One-Pot Oxidative Amidation of Aldehydes via the Generation of Nitrile Imine Intermediates.

A one-pot procedure for the oxidative amidation of aldehydes via the in situ generation of reactive nitrile imine (NI) intermediates has been developed. Distinct from our progenitor processes, mechanistic and control experiments revealed that the NI undergoes rapid oxidation to an acyl diazene species, which then facilitates N-acylation of an amine. A range of substrates have been explored, including application in the synthesis of pharmaceutically relevant compounds.

Novel WRN Helicase Inhibitors Selectively Target Microsatellite Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology.

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome.

Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome.

Kinetic Optimization of Lysine-Targeting Covalent Inhibitors of HSP72.

The covalent inhibition mechanism of action, which overcomes competition with high-affinity, high-abundance substrates of challenging protein targets, can deliver effective chemical probes and drugs. The success of this strategy has centered on exposed cysteine residues as nucleophiles but the low abundance of cysteine in the proteome has limited its application. We have recently reported our discovery that lysine-56 in the difficult-to-drug target HSP72 could form a covalent bond with a small-molecule inhibitor. We now disclose the optimization of these targeted covalent inhibitors using rational design. Essential to our optimization was the development of a new covalent fluorescence polarization assay, which allows for the direct measurement of the key kinetic parameter in covalent inhibitor design, kinact/KI, extrapolation of the underlying parameters, kinact and Ki, and direct comparison to reversible analogues. Using our approach, we demonstrate a >100-fold enhancement in covalent efficiency and key learnings in lysine-selective electrophile optimization.

Lysine-Targeting Covalent Inhibitors.

Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncology. This strategy exploits small-molecule/protein crystal structures to design tightly binding ligands with appropriately positioned electrophilic warheads. Whilst most focus has been on targeting binding-site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition. However, owing to the basicity of the ϵ-amino group in lysine, this strategy generates a number of specific challenges. Herein, we review the key principles for inhibitor design, give historical examples, and present recent developments that demonstrate the potential of lysine targeting for future drug discovery.

An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine-56.

The stress-inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine-targeted irreversible inhibitor. Using rational design, we adapted a validated 8-N-benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine-56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.