Protein-templated ligand discovery via the selection of DNA-encoded dynamic libraries and to identify YEATS2 YEATS domain inhibitors through the selection of DNA-encoded Libraries

Abstract

Finding small molecule ligands, capable of specifically binding to the protein target of interest, remains a major challenge for both academic and industrial research. For decades, high-throughput screening (HTS) represents a mainstay for the provision of hits in the ligand discovery field. Screening by using HTS assay provide a reliable starting point for further investigation. Many So-called ‘lead compounds’ optimized through several cycles of pre- or clinical development finally come on market. Despite the achievement made so far, one of the major limitations of HTS platform is the requirement of individual synthesizing and testing the library members, which therefore results in prohibitive cost and complex logistics. Meanwhile, with the rise of genomics era, there is an urgent need for alternative methodologies toward targets with more complexity for example targets from protein-protein interactions (PPI), membrane proteins and transcription factors. During the past three decades, DNA-encoded library (DEL), a concept originally proposed by Brenner and Lerner in 1992, has growth rapidly and become a powerful tool. Compared with HTS methodologies, DEL can achieve simultaneous selection for millions, billions or even trillions of compounds in a single vessel, significantly simplifying the selection procedure against protein targets either immobilized or in solution phase. The decoding of genetic information via PCR technology followed by the next generation sequencing (NGS) allows for the identification of high-affinity binder. Generally, based on different encoding strategies, DEL can be divided into two categories: single-pharmacophore libraries and dual-pharmacophore encoded self-assembling chemical (ESAC) libraries. In this thesis, practical applications of these two branches will be involved in Part I & Part II separately.

In contrast to single-pharmacophore libraries, ESAC libraries with individual fragments attached at opposite end of two partially complementary DNA strands, can realize the simultaneous display of a ligand pair to interact with protein target of interest. High affinity binders selected out by ESAC require a post-selection tethering process to yield a full ligand, which is considered to be highly challenging and usually takes more time than selection itself. Of the first project in the thesis, we proposed an innovation in-situ synthesis approach of full ligand by combination of DELs with Dynamic Combinatorial Libraries (DCL). By addition of the target protein, the identification of best linkers can be done simultaneously together with ligand pair. To demonstrate the performance of this method, a 4.35-million- and a 3-million-member DEDLs with different library architectures have been prepared and selected against various targets, including SARS-CoV-2 spike protein, an important anti-COVID-19 drug target. A series of novel and selective binders have been identified and tested by biological assays. Our study has circumvented a significant obstacle in DELs and may provide a broadly applicable method for ligand discovery against biological targets.

Single-pharmacophore libraries are commonly constructed by DNA-recorded synthesis (split-and-pool) and different chemical moieties are added to either double-stranded or single-stranded DNA segment (dsDNA or ssDNA) that uniquely identify them. In the project, we adopted a robust ‘Headpiece (HP)’-based double stranded format as our starting point of library construction. YEATS domains are newly identified epigenetic readers of histone Kac and Kcr marks. It has been reported that the dysregulated interactions between YEAST2 YEATS domain and histone Kac marks are associated with the tumorigenesis of NSCLC, suggesting the therapeutic potential of YEATS2 YEATS domain inhibition. However, no inhibitors targeting the YEATS domain of YEATS2 have been reported. Based on a known peptide-based inhibitor LS-1-124, we then design and synthesize a medium-size focused library as well as a large-scale diversity library for selection against YEATS2 YEATS domain. Testing of identified molecules by biological assay is still in progress. Further modification is needed to achieve low nM binder.