Design, synthesis and biophysical evaluation of peptides targeting pharmacologically relevant proteins

Abstract

Protein-protein interactions (PPIs) and protein surfaces are considered challenging targets for drug discovery. In this field, conventional medicinal chemistry (i.e. small molecules) has largely failed to provide effective hits. On the other hand, peptides are endowed with a higher degree of structural flexibility (which allows them to better adapt to irregular targets) and are able to display a variety of tailor-made topologies, emerging as an alternative to target proteins that were considered undruggable. In this thesis, we have explored the potential of designed peptides to modulate the function of two therapeutically relevant protein targets involved in cancer (epidermal growth factor, EGF) and cognitive disorders (prolyloligopeptidase, POP). Regarding the discovery of peptide ligands against EGF, docking tools have allowed the de novo design of a family of small peptides, which have shown a reproducible, albeit weak, binding to EGF. In order to obtain more active candidates, the relevant interacting regions of EGFR have been identified and mimicked with a diversity of cycle-constrained peptides. These are larger and structurally richer scaffolds that have proved more efficient in targeting a small and featureless protein, such as EGF. The best peptide hit, a 28-mer cyclic miniprotein (cp28), has served as the starting point in a computer-guided optimization process that strived for more active and structurally constrained analogues. Our design effort has resulted in a series of bicyclic peptides that mimic the mode of binding of cp28 to EGF, albeit with reduced size, increased hydrophilicity and a more restrained topology. The chemical synthesis of these complex bicyclic molecules was enabled by state-of-the-art native chemical ligation techniques. In order to assess the binding of our peptides with EGF, an array of suitable biophysical techniques was explored, and the most suitable ones were implemented to our discovery process. In particular, NMR spectroscopy (combined with expression of recombinant 15N-EGF) has allowed the monitoring of ligand-induced changes on the protein NMR spectra. In parallel, a recently developed acoustic biosensor (SAW) was set up as a low-cost, label-free, and highly sensitive technique to quantify the interactions with EGF. Moreover, our best peptide candidates (cp28 and cp23G) were able to disrupt the EGF-EGFR interaction, an effect that has been tested in several cell-like and living cell assays. Indeed, these peptides were able to halt the proliferation of EGFR(+) human carcinoma cells, an effect that underlines their biological efficacy. Moreover, the bicycle-constrained analogues display an exceptional level of biological stability, especially in serum and hepatic media.

In the last part of this thesis, a series of bioactive peptides has been designed with a fundamentally different mechanism of action. Peptides typically possess fast dissociation rates from the protein target, a feature that represents an obstacle when competing with endogenous ligands for the binding to cavities, such as catalytic sites in enzymes. As a proof of concept, a novel class of covalent-acting peptidomimetics were developed to supress the activity of POP, a protease involved in neurodegenerative disorders. In these bifunctional molecules, the peptide backbone and side chains formed a template that selectively binds to the POP active site, whereas a novel sulfonyl fluoride electrophile was optimally positioned to react with the catalytic Ser residue. These compounds showed a high potency in vitro, being able to inactivate POP at low nM concentrations, and their mechanism of action as irreversible inhibitors was confirmed by kinetic assays. Moreover, they displayed a remarkable selectivity against closely related proteases, and they were able to permeate through a lipid bilayer that mimics the composition of the blood- brain barrier. In summary, our findings show how two completely different classes of peptides, bicycle-constrained miniproteins and covalent-acting peptidomimetics, with binding affinities several orders of magnitude apart, can be efficiently designed to target specific protein surfaces. With PPIs and challenging binding sites becoming the focus of current drug discovery projects, these type of ligands are ideally positioned to deliver new drugs for the treatment of disease.

Les interaccions proteïna-proteïna representen un dels principals reptes pel desenvolupament de nous fàrmacs, donat el fracàs de la química terapèutica convencional per adreçar aquest tipus de dianes. En aquesta tesi, hem modelitzat (mitjançant tècniques de docking) una sèrie de pèptids petits capaços d’unir-se a EGF, una proteïna que juga un paper crític pel desenvolupament dels tumors. Per tal d’estudiar les interaccions dels lligands, s’han explorat diverses tècniques biofísiques de les quals la RMN i el biosensor SAW han sigut les més adients. Per tal d’aconseguir candidats més actius que els identificats per docking, s’han dissenyat pèptids cíclics i bicíclics que mimetitzen epítops d’EGFR rellevants pel reconeixement molecular d’EGF. Aquests pèptids, que incorporen aminoàcids no naturals, tenen una gran estabilitat biològica i han estat capaços de disminuir la proliferació de cèl·lules tumorals fins a un 80%. D’altra banda, per tal de modular l’activitat d’una proteasa (la prolil- oligopeptidasa, o POP) que està involucrada en trastorns neurològics, s’han dissenyat diferents peptidomimètics amb acció covalent. Aquests composts són capaços d’inactivar la POP a concentracions nanomolars, a la vegada que són selectius per aquest enzim. A més, aconsegueixen valors elevats de permeabilitat en un assaig de difusió passiva a través de lípids cerebrals, el que posa de manifest el seu potencial com a fàrmacs. Els nostres resultats mostren com diferents classes de pèptids, amb afinitats i mecanismes d’acció dispars, són capaços de modular l’activitat de proteïnes amb interès terapèutic, com ara l’EGF i la POP. Aquest tipus de molècules expandeix les fronteres de la química mèdica convencional per al descobriment de nous fàrmacs.