Sam M. Rowe

We report a ‘direct-to-biology’ (D2B) approach for optimising covalent acrylamide binders of protein targets and apply this to the identification of a selective and cell-active inhibitor of Werner (WRN) helicase. Inhibition of WRN helicase activity exhibits a synthetic lethal relationship with cancers displaying high microsatellite instability (MSI-H) and is being pursued as a therapeutic strategy in the clinic. Using intact-protein liquid chromatography-mass spectrometry (LC-MS) screening, we identified acrylamide fragment binders of the WRN helicase domain and then used covalent D2B chemistry to optimise these initial hits. Our efforts ultimately afforded a potent covalent inhibitor of WRN-mediated DNA unwinding, which displays selective, concentration-dependent cellular engagement of WRN, and demonstrates synthetic lethality in an MSI-H setting. Furthermore, our inhibitor targets a distinct conformation of WRN helicase compared to the current clinical covalent inhibitor, presenting a complementary approach for covalent inhibition of WRN helicase. This work demonstrates how D2B chemistry platforms can be used to explore structure-activity relationships in a modular fashion, while reducing investment of human and material resources.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low‐throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for ‘direct‐to‐biology’ (D2B) screening of cysteine‐targeting chloroacetamide RFs, wherein synthesis is performed in 384‐well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS‐CoV‐2 main protease (M^Pro), an established drug target for the treatment of COVID‐19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a ‘switch’ to a reversible inhibitor series. This example of ligand discovery for M^Pro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non‐covalent ligands.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low‐throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for ‘direct‐to‐biology’ (D2B) screening of cysteine‐targeting chloroacetamide RFs, wherein synthesis is performed in 384‐well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS‐CoV‐2 main protease (M^Pro), an established drug target for the treatment of COVID‐19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a ‘switch’ to a reversible inhibitor series. This example of ligand discovery for M^Pro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non‐covalent ligands.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target’s perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low-throughput, constrained by the need for synthesis and purification of target compounds. We report a high-throughput platform for ‘direct-to-biology’ (D2B) screening of cysteine-targeting chloroacetamide RFs, wherein synthesis is performed in 384-well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS-CoV-2 main protease (MPro), an established drug target for the treatment of COVID-19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a ‘switch’ to a reversible inhibitor series. This example of ligand discovery for MPro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non-covalent ligands.

Cyclopropanes are highly useful motifs that are often incorporated into drug candidates to improve potency, metabolic stability, or pharmacokinetic properties. An expedient method for the α-cyclopropanation of ketones using hydrogen borrowing (HB) catalysis is described. The transformation occurs via HB alkylation of a hindered ketone with subsequent intramolecular displacement of a pendant leaving group affording the cyclopropanated product. The leaving group can be installed in either the ketone or alcohol component of the HB system, giving access to α-cyclopropyl ketones via two complementary approaches. Conversion to the corresponding carboxylic acids can be achieved in a simple two-step sequence to afford synthetically useful 1,1-substituted spirocyclopropyl acid building blocks.

The ABC multidrug exporter MsbA mediates the translocation of lipopolysaccharides and phospholipids across the plasma membrane in Gram-negative bacteria. Although MsbA is structurally well characterised, the energetic requirements of lipid transport remain unknown. Here, we report that, similar to the transport of small-molecule antibiotics and cytotoxic agents, the flopping of physiologically relevant long-acyl-chain 1,2-dioleoyl (C18)-phosphatidylethanolamine in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and the chemical proton gradient as sources of metabolic energy. In contrast, the flopping of the large hexa-acylated (C12-C14) Lipid-A anchor of lipopolysaccharides is only ATP dependent. This study demonstrates that the energetics of lipid transport by MsbA is lipid dependent. As our mutational analyses indicate lipid and drug transport via the central binding chamber in MsbA, the lipid availability in the membrane can affect the drug transport activity and vice versa.

This review discusses the diversity of structure and physicochemical properties of antimicrobial peptides and their derivatives, various chemical synthetic strategies that have been applied in their development, and how this links to their activity.

This tutorial review discusses the potential of ribosomally synthesised and post-translationally modified peptides (RiPPs) as antimicrobials and looks at the chemical synthesis of three classes of RiPP: lasso peptides, cyclotides, and lanthipeptides.

A functionalisable divinyltriazine linker was developed for two-component peptide stapling and macrocyclisation of cysteine residues.