Final Report Summary - CYPEPUTICS (Tricyclic Peptides for the Development of Therapeutics)
Summary of the research objectives
Peptide macrocycles can bind with high affinity and selectivity to protein targets and are an attractive class of molecules for the development of therapeutics. Recently, a phage display-based strategy was developed that allowed to generate potent bicyclic peptide antagonists (Heinis, C., et al., Nat. Chem. Biol., 2009). While bicyclic peptides with nanomolar affinities to a range of protein targets could be generated, it was more difficult to obtain high-affinity binders to some proteins, particularly to those having flat surfaces and no clefts or cavities. In this project, we propose to develop rigid, tricyclic peptide scaffolds that should, due to a defined three-dimensional structure, bind to flat surfaces similar to antibodies. The format envisioned for the synthesis of tricyclic peptide scaffolds consist of a cyclic hexa-peptide which is anchored to a linear peptide consisting three cysteine residues. Phage-encoded combinatorial libraries of these peptide folds were generated and subjected to affinity selections. Tricyclic peptide binding to a variety of biological targets e.g. urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF) were identified.
Description of work performed
Mimicking the binding site of antibodies by using small synthetic molecules has been a longstanding challenge. In this work, we report a novel antibody-mimicking structure that is based on a synthetic cyclic hexa-peptide scaffold to which two peptide loops are anchored. The synthetic cyclic hexa-peptide scaffold is made of 6 amino acids (consisting of 3 D and 3 L-amino acids). The latter peptidic loops are supposed to mimic the complementarity determining regions (CDRs) of antibodies. The structure was assembled by linking a linear peptide of the format CXXXXCXXXXC (C cysteine, X random amino acid) to a cyclic hexa-peptide functionalized with three thiol-reactive groups. We generated and screened large combinatorial libraries of such antibody mimetics following a phage display based approach developed in the lab. In brief, random peptides of the format CXXXXCXXXXC were displayed on phage and chemically linked to the cyclic hexa-peptide. Binders against urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF) were isolated by phage selection and identified by DNA sequencing. The identified peptides showed strong consensus sequences for uPA while for VEGF diverse peptide sequences were isolated. The isolated peptides were then chemically synthesized using SPPS. The synthesized peptides were then tested against the target (uPa or VEGF) using protease inhibition assay or ELISA.
Main results achieved so far
The major challenge of the project was to establish the chemistry to develop cyclic hexa-peptide scaffold. A multi-step synthesis strategy was developed and optimized to give the hexa-peptide scaffolds in large amount. The reaction between the cyclic hexa-peptide and model peptide with 3 cysteine was optimized at pH 8 to give a multi- cyclic peptide architecture. The reaction was further transformed to produce multi cyclic peptide libraries on phage. As proof of concept these multi cyclic peptide libraries were then screened against 2 therapeutically important targets: uPA and VEGF. Good binders could be isolated against both targets. The identified multi-cyclic peptide against uPA showed low to high µM inhibition. The tricyclic peptides isolated against VEGF bound to the target in a phage ELISA assay. This clearly shows that tricyclic peptides developed using the novel hexa-cyclic scaffold is a valuable format for the development of ligands.
Expected final results and its potential impact
The project will be continued in the lab where the activity of the identified peptides will be further optimized by affinity maturation and rational design. In addition the chemistry developed to synthesize cyclic hexa-peptide scaffolds will also be used by co-workers to further develop multi-cyclic peptides against various antibody targets like HER2, EGFR. An array of hexa-cyclic scaffold in a combinatorial manner can also be synthesized to generate libraries of different multi cyclic peptides.
The long term socio economic aim of the project is to develop synthetic antibody mimetic for therapeutic application. Because of the small size, the antibody mimetics will better penetrate into tissues to reach targets. Furthermore, the synthetic antibody mimetics are easier to produce than antibodies. Due to its synthetic background the antibody mimetic solution will be more homogenous and its quality control and production will be much cheaper and faster as compared to antibodies. The synthetic antibody mimetics will be much cheaper than available antibody therapies and will lead to reduction of medicine cost and ultimately will impact the effective cost of therapy to the patient and reduce the burden of the disease to the country.
Peptide macrocycles can bind with high affinity and selectivity to protein targets and are an attractive class of molecules for the development of therapeutics. Recently, a phage display-based strategy was developed that allowed to generate potent bicyclic peptide antagonists (Heinis, C., et al., Nat. Chem. Biol., 2009). While bicyclic peptides with nanomolar affinities to a range of protein targets could be generated, it was more difficult to obtain high-affinity binders to some proteins, particularly to those having flat surfaces and no clefts or cavities. In this project, we propose to develop rigid, tricyclic peptide scaffolds that should, due to a defined three-dimensional structure, bind to flat surfaces similar to antibodies. The format envisioned for the synthesis of tricyclic peptide scaffolds consist of a cyclic hexa-peptide which is anchored to a linear peptide consisting three cysteine residues. Phage-encoded combinatorial libraries of these peptide folds were generated and subjected to affinity selections. Tricyclic peptide binding to a variety of biological targets e.g. urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF) were identified.
Description of work performed
Mimicking the binding site of antibodies by using small synthetic molecules has been a longstanding challenge. In this work, we report a novel antibody-mimicking structure that is based on a synthetic cyclic hexa-peptide scaffold to which two peptide loops are anchored. The synthetic cyclic hexa-peptide scaffold is made of 6 amino acids (consisting of 3 D and 3 L-amino acids). The latter peptidic loops are supposed to mimic the complementarity determining regions (CDRs) of antibodies. The structure was assembled by linking a linear peptide of the format CXXXXCXXXXC (C cysteine, X random amino acid) to a cyclic hexa-peptide functionalized with three thiol-reactive groups. We generated and screened large combinatorial libraries of such antibody mimetics following a phage display based approach developed in the lab. In brief, random peptides of the format CXXXXCXXXXC were displayed on phage and chemically linked to the cyclic hexa-peptide. Binders against urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF) were isolated by phage selection and identified by DNA sequencing. The identified peptides showed strong consensus sequences for uPA while for VEGF diverse peptide sequences were isolated. The isolated peptides were then chemically synthesized using SPPS. The synthesized peptides were then tested against the target (uPa or VEGF) using protease inhibition assay or ELISA.
Main results achieved so far
The major challenge of the project was to establish the chemistry to develop cyclic hexa-peptide scaffold. A multi-step synthesis strategy was developed and optimized to give the hexa-peptide scaffolds in large amount. The reaction between the cyclic hexa-peptide and model peptide with 3 cysteine was optimized at pH 8 to give a multi- cyclic peptide architecture. The reaction was further transformed to produce multi cyclic peptide libraries on phage. As proof of concept these multi cyclic peptide libraries were then screened against 2 therapeutically important targets: uPA and VEGF. Good binders could be isolated against both targets. The identified multi-cyclic peptide against uPA showed low to high µM inhibition. The tricyclic peptides isolated against VEGF bound to the target in a phage ELISA assay. This clearly shows that tricyclic peptides developed using the novel hexa-cyclic scaffold is a valuable format for the development of ligands.
Expected final results and its potential impact
The project will be continued in the lab where the activity of the identified peptides will be further optimized by affinity maturation and rational design. In addition the chemistry developed to synthesize cyclic hexa-peptide scaffolds will also be used by co-workers to further develop multi-cyclic peptides against various antibody targets like HER2, EGFR. An array of hexa-cyclic scaffold in a combinatorial manner can also be synthesized to generate libraries of different multi cyclic peptides.
The long term socio economic aim of the project is to develop synthetic antibody mimetic for therapeutic application. Because of the small size, the antibody mimetics will better penetrate into tissues to reach targets. Furthermore, the synthetic antibody mimetics are easier to produce than antibodies. Due to its synthetic background the antibody mimetic solution will be more homogenous and its quality control and production will be much cheaper and faster as compared to antibodies. The synthetic antibody mimetics will be much cheaper than available antibody therapies and will lead to reduction of medicine cost and ultimately will impact the effective cost of therapy to the patient and reduce the burden of the disease to the country.
