the final structure calculations. Bottom left and RHS panels show angular order parameters (S) for backbone (φ, ψ) and sidechain (χ1) dihedral angles.

µ-KIIIA Structure Determination

  • K. K. Khoo, K. Gupta, B. R. Green, M.-M. Zhang, M. Watkins, B. M. Olivera, P. Balaram, D. Yoshikami, G. Bulaj, R. S.

Norton, Biochemistry, 2012, 51, 9826–9835.

slide-13
SLIDE 13

20 final structures for µ-KIIIA

µ-KIIIA Structure Determination

  • K. K. Khoo, K. Gupta, B. R. Green, M.-M. Zhang, M. Watkins, B. M. Olivera, P. Balaram, D. Yoshikami, G. Bulaj, R. S.

Norton, Biochemistry, 2012, 51, 9826–9835.

slide-14
SLIDE 14

µ-KIIIA Structure Determination

  • K. K. Khoo, K. Gupta, B. R. Green, M.-M. Zhang, M. Watkins, B. M. Olivera, P. Balaram, D. Yoshikami, G. Bulaj, R. S.

Norton, Biochemistry, 2012, 51, 9826–9835.

slide-15
SLIDE 15

µ-KIIIA Structure Determination

  • K. K. Khoo, K. Gupta, B. R. Green, M.-M. Zhang, M. Watkins, B. M. Olivera, P. Balaram, D. Yoshikami, G. Bulaj, R. S.

Norton, Biochemistry, 2012, 51, 9826–9835.

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SLIDE 16

Conotoxin Proteomimetic

  • Synthesis of knottin proteins is extremely difficult.
  • A. Van Der Haegen et al, FEBS J., 2011, 278, 3408–3418.

S S S C C N C S S K W C R D H S R C C S S S SH SH SH C C N C S S K W C R D H S R C C SHSH SH

  • xidation
slide-17
SLIDE 17

µ-conotoxin KIIIA µ-KIIIA mimetic

NH2 Ac S K W X R D H X R

S S S C C N C S S K W C R D H S R C C S S S

Conotoxin Proteomimetic

slide-18
SLIDE 18

S S S C C N C S S K W C R D H S R C C S S S

µ-conotoxin KIIIA µ-KIIIA mimetic

NH2 Ac S K W X R D H X R

  • Simple synthesis
  • Easy purification
  • α-helical

Conotoxin Proteomimetic

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SLIDE 19

Synthesis Purification

75% yield >99% Purity

Conotoxin Proteomimetic

FmocHN FmocHN Rink Amide Resin Grubb's 1st

  • Gen. Cat.

DCM, 2 h TFA/TIS/H2O, (95:2.5:2.5), 3 h 1) 20% piperidine/DMF 2) Fmoc-AA-OH HCTU, DIEA DMF, MW 100% conversion Ac-Ser(tBu)-Lys(Boc)-Trp(Boc)-X-Arg(Pbf)-Asp(tBu)-His(Trt)-X-Arg(Pbf)-NH Ac-Ser(tBu)-Lys(Boc)-Trp(Boc)-X-Arg(Pbf)-Asp(tBu)-His(Trt)-X-Arg(Pbf)-NH Ac-Ser-Lys-Trp-X-Arg-Asp-His-X-Arg-NH2

slide-20
SLIDE 20

NH2 Ac S K W X R D H X R NH2 Ac S K W A R D H S R

S S S C C N C S S K W C R D H S R C C S S S

Conotoxin Proteomimetic

slide-21
SLIDE 21
  • Simple synthesis
  • Easy purification
  • α-helical

NH2 Ac S K W X R D H X R NH2 Ac S K W A R D H S R

S S S C C N C S S K W C R D H S R C C S S S

Conotoxin Proteomimetic

slide-22
SLIDE 22

NH2 Ac X W A R X H S R NH2 Ac K X A R D X S R NH2 Ac K W X R D H X R NH2 Ac K W A X D H S X NH2 Ac K W A R D H S R

KIIIA Short Native Sequence KIIIA Staple Scan

CT1 CT4 CT5 CT2 CT3

Sunny Hanspal

Staple Scan

slide-23
SLIDE 23

NH2 Ac X W A R X H S R NH2 Ac K X A R D X S R NH2 Ac K W X R D H X R NH2 Ac K W A X D H S X NH2 Ac K W A R D H S R

KIIIA Short Native Sequence KIIIA Staple Scan

CT1 CT4 CT5 CT2 CT3

Two isomers in HPLC?

Sunny Hanspal

Staple Scan

slide-24
SLIDE 24
  • Tate. E et al, ACS Chem. Biol., 2014, 9(10), 2204-2209

Sunny Hanspal

Cis/trans isomers

slide-25
SLIDE 25

Conformational Analysis Circular Dichroism

Peptide Helicity (%) Conotoxin 1 16 Conotoxin 2 35 Conotoxin 3-cis 43 Conotoxin 3-trans 22 Conotoxin 4 31 Conotoxin 5 18

  • 11200
  • 6200
  • 1200

3800 8800 13800 180 200 220 240 260 Ellip&city θ Wavelength (nm) CT1 CT5 CT3 trans CT3 Cis CT4 CT2 Prod 2

NH2 Ac X W A R X H S R NH2 Ac K X A R D X S R NH2 Ac K W X R D H X R NH2 Ac K W A X D H S X NH2 Ac K W A R D H S R

KIIIA Short Native Sequence KIIIA Staple Scan

CT1 CT4 CT5 CT2 CT3

Sunny Hanspal

i – i + 4 staple – cis alkene required

slide-26
SLIDE 26

Si, i+3R(8) G.L. Verdine

  • Org. Lett., 2010, 12, 3046

Si, i+4S(8) Y.-W. Kim & G.L. Verdine

  • Bioorg. Med. Chem. Lett.,

2009, 19, 2533 Ri, i+7S(11) G.L. Verdine JACS, 2000, 122, 5891 Stitched staple G.L. Verdine JACS, 2014, 136, 12314 Double staple L.D. Walensky

  • Proc. Natl. Acad. Sci. USA, 2010,

107, 14093

  • Binding affinity
  • Specificity
  • Protease resistant
  • Cell permeable?
  • N. S. Robertson, A. G. Jamieson, Rep. Org Chem., 2015, 5, 65 - 74.

All-Hydrocarbon Stapled Peptides

slide-27
SLIDE 27

Astrid Knuhtsen

Two isomers in HPLC!

FmocHN NH2 Ac X K W A R D H X R

SPPS

  • NMR structure required for design

S S S C C N C S S K W C R D H S R C C S S S

µ-KIIIA i - i+7 Stapled Peptide

slide-28
SLIDE 28

cis

Decoupled

Astrid Knuhtsen James Jones (Dstl)

µ-KIIIA i - i+7 Stapled Peptide

slide-29
SLIDE 29

trans

Decoupled

Astrid Knuhtsen James Jones (Dstl)

µ-KIIIA i - i+7 Stapled Peptide

slide-30
SLIDE 30

Circular Dichroism

200 220 240 260

  • 10000

10000 20000

Cis Trans nm θ (deg*cm2*dmol-1)

NH2 Ac X K W A R D H X R NH2 Ac X K W A R D H X R H H H H

helicity (222 nm) 25% 40%

(hNaV1.4 ion channel)

µ-KIIIA i - i+7 Stapled Peptide

Astrid Knuhtsen

  • 7
  • 6
  • 5
  • 4

25 50 75 100

[Mimetic] M Channel activity (% of control)

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SLIDE 31

Circular Dichroism

200 220 240 260

  • 10000

10000 20000

Cis Trans nm θ (deg*cm2*dmol-1)

NH2 Ac X K W A R D H X R NH2 Ac X K W A R D H X R H H H H

helicity (222 nm) 25% 40%

(hNaV1.4 ion channel)

µ-KIIIA i - i+7 Stapled Peptide

Astrid Knuhtsen

i – i + 7 staple – trans alkene required

  • 7
  • 6
  • 5
  • 4

25 50 75 100

[Mimetic] M Channel activity (% of control)

slide-32
SLIDE 32
  • Peptides make great tools, peptidomimetics can improve

the physicochemical properties

  • Conformational analysis of peptides/peptidomimetics

using NMR provides crucial structural information required for molecular design

  • i – i + 4 staple – cis alkene required
  • i – i + 7 staple – trans alkene required
  • Conotoxins are hard to mimic…
  • Rapid method for the conformational analysis of

peptidomimetics is urgently required (not CD!)

Summary