Fluorine in Peptide and Protein Engineering

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1 Fluorine in Peptide and Protein Engineering Rita Fernandes Porto, February 11 th 2016 Supervisor: Prof. Dr. Beate Koksch 1

2 Fluorine a unique element for molecule design The most abundant halogen in earth s crust Relatively low abundance (only ~10 biogenically produced organofluorine compounds) Currently 30% of the top thirty drugs by sales contain a fluorine atom O Hagan, D.; J. Fluor. Chem. 2010, 131, O Hagan, D.; B. Harper, D.; J. Fluor. Chem. 1999, 100,

3 Fluorine a unique element for molecule design Size High electronegativity H 3 C CH 3 Very low polarizability CHH 3 CFF 3 OC Strong inductive 21.6 effect 1.20 Å 3 Å Å Å Å 3 Bioisosteric substitutions Fluorination opens a universe of options for peptide and protein engineering 19 F NMR Metabolic stability Conformational stability Lipophilicity and polarity O Hagan, D.; B. Harper, D., J. Fluor. Chem. 1999, 100, ; O Hagan, D., J. Fluor. Chem. 2010, 131,

4 Incorporation of fluorine in peptides and proteins AG Koksch work Synthesis of fluorinated amino acids, peptides, and proteins; Peptide/protein interactions; Preferred binding partners for fluorinated amino acids by phage display; Conformation and folding kinetics of amyloid formation; Protease stability; Fluorine in biologically relevant systems. Salwiczek, M.; Nyakatura, E. K.; Gerling, U. I. M.; Ye, S.; Koksch, B.; Chem. Soc. Rev. 2012, 41, Gerling, U. I. M.; Salwiczek, M.; Cadicamo, C. D.; Erdbrink, H.; Czekelius, C.; Grage, S. L.; Wadhwani, P.; Ulrich, A. S.; Behrends, M.; Haufe, G.; Koksch, B.; Chem. Sci. 2014, 5, Asante, V.; Mortier, J.; Wolber, G.; Koksch, B.; Amino Acids 2014, 46, Ye, S.; Loll, B.; Berger, A. A.; Mülow, U.; Alings, C.; Wahl, M. C.; Koksch, B.; Chem. Sci. 2015, 6,

5 Hydrophobicity and α-helix propensity of fluorinated amino acids Gerling, U.I.M.; Salwiczek, M.; Cadicamo, C.D.; Erdbrink, H.; Grage, S.; Wadhwani, P.; Ulrich, A.; Behrens, M; Haufe, G.; Czekelius, C; Koksch, B., Chem. Sci. 2014, 5 (2),

6 PART I Influence of fluorine on the folding of amyloid forming peptides Gerling, U. I. M.; Salwiczek, M.; Cadicamo, C. D.; Erdbrink, H.; Czekelius, C.; Grage, S. L.; Wadhwani, P.; Ulrich, A. S.; Behrends, M.; Haufe, G.; Koksch, B.; Chem. Sci. 2014, 5,

7 Peptide design: structure of the amyloid forming model peptide K 21 V 14 K 7 d a S 24 S 17 S 10 VV 3 3 f f V 3 S 10 S 17 S 24 V 3 b E 6 V E 20 E 25 E 18 E 11 L V E L 4 15 L 19 K V 13 2 L 8 L 12 E 6 c g L 1 L 5 e b e K 2 K 9 a L 5 L 12 L 19 L 26 d L 1 L 8 L 15 K 23 K 16 K 9 g E 4 E 20 c K 7 V 13 L K 26 L E V E 14 K K E 25 a, d e, g b, c, f Hydrophobic interactions Electrostatic interactions Polar/ charged residues free for substitutions Fluorinated amino acids (positions 13 or 14) Residues which promote amyloid formation Residues involved in α-helical coiled coil formation 7

8 Peptide library 8

9 Influence of fluorine on the folding of amyloid forming peptides Single substitution at position 13 Single substitution at position 14 Fluorine content Rate of amyloid formation 9

10 PART II Impact of fluorination on protease stability of peptides Asante, V.; Mortier, J.; Wolber, G.; Koksch, B.; Amino Acids 2014, 46,

11 Fluorine in drug design Weak metabolic stability ENZYME Low bioavailalibity in vivo Development of new therapeutically active peptides Low biological potency Proteolytic stability Incorporation of non-natural amino acids with new functional groups Jäckel, C.; Salwiczek, M.; Koksch, B. Angew. Chem. Int. Ed. 2006,45, ; Asante, V.; Mortier, J.; Wolber, G.; Koksch, B., Amino Acids 2014, 46 (12),

12 Peptide library ENZYME S3 S2 S1 S1' S2' S3' Peptide P4 P3 P2 P1 P1' P2' P3' P4' P5' cleavage site P2 control Abz- Phe P2-TfeGly P2-DfeGly P2-Abu Abz- Abz- Abz- TfeGly DfeGly Abu Phe Phe Phe P1 P1'-TfeGly P1'-DfeGly Abz- Abz- Phe Phe TfeGly DfeGly P1'-Abu Abz- Phe Abu P2 P2'-TfeGly P2'-DfeGly Abz- Abz- Phe Phe TfeGly DfeGly P2'-Abu Abz- Phe Asante, V.; Mortier, J.; Wolber, G.; Koksch, B., Amino Acids 2014, 46 (12), Abu 12

13 Proteolytic Stability -Chymotrypsin: Pepsin: TfeGly DfeGly TfeGly DfeGly TfeGly P2 P 1 P 2 DfeGly TfeGly DfeGly TfeGly DfeGly TfeGly DfeGly Fluorination significantly affects the stability of peptides against enzymes but with different outcomes Fluorine substituents in the context of a peptide environment do not lead to a general increase in proteolytic stability Depends on: position of substitution, type of enzyme, degree of fluorination Asante, V.; Mortier, J.; Wolber, G.; Koksch, B., Amino Acids 2014, 46 (12),

14 PART III Impact of fluorination on activity of the serine protease inhibitor BPTI 14

15 BPTI (Basic (bovine) Pancreatic Trypsin Inhibitor) 58 amino acid sequence High thermal and chemical resistance β-hairpin (twisted anti-parallel sheet), two helical regions Three disulfide bonds Serine protease inhibitor family (trypsin, chymotrypsin, plasmin) 15 is crucial residue of interacting loop ENZYME BPTI-trypsin interaction Ascenzi, P.; Bocedi, A.; Bolognesi, M.; Spallarossa, A.; Coletta, M.; De Cristofaro, R.; Menegatti, E. Curr. Protein Pept. Sci. 2003, 4(3):231; Ye, S.; Loll, B.; Berger, A.A.; Mülow, U.; Alings, C.; Wahl, M.; Koksch, B., Chem.Sci. 2015, 6,

16 Impact of Fluorine on structure and stability Thermal denaturation values for BPTI variants Tm/GdmCl Tm/Urea 15(wt) Abu DfeGly TfeGly Minimal structural perturbation Increase of thermal stability Ye, S.; Loll, B.; Berger, A.A.; Mülow, U.; Alings, C.; Wahl, M.; Koksch, B., Chem.Sci. 2015, 6,

17 Inhibitory activity Binding disassociation constant (K d M) of BPTI species with β-trypsin BPTI β-trypsin 15(wt) 5.17 x Abu n.d. 15DfeGly 3.88 x TfeGly 5.20 x 10-7 n.d. = not obtained from curves Reduction of inhibition with the incorporation of Abu Restoration of the inhibition levels upon incorporation of fluorine Ye, S.; Loll, B.; Berger, A.A.; Mülow, U.; Alings, C.; Wahl, M.; Koksch, B., Chem.Sci. 2015, 6,

18 Protein crystallographic structure Vapor diffusion (sitting drop) method Protein complexes Resolution β-trypsin ENZYME BPTI (wild-type) 1.25 Å β-trypsin BPTI (15Abu) β-trypsin BPTI (15DfeGly) β-trypsin BPTI (15TfeGly) 1.37 Å 1.37 Å 1.30 Å In collaboration with Dr. Bernhard Loll, Prof. Dr. Markus Wahl, FU-Berlin Ye, S.; Loll, B.; Berger, A.A.; Mülow, U.; Alings, C.; Wahl, M.; Koksch, B., Chem.Sci. 2015, 6,

19 Distance analysis between side chain substituents of BPTI and the β-trypsin 15Abu 15DfeGly 15TfeGly FG1 and FG2 of DfeGly FAD and FAE of TfeGly Closer to the S1 contacts This trend appears to offer evidence of the type of fluorophilic environment Ye, S.; Loll, B.; Berger, A.A.; Mülow, U.; Alings, C.; Wahl, M.; Koksch, B., Chem.Sci. 2015, 6,

20 Distance analysis between side chain substituents of BPTI and the structural waters ENZYME 15Abu 15DfeGly 15TfeGly Distance to water C 3.6 Å in Abu, 3.7 Å in DfeGly 3.4 Å in TfeGly Favorable interaction, a weak OH... FC H- bond, exists between the TfeGly side chain (FAC atom) and water C. Ye, S.; Loll, B.; Berger, A.A.; Mülow, U.; Alings, C.; Wahl, M.; Koksch, B., Chem.Sci. 2015, 6,

21 Water mediated H-Bond network β-trypsin-bpti complexes B-factor (Å) Wild-type 15Abu 15DfeGly 15TfeGly ENZYME Average B-factor of protein complex All water molecules on average BPTI Pro13-Arg17 (interacting loop) All atoms Main chain Side Chain BPTI Xaa15 All atoms Main chain Side Chain Structural water molecules in S 1 pocket A/A -/ /- 17.5/- 18.4/- B C D/D -/ /- 17.8/- 17.9/- E/E -/ /- 19.1/- 19.4/- More tightly hold waters B and C in the fluorinated variants than does the hydrocarbon parent side chain of Abu B-factor (Debye-Walle factor) indicates the flexibility of atoms in crystal structure Ye, S.; Loll, B.; Berger, A.A.; Mülow, U.; Alings, C.; Wahl, M.; Koksch, B., Chem.Sci. 2015, 6,

22 Acknowledgements Prof. Dr. Beate Koksch AG Koksch group members 22

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24 Hydrophobicity of fluorinated amino acids Non-fluorinated amino acids: Aliphatic side chain hydrophobicity Retention time side chain volume Fluorinated amino acids: x No correlation between side chains volume and retention time Single fluor substitution on Abu hydrophobocity Ac-Tyr-Gly-Gly Xaa NH 2 Why? More than 1 fluor substitution on Abu hydrophobocity the substitution of hydrogen by fluorine increases the solvent accessible surface area and leads to an increase in hydration energy C F bond is more polarized than the C H bond, and thus, electrostatic interactions of the fluorinated group with the solvent are energetically more favored 24

25 BPTI: protein crystallographic structure 15(wt) Superimposition of all P1 side chains All four β-trypsin-bpti complexes are highly similar in conformation Good superimposition except 15 side chain and number of water molecules within the S1 binding site Shorter side chains in 15Abu, 15DfeGly and 15TfeGly more two waters in the binding site (B and C) Waters A, B, C and D form a hydratation shell around the side chains of the unnatural amino acids 25

26 BPTI: protein crystallographic structure 15Abu 15DfeGly 15TfeGly Why water C is closer to TfeGly? 1. The closer 3EG FAC -water C distance is a consequence of the slight shift in conformation within the side chain that occurs due to the electrostatic repulsion between 3EG FAC and 3EG N (3.0 Å apart). 2. Greater van der Waals radius of fluorine compared to hydrogen; however no such phenomenon is seen between water molecule C and atoms OBF FG2 (3.1 Å) or 3EG FAE (3.2 Å), compared to ABA HG2 (3.1 Å) 3. Favorable interaction, a weak OH... FC H-bond, exists between the TfeGly side chain and water C 26

27 BPTI: protein crystallographic structure 27