scholarly journals Biological effects of simple changes in functionality on rhodium metalloinsertors

2013 ◽  
Vol 371 (1995) ◽  
pp. 20120117 ◽  
Author(s):  
Alyson G. Weidmann ◽  
Alexis C. Komor ◽  
Jacqueline K. Barton
Keyword(s):  
Cellular Proliferation ◽  
Biological Effects ◽  
High Specificity ◽  
Selective Inhibition ◽  
Binding Affinities ◽  
Structural Modifications ◽  
Dna Mismatch ◽  
Dna Mismatches ◽  
Sensitive Structure ◽  
Function Relationship

DNA mismatch repair (MMR) is crucial to ensuring the fidelity of the genome. The inability to correct single base mismatches leads to elevated mutation rates and carcinogenesis. Using metalloinsertors–bulky metal complexes that bind with high specificity to mismatched sites in the DNA duplex–our laboratory has adopted a new chemotherapeutic strategy through the selective targeting of MMR-deficient cells, that is, those that have a propensity for cancerous transformation. Rhodium metalloinsertors display inhibitory effects selectively in cells that are deficient in the MMR machinery, consistent with this strategy. However, a highly sensitive structure–function relationship is emerging with the development of new complexes that highlights the importance of subcellular localization. We have found that small structural modifications, for example a hydroxyl versus a methyl functional group, can yield profound differences in biological function. Despite similar binding affinities and selectivities for DNA mismatches, only one metalloinsertor shows selective inhibition of cellular proliferation in MMR-deficient versus -proficient cells. Studies of whole-cell, nuclear and mitochondrial uptake reveal that this selectivity depends upon targeting DNA mismatches in the cell nucleus.

scholarly journals Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells

2017 ◽  
Vol 114 (27) ◽  
pp. 6948-6953 ◽  
Author(s):  
Julie M. Bailis ◽  
Alyson G. Weidmann ◽  
Natalie F. Mariano ◽  
Jacqueline K. Barton
Keyword(s):  
Mismatch Repair ◽  
Genome Stability ◽  
High Specificity ◽  
Standard Of Care ◽  
Cellular Mechanism ◽  
Selective Cytotoxicity ◽  
Dna Mismatch ◽  
Repair Enzymes ◽  
Damage Response ◽  
Dna Mismatches

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)]2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.

scholarly journals TP53 Abnormalities and MMR Preservation in 5 Cases of Proliferating Trichilemmal Tumours

2021 ◽  
Vol 8 (2) ◽  
pp. 147-158
Author(s):  
Raquel Martín-Sanz ◽  
José María Sayagués ◽  
Pilar García-Cano ◽  
Mikel Azcue-Mayorga ◽  
María del Carmen Parra-Pérez ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Ultraviolet Radiation ◽  
Mismatch Repair ◽  
Squamous Cell ◽  
Cellular Proliferation ◽  
P53 Expression ◽  
Dna Mismatch ◽  
Molecular Alterations ◽  
Root Sheath ◽  
Close Relationship

Proliferating trichilemmal tumours (PTT) are defined by a benign squamous cell proliferation inside a trichilemmal cystic (TC) cavity. A possible explanation of this proliferative phenomenon within the cyst may be molecular alterations in genes associated to cell proliferation, which can be induced by ultraviolet radiation. Among other genes, alterations on TP53 and DNA mismatch repair proteins (MMR) may be involved in the cellular proliferation observed in PTT. Based on this assumption, but also taking into account the close relationship between the sebaceous ducts and the external root sheath where TC develop, a MMR, a p53 expression assessment and a TP53 study were performed in a series of 5 PTT cases, including a giant one. We failed to demonstrate a MMR disorder on studied PTT, but we agree with previous results suggesting increased p53 expression in these tumours, particularly in proliferative areas. TP53 alteration was confirmed with FISH technique, demonstrating TP53 deletion in most cells.

Protein kinase C isotypes in human erythroleukemia cell proliferation and differentiation

1992 ◽  
Vol 101 (3) ◽  
pp. 671-679
Author(s):  
B.A. Hocevar ◽  
D.M. Morrow ◽  
M.L. Tykocinski ◽  
A.P. Fields
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cellular Proliferation ◽  
Biological Effects ◽  
Growth Arrest ◽  
Tumor Promoter ◽  
Induced Differentiation ◽  
Megakaryocytic Differentiation ◽  
Kinase C ◽  
Dose Dependent

The human erythroleukemia (K562) cell line is induced to differentiate into megakaryocytic cells by treatment with the tumor promoter phorbol myristate acetate (PMA). PMA-induced differentiation is characterized by (1) almost complete cessation of cellular proliferation, (2) expression of the megakaryocytic cell surface marker glycoprotein IIb/IIIa (gpIIIa), (3) increased secretion of granulocyte/macrophage-colony stimulating factor (GM-CSF) and (4) increased secretion of interleukin-6 (IL-6). PMA-induced differentiation is dose-dependent with maximal activity seen at 10 nM PMA. In contrast, bryostatin (bryo), a structurally distinct protein kinase C (PKC) activator, fails to induce megakaryocytic differentiation or growth arrest at the concentrations tested (0.01-100 nM). Rather, bryo inhibits PMA-induced growth arrest and megakaryocytic differentiation in a dose-dependent fashion (full inhibition at 100 nM). The divergent biological effects of PMA and bryo correspond to the differential activation and translocation of PKC isotypes in K562 cells. PKC isotype analysis demonstrates that undifferentiated cells express both alpha and beta II PKC but no detectable beta I, gamma or epsilon PKC. Treatment of cells with either PMA or bryo leads to rapid translocation of both alpha and beta II PKC from the cytosol to the non-nuclear particulate fraction. However, bryo also induces selective translocation of beta II PKC to the nuclear membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact

2021 ◽  
Vol 11 ◽  
Author(s):  
Natali Vega-Magaña ◽  
Rocío Sánchez-Sánchez ◽  
Jorge Hernández-Bello ◽  
Alberto Antony Venancio-Landeros ◽  
Marcela Peña-Rodríguez ◽  
...  
Keyword(s):  
Biological Effects ◽  
Low Cost ◽  
Gene Mutations ◽  
High Specificity ◽  
Screening Strategy ◽  
Clinical Impact ◽  
Mexican Population ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Reverse Transcription Pcr

BackgroundSeveral variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population.MethodsTargeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico.ResultsIn silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico.ConclusionThis work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.

scholarly journals Selective Inhibition of Bakuchicin Isolated fromPsoralea corylifoliaon CYP1A in Human Liver Microsomes

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Sun Joo Kim ◽  
Heung Chan Oh ◽  
Youn-Chul Kim ◽  
Gil-Saeng Jeong ◽  
Sangkyu Lee
Keyword(s):  
Human Liver ◽  
Competitive Inhibition ◽  
Biological Activities ◽  
Biological Effects ◽  
Liver Microsomes ◽  
Selective Inhibition ◽  
Human Liver Microsomes ◽  
Inhibition Mechanism ◽  
Inhibitory Effects ◽  
Cyp Isoforms

Bakuchicin is a furanocoumarin isolated fromPsoralea corylifoliaand shows several biological activities. Although there have been studies on the biological effects of bakuchicin, its modulation potency of CYP activities has not been previously investigated. Here, we investigated the inhibitory effects of bakuchicin on the activities of CYP isoforms by using a cocktail of probe substrates in pooled human liver microsomes (HLMs) and human recombinantcDNA-expressedCYP. Bakuchicin strongly inhibited CYP1A-mediated phenacetinO-deethylation with an IC50value of 0.43 μM in HLMs. It was confirmed by human recombinantcDNA-expressedCYP1A1 and CYP1A2 with aKivalue of 0.11 μM and 0.32 μM, respectively. A Lineweaver-Burk plot indicated that the inhibition mechanism of bakuchicin was competitive inhibition. Overall, this is the first study to investigate the potential CYP1A1 and CYP1A2 inhibition associated with bakuchicin and to report its competitive inhibitory effects on HLMs.

scholarly journals Phosphoproteomic analysis sheds light on intracellular signaling cascades triggered by Formyl-Peptide Receptor 2

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Fabio Cattaneo ◽  
Rosita Russo ◽  
Martina Castaldo ◽  
Angela Chambery ◽  
Cristiana Zollo ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Drug Targets ◽  
Large Scale ◽  
Cellular Proliferation ◽  
Biological Effects ◽  
Formyl Peptide Receptor ◽  
Ros Generation ◽  
Systematic Analysis ◽  
Phosphorylated Proteins ◽  
Formyl Peptide

AbstractFormyl peptide receptors (FPRs) belong to the family of seven transmembrane Gi-protein coupled receptors (GPCR). FPR2 is considered the most promiscuous member of this family since it recognizes a wide variety of ligands. It plays a crucial role in several physio-pathological processes and different studies highlighted the correlation between its expression and the higher propensity to invasion and metastasis of some cancers. FPR2 stimulation by its synthetic agonist WKYMVm triggers multiple phosphorylations of intracellular signaling molecules, such as ERKs, PKC, PKB, p38MAPK, PI3K, PLC, and of non-signaling proteins, such as p47phox and p67phox which are involved in NADPH oxidase-dependent ROS generation. Biological effects of FPR2 stimulation include intracellular Ca2+ mobilization, cellular proliferation and migration, and wound healing. A systematic analysis of the phosphoproteome in FPR2-stimulated cells has not been yet reported. Herein, we describe a large-scale phosphoproteomic study in WKYMVm-stimulated CaLu-6 cells. By using high resolution MS/MS we identified 290 differentially phosphorylated proteins and 53 unique phosphopeptides mapping on 40 proteins. Phosphorylations on five selected phospho-proteins were further validated by western blotting, confirming their dependence on FPR2 stimulation. Interconnection between some of the signalling readout identified was also evaluated. Furthermore, we show that FPR2 stimulation with two anti-inflammatory agonists induces the phosphorylation of selected differentially phosphorylated proteins, suggesting their role in the resolution of inflammation. These data provide a promising resource for further studies on new signaling networks triggered by FPR2 and on novel molecular drug targets for human diseases.

scholarly journals Inhibition of Cellular Proliferation through IκB Kinase-Independent and Peroxisome Proliferator-Activated Receptor γ-Dependent Repression of Cyclin D1

2001 ◽  
Vol 21 (9) ◽  
pp. 3057-3070 ◽  
Author(s):  
Chenguang Wang ◽  
Maofu Fu ◽  
Mark D'Amico ◽  
Chris Albanese ◽  
Jian-Nian Zhou ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Nuclear Receptor ◽  
Cellular Proliferation ◽  
Biological Effects ◽  
S Phase ◽  
In Vivo Studies ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Iκb Kinase ◽  
Anti Inflammatory

ABSTRACT The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARγ exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARγ ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARγ ligands on cellular proliferation. Recent findings that PPARγ ligands convey PPARγ-independent activities through IκB kinase (IKK) raises important questions about the specific mechanisms through which PPARγ ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARγ. Herein PPARγ, liganded by either natural (15d-PGJ2 and PGD2) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ2 was not observed in PPARγ-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ2. Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARγ but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARγ involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ2 enhanced recruitment of p300 to PPARγ but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.

scholarly journals High Specificity in Protein Recognition by Hydrogen-Bond-Surrogate α-Helices: Selective Inhibition of the p53/MDM2 Complex

ChemBioChem ◽  
2010 ◽  
Vol 11 (15) ◽  
pp. 2104-2107 ◽  
Author(s):  
Laura K. Henchey ◽  
Jason R. Porter ◽  
Indraneel Ghosh ◽  
Paramjit S. Arora
Keyword(s):  
Hydrogen Bond ◽  
High Specificity ◽  
Selective Inhibition ◽  
Protein Recognition ◽  
Hydrogen Bond Surrogate

scholarly journals New multiscale characterisation methodology for effective determination of isolation-structure-function relationship of extracellular vesicles

2021 ◽  
Author(s):  
Thanh Huyen Phan ◽  
Shiva Kamini Divakarla ◽  
Jia Hao Yeo ◽  
Qingyu Lei ◽  
Priyanka Tharkar ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biological Effects ◽  
Dry Mass ◽  
Experimental Methodology ◽  
Wide Range ◽  
Isolation Methods ◽  
Function Relationship ◽  
Relationship Of

AbstractExtracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass-produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterisation approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

scholarly journals Targeted enhancement of the therapeutic window of L19-TNF by transient and selective inhibition of RIPK1-signaling cascade

2019 ◽  
Author(s):  
Sheila Dakhel ◽  
Tiziano Ongaro ◽  
Baptiste Gouyou ◽  
Mattia Matasci ◽  
Alessandra Villa ◽  
...  
Keyword(s):  
Small Molecule ◽  
Biological Effects ◽  
Clinical Stage ◽  
Selective Inhibition ◽  
Therapeutic Window ◽  
Tumor Bearing ◽  
Hemorrhagic Necrosis ◽  
Molecule Inhibitor ◽  
Selective Localization ◽  
Pro Inflammatory Cytokines

AbstractIntroductionCytokine-based products are gaining importance for cancer immunotherapy. L19-TNF is a clinical-stage antibody-cytokine fusion protein that selectively accumulates to tumors and displays potent anticancer activity in preclinical models. Here, we describe an innovative approach to transiently inhibit off-target toxicity of L19-TNF, while maintaining antitumor activity.MethodsGSK’963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice for its ability to reduce acute toxicity associated with TNF signaling. The biological effects of L19-TNF on tumor cells, lymphocytes and tumor vessels were investigated with the aim to enable the administration of TNF doses, which would otherwise be lethal.ResultsTransient inhibition of RIPK1 allowed to increase the maximal tolerated dose of L19-TNF. The protective effect of GSK’963 did not affect the selective localization of the immunocytokine to tumors as evidenced by quantitative biodistribution analysis and allowed to reach high local TNF concentrations around tumor blood vessels, causing diffused vascular shutdown and hemorrhagic necrosis within the neoplastic mass.ConclusionsThe selective inhibition of RIPK1 with small molecule inhibitors can be used as a pharmaceutical tool to transiently mask TNF activity and improve the therapeutic window of TNF-based biopharmaceuticals. Similar approaches may be applicable to other pro-inflammatory cytokines.

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