Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons

Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice

Variant PRC1 Complex-Dependent H2A Ubiquitylation Drives PRC2 Recruitment and Polycomb Domain Formation

Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.This study was funded by the Wellcome Trust (WT0834922 and WT081385), CRUK (C28585/A10839), NIHR, EMBO, Lister Institute of Preventative Medicine, RIKEN, MEXT, and JST CRES

Probing the Binding Requirements of Modified Nucleosides with the DNA Nuclease SNM1A

SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However, the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester, carboxylate and hydroxamic acid malonate derivatives which were installed in the 5′-position or 3′-position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration. The most potent compound contains a hydroxamic acid malonate group at the 5′-position. Overall, our studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and indicate that groups containing a negatively charged group in close proximity to a metal chelator, such as hydroxamic acid malonates, are promising structures in the design of inhibitors

Strategies to Target Specific Components of the Ubiquitin Conjugation/Deconjugation Machinery

The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery

Exploring the binding pockets of the DNA damage repair enzyme SNM1A through nucleobase modification

The DNA damage repair enzyme SNM1A is a potential target for anti-cancer chemotherapy. SNM1A has a metal-containing active site that has previously been successfully targeted with nucleosides/oligonucleotides modified with metal–binding groups. Nucleosides with modifications in the 3′- and 5′-positions of the ribose ring have been shown to inhibit SNM1A. To date, modification of the nucleobase has yet to be explored for targeting SNM1A. Herein, we describe the generation of a nucleoside modified with a 5′-metal-binding group and an alkyne handle on the nucleobase. Incorporation of the alkyne handle enables functionalisation for the exploration of peripheral binding pockets. This is the first time that nucleobase modification has been explored for targeting SNM1A. However, the novel nucleoside derivative displayed no significant recognition by SNM1A

Probing the Binding Requirements of Modified Nucleosides with the DNA Nuclease SNM1A

SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However, the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester, carboxylate and hydroxamic acid malonate derivatives which were installed in the 5′-position or 3′-position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration. The most potent compound contains a hydroxamic acid malonate group at the 5′-position. Overall, our studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and indicate that groups containing a negatively charged group in close proximity to a metal chelator, such as hydroxamic acid malonates, are promising structures in the design of inhibitors

Flipping the switch: innovations in inducible probes for protein profiling

Over the past two decades, activity-based probes have enabled a range of discoveries, including the characterization of new enzymes and drug targets. However, their suitability in some labeling experiments can be limited by nonspecific reactivity, poor membrane permeability, or high toxicity. One method for overcoming these issues is through the development of “inducible” activity-based probes. These probes are added to samples in an unreactive state and require in situ transformation to their active form before labeling can occur. In this Review, we discuss a variety of approaches to inducible activity-based probe design, different means of probe activation, and the advancements that have resulted from these applications. Additionally, we highlight recent developments which may provide opportunities for future inducible activity-based probe innovations

Two-step validation approach for tools to study the DNA repair enzyme SNM1A

We report a two-step validation approach to evaluate the suitability of metal-binding groups for targeting DNA damage-repair metalloenzymes using model enzyme SNM1A. A fragment-based screening approach was first used to identify metal-binding fragments suitable for targeting the enzyme. Effective fragments were then incorporated into oligonucleotides using the copper-catalysed azide–alkyne cycloaddition reaction. These modified oligonucleotides were recognised by SNM1A at >1000-fold lower concentrations than their fragment counterparts. The exonuclease SNM1A is a key enzyme involved in the repair of interstrand crosslinks, a highly cytotoxic form of DNA damage. However, SNM1A and other enzymes of this class are poorly understood, as there is a lack of tools available to facilitate their study. Our novel approach of incorporating functional fragments into oligonucleotides is broadly applicable to generating modified oligonucleotide structures with high affinity for DNA damage-repair enzymes