Target Cysteine Residues Research Articles

Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

▪IL2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) are two members of the Tec family of non-receptor tyrosine kinases which are essential mediators of intracellular signaling in both normal and neoplastic T and natural killer (NK)-cells. ITK-deficient mice display alterations in T cell receptor (TCR) responses including proliferation, cytokine production and activation of downstream pathways, and these defects are exacerbated by RLK deficiency. Given their critical roles, inhibitors of ITK and RLK have therapeutic potential in a number of human autoimmune diseases, inflammatory conditions, and malignancies. Specific inhibitors targeting both ITK and RLK have not been reported and until very recently it has been challenging to develop specific inhibitors with clinical potential given the homology to many other human kinases. Targeting cysteine residues within the ATP binding pocket of kinases is a safe and effective way to design and optimize highly potent and selective kinase inhibitors. In this study, we describe preclinical mechanism of action studies of a novel covalent ITK/RLK inhibitor, PRN694, which covalently binds to cysteine moieties 442 of ITK and 350 of RLK and blocks their kinase activities.We undertook the design of highly selective ITK/RLK inhibitors using a combination of tailored covalency and structure-based design. Molecular modeling and a synthetic feasibility assessment led to the prioritization of several small focused compound libraries. The structure-based medicinal chemistry optimization of one such library culminated in the identification of PRN694. The in vitro selectivity and potency of PRN694 against a panel of 250 kinases revealed only small number of kinase targets, and in a microfluidic kinase assay, PRN694 exhibited high selectivity and potency against ITK and RLK with IC50 values of 0.3 nM and 1.3 nM, respectively. This selectivity was validated in Jurkat T cells with mutated ITK or overexpressed RLK. As expected, PRN694 suppressed a variety of downstream TCR pathways including nuclear activation of NFAT1 and cytoplasmic PLCγ1 and IKBα in TCR-activated Jurkat cells expressing wild type ITK. However, when the covalent binding site for PRN694 was removed, using ITK-C442A, this suppressive effect was lost, indicating specific covalent interaction with ITK. Furthermore, we demonstrated that only PRN694 was able to ablate TCR-induced activation in Jurkat cells which stably express RLK in addition to ITK. These data confirmed that PRN694 was a dual inhibitor of ITK and RLK and could block downstream TCR-activation in T-cells that express both complementary TEC-kinases. In vitro cellular assays also confirmed that PRN694 could prevent TCR or Fc receptor (FcR) - induced cellular and molecular activation in primary T or NK cells, inhibit TCR-induced T-cell proliferation without direct cytotoxicity, and block pro-inflammatory cytokine (including IL-2, TNFα, and IFNγ) release as well as activation of Th17 cells. Additionally, in vivo assays demonstrated durable pharmacodynamic effects of PRN694 on ITK. At 1 hour, 6 hour and 14 hour time points, the percentages of ITK occupancy were 98%, 95% and 54% respectively, and the concentrations of PRN694 in the plasma were 1530 ng/ml, 359.3 ng/ml and 14.8 ng/ml respectively.Further, we examined potential therapeutic applications of PRN694 in the treatment of T-cell leukemias or T-cell driven inflammation. Our results demonstrated an effective blockade of TCR stimulation by PRN694 in the HH cutaneous T-cell lymphoma cell line and primary T-cell prolymphocytic leukemia (T-PLL) cells, suggesting a potential clinical application of this compound in the treatment of T-cell leukemias. ITK and RLK play a critical role in T-cell driven inflammation. Therefore, we also tested the effect of PRN694 on an oxazolone induced delayed type hypersensitivity (DTH)reaction using a well-established in vivo model of inflammation driven by cell-mediated immunity, and found that PRN694 treatment significantly inhibited the DTH reaction.In conclusion, we designed and characterized a novel covalent ITK/RLK inhibitor, PRN694, which has direct therapeutic potential for the treatment of T or NK cell related diseases including inflammation, autoimmune diseases, and malignancy. DisclosuresPorcu:Actelion: Honoraria; Infinity: Research Funding; Seattle genetics: Research Funding; Celgene: Honoraria; United States Cutaneous Lymphoma Consortium: Membership on an entity’s Board of Directors or advisory committees; Cutaneous Lymphoma Foundation: Membership on an entity’s Board of Directors or advisory committees. Bradshaw:Principia Biopharma: Employment, Equity Ownership. Owens:Principia Biopharma, Inc: Employment, Equity Ownership. Brameld:Principia Biopharma: Employment, Equity Ownership. Funk:Principia Biopharma Inc.: Employment, Equity Ownership. Hill:Principia Biopharma: Employment. Johnson:Principia Biopharma: Research Funding. Dubovsky:Principia Inc.: Research Funding.

Computational prediction of candidate proteins for S-nitrosylation in Arabidopsis thaliana.

Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments “chloroplast,” “CUL4-RING ubiquitin ligase complex,” and “membrane” more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.

Hydrogen sulfide and polysulfides as biological mediators.

Hydrogen sulfide (H2S) is recognized as a biological mediator with various roles such as neuromodulation, regulation of the vascular tone, cytoprotection, anti-inflammation, oxygen sensing, angiogenesis, and generation of mitochondrial energy. It is produced by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). The activity of CBS is enhanced by S-adenosyl methionine (SAM) and glutathionylation, while it is inhibited by nitric oxide (NO) and carbon monoxide (CO). The activity of CSE and cysteine aminotransferase (CAT), which produces the 3MST substrate 3-mercaptopyruvate (3MP), is regulated by Ca2+. H2S is oxidized to thiosulfate in mitochondria through the sequential action of sulfide quinone oxidoreductase (SQR), sulfur dioxygenase, and rhodanese. The rates of the production and clearance of H2S determine its cellular concentration. Polysulfides (H2Sn) have been found to occur in the brain and activate transient receptor potential ankyrin 1 (TRPA1) channels, facilitate the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus, and suppress the activity of phosphatase and tensin homolog (PTEN) by sulfurating (sulfhydrating) the target cysteine residues. A cross talk between H2S and NO also plays an important role in cardioprotection as well as regulation of the vascular tone. H2S, polysulfides, and their cross talk with NO may mediate various physiological and pathophysiological responses.

Abstract LB-324: First isoform selective inhibitor of FGFR4 for the treatment of genomically defined patients with hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. Chemotherapy has proven ineffective, and Sorafenib remains the only approved targeted drug with no second or third line treatment options. Sorafenib slows the growth of advanced liver cancers and helps some patients live longer - by an average of about three months. There is a pressing need for more effective therapies. FGF19 is a highly controlled hormone normally expressed in the intestine, that acts in the liver to regulate bile acid synthesis and hepatocyte proliferation via activation of FGFR4. In 7% of patients with HCC, FGF19 is contained within a focal amplification on chromosome 11q13.3. Overexpression of FGF19 in transgenic mice produces liver tumors which are sensitive to treatment with a FGFR4 tool antibody. Additionally, the growth of tumor cells in xenograft models with FGF19 amplification is dependent on FGFR4 signaling. Thus, selective inhibition of FGFR4 might represent a viable strategy for treating this genetically defined subgroup of HCC patients. Herein, we describe our efforts to identify an ultraselective small molecule inhibitor of FGFR4 which spares the other FGFR isoforms with the intent to avoid FGFR1-3 driven, dose limiting toxicities like soft tissue mineralization. Utilizing structure based drug design, we prepared a series of inhibitor templates designed to covalently modify a target cysteine residue present in FGFR4, but not the other FGFR isoforms. Optimization of one of these templates led to the identification of BLU9931, a highly potent and exquisitely selective, covalent inhibitor of FGFR4. BLU9931 persistently inhibits FGFR4 mediated signaling in cancer cells as evidenced by decreased phosphorylation of FRS2 and ERK. Importantly, BLU9931 does not block signaling driven by FGFR1. Upon oral dosing in mice, BLU9931 is well tolerated and demonstrates robust and dose dependent induction of the FGFR4 target gene CYP7a1 in Hep3B cells, a FGF19 amplified HCC xenograft model. Upon extended dosing, BLU9931 causes sustained regression of tumors, including complete responses. We then explored if HCCs with alterations other than FGF19 amplification are also dependent on FGFR4 signaling. Dosing of molecularly annotated patient derived HCC xenografts with BLU9931 suggests that selective targeting of FGFR4 represents a viable option for the treatment of an expanded population segment of genomically defined HCC patients, much larger than originally anticipated. Citation Format: Margit Hagel, Chandra Miduturu, Mike Sheets, Weifan Weng, Nooreen Rubin, Neil Bifulco, Lucian DiPietro, Joseph Kim, Natasja Brooijmans, Nicolas Stransky, Christopher Winter, Christoph Lengauer, Timothy Guzi. First isoform selective inhibitor of FGFR4 for the treatment of genomically defined patients with hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-324. doi:10.1158/1538-7445.AM2014-LB-324

Arsenite binds to the RING finger domains of RNF20-RNF40 histone E3 ubiquitin ligase and inhibits DNA double-strand break repair.

Arsenic is a widespread environmental contaminant. However, the exact molecular mechanisms underlying the carcinogenic effects of arsenic remain incompletely understood. Core histones can be ubiquitinated by RING finger E3 ubiquitin ligases, among which the RNF20-RNF40 heterodimer catalyzes the ubiquitination of histone H2B at lysine 120. This ubiquitination event is important for the formation of open and biochemically accessible chromatin fiber that is conducive for DNA repair. Herein, we found that arsenite could bind directly to the RING finger domains of RNF20 and RNF40 in vitro and in cells, and treatment with arsenite resulted in substantially impaired H2B ubiquitination in multiple cell lines. Exposure to arsenite also diminished the recruitment of BRCA1 and RAD51 to laser-induced DNA double-strand break (DSB) sites, compromised DNA DSB repair in human cells, and rendered cells sensitive toward a radiomimetic agent, neocarzinostatin. Together, the results from the present study revealed, for the first time, that arsenite may exert its carcinogenic effect by targeting cysteine residues in the RING finger domains of histone E3 ubiquitin ligase, thereby altering histone epigenetic mark and compromising DNA DSB repair. Our results also suggest arsenite as a general inhibitor for RING finger E3 ubiquitin ligases.

Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins

AbstractIn this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescent‐labeling studies demonstrated that the installation of a C‐terminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site‐specific labeling of cysteine‐containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.

Allenamides as orthogonal handles for selective modification of cysteine in peptides and proteins.

In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescent-labeling studies demonstrated that the installation of a C-terminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site-specific labeling of cysteine-containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.

Reactivity of C-Terminal Cysteines with HNO

Nitroxyl (HNO), a potential heart failure therapeutic, is known to target cysteine residues to form sulfinamides and/or disulfides. Because HNO-derived modifications may depend on their local environment, we have investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-containing peptides at physiological pH and temperature. Our findings indicate that the nature of HNO-derived modifications of C-terminal cysteines is affected by the C-terminal carboxylate. Apart from the lack of sulfinamide formation, these studies have revealed the presence of new products, a sulfohydroxamic acid derivative (RS(O)2NHOH) and a thiosulfonate (RS(O)2SR), presumably produced under our experimental conditions via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH). Moreover, these modifications are formed independent of oxygen.

Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

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.

Nitric oxide-based protein modification: formation and site-specificity of protein S-nitrosylation

Nitric oxide (NO) is a reactive free radical with pleiotropic functions that participates in diverse biological processes in plants, such as germination, root development, stomatal closing, abiotic stress, and defense responses. It acts mainly through redox-based modification of cysteine residue(s) of target proteins, called protein S-nitrosylation.In this way NO regulates numerous cellular functions and signaling events in plants. Identification of S-nitrosylated substrates and their exact target cysteine residue(s) is very important to reveal the molecular mechanisms and regulatory roles of S-nitrosylation. In addition to the necessity of protein–protein interaction for trans-nitrosylation and denitrosylation reactions, the cellular redox environment and cysteine thiol micro-environment have been proposed important factors for the specificity of protein S-nitrosylation. Several methods have recently been developed for the proteomic identification of target proteins. However, the specificity of NO-based cysteine modification is still less defined. In this review, we discuss formation and specificity of S-nitrosylation. Special focus will be on potential S-nitrosylation motifs, site-specific proteomic analyses, computational predictions using different algorithms, and on structural analysis of cysteine S-nitrosylation.

From Sulfenylation to Sulfhydration: What a Thiolate Needs to Tolerate

There is a growing appreciation that oxidants such as hydrogen peroxide (H(2)O(2)) and gases such as nitric oxide (NO) and hydrogen sulfide (H(2)S) can act as modulators of various signaling pathways. Much of this signaling occurs through the modifications of specific, critical cysteine residues in target proteins. How such small, diffusible molecules (H(2)O(2), NO, H(2)S) can achieve the required specificity is incompletely understood. Now, new findings provide considerable insight into these and related questions.

Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO

BackgroundEndothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.MethodsGolgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.ResultsSeventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.ConclusionOur data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.

A change in the sensitivity of elongation factor G to oxidation protects photosystem II from photoinhibition in Synechocystis sp. PCC 6803

The repair of photosystem II (PSII) after photodamage is particularly sensitive to oxidative stress and inhibition of such repair is associated with the oxidation of specific cysteine residues in elongation factor G (EF-G), a key translation factor, in the cyanobacterium Synechocystis sp. PCC 6803. Expression of mutated EF-G with a target cysteine residue replaced by serine in Synechocystis resulted in the protection of PSII from photoinhibition. This protection was attributable to the enhanced repair of PSII via acceleration of the synthesis of the D1 protein, which might have been due to reduced sensitivity of protein synthesis to oxidative stress.

HCB2 Ligand-Interaction Landscape: Cysteine Residues Critical to Biarylpyrazole Antagonist Binding Motif and Receptor Modulation

The human cannabinoid 2 GPCR (hCB2) is a prime therapeutic target. To define potential cysteine-related binding motifs critical to hCB2-ligand interaction, a library of hCB2 cysteine-substitution mutants and a novel, high-affinity biarylpyrazole hCB2 antagonist/inverse agonist (AM1336) functionalized to serve as a covalent affinity probe to target cysteine residues within (or in the microenvironment of) its hCB2 binding pocket were generated. The data provide direct experimental demonstration that both hCB2 TMH7 cysteines [i.e., C7.38(284) and C7.42(288)] are critical to optimal hCB2-AM1336 binding interaction and AM1336 pharmacological activity in a cell-based functional assay (cAMP formation). Elongating the AM1336 aliphatic side chain generated another novelhCB2 inverse agonist that binds covalently and selectively to C7.42(288) only. Identification of specific cysteine residues critical to hCB2 ligand interaction and function informs the structure-based design of hCB2-targeted medicines.

Regulatory Effect of Cinnamaldehyde on Monocyte/Macrophage-Mediated Inflammatory Responses

Cinnamaldehyde (CA) has been known to exhibit anti-inflammatory and anticancer effects. Although numerous pharmacological effects have been demonstrated, regulatory effect of CA on the functional activation of monocytes and macrophages has not been fully elucidated yet. To evaluate its monocyte/macrophage-mediated immune responses, macrophages activated by lipopolysaccharide (LPS), and monocytes treated with proaggregative antibodies, and extracellular matrix protein fibronectin were employed. CA was able to suppress both the production of nitric oxide (NO) and upregulation of surface levels of costimulatory molecules (CD80 and CD69) and pattern recognition receptors (toll-like receptor 2 (TLR2) and complement receptor (CR3)). In addition, CA also blocked cell-cell adhesion induced by the activation of CD29 and CD43 but not cell-fibronectin adhesion. Immunoblotting analysis suggested that CA inhibition was due to the inhibition of phosphoinositide-3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK)1 as well as nuclear factor-(NF-) κB activation. In particular, thiol compounds with sulphydryl group, L-cysteine and dithiothreitol (DTT), strongly abrogated CA-mediated NO production and NF-κB activation. Therefore, our results suggest that CA can act as a strong regulator of monocyte/macrophage-mediated immune responses by thiolation of target cysteine residues in PI3K or PDK1.

Genome‐wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol‐dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR‐family regulator YraB (AdhR)

Quinones and alpha,beta-unsaturated carbonyls are naturally occurring electrophiles that target cysteine residues via thiol-(S)-alkylation. We analysed the global expression profile of Bacillus subtilis to the toxic carbonyls methylglyoxal (MG) and formaldehyde (FA). Both carbonyl compounds cause a stress response characteristic for thiol-reactive electrophiles as revealed by the induction of the Spx, CtsR, CymR, PerR, ArsR, CzrA, CsoR and SigmaD regulons. MG and FA triggered also a SOS response which indicates DNA damage. Protection against FA is mediated by both the hxlAB operon, encoding the ribulose monophosphate pathway for FA fixation, and a thiol-dependent formaldehyde dehydrogenase (AdhA) and DJ-1/PfpI-family cysteine proteinase (YraA). The adhA-yraA operon and the yraC gene, encoding a gamma-carboxymuconolactone decarboxylase, are positively regulated by the MerR-family regulator, YraB(AdhR). AdhR binds specifically to its target promoters which contain a 7-4-7 inverted repeat (CTTAAAG-N4-CTTTAAG) between the -35 and -10 elements. Activation of adhA-yraA transcription by AdhR requires the conserved Cys52 residue in vivo. We speculate that AdhR is redox-regulated via thiol-(S)-alkylation by aldehydes and that AdhA and YraA are specifically involved in reduction of aldehydes and degradation or repair of damaged thiol-containing proteins respectively.

Induction of apoptosis by phenethyl isothiocyanate in cells overexpressing Bcl-X L

Isothiocyanates are a class of phytochemicals able to induce apoptosis in numerous cells including Jurkat T-lymphoma cells overexpressing the oncoprotein Bcl-2. To test if isothiocyanates are also effective against other anti-apoptotic members of the Bcl-2 family we generated Jurkat cells stably overexpressing Bcl-X L. Phenethyl isothiocyanate (PEITC) was cytotoxic to these cells, with an LD 50 ranging from 9 to 18 μM depending on the level of Bcl-X L expression. Apoptosis induction in response to PEITC was confirmed by caspase activation and phosphatidylserine exposure. Isothiocyanates specifically target cysteine residues, therefore we tested the hypothesis that PEITC directly impairs Bcl-2 and Bcl-X L activity by interacting with their conserved cysteine residues. Jurkat cells overexpressing double cysteine mutants of Bcl-2 were generated, but they remained sensitive to PEITC. We conclude that PEITC antagonizes the action of anti-apoptotic Bcl-2 family members via an indirect mechanism.

The Stability and Functionality of Chemically Crosslinked Microtubules

A variety of bifunctional crosslinking agents have been explored for stabilizing microtubule shuttles used for the active transport of nanomaterials in artificial environments. Crosslinking agents that target amine residues form intertubulin crosslinks that produce crosslinked microtubules (CLMTs) with structural and functional lifetimes that can be up to four times as long as those achieved with taxol stabilization. Such CLMTs are stable at temperatures down to -10 degrees C, are resistant to depolymerization induced by metal ions such as Ca2+, and yet continue to be adsorbed and transported by self-assembled monolayers containing the motor protein kinesin. However, crosslinkers that target cysteine residues depolymerize the MTs, probably by interfering with the guanosine triphosphate binding site. The impact of crosslink attributes, including terminal group chemistry, chain length, crosslink density, and specific location on the tubulin surface, on microtubule stability and functionality are discussed.

Allosteric Inhibition of PTP1B Activity by Selective Modification of a Non-Active Site Cysteine Residue

The fluorogenic reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABDF) attenuates the functional activity of the protein tyrosine phosphatase PTP1B by reacting selectively with a single cysteine residue, leaving other cysteines in the protein unmodified. This modification reduces Vmax without substantially affecting substrate binding (Km), indicative of an allosteric mode of inhibition. Consistent with this, the cysteine residue modified by ABDF, Cys 121, lies outside the catalytic site but makes interactions with residues that contact His 214, which has been shown to be important for catalysis. Cys 121 is highly conserved among phosphatases, and ABDF also inhibits TC-PTP and LAR. These findings illustrate that targeting cysteine residues outside catalytic sites may be exploited in allosterically regulating enzymes. Moreover, these results suggest a new strategy for inhibiting a promising diabetes target.

Diethylmaleate activates the transcription factor Pap1 by covalent modification of critical cysteine residues

During the last decade, much has been learnt about the mechanisms by which oxidative stress is perceived by aerobic organisms. The Schizosaccharomyces pombe Pap1 protein is a transcription factor localized at the cytoplasm, which accumulates in the nucleus in response to different inducers, such as the pro-oxidant hydrogen peroxide (H2O2) or the glutathione-depleting agent diethylmaleate (DEM). As described for other H2O2 sensors, our genetic data indicates that H2O2 reversibly oxidizes two cysteine residues in Pap1 (Cys278 and Cys501). Surprisingly, our studies demonstrate that DEM generates a non-reversible modification of at least two cysteine residues located in or close to the nuclear export signal of Pap1 (Cys523 and Cys532). This modification impedes the interaction of the nuclear exporter Crm1 with the nuclear export signal located at the carboxy-terminal domain of Pap1. Mass spectrometry data suggest that DEM binds to the thiol groups of the target cysteine residues through the formation of a thioether. Here we show that DEM triggers Pap1 nuclear accumulation by a novel molecular mechanism.