Generating macrocyclic inhibitors of protein-protein interactions

Protein-protein interactions (PPIs) play important roles in many diseases and their modulation is an attractive strategy for developing new therapeutics. However, the relatively large and often flat binding surfaces of PPIs makes the development of inhibitors based on classical small molecule drugs rather challenging. Macrocycles, a class of ring-shaped chemical structures, are able to bind to challenging targets such as PPIs, while having the potential to be cell-permeable or even orally available. The potential of targeting intracellular PPIs by macrocyclic compounds has triggered a great deal of interest in the pharmaceutical industry. A challenge with developing macrocycle-based drugs to new targets, however, is the identification of ligands, the reason for this mainly being the lack of large libraries of macrocyclic compounds that could be screened.The goal of my Ph.D. thesis project was to develop macrocyclic inhibitors for several protein-protein interactions. Towards this end, I contributed to establishing novel strategies for synthesizing and screening large macrocyclic compound libraries. Additionally, I established reported or developed new bioassays suited for the high-throughput screening of compound libraries, and I applied them for screening macrocycle libraries generated with the new methods.In my first project, I applied acoustic droplet ejection technology (ADE) for the synthesis and screening of large macrocyclic compound libraries at a picomole scale. Towards this aim, I used a synthetic strategy based on the combinatorial diversification of m bromoacetamide linear peptides with n primary amines followed by cyclization with o bis-electrophile linkers yielding m x n x o macrocycles. I used ADE to perform all liquid transfers, which proved efficient and yielded macrocycles at picomole scale. I performed the screening by directly adding assay reagents to the synthesis plates containing crude macrocycle products. As a proof-of-concept, I assembled a 2,700-member target-focused macrocycle library and screened it against the MDM2:p53 protein-protein interaction, and was able to identify macrocyclic inhibitors with micromolar to sub-micromolar affinity. Most importantly, this work validated the feasibility to synthesize combinatorial macrocycle libraries using ADE at the picomole scale, which was subsequently applied to several other approaches later developed in our lab.In my second project, I aimed at finding macrocyclic inhibitors of the PPI IL-23:IL-23R, which is an important target of several autoimmune disorders. To that end, I developed a high-throughput screening assay for identifying inhibitors of the interaction. I initially developed a screening assay based on fluorescence polarization, which turned out to be prone to artifacts. In a second attempt, I established an assay based on TR-FRET that proved reliable for the identification of IL-23:IL-23R inhibitors, and in particular also for the screening of crude products of macrocyclization reactions. I subsequently synthesized a pilot-scale library by combinatorially cyclizing 384 linear peptides with 8 cyclization linkers to generate 3,072 target-focused macrocyclic compounds. Screening the library using the TR-FRET-based assay identified single-digit micromolar inhibitors of the IL-23:IL-23R PPI, that however, were based on linear side-products of the macrocyclization reactions rather than macrocyclic structures. While no potent ma

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

Phage Selection of Bicyclic Peptide Ligands and Development of a New Peptide Cyclisation Method

Jeremy Touati

Bicyclic peptides binding to targets of interest can be isolated from combinatorial libraries using a phage display-based approach. In a first project of this thesis, we aimed at generating bicyclic peptide inhibitors of matrix metalloproteinases (MMPs), a family of proteases that share high structural similarities and have been difficult to target in a specific manner. From phage-encoded combinatorial libraries, we isolated a bicyclic peptide that inhibits specifically the gelatinase MMP-2 with a Ki of 2 μM. This inhibitor was less potent than the best peptidic MMP-2 inhibitor, the linear APP-derived inhibitory peptide (Ki = 13 nM). However, due to its conformational constraint, the bicyclic peptide is expected to be significantly more stable and hence more suitable for applications in biological systems. It may be used as a research tool alternatively to the broadly applied monocyclic peptide MMP-2 inhibitor CTT which is less potent (Ki of 142 μM). If affinity matured, the bicyclic peptide MMP-2 inhibitor might even be developed into an anti-cancer therapeutic. In a second project, bicyclic peptide binders to the centriolar protein SAS-6 were generated. SAS-6 plays an important structural role in centrioles and bicyclic peptide binders are currently applied in cellular assays by the laboratory of Professor Pierre Gönczy (EPFL) to study the process of centriole formation. In a third project of this thesis, we established an enzymatic method to selectively cyclise peptides with unprotected side chains using a transglutaminase (TGase). TGases catalyse the formation of stable amide bonds between the side chains of glutamine and primary amines. They have been used extensively in biotechnological applications to cross-link peptides and proteins or to attach labels to proteins. We found that the microbial transglutaminase (MTGase) of Streptomyces mobaraensis can cyclise quantitatively peptides with an N- terminal glutamine-donor sequence and a C-terminal lysine residue in an intramolecular reaction. Experiments with minimised glutamine-donor substrates revealed that peptides with only an Ala-Leu dipeptide N-terminal to the glutamine and a randomly chosen peptide between the glutamine and lysine residues are efficiently cyclised. By combining the MTGase-based cyclisation strategy with a chemical thiol-based cyclisation reaction, we were able to generate tricyclic peptide structures. It remains to be tested if this method can be applied to cyclise combinatorial peptide libraries on the surface of phage. In a fourth and last project, we developed a method to follow qualitatively and quantitatively chemical or enzymatic modifications applied to peptides displayed on phage. In this method, peptides on phage were chemically modified, cleaved off by a protease, purified, concentrated and analysed by mass spectrometry. The method will help to assess new reactions applied to phage peptides such as the MTGase-based enzymatic cyclisation reaction.

EPFL2012

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.