Abstract A026: Synthesis and structure-activity studies of DNA- and RNA-binding nucleopeptides with a cell penetrating potential

Abstract

In the field of modern biomedicine, significant progress has been made in countering or restoring faulty genes using exogenous nucleic acids or their mimetics (NAMs). This approach enables targeted and long-lasting therapeutic effects by circumventing the need of interventions at a protein level. Despite the boom of clinical approvals, the development of NAMs still faces crucial challenges, including susceptibility to metabolic breakdown, limited uptake by cells, self-aggregation, and the potential for immune reactions. In addition to employing advanced delivery strategies, alternative methods to address these issues involve modifying NAMs chemically or combining oligonucleotides (ONs) with cellular carriers, including cell-penetrating peptides. Against this background, herein we present the nucleopeptides (NPs) as a family of unexplored NAMs that bridge the gap between ONs and peptide structures. By incorporating two consecutive amino acids—one bearing a nucleobase on the side chain and the other remaining unaltered—we created a monomer that spans the length of a single nucleotide. This functional module possesses nucleobase recognition elements along with additional functional groups eligible to modulate physio-chemical properties. We investigated this class of macromolecules by preliminarily devising an efficient solid-phase peptide synthesis technique profitable for homo- and hetero-sequences and sparing the monomer assembly in solution. [1] The influence of the amino acid composition on target binding properties, metabolic stability, and cellular penetration, were assessed through the synthesis of a series of homothymine hexameric derivatives. This simplified model was also used to investigate the distance between the nucleobase and the backbone, the absolute configuration of the nucleobase- bearing element, and the polycationic/anionic nature of the unmodified amino acid. [2-4] We evaluated the recognition and the thermodynamic stability of NPs/DNA and NPs/RNA hetero-duplexes through circular dichroism and UV spectroscopy experiments, while we employed confocal microscopy with suitable FITC- labeled derivatives to determine the intracellular localization in target cells. [1,3] Our results suggested that NPs exhibit distinct binding properties towards complementary RNA and DNA sequences according to their aminoacidic composition, and are characterized by a higher affinity compared to ONs and PNAs, thus arising as promising cell penetrating NAMs. [1] Mercurio ME, Tomassi S, et al. J Org Chem. 2016, 81(23), 11612- 11625. [2] Tomassi S, Ieranò C, et al. Bioorg Med Chem. 2018, 26(9), 2539-2550. [3] Tomassi S, Montalban FF, et al. Symmetry  2019, 11(4), 567-582. [4] Tomassi S, Ieranò C, et al. Int J Mol Sci. 2022, 23(15), 8504-8518.

Citation Format: Stefano Tomassi, Caterina Ieranò, Stefania Scala, Anna Messere, Salvatore Di Maro. Synthesis and structure-activity studies of DNA- and RNA-binding nucleopeptides with a cell penetrating potential [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A026.