Catechols and 3-hydroxypyridones as inhibitors of the DNA repair complex ERCC1-XPF.

Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 µM, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and γH2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of <10 µM.

Cysteine specific bioconjugation with benzyl isothiocyanates.

Protein labelling has a wide variety of applications in medicinal chemistry and chemical biology. In addition to covalent inhibition, specific labelling of biomolecules with fluorescent dyes is important in both target discovery, validation and diagnostics. Our research was conducted through the fragment-based development of a new benzyl-isothiocyanate-activated fluorescent dye based on the fluorescein scaffold. This molecule was evaluated against fluorescein isothiocyanate, a prevalent labelling agent. The reactivity and selectivity of phenyl- and benzyl isothiocyanate were compared at different pHs, and their activity was tested on several protein targets. Finally, the clinically approved antibody trastuzumab (and it's Fab fragment) were specifically labelled through reaction with free cysteines reductively liberated from their interchain disulfide bonds. The newly developed benzyl-fluorescein isothiocyanate and its optimized labelling protocol stands to be a valuable addition to the tool kit of chemical biology.

Enabling the controlled assembly of antibody conjugates with a loading of two modules without antibody engineering.

The generation of antibody conjugates with a loading of two modules is desirable for a host of reasons. Whilst certain antibody engineering approaches have been useful in the preparation of such constructs, a reliable method based on a native antibody scaffold without the use of enzymes or harsh oxidative conditions has hitherto not been achieved. The use of native antibodies has several advantages in terms of cost, practicality, accessibility, time and overall efficiency. Herein we present a novel, reliable method of furnishing antibody conjugates with a loading of two modules starting from a native antibody scaffold.

Next-generation disulfide stapling: reduction and functional re-bridging all in one.

Herein we present a significant step towards next-generation disulfide stapling reagents. A novel class of reagent has been designed to effect both disulfide reduction and functional re-bridging. The strategy has been applied to great success across various peptides and proteins. Moreover, application to a multi-disulfide system resulted in functional re-bridging without disulfide scrambling.

A mild TCEP-based para-azidobenzyl cleavage strategy to transform reversible cysteine thiol labelling reagents into irreversible conjugates.

It has recently emerged that the succinimide linkage of a maleimide thiol addition product is fragile, which is a major issue in fields where thiol functionalisation needs to be robust. Herein we deliver a strategy that generates selective cysteine thiol labelling reagents, which are stable to hydrolysis and thiol exchange.