Decreasing nitrogen assimilation under drought stress by suppressing DST-mediated activation of Nitrate Reductase 1.2 in rice.

Nitrogen is an essential nutrient for plant growth and development, and plays vital roles in crop yield. Assimilation of nitrogen is thus fine-tuned in response to heterogeneous environments. However, the regulatory mechanism underlying this essential process remains largely unknown. Here, we report that a zinc-finger transcription factor, drought and salt tolerance (DST), controls nitrate assimilation in rice by regulating the expression of OsNR1.2. We found that loss of function of DST results in a significant decrease of nitrogen use efficiency (NUE) in the presence of nitrate. Further study revealed that DST is required for full nitrate reductase activity in rice and directly regulates the expression of OsNR1.2, a gene showing sequence similarity to nitrate reductase. Reverse genetics and biochemistry studies revealed that OsNR1.2 encodes an NADH-dependent nitrate reductase that is required for high NUE of rice. Interestingly, the DST-OsNR1.2 regulatory module is involved in the suppression of nitrate assimilation under drought stress, which contributes to drought tolerance. Considering the negative role of DST in stomata closure, as revealed previously, the positive role of DST in nitrogen assimilation suggests a mechanism coupling nitrogen metabolism and stomata movement. The discovery of this coupling mechanism will aid the engineering of drought-tolerant crops with high NUE in the future.

The Benzene Hematotoxic and Reactive Metabolite 1,4-Benzoquinone Impairs the Activity of the Histone Methyltransferase SET Domain Containing 2 (SETD2) and Causes Aberrant Histone H3 Lysine 36 Trimethylation (H3K36me3).

Human SETD2 is the unique histone methyltransferase that generates H3K36 trimethylation (H3K36me3), an epigenetic mark that plays a key role in normal hematopoiesis. Interestingly, recurrent inactivating mutations of SETD2 and aberrant H3K36me3 are increasingly reported to be involved in hematopoietic malignancies. Benzene (BZ) is a ubiquitous environmental pollutant and carcinogen that causes leukemia. The leukemogenic properties of BZ depend on its biotransformation in the bone marrow into oxidative metabolites, in particular 1,4-benzoquinone (BQ). This hematotoxic metabolite can form DNA and protein adducts that result in the damage and the alteration of cellular processes. Recent studies suggest that BZ-dependent leukemogenesis could depend on epigenetic perturbations, notably aberrant histone methylation. We investigated whether H3K36 trimethylation by SETD2 could be impacted by BZ and its hematotoxic metabolites. Herein, we show that BQ, the major leukemogenic metabolite of BZ, inhibits irreversibly the human histone methyltransferase SETD2, resulting in decreased H3K36me3. Our mechanistic studies further indicate that the BQ-dependent inactivation of SETD2 is due to covalent binding of BQ to reactive Zn-finger cysteines within the catalytic domain of the enzyme. The formation of these quinoprotein adducts results in loss of enzyme activity and protein crosslinks/oligomers. Experiments conducted in hematopoietic cells confirm that exposure to BQ results in the formation of SETD2 crosslinks/oligomers and concomitant loss of H3K36me3 in cells. Taken together, our data indicate that BQ, a major hematotoxic metabolite of BZ, could contribute to BZ-dependent leukemogenesis by perturbing the functions of SETD2, a histone lysine methyltransferase of hematopoietic relevance. SIGNIFICANCE STATEMENT: Benzoquinone is a major leukemogenic metabolite of benzene. Dysregulation of histone methyltransferase is involved in hematopoietic malignancies. This study found that benzoquinone irreversibly impairs SET domain containing 2, a histone H3K36 methyltransferase that plays a key role in hematopoiesis. Benzoquinone forms covalent adducts on Zn-finger cysteines within the catalytic site, leading to loss of activity, protein crosslinks/oligomers, and concomitant decrease of H3K36me3 histone mark. These data provide evidence that a leukemogenic metabolite of benzene can impair a key epigenetic enzyme.

Interaction of Natural Compounds in Licorice and Turmeric with HIV-NCp7 Zinc Finger Domain: Potential Relevance to the Mechanism of Antiviral Activity.

Nucleocapsid proteins (NCp) are zinc finger (ZF) proteins, and they play a central role in HIV virus replication, mainly by interacting with nucleic acids. Therefore, they are potential targets for anti-HIV therapy. Natural products have been shown to be able to inhibit HIV, such as turmeric and licorice, which is widely used in traditional Chinese medicine. Liquiritin (LQ), isoliquiritin (ILQ), glycyrrhizic acid (GL), glycyrrhetinic acid (GA) and curcumin (CUR), which were the major active components, were herein chosen to study their interactions with HIV-NCp7 C-terminal zinc finger, aiming to find the potential active compounds and reveal the mechanism involved. The stacking interaction between NCp7 tryptophan and natural compounds was evaluated by fluorescence. To elucidate the binding mode, mass spectrometry was used to characterize the reaction mixture between zinc finger proteins and active compounds. Subsequently, circular dichroism (CD) spectroscopy and molecular docking were used to validate and reveal the binding mode from a structural perspective. The results showed that ILQ has the strongest binding ability among the tested compounds, followed by curcumin, and the interaction between ILQ and the NCp7 zinc finger peptide was mediated by a noncovalent interaction. This study provided a scientific basis for the antiviral activity of turmeric and licorice.

Arsenic co-carcinogenesis: Inhibition of DNA repair and interaction with zinc finger proteins.

Arsenic is widely present in the environment and is associated with various population health risks including cancers. Arsenic exposure at environmentally relevant levels enhances the mutagenic effect of other carcinogens such as ultraviolet radiation. Investigation on the molecular mechanisms could inform the prevention and intervention strategies of arsenic carcinogenesis and co-carcinogenesis. Arsenic inhibition of DNA repair has been demonstrated to be an important mechanism, and certain DNA repair proteins have been identified to be extremely sensitive to arsenic exposure. This review will summarize the recent advances in understanding the mechanisms of arsenic carcinogenesis and co-carcinogenesis, including DNA damage induction and ROS generation, particularly how arsenic inhibits DNA repair through an integrated molecular mechanism which includes its interactions with sensitive zinc finger DNA repair proteins.

Leveraging on Active Site Similarities; Identification of Potential Inhibitors of Zinc-Finger and UFSP domain Protein (ZUFSP).

BACKGROUND: ZUFSP (Zinc-finger and UFSP domain protein) is a novel representative member of the recently characterized seventh class of deubiquitinating enzymes (DUBs). Due to the roles DUBs play in genetic instability, they have become a major drug target in cancer and neurodegenerative diseases. ZUFSP, being a DUB enzyme has also been implicated in genetic stability. However, no lead compound has been developed to target ZUFSP. OBJECTIVE/METHODS: Therefore, in this study, we used a combined drug repurposing, virtual screening and per-Residue Energy Decomposition (PRED) to identify ZUFSP inhibitors with therapeutic potential. 3-bromo-6-{[4-hydroxy-1-3(3-phenylbutanoyl)piperidin-4-yl]methyl}-4H,5H,6H,7H-thieno[2,3- C]pyridine-7-one (BHPTP) which is an inhibitor of USP7 was repurposed to target ZUFSP. The rationale behind this is based on the similarity of the active between USP7 and ZUFSP. RESULTS: PRED of the binding between BHPTP and ZUFSP revealed Cys223, Arg408, Met410, Asn460, and Tyr465 as the crucial residues responsible for this interaction. The pharmacophoric moieties of BHPTP responsible for this binding along with other physiochemical properties were used as a filter to retrieve potential ligands. 799 compounds were retrieved, ZINC083241427, ZINC063648749, and ZINC063648753 were selected due to the binding energy they exhibited. Cheminformatics analysis revealed that the compounds possess high membrane permeability, however, BHPTP had a low membrane permeability. Furthermore, the compounds are drug like, having obeyed Lipinski's rule of five. CONCLUSION: Taken together, findings from this study put ZINC083241427, ZINC063648749, and ZINC063648753 as potential ZUFSP inhibitor, however, more experimental validation is required to unravel the mechanism of actions of these compounds.

Zinc finger protein-440 promotes cartilage degenerative mechanisms in human facet and knee osteoarthritis chondrocytes.

OBJECTIVES: To investigate the role of zinc finger protein 440 (ZNF440) in the pathophysiology of cartilage degeneration during facet joint (FJ) and knee osteoarthritis (OA). METHODS: Expression of ZNF440 in FJ and knee cartilage was determined by immunohistochemistry, quantitative (q)PCR, and Western blotting (WB). Human chondrocytes isolated from FJ and knee OA cartilage were cultured and transduced with ZNF440 or control plasmid, or transfected with ZNF440 or control small interfering RNA (siRNA), with/without interleukin (IL)-1ß. Gene and protein levels of catabolic, anabolic and apoptosis markers were determined by qPCR or WB, respectively. In silico analyses were performed to determine compounds with potential to inhibit expression of ZNF440. RESULTS: ZNF440 expression was increased in both FJ and knee OA cartilage compared to control cartilage. In vitro, overexpression of ZNF440 significantly increased expression of MMP13 and PARP p85, and decreased expression of COL2A1. Knockdown of ZNF440 with siRNA partially reversed the catabolic and cell death phenotype of human knee and FJ OA chondrocytes stimulated with IL-1ß. In silico analysis followed by validation assays identified scriptaid as a compound with potential to downregulate the expression of ZNF440. Validation experiments showed that scriptaid reduced the expression of ZNF440 in OA chondrocytes and concomitantly reduced the expression of MMP13 and PARP p85 in human knee OA chondrocytes overexpressing ZNF440. CONCLUSIONS: The expression of ZNF440 is significantly increased in human FJ and knee OA cartilage and may regulate cartilage degenerative mechanisms. Furthermore, scriptaid reduces the expression of ZNF440 and inhibits its destructive effects in OA chondrocytes.

Integrity of zinc finger motifs in PML protein is necessary for inducing its degradation by antimony.

Antimony (Sb) belongs to the same group as arsenic (As) in the periodic table, and both share similar characteristics. However, Sb2O3 (SbIII) has no methylation capacity, unlike arsenic trioxide (As2O3). In the present study, we determined the effect of SbIII on NB4 cells and found that antimony could induce PML-RARα fusion protein degradation, reorganization of PML-NBs, and NB4 cell differentiation with low cytotoxicity. On the other hand, zinc finger motifs in PML protein are considered to be a key target binding site for arsenic-induced PML-RARα protein degradation. Interestingly, antimony and arsenic lost their ability to degrade PML-RARα fusion protein in NB4 cells following pretreatment with phenanthroline (i.e., chelator of zinc ions), indicating that the integrity of zinc finger motifs in PML-RARα fusion protein is a fundamental condition for inducing the protein's degradation by antimony and arsenic. Moreover, we found that SbIII could not induce mutant PML (e.g., A126V and L218P) solubility change and degradation, similar to As2O3. In contrast, we found that the organic antimony compound phenylstibine oxide (PSO) could induce mutant PML protein degradation. In conclusion, our results indicate that SbIII might also be a promising agent to treat acute promyelocytic leukemia, in the same manner as As2O3.

Increased expression of ZNF 703 in breast cancer tissue: An opportunity for RNAi-NSAID combinatorial therapy.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to exhibit antitumor activities. Among the very well-known oncogenes in breast cancer is zinc finger protein 703 (ZNF703) and cyclooxygenase-2 (COX-2). Numerous reports indicate a direct link among apoptosis resistance, chemotherapy resistance, and increased expression of ZNF703. In the present study, the expression level of ZNF703 was compared in human breast cancer tissue, healthy breast tissue, and MCF-7 breast cancer cell line by a real-time PCR. We also investigated the inhibitory effect of anti-ZNF703 RNAi interference (RNAi) and ibuprofen, either individually or in combination, on MCF-7 cell survival and apoptosis. Results showed a 93.3% and fourfold increase in the expression of ZNF703 in breast cancer tissue and MCF-7 cell line, respectively. Ibuprofen inhibited the viability of MCF-7 cells in a concentration-dependent manner. Ibuprofen alone or in combination with anti-ZNF703 RNA reduced the expression of ZNF703, induced apoptosis, reduced mitochondrial membrane potential, and elevated BAX and LC3A in MCF-7 cells. Our results show that the combination of ibuprofen and anti-ZNF703 siRNA is more effective in promoting apoptosis than each treatment alone. We report that the combination of anti-ZNF703 RNAi with ibuprofen as the inhibitor of COX-2 is highly effective in inhibiting MCF-7 as a breast cancer cell line and shows therapeutic potential for breast cancer.

Peroxynitrite contributes to arsenic-induced PARP-1 inhibition through ROS/RNS generation.

Arsenic, in the trivalent form (AsIII), is a human co-carcinogen reported to enhance mutagenesis effects of other carcinogens such as UV radiation by inhibiting DNA repair. The zinc finger DNA repair protein Poly (ADP-ribose) polymerase 1 (PARP-1) is a sensitive target of AsIII and both reactive oxygen and nitrogen species (ROS/RNS) generated by AsIII contribute to PARP-1 inhibition. However, the mechanisms of ROS/RNS-mediated PARP inhibition and how AsIII-generated ROS/RNS may be interconnected are still unclear. In this study, we found AsIII exposure of normal human keratinocyte (HEKn) cells generated peroxynitrite through superoxide and nitric oxide production in an AsIII concentration dependent manner. Peroxynitrite inhibited PARP-1 activity and caused zinc loss from PARP-1 protein while scavenging peroxynitrite was protective of the impacts on PARP-1. We identified peroxynitrite was responsible for S-nitrosation on cysteine residues resulting in PARP-1 zinc finger conformational changes. Taken together, the evidence indicates AsIII generates peroxynitrite through superoxide and nitric oxide production, induces S-nitrosation on PARP-1, leading to zinc loss and activity inhibition of PARP-1, thus enhancing DNA damage caused by UV radiation. These findings highlight a role for peroxynitrite as a key molecule of ROS/RNS mediated DNA repair inhibition by AsIII which should inform the development of prevention and intervention strategies against AsIII co-carcinogenesis.

Zinc finger domains as therapeutic targets for metal-based compounds - an update.

Zinc finger proteins are one of the most abundant families of proteins and present a wide range of structures and functions. The structural zinc ion provides the correct conformation to specifically recognize DNA, RNA and protein sequences. Zinc fingers have essential functions in transcription, protein degradation, DNA repair, cell migration, and others. Recently, reports on the extensive participation of zinc fingers in disease have been published. On the other hand, much information remains to be unravelled as many genomes and proteomes are being reported. A variety of zinc fingers have been identified; however, their functions are still under investigation. Because zinc fingers have identified functions in several diseases, they are being increasingly recognized as drug targets. The replacement of Zn(ii) by another metal ion in zinc fingers is one of the most prominent methods of inhibition. From one side, zinc fingers play roles in the toxicity mechanisms of Ni(ii), Hg(ii), Cd(ii) and others. From the other side, gold, platinum, cobalt, and selenium complexes are amongst the compounds being developed as zinc finger inhibitors for therapy. The main challenge in the design of therapeutic zinc finger inhibitors is to achieve selectivity. Recently, the design of novel compounds and elucidation of the mechanisms of zinc substitution have renewed the possibilities of selective zinc finger inhibition by metal complexes. This review aims to update the status of novel strategies to selectively target zinc finger domains by metal complexes.

Signaling pathways involved in the expression of SZNF and the target genes binding with SZNF related to cyadox.

SZNF (Sus scrofa zinc finger CCHC domain containing 3) is a post-transcription regulation factor, belonging to CCHC zinc finger proteins. Cyadox is a novel quinoxaline drug with antibacterial and growth promotion effects. In this study, we investigated the pharmacological mechanism of cyadox mediated by SZNF. Firstly, signaling pathways related to cyadox-induced SZNF expression were studied. The results showed that the mRNA level of SZNF reached the peak as early as 4 h after 2 µM cyadox treatment in swine hepatocytes. Several signaling pathways, including JAK2/STAT1, PI3K/Akt, TGF-ß/Smad3 and p38, might play critical roles in regulation of SZNF. The JAK2/STAT1, JAK2/PI3K/Akt, PI3K/Akt and myD88 & TAK1 & ASK1 /P38 signaling pathways were firstly activated after cyadox treatment in swine hepatocyte, the TGF-ß/Smad3 signaling pathway was activated later. Then given the characteristic of RNA binding of CCHC zinc finger proteins, the target mRNAs binding with SZNF were detected by RNA immunoprecipitation coupled to sequencing (RIP-seq) in PK-15 cells treated with cyadox. The RIP-Seq results showed that the bound mRNAs of 45 genes and 93 genes by SZNF protein were increased and decreased, respectively in cyadox-treated PK-15 cells compared with blank sample. With bioinformatics analysis, we showed that cyadox might exert its antibacterial and growth promotion effect by regulating SZNF-associated target genes in post-transcriptional level, such as genes related to growth (MLXIP, CKS2) and inflammation (LGALS3, PLAU). Thus, our results indicated that SZNF can post-transcriptionally regulate its target genes related to growth and inflammatory in cyadox-treated cells, which may explain the pharmacological mechanism of this drug.

Golgi-specific DHHC type zinc finger protein is decreased in neurons of intractable epilepsy patients and pentylenetetrazole-kindled rats.

Golgi-specific DHHC type zinc finger protein (GODZ) is a member of the DHHC protein family, and its enzymatic activity is regulated by fibroblast growth factor or Src kinase-mediated tyrosine phosphorylation. In cultured neurons, GODZ affects the numbers of calcium ions channels, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors, N-methy-D-aspartate receptors, and γ-aminobutyric acid A receptors on postsynaptic membrane by palmitoylation, thus modulating synaptic plasticity. As the change in synaptic plasticity plays a role in epilepsy, GODZ may play roles in epilepsy. However, the expression of GODZ has never been investigated in brain tissues in vivo, and its change during epilepsy is still unclear. In this study, the cellular distribution of GODZ in brain tissues of both patients and rats was determined using double-labeled immunofluorescence and the levels of GODZ protein and mRNA among intractable epilepsy patients, pentylenetetrazole (PTZ)-kindled rats, and controls were measured using immunohistochemistry, Western blot, and real-time quantitative polymerase chain reaction. GODZ expression was identified on cytomembranes and in the cytoplasm of neurons in the temporal neocortex of intractable epilepsy patients and in the hippocampus and the adjacent temporal cortex of PTZ-kindled rats, but not in astrocytes. Decreased GODZ protein and mRNA were identified in brain tissues of intractable epilepsy patients and PTZ-kindled rats compared with the controls. In conclusion, GODZ is expressed in neurons, but not astrocytes, and epilepsy may reduce the protein and mRNA levels of GODZ, indicating a possible role of GODZ in the pathogenesis or the pathophysiology of epilepsy.

Inhibition of histone lysine methyltransferases G9a and GLP by ejection of structural Zn(II).

Histone lysine methyltransferases G9a and GLP are validated targets for the development of new epigenetic drugs. Most, if not all, inhibitors of G9a and GLP target the histone substrate binding site or/and the S-adenosylmethionine cosubstrate binding site. Here, we report an alternative approach for inhibiting the methyltransferase activity of G9a and GLP. For proper folding and enzymatic activity, G9a and GLP contain structural zinc fingers, one of them being adjacent to the S-adenosylmethionine binding site. Our work demonstrates that targeting these labile zinc fingers with electrophilic small molecules results in ejection of structural zinc ions, and consequently inhibition of the methyltransferase activity. Very effective Zn(II) ejection and inhibition of G9a and GLP was observed with clinically used ebselen, disulfiram and cisplatin.

Divergent synthesis and identification of the cellular targets of deoxyelephantopins.

Herbal extracts containing sesquiterpene lactones have been extensively used in traditional medicine and are known to be rich in α,ß-unsaturated functionalities that can covalently engage target proteins. Here we report synthetic methodologies to access analogues of deoxyelephantopin, a sesquiterpene lactone with anticancer properties. Using alkyne-tagged cellular probes and quantitative proteomics analysis, we identified several cellular targets of deoxyelephantopin. We further demonstrate that deoxyelephantopin antagonizes PPARγ activity in situ via covalent engagement of a cysteine residue in the zinc-finger motif of this nuclear receptor.

Elevated Snail expression in human gingival fibroblasts by cyclosporine A as the possible pathogenesis for gingival overgrowth.

BACKGROUND/PURPOSE: Cyclosporine A (CsA) is used as an immunosuppressive agent, and its prominent side effect is the induction of gingival overgrowth. Snail is a master regulator of epithelial-mesenchymal transition (EMT). EMT under pathological processes could lead to fibrotic changes. The purpose of this study was to investigate the role of Snail in the pathogenesis of CsA-induced gingival overgrowth. METHODS: The effect of CsA on normal human gingival fibroblasts (HGFs) was used to elucidate whether Snail expression could be induced by CsA by using quantitative real-time reverse transcription-polymerase chain reaction and western blot. The cell proliferation rate in CsA-treated HGFs with Snail lentiviral-mediated short hairpin RNA interference (shRNAi) knockdown was evaluated by tetrazolium bromide reduction assay. RESULTS: CsA increased the Snail transcript and Snail protein expression in HGFs in a dose-dependent manner (p < 0.05). In addition, downregulation of Snail by lentiviral infection significantly reduced CsA-stimulated cell proliferation in HGFs (p < 0.05). CONCLUSION: CsA stimulated Snail expression and cell proliferation in HGFs, while silencing Snail could effectively reverse these phenomena. These results may provide new avenues for the design of novel antifibrotic therapies for CsA-induced gingival overgrowth through targeting Snail.

In silico structure-based design and synthesis of novel anti-RSV compounds.

Respiratory syncytial virus (RSV) is the major cause for respiratory tract disease in infants and young children. Currently, no licensed vaccine or a selective antiviral drug against RSV infections are available. Here, we describe a structure-based drug design approach that led to the synthesis of a novel series of zinc-ejecting compounds active against RSV replication. 30 compounds, sharing a common dithiocarbamate moiety, were designed and prepared to target the zinc finger motif of the M2-1 protein. A library of ∼ 12,000 small fragments was docked to explore the area surrounding the zinc ion. Among these, seven ligands were selected and used for the preparation of the new derivatives. The results reported here may help the development of a lead compound for the treatment of RSV infections.

miR-128 modulates chemosensitivity and invasion of prostate cancer cells through targeting ZEB1.

OBJECTIVE: Recent reports strongly suggest the profound role of miRNAs in cancer therapeutic response and progression, including invasion and metastasis. The sensitivity to therapy and invasion is the major obstacle for successful treatment in prostate cancer. We aimed to investigate the regulative effect of miR-128/zinc-finger E-box-binding homeobox 1 axis on prostate cancer cell chemosensitivity and invasion. METHODS: The miR-128 expression pattern of prostate cancer cell lines and tissues was detected by real-time reverse transcriptase-polymerase chain reaction, while the mRNA and protein expression levels of zinc-finger E-box-binding homeobox 1 were measured by real-time reverse transcriptase-polymerase chain reaction and western blot assay, respectively. Dual-luciferase reporter gene assay was used to find the direct target of miR-128. Furthermore, prostate cancer cells were treated with miR-128 mimic or zinc-finger E-box-binding homeobox 1-siRNA, and then the cells' chemosensitivity and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and transwell assay, respectively. RESULTS: We found miR-128 expression obviously decreased in prostate cancer tissues compared with paired normal tissues. Restored miR-128 expression sensitized prostate cancer cells to cisplatin and inhibited the invasion. Furthermore, there was an inverse expression pattern between miR-128 and zinc-finger E-box-binding homeobox 1 in prostate cancer cells and tissues, and zinc-finger E-box-binding homeobox 1 was identified as a direct target of miR-128 in prostate cancer. Knockdown of zinc-finger E-box-binding homeobox 1 expression efficiently sensitized prostate cancer cells to cisplatin and inhibited the invasion. However, ectopic zinc-finger E-box-binding homeobox 1 expression impaired the effects of miR-128 on chemosensitivity and invasion in prostate cancer cells. CONCLUSIONS: miR-128 functions as a potential cancer suppressor in prostate cancer progression and rational therapeutic strategies for prostate cancer would be developed based on miR-128/zinc-finger E-box-binding homeobox 1 axis.

Cyclosporine A up-regulates Sonic hedgehog in gingiva: role of the up-regulation on gingival cell proliferation.

BACKGROUND AND OBJECTIVE: Sonic hedgehog protein (SHH) is a mitogen that stimulates cell proliferation. Cyclosporine A enhances the proliferation of gingival cells; however, the relationships of SHH to cyclosporine A or to cyclosporine A-enhanced gingival cell proliferation have not been described. MATERIAL AND METHODS: Here, we investigated SHH expression in gingiva in vitro and in vivo after cyclosporine A treatment and tested the effect of SHH inhibition on cyclosporine A-enhanced gingival fibroblast proliferation in vitro. RESULTS: In human gingival fibroblasts, cyclosporine A treatment increased the expression of SHH transcripts and SHH protein, and stimulated cell proliferation; the addition of cyclopamine, an SHH signaling inhibitor, suppressed cyclosporine A-enhanced cell proliferation. Up-regulated expression of SHH and up-regulation of proliferating cell nuclear antigen transcripts and protein were observed in the edentulous gingiva of cyclosporine A-treated rats. CONCLUSION: Cyclosporine A up-regulates gingival SHH expression in vitro and in vivo, and the inhibition of the SHH pathway counteracts the stimulatory effect of cyclosporine A on gingival fibroblast proliferation. Therefore, we suggest that SHH mediates a novel molecular mechanism for cyclosporine A-induced gingival complications.

The PA207 peptide inhibitor of LIM-only protein 2 (Lmo2) targets Zinc Finger domains in a non-specific manner.

Peptide aptamers of LIM-only protein 2 (Lmo2) were previously used to successfully treat Lmo2-induced tumours in a mouse model of leukaemia. Here we show that the Lmo2 aptamer PA207, either as a free peptide or fused to thioredoxin Trx-PA207, causes purified Lmo2 to precipitate rather than binding to a defined surface on the protein. Stabilisation of Lmo2 through interaction with LIM domain binding protein 1 (Ldb1), a normal binding partner of Lmo2, abrogates this effect. The addition of free zinc causes Trx-PA207 to self associate, suggesting that PA207 destabilises Lmo2 by modulating normal zinc-coordination in the LIM domains. GST-pulldown experiments with other Lmo and Gata proteins indicates that PA207 can bind to a range of zinc finger proteins. Thus, PA207 and other cysteine-containing peptide aptamers for Lmo2 may form a class of general zinc finger inhibitors.

Targeting low-druggability bromodomains: fragment based screening and inhibitor design against the BAZ2B bromodomain.

Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable.