DNA-ENCODED CHEMICAL LIBRARIES: ADVANCES AND APPLICATIONS TO DRUG DIFFICULT TARGETS

The discovery of small organic ligands or biologics capable of modulating biological processes remains one of the biggest challenges in developing new medicines. Different technologies have been implemented over the last decades to ease this process and make it more efficient. In this regard, encoded display technologies have played a major role in the discovery of new antibodies, peptides, and proteins. However, the efficient exploitation of automated high-throughput screening to discover small organic ligands has mainly been limited to big pharmaceutical companies. DNA-Encoded Chemical Libraries (DELs) have emerged as a powerful and cost-effective alternative to solve this issue. The technology has been established during the last 25 years and has become one of the best methods to synthesize and screen libraries of unprecedented size, promising a bright future in the early drug discovery stages. DELs are collections of small molecules individually coupled to oligonucleotide fragments, serving as amplifiable identification barcodes. In the first part of this thesis new DEL designs, displaying molecules capable of targeting challenging therapeutic targets while keeping library-quality at the highest grade, were investigated. A novel single-pharmacophore library, termed AG-DEL, was synthesized. The library was constructed using split-and-pool procedures on single-stranded DNA. The modularity of this library design allowed the creation of different dual-pharmacophore libraries in an encoded self-assembling chemical library format (ESAC 2+1 and ESAC Plus). Furthermore, the new AG-DEL facilitated the use of novel screening methodologies (e.g., photo-crosslinking) to efficiently discover new small organic ligands. DEL synthesis mainly relies on the chemical diversity of building blocks and the efficiency of the chemical reactions to link them. Following this trend, many different groups have made great efforts during the last years to develop new mild and efficient DNA-compatible reactions. One of the most used reactions for DEL synthesis is the amide bond formation, thanks mainly to various reliable reaction protocols and the big commercially available collections of amino acids. Nevertheless, the current availability of DNA-compatible post-functionalization of amino acids is still quite limited due to some restrictions inherent to the presence of the DNA. In the second part of this thesis, a new DNA-compatible diazo-transfer reaction was successfully optimized and implemented. This reaction has shown to be efficient, both in reaction times and reaction yields, as well as to be mild and fully compatible with DNA, as demonstrated by subsequent enzyme-mediated ligation of the oligonucleotide template to a new fragment, and has served for the synthesis of new ESAC Plus libraries within our group. The modulation of protein-protein interactions (PPIs) represents another formidable challenge. These interactions are often characterized by large and flat protein surfaces that are composed of many different interacting groups. Therefore, these interactions are usually targeted using large macrocyclic peptides or antibodies. Notwithstanding this challenge, some examples have been reported during the last years in which small organic ligands or peptidomimetics were specifically designed for targeting this class of proteins. Some of these examples have successfully reached clinical trials and even marketing authorization, showing the critical importance of PPI modulators and indicating broad prospects. In PPI modulation, the discovery of ligands targeting cytokines is even more challenging, due to the small size and particularly flat surface of these proteins. Nevertheless, different small molecule ligands targeting cytokines have been described over the years. Among all these proteins, Interleukin-2 (IL2) represents one of the best examples. IL2 is a pro-inflammatory cytokine, that plays a crucial role in immunity, and different therapeutic approaches using IL2 are increasingly being used for the treatment of a variety of malignancies, like melanoma and renal cell carcinoma. However, the use of IL2 has been limited due to strong side effects related to the high doses of cytokine necessary to achieve a pharmacological effect. Side effects have been linked to the release of pro-inflammatory cytokines as well as to CD25-mediated endothelial damage induced by IL2 binding to endothelial surface receptors, leading to a vascular leak syndrome. The interaction between IL2 and its alpha subunit receptor (IL2Ra or CD25) activates immunosuppressive regulatory T cells (Tregs) and reduces its antitumor activity. Thus, avoiding the formation of the multimeric IL2/IL2R complex can enhance the antitumor response. The last chapter of this thesis was focused on the development of novel DEL-derived IL2 ligands capable of interacting at the CD25 binding domain of IL2. During these studies, a tumor-targeting antibody-IL2 fusion protein, L19-IL2, was used to find ligands masking the IL2 moiety. The ligands were optimized by a medicinal chemistry approach and characterized by fluorescence polarization. Furthermore, the best ligand showed binding at the CD25 binding epitope of IL2, as evidenced by competition experiments using an anti-IL2 antibody. The use of one of the discovered compounds or an affinity matured derivative can allow the generation of a new class of biopharmaceutical-small molecule complexes that localize at the site of the disease and regain activity of the cytokine only at the tumor site.