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Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides.

Cromm PM, Schaubach S, Spiegel J, Fürstner A, Grossmann TN, Waldmann H - Nat Commun (2016)

Bottom Line: The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand.This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far.The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.

ABSTRACT
Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

No MeSH data available.


Sequence and binding studies of peptide 25.(a) Sequence of bicyclic peptide 25 showing highest affinity for Rab8a6–176(GppNHp). (b) Competition of fluorescein-labelled peptide 25 (60 nM) bound to Rab8a(GppNHp; 15 μM) with increasing concentrations of acetylated peptides StRIP3 and wt-R6IP (competitors). Errors represent 1σ of triplicates.
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f5: Sequence and binding studies of peptide 25.(a) Sequence of bicyclic peptide 25 showing highest affinity for Rab8a6–176(GppNHp). (b) Competition of fluorescein-labelled peptide 25 (60 nM) bound to Rab8a(GppNHp; 15 μM) with increasing concentrations of acetylated peptides StRIP3 and wt-R6IP (competitors). Errors represent 1σ of triplicates.

Mentions: In addition, dibrominated (entry 6–8) and bicyclic peptides (entry 9–14) were synthesized resulting in a total of 12 StRIP3 derivatives grouped into four subfamilies (Table 1, Supplementary Table 3): (i) alkyne mono-macrocyclic peptides 18–20 (entry 3–5); (ii) dibrominated olefin macrocyclic peptides 21–23 (entry 6–8); (iii) orthogonally macrocyclized peptides 24–26 carrying the original olefin crosslink and an additional alkyne crosslink at the C terminus (entry 9–11); and (iv) bicyclic peptides 27–29 with exchanged positions for the alkyne and the olefin crosslink (entry 12–14). Since the N-terminal part of parent peptide StRIP3 is already constrained by the olefin macrocycle, we aimed for the introduction of a new macrocycle in the C-terminal part. We reasoned that additional constraint could further stabilize the bioactive peptide conformation. Owing to a lack of structural information, it is not obvious which amino acids are directly involved in Rab-binding. For this reason, we selected two amino acids with hydrophobic side chains (L911 and A915) for macrocycle introduction as their non-polar side chains are potentially mimicked by the hydrocarbon macrocycle. All the peptides were synthesized via SPPS and modified with an N-terminal fluorescein–polyethyleneglycol label (Supplementary Table 3) to enable determination of their binding affinity towards activated Rab8a6-176(GppNHp) in a fluorescence polarization (FP) assay (Supplementary Fig. 13). After initial ranking of the peptides by means of relative Kd values (rel. Kd, Table 1, Supplementary Table 4), the affinity of the best binders (peptide 21, 25 and 28) was determined in an independent FP assay run in triplicates (Table 1, Supplementary Fig. 14). Replacement of the olefin by an alkyne crosslink yields peptides 18–20 with affinities comparable to StRIP3. In contrast, the dibrominated olefin derivatives 21–23 show improved affinity towards Rab8a6–176 with peptide 21 being the most potent binder within this subfamily (Kd=10.7 μM, Table 1). Peptide 21 shows a 2-fold increased binding affinity when compared with StRIP3. Notably, an even higher improvement in binding affinity to Rab8a6–176(GppNHp) is observed for two of the bicyclic peptides 24–29, namely peptide 25 and 28 (Table 1 and Supplementary Table 4). In both the cases, the nine-carbon alkyne crosslink (with 1 at N-terminal and 2 at C-terminal position within the sequence) provides the most potent architecture resulting in two significantly improved ligands for activated Rab8a6–176 (Kd[25]=6.6 μM; Kd[28]=9.6 μM). Bicyclic peptide 25 (Fig. 5a) is more than three times more potent than the parent hydrocarbon stapled peptide StRIP3 and displays a more than 15-fold increased binding affinity compared with the unmodified wild-type peptide wt-R6IP. Binding affinity of peptide 25 was confirmed in microscale thermophoresis measurements. On the basis of fluorescence intensity, an affinity for Rab8a6–176(GppNHp) was observed (Kd[25]=11 μM, Supplementary Table 5, Supplementary Figs 15 and 16), which is in the range of our FP measurements (Kd[25]=6.6 μM, see above). In addition, FP competition experiments were performed using a complex between labelled peptide 25 and Rab8a6–176(GppNHp), which was treated with an excess of acetylated StRIP3. In this setup, we observed full displacement of peptide 25 (IC50=33 μM, red Fig. 5b). As one would expect, the acetylated low-affinity peptide wt-R6IP does not compete with peptide 25 (black, Fig. 5b). These results verify reversible binding of peptide 25 to the same site on Rab8 as parent peptide StRIP3.


Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides.

Cromm PM, Schaubach S, Spiegel J, Fürstner A, Grossmann TN, Waldmann H - Nat Commun (2016)

Sequence and binding studies of peptide 25.(a) Sequence of bicyclic peptide 25 showing highest affinity for Rab8a6–176(GppNHp). (b) Competition of fluorescein-labelled peptide 25 (60 nM) bound to Rab8a(GppNHp; 15 μM) with increasing concentrations of acetylated peptides StRIP3 and wt-R6IP (competitors). Errors represent 1σ of triplicates.
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Related In: Results  -  Collection

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f5: Sequence and binding studies of peptide 25.(a) Sequence of bicyclic peptide 25 showing highest affinity for Rab8a6–176(GppNHp). (b) Competition of fluorescein-labelled peptide 25 (60 nM) bound to Rab8a(GppNHp; 15 μM) with increasing concentrations of acetylated peptides StRIP3 and wt-R6IP (competitors). Errors represent 1σ of triplicates.
Mentions: In addition, dibrominated (entry 6–8) and bicyclic peptides (entry 9–14) were synthesized resulting in a total of 12 StRIP3 derivatives grouped into four subfamilies (Table 1, Supplementary Table 3): (i) alkyne mono-macrocyclic peptides 18–20 (entry 3–5); (ii) dibrominated olefin macrocyclic peptides 21–23 (entry 6–8); (iii) orthogonally macrocyclized peptides 24–26 carrying the original olefin crosslink and an additional alkyne crosslink at the C terminus (entry 9–11); and (iv) bicyclic peptides 27–29 with exchanged positions for the alkyne and the olefin crosslink (entry 12–14). Since the N-terminal part of parent peptide StRIP3 is already constrained by the olefin macrocycle, we aimed for the introduction of a new macrocycle in the C-terminal part. We reasoned that additional constraint could further stabilize the bioactive peptide conformation. Owing to a lack of structural information, it is not obvious which amino acids are directly involved in Rab-binding. For this reason, we selected two amino acids with hydrophobic side chains (L911 and A915) for macrocycle introduction as their non-polar side chains are potentially mimicked by the hydrocarbon macrocycle. All the peptides were synthesized via SPPS and modified with an N-terminal fluorescein–polyethyleneglycol label (Supplementary Table 3) to enable determination of their binding affinity towards activated Rab8a6-176(GppNHp) in a fluorescence polarization (FP) assay (Supplementary Fig. 13). After initial ranking of the peptides by means of relative Kd values (rel. Kd, Table 1, Supplementary Table 4), the affinity of the best binders (peptide 21, 25 and 28) was determined in an independent FP assay run in triplicates (Table 1, Supplementary Fig. 14). Replacement of the olefin by an alkyne crosslink yields peptides 18–20 with affinities comparable to StRIP3. In contrast, the dibrominated olefin derivatives 21–23 show improved affinity towards Rab8a6–176 with peptide 21 being the most potent binder within this subfamily (Kd=10.7 μM, Table 1). Peptide 21 shows a 2-fold increased binding affinity when compared with StRIP3. Notably, an even higher improvement in binding affinity to Rab8a6–176(GppNHp) is observed for two of the bicyclic peptides 24–29, namely peptide 25 and 28 (Table 1 and Supplementary Table 4). In both the cases, the nine-carbon alkyne crosslink (with 1 at N-terminal and 2 at C-terminal position within the sequence) provides the most potent architecture resulting in two significantly improved ligands for activated Rab8a6–176 (Kd[25]=6.6 μM; Kd[28]=9.6 μM). Bicyclic peptide 25 (Fig. 5a) is more than three times more potent than the parent hydrocarbon stapled peptide StRIP3 and displays a more than 15-fold increased binding affinity compared with the unmodified wild-type peptide wt-R6IP. Binding affinity of peptide 25 was confirmed in microscale thermophoresis measurements. On the basis of fluorescence intensity, an affinity for Rab8a6–176(GppNHp) was observed (Kd[25]=11 μM, Supplementary Table 5, Supplementary Figs 15 and 16), which is in the range of our FP measurements (Kd[25]=6.6 μM, see above). In addition, FP competition experiments were performed using a complex between labelled peptide 25 and Rab8a6–176(GppNHp), which was treated with an excess of acetylated StRIP3. In this setup, we observed full displacement of peptide 25 (IC50=33 μM, red Fig. 5b). As one would expect, the acetylated low-affinity peptide wt-R6IP does not compete with peptide 25 (black, Fig. 5b). These results verify reversible binding of peptide 25 to the same site on Rab8 as parent peptide StRIP3.

Bottom Line: The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand.This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far.The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.

ABSTRACT
Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

No MeSH data available.