Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a recently discovered enzyme that catalyzes the hydrolysis of 3'-phosphotyrosyl bonds. Such linkages form in vivo following the DNA processing activity of ...topoisomerase I (Top1). For this reason, Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes, which can be generated by either exogenous or endogenous factors. Tdp1 has been regarded as a potential therapeutic co-target of Top1 in that it seemingly counteracts the effects of Top1 inhibitors, such as camptothecin and its clinically used derivatives. Thus, by reducing the repair of Top1-DNA lesions, Tdp1 inhibitors have the potential to augment the anticancer activity of Top1 inhibitors provided there is a presence of genetic abnormalities related to DNA checkpoint and repair pathways. Human Tdp1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolates and 3'-abasic sites indicating it may function as a general 3'-DNA phosphodiesterase and repair enzyme. The importance of Tdp1 in humans is highlighted by the observation that a recessive mutation in the human TDP1 gene is responsible for the inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1). This review provides a summary of the biochemical and cellular processes performed by Tdp1 as well as the rationale behind the development of Tdp1 inhibitors for anticancer therapy.
Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a recently discovered enzyme specifically repairing topoisomerase II (TOP2)-mediated DNA damage. It has been shown that inhibition of TDP2 synergize with ...TOP2 inhibitors. Herein, we report the discovery of the furoquinolinedione chemotype as a suitable skeleton for the development of selective TDP2 inhibitors. Compound 1 was identified as a TDP2 inhibitor as a result of screening our in-house compound library for compounds selective for TDP2 vs. TDP1. Further SAR studies provide several selective TDP2 inhibitors at low-micromolar range. The most potent compound 74 shows inhibitory activity with IC50 of 1.9 and 2.1 μM against recombinant TDP2 and TDP2 in whole cell extracts (WCE), respectively.
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•A novel TDP2 hit (1) was found through screening from in-house chemical library.•Seventy seven furoquinolinedione analogues were synthesized.•Compound 74 showed the most TDP2 inhibition with IC50 at low micromolar range.•The SAR was analyzed.•Furoquinolinedinone chemotype represents a novel skeleton for novel TDP2 inhibitors.
Tyrosyl-DNA phosphodiesterase I (Tdp1) is a cellular enzyme that repairs the irreversible topoisomerase I (Top1)-DNA complexes and confers chemotherapeutic resistance to Top1 inhibitors. Inhibiting ...Tdp1 provides an attractive approach to potentiating clinically used Top1 inhibitors. However, despite recent efforts in studying Tdp1 as a therapeutic target, its inhibition remains poorly understood and largely underexplored. We describe herein the discovery of arylidene thioxothiazolidinone as a scaffold for potent Tdp1 inhibitors based on an initial tyrphostin lead compound 8. Through structure-activity relationship (SAR) studies we demonstrated that arylidene thioxothiazolidinones inhibit Tdp1 and identified compound 50 as a submicromolar inhibitor of Tdp1 (IC₅₀ = 0.87 μM). Molecular modeling provided insight into key interactions essential for observed activities. Some derivatives were also active against endogenous Tdp1 in whole cell extracts. These findings contribute to advancing the understanding on Tdp1 inhibition.
Cysteines (Cys) made acidic by the protein environment are generally sensitive to pro-oxidant molecules. Glutathionylation is a post-translational modification that can occur by spontaneous reaction ...of reduced glutathione (GSH) with oxidized Cys as sulfenic acids (-SOH). The reverse reaction (deglutathionylation) is strongly stimulated by glutaredoxins (Grx) and requires a reductant, often GSH.
Here, we show that chloroplast GrxS12 from poplar efficiently reacts with glutathionylated substrates in a GSH-dependent ping pong mechanism. The pK(a) of GrxS12 catalytic Cys is very low (3.9) and makes GrxS12 itself sensitive to oxidation by H(2)O(2) and to direct glutathionylation by nitrosoglutathione. Glutathionylated-GrxS12 (GrxS12-SSG) is temporarily inactive until it is deglutathionylated by GSH. The equilibrium between GrxS12 and glutathione (E(m(GrxS12-SSG))= -315 mV, pH 7.0) is characterized by K(ox) values of 310 at pH 7.0, as in darkened chloroplasts, and 69 at pH 7.9, as in illuminated chloroplasts.
Based on thermodynamic data, GrxS12-SSG is predicted to accumulate in vivo under conditions of mild oxidation of the GSH pool that may occur under stress. Moreover, GrxS12-SSG is predicted to be more stable in chloroplasts in the dark than in the light.
These peculiar catalytic and thermodynamic properties could allow GrxS12 to act as a stress-related redox sensor, thus allowing glutathione to play a signaling role through glutathionylation of GrxS12 target proteins.
Integrase (IN) is a clinically validated target for the treatment of human immunodeficiency virus infections and raltegravir exhibits remarkable clinical activity. The next most advanced IN inhibitor ...is elvitegravir. However, mutant viruses lead to treatment failure and mutations within the IN coding sequence appear to confer cross-resistance. The characterization of those mutations is critical for the development of second generation IN inhibitors to overcome resistance. This review focuses on IN resistance based on structural and biochemical data, and on the role of the IN flexible loop i.e., between residues G140-G149 in drug action and resistance.
Triosephosphate isomerase (TPI) catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in ...both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii (Cr). The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 A. CrTPI is a homodimer with subunits containing the typical (β/α)8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications.
The importance of redox-regulation in Arabidopsis thaliana roots has been investigated through the identification of the proteins interacting with thioredoxin (TRX), an ubiquitous thiol-disulfide ...reductase. We have applied a proteomic approach based on affinity chromatography on a monocysteinic mutant of plastidial y-type TRX used as a bait to trap putative partners in a crude extract of root proteins. Seventy-two proteins have been identified, functioning mainly in metabolism, detoxification and response to stress, protein processing and signal transduction. This study allowed us to isolate 24 putative new targets and to propose the mevalonic acid-dependent biosynthesis of isoprenoids as a new redox-mediated process. The redox-regulation of phenylpropanoid biosynthesis is also suggested, three enzymes of this pathway being retained on the column. We also provided experimental evidence that phenylammonia-lyase was enzymatically more active when reduced by TRXy in root crude extract. Among the high number of partners involved in defense against stress we isolated from the column, we focused on plastidial monodehydroascorbate reductase and showed that its activity was dramatically increased in vitro in the presence of DTT-reduced TRXy1 in root crude extracts. Our data strongly suggest that TRXy1 could be the physiological regulator of monodehydroascorbate reductase in root plastids.
Le statut scolaire local dans l’académie de Strasbourg, adossé au statut concordataire du territoire, est original et hérité d’une histoire complexe et singulière. Interrogeant le principe de laïcité ...à l’école, il reste méconnu et suscite des points de crispation – notamment autour de l’enseignement religieux qui fait débat. Mais le particularisme local, en ce qui concerne le rapport au religieux, comporte également des avantages défendus et valorisés par beaucoup d’acteurs locaux, dans la mesure où il est constitutif de l’identité régionale, vécu le plus souvent comme parfaitement compatible avec la République et favorable à un dialogue interreligieux porteur de sens.
The local school status in the Academy of Strasbourg, which is based on the concordat status of the territory, is an original one and was inherited from a complex and singular history. It questions the principle of secularism in schools, is still not well known and can be a source of tension, particularly with regard to religious education, which is a subject of debate. However, local particularism, with regard to the relationship with religion, also involves advantages that are defended and valued by many local actors, insofar as it is part of the regional identity, and is most often experienced as perfectly compatible with the Republic and favourable for meaningful inter-religious dialogue.
Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein ...and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled 35Scysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.