Methods for the generation of large combinatorial macrocycle libraries

Macrocycles are an attractive class of molecules due to their good binding properties and yet rather small size that allows, in many cases, crossing membranes to reach intracellular targets. In comparison to classical small molecule drugs, macrocycles are generally better at binding to challenging targets such as proteins having flat, featureless surfaces or protein-protein interactions. However, the development of macrocycle-based ligands has been challenging due to the lack of sufficiently large libraries of macrocyclic compounds for screening. The preparation of large numbers of macrocycles is limited due to the chromatographic purification that is typically required following low-yielding cyclization reactions. As a result, the full potential of macrocycles cannot be exploited in drug discovery efforts.The goal of my thesis was to develop new methods for the efficient generation of large macrocycle libraries. Towards this end, I envisioned to establish a new macrocycle library synthesis principle in which a large panel of m small cyclic peptides are combinatorially ligated in microwell plates with a large number of n chemical fragments, and the resulting m x n products screened without purification. For accessing large libraries, I planned to perform the reactions and screens at a nanomole scale and using contact-less liquid transfer.In a first project, I developed a method for obtaining readily pure macrocyclic peptides directly from solid phase. Peptide synthesis took place on a disulfide linker to solid phase, which allowed for side chain deprotection with acid without simultaneous peptide cleavage. When a thiol group was included at the N-terminus, treatment with base in DMSO resulted in an intramolecular disulfide exchange to release peptides cyclized by a disulfide bond. The method was tolerant of diverse peptide sequences, and released all tested compounds in high purity. With this method, I was able to synthesize cyclic peptides in 96-well plates, and thus hundreds of peptides in parallel with a moderate effort. I screened a test library and identified a weak inhibitor of thrombin (Ki = 13 ± 1 µM), which validated the method. In the second project, I intended to combinatorially diversify the above macrocyclic peptides with chemical fragments in nanoliter volumes, as described above. For chemical ligation, I chose to acylate amines built into the macrocyclic peptides with carboxylic acid building blocks. Following proof-of-concept experiments, I diversified a 384-member library with 10 carboxylic acids in nanoliter volumes to give 3,840 macrocyclic compounds, which I screened against thrombin. I identified a potent thrombin inhibitor (Ki = 44 ± 1 nM), and a co-crystal structure was obtained which confirmed binding to the active site of the protease. In a final application, I generated and screened a library of 19,968 macrocycles against MDM2, an important oncology target forming a protein-protein interaction with p53. The screen identified a high nanomolar binder (KD = 394 ± 41 nM) that I optimized in two rounds of iterative library synthesis and screening (KD = 31 ± 6 nM).The methods developed in the two projects are currently applied by our laboratory, and for the development of macrocycle-based ligands to diverse disease targets.

Synthesis of DNA‐encoded disulfide‐ and thioether‐cyclized peptides

Christian Heinis, Van Manh Pham

DNA-encoded chemical library technologies enable the screening of large combinatorial libraries of chemically and structurally diverse molecules, including short cyclic peptides. A challenge in the combinatorial synthesis of cyclic peptides is the final step, the cyclization of linear peptides that typically suffers from incomplete reactions and large variability between substrates. Several efficient peptide cyclization strategies rely on the modification of thiol groups, such as the formation of disulfide or thioether bonds between cysteines. In this work, we established a strategy and reaction conditions for the efficient chemical synthesis of cyclic peptide-DNA conjugates based on linking the side chains of cysteines. We tested two different thiol-protecting groups and found that tert-butylthio (S-tBu) works best for incorporating a pair of cysteines, and we show that the DNA-linked peptides can be efficiently cyclized through disulfide and thioether bond formation. In combination with established procedures for DNA encoding, the strategy for incorporation of cysteines may be readily applied for the generation and screening of disulfide- and thioether-cyclized peptide libraries.

2019

Development of Photocatalysts towards the Visible-Light driven C-Terminal Bioconjugation of Peptides and Proteins

Marion Marie-Agnès Garreau

If small molecules have led to tremendous progress in curing diseases, new therapeutic agents such as peptides or antibody-drugs conjugates have emerged as a highly promising next generation of pharmaceuticals. As for small molecules, their derivatization is often the key to custom activity or physicochemical properties. Peptide and protein modification methods are thus in important. This is however particularly challenging as it requires physiological conditions, as well as excellent chemoselectivities. Mild activation techniques are thus under special attention. For that purpose, photoredox catalysis stands as a mild strategy to generate radicals selectively. Visible-light is thus attractive for biomolecule functionalization and impressive reports have been disclosed in the last years, even if the field is still at an early stage. Another mild strategy towards protein functionalization is the use of hypervalent iodine reagents as an electrophilic alkyne source. This has been successful for biomolecule bioconjugation on several residues. Gratifyingly, those two concepts could be combined and a decarboxylative alkynylation of amino acids using photoredox catalysis and hypervalent iodine reagents was previously released. The goal of my PhD was to contribute to the field of biomolecules site-selective chemical modification by providing a novel labeling of carboxylic acids. To this end, it was envisioned to extend the existing photoredox-catalyzed decarboxylative alkynylation, from simple carboxylic acids to peptides, and later proteins. Among the diverse possible positions for protein bioconjugation, the C-terminal extremity is extremely attractive as it allows for a single-site selectivity. Available methodologies remain however scarce and are not general. Our investigations started with the development of novel fine-tuned organic dyes. A rational design allowed us to report organophotocatalysts filling existing gaps in redox properties. The following extensions of this concept and the use of this library to unlock novel reactivities demonstrated the impact of this contribution. Those organophotocatalysts were precious in the success of our peptide decarboxylative alkynylation. This reaction was found to proceed with an excellent regio and chemo-selectivity towards the C-terminal position. Native peptides up to hexamers were suitable substrates and the potential was confirmed by the introduction of bioorthogonal functional groups. Unfortunately, larger native peptides could not be modified under those conditions, leading us to design a novel strategy. We envisioned that a covalent linkage of an organophotocatalyst on proteins would allow an alkynylation at the C-terminus through a proximity-induced photoredox-catalyzed decarboxylation. For this unprecedented concept, 4CzIPN derivatives were thus designed to be introduced on a single-site on peptides. Ongoing work aims to assess the success of this approach. Finally, another direction explored during this thesis was a decarboxylative oxidation of peptides towards the formation of N,O-acetals. They were employed as a platform for derivatization. Through Friedel-crafts arylations, phenols and indoles could be successfully introduced. This strategy was further extended towards the cross-linking of peptides, providing a novel scaffold of unnatural tetramers of potential interest for drug discovery.

EPFL2020

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

Cyclic peptide ligands are a promising molecular format for the development of therapeutics. They combine some of the advantages of large protein therapeutics (high affinity and specificity) and of small molecule drugs (accessibility to chemical synthesis and low immunogenicity). With phage display, large combinatorial libraries of more than one billion mono- and polycyclic peptides can be developed and screened against virtually any target. During phage affinity selections, the physical link between phenotype (encoded peptide) and genotype (phage DNA) allows the identification of enriched peptide binders by sequencing of the phage vector DNA. Based on these concepts, our laboratory has established a robust chemistry in which phage displayed peptides containing two or three cysteines are cyclized or bi-cyclized with thiol-reactive linkers. This procedure has been generally applied for the isolation of potent and selective (bi)cyclic peptide ligands with therapeutic potential. The isolation of binders with optimal properties is highly dependent on the possibility to generate (bi)cyclic peptide libraries with high structural diversity and on the ability to thoroughly decode the phage selection output. In my PhD work, I developed new methods to generate large and structurally diverse libraries and to decode more efficiently phage selected peptides. In my first project, I explore the cyclization of peptides with two chemical bridges as a method to provide rapid access to phage-encoded libraries with high scaffold diversity. To this end, a range of twelve structurally diverse chemicals were tested for thiol-reactivity and phage compatibility. Subsequently, peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) were cyclized with six of these chemical linkers and panned against the protease plasma kallikrein. The selection yielded inhibitors with remarkable affinity (sub-nM KI), target selectivity (>1000-fold) and proteolytic stability (t1/2 in plasma > 3 days) despite a relatively small molecular mass (~ 1200 Da). In the second project, I developed phage-encoded libraries of small cyclic peptides in order to identify ligands that have the potential to be orally absorbed. A major challenge for the generation of such library is the usually low diversity of compounds due to the limited number of amino acids. In this work, we addressed this limitation by taking advantage of a recently discovered peptide cyclization reaction in which the thiol of a cysteine is ligated with the N-terminal amino group using a chemical reagent. We applied this strategies to two phage-encoded peptide libraries of four and five amino acids (XXXC and XXXXC) that were cyclized with eight chemical linkers yielding a collection of more than 1.3 million compounds. Panning the libraries against the two disease targets plasma kallikrein and coagulation factor XIa yielded ligands with single-digit micromolar affinity. In a third project, I developed a strategy to bypass the bacterial infection step in phage display. Bacterial infection is usually crucial for the propagation and identification of enriched clones by DNA sequencing. However, mutations or chemical modifications of the phage coat proteins, sometimes lead to decreased infection rates and therefore compromise the entire procedure. Consequently, there is an interest for developing methods in which this step could be completely omitted.