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)
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.
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.
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.
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.
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.
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.
Conotoxin Proteomimetic
• 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 SC C N C S S K W C R D H S R C C
SS
S
Conotoxin Proteomimetic
SS SC C N C S S K W C R D H S R C C
SS
S
µ-conotoxin KIIIA
µ-KIIIA mimetic
NH2Ac S K W X R D H X R
• Simple synthesis • Easy purification • α-helical
Conotoxin Proteomimetic
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
NH2Ac S K W X R D H X R
NH2Ac S K W A R D H S R
SS SC C N C S S K W C R D H S R C C
SS
S
Conotoxin Proteomimetic
• Simple synthesis • Easy purification • α-helical
NH2Ac S K W X R D H X R
NH2Ac S K W A R D H S R
SS SC C N C S S K W C R D H S R C C
SS
S
Conotoxin Proteomimetic
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
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 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
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
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 SC C N C S S K W C R D H S R C C
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
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)
NH2Ac X K W A R D H X R
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
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