Ligand Discovery and Affinity Maturation with Single Stranded DNA Encoded Chemical Libraries

Abstract

Drugs are chemical or biological compounds, able to specific interact with one or more target proteins, hence driving a pharmacological benefit for the prevention, diagnosis or treatment of a pathology. In the past, the isolation of novel drugs has often started from the screening of bioactive compounds from nature, on the basis of trial and error approaches. More recently, however, also thanks to advances in molecular biology and in the production of recombinant proteins, it has become important to discover protein ligands by screening organic molecules in large compound collections, called “chemical libraries”. At present, it is customary for large pharmaceutical companies to try and identify specific protein ligands (called “hits”) starting from chemical libraries that may contain up to one million compounds. Similar activities are prohibitively expensive for academic groups. In many cases, high-throughput screening assays are used. Once a hit compound is identified, suitable modifications using medicinal chemistry are performed in order to generate improved molecules, which may deserve to be tested both in vitro and in vivo. If the so-called “lead compounds” exhibit the required specificity and potency, alongside other parameters which are important for clinical development, the product candidates may progress towards late-stage industrial activities. In many cases, however, screening campaigns based on high-throughput screening fail to delivery hits. For this reason, there is a considerable interest in the development of novel methodologies for the construction and screening of large chemical libraries. DNA-Encoded chemical libraries (DELs) are collections of small organic molecules, individually coupled to DNA fragments which act as amplifiable identification barcodes. DELs can be very large (i.e., containing millions to billions of compounds), as they can be synthesized using split&pool procedures, starting from a moderate number of starting building blocks. In this thesis, I describe the synthesis and validation of a novel DEL (termed GB-DEL), featuring DNA in single-stranded format and containing 366.600 compounds. The library was based on a stereoisomeric scaffold (i.e., a derivative of glutamic acid), whose compact structure facilitated chemical transformations and yielded library members of small size (i.e., typically smaller than 500 Dalton). The GB-DEL library was screened against a number of different proteins and provided ligands against targets of pharmaceutical interest. I have discovered and characterized ligands directed against carbonic anhydrase IX (CAIX), tyrosinase (TYR), tyrosinase-related protein 1 (TYRP-1) and placental alkaline phosphatase, which are tumorassociated antigens suitable for pharmacodelivery applications. Be I have isolated small organic binders against proteins involved in DNA repair and cell metabolism such as FANCD2/FANCI-associated nuclease I (FAN1) and alpha amino adipic semi aldehyde synthase (ASS), as well as enzymes involved in cellular signaling (such as PI3K and CREBBP). GB-DEL hybridization with chemically-modified complementary DNA strands, carrying a protein-binding moiety resulted in self-assembled chemical structures displaying three sets of building block and facilitated the identification of affinity-matured compounds. In a dedicated study, I have investigated whether it would be more convenient to use DELs in single-stranded DNA format, or rather convert the DNA into a double-stranded format using Klenow polymerization. Comparative selection experiments revealed that both approaches could be productive in yielding specific protein ligands, but the use of single-stranded DNA barcodes produced slightly superior selection fingerprints. In one chapter of the thesis, I have reported the discovery and validation of a potent and selective inhibitor of placental alkaline phosphatase (PLAP), a tumor associated antigen frequently expressed in tumors of the female reproductive tract. One of the compounds isolated from a DEL (i.e., from the NF-DEL library, produced by my colleague Nicholas Favalli) inhibited PLAP with an IC50 = 32 nM. Importantly, the product had not detectable inhibition of the closely related tissue non-specific alkaline phosphatase (TNAP), which is expressed on the surface of many normal cells. The PLAP ligand was conjugated to fluorescein and specifically bound to PLAP-positive tumors in vitro. When injected into tumor-bearing mice, the fluoresceinated molecule targeted cervical cancer in vivo, as evidenced by ex vivo fluorescence microscopy investigations. Finally, I have also screened the GB-DEL against tyrosinase and TYRP-1, two proteins selectively expressed in melanocytes and, in pathological context, in melanoma lesions. We discovered novel ligands capable of selective binding to both proteins and used a previously described tyrosinase inhibitor (Thiamidol™) as starting point for affinity maturation methodologies. Collectively, the thesis shows that DEL technology can facilitate the discovery of ligands to a variety of different proteins of biological or pharmaceutical interest.