A releasable disulfide-linked peptide tag facilitates the synthesis and purification of short peptides

Combinatorial cyclization of hundreds to thousands of random linear peptides by structurally diverse chemical linkers offers access to large macrocyclic compound libraries. A bottleneck in the development of such libraries is the preparation of large numbers of short random linear peptides. Herein, we present a tag-based strategy that is not dependent on a throughput-limiting chromatographic purification step and thus enables parallel production of short peptides. In brief, peptides are synthesized on solid phase as conjugates with a disulfide-linked Cys-Gly-Arg-Trp tetra-peptide tag. The charged arginine residue in the tag allows for purification of the peptides by diethyl ether-precipitation and the tryptophan allows for quantification of the product by absorption measurement. Addition of a reducing agent releases the short peptides from the tag. The released sulfhydryl group in the peptide can readily be used for cyclization of the peptide library with bis-electrophilic linker reagents.

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.

EPFL2017

Methods for the generation of large combinatorial macrocycle libraries

Sevan Mleh Habeshian

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.

EPFL2021

Structurally Diverse Cyclisation Linkers Impose Different Backbone Conformations in Bicyclic Peptides

Alessandro Angelini, Shiyu Chen, Christian Heinis, Julia Morales Sanfrutos

Combinatorial libraries of structurally diverse peptide macrocycles offer a rich source for the development of high-affinity ligands to targets of interest. In this work we have developed linkers for the generation of genetically encoded bicyclic peptides and tested whether the peptides cyclised by them have significant variations in their backbone conformations. Two new cyclisation reagents, each containing three thiol-reactive groups, efficiently and selectively cyclised linear peptides containing three cysteine moieties. When the mesitylene linker of the bicyclic peptide PK15, a potent inhibitor of plasma kallikrein (Ki=2 nM), was replaced by the new linkers, its inhibitory activity dropped by a factor of more than 1000, suggesting that the linkers impose different conformations on the peptide. Indeed, structural analysis by solution-state NMR revealed different NOE constraints in the three bicyclic peptides, indicating that these relatively small linkers at the centres of bicyclic peptide structures significantly influence the conformations of the peptides. These results demonstrate the prominent structural role of linkers in peptide macrocycles and suggest that application of different cyclisation linkers in a combinatorial fashion could be an attractive means to generate topologically diverse macrocycle libraries.