A yeast-based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis

Chidley, Christopher; Haruki, Hirohito; Pedersen, Miriam Grønlund; Müller, Emil; Johnsson, Kai

doi:10.1038/nchembio.557

Cited by 114 publications

(141 citation statements)

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“…The fact that BH4 was able to reverse the inhibitory effects of sulfathiazole on catecholamine/monoamine production in PC12 cells indicated that the sulfa drug was targeting SPR. Our data are consistent with earlier studies showing that various sulfa drugs are effective inhibitors of BH4 biosynthesis in cytokine-stimulated primary human dermal fibroblasts, PC12 cells, and human neuroblastoma cells SK-N-BE(2), as well as the formation of L-3,4-dihydroxyphenylalanine and 4-methoxytyramine in SK-N-BE(2) cells (Chidley et al, 2011;Haruki et al, 2013).…”

Section: Discussionsupporting

confidence: 81%

“…In the case of a nonsteroidal anti-inflammatory agent such as indomethacin, this may contribute to its therapeutic activity; alternatively, this may represent an "off-target" action of the drug, potentially resulting in toxicity. Recently, using a yeasthybrid system, an anti-inflammatory sulfonamide, sulfasalazine, was identified as an inhibitor of SPR (Chidley et al, 2011). Subsequently, sulfasalazine metabolites, as well as structurally related sulfonamide-and sulfonylurea-based antimicrobial/ anti-inflammatory and antidiabetic drugs, were shown to be inhibitors of SPR (Chidley et al, 2011;Haruki et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, using a yeasthybrid system, an anti-inflammatory sulfonamide, sulfasalazine, was identified as an inhibitor of SPR (Chidley et al, 2011). Subsequently, sulfasalazine metabolites, as well as structurally related sulfonamide-and sulfonylurea-based antimicrobial/ anti-inflammatory and antidiabetic drugs, were shown to be inhibitors of SPR (Chidley et al, 2011;Haruki et al, 2013). Inhibition of SPR by these drugs could result in reduced levels of BH2 and BH4, causing inhibition of enzymes that require the biopterin cofactor and leading to toxicity.…”

Section: Introductionmentioning

confidence: 99%

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Sulfa Drugs Inhibit Sepiapterin Reduction and Chemical Redox Cycling by Sepiapterin Reductase

Yang

Jan

Mishin

et al. 2015

The Journal of Pharmacology and Experimental Therapeutics

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Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), the precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one-electron reduction of redox-active chemicals, including quinones and bipyridinium herbicides (e.g., menadione, 9,10-phenanthrenequinone, and diquat); rapid reaction of the reduced radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide-and sulfonylurea-based sulfa drugs were found to be potent noncompetitive inhibitors of both sepiapterin reduction and redox cycling. The most potent inhibitors of sepiapterin reduction (IC 50 s 5 31-180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole, and chlorpropamide. Higher concentrations of the sulfa drugs (IC 50 s 5 0.37-19.4 mM) were required to inhibit redox cycling, presumably because of distinct mechanisms of sepiapterin reduction and redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4-dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling mediated by SPR. Inhibition of BH2/BH4 resulted in decreased production of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 5-hydroxytryptamine. Sulfathiazole (200 mM) markedly suppressed neurotransmitter production, an effect reversed by BH4. These data suggest that SPR and BH4-dependent enzymes, are "off-targets" of sulfa drugs, which may underlie their untoward effects. The ability of the sulfa drugs to inhibit redox cycling may ameliorate ROS-mediated toxicity generated by redox active drugs and chemicals, contributing to their anti-inflammatory activity.

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Section: Discussionsupporting

confidence: 81%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sulfa Drugs Inhibit Sepiapterin Reduction and Chemical Redox Cycling by Sepiapterin Reductase

Yang

Jan

Mishin

et al. 2015

The Journal of Pharmacology and Experimental Therapeutics

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“…Recent applications include its use for the analysis of protein complexes, 3 super-resolution microscopy, 4 the identification of protein−protein interactions, 5 drug target identification, 6 and the determination of protein half-life in animals. 7 The appeal of self-labeling tags such as SNAP-tag is the ease with which fusion proteins can be labeled with synthetic probes even in living cells.…”

mentioning

confidence: 99%

Directed Evolution of the Suicide Protein O⁶-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability

et al. 2012

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Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to higher stability, especially of the alkylated protein, suggesting that the reactivity of the suicide enzyme can be influenced by stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key α helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure−function relationship of the DNA repair protein.T he specific labeling of proteins with synthetic probes is a powerful approach for studying protein function. One way to achieve such a specific labeling is based on so-called selflabeling protein tags.1 In this approach, the protein of interest is expressed as a fusion protein with a peptide or protein (i.e., tag) whose role is to specifically bind to a synthetic probe in vitro or in vivo. A well-established example of a self-labeling protein tag is SNAP-tag.2 SNAP-tag specifically reacts with substituted O 6 -benzylguanine derivatives and thereby permits the labeling of SNAP-tag fusion proteins with a wide variety of different synthetic probes. Recent applications include its use for the analysis of protein complexes, 3 super-resolution microscopy, 4 the identification of protein−protein interactions, 5 drug target identification, 6 and the determination of protein half-life in animals. 7 The appeal of self-labeling tags such as SNAP-tag is the ease with which fusion proteins can be labeled with synthetic probes even in living cells. A conceptual limitation of the approach is the fact that the tag can affect the properties of its fusion partner. It is therefore important that the properties of the tag be as thoroughly characterized as possible.SNAP-tag was generated in a stepwise manner from human O 6 -alkylguanine-DNA alkyltransferase (hAGT) by introduction of a total of 19 point mutations (Figure 1) and deletion of 25 C-terminal residues. Saturation mutagenesis of four active-site residues followed by phage display and selection for activity against BG derivatives resulted in GE AGT, a mutant with 20-fold increased activity toward such substrates ( Figure 1B). 8Subsequent saturation mutagenesis of four additional residues involved in substrate binding followed by phage selection resulted in AGT-54, a mutant with 1.5-fold higher activity than GE AGT. To further optimize the protein for applications in protein labeling, mutations were introduced to suppress DNA binding and reactivity toward nucleosides, to remove nonessential cysteines, and to truncate the last 25 residues. 9The resulting mutant M AGT displayed relatively low activ...

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“…Expression-based trapping methods, such as yeast three-hybrid and MASPIT as a mammalian variant, have shown to be valuable alternatives. A modified yeast three-hybrid approach which was used to profile a number of clinically approved drugs was developed by the Johnsson group (55). We previously established the utility of MASPIT to identify known and novel targets of a number of kinase inhibitors, using a cDNA library screening platform (5).…”

Section: Discussionmentioning

confidence: 99%

Proteome-scale Binary Interactomics in Human Cells

Lievens

Heyden

Masschaele

et al. 2016

Molecular & Cellular Proteomics

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Because proteins are the main mediators of most cellular processes they are also prime therapeutic targets. Identifying physical links among proteins and between drugs and their protein targets is essential in order to understand the mechanisms through which both proteins themselves and the molecules they are targeted with act. Thus, there is a strong need for sensitive methods that enable mapping out these biomolecular interactions. Here we present a robust and sensitive approach to screen proteome-scale collections of proteins for binding to proteins or small molecules using the well validated MAPPIT (Mammalian Protein-Protein Interaction Trap) and MASPIT (Mammalian Small Molecule-Protein Interaction Trap) assays. Using high-density reverse transfected cell microarrays, a close to proteome-wide collection of human ORF clones can be screened for interactors at high throughput. The versatility of the platform is demonstrated through several examples. With MAPPIT, we screened a 15k ORF library for binding partners of RNF41, an E3 ubiquitin protein ligase implicated in receptor sorting, identifying known and novel interacting proteins. The potential related to the fact that MAPPIT operates in living human cells is illustrated in a screen where the protein collection is scanned for interactions with the glucocorticoid receptor (GR) in its unliganded versus dexamethasone-induced activated state. Several proteins were identified the interaction of which is modulated upon ligand binding to the GR, including a number of previously reported GR interactors. Finally, the screening technology also enables detecting small molecule target proteins, which in many drug discovery programs represents an important hurdle. We show the efficiency of MASPITbased target profiling through screening with tamoxifen, a first-line breast cancer drug, and reversine, an investigational drug with interesting dedifferentiation and antitumor activity. In both cases, cell microarray screens yielded known and new potential drug targets highlighting the utility of the technology beyond fundamental biology. Molecular & Cellular

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A yeast-based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis

Cited by 114 publications

References 50 publications

Sulfa Drugs Inhibit Sepiapterin Reduction and Chemical Redox Cycling by Sepiapterin Reductase

Sulfa Drugs Inhibit Sepiapterin Reduction and Chemical Redox Cycling by Sepiapterin Reductase

Directed Evolution of the Suicide Protein O⁶-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability

Proteome-scale Binary Interactomics in Human Cells

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A yeast-based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis

Cited by 114 publications

References 50 publications

Sulfa Drugs Inhibit Sepiapterin Reduction and Chemical Redox Cycling by Sepiapterin Reductase

Sulfa Drugs Inhibit Sepiapterin Reduction and Chemical Redox Cycling by Sepiapterin Reductase

Directed Evolution of the Suicide Protein O6-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability

Proteome-scale Binary Interactomics in Human Cells

Contact Info

Product

Resources

About

Directed Evolution of the Suicide Protein O⁶-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability