Seamless Integration of Dose-Response Screening and Flow Chemistry: Efficient Generation of Structure-Activity Relationship Data of β-Secretase (BACE1) Inhibitors

Drug discovery is a multifaceted endeavor encompassing as its core element the generation of structure-activity relationship (SAR) data by repeated chemical synthesis and biological testing of tailored molecules. Herein, we report on the development of a flow-based biochemical assay and its seamless integration into a fully automated system comprising flow chemical synthesis, purification and in-line quantification of compound concentration. This novel synthesis-screening platform enables to obtain SAR data on b-secretase (BACE1) inhibitors at an unprecedented cycle time of only 1 h instead of several days. Full integration and automation of industrial processes have always led to productivity gains and cost reductions, and this work demonstrates how applying these concepts to SAR generation may lead to a more efficient drug discovery process.

"Switch peptides"

Lydiane Jeanine Saucède

Despite of considerable progress in the research at the interface of Chemistry, Biology and Medicine, neurodegenerative diseases affect more and more seriously our ageing population and remain a real therapeutic challenge for researcher. Nevertheless, advances made in this field allow us to better understand the molecular mechanisms responsible for these disorders and to stress the involvement and the key-role of conformational changes of some proteins. Thought to be at the origin of Alzheimer's disease, amyloid-β peptide undergoes structural modifications leading to β-sheet structures, which further aggregate into toxic fibrils and plaques. However, intrinsic properties of these structures result in problems such as insolubility and self-aggregation limiting their experimental access. Among the main research lines to find an efficient therapeutic agent to fight against Alzheimer's disease, fibrillar inhibition is considered as promising strategy. It requires the use of a molecule, called β-breaker, which would be able to block the misfolding triggered by conformational transitions. Consequently, the present thesis is focusing on the design and chemical synthesis of potential inhibitors of amyloid-β fibrillogenesis. As a specific common feature, the designed compounds integrate in their chemical structure a short sequence ("nucleation site") derived from the central part of Aβ(1-42), allowing to specifically interact with the pathogenic peptide. We first envisaged to design cyclic peptides as potential inhibitors containing proline residues, known for its destabilizing effect upon secondary structures (way A). In collaboration with C. Soto et al., Univ. Galveston, the biological activity of the prototype cyclo(Lys-Leu-Pro-Phe-Phe-Glu) was tested. Most notably, electron microscopy, in collaboration with J. Dubochet and M. Adrian, UNIL, Congo red and thioflavin T staining, and CD studies pointed to the potential of cyclic peptides as Aβ inhibitors. Subsequently, conjugate peptides, assembling the recognition sequence of amyloid-β and organic molecules with potential to block fibrillogenesis were synthesized specifically. (+/-)-Trans-4-cotininecarboxylic acid, 3-indolebutyric acid and a tripeptide β-strand mimic were chosen. The biological assessment of these molecules allowed us to highlight their potential in this therapeutic strategy as well as the advantage given by the intercalation of a proline between the two components of these conjugates. In an effort to introduce in the structure of β-breakers a dynamization element, comparable to the cis/trans isomerization of proline, we designed a new type of β-breaking molecules according to strategy B (Figure). The elaboration of this innovating concept termed "switch-peptides" allows to controll the function of a polypeptide by using intramolecular acyl migration as switch-element for the in situ induction of structure and function. By combining an conformational induction unit σ, a switch element S (cysteine or serine) and an amyloid recognition sequence, we obtained a new generation of dynamic β-breakers. At the Soff state, the σ part was linked to the switch element S via an ester bond, deactivating the structural influence of the conformational induction unit on the target peptide, resulting in the absence of biological activity. By removing the protecting group Y from the amino function of the switch element, intramolecular acyl migration was triggered, restoring the native amide bond, setting off the impact of σ (Son state). In applying pseudo-prolines as β-breaking σ-elements (resulting in a "kink" conformation), the corresponding switch-peptide can adopt a recognition state (Soff) and a β-sheet disrupting state (Son), triggered by controlled acyl migration. We explored the use of cysteine as switch element, with special attention to the chemoselective synthesis, kinetics of acyl migration and potential for β-breaking. Interestingly, the S→N acyl migration proceeded very fast at physiological pH, in comparison to serine derived O→N migrations. Of utmost importance for further extensions of the switch-concept, the design of non peptidic S-elements such as trifunctionalized aromatic compounds would allow us to generate the β-breaking element in situ. As a first step, the intramolecular acyl migration O→N via an intermediate of 5, 6, 7 or 9 membered rings, as well as the reversibility of this reaction, was successfully demonstrated. In addition, enzymatically cleavable protecting groups Y for triggering acyl migrations were established. In conclusion, the present thesis presents some promising concepts in the design of fibril disrupting compounds of considerable therapeutical potential.

EPFL2006

Peptide ligands stabilized by small molecules

Alessandro Angelini, Davide Bertoldo, Shiyu Chen, Christian Heinis, Florence Pojer

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle-free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.

Wiley-V C H Verlag Gmbh2014

Development of Stapling Methods by Selective Side Chain Modifications of Bioactive Peptides

Christopher Matthew Bandeli Kourra

Cyclic peptide therapeutics fill the gap between small molecules (5000 Da) as a separate class of drugs, combining the advantages of both in terms of high selectivity, bioavailability, synthetic accessibility and low toxicity. However, their conformational flexibility and proteolytic instability results in fast metabolism. Therefore, modification of their chemical and structural properties at the proteolytically vulnerable sites can circumvent these deficiencies. Several strategies exist to stabilise peptides through local or global constraints, side chain functionalisation or amide bond surrogates. This thesis describes the development and application of two chemical transformations to peptides with the aim of greatly enhancing their metabolic stability either via side chain to side chain cyclisation or disulfide modification. Following the functionalisation, a comparative evaluation of the pharmacological properties of the modified peptides was conducted with respect to the parent peptides. The first cyclisation strategy aimed to apply the redox-neutral chemistry developed in the burgeoning field of borrowing hydrogen technology to peptides, as a new class of tolerable substrates. These would contain the requisite amine and alcohol functional groups enabling their direct coupling to form the lactam or N-alkylated cycle. Numerous investigations into this transformation with a plethora of substrates and catalysts did not lead to the desired cyclisation. For the second approach, a simple one-pot procedure for the 'stapling' of disulfide bonds in native peptides was developed. Tris(2-carboxyethyl)phosphine mediated reduction of the disulfide bond was followed by concomitant insertion of a 'doubly electrophilic' carbon bridge to form a physiologically stable dithioacetal bond. The reaction was performed under mild, biocompatible reaction conditions, allowing for the facile conversion of native peptides into more stable analogues. The protocol was applicable to a range of bioactive peptides with a multitude of reactive functional groups demonstrating the high versatility of this approach. The importance and utility of the modified analogues were verified by testing their binding affinity and biological activity against the corresponding parent peptides. The impact of the disulfide modification on these properties for each peptide were analysed on a case-by-case basis. The SCS modified analogues of Oxytocin and Vasopressin maintained binding affinities in the same order of magnitude as their respective parent compounds across all corresponding receptors. Nevertheless, this was not the case for SCS-Octreotide and SCS-Somatostatin. Those peptides which maintained comparable binding affinities were then tested for their functional activity at the receptors of biological interest. SCS-Oxytocin retained its agonist potency displaying sub-nanomolar activity at the Oxytocin receptor, whilst SCS-Vasopressin exhibited sub micromolar functional response at the Vasopressin 1A receptor. Then the stability of the modified peptides in human serum was evaluated. In some cases, the enhancement in serum stability was vastly increased. In particular our modified SCS-Oxytocin analogue clearly demonstrated a significant improvement in serum half-life, as well as heat and pH stability over its native parent form. This disulfide stapling method could have wide-reaching application for the development of more stable therapeutic analogues.