Selective Inhibition of Cysteine-Dependent Enzymes by Bioorthogonal Tethering.

A general approach for the rapid and selective inhibition of enzymes in cells using a common tool compound would be of great value for research and therapeutic development. We previously reported a chemogenetic strategy that addresses this challenge for kinases, relying on bioorthogonal tethering of a pan inhibitor to a target kinase through a genetically encoded non-canonical amino acid. However, pan inhibitors are not available for many enzyme classes. Here, we expand the scope of the chemogenetic strategy to cysteine-dependent enzymes by bioorthogonal tethering of electrophilic warheads. For proof of concept, selective inhibition of two E2 ubiquitin-conjugating enzymes, UBE2L3 and UBE2D1, was demonstrated in biochemical assays. Further development and optimization of this approach should enable its use in cells as well as with other cysteine-dependent enzymes, facilitating the investigation of their cellular function and validation as therapeutic targets.

ß-Lactam TRPM8 Antagonist RGM8-51 Displays Antinociceptive Activity in Different Animal Models.

Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a ß-lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases.

Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells.

Genetic code expansion allows unnatural (non-canonical) amino acid incorporation into proteins of interest by repurposing the cellular translation machinery. The development of this technique has enabled site-specific incorporation of many structurally and chemically diverse amino acids, facilitating a plethora of applications, including protein imaging, engineering, mechanistic and structural investigations, and functional regulation. Particularly, genetic code expansion provides great tools to study mammalian proteins, of which dysregulations often have important implications in health. In recent years, a series of methods has been developed to modulate protein function through genetically incorporated unnatural amino acids. In this review, we will first discuss the basic concept of genetic code expansion and give an up-to-date list of amino acids that can be incorporated into proteins in mammalian cells. We then focus on the use of unnatural amino acids to activate, inhibit, or reversibly modulate protein function by translational, optical or chemical control. The features of each approach will also be highlighted.