Synthesis and direct assay of large macrocycle diversities by combinatorial late-stage modification at picomole scale

Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology. In a proof-of-concept, we generate a target-tailored library of 19,968 macrocycles by conjugating 104 carboxylic-acid fragments to 192 macrocyclic scaffolds. The high reaction efficiency and small number of side products of the acylation reactions allowed direct assay without purification and thus a large throughput. In screens, we identify nanomolar inhibitors against thrombin (Ki = 44 ± 1 nM) and the MDM2:p53 protein-protein interaction (Kd MDM2 = 43 ± 18 nM). The increased efficiency of macrocycle synthesis and screening and general applicability of this approach unlocks possibilities for generating leads against any protein target

From phage display and venoms to protease-resistant peptides: Design of BBB-shuttles and peptides targeting EGF

[eng] Peptides play a critical role in human physiology and harbour a huge potential as therapeutic agents. In this thesis, new peptides have been discovered as ligands for the epidermal growth factor (EGF) and as new BBB-shuttles, using phage display and chemical synthesis of peptides and proteins. EGF is overexpressed in several cancers, inducing the proliferation and survival of these cells. By inhibiting EGF, we will prevent the activation of the receptor and, consequently, its negative effects. Moreover, phage display is a powerful tool to identify peptide ligands. However, only L-peptides can be displayed, which are protease unstable. Hence, mirror image phage display was developed to identify D-peptides, more stable in front of proteases. In this methodology, the selection is carried out against the mirror image of the original target and, after the panning selection and the synthesis of the enantiomer of the ligand, D-peptides are obtained. In this regard, the enantiomer of EGF was synthesised using a combination of solid-phase peptide synthesis and native chemical ligation. After the panning of two phage display peptide libraries against the immobilised protein, nine sequences were selected and synthesised with D-amino acids. Three of these peptides have high affinities for EGF and are stable in serum proteases for more than 24h. Most potential drugs for the treatment of central nervous system disorders (such as brain cancer and Alzheimer’s disease) do not cross the blood-brain barrier (BBB). Much effort has been devoted to the discovery of BBB-shuttle peptides – entities that have the capacity to carry cargoes across the BBB. The sources of BBB-shuttle peptides are diverse, ranging from the mimicry of endogenous proteins to the use of phage display. Phage display has been applied against a human BBB cellular model which consists in the co-culture of human brain capillary endothelial cells and bovine pericytes. From the screening of a phage display library containing random 12-amino acid sequences, SGVYKVAYDWQH (SGV) was selected. Validation studies were performed confirming that SGV is able to increase the uptake of a model protein in endothelial cells. When a BBB-shuttle is conjugated to a cargo, the ratio BBB-shuttle:cargo can range from 1:1 to 400:1, depending on the cargo. However, there are cases where the modification of the cargo with one copy of the BBB-shuttle is not sufficient to promote its passage through the BBB. More copies can be introduced, but a mixture of ratios of BBB-shuttle:cargo conjugates may be obtained. In those cases, it may be interesting to use multivalent BBB-shuttles, where more than one copy of the shuttle is attached to a core, which is linked to the cargo at one position. In this regard, branched dimerisation of a known BBB-shuttle was explored to enhance BBB permeability of model proteins. The results obtained with THRre peptide as the BBB-shuttle suggest that the mild improvement in permeability may not compensate the increased synthetic effort that these constructs represent. Moreover, chlorotoxin (CTX), a disulphide-rich peptide found in the venom of a scorpion, is reported to be able to enter the brain and bind specifically to tumour tissue. However, CTX is a relatively large peptide (36 amino acids) to be used as a BBB-shuttle, where we may need to scale it up or modify it. In this regard, we designed minimised versions of CTX (MiniCTXs) and evaluated their BBB properties, as well as their toxicity and stability. The results from these experiments produced two peptides as good BBB-shuttles, with similar stability, cellular uptake and BBB permeability than the best BBB-shuttles.[cat] En aquesta tesi doctoral, s’han descobert nous pèptids com a lligands del factor de creixement endotelial (EGF) i com a noves llançadores peptídiques, usant les tècniques de phage display i la síntesi química de pèptids. EGF es troba sobre-expressat en diversos càncers i, mitjançant la seva inhibició, la proliferació d’aquestes cèl·lules es podria reduir. Amb phage display es poden trobar aquests lligands, però els fags només poden expressar pèptids L, els quals són degradats per proteases. En el mirror image phage display, la selecció es duu a terme contra la imatge especular de la diana original i, després de la selecció i la síntesi dels enantiòmers dels millors candidats, s’obtenen pèptids D com a lligands. En aquest context, l’enantiòmer de EGF s’ha sintetitzat usant una combinació de síntesi en fase sòlida i lligació química nativa. Després del procés de selecció amb dos biblioteques de fags contra la proteïna immobilitzada, nou seqüencies s’han seleccionat i sintetitzat amb aminoàcids D. Tres d’aquests pèptids presenten altes estabilitats i afinitats per EGF. La majoria de fàrmacs pel tractament de malalties del sistema nerviós central no creuen la barrera hematoencefàlica. Una alternativa és l’ús de llançadores, que tenen la capacitat de transportar fàrmacs al cervell. La metodologia de phage display s’ha aplicat en un model humà de la barrera hematoencefàlica i, després del cribratge, la seqüència SGVYKVAYDWQH (SGV) s’ha seleccionat. Estudis de validació s’han dut a terme confirmant que SGV és capaç d’incrementar el transport d’una proteïna en cèl·lules endotelials. Hi ha casos en que la modificació del fàrmac amb una sola llançadora no és suficient per creuar la barrera hematoencefàlica. En aquest sentit, s’ha explorat la dimerització d’una llançadora (THRre) per incrementar el transport de proteïnes. Els resultats obtinguts suggereixen un moderat increment en el transport, el qual pot no compensar l’esforç sintètic requerit. La clorotoxina (CTX), pèptid trobat en el verí d’un escorpí, i versions reduïdes d’aquesta (MiniCTXs), s’han estudiat com a nova font de llançadores. Les propietats d’aquests pèptids de creuar la barrera hematoencefàlica han estat avaluades. Al final d’aquest procés, dos MiniCTXs han estat seleccionades, amb uns valors de transport semblants a les millors llançadores

Chemically Enhanced Peptide and Protein Therapeutics

Proteins and peptides are on the rise as therapeutic agents and represent a higher percentage of approved drugs each year: 24% in 2021 vs [...

Generation of a Large Peptide Phage Display Library by Self-Ligation of Whole-Plasmid PCR Product

The success of phage display, used for developing target-specific binders based on peptides and proteins, depends on the size and diversity of the library screened, but generating large libraries of phage-encoded polypeptides remains challenging. New peptide phage display libraries developed in recent years rarely contained more than 1 billion clones, which appears to have become the upper size limit for libraries generated with reasonable effort. Here, we established a strategy based on whole-plasmid PCR and self-ligation to clone a library with more than 2 × 1010 members. The enormous library size could be obtained through amplifying the entire vector DNA by PCR, which omitted the step of vector isolation from bacterial cells, and through appending DNA coding for the peptide library via a PCR primer, which enabled efficient DNA circularization by end-ligation to facilitate the difficult step of vector-insertion of DNA fragments. Panning the peptide repertoires against a target yielded high-affinity ligands and validated the quality of the library and thus the new library cloning strategy. This simple and efficient strategy places larger libraries within reach for nonspecialist researchers to hopefully expand the possible targets of phage display applications

BrainBike peptidomimetic enables efficient transport of proteins across brain endothelium

International audienceProtein therapeutics cannot reach the brain in sufficient amounts because of their low permeability across the blood-brain barrier. Here we report a new family of bicyclic peptide shuttles, BrainBikes, capable of increasing transport of proteins, including antibody derivatives, in a human cell-based model of the blood-brain barrier