The Hybrid Approach for Rediscovering Bioactive Drug Candidates and Off-Targets Rapidly (HARBOR) project aims to address the 90% failure rate in modern G-quadruplex (G4)-drug development, often attributed to insufficient knowledge of identified therapeutic targets, a problem that is even more relevant in the field of G4, non-canonical DNA structures linked to cancer and antimicrobial resistance (AMR).1,2
HARBOR’s central hypothesis is that an integrated "hybrid approach" can bridge the gap between in silico, biophysical, and in vitro stages to accelerate the discovery of selective ligands for G4s. The primary research question is whether a streamlined pipeline can effectively bridge the gap between computational predictions and biological validation, thereby reducing the lack of correlation between these phases and increasing success in vitro and in vivo. The project focuses on identifying new G4 therapeutic targets in human cancer-related genes, including FOXA1, NR2F1, GALNT6, MUC4, ZMYM3, and RNF145, with the aim of identifying new anticancer compounds.3–5
Furthermore, HARBOR aims to expand knowledge and find alternative targets to counteract AMR. It focuses on the search for novel G4s in microbial genomes identified as priorities by the WHO reports in 2025. These include methicillin-resistant S. aureus, resistant to fluoroquinolones and cephalosporins; carbapenem-resistant P. aeruginosa and E. coli; and rifampicin/isoniazid-resistant M. tuberculosis. The study will explore new compounds and lay the groundwork for G4-based AMR studies.6–8 Another key step is the discovery and repurposing of selective ligand candidates arising from public repositories and internal databases at my Hosting Institution, from which a standardized screening pipeline is already established. The overall methodology follows a three-task research design: (1) Target and ligand identification using bioinformatics coupled with Nuclear Magnetic Resonance (NMR) spectroscopy and Taq-polymerase stop assay; (2) Biophysical investigation of binding affinity and selectivity via ESI-mass spectrometry analysis, NMR titration and interaction profile of the complex under evaluation; and (3) in vitro validation in cancer and control cell lines in which the cancer genes under investigation are expressed. From here, the expected result is the identification of new G4 targets in human genes involved in prostate, lung, and hepatic cancer, and after in silico studies, the identification of a maximum of 18 selective ligands, which need to be analyzed in cellular cell lines, thereby leading to the identification of 3 best performing ligands. Furthermore, identification of G4s in microbial genomes will be pivotal for initiating further studies on G4s in AMR. Specifically, some of the employed predictive methods include QGRS Mapper for G4-forming sequence prediction in investigated genes or genomes, molecular docking to evaluate the binding profile of selected compounds, molecular dynamics simulations to investigate complex formation from a structural perspective, and biological studies in which viability, cytotoxicity, and gene and protein profiles will be assessed through different assays for cellular validation. The project exhibits strong interdisciplinarity by merging computational biology, medicinal chemistry, biophysics, and pharmacology. Main expected results include identifying and characterizing new therapeutic targets in the anticancer and antimicrobial areas; discovering at least three lead compounds for anticancer therapeutic targets for further research; and identifying G4s in the genomes of bacterial strains under investigation, all through establishing a validated G4-drug-discovery pipeline. In this context, translational outcomes include identifying candidates for in vivo studies from which the application of drug repurposing could also speed the process, and generating patentable compounds. HARBOR advanc