Epigenetic evolution is a common mechanism used by cancer cells to evade the therapeutic effects of drug treatment. In ovarian cancers, epigenetically driven resistance is thought to be responsible for many late-stage patient deaths. DNA secondary structures called G-quadruplexes (G4s) are emerging as potential epigenetic marks of relevance to cancer evolution, but their prevalence and distribution in ovarian cancer models have never been investigated before. Here, we describe the first investigation of the role of G4s in the epigenetic regulation of drug-resistant ovarian cancer cells. Through genome-wide mapping of G4s in paired drug-sensitive and drug-resistant cell lines, we find that increased G4 accumulation is associated with enhanced transcription of signalling pathways previously established to promote drug-resistant states, including genes involved in the epithelial to mesenchymal transition and WNT signalling. In contrast to previous studies, the expression-enhancing effects of G4s are not found at gene promoters, but intergenic and intronic regions, indicating that G4s can promote long-range transcriptional regulation in drug-resistant cells. Furthermore, we discover that clusters of G4s (super-G4s) are associated with particularly high levels of transcriptional enhancement that surpass the effects of super-enhancers, which act as well-established regulatory sites in many cancers. Finally, we demonstrate that targeting G4s with small molecules results in significant downregulation of pathways associated with drug resistance, resulting in resensitization of resistant cells to chemotherapy agents. These findings indicate that G4 structures are critical for the epigenetic regulatory networks of drug-resistant cells and represent a promising target to treat drug-tolerant ovarian cancer.

The discovery of new compounds with pharmacological properties is usually a lengthy, laborious and expensive process. Thus, there is increasing interest in developing workflows that allow for the rapid synthesis and evaluation of libraries of compounds with the aim of identifying leads for further drug development. Herein we report on such a workflow for the synthesis and evaluation of libraries of over 80 iridium(III) complexes as phototoxic agents against cancer cells. We demonstrate that it is possible to complete the whole semiautomated workflow within 72 hours, allowing for rapid library iteration. This includes all steps from the synthesis of a library of new complexes and their characterisation, to determining their cellular uptake, localisation and phototoxicity. We demonstrate the power of this rapid synthesise-and-test cycle by identifying highly active complexes which are well-tolerated in the dark but display phototoxicity at very low nM concentrations against cancer cells.

The development of selective ligands to target DNA G-quadruplexes (G4s) has been pivotal in revealing their role in transcriptional regulation. However, most of the ligands described to date lack intra-G4 selectivity, severely limiting their potential for uncovering the biological function of individual G4s across the genome. To overcome these limitations, we developed ATENA (Approach to Target Exact Nucleic Acid alternative structures). ATENA relies on the chemical modification of established G4-ligands to enable their conjugation onto a catalytically inactive Cas9 protein (dCas9) using HaloTag, allowing for the targeting of individual G4s in living cells. We have systematically screened the length of the PEG-linkers connecting the G4-ligands to the HaloTag and sgRNA sequences to attain optimal G4 engagement both in vitro and in cells. Using optimized conditions, we leveraged ATENA to demonstrate how the selective targeting of the well-studied G4 in the promoter of the oncogene c-MYC suppresses its transcription exclusively from the P1 promoter. We also show that positioning ligands in the proximity of regulatory elements suppresses c-MYC transcription in a G4-independent manner, highlighting the importance of appropriate design to measure genuine G4-mediated transcriptional changes. We also demonstrate that selective targeting of a G4 in the PVT1 promoter can either stimulate or repress its transcription depending on the type of G4-ligand used, indicating that functional responses associated with G4-stabilization can highly depend on the type of ligand used. We further harnessed ATENA to study transcriptional perturbation associated with cell-specific G4s, revealing that the functional responses associated with these structures are tightly linked with the expression levels of the targeted gene. Our study provides critical insights into G4-based therapeutic design, offering an innovative platform to investigate G4 biology with high precision. ### Competing Interest Statement Marco Di Antonio is a co-funder of Project Maestro.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC) n in C9orf72 . Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC) n , which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.

Unprecedented bacterial targets are urgently needed to overcome the resistance crisis. Herein we systematically mine pyridoxal phosphate-dependent enzymes (PLP-DEs) in bacteria to focus on a target class which is involved in crucial metabolic processes. For this, we tailored eight pyridoxal (PL) probes bearing modifications at various positions. Overall, the probes exceeded the performance of a previous generation and provided a detailed map of PLP-DEs in clinically relevant pathogens including challenging Gram-negative strains. Putative PLP-DEs with unknown function were exemplarily characterized via in-depth enzymatic assays. Finally, we screened a panel of PLP binders for antibiotic activity and unravelled the targets of hit molecules. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP-dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP-DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.

Targeted covalentinhibitors are powerful entities in drug discovery, but their application hasso far mainly been limited to addressing cysteine residues. The development ofcysteine-directed covalent inhibitors has largely profited from determiningtheir proteome-wide selectivity using competitive residue-specific proteomics. Severalprobes have recently been described to monitor other amino acids using thistechnology and many more electrophiles exist to modify proteins. Nevertheless,a direct, proteome‑wide comparison of the selectivity of diverse probes isstill entirely missing. Here, we developed a completely unbiased workflow to analyseelectrophile selectivity proteome‑wide and applied it to directly compare 54 alkyneprobes containing diverse reactive groups. In this way, we verified and newly identifiedprobes to monitor a total of nine different amino acids as well as the N‑terminusproteome‑wide. This selection includes the first probes to globally monitortryptophans, histidines and arginines as well as novel tailored probes formethionines, aspartates and glutamates.