Application of NMR in the Design of Peptide Tools for Chemical - PowerPoint PPT Presentation

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 β -Strand Mimetic O i + 1 i + 3 O O R O R O N O N H H H N N N N N N N N N H H O R 2 O R O R O R R 1 R 3 i i + 2 i + 4 BocHN O Chem. Comm., 2012 , 48 , 3709-3711. Stapled α -Helix Peptides Zinc Dependent Enzyme Inhibitors Aurora-A/TPX2 HDAC/DUB A" B" O ZBG HN C" His187" O O H AcHN N N NH 2 H R 1 R 2 O R 1 , R 2 = amino acid side chain ACS Chem. Bio ., 2016 , 11 , 3383-3390. Rep. Org Chem. , 2015 , 5 , 65 – 74. Nat. Commun. , 2016 , 7 , 11262 Org. Bio. Chem ., 2014 , 12 , 8775-8782.

Do Peptides Make Good Drugs? • 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)

Problems with Peptide Drugs • Limited orally bioavailability • Low membrane permeability (dissociation of water) H H O O R 2 H O H 3 N N N O R 1 H O R 3 H O H O H H • Approximately 75% of peptide drugs are administered intravenously • Short circulating plasma half-life - Proteases O O H 2 N N OH H

Peptidomimetic Design Br R Design synthetic High-throughput R N mimic of important O screen to identify Br side-chain residues small molecule inhibitors N R N OH N O O J. Am. Chem. Soc. , 2001, 123 , 5382 Science , 2004, 303 , 844. Designed Peptidomimetics for the disruption of protein-protein interactions • Binding affinity Conformationally constrain native peptide • Specificity • Protease resistant J. Am. Chem. Soc. , 1997, 119 , 455. • Cell permeable?

Stapled Helices Salt Bridge Lactam Disulfide O O NH O S NH S NH 3 O J. C. Phelan R. L. Baldwin P. G. Schultz J. Am. Chem. Soc. , 1997, 119 , 455 Biochemistry , 1993, 32 , 9668 J. Am. Chem. Soc. , 1991, 113 , 9391 Hydrophobic Metal ligation Hydrocarbon interactions O 2 N L H NO 2 M N O L O N NO 2 H O 2 N P. B. Hopkins R. H. Grubbs A. D. Hamilton J. Am. Chem. Soc. , 1990, 112 , 9403 Angew. Chem. Int. Ed. , 1998, 37 , 3281 Biochemistry , 1995, 34 , 984 M. R. Ghadiri J. Am. Chem. Soc. , 1990, 112 , 9633

All-Hydrocarbon Stapled Peptides S i, i+3 R (8) S i, i+4 S (8) R i, i+7 S (11) G.L. Verdine Y.-W. Kim & G.L. Verdine G.L. Verdine Org. Lett., 2010 , 12 , 3046 Bioorg. Med. Chem. Lett., JACS, 2000 , 122 , 5891 2009 , 19 , 2533 • Hydrocarbon length • Stereochemistry • α -methyl- α -AA Double staple Stitched staple L.D. Walensky G.L. Verdine Proc. Natl. Acad. Sci. USA, 2010 , JACS, 2014 , 136 , 12314 107 , 14093 N. S. Robertson, A. G. Jamieson, Rep. Org Chem. , 2015 , 5 , 65 - 74.

Conotoxin Proteomimetic Conus Kinoshitai • Conotoxins are a family mini-proteins • Isolated from marine cone snails • Predatory sea animal www.coneshell.net • Produces 100s of neurotoxic peptides • Conotoxin µ -KIIIA • Voltage-gated sodium channels, Na V 1.1-1.9 • Potential as analgesic • Knottin or cystine knot scaffold Chem. Rev. , 2014, 114 , 5815–5847.

µ -KIIIA Structure Determination • 15 possible foldamers of µ -KIIIA • Structural initially assigned as wrongly ( Biochemistry , 2009 , 48 , 1210–1219 ) 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 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) 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 Amide and aromatic region of NOESY spectra (blue) overlayed with TOCSY spectra (red) at 5 °C for µ-KIIIA (pH 4.8). 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 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 ( χ 1 ) dihedral angles. 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 20 final structures for µ -KIIIA 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. SH SH SH SH SHSH C C N C S S K W C R D H S R C C oxidation S S S S S S C C N C S S K W C R D H S R C C A. Van Der Haegen et al , FEBS J. , 2011 , 278 , 3408–3418.

Conotoxin Proteomimetic S S S S S S C C N C S S K W C R D H S R C C Ac S K W X R D H X R NH 2 µ -KIIIA mimetic µ -conotoxin KIIIA

Conotoxin Proteomimetic S S S S S S C C N C S S K W C R D H S R C C Ac S K W X R D H X R NH 2 µ -KIIIA mimetic • Simple synthesis • Easy purification µ -conotoxin KIIIA • α -helical

Conotoxin Proteomimetic Synthesis FmocHN 1) 20% piperidine/DMF FmocHN 2) Fmoc-AA-OH HCTU, DIEA Purification Rink Amide Resin DMF, MW Ac-Ser( t Bu)-Lys(Boc)-Trp(Boc)- X -Arg(Pbf)-Asp( t Bu)-His(Trt)- X -Arg(Pbf)-NH Grubb's 1 st Gen. Cat. 100% conversion DCM, 2 h Ac-Ser( t Bu)-Lys(Boc)-Trp(Boc)- X -Arg(Pbf)-Asp( t Bu)-His(Trt)- X -Arg(Pbf)-NH TFA/TIS/H 2 O, (95:2.5:2.5), 3 h >99% Purity Ac-Ser-Lys-Trp- X -Arg-Asp-His- X -Arg-NH 2 75% yield

Conotoxin Proteomimetic S S S S S S C C N C S S K W C R D H S R C C Ac S K W A R D H S R NH 2 Ac S K W X R D H X R NH 2

Conotoxin Proteomimetic S S S S S S C C N C S S K W C R D H S R C C Ac S K W A R D H S R NH 2 Ac S K W X R D H X R NH 2 • Simple synthesis • Easy purification • α -helical

Staple Scan Sunny Hanspal KIIIA Short Native Sequence Ac K W A R D H S R NH 2 CT1 KIIIA Staple Scan Ac X W A R X H S R NH 2 CT2 Ac K X A R D X S R NH 2 CT3 CT4 Ac K W X R D H X R NH 2 Ac K W A X D H S X NH 2 CT5

Staple Scan Sunny Hanspal KIIIA Short Native Sequence Ac K W A R D H S R NH 2 CT1 KIIIA Staple Scan Ac X W A R X H S R NH 2 CT2 Two isomers in HPLC? Ac K X A R D X S R NH 2 CT3 CT4 Ac K W X R D H X R NH 2 Ac K W A X D H S X NH 2 CT5

Cis/trans isomers Sunny Hanspal Tate. E et al, ACS Chem. Biol., 2014 , 9(10), 2204-2209

Conformational Analysis Sunny Hanspal Circular Dichroism 13800 KIIIA Short Native Sequence CT1 8800 CT5 Ac K W A R D H S R NH 2 CT1 Ellip&city θ 3800 CT3 trans Wavelength (nm) KIIIA Staple Scan CT3 Cis -1200 180 200 220 240 260 CT4 -6200 CT2 Prod 2 Ac X W A R X H S R NH 2 CT2 -11200 Peptide Helicity (%) Ac K X A R D X S R NH 2 CT3 Conotoxin 1 16 Conotoxin 2 35 CT4 Ac K W X R D H X R NH 2 Conotoxin 3-cis 43 Conotoxin 3-trans 22 Conotoxin 4 31 Ac K W A X D H S X NH 2 CT5 Conotoxin 5 18 i – i + 4 staple – cis alkene required

All-Hydrocarbon Stapled Peptides S i, i+3 R (8) S i, i+4 S (8) R i, i+7 S (11) G.L. Verdine Y.-W. Kim & G.L. Verdine G.L. Verdine Org. Lett., 2010 , 12 , 3046 Bioorg. Med. Chem. Lett., JACS, 2000 , 122 , 5891 2009 , 19 , 2533 • Binding affinity • Specificity • Protease resistant • Cell permeable? Double staple Stitched staple L.D. Walensky G.L. Verdine Proc. Natl. Acad. Sci. USA, 2010 , JACS, 2014 , 136 , 12314 107 , 14093 N. S. Robertson, A. G. Jamieson, Rep. Org Chem. , 2015 , 5 , 65 - 74.

Astrid Knuhtsen µ -KIIIA i - i +7 Stapled Peptide S S S S S S C C N C S S K W C R D H S R C C • NMR structure required for design SPPS FmocHN Ac X K W A R D H X R NH 2 Two isomers in HPLC!

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

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

Astrid Knuhtsen µ -KIIIA i - i +7 Stapled Peptide Circular Dichroism H helicity (222 nm) H 20000 θ (deg * cm 2 * dmol -1 ) 25% 10000 Ac X K W A R D H X R NH 2 H Cis 0 Trans H 200 220 240 260 40% nm -10000 Ac X K W A R D H X R NH 2 ( h Na V 1.4 ion channel) 100 Channel activity (% of control) 75 50 25 0 -7 -6 -5 -4 [Mimetic] M