DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras.

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

Erratum: Author Correction: High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity.

[This corrects the article DOI: 10.1038/s42003-019-0587-z.].

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity.

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 µM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

ATP Acyl Phosphate Reactivity Reveals Native Conformations of Hsp90 Paralogs and Inhibitor Target Engagement.

Hsp90 is an ATP-dependent chaperone of widespread interest as a drug target. Here, using an LC-MS/MS chemoproteomics platform based on a lysine-reactive ATP acyl phosphate probe, several Hsp90 inhibitors were profiled in native cell lysates. Inhibitor specificities for all four human paralogs of Hsp90 were simultaneously monitored at their endogenous relative abundances. Equipotent inhibition of probe labeling in each paralog occurred at sites both proximal to and distal from bound ATP observed in Hsp90 cocrystal structures, suggesting that the ATP probe is assaying a native conformation not predicted by available structures. Inhibitor profiling against a comprehensive panel of protein kinases and other ATP-binding proteins detected in native cell lysates identified PMS2, a member of the GHKL ATPase superfamily as an off-target of NVP-AUY922 and radicicol. Because of the endogenously high levels of Hsp90 paralogs in typical cell lysates, the measured potency of inhibitors was weaker than published IC50 values. Significant inhibition of Hsp90 required inhibitor concentrations above a threshold where off-target activity was detectable. Direct on- and off-target engagement was measured by profiling lysates derived from cells treated with Hsp90 inhibitors. These studies also assessed the downstream cellular pathway effects of Hsp90 inhibition, including the down regulation of several known Hsp90 client proteins and some previously unknown client proteins. Overall, the ATP probe-based assay methodology enabled a broad characterization of Hsp90 inhibitor activity and specificity in native cell lysates.

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways.

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.

Transiently misacylated tRNA is a primer for editing of misactivated adenylates by class I aminoacyl-tRNA synthetases.

The genetic code depends on amino acid fine structure discrimination by aminoacyl-tRNA synthetases. For isoleucyl- (IleRS) and valyl-tRNA synthetases (ValRS), reactions that hydrolyze misactivated noncognate amino acids help to achieve high accuracy in aminoacylation. Two editing pathways contribute to aminoacylation fidelity: pretransfer and post-transfer. In pretransfer editing, the misactivated amino acid is hydrolyzed as an aminoacyl adenylate, while in post-transfer editing a misacylated tRNA is deacylated. Both reactions are dependent on a tRNA cofactor and require translocation to a site located approximately 30 A from the site of amino acid activation. Using a series of 3'-end modified tRNAs that are deficient in either aminoacylation, deacylation, or both, total editing (the sum of pre- and post-transfer editing) was shown to require both aminoacylation and deacylation activities. These and additional results with IleRS are consistent with a post-transfer deacylation event initiating formation of an editing-active enzyme/tRNA complex. In this state, the primed complex processively edits misactivated valyl-adenylate via the pretransfer route. Thus, misacylated tRNA is an obligatory intermediate for editing by either pathway.

Plasticity of recognition of the 3'-end of mischarged tRNA by class I aminoacyl-tRNA synthetases.

Certain aminoacyl-tRNA synthetases prevent potential errors in protein synthesis through deacylation of mischarged tRNAs. For example, the close homologs isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) deacylate Val-tRNA(Ile) and Thr-tRNA(Val), respectively. Here we examined the chemical requirements at the 3'-end of the tRNA for these hydrolysis reactions. Single atom substitutions at the 2'- and 3'-hydroxyls of a variety of mischarged RNAs revealed that, while acylation is at the 2'-OH for both enzymes, IleRS catalyzes deacylation specifically from the 3'-OH and not from the 2'-OH. In contrast, ValRS can deacylate non-cognate amino acids from the 2'-OH. Moreover, for IleRS the specificity for a 3'-O location of the scissile ester bond could be forced to the 2'-position by introduction of a 3'-O-methyl moiety. Cumulatively, these and other results suggest that the editing sites of these class I aminoacyl-tRNA synthetases have a degree of inherent plasticity for substrate recognition. The ability to adapt to subtle differences in mischarged RNAs may be important for the high accuracy of aminoacylation.