"Targeting the ""untargetable"" by merging biological and chemical compound libraries"

Project Information

TARGET

Grant agreement ID: 101020521

DOI

10.3030/101020521

EC signature date 22 May 2021

Start date 1 January 2022

End date 31 December 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 239 500,00

EU contribution

€ 2 239 500,00

Coordinated by

ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE
Switzerland

Periodic Reporting for period 2 - TARGET (Targeting the "untargetable" by merging biological and chemical compound libraries)

Reporting period: 2023-07-01 to 2024-12-31

Summary of the context and overall objectives of the project

The TARGET project aims to develop a novel strategy for targeting ‘undruggable’ proteins by merging biological and chemical compound libraries. This approach seeks to generate synthetic ligands capable of binding to challenging intracellular protein-protein interactions, providing a foundation for new therapeutics addressing unmet medical needs.

The Challenge
Many critical drug targets lack deep binding pockets required for small molecule drugs, limiting treatment options. Despite advancements, many intracellular and extracellular proteins remain undruggable. Notable examples include TNFa, KRASG12D, and MYC. Macrocyclic compounds offer a potential solution, as their cyclic structure improves binding efficiency while maintaining small molecular size for cell permeability and oral delivery. However, the development of such ligands is hindered by limited chemical diversity in biological libraries and difficulties in screening large synthetic macrocyclic libraries.

Our Solution
TARGET proposes merging biological libraries (e.g. phage display peptides) with chemically synthesized compounds to create a highly diverse screening space. Biological libraries offer vast peptide variation but limited chemical diversity, while chemical libraries provide structural variety but are harder to screen. By combining these approaches, TARGET aims to generate novel ligands with enhanced binding properties.

Impact
This strategy could overcome key barriers in drug development, particularly for intracellular disease targets. If successful, macrocyclic peptide-based ligands could be translated into therapeutics, benefiting patients with conditions such as cancer and other hard-to-treat diseases.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Over the past 2.5 years, the TARGET project has successfully developed high-throughput phage display methods and established strategies for chemically modifying phage-displayed peptides (WP1). Additionally, we have synthesized and diversified macrocyclic peptide libraries via amino groups (WP2). The following summarizes key advancements.

High-Throughput Phage Display & Chemical Modification (WP1a & WP1b)
We established a method to chemically modify phage-encoded peptide libraries, expanding their diversity beyond natural amino acids. By using a 96-well plate format, we encoded chemical modifications by well position, enabling parallel screening. A critical innovation was capturing phage on a solid phase, allowing automated purification and sequential chemical treatments.
Initially, we explored alkyne-azide click reactions but found them inconsistent. We instead adopted thiol-based modifications, which proved robust for diverse electrophilic reagents. Using this approach, we constructed and screened peptide libraries with appended chemical fragments and validated the method on challenging protein targets such as MCL-1.

Expanding Chemical Diversity via Amine Modification (WP2a & WP2b)
To further increase chemical diversity, we developed acylation methods to conjugate carboxylic acid-functionalized fragments to peptide amino groups. This approach leverages the vast availability of carboxyl-containing chemicals and creates stable amide bonds common in therapeutics.
While initial attempts to modify phage-displayed peptides faced challenges due to lysine-rich phage surfaces, we are evaluating alternative genetically encoded peptides with chemically inert surfaces. In parallel, we successfully applied high-throughput amine modification in 384-well plates to generate and screen chemically diversified cyclic peptide libraries.
These advancements pave the way for discovering novel ligands for previously undruggable targets.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our efforts to generate and screen compound libraries of unprecedented size and structural diversity are progressing well with so far the following two highlights:

1. Development of a method to capture phage on solid phase for chemical diversification of the displayed peptides. This method works efficiently and even better than we anticipated initially. It allows generating and handling a library in a single well of a 96-well plate and thus the parallel and high-throughput diversification of phage-encoded peptide libraries.

2. First proof of concept for phage display selection of peptides diversified with a large number of different chemical building blocks (fragments). Importantly, the peptides isolated in the proof-of-concept study bound much more strongly to the targets (MCL-1) when chemically modified with the corresponding fragment, underscoring the importance of the expanded chemical diversity.

As next steps, we will expand the library sizes and screen the libraries against several challenging targets. We are optimistic that we can generate high-affinity macrocyclic compounds to at least some of the targets.

Strategy (a), instrumentation (b) and results (c)