The anticancer properties of cruciferous vegetables are well known and attributed to an abundance of isothiocyanates (ITCs) such as benzyl ITC (BITC) and phenethyl ITC (PEITC). While many potential targets of ITCs have been proposed, a full understanding of the mechanisms underlying their anticancer activity has remained elusive. Here we report that BITC and PEITC effectively inhibit deubiquitinating enzymes (DUBs), including the enzymes USP9x and UCH37, which are associated with tumorigenesis, at physiologically relevant concentrations and time scales. USP9x protects the anti-apoptotic protein Mcl-1 from degradation, and cells dependent on Mcl-1 were especially sensitive to BITC and PEITC. These ITCs increased Mcl-1 ubiquitination and either ITC treatment or RNAi-mediated silencing of USP9x decreased Mcl-1 levels, consistent with the notion that USP9x is a primary target of ITC activity. These ITCs also increased ubiquitination of the oncogenic fusion protein Bcr-Abl, resulting in degradation under low ITC concentrations and aggregation under high ITC concentrations. USP9x inhibition paralleled the decrease in Bcr-Abl levels induced by ITC treatment, and USP9x silencing was sufficient to decrease Bcr-Abl levels, further suggesting that Bcr-Abl is a USP9x substrate. Overall, our findings suggest that USP9x targeting is critical to the mechanism underpinning the well established anticancer activity of ITC. We propose that the ITC-induced inhibition of DUB may also explain how ITCs affect inflammatory and DNA repair processes, thus offering a unifying theme in understanding the function and useful application of ITCs to treat cancer as well as a variety of other pathological conditions.

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 μM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.

Cruciferous vegetables such as broccoli and kale have well documented chemopreventative and anticancer effects that are attributed to the presence of isothiocyanates (ITCs). ITCs modulate the levels of many oncogenic proteins, but the molecular mechanisms of ITC action are not understood. We previously reported that phenethyl isothiocyanate (PEITC) inhibits two deubiquitinases (DUBs), USP9x and UCH37. DUBs regulate many cellular processes and DUB dysregulation is linked to the pathogenesis of human diseases including cancer, neurodegeneration, and inflammation. Using SILAC assisted quantitative mass spectrometry, here we identify 9 new PEITC-DUB targets: USP1, USP3, USP10, USP11, USP16, USP22, USP40, USP48 and VCPIP1. Seven of these PEITC-sensitive DUBs have well-recognized roles in DNA repair or chromatin remodeling. PEITC both inhibits USP1 and increases its ubiquitination and degradation, thus decreasing USP1 activity by two mechanisms. The loss of USP1 activity increases the level of mono-ubiquitinated DNA clamp PCNA, impairing DNA repair. Both the inhibition/degradation of USP1 and the increase in mono-ubiquitinated PCNA are new activities for PEITC that can explain the previously recognized ability of ITCs to enhance cancer cell sensitivity to cisplatin treatment. Our work also demonstrates that PEITC reduces the mono-ubiquityl histones H2A and H2B. Understanding the mechanism of action of ITCs should facilitate their use as therapeutic agents.

Deubiquitinating enzymes (DUBs) have emerged as promising drug targets, but few small molecule DUB inhibitors have been identified. Herein we introduce carbonates as a novel class of mechanism‐based DUB inhibitors. Activity profiling was used to determine inhibitor potency and specificity in cell lysates. The most potent inhibitor, C17, has a preference for USP9x and USP7. In K562 cells, C17 (50 micromolar) causes the degradation of Bcr‐Abl kinase, as expected when USP9x is inhibited. In MCF7 cells, C17 (25 micromolar) causes the degradation of Mdm2 and upregulation of P53, as expected when USP7 is inhibited. We believe that carbonates will be useful tools for studying the ubiquitination pathways and provide a new strategy for the design of cysteine protease inhibitors. Grant Funding Source: Supported by the NIH R01 GM100921(LH), HHMI International Student Fellowship (MJCL).

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The chemoprotective effects of a diet rich in broccoli or kale has been appreciated for several decades. Such cruciferous vegetables are a rich source of isothiocyanates (ITCs) such as benzyl ITC (BITC) and phenethyl ITC (PEITC). Each of these ITCs have antiproliferative activity against various tumors and PEITC is in clinical trials for lung and oral cancers. However, the mechanism by which ITCs suppress carcinogenesis has been the subject of much debate and numerous potential targets have been proposed. Here we show that BITC and PEITC inhibit the deubiquitinating enzyme (DUB) USP9x in vitro and in living cells. Both ITC treatment and USP9x knockdown decrease the levels of the oncogenic proteins MCL1 and Bcr‐Abl kinase. BITC and PEITC also inhibit UCH37, a proteasome associated DUB involved in the degradation of many proteins. Competitive activity profiling in cells pre‐treated with these ITCs suggests that other DUBs may also be inhibited. Inhibition occurs at physiologically relevant concentrations and time scales, and thus can explain many of the anticancer properties of dietary ITCs.

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