Transthiocarbamoylation of proteins by thiolated isothiocyanates.
ABSTRACT Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits. Here we shed light on isothiocyanate-thiol conjugates and studied their electrophilic potential transferring an isothiocyanate moiety to cellular proteins. When we examined the effect of sulfhydryl molecules on cellular response induced by 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analog of sulforaphane isolated from broccoli, we observed significant induction of heme oxygenase-1 by 6-HITC even in the presence of N-acetyl-L-cysteine or glutathione (GSH). In addition, the authentic 6-HITC-β-mercaptoethanol (6-HITC-ME) conjugate markedly up-regulated the enzyme expression, suggesting the electrophilic potential of thiolated isothiocyanates. To gain a chemical insight into the cellular response induced by thiolated isothiocyanates, we studied the occurrence of transthiocarbamoylation of sulfhydryl molecules by 6-HITC-ME and observed that, upon incubation of 6-HITC-ME with GSH, a single product corresponding to the GSH conjugate of 6-HITC was generated. To test the functional ability of thiolated isothiocyanates to thiocarbamoylate proteins in living cells, we designed a novel probe, combining an isothiocyanate-reactive group and an alkyne functionality, and revealed that the transthiocarbamoylation of proteins occurred in the cells upon exposure to 6-HITC-ME. The target of thiocarbamoylation included heat shock protein 90 β (Hsp90β), a chaperone ATPase of the Hsp90 family implicated in protein maturation and targeting. To identify the sites of the Hsp90β modification, we utilized nano-LC/MALDI-TOF MS/MS and suggested that a thiol group on the peptide containing Cys-521 reacted with 6-HITC, resulting in a covalent adduct in a 6-HITC-treated recombinant Hsp90β in vitro. The site-selective binding to Cys-521 was supported by in silico modeling. Further study on the thiocarbamoylation of Hsp90β suggested that the formation of 6-HITC-Hsp90β conjugate might cause activation of heat shock factor-1, rapidly signaling a potential heat shock response. These data suggest that thiolated isothiocyanates are an active metabolite that could contribute to cellular responses through transthiocarbamoylation of cellular proteins.
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ABSTRACT: Background:Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation.Methods:Combining chemical synthesis, biological evaluation, and structure-activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone.Results:The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives.Conclusion:By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.British Journal of Cancer advance online publication, 5 December 2013; doi:10.1038/bjc.2013.710 www.bjcancer.com.British Journal of Cancer 12/2013; 110(1). DOI:10.1038/bjc.2013.710 · 4.82 Impact Factor
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ABSTRACT: Bifunctional electrophiles have been used in various chemopreventive, chemotherapeutic, and bioconjugate applications. Many of their effects in biological systems are traceable to their reactive properties, whereby they can modify nucleophilic sites in DNA, proteins, and other cellular molecules. Previously, we found that two different bifunctional electrophiles-diethyl acetylenedicarboxylate and divinyl sulfone-exhibited a strong enhancement of toxicity when compared with analogous monofunctional electrophiles in both human colorectal carcinoma cells and baker's yeast. Here, we have compared the toxicities for a broader panel of homobifunctional electrophiles bearing diverse electrophilic centers (e.g., isothiocyanate, isocyanate, epoxide, nitrogen mustard, and aldehyde groups) to their monofunctional analogs. Each bifunctional electrophile showed at least a three-fold enhancement of toxicity over its monofunctional counterpart, although in most cases the differences were even more pronounced. To explain their enhanced toxicity, we tested the ability of each bifunctional electrophile to cross-link recombinant yeast thioredoxin 2 (Trx2), a known intracellular target of electrophiles. The bifunctional electrophiles were capable of cross-linking Trx2 to itself in vitro and to other proteins in cells exposed to toxic concentrations. Moreover, most cross-linkers were preferentially reactive with thiols in these experiments. Collectively, our results indicate that thiol-reactive protein cross-linkers in general are much more potent cytotoxins than analogous monofunctional electrophiles, irrespective of the electrophilic group studied.Chemical Research in Toxicology 10/2013; 26(11). DOI:10.1021/tx400290j · 4.19 Impact Factor
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ABSTRACT: The KEAP1/NRF2 pathway and the heat shock response are two essential cytoprotective mechanisms that allow adaptation and survival under conditions of oxidative, electrophilic, and thermal stress by regulating the expression of elaborate networks of genes with versatile protective functions. The two pathways are independently regulated by the transcription factor nuclear factor-erythroid 2 p45-related factor 2 (NRF2) and heat shock factor 1 (HSF1), respectively. The activity of these transcriptional master regulators increases during conditions of stress and also upon encounter of small molecules (inducers), both naturally occurring as well as synthetically produced. Inducers have a common chemical property: the ability to react with sulfhydryl groups. The protein targets of such sulfhydryl-reactive compounds are equipped with highly reactive cysteine residues, which serve as sensors for inducers. The initial cysteine-sensed signal is further relayed to affect the expression of large networks of genes, which in turn can ultimately influence complex cell fate decisions such as life and death. The paper summarizes the multiple lines of experimental evidence demonstrating that the reactivity with sulfhydryl groups is a major determinant of the mechanism of action of small molecule dual activators of the KEAP1/NRF2 pathway and the heat shock response.12/2012; 2012:606104. DOI:10.6064/2012/606104