The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments.At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G Saccharomyces cerevisiae, Cdc5 is an only apparent Plk homologue, plays multiple roles in mitotic progression, and thus appears to be functionally relevant to Plk1. Consistent with its diverse roles, Plk1/Cdc5 phosphorylates various substrates in the cell (The polo-like kinases (Plks)1–the cell , 5, 6, acdc5 mutant defective in PBD-dependent substrate targeting has shown that the phosphopeptide binding mediated by PBD is not essential for mitotic progression and cell viability but for maintenance of spindle pole body integrity (One of the characteristic features of the Plks is the presence of the polo-box domain (PBD) in the C-terminal non-catalytic region. PBD functions in targeting the catalytic activity of Plk to its substrate proteins at specific subcellular sites . Biochem8–ntegrity . Therefo2 phase. This sister chromatid cohesion is essential for successful chromosome segregation, because it allows the two kinetochores on the sister chromatids to be attached to opposite spindle poles. In mitotic anaphase, proteolytic cleavage of a cohesin subunit Rad21 (Scc1 or Mcd1 in yeast) by a protease separase (Esp1 in yeast) liberates sister chromatids from cohesion and allows chromosome segregation to begin   , 26. Henscc1-15A) impaired yeast cell growth only moderately when combined with pds1 gene deletion. Interestingly, the same double mutant (scc1-15A pds1Δ) revealed marked sensitivity to the DNA-damaging agent phleomycin, indicating that the priming phosphorylation of Scc1 plays a more significant role under specific circumstances.In this study, we discovered using chromatin immunoprecipitation (ChIP) analysis that budding yeast polo-like kinase Cdc5 was associated with cohesin complex that is bound to mitotic chromosome arms in a PBD-dependent manner. In cohesin-depleted cells, Cdc5 binding to chromosomes was almost completely abolished, revealing that among chromosome-bound proteins, cohesin is the only major target of PBD-dependent Cdc5 binding. Biochemical analysis unveiled that Cdc5 recognized a middle region of cohesin subunit Scc1 that multiple Ser/Thr residues in this region  were phosphorylated in mitosis and that the phosphorylation is required for PBD recruitment. Preventing the phosphorylation by alanine substitutions of these Ser/Thr residues (ChIP followed by DNA sequencing) technique, which produces less noisy and quantitatively more accurate data than ChIP-chip. Yeast cells containing the FLAG-tagged CDC5 (Cdc5-FL) gene were arrested at G2/M phase by the microtubule-destabilizing reagent benomyl, fixed, and lysed. Then DNA fragments bound to Cdc5 protein were immunopurified, and the obtained DNA was sequenced by a massively parallel DNA sequencing instrument. The resultant ChIP-seq profile indicates that Cdc5 is located mostly at transcriptional convergent regions, which are characteristic of budding yeast cohesin-binding sites (A) (A). Correlation plot of ChIP-seq signal intensity reveals that the co-localization was observed universally along chromosome arms . A notable exception is pericentromeric regions, where cohesin is highly enriched, but Cdc5 binding was less pronounced .ChIP-chip (ChIP on DNA chip) analysis of budding yeast Cdc5 revealed that its genome-wide distribution resembled that of cohesin, as we reported previously . To undeng sites A 28). I. IChIP fites (A) A. Correlauxin-inducible degron) module, and selective degradation of the fused protein was induced by addition of an auxin derivative, indole-3-acetic acid (IAA) (C) (D). ChIP-seq analysis of Cdc5 in these cohesin-depleted cells revealed the disappearance of Cdc5 from the cohesin-binding sites. The peaks newly emerged in the cohesin-depleted cells were most likely to be a false-positive signal at “hyper-chippable” regions, because these peaks were observed also in cells without epitope tagging   analysis at multiple genomic loci of DNA obtained in Cdc5-PK ChIP . Although Cdc5-PK was highly enriched at representative cohesin sites along the chromosome arms  in cells with an intact cohesin (+ vehicle), it was dissociated from these sites upon cohesin depletion (+ IAA). Consistent with these results, Cdc5 dissociation from the cohesin-binding sites was also observed in ChIP-qPCR analysis in temperature-sensitive cohesin mutant . In contrast, we observed that chromosome binding of cohesin complex was not affected in Cdc5-depleted cells where expression of Cdc5 was shut off . This result is consistent with the notion that unlike vertebrates, Plk1-dependent cohesin dissociation from prophase chromosomes is not seen in budding yeast. Taken together, we conclude that Cdc5 protein is associated with the cohesin complex that is bound to G2/M phase chromosome arms.We then examined whether Cdc5 binding to the cohesin localization sites is dependent on the integrity of the cohesin complex. Scc1 or Smc3, a subunit of cohesin, was C-terminally fused to the aid (id (IAA) C 29). D. Dauxin-IAA) (C) D. ChIP-sFig. 1A) , and werA) (B). A Cdc5 PBD mutant consisting of three point mutations, W517F, V518A, and L530A  was shown to abolish the ability of Cdc5 to localize at subcellular targets and to support yeast cell growth . The other mutant protein, Cdc5-mut3 also revealed decreased binding, although the degree of decrease is about 30–50%. ChIP-seq analysis of Cdc5-mut1, -mut2, and -mut3 proteins indicated that the dissociation of the PBD-deficient Cdc5 proteins was universally observed throughout the entire genome (D and E). Almost all the genome sites with which wild-type Cdc5 is associated  exhibited greatly reduced binding of Cdc5-mut1 mutant protein. In conclusion, these data demonstrate that Cdc5 employs PBD to associate with the chromosome-bound cohesin complex.We next addressed how Cdc5 recognizes the chromatin-bound cohesin complex. Polo-like kinase is characterized by the presence of the polo-box domain (PBD), which functions as a phosphoserine/threonine-binding domain and promotes substrate targeting of polo-like kinase A 8). St. StA) (8A) , 10. To A) B. A Cdc5l growth . Other min vitro . Yeast con sites C. The ote genome , D and EA). As shown in Fig. 3B, the Scc1 subunit was co-purified with GST-PBD. Importantly, co-precipitation was not seen in control assays, where GST tag only (unfused GST) or GST-PBD with the mut1 mutation was expressed instead of the wild-type GST-PBD. Although the expression level of wild-type and mutant GST-PBD in yeast cells was almost identical, the wild-type GST-PBD was consistently recovered with less efficiency than the mutant one, due to an unknown reason. Nevertheless, significantly more HA-Scc1 was co-purified with the wild-type GST-PBD than with the mutant, indicating high affinity of Scc1 for functional PBD. In contrast to the Scc1 subunit, Smc1, Smc3 and Scc3 showed no co-purification specific to wild-type PBD. For Smc1 and Smc3, a significant amount of protein was co-purified with the mutant GST-PBD, implying that the observed co-precipitation was independent of the integrity of PBD and was presumably due to nonspecific interactions. The specific interaction between Scc1 and functional PBD was verified in the experiment where the full-length Cdc5 fused with GST was overexpressed instead of GST-PBD. In this experiment, slowly migrating forms of Scc1 appeared upon expression of GST-Cdc5, and remarkably, these presumably hyper-phosphorylated Scc1 species showed higher affinity to Cdc5 , which is consistent with the notion that PBD binds to a phosphorylated protein.Because it is now evident that Cdc5 recruitment to chromatin-bound cohesin is dependent on its PBD, we sought for which cohesin subunit mediates the interaction with PBD. For this purpose, either of the cohesin subunits  fused with the HA epitope was overexpressed simultaneously with GST-fused PBD in yeast cells. GST-PBD was affinity-purified from the cell lysate by glutathione (GSH)-Sepharose beads, and co-purified cohesin subunits were visualized by Western blotting detecting HA epitope A. As shoA). Each of the Scc1 fragments was co-overexpressed with GST-PBD in yeast cells, and its association with PBD was examined using GST pulldown assay. Scc1 can be divided into three parts  by the two separase-dependent cleavage sites  revealed specific co-purification with wild-type GST-PBD. Similarly, two other fragments sharing the entire middle part (M)  showed interaction with wild-type GST-PBD but not with GST only nor with mutated GST-PBD. Multiple bands seen in these fragments were likely to be due to phosphorylation of the fragments (see below). Fragment MC1 (amino acids 172–433) was co-purified both with wild-type and mutated GST-PBD. As described above, however, the amount of the purified wild-type GST-PBD protein was significantly smaller than that of mutant GST-PBD, indicating that MC1 also exhibited higher affinity to wild-type PBD. In contrast, the fragments that do not span the middle part  were not co-purified specifically with wild-type GST-PBD . We therefore conclude that the middle part is an essential region for Scc1 to be recognized by PBD. It should be noted that the two Cdc5 phosphorylation sites for efficient cleavage, Ser-175 and Ser-263, locate within or very close to the middle region . We therefore examined whether the slow migration was due to phosphorylation of the fragment. The middle part (M) of Scc1 was co-overexpressed with GST-PBD, and the slowly migrating form of the M fragment was purified by GSH-Sepharose. Treatment of the obtained material with λ protein phosphatase (λPP) converted the slowly migrating form to a faster migrating form, indicating that slow migration was due to phosphorylation . Collectively, our data suggest that PBD recognizes phosphorylated forms of the Scc1 M fragment.Some of the PBD-interacting Scc1 fragments showed multiple bands in Western blot analysis, and slowly migrating forms were selectively co-purified with GST-PBD ) and examined whether this substitution impairs Cdc5 binding to Scc1. ChIP-qPCR analysis revealed that scc1-cdk(−) mutation did not affect the binding of Cdc5 (A), indicating that phosphorylation at other site(s) has an important role in PBD recruitment. To determine the essential priming phosphorylation site(s) in the Scc1 middle region, we generated a set of mutated M fragments, in which all Ser/Thr residues within this region were systematically substituted with alanine (B). There are 17 Ser/Thr residues in this region. It was shown that among them, Ser-175 and Ser-263 were phosphorylated by Cdc5 kinase itself, and these phosphorylations promote efficient cleavage of Scc1 by separase (C). Consistently, chromosomal binding of Cdc5 measured by ChIP-qPCR was not changed in cells expressing Scc1-plk(−) mutant protein (A). In contrast, alanine substitution of all the other 15 Ser/Thr residues in the M fragment (M-15A) impaired PBD binding to the fragment (C). ChIP-qPCR analysis also revealed that the same substitution in the full-length Scc1 caused almost complete dissociation of Cdc5 from the cohesin-binding sites without affecting chromosome binding of cohesin itself (D) was no longer detected for the M-15A mutant fragment. These data strongly suggest that the priming phosphorylation essential for PBD recruitment to Scc1 has occurred at some of these 15 Ser/Thr residues .As described already, PBD targets phosphoserine- or phosphothreonine-containing peptide, and this phosphorylation to be targeted by PBD is called priming phosphorylation. It is shown that the priming phosphorylation is catalyzed by Cdk kinase in many cases. For instance, Cdk1/cyclin B phosphorylates a structural protein of the Golgi apparatus, Grasp65, in its C-terminal domain at four consensus sites, which in turn become recognition sites by Plk1 PBD , 33. Bec of Cdc5 A, indica alanine B. There separase . We founseparase C. Consis protein A. In confragment C. ChIP-qn itself . Notablyn itself D was no B). Among them, M-5A mutant fragment, in which Ser-183, Ser-194, Ser-195, Ser-219, and Ser-220 are substituted to alanine, diminished the interaction with PBD almost completely (D). ChIP-qPCR analysis confirmed Cdc5 dissociation from the chromosomal cohesin sites in vivo by the same 5A substitutions (B), showed very little interaction with PBD, like M-5A (D). These results suggest that multisite phosphorylation of the Scc1 middle region is required for recruiting PBD to cohesin.To narrow down further the priming phosphorylation sites, we tested other Scc1 M fragments with different sets of alanine substitutions B. Among mpletely D. ChIP-qitutions . The fivitutions B, showedike M-5A D. These A and E). In wild-type (SCC1) cells, a cleaved form of Scc1 became visible by 15 min after induction of Cdc20, an activator of the anaphase-promoting complex, and the amount of the intact Scc1 protein was reduced to less than 20% of the initial level by 30 min (B and C). Because the cleaved form is targeted for degradation by the N-end rule pathway . We also monitored dissociation of cohesin from chromosomes by ChIP-qPCR. The amount of chromosome-bound wild-type Scc1 as well as Scc1-15A mutant protein decreased very similarly when cells proceed through anaphase (D). These results indicate that the priming phosphorylation in the Scc1 middle part plays little, if any, role in Scc1 anaphase cleavage in wild-type cells.It was shown that Cdc5 polo-like kinase phosphorylates the Scc1 subunit at Ser-175 and Ser-263, and this phosphorylation promotes efficient Scc1 cleavage by separase in anaphase . In Cdc5y 30 min , B and C pathway , the cleild type , B and Canaphase D. These et al. (i.e. scc1-plk(−) mutation) resulted in chromosome segregation defects and eventual cell death when combined with pds1 gene deletion (pds1Δ), whereas scc1-plk(−) mutation on its own did not hinder cell proliferation. This genetic interaction is consistent with the notion that Pds1 and the phosphorylation of Scc1 at Ser-175/Ser-263 by Cdc5 function redundantly in promoting timely and efficient cleavage of Scc1 in anaphase (scc1-plk(−) mutation enhanced temperature sensitivity of the pds1Δ mutant (B) but did not affect cell growth of wild-type cells (A) when expression of an ectopic wild-type SCC1 was repressed by transferring to galactose-free medium. At 23 °C, the scc1-plk(−) pds1Δ double mutant formed no colonies, although the pds1Δ single mutant grew as well as wild type. We then examined genetic interaction between scc1-15A and pds1Δ mutants. The scc1-15A mutant showed no growth defect on its own (A). Unlike scc1-plk(−) mutant, scc1-15A mutant revealed only a modest synthetic growth defect when combined with pds1Δ mutation (B). At 23 and 26 °C, the double mutant was able to form colonies, although the number of visible colonies was reduced ∼10-fold compared with the pds1Δ single mutant. This result implies that the Scc1 priming phosphorylation plays only a marginal role in enhancing anaphase Scc1 cleavage in cells lacking securin, where Scc1 phosphorylation by Cdc5 is indispensable.We next utilized genetic approach to assess physiological relevance of the Scc1 priming phosphorylation. Alexandru et al.  reportedanaphase . First, Δ mutant B but didpe cells A when ex its own A. Unlikemutation B. At 23 scc1-15A and scc1-15A pds1Δ cells on plates containing drugs was measured by spotting serial dilutions of liquid cultures. None of methylmethane sulfonate , hydroxyurea , benomyl (microtubule-destabilizing reagent), or H2O2 (an oxidant) caused a growth defect specific to scc1-15A or scc1-15A pds1Δ strains (A). Interestingly, we found that growth of scc1-15A pds1Δ cells was severely impaired in the presence of phleomycin, a bleomycin-related antibiotic, whereas wild-type and scc1-15A cells grew normally, and pds1Δ cells showed only modest growth defect under the same conditions (3 and 6 μg/ml phleomycin) (B). The scc1-15A pds1Δ cells also exhibited a growth defect on a plate containing BleocinTM, a formulation containing bleomycin (B), indicating that the double mutant was generally sensitive to this family of antibiotics.Cohesin is known to be involved not only in sister chromatid cohesion, but also in the DNA double-stranded break repair process , 36. We  strains A. Intereeomycin) B. The scleomycin B, indicascc1-15A pds1Δ cells show a growth defect in the presence of DSB caused by HO endonuclease, a site-specific double-stranded endonuclease. HO endonuclease was expressed from galactose-inducible promoter in wild-type, pds1Δ, scc1-15A, and scc1-15A pds1Δ cells in which an HO endonuclease cleavage site was integrated into chromosome V. HO induction by 0.05% or a higher concentration of galactose impeded colony formation in spotting assay, regardless of genotype (data not shown). At a lower expression level of HO induced by 0.01% galactose, however, the scc1-15A pds1Δ double mutant specifically showed growth defect (C). This result is consistent with the notion that scc1-15A pds1Δ is defective in cellular response to DSB.Phleomycin and bleomycin is known to induce double-stranded breaks (DSB) in DNA , 38. We h defect C. This rscc1-15A pds1Δ cells were also sensitive to ultraviolet (UV) irradiation, which forms a thymine dimer lesion in DNA. When irradiated with 254 nm UV light at 50 J/m2, the pds1Δ mutant showed reduced viability, compared with wild type (B). The scc1-15A pds1Δ double mutant exhibited further reduced viability, implying that the Scc1 priming phosphorylation and Pds1 synergistically function in response to UV-induced DNA lesions. Taken together, the priming phosphorylation of Scc1 seems to have an important role in responding to DNA lesions, including DSB.In addition to DSB, we observed that ild type B. The sceco1-1 mutant. Eco1 acetylates cohesin in S phase, thereby enabling it to bind chromosomes stably and to establish sister chromatid cohesion  on chromosome arms (eco1-1 cells (by ∼70% on average). Interestingly, Cdc5 binding to the centromeric loci was not affected by eco1-1 mutation. These results support the above-mentioned hypothesis that Cdc5 exhibits higher affinity to cohesin engaged in sister chromatid cohesion, which may explain the disfavored association of Cdc5 with centromeric cohesin.We finally addressed why Cdc5 showed preferential binding to cohesin on chromosome arms and not to that around the centromeres. It is known that chromosome binding of cohesin is more dynamic around the centromeres, implying that the majority of centromeric cohesin is not engaged in sister chromatid cohesion , 40. Cdccohesion . In the ome arms . This rein vitro. These results suggest that the observed Cdc5-Scc1 association reflects PBD-dependent targeting of Cdc5 kinase to its substrate. This notion is consistent with the previous reports that Scc1 is phosphorylated by Cdc5 in mitotic yeast cells . Cdc5 may interact with a telomeric protein in a PBD-independent manner.In this study, we discovered that Cdc5 polo kinase is associated with cohesin complex localized on budding yeast chromosome arms prior to anaphase onset. The PBD mutations impairing the binding of PBD to phosphopeptide abolished the Cdc5 association with cohesin, and PBD was preferentially bound to phosphorylated Scc1 fragments st cells , 22. In ome arms E. Cdc5 mChromosomal association of Cdc5 was greatly diminished in a mutant of Eco1, which acetylates cohesin specifically during S phase and promotes establishment of sister chromatid cohesion. Hornig and Uhlmann  reportedin vitro at several additional sites, and three of them  are located within the middle part . Notably, we also observed that among several phosphorylated forms of Scc1 M fragments, only a molecular species with relatively slow mobility on SDS-PAGE showed high affinity to PBD (D). These results strongly suggest that multisite phosphorylation in the middle region is required for promoting efficient PBD binding to cohesin. Multisite phosphorylation is a common feature of many protein kinase substrates. In previous studies of such an example, a Cdk inhibitor, Sic1, multisite phosphorylation is proposed to enable an ultrasensitive switch-like response to a gradually increasing kinase activity , which include two of the abovementioned y to PBD D. These activity , 47. Sccscc1-15A cells. This result is not unexpected, because Cdc5-dependent phosphorylation and the securin Pds1 are known to play a redundant role in promoting efficient and timely cleavage of Scc1 in anaphase (scc1-plk(−) mutant, which lacks the target sites of Cdc5 phosphorylation for efficient cleavage, showed synthetic lethality with a pds1Δ mutant at 23 °C or higher temperatures (B). We therefore addressed how scc1-15A mutant cells grow when combined with pds1 deletion, and we revealed that they showed only moderate sickness. This result indicates that the priming phosphorylation is not an absolute requirement for Ser-175 and Ser-263 phosphorylation of Scc1 for anaphase cleavage. Based on the cellular phenotype of the Cdc5 PBD mutant, PBD-substrate interaction, but not phosphorylation by Cdc5, is thought to be largely dispensable for mitotic progression in budding yeast  plasmid (empty vector) was used as a negative control to express the GST tag only (unfused GST).For GST pulldown assay in yeast cells, the DNA fragment encoding the intact Cdc5 or PBD of Cdc5 (amino acids 357–705) was cloned into pEG(KT) multicopy plasmid , which c1 phase by culturing in medium containing 2 μm α-factor (peptide synthesized by Sigma) for 2.5 h. Release from the G1 arrest was induced by adding 150 μg/ml Pronase  to the medium. To be arrested in G2/M phase, cells were cultured in medium containing 80 μg/ml benomyl (Sigma) for 2.5 h. For preparation of G2/M phase-arrested cells lacking a cohesin subunit Scc1 or Smc3 by use of aid system, cells were arrested in G1 phase by cultivating in α-factor-containing medium for 2 h, and then selective degradation of the aid-fused Scc1/Smc3 was induced by adding 1 mm IAA  and culturing for another 1 h. Subsequently, the cells were released from G1 arrest and re-arrested in G2/M phase by changing the medium to fresh YPD containing Pronase, benomyl, and IAA and culturing for additional 2 h. In preparation of cells progressing through anaphase synchronously, cells with PGAL-3HA-CDC20, where the endogenous Cdc20 is N-terminally tagged with 3HA epitope and controlled by the GAL1 promoter, were grown in YPR (YPD containing 2% raffinose instead of glucose) containing 2% galactose and then transferred to and incubated in YPR for 3 h to promote Cdc20 depletion and metaphase arrest. Proper arrest was confirmed by accumulation of large-budded cells under microscopy. Finally, the cells were released synchronously from the arrest by adding 2% galactose to the medium.Yeasts were cultured in complete YPD  medium aGAL1–3HA-CDC5 cells, where the endogenous Cdc5 is N-terminally tagged with 3HA epitope and controlled by the GAL1 promoter. For preparation of cells arrested in G2/M phase and lacking the endogenous Cdc5, the cells were first cultured in SGal-L  and arrested in G1 phase by addition of 2 μm α-factor for 3 h. Then the cells were transferred to and incubated in YPD containing α-factor for 30 min to promote Cdc5 depletion and finally re-arrested in G2/M phase by adding Pronase and benomyl to the medium and culturing for an additional 2 h. In analysis of Scc1-15A and Scc1-5A proteins in G2/M phase-arrested scc1-aid strains, the cells were first grown in SC medium lacking tryptophan and arrested in G1 phase by adding 2 μm α-factor for 2 h. Then the cells were transferred to and incubated in YPD containing IAA and α-factor for 1 h to promote Scc1-aid depletion and finally re-arrested in G2/M phase by adding Pronase and benomyl to the medium and culturing for an additional 2 h. For GST pulldown assay, cells with overexpression plasmids were grown in SRaf-UraTrp  at 30 °C and then cultured in YPR for 2 h. Subsequently protein expression and G2/M phase arrest were promoted by including 2% galactose and benomyl in the medium for 3 h. Strains harboring the galactose-inducible SCC1 gene (PGAL1-SCC1) at AUR1 locus were created, maintained, and grown in YPRG  medium before spotting assay.For selection and maintenance of transformants with auxotrophic marker(s), synthetic complete (SC) medium lacking appropriate nutrient(s)  was usedGAL1-SCC1 harboring strains) or YPD and allowed to grow overnight at 23 °C. Medium was washed out, and the cells were resuspended in 10% glycerol before counting by a hemocytometer. The initial density of cells was adjusted to 2.4 × 106 cells/ml, and 7-fold dilution was performed sequentially four times. 5 μl of each diluted cell suspension was spotted onto YPRG or YPD solid media . The following chemical agents were used in plate assay to challenge yeast cell cycle and/or cell growth: 3 or 6 μg/ml phleomycin (Sigma), 0.25 or 0.5 μg/ml BleocinTM , 0.02% MMS (Sigma), 40 mm HU , 10 μg/ml benomyl (Sigma), 3 mm H2O2 (Wako). These agents were added to melted warm YPD agar before pouring into sterile Petri dishes. To induce DNA lesions by UV radiation, the cells spotted on solid media were exposed to UV-C light (at 254 nm wavelengths) generated by UV cross-linkers (Ultra-Violet Products Ltd.) at a dose of 40 or 50 J/m2. Strains harboring PGAL1-HO were pre-cultured in YPR and spotted onto YPD or YPR with 0.01% galactose. After spotting, the plates were incubated at the indicated temperature until colonies of wild-type control were formed.Yeasts were inoculated in either YPRG , monoclonal anti-FLAG , monoclonal anti-GST , and polyclonal anti-aid  antibodies were used for ChIP and/or Western blotting. For the HA epitope, monoclonal anti-HA antibody clones 12CA5  and 16B12  were used for Western blot and ChIP, respectively. Antibody dilution in Western blot analysis was 1:1,000 for all antibodies. Peroxidase-conjugated antibodies against mouse and rabbit IgG  were used as secondary antibodies in Western blot analysis.ChIP analysis was performed as described previously . For antm/ImC, where mC is the mean of the ChIP DNA measurements, and mI is the mean of the input DNA measurements. The primers used in qPCR are listed in ChIP-purified DNA was quantified using KAPA SYBR Fast qPCR kit (KAPA Biosystems) and real time PCR systems 7500 and StepOnePlus  as per the manufacturer's instructions. Both input and ChIP DNA, or DNA before and after ChIP, were measured in duplicate. The level of enrichment in ChIP was calculated as a ratio of the amount of ChIP DNA over that of input, or S. cerevisiae reference genome using Bowtie (version 1.1.0) (www.ncbi.nlm.nih.gov), respectively. Sequencing and mapping statistics are summarized in High-throughput sequencing and analysis of ChIP-purified DNA was conducted, as described previously , with sen 1.1.0) , with “—n 1.1.0)  with “PEm NaCl, 1 mm NaN3, 50 mm NaF, 10 mm EDTA, pH 8.0), and stored on ice until all samples were harvested. Then, the cells were washed once with 0.5 ml of in situ buffer , resuspended in 40 μl of extraction buffer , 1 mm phenylmethylsulfonyl fluoride (PMSF)), mixed with 120 μl of 2× SDS-PAGE sample buffer containing 0.5-mm diameter glass beads (Sigma), and boiled at 95 °C for 5 min with continuous mixing. Subsequently, the cells were broken by 4 min of vigorous shaking  in a Multi-beads Shocker (Yasui Kikai), and the collected crude lysate was boiled for another 5 min at 95 °C. The lysate was clarified by centrifugation at 14,000 rpm for 10 min, and the supernatant was subjected to Western blot analysis.Yeast protein extract was prepared from trichloroacetic acid (TCA)-treated yeast cells as described . For timSDS-PAGE and Western blotting were performed as described previously . Total p2/M phase-arrested cells co-overexpressing GST-fused PBD (or Cdc5) and HA-tagged Scc1 fragments (or intact cohesin subunit) was harvested and washed once with ice-cold PBS, before flash freezing with liquid N2 and storage at −80 °C. Cells expressing mutant PBD fused with GST or unfused GST, instead of GST-PBD, were used as a negative control. The following procedures were performed on ice or at 4 °C. Lysis buffer consisting of 40 mm Hepes-KOH, pH 7.4, 140 mm NaCl, 10% glycerol, 0.1% Triton X-100, 1 mm dithiothreitol, 0.5 mm PMSF, 1× cOmplete protease inhibitor mixture (Roche Applied Science), 1× Protease inhibitor mixture for fungal and yeast extracts (Sigma), 1× PhosSTOP Phosphatase inhibitor mixture (Roche Applied Science) was used for lysate preparation and preparation/wash of affinity beads. The frozen cell pellet was thawed and lysed in 500 μl of Lysis buffer containing 0.5-mm diameter glass beads (Sigma) with 6 min of vigorous shaking  at 0 °C. The recovered crude lysate was clarified by centrifugation  repeated twice, and then the resultant lysate was mixed with 30 μl of glutathione-Sepharose 4B beads  that were pre-equilibrated with Lysis buffer, and incubated for 1 h on a rotor. The beads were subsequently washed three times by spinning down the beads , discarding the supernatant, and resuspending the beads in 800-μl fresh Lysis buffer. Finally, the supernatant was discarded, and the bead-bound proteins were solubilized by boiling with 40 μl of 2× SDS-PAGE sample buffer at 95 °C for 3 min (“pulldown” fraction). An aliquot of the clarified lysate before mixing with GSH beads was used as “input” fraction. To identify a cohesin subunit or Scc1 fragments that are able to bind to functional PBD, the amount of protein under test co-purified with GST-PBD was compared with those co-purified with GST-fused mutant PBD or GST only. The pulldown fractions that correspond to the same number (∼108) of yeast cells at the start were loaded onto a single polyacrylamide gel and subjected to anti-HA Western blotting analysis.The 40-ml culture of GThe λPP treatment of the purified proteins was carried out on GSH beads using λPP (New England Biolabs) and NEBuffer for protein metallophosphatase (New England Biolabs). The protein-bound GSH beads after the wash step were subsequently washed twice with NEBuffer for protein metallophosphatase and resuspended in 50 μl of the same buffer. 400 units of λPP was added and incubated at 30 °C for 1 h. After the reaction, the beads were spun down and boiled with 40 μl of 2× SDS-PAGE sample buffer at 95 °C for 3 min.S. P., K. S., and T. S. designed the study. S. P. conducted all the experiments, following the early-phase study of Cdc5-Scc1 co-localization by E. T. M. I. analyzed high-throughput sequencing data. S. P., K. S., and T. S. interpreted the data and prepared the manuscript.


Renealmia alpinia (Zingiberaceae), a medicinal plant of tropical rainforests, is used to treat snakebites and other injuries and also as a febrifuge, analgesic, antiemetic, antiulcer, and anticonvulsant. The dichloromethane extract of R. alpinia leaves showed potent inhibition of human monoamine oxidases- (MAOs-) A and B. Phytochemical studies yielded six known compounds, including pinostrobin 1, 4′-methyl ether sakuranetin 2, sakuranetin 3, pinostrobin chalcone 4, yashabushidiol A 5, and desmethoxyyangonin 6. Compound 6 displayed about 30-fold higher affinity for MAO-B than MAO-A, with Ki values of 31 and 922 nM, respectively. Kinetic analysis of inhibition and equilibrium-dialysis dissociation assay of the enzyme-inhibitor complex showed reversible binding of desmethoxyyangonin 6 with MAO-A and MAO-B. The binding interactions of compound 6 in the active site of the MAO-A and MAO-B isoenzymes, investigated through molecular modeling algorithms, confirmed preferential binding of desmethoxyyangonin 6 with MAO-B compared to MAO-A. Selective reversible inhibitors of MAO-B, like desmethoxyyangonin 6, may have important therapeutic significance for the treatment of neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease. Renealmia genus (Zingiberaceae) belongs to the rare class of amphi-Atlantic plants well represented in tropical Africa and Americas  were tested. Results are presented as double reciprocal Lineweaver-Burk plots. The kinetic data, namely, KM, Vmax, and Ki values, were calculated by SigmaPlot 12.3 with enzyme-kinetics module using Michaelis-Menten equation. The results were also analyzed for the type of inhibition.The potential inhibitor desmethoxyyangonin 6 were further examined by incubating the enzyme with high concentrations of the inhibitor followed by extensive dialysis of the enzyme-inhibitor complex and recovery of enzyme activities. MAO-A (0.05 mg/mL protein) was incubated with desmethoxyyangonin 6 (20 μM and 100.0 μM) and MAO-B (0.05 mg/mL protein) was incubated with desmethoxyyangonin 6 (1.50 μM and 20.0 μM) in an enzyme incubation mixture of 1 mL containing 100 mM potassium phosphate buffer (pH 7.4). Formation of enzyme-inhibitor complex was allowed by incubation of the reaction mixtures for 20 min at 37°C. The enzyme-inhibitor mixtures after incubation were chilled, transferred to the dialysis bags, and dialyzed against 25 mM potassium phosphate buffer (pH 7.4) for 14–16 hours at 4°C . The enzyme catalytic activities were measured before and after dialysis.Binding and inhibition of MAO-A and MAO-B with desmethoxyyangoninμg/mL MAO-A were 7.50 μM (desmethoxyyangonin 6) and 0.600 μM (phenelzine) and with 50 μg/mL MAO-B were 0.40 μM (desmethoxyyangonin 6) and 0.100 μM (phenelzine). Controls without inhibitors were also run simultaneously. The enzyme activities were determined as described above.MAO-A and MAO-B enzymes were preincubated for different time periods (0–15 min) with the inhibitor. Concentrations of the inhibitor tested for time-dependent inhibition with 20  6 was prepared using the LigPrep [The crystal structures of MAO-A and MAO-B were downloaded from the Protein Data Bank with PDB IDs 2Z5X for MAO-A  and 1OJ9 LigPrep . Molecul LigPrep , using P LigPrep  of the S LigPrep . All resR. alpinia dichloromethane extract showed significant inhibitory effect with IC50 values of 3.75 and 1.70 μg/mL for the MAO-A and MAO-B, respectively. The extract was subjected to purification using a silica gel column chromatography, yielding six purified compounds 1–6 , by comparison with those reported in the literature [ 1–6 were evaluated against MAO-A and MAO-B inhibition assays. Compounds 1–5 showed moderate inhibition effect; however compound 6 exhibited potent MAO inhibition  buffer. The enzyme activities were examined before and after dialysis. Through overnight dialysis, the recombinant human MAO-A enzyme lost about 10–15% of activity. Incubation of MAO-A with 20.0 and 100 μM concentrations of desmethoxyyangonin 6 inhibited more than 65% of the enzyme activity  with MAO-B caused more than 90% inhibition of enzyme activity, which was fully recovered after dialysis  at concentrations that caused nearly 60–70% inhibition . The con complex . MAO-A aactivity . After tdialysis . These o 6 led us to the investigations on interactions of compound 6 with the human MAO-A and MAO-B employing computational molecular modeling algorithms. The preferred binding poses of desmethoxyyangonin 6 in MAO-A and MAO-B are shown in  6 to MAO-B than to MAO-A and support the experimental observations on selective inhibition of MAO-B compared to MAO-A, with desmethoxyyangonin 6. MM/GBSA binding energies were computed as −35.10 and −62.95 Kcal/mol for the MAO-A and MAO-B complexes, respectively. These observations are consistent with their docking scores and observed experimental IC50 and Ki values. Further analysis of these interactions indicates that the pyranone moiety of compound 6  π-π stacks with the phenol moiety of residue Y326 in MAO-B complex -methysticin through inhibition of platelet MAO-B [ 6 has been reported to prevent inflammation and hepatitis in mice through attenuation of lipopolysaccharide- (LPS-) stimulated inflammation in murine macrophages and LPS/D-galactosamine-  induced fulminant hepatitis in mice [ 6 have not been listed under PAINS (pan assay interference), and 6 does not contain any PAIN-like motif [Many herbs remedies contain MAO inhibitors without the unpleasant side effects. Recently, Carradori et al.  have lis anxiety . The neu anxiety . Isolateet MAO-B . Similaret MAO-B . Methystet MAO-B . Desmeth in mice . Moreoveke motif , 40. 1 and desmethoxyyangonin 6 in R. alpinia further explains the potential benefits of this plant for human health and nutrition. Anti-inflammatory properties and selective reversible inhibition of human MAO-B by desmethoxyyangonin 6 suggest potential therapeutic use of this kavalactone for treatment of neurodegenerative diseases like Parkinson's disease.The presence of pinostrobin R. alpinia after fractionation and purification produced pure desmethoxyyangonin 6, which was further evaluated for the inhibitory effects against recombinant human MAO-A and MAO-B enzymes. Desmethoxyyangonin 6 was identified as a prominent MAO-A and MAO-B inhibitory constituent from R. alpinia. The computational docking and thermodynamic analysis of MAO-A and MAO-B complexed with desmethoxyyangonin 6 support the experimental results regarding 29-fold selective inhibition of MAO-B compared to MAO-A. The presence of prominent MAO-B constituent supports the R. alpinia folkloric use, and desmethoxyyangonin 6 can be used as a lead compound for rational design of anti-Parkinson's disease agents.In this study, phytochemical analysis of dichloromethane extract of


We compared linear Patlak’s analysis to non-linear three-compartment three-parameter kinetic model analysis (3P-KMA) providing Ki, kb, and fraction of free 18F-FDG in blood and interstitial volume (Vb).Patlak’s graphical analysis can provide tracer net influx constant (Ki) with limitation of assuming irreversible tracer trapping, that is, release rate constant (k18F-FDG input function (IF) and a tissue time-activity curve. The former was fitted with a three-exponentially decreasing function, and the latter was fitted with an analytical formula involving the fitted IF data . Bland-Altman analysis was used for Ki comparison between Patlak’s analysis and 3P-KMA. Additionally, a three-compartment five-parameter KMA (5P-KMA) was implemented for comparison with Patlak’s analysis and 3P-KMA.Dynamic PET data of 21 lung cancer patients were retrospectively analyzed, yielding for each patient an b > 0 deleted this difference. A strong correlation was found between Ki ratio (=3P-KMA/Patlak) and kb . No significant difference in Ki was found between 3P-KMA versus 5P-KMA, and between 5P-KMA versus Patlak’s analysis, with limits of agreement of ± 23.0 and ± 31.7% (95% confidence), respectively.We found that 3P-KMA Ki was significantly greater than Patlak’s Ki over the whole patient series, + 6.0% on average, with limits of agreement of ± 17.1% (95% confidence). Excluding 8 out of 21 patients with kb to zero: the greater the kb value, the greater the Ki underestimation. This underestimation was not revealed when comparing 5P-KMA and Patlak’s analysis. We suggest that further studies are warranted to investigate the 3P-KMA efficiency in various tissues showing greater 18F-FDG trapping reversibility than lung cancer lesions.Comparison between 3P-KMA and Patlak’s analysis significantly showed that the latter underestimates Ki because it arbitrarily set k Among these KMAs, Patlak’s analysis is usually considered as a gold standard that provides the 18F-FDG net influx constant  from a linear fitting of graphical data [Positron emission tomography using 8Ffluoroder cells , 3. Howeer cells . This iser cells –13. Theser cells . The KMAcal data . Howevercal data –11, 15.b) in an exponential term [−1 mL−1), kb (in min−1), and fraction of free 18F-FDG in blood and interstitial volume  [18F-FDG IF [Assuming that there may be a slow loss of the trapped tracer to the blood, i.e., a reversible trapping, Patlak and Blasberg derived a generalized non-linear equation including a release rate constant  , 15. It F-FDG IF , 17. Theb, and Vb outcomes obtained from 3P-KMA were compared to those obtained from a three-compartment five-parameter KMA (5P-KMA) that is usually considered as a reference model when tracer trapping is reversible. Actually, 5P-KMA provides four kinetic (micro)parameters (and Vb) from which Ki and kb may be computed, whereas the non-linear fitting of the 3P-KMA provides Ki and kb without any additional computing.To the best of our knowledge, comparison between non-linear fitting by 3P-KMA and linear fitting by standard Patlak’s analysis that assumes an irreversible tracer trapping has not been reported so far, whatever the tissue either under physiological or pathological conditions. Therefore, the primary aim of this study was to make this comparison in a series of lung cancer patients that was previously acquired . Additio−1 . The lesion mean size was 31.8 mm .Dynamic data of 21 patients  with non-small cell lung cancer obtained from a previous prospective study were retrospectively analyzed [18F-FDG  in a cannula previously inserted in the vein of the arm, a 3D thorax dynamic list-mode acquisition protocol was started lasting 60 min . Images were reconstructed using the iterative method RAMLA LOR-3D, with a 144 × 144 matrix and pixel size of 4 × 4 × 4 mm3. In particular, this dynamic acquisition provided 11 frames of 5 min each, leading to 11 data points of the experimental 18F-FDG IF and of the experimental cancer tissue TAC, ranging 7.5–57.5 min post-injection. For determining the experimental 18F-FDG IF, in each patient, a volume of interest (VOI) was drawn over the descending thoracic aorta in each frame of the dynamic acquisition yielding an intermediate 18F-FDG blood TAC . Then, the final IF was obtained through a calibration of the intermediate IF with the 18F-FDG plasma value measured in a venous blood sampling performed at 45 min post-injection, that is, when an equilibrium is reached between 18F-FDG concentration in arterial and vein blood [PET imaging procedure has been previously described in details . Brieflyin blood . VOIs fob) [Assuming that there may be a slow loss of the trapped tracer to the blood and when the analysis remains limited to data collected for the period t > t* after injection, that is, when the reversible compartments are in effective steady state with the blood plasma, Patlak and Blasberg derived a non-linear equation including a release rate constant (kb) :T(t) (in kBq mL−1) is defined as the total tracer activity at time t per tissue volume unit that includes both trapped tracer and free tracer in the blood and interstitial volumes. Ap(t) (in kBq mL−1) is the blood activity at time t per blood volume unit, that is, the 18F-FDG IF.Ab = 0. Eleven cancer tissue TAC data points and the corresponding 11 data points of the experimental 18F-FDG IF, ranging 7.5–57.5 min post-injection, were used. The lower limit of 7.5 min for this range was chosen in order to limit the analysis to data collected for the period t > t* after injection, as required by Eq. T(t)/Ap(t) versus the ratio of time integral of the right hand side of Eq. p(t), i.e., the so-called stretched time.In each patient, Patlak’s graphical analysis was implemented from Eq. 18F-FDG IF with a three exponentially decreasing function derived from Hunter’s results, after data were uncorrected for the 18F physical decay. Hunter’s results were used, and not Vriens’ ones as in previous studies, because the former were established with blood sampling performed at 55 min post-injection, in comparison with 25 min for the latter [In each patient, 3P-KMA was implemented by first fitting the 11 data points of the experimental e latter , 15–17:2A0 , α2, and α3  were obtained by fitting the experimental 18F-FDG IF data points . Then, in each patient, a formula was established by analytically solving integral of Eq. b [In Eq. 2, the amplitude ratios of the three exponential functions and the time constant of the first exponential function  were available from Hunter’s results . In eachof Eq. b , 15:3\do18F physical decay constant is λ, and Ki, kb, and F were obtained in each patient by fitting the 18F-FDG tissue TAC , ranging 7.5–57.5 min post-injection, uncorrected for 18F physical decay. Note that previous studies used 18F-FDG tissue data obtained at late dynamic PET imaging, i.e., beyond 2 h after injection, in comparison with the current ones obtained at early imaging (7.5–57.5 min post-injection) [In Eq. jection) , 15. How1, k2-3-4 and Vb: K1 and k2 account for forward and reversed transport between blood and reversible compartment, and k3 and k4 account for forward and reversed transport between reversible and trapped compartment, respectively [b may be computed from K1, k2-3-4, as:In each patient, the 5P-KMA model was implemented on PMOD software by using the whole experimental IF and tissue TAC data points acquired from injection, with very short frames including the bolus injection  . The 5P-ectively , 15. Theα2 and α3 values : P < 0.0001 for the two comparisons.Figure b, and Vb obtained from 3P-KMA are presented for each patient in Table R = 0.631; P < 0.01; graph not shown).Figures R = 0.995; P < 0.001; graph not shown). Figure b > 0 were excluded .3P-KMA Ki was found to be strongly correlated with Patlak’s Ki  obtained from 5P-KMA are presented in Table R = 0.989; P < 0.001). No significant difference was found between 3P-KMA Ki and 5P-KMA Ki: Ki ratio  which was 1.017 ± 0.054 on average (95% confidence limits), with 95% limits of agreement of 0.230. No significant difference was found between 5P-KMA Ki and Patlak’s Ki: Ki ratio . No significant difference was found between 3P-KMA kb and 5P-KMA kb: kb difference  which was 0.00041 ± 0.00083 min−1 on average (95% confidence limits), with 95% limits of agreement of 0.00359 min−1. No significant correlation was found between 3P-KMA Vb and 5P-KMA Vb (R = 0.12).Values of Ki, kb, Vb) obtained over the current lung cancer series.Figure 18F-FDG IF were fitted by using a three-exponential decreasing function. These data points ranged 7.5–57.5 min post-injection, that is, after an equilibrium has been reached between compartments in order to satisfy the Patlak’s condition t > t*. This function was derived from Hunter’s results, of which A0, α2, and α3 were obtained by fitting (Eq. 2) [α1 by Hunter and of α2 and α3 reported in Table α1 reported by Hunter may be used in each individual [α2 and α3 values were significantly lower than the latter ones (P < 0.0001) [P < 0.001; Fig. R = 0.631; P < 0.01). It is noteworthy that, although the current study with 18F-FDG used a three-exponential decreasing function, 3P-KMA may also be efficient with either a mono- or a bi-exponential IF depending on the tracer. Moreover, the ways the exponentially decaying IF can be obtained may be various: either from arterial or venous blood sampling, or image-derived, or from population-based IF models possibly scaled to later dynamic measurements on blood pool ROIs [In each patient, 11 data points of the  (Eq. 2) . Indeed,dividual . Compari 0.0001) . The IF ool ROIs .b > 0 were excluded, 3P-KMA Ki was no more significantly greater than Patlak’s Ki, with 95% limits of agreement of 9.8%. These findings were in agreement with Patlak and Blasberg’s comment suggesting that, in case of reversible trapping (kb > 0), linear fitting of a concave curve rather than that of a (true) linear one (kb = 0), results in an underestimation of the slope and hence to Ki underestimation [18F-FDG uptake in lung cancer lesions. However, the strong correlation between Ki ratio  and kb  and kb = 0.0009 ± 0.0016 min−1 (SD) for 3P-KMA parameters (Eqs. 4 and 5). As a result, the measurement uncertainty of Ki and kb from 5P-KMA combines that of the three (micro)parameters, whereas the measurement uncertainty of Ki and kb from 3P-KMA may be obtained without any further combination [b and 5P-KMA Vb (R = 0.12), and the Vb mean value over the current series was found to be 0.16 ± 0.09 (SD) and 0.05 ± 0.03 (SD), respectively  with that of 0.17 ± 0.07 (SD) previously published for 5P-KMA by Dimitrakopoulou-Strauss et al. further strengthens the findings of the current study [No significant difference over the current series was found between 3P-KMA Ki and 5P-KMA Ki and between 3P-KMA kectively . Howeverbination .  to zero: the greater the kb value, the greater the Ki underestimation. This underestimation was not revealed when comparing 5P-KMA and Patlak’s analysis. We suggest that further studies are warranted to investigate 3P-KMA efficiency in various tissues, either physiological or pathological, showing greater 18F-FDG trapping reversibility than lung cancer lesions.Comparison between 3P-KMA and standard Patlak’s analysis showed that the latter significantly underestimates, on average, the net influx constant (Ki) value in comparison with the former, because it arbitrarily set the release rate constant (k


GluN2A subunit levels were concurrently increased following lithium treatment. Similar alterations were also demonstrated in vivo; lithium administration increased GSK3β serine 9 phosphorylation and GluN2A levels, suggesting a reduced GSK3β activity and augmented GluN2A expression. Correspondingly, we observed that the amplitudes of evoked GluN2A-mediated excitatory postsynaptic currents in mPFC pyramidal neurons were significantly increased following lithium administration. Our data suggest that GSK3β activity negatively regulates GluN2A expression, likely by mediating upstream β-catenin phosphorylation, in prefrontal cortical neurons. Furthermore, our biochemical and electrophysiological experiments demonstrate that lithium mediates a specific increase in GluN2A subunit expression, ultimately augmenting GluN2A-mediated currents in the prefrontal cortex.Glycogen synthase kinase 3β (GSK3β) is a highly conserved serine/threonine kinase that has been implicated in both psychiatric and neurodegenerative diseases including schizophrenia, bipolar disorder, and Alzheimer's disease; therefore regulating its activity has become an important strategy for treatment of cognitive impairments in these disorders. This study examines the effects of lithium on GSK3β and its interaction with β-catenin and NMDA receptors within the prefrontal cortex. Lithium, a clinically relevant drug commonly prescribed as a mood stabilizer for psychiatric disorders, significantly increased levels of phosphorylated GSK3β serine 9, an inhibitory phosphorylation site, and decreased β-catenin ser33/37/thr41 phosphorylation  Glycogen synthase kinase 3β (GSK3β) plays an important role in both psychiatric and neurodegenerative disorders , CK (casein kinase), GSK3β, and β-catenin  animal guidelines and the experimental protocols (#20280) were approved by the Institutional Animal Care and Use Committee at Drexel University College of Medicine. Timed pregnant (TP16) and male (200–250 g) Sprague-Dawley (SD) rats were purchased from Charles River Laboratories .in vitro (DIV) four, half of the media was replaced with fresh serum-free media. Cultures were maintained at 37°C for at least 2 weeks before use.Preparation of rat prefrontal cultures was modified from previously described methods  published a dose-response curve  demonstrating lithium's effect on GSK3β activity, comparing phosphorylation of GSK3β serine 9 to total protein levels. Lithium chloride treatment in the dose range of 5–10 mM was shown to inhibit the enzymatic activity of GSK3β by more than 80%. A 5 mM dose of lithium was sufficient to reduce GSK3 kinase activity, while concurrently increasing total β-catenin levels starting at 6 h following treatment  of male SD rats weighing between 200 and 250 g. Control animals were injected with an equal volume of saline. These doses were chosen because serum levels have been reported to fall within the therapeutic range of drug in patients (0.8–1.0 mmol/L) and higher doses were demonstrated to produce adverse side effects  was added to each well, and incubated for 15 min at 4°C with gentle rocking. The RIPA buffer contained the following inhibitors: sodium pyrophosphate tetrabasic , ethylenediaminetetraacetic acid , sodium fluoride , sodium orthovanadate , leupeptin (inhibits serine and cysteine proteases), aprotinin (inhibits serine proteases), PMSF (inhibits serine proteases), and pepstatin A (inhibits aspartic acid proteases). Adherent cells were collected with a cell scraper, and the cell lysate was transferred to a microcentrifuge tube (combining wells of the same treatment). Lysates were homogenized with a mortar and pestle for 20 strokes and then incubated for 45 min on ice. After incubation, samples were centrifuged at 10,000 g for 10 min at 4°C, pellets discarded, and the supernatant collected and stored at −20°C. Western samples (20 μg/15 μl) were prepared, heated at 95°C for 5 min, cooled to room temperature, and stored at −20°C.Plates were set on ice, and all media was removed using vacuum suction. Wells were washed twice with cold 0.1 M PBS, all solution was removed, 50 μl of RIPA buffer  was thawed, weighed, and homogenized in 9 volumes of RIPA buffer containing inhibitors using a mortar and pestle. Lysates were incubated on ice for 30 min. After incubation, samples were centrifuged at 10,000 g for 15 min at 4°C, and the supernatant was collected and stored at −20°C. Western samples (15 μg/15 μl) were prepared, heated at 95°C for 5 min, cooled to room temperature, and stored at −20°C.in vitro; 15 μg/15 μl in vivo) were run on acrylamide gels comprised of a 3.0% acrylamide stacking gel on top of a 7.5% acrylamide separating gel. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane  with a 5% methanol transfer buffer at 100 V for 1 h at 4°C. Membranes were trimmed, rinsed in Tris-buffered saline with 1% Tween-20 (TBST) and blocked for 1 h with 5% milk in TBST. Then, primary antibodies were incubated overnight at 4°C with agitation. The following antibodies were applied at the approximate concentrations listed: GSK3β , pGSK3β serine 9 , GluN1 , GluN2A , GluN3A , GluN2B , β-catenin  and pβ-cateninS33/37/T41 . β-actin  was used as a loading control. After primary antibody incubation, membranes were rinsed with TBST three times for 20 min, blocked for 1 h with 5% milk in TBST, incubated with secondary antibody  for 1 h, and rinsed with TBST three times for 20 min at room temperature with agitation. Membranes were developed with ECL detection kit . After developing, membranes were rinsed with TBST for 5 min and stripped with Restore Western Blot Stripping Buffer  for an additional 15–30 min, depending on the antibody intensity. Membranes were then rinsed with TBST 3 times for 10 min. To probe the membrane for additional proteins the entire process, the described sequence was repeated from primary antibody incubation; membranes were blocked with 5% milk in TBST for 1 h and then incubated with primary antibody overnight at 4°C with agitation. Protein expression was determined using Bio-Sciences, Piscataway, NJ ImageJ64 software.Whole cell fraction samples  or an equivalent dose of physiological saline, and were sacrificed for slice collection after 24 h. Animals were perfused with ice-cold sucrose buffer, decapitated, and the brain was extracted. The mPFC was dissected in ice-cold sucrose solution and glued to a block in a vibratome bath containing the same solution . Slices containing the mPFC (300 μm) were collected and transferred to a slice chamber containing oxygenated ACSF that was bubbled continuously with 95% O+)-based intracellular solution  and in the presence of picrotoxin (50 μM), DNQX (20 μM), and Ro-25-6981 (0.5 μM), to block GABAA, AMPA, and GluN2B-containing NMDA receptor-mediated currents respectively . Recordings were obtained using a Cesium . All data underwent Shapiro-Wilk normality tests and Levene's Test of Equality of Error Variances when appropriate. The data without normal distribution were analyzed with a non-parametric Mann-Whitney U-test using SPSS. All data was presented as a mean ± standard error (S.E). Level for significance was set at p ≤ 0.05 for all comparisons.Western blot protein expression was determined using the ImageJ64 software. Each sample was taken as a ratio to the loading control . In the case of phosphorylation, each sample was taken as a ratio of its respective total protein levels . Total protein in the text refers to an antibody that tags the target protein without specificity for a particular post-translational modification such as phosphorylation, accounting for cellular levels of that protein under all conditions. For example, total GSK3β would encompass phosphorylated as well as unphosphorylated levels. To replicate prior research findings  = 2.163, p = 0.180, or time, F = 0.020, p = 0.891, on total GSK3β protein levels. There was no significant interaction between treatment group and time point on GSK3β protein levels, F = 0.069, p = 0.799. Lithium treatment significantly increased GSK3β ser9 phosphorylation protein levels, F = 40.593, p = 0.000. There was no significant effect of time on GSK3β ser9 phosphorylation, F = 0.467, p = 0.514. There was no significant interaction between treatment group and time point on GSK3β ser9 phosphorylation protein levels, F = 0.037, p = 0.853  = 3.710, p = 0.090, or time, F = 0.017, p = 0.899, on total β-catenin protein levels. There was no significant interaction between treatment group and time point on β-catenin protein levels, F = 0.269, p = 0.618. Lithium treatment significantly decreased β-catenin ser33/37thr41 phosphorylation protein levels, F = 33.341, p = 0.000. There was no significant effect of time on β-catenin ser33/37thr41 phosphorylation, F = 2.652, p = 0.142. There was no significant interaction between treatment group and time point on β-catenin ser33/37thr41 phosphorylation protein levels, F = 1.896, p = 0.206  for two time points, 4 or 24h, and Western blot analysis was used to determine protein expression levels of β-catenin and pβ-catenin ser33/37/thr41, the GSK3β target regulatory site. A two-way ANOVA was conducted to determine the effects of treatment group and time on total β-catenin as well as β-catenin ser33/37thr41 phosphorylation protein levels. There was no significant effect of treatment group, t-test was run to determine if there were differences in total or phosphorylated β-catenin ser33/37/thr41 protein levels between saline and lithium-treated animals. Lithium administration had no effect on either β-catenin, t(10) = 0.518, p = 0.616, or β-catenin ser33/37/thr41 phosphorylation, t(10) = −0. 431, p = 0.675  for two time points, 4 or 24 h and Western blot analysis was used to determine protein expression levels of NMDAR subunits GluN2A, GluN2B, GluN3A, and GluN1.F = 5.343, p = 0.050. There was a significant effect of time on GluN2A expression, F = 13.559, p = 0.006, increasing after 24 h; but no significant interaction between treatment group and time point on GluN2A protein levels, F = 1.627, p = 0.238. There was no significant effect of treatment group, F = 1.728, p = 0.225, or time, F = 4.029, p = 0.080, as well as no significant interaction between treatment group and time point on GluN2B protein levels, F = 0.437, p = 0.527. Additionally, there was also no significant effect of treatment group, F = 0.999, p = 0.347, or time, F = 0.714, p = 0.423, as well as no significant interaction between treatment group and time point on GluN3A protein levels, F = 0.561, p = 0.475  = −0.151, p = 0.883, or GluN1 t(8) = −1.333, p = 0.219, protein levels compared to saline-treated animals  = −2.387, p = 0.038  = −0.227, p = 0.822 , we treated rats with lithium (50 mg/kg) and measured evoked GluN2A-mediated excitatory postsynaptic currents (eEPSCs) in the layer 5 pyramidal neurons of the mPFC. Based on our previous reports . Inositol and GSK3 are the two major targets of lithium, which are both argued to underlie its effects. The inositol depletion model proposes that lithium inhibits IMPase, depleting the cell of endogenous inositol, thereby blocking the production of inositol 1,4,5 triphosphate (InsP3) and preventing extracellular signaling by this pathway. Albeit some researchers suggest inositol depletion mediates the effects of lithium  in cultured neurons reported similar findings regarding the relationship between GSK3β and NMDA receptors. Induced activation of GSK3β, either pharmacologically or genetically, led to decreased GluN2A/B expression that could then be mitigated by the simultaneous inhibition of the kinase. This study demonstrates the negative regulation that GSK3β imparts over GluN2A and GluN2B (Zhu et al.,  in cultuSpecifically, PFC persistent neuronal firing is the foundation of working memory with NMDA receptor activity playing a substantial role in this process Wang, . NMDA anNMDARs and PFC-dependent cognition also share common ties to the pathology observed in schizophrenia. NMDA receptor disruption has been well characterized in animal models of schizophrenia, demonstrating the paramount role these receptors play in disease. Mutations in high-risk gene candidates for schizophrenia such as neuregulin 1, DISC1, and dysbindin have all been demonstrated to disturb NMDA receptor regulation (Geddes et al., Surmounting evidence links NMDA receptor hypofunctioning in the PFC, particularly of GluN2A and GluN2B-containing receptors, as an underlying pathological origin of cognitive impairments (Wang et al., in vitro and in vivo with effects possibly mediated by β-catenin phosphorylation in the PFC. It is important to note that a limitation with our study is that our findings are largely correlational since we do not directly test whether lithium inhibits GSK3β kinase activity or selectively manipulate GSK3β in the mPFC, such as the utilization of shRNA. Therefore, our current study has set an essential framework, but future studies are warranted to further untangle the nature of lithium and its possible therapeutic actions of NMDA receptor augmentation in the PFC and the regulation of GSK3β.In summary, pharmacological inhibition of GSK3β with lithium significantly increased pGSK3β ser9 and decreased pβ-catenin ser33/37/thr41, thereby suggesting GSK3β inhibition and reduced β-catenin degradation. Total GluN2A levels concurrently increased after 24 h following lithium, demonstrating preferential upregulation of the GluN2A gene expression. These changes also corresponded with a physiological change in the mPFC, augmenting GluN2A-mediated eESPCs. We speculate this could be due to an increase in GluN2A expression at the postsynaptic density, which could increase the GluN2A-mediated inward current of mPFC pyramidal neurons, leading to increased amplitude of excitatory events. Conclusively, GSK3β activity influences the expression of GluN2A both The authors have read and have abided by the statement of ethical standards for manuscripts submitted to the Frontiers in Cellular Neuroscience. We declare that submitted manuscript does not contain previously published materials and are not under consideration for publication elsewhere. Each author has made a significant scientific contribution to the study, and is familiar with the primary data. All authors listed have read the complete manuscript and have approved submission of the paper. The manuscript is original work without fabrication, fraud, or plagiarism. All authors declare no conflicts of interest.SM contributed to the design, acquisition, analysis, and interpretation of the data; BF contributed to collection and interpretation of the electrophysiological data; and W-JG contributed to the conception of the work. All authors contributed to writing and editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A large number of enzyme inhibitors are used as drugs to treat several diseases such as gout, diabetes, AIDS, depression, Parkinson’s and Alzheimer’s diseases. Electrochemical biosensors based on enzyme inhibition are useful devices for an easy, fast and environment friendly monitoring of inhibitors like drugs. In the last decades, electrochemical biosensors have shown great potentials in the detection of different drugs like neostigmine, ketoconazole, donepezil, allopurinol and many others. They attracted increasing attention due to the advantage of being high sensitive and accurate analytical tools, able to reach low detection limits and the possibility to be performed on real samples. This review will spotlight the research conducted in the past 10 years (2007–2017) on inhibition based enzymatic electrochemical biosensors for the analysis of different drugs. New assays based on novel bio-devices will be debated. Moreover, the exploration of the recent graphical approach in diagnosis of reversible and irreversible inhibition mechanism will be discussed. The accurate and the fast diagnosis of inhibition type will help researchers in further drug design improvements and the identification of new molecules that will serve as new enzyme targets. The living organisms are the site of many reactions driven by enzymes. The aberrant activity of one of these enzymes can result in disruption of the normal biological process leading to the occurrence of diseases. One of the main therapeutic approaches in the treatment and prevention of several diseases such as cancer, AIDS, Alzheimer’s and Parkinson’s diseases, depression and hyperthyroidism is based on the use of drugs that act on enzyme activity, especially as inhibitors ,2,3,4,5.Drugs are considered as xenobiotics, so they are metabolized by enzymatic reactions into one or more other active/inactive pharmacological compounds ,7. The fIn analytical biochemistry, researchers use in vitro tests to determine the influence of drugs on enzymes to test their effectiveness in single use and sometimes in drug mixtures. Enzymatic inhibition studies are used to follow pharmacokinetic changes that may affect drug effectiveness and safety, especially if they have a low therapeutic index. Furthermore, in silico method showed a great interest for the development of new drugs by predicting the metabolic pathways of physiologically active substances and their interactions on a molecular level .Development of new analytical tools for simultaneously monitoring of drug concentration and enzymatic inhibition rates for in vitro experiments is necessary before proceeding to in vivo assays, especially for testing newly designed drugs . AccordiOver the last decade, considerable interest has been focused on enzyme-based electrochemical biosensing systems as simple and effective tools for analytical investigations as an alternative to conventional methods. The utility of these bio-devices comes from their simple sensing systems to analyze the substrate, the generated product, or even the screening and measurement of other compounds such as drugs that act as inhibitors of enzymatic activity. Remarkable developments in enzymatic electrochemical biosensors designed for screening of therapeutic compounds with required analytical performances have been reported ,17,18,19The importance of using enzymes for analytical purposes is due to their numerous properties as precision in the measurement of enzymatic reaction rates, the sensitivity towards the substrate due to the ‘key-lock’ configuration, the suitability for the needs and the wide diversity of analytical enzymes available on a large-scale. In clinical and pharmaceutical fields, enzymatic electrochemical biosensors based on inhibition are very promising and offer numerous advantages such as robustness, portability and cost-effectiveness.Over the past years, our group has published reviews dealing with electrochemical biosensors based on enzyme inhibition for the determination of different compounds such as pesticides, food contaminants and drugs ,26. HoweEnzymatic inhibition is considered as the key point in clinical applications and drug therapy. Indeed, several drugs such as neostigmine, donepezil, allopurinol, methimazole and moclobemide induce the inhibition of target enzymes involved in various diseases ,43,44. AThe response of biosensor based on enzyme inhibition is recorded prior addition of inhibitor  and carbon nanofibers (CNFs) are some of the novel nanomaterial components used in transducer design to improve the electrical signals from enzymes to the electrode ,65.Recently, electrochemical biosensors based on screen-printed electrodes were reported as encouraging analysis tools meeting the requirements of in situ screening devices, since all the equipment needed for the electrochemical analysis is portable. They have all the major performance characteristics of biosensors, such as good sensitivity, minimum sample preparation, simplicity of the apparatus and fast results readout. Moreover, they are cost effective, small and tend to be increasingly miniaturized thanks to new technologies .The development of microfluidic lab-on-a-chip systems for analytical chemistry, biology, biomedical and clinical diagnostics applications has led to interesting analytical tools since these devices offer many potential advantages, including reduced reagent consumption, smaller analysis volumes, faster analysis times, and increased instrument portability .Acetylcholinesterase (AchE) is considered as neurophysiological enzyme that controls the concentration of the neurotransmitter acetylcholine (Ach) in the synaptic cleft via the hydrolysis of acetylcholine into choline and acetate . A decreDu et al. suggested in their work an electrochemical biosensor for the detection of galantamine and neostigmine. A homogenous film of silica sol-gel incorporating gold nanoparticles (AuNPs-Si-SG) was deposited electrochemically on a glassy carbon electrode. Then, acetylcholinesterase (AchE) was coated onto the modified glassy carbon electrode. The use of silica sol-gel with AuNPs not only prevents acetylcholinesterase from leaking out of the electrode surface but also creates a stable microenvironment for the enzyme activity .Pohanka et al. proposed an electrochemical biosensor with acetylcholinesterase immobilized on a surface of the platinum working electrode. This biosensor was used to test two different drugs namely, tacrine and 7-MEOTA (the 7-methoxy derivative) used in treatment of cognitive manifestations of AD. The comparison of the inhibition effect of these two drugs revealed that 7-MEOTA is a 400 times stronger inhibitor than tacrine. This finding suggests the use of 7-MEOTA in treatment of AD cognitive manifestation at lower doses and with the same effect as tacrine, which make it a non-toxic alternative drug. This study showed that the biosensor technology could be used as a cheap and quick device for testing AchE inhibitors .−8–10−5 M with −7 M [Parsajoo and Kauffmann developed a microfluidic device to monitor AchE activity and its inhibition. They successfully implemented a silver electrode for the amperometric detection of the artificial substrate thiocholine. They suggested that the developed system has a good sensitivity and selectivity towards compounds that react with silver chloride such as free thiol or sulfide. Furthermore, neostigmine inhibition effect was followed with the developed system in the range of 10× 10−7 M .Chemical mediators are widely used with screen-printed electrodes (SPE) to reduce the applied potentials, to increase the sensitivity of detection and to avoid interferences. Moreover, it is easy to incorporate the mediator in the ink for the preparation of screen-printed electrodes. Asturias-Arribas et al. developed AchE biosensor-based on screen-printed electrode (SPEs) using tetrathiafulvalene (TTF) as a mediator. A cross-linking method was used to immobilize AchE on TTF modified SPEs (TTF-SPEs). The suggested biosensor was optimized for chronoamperometric detection of codeine in urine samples and in pharmaceutical tablets. Codeine binds to AchE in a competitive way .In another work, Zamfir et al. fabricated an AchE biosensor based on tetrathiafulvalene-tetracynoquinodimethane ionic liquid gel (TTF-TCNQ-IL). They characterized the biosensor using FT-IR spectroscopy and electrochemical impedance spectroscopy. This biosensor was used for the detection of carbamate drugs: neostigmine and eserine with limits of detection of 0.3 nM and 26 pM respectively. The proposed biosensor can be also applied in the screening of carbamates drug in wastewater .Turan et al. reported a bi-enzymatic biosensor of acetylcholinesterase (AchE) and choline oxidase (ChO) based on graphite electrode coated with poly (BODT-co-FMOC) a conducting copolymer to monitor anti-dementia drugs (neostigmine and donepezil). They used the copolymer as an immobilization matrix for AchE and ChO. The authors used Scanning Electron Microscopy (SEM) and cyclic voltammetry as characterization techniques for the constructed biosensor. Neostigmine and donepezil inhibition effects on acetylcholinesterase were studied with the developed biosensor and the detection limits of 2.5 nM and 0.071 nM were reported respectively .In their work, Vandeput et al. developed an amperometric biosensor based on immobilized acetylcholinesterase for the screening of several drugs known as AchE inhibitors. They used glutaraldehyde (GA) and cysteamine for a covalent immobilization of acetylcholinesterase on the surface of the gold microelectrode. They suggested that this type of immobilization allowed the retention of acetylcholinesterase activity, which facilitated the application of this system with reversible inhibitors of acetylcholinesterase .For many years, magnetic particles (MPs) have been used as a good platform for the immobilization of enzymes through their attachment with amino, hydroxyl or carboxyl groups on the surface of MPs. Kostelink et al. suggested an acetylcholinesterase/MPs screen-printed electrode based biosensor for the detection of an anti-Alzheimer’s drug . They immobilized acetylcholinesterase covalently using MPs and glutaraldehyde (GA). Under the optimized conditions, acetylcholinesterase inhibition by galantamine was studied in the range from 2.5 to 10 µM with a detection limit of 1.5 µM .Monoamine oxidase (MAO) catalyzes the production of hydrogen peroxide through the deamination of monoamine such as dopamine, adrenaline, serotonin, etc. This class of enzyme have two different isomers: monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) ,76.Monoamine oxidase inhibitors (IMAO) have shown potential uses in the treatment of several diseases such as depression and Parkinson’s disease . The firIn the last decade, the Medyantseva group have developed different amperometric biosensors based on immobilized MAO to monitor different drugs used as antidepressants ,84,85,86−7 M, 8 × 10−9 M and 8 × 10−10 M for pyrazidol, petylyl and fluoxetine respectively [The first biosensor for analyzing pyrazidol, petylyl and fluoxetine (antidepressants) proposed by Medyantseva group was based on immobilized MAO with gelatin on platinum screen-printed electrode (SPE). These antidepressants act as inhibitors of MAO. The developed biosensor allowed the measurement of these inhibitors with low detection limits, which were 8 × 10ectively ,86.−8 M (furadonine), 8.3 × 10−9 M (furazolidone) and 9.4 × 10−10 M (furagin) [Nitrofuran drugs are another class of drugs typically used as antibiotics and antimicrobials, which act as inhibitors of MAO. Volotskaya et al. suggested an amperometric biosensor based on immobilized MAO on screen-printed electrode for the assessment of nitrofuran drugs  in pharmaceutical tablets and biological fluids such as urine. These drugs were assessed with the proposed biosensor with low detection limits of 8.5 × 10furagin) .In another work the Medyantseva group developed a MAO biosensor based on a screen-printed electrode (SPE) modified with multiwalled carbon nanotubes (MWCNTs) for determination of antidepressants. The monoamine oxidase (MAO) was immobilized on SPE by a cross-linking method using BSA and glutaraldehyde (GA). In this study, they used the developed biosensor to monitor the concentration of the antidepressants like imipramine, afobazole and phenazepam in drugs formulation and biological fluids (urine) ,84.−8–10−4 M for both imipramine and amitryptline with detection limits of 7 × 10−9 M and 8 × 10−9 M respectively. The use of MWCNTs and AgNPs improved the sensitivity of the biosensor and extended the concentration range of the tested drugs [On the other hand, the same group proposed a screen-printed electrode (SPE) modified with MWCNTs, silver nanoparticles (AgNPs) and immobilized MAO for determination of imipramine and amitryptline in urine. The inhibition effect of these antidepressants on MAO was studied in the concentration range of 10ed drugs .−8–10−4 M.In order to improve the analytical performance of MAO biosensor Brusnitsyn et al. suggested an amperometric MAO biosensor based on a screen-printed electrode (SPE) modified with nanomaterials such as carbon nanotubes (CNTs) and graphene oxide (GO) for monitoring antidepressants (moclobemide and amitriptyline) in pharmaceutical tablets . The dru−9–10−4 M with a detection limit of 8 × 10−10 M. In the other biosensor, they modified screen-printed electrode with carbon nanotubes (CNTs) and AgNPs. These nanomaterials were stabilized by the use of functionalized hyperbranched polyesterpolyols [−8–10−4 M and with a detection limit of 3 × 10−9 M.Recently the Medyantseva group developed two different MAO biosensors modified with nanomaterials for monitoring moclobemide, tianeptine and amitriptyline antidepressants drugs in urine and in pharmaceutical tablets. In the first biosensor, they used screen-printed electrode modified with MWCNTs, gold and silver nanoparticles . Using trpolyols . The ant2) for measurement of selegiline hydrochloride, which is an irreversible inhibitor of MAO [−3 M to 3.5 × 10−4 M with low detection limit of 0.67 × 10−3 M.Another group reported recently a MAO biosensor based on a SPE modified with manganese dioxide  is one of the xanthine oxidase inhibitors in clinical use to treat gout. Therefore, xanthine oxidase is the target enzyme in the implementation of biosensors to screen the drug allopurinol (Xanthine oxidase (XO) is an important enzyme that can catalyze both hypoxanthine and xanthine. It allows the oxidation of hypoxanthine (HYP) to xanthine (XAN), which in the next step is oxidized to uric acid (UA) as the final degradation product in the human purine catabolic pathway. The overproduction of these products lead to metabolic disorders such as xanthinuria, hyperuricemia, gout and renal failure ,89. The opurinol .A xanthine oxidase biosensing system was used for the first time for simultaneously monitoring allopurinol and oxypurinol at nanomolar concentrations by Hason and coworkers . Allopur−6 M to 2 × 10−4 M with a detection limit of 10−7 M. Using the same biosensor, xanthine oxidase inhibition by allopurinol was investigated and the obtained calibration curve was recorded in presence and in absence of the inhibitor. It was determined that allopurinol exerted a quasi-reversible competitive inhibition, which can compete with xanthine for the active site of xanthine oxidase to form the xanthine oxidase—inhibitor complex [A novel xanthine biosensor based on immobilization of xanthine oxidase by attractive materials layered double hydroxides was developed in 2009 . Xanthin complex .Sida rhombifolia L. has attracted increasing attention due to its anti-gout effects. The inhibition kinetics of Sida rhombifolia L. extracts toward xanthine oxidase were investigated using an electrochemical biosensing method [The side effects caused by synthetic drugs like allopurinol have prompted people to turn to traditional medicine using medicinal plants that are considered safer and more effective. Accordingly, many studies have been focused on the inhibitory effects of medicinal plants on xanthine oxidase ,93,94,95g method . Based o2O2. The developed biosensor was tested first for allopurinol analysis. A linear range of allopurinol concentrations is obtained from 0.125 to 2.5 µM with an estimated 50% of inhibition Recently, our group developed a simple and sensitive amperometric biosensor for the screening of medicinal plants for potential xanthine oxidase inhibitors . In thisCytochrome P450s (CYPs) are a super-family of enzymes called hemoproteins due to the presence of a heme in their active site. They are involved in the metabolism of a variety of molecules such as drugs ,98. ThusConsiderable efforts have been focused on the development of biosensors based on cytochrome P450 activity measurement. Many techniques have been used to improve the efficiency of these biosensors. To increase the electron transfer between the cytochrome P450 and the electrode, the use of different electrode type and the modification of surface transducers are of high relevance .Among different isomers of cytochrome P450, cytochrome P450-3A4 (CYP3A4) is the most used target enzyme in pharmaceutical fields as it metabolizes a majority of drugs ,108. MieCarbon nanotubes (CNTs) and carbon nanofibers (CNFs) have attracted great interest recently as a new platform for biosensor assembly. The immobilization of a number of enzymes, including CYP enzymes, for the design of electrochemical biosensors using this new platform has been described ,103.Using a carbon nanofibers (CNFs)-based CYP3A4 biosensor the inhibition effect of ketoconazole was also reported . The immThe use of nanomaterials such as multiwalled carbon nanotubes (MWCNT) in the design of cytochrome P450 biosensors improves the sensitivity, which allows one to reach low detection limits and consequently enhance drug screening and monitoring. Carrara group used multiwalled carbon nanotubes (MWCNT) for the design of cytochrome P450 (CYPs)-based biosensors to monitor multiple drugs . For thiLater, MCNWTs were used for CYP3A4 sensor design. The enzymatic biosensor was employed for the study of abiraterone, a drug used in prostate cancer therapy . The senBaj-Rossi and coworkers modified a SPE surface with MWCNTs to immobilize microsomal CYP1A2 for naproxen detection. Naproxen (NAP) is non-steroidal anti-inflammatory drug widely used in osteo- and rheumatoid arthritis therapy. The proposed electrochemical biosensor was used for 16h NAP monitoring using continuous flow system and was calibrated to measure NAP in mouse serum . The MWCThe research team of Ajayi studied the competitive inhibition by paroxetine of CYP2D6 encapsulated in a nanostructured material . ParoxetAnother promising method to build an enzyme biosensor is based on the enzyme adsorption onto a dendrimer layer . PANAM dA new biosensor coupling DNA to enzymes was designed for the study of enzyme inhibition and consequently to prevent DNA damage due to CYP 450cam (CYP101) metabolism of styrene . CYP/DNAo-quinones [Tyrosinase is an enzyme that holds two copper on its active site and catalyzes the production of quinones ,110. Thiquinones ,111,112 Methimazole (MT) is one of the active drugs used in the therapy of hyperthyroidism. This drug inhibits the production of thyroid hormones due to its action that decreases the iodide incorporation into tyrosine. Therefore, recent electrochemical biosensors have been reported for the analysis of methimazole as an antithyroidal drug ,24.o-benzoquinone due to the formation of the corresponding thioquinone derivatives. Furthermore, the application of this biosensor to different pharmaceutical samples containing methimazole supports the utility of this method.Martinez and coworkers presented and discussed an electrochemical biosensor based on tyrosinase inhibition for methimazole analysis. The resulting biosensor was constructed using a screen-printed electrode modified with carbon nanotubes and tyrosinase, providing a sensitive, fast and inexpensive analytical method. Using this method, methimazole determination was performed in the range of 0.074–63.5 µM with a detection limit of 0.056 µM . The addAnother biosensor was developed by Kurbanoglu and coworkers, for the investigation of methimazole, using a nanoparticles based lab-on-a-chip system . In thiso-benzoquinone, the electrochemical reduction of o-benzoquinone was detected at a gold electrode surface at 0 V vs. Ag/AgCl before and after enzyme inhibition by pipemidic acid. Upon the addition of pipemidic acid, the reduction current decreases due to the formation of amino-quinone derivatives. The usefulness of this microfluidic-enzymatic sensor coupled with electrochemical method toward the detection of pipemidic acid at very low concentrations in pharmaceutical samples has been confirmed in this work. The recovery of pipemidic acid from four pharmaceutical samples ranged from 97.50% to 102.50%. The proposed method could be used to determine the pipemidic acid concentration in the range of 0.02–70 µM with a limit of detection of 18 nM.Pipemidic acid (PA) is a synthetic quinolone drug used as an antibacterial agent. Bertolino and coworkers developed a microfluidic-enzymatic sensor combined with electrochemical detection designed for the quantification of pipemidic acid . The detA novel biosensor based on the inhibition of tyrosinase activity by captopril, which is generally used to treat congestive heart failure, has been developed by Kurbanoglu and coworkers . The desMany researchers have linked the presence of protease to diseases  ,115,116.–8 M for naproxen and diclofenac and 0.5 × 10–8 M for ibuprofen and tolmetin  are widely used against several diseases, especially arthrosis since they compete with arachidonic acid that binds to cyclooxygenase isoforms (COx1 and COx2). COx1 exists all over the human biological system, while COx2 is rarely present in the stomach. The cyclooxygenase-based biosensor have been described for the detection of NSAIDs drugs including naproxen, diclofenac, ibuprofen and tolmetin . The enztolmetin .DNA methylation phenomena are correlated with various diseases, especially cancer. Aberrant methyltransferase enzyme (MTase) is associated to the pathogenesis of leukemia. Therefore, the study of the MTase activity and its inhibitors is important in the cancer curing process. Yin and coworkers  developeAberrant O-linked N-acetylglucosamine transferase OGT activity has been found to be involved in many diseases such as diabetes , Alzheimp-nitrophenyl α-D-glucopyranoside (PNPG) which is converted to 4-nitrophenol and measured by chronoamperometry. The results showed that the inhibition of α-glycosidase (AG) with amaryl and acarbose was reversible. Therefore, it will be convenient to use the developed biosensor for the screening of medicinal plants having the ability to inhibit α-glycosidase.Diabetes is one the most common diseases in the world. In traditional medicine, the use of natural plants with anti-diabetic activities is widely used in the treatment of type 2 diabetes mellitus, called also non-insulin-dependent diabetes (NIDDM). The biological activity of these natural compounds was verified by inhibition of α-glycosidase (AG). In 2007, an electrochemical biosensor was used for the detection of two different drugs (amaryl and acarbose) that act as α-glycosidase inhibitors . The bio2)-based electrode [p-nitrophenyl α-D-glucopyranoside on a screen-printed carbon electrode at low pH to prevent the premature reaction between substrate and enzyme. In the presence of Tebengau plant extracts, the α-glycosidase enzymatic activity was inhibited, suggesting the application of the developed biosensor in the rapid screening of inhibitors from medicinal plants, which will prevent the enzymatic production of glucose.Furthermore, α-glycosidase enzyme was immobilized onto an amine-functionalized multiwalled carbon nanotubes (MWCNTs-NHlectrode . The bioSulfonamides (SA’s) are a superfamily of drugs used in human and veterinary medicine. In the body, they inhibit carbonic anhydrase enzyme. The inhibition reaction can be used as tool for the detection of SA’s pharmaceutical residues in biological and environmental samples. Our research group developed an electrochemical carbonic anhydrase (CA)-based biosensor for the detection of sulfanilamide (SAD) . The enzThis review compiles research activities realized during the last decade using electrochemical biosensors for drug detection based on enzymatic inhibition. The usefulness and the importance of these biosensors in analytical biochemistry for the screening of drugs and their implication in the therapy of various diseases were reported. Moreover, since the phenomena of enzyme inhibition is unavoidably associated with drug therapy, the inhibition mechanisms were also discussed.Some of the most important steps in drug-metabolizing phenomena are based on enzymatic reactions. The enzymes most involved in this biotransformation include CYP enzymes. Other enzymes, including acetylcholinesterase, xanthine oxidase, monoamine oxidase, etc., are considered as drug targets. In general, the consequences of prescribing drugs may result on enzymatic inhibition.In this paper, we focus our review on monitoring enzyme inhibition for pharmaceutical and clinical applications using electrochemical biosensors. Aberrant activity of some enzymes is associated with various diseases. Direct measurement of enzyme activity in biological samples before and after administration of drug is an obvious method for assessing inhibition effects, among others, before proceeding to in vivo novel drug administration studies. Numerous drugs are analyzed in this way, such as antitumoral, anti-epilepsy, anti-infectious agents, etc. Therefore, it is important to develop a simple and sensitive method for enzymatic activity assays. More importantly, for curing diseases caused by aberrant enzymes, the screening of drugs as enzyme inhibitors is also necessary and interesting.The electrochemical biosensors based on enzyme inhibition are more and more used in drug screening as alternative techniques to conventional methods. They are simple, miniaturized, fast and sensitive. For all these reasons and others, the present review reports the latest electrochemical biosensors designed in the clinical and pharmaceutical field. The enzymes employed, inhibition type induced by various drugs, the transducers used and their applications were discussed. The recent works reported in this review indicate the potential applications of biosensors based on enzyme inhibition for drug screening and monitoring. Hence, more attention should be focus on the application of biosensors on real samples and clinical cases. Accordingly, biosensor development should be more focused on miniaturized, sensitive and portable devices for analyzing real samples.In addition, this review showed that in the inhibition studies, the kinetics of enzymatic reaction in absence and presence of different inhibitors are poorly reported. The novel graphical approach proposed a few years ago by Amine et al.  can be uThe personalized screening methods such as biosensors based on enzyme inhibition are a promising technology in the pharmaceutical and clinical fields, especially to improve the quality of life of patients with several diseases and subjected to numerous drug therapies.