Application of NMR in the Design of Peptide Tools for Chemical Biology and Drug Discovery ·...

Application of NMR in the Design of Peptide Tools for Chemical Biology and Drug Discovery

Dr Andrew Jamieson School of Chemistry

University of Glasgow [email protected]

@jamiesonlab

Research Programme Peptides/peptidomimetics

AcHN NH

HN

HNO

O

O

NH2

O

R2R1

RHN

O

RHN

SO

O

N

R

HNO

OH

RHN

HNO

OH

FmocHN OH

O

ZBG

61 2 3

RHN

11

OH

OHR

B

12

HOOH

R

O

7R

OP

13

OO

O

ZBG

R1, R2 = amino acid side chain

R

SH

5

RN

NN

10

RN

O

8

RHN

CF3

9

RHN

HNS

4

Amide BioisosteresZinc Binding Groups Transition State Analogues

automated solid phase

peptide synthesis

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

NN

N

O

O

N

O

ON

N

O

OBocHN R1

R2R3

HN N

H

HN N

H

HN

O

O

O

O

O

R R R

R R

i

i + 1

i + 2

i + 3

i + 4

•  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?

•  Limited orally bioavailability •  Low membrane permeability (dissociation of water)

•  Approximately 75% of peptide drugs are administered intravenously •  Short circulating plasma half-life - Proteases

O

NH

O

OH H2N

H3N N NO

OR3

H

O

R2

H

O

R1

OH H

HOH H

OH

H3N N NO

OR3

H

O

R2

H

O

R1

H3N N NO

OR3

H

O

R2

H

O

R1Problems with Peptide Drugs

Peptidomimetic Design

Designed Peptidomimetics for the disruption of

protein-protein interactions

High-throughput screen to identify

small molecule inhibitorsN

N

ON N OH

O

O

Br

Br

Design syntheticmimic of important side-chain residues

R

R

R

Conformationallyconstrain

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?

Stapled Helices

NH3

O

O

R. L. BaldwinBiochemistry, 1993, 32, 9668

Salt Bridge

O

NH

O

NH

Lactam

J. C. PhelanJ. Am. Chem. Soc., 1997, 119, 455

L

L

S

S

Disulfide

P. G. SchultzJ. Am. Chem. Soc., 1991, 113, 9391

NH

O

NO2

O2N

HN

O

NO2

O2N

Hydrophobicinteractions

A. D. HamiltonBiochemistry, 1995, 34, 984

Metal ligation

M

P. B. HopkinsJ. Am. Chem. Soc., 1990, 112, 9403

M. R. GhadiriJ. Am. Chem. Soc., 1990, 112, 9633

Hydrocarbon

R. H. GrubbsAngew. Chem. Int. Ed., 1998, 37, 3281

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

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.

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 )

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)



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



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.



20 final structures for µ-KIIIA




•  Synthesis of knottin proteins is extremely difficult.

A. Van Der Haegen et al, FEBS J., 2011, 278, 3408–3418.

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

SS

S

SHSH SH

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

SHSHSH

oxidation

µ-conotoxin KIIIA

µ-KIIIA mimetic

NH2Ac S K W X R D H X R


SS

S



SS

S

µ-conotoxin KIIIA

µ-KIIIA mimetic


•  Simple synthesis •  Easy purification •  α-helical


Synthesis

Purification

75% yield

>99% Purity


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


NH2Ac S K W A R D H S R


SS

S


•  Simple synthesis •  Easy purification •  α-helical


NH2Ac S K W A R D H S R


SS

S


NH2Ac X W A R X H S R

NH2Ac K X A R D X S R

NH2Ac K W X R D H X R

NH2Ac K W A X D H S X

NH2Ac K W A R D H S R

KIIIA Short Native Sequence

KIIIA Staple Scan

CT1

CT4

CT5

CT2

CT3

Sunny Hanspal Staple Scan







KIIIA Staple Scan

CT1

CT4

CT5

CT2

CT3 Two isomers in HPLC?

Sunny Hanspal Staple Scan

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

Sunny Hanspal Cis/trans isomers

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

CT3trans

CT3Cis

CT4

CT2Prod2







KIIIA Staple Scan

CT1

CT4

CT5

CT2

CT3

Sunny Hanspal

i – i + 4 staple – cis alkene required

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

Astrid Knuhtsen

Two isomers in HPLC!

FmocHNNH2Ac X K W A R D H X R

SPPS

•  NMR structure required for design


SS

S

µ-KIIIA i - i+7 Stapled Peptide

cis

Decoupled

Astrid Knuhtsen James Jones (Dstl) µ-KIIIA i - i+7 Stapled Peptide

trans

Decoupled

Astrid Knuhtsen James Jones (Dstl) µ-KIIIA i - i+7 Stapled Peptide

Circular Dichroism

200 220 240 260

-10000

0

10000

20000

CisTrans

nm

θ (d

eg* c

m2 * d

mol

-1)

NH2Ac X K W A R D H X R


H

H

HH helicity (222 nm)

25%

40%

(hNaV1.4 ion channel)

µ-KIIIA i - i+7 Stapled Peptide Astrid Knuhtsen

-7 -6 -5 -40

25

50

75

100

µ-KIIIA mimetics

[Mimetic] M

Cha

nnel

act

ivity

(% o

f con

trol

)

Circular Dichroism

200 220 240 260

-10000

0

10000

20000

CisTrans

nm

θ (d

eg* c

m2 * d

mol

-1)



H

H

HH 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 -40

25

50

75

100

µ-KIIIA mimetics

[Mimetic] M

Cha

nnel

act

ivity

(% o

f con

trol

)

•  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

Application of NMR in the Design of Peptide Tools for Chemical Biology and Drug Discovery ·...

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