scholarly journals Structural studies on 14-3-3ζ: Compounds that target the dimer interface

2014 ◽  
Vol 70 (a1) ◽  
pp. C808-C808
Author(s):  
Urmi Dhagat ◽  
Joanna Woodcock ◽  
Chrystal Tiong ◽  
Jessica Holien ◽  
Carl Coolen ◽  
...  
Keyword(s):  
Small Molecules ◽  
Disease Recurrence ◽  
Therapeutic Benefit ◽  
Leukemic Cells ◽  
Structural Studies ◽  
Salt Bridges ◽  
Dimer Interface ◽  
Novel Approach ◽  
Apoptotic Activity

14-3-3 proteins are a highly conserved family of dimeric phospho-serine binding proteins that modulate the functions of key cellular proteins involved in signaling. 14-3-3ζ plays a prominent role in signaling pathways leading to inhibition of apoptosis, sequestration of tumor suppressor proteins and activation of signalling pathways that promote growth. 14-3-3ζ expression is up-regulated in many human cancers and associated with enhanced survival of cancer cells. The significant association of 14-3-3ζ over expression with disease recurrence and chemo-resistance makes this protein an attractive candidate for anti-cancer therapy. The anti-apoptotic activity of 14-3-3ζ is entirely dependent on the dimeric state of the protein. Our studies have shown that 14-3-3ζ activity is regulated by sphingosine and other lipid analogs that render 14-3-3 phosphorylatable, disrupting its dimeric state thereby leading to apoptosis [1]. Structural studies and mutagenesis on 14-3-3ζ confirm that the dimeric state of 14-3-3ζ is stabilized by salt bridges that form across the dimer interface. Based on this we have carried out an in silico screen of a virtual library of drug-like small molecules to identify compounds that bind to the dimer interface of 14-3-3ζ. Candidate small molecules have been assessed for their ability to render 14-3-3ζ phosphorylatable in vitro and consequently we have identified a family of small molecules with 14-3-3ζ dimer-destabilizing properties. These small molecules induce apoptosis in leukemic cells by activating apoptotic mediators known to be regulated by dimeric 14-3-3. We have recently solved the crystal structure of 14-3-3ζ with one of our hit compounds bound at the dimer interface. Our results suggest that relatively small perturbations at the dimer interface, can destabilize the salt bridges that hold 14-3-3 dimers together, thus providing a novel approach to targeting 14-3-3 proteins for therapeutic benefit.

Activity of Anti-Tumor Endoribonucleases, Onconase (ranpirnase) and R-Amphinase in Chronic Lymphocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4205-4205
Author(s):  
Piotr Smolewski ◽  
Anna Linke-Szewczyk ◽  
Barbara Cebula ◽  
Kuslima Shogen ◽  
Wojciech Ardelt ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Caspase 3 ◽  
Synergistic Effects ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Purine Analogues ◽  
Mitochondrial Potential ◽  
Bax Protein ◽  
Apoptotic Activity

Abstract Despite of evident progress in treatment of chronic lymphocytic leukemia (CLL), the disease still remains incurable. Several attempts have been made therefore to develop most effective and selective therapeutical approaches. A promising approach that involves targeting RNA either by the use of specific antisense oligonucleotides or cytostatic/cytotoxic ribonucleases is recently being promoted. Two such ribonucleases, onconase (ONC; ranpirnase) and R-amphinase (R-Amph), derived from Rana pipiens oocytes, have been developed. ONCdemonstrated preferential toxicity to tumor cells and was shown to be effective in vivo in animal tests as well as in clinical trials in treatment of malignant mesothelioma. Moreover, ONC is synergistic when used in combination with a variety of antitumor modalities including anthracyclines. R-Amph was developed only recently and thus far there is only a single report demonstrating its cytostatic and cytotoxic activity against human promyelocytic HL-60-, Jurkat T-cell- and U-937 histiomonocytic leukemic cells in vitro. In the present study we aimed to assess potential cytotoxicity of ONCand R-Amph against CLL cells. Toward this aim, leukemic cells were isolated from 36 untreated patients with CLL and were cultured for 24–72 h with either ONC or R-Amph alone and in combination with purine analogues, cladribine (2-CdA) and fludarabine (FA), two drugs routinely used in treatment of CLL, as well as with doxorubicin (DOX), the drug reported to show synergy with ONC in solid tumors. Cytotoxicity of the study drugs was assessed by the propidium iodide exclusion assay using flow cytometry. Their pro-apoptotic activity was examined by the Annexin-V (Ann-V) binding test, detection of caspase-3, -8, and -9 activation, a decrease of mitochondrial potential and the expression of apoptosis–regulating proteins from the Bcl-2 family. Compensated apoptotic index (CAI) has been calculated based on Ann-V assay as a difference in the percentage of apoptotic cells between the drug-treated sample and spontaneous apoptosis in the parallel untreated control. After preliminary experiments the optimal concentrations of both ONC and R-Amph were found to be 20 μg/ml; these were the lowest doses that induced significant cytotoxicity during 24–72 h of incubation, in comparison with parallel controls. The significant effect of ONC was evident after 48 h of treatment (median CAI=11.5%; p=0.035 versus control). After 72 h of incubation the median CAI for ONC was 17.1% (p=0.009). The significant cytotoxicity of R-Amph was seen after 72 h incubation (median CAI =19.9%; p=0.007, respectively). The mechanism of this cytotoxicity involved the induction of apoptosis along its mitochondrial pathway, with the drop of mitochondrial potential and activation of caspase-9 and caspase-3, concurrent with an increase in expression of pro-apoptotic Bax protein (p=0.035 versus control; after 72h) and a decrease of anti-apoptotic Bcl-2 expression (p=0.006; after 72 h). No significant changes in expression of Bak and Mcl-1 were observed. Synergistic effect was found for both, ONC plus 2-CDA and ONC plus FA (combination indices, CI; <0.8). Also the combination of R-Amph with 2-CDA or with FA exerted synergistic cytotoxiciy (both CI <0.8). Although, the combination of DOX with ONC or R-Amph demonstrated an increased in pro-apoptotic activity when compared to single agents, the effect was not statistically significant. In conclusion, this is the first study showing cytotoxic, pro-apoptotic affect of RNA-targeting agents, Onconase and R-Amphinase, in CLL. This promising anti-leukemic activity of both ribonucleases, especially their synergistic effects exerted in combination with purine analogues warrant further intensive preclinical and, subsequently, clinical study in this disease.

scholarly journals Preparation of Liposomal Raloxifene-Graphene Nanosheet and Evaluation of Its In Vitro Anticancer Effects

Dose-Response ◽  
2022 ◽  
Vol 20 (1) ◽  
pp. 155932582110639
Author(s):  
Ahmed E. Altyar ◽  
Omar Fahmy
Keyword(s):  
Thermodynamic Stability ◽  
In Vitro Release ◽  
Annexin V ◽  
Liposomal Formulation ◽  
Prolonged Release ◽  
Graphene Nanosheet ◽  
Anticancer Effects ◽  
Novel Approach ◽  
Apoptotic Activity

Background In current years, researchers have shown their prime interest in developing multifunctional drug delivery systems, especially against cancers, for effective anticancer outcomes. Methodology Raloxifene (RLX) loaded liposomal-graphene nanosheet (GNS) was developed. The novelty of this work was to enhance the solubilization of RLX and improvement of its bioavailability in the disease area. So, the selection of optimized formula design of experiment was implemented which produced the desired formula with the particle size of 156.333 nm. Further, encapsulation efficiency, in vitro release, and thermodynamic stability of optimized formulation were evaluated. The optimized formulation exhibited prolonged release of RLX for a longer period of 24 h, which can minimize the dose-related toxicity of the drug. Furthermore, optimized formulation demonstrated remarkable thermodynamic stability in terms of phase separation, creaming, and cracking. Results The cytotoxicity study on the A549 cell line exhibited significant ( P < .05) results in favor of optimized formulation than the free drug. The apoptotic activity was carried out by Annexin V staining and Caspase 3 analysis, which demonstrated remarkable promising results for optimized liposomal formulation. Conclusion From the findings of the study, it can be concluded that the novel optimized liposomal formulation could be pondered as a novel approach for the treatment of lung cancer.

Targeting Micrornas With Small Molecules: A Novel Approach to Treating Breast Cancer

2010 ◽  
Author(s):  
George A. Calin ◽  
Shuxing Zhang ◽  
Waldemar Priebe
Keyword(s):  
Breast Cancer ◽  
Small Molecules ◽  
Novel Approach

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

Phytosomal Curcumin Elicits Anti-tumor Properties Through Suppression of Angiogenesis, Cell Proliferation and Induction of Oxidative Stress in Colorectal Cancer

2019 ◽  
Vol 24 (39) ◽  
pp. 4626-4638 ◽  
Author(s):  
Reyhaneh Moradi-Marjaneh ◽  
Seyed M. Hassanian ◽  
Farzad Rahmani ◽  
Seyed H. Aghaee-Bakhtiari ◽  
Amir Avan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Colorectal Cancer ◽  
Therapeutic Potential ◽  
Vegf Signaling ◽  
Anticancer Effects ◽  
Inhibition Of Angiogenesis ◽  
Underlying Mechanisms ◽  
Apoptotic Activity

Background: Colorectal cancer (CRC) is one of the most common causes of cancer-associated mortality in the world. Anti-tumor effect of curcumin has been shown in different cancers; however, the therapeutic potential of novel phytosomal curcumin, as well as the underlying molecular mechanism in CRC, has not yet been explored. Methods: The anti-proliferative, anti-migratory and apoptotic activity of phytosomal curcumin in CT26 cells was assessed by MTT assay, wound healing assay and Flow cytometry, respectively. Phytosomal curcumin was also tested for its in-vivo activity in a xenograft mouse model of CRC. In addition, oxidant/antioxidant activity was examined by DCFH-DA assay in vitro, measurement of malondialdehyde (MDA), Thiol and superoxidedismutase (SOD) and catalase (CAT) activity and also evaluation of expression levels of Nrf2 and GCLM by qRT-PCR in tumor tissues. In addition, the effect of phytosomal curcumin on angiogenesis was assessed by the measurement of VEGF-A and VEGFR-1 and VEGF signaling regulatory microRNAs (miRNAs) in tumor tissue. Results: Phytosomal curcumin exerts anti-proliferative, anti-migratory and apoptotic activity in-vitro. It also decreases tumor growth and augmented 5-fluorouracil (5-FU) anti-tumor effect in-vivo. In addition, our data showed that induction of oxidative stress and inhibition of angiogenesis through modulation of VEGF signaling regulatory miRNAs might be underlying mechanisms by which phytosomal curcumin exerted its antitumor effect. Conclusion: Our data confirmed this notion that phytosomal curcumin administrates anticancer effects and can be used as a complementary treatment in clinical settings.

scholarly journals Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽  
1978 ◽  
Vol 52 (4) ◽  
pp. 712-718 ◽  
Author(s):  
SD Smith ◽  
EM Uyeki ◽  
JT Lowman
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Lymphoid Cells ◽  
Assay System ◽  
Leukemic Cells ◽  
Specific Cell ◽  
Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

scholarly journals Evidence for the interleukin-2 dependent expansion of leukemic cells in adult T cell leukemia

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1407-1411 ◽  
Author(s):  
M Maeda ◽  
N Arima ◽  
Y Daitoku ◽  
M Kashihara ◽  
H Okamoto ◽  
...  
Keyword(s):  
T Cell ◽  
Receptor Gene ◽  
Interleukin 2 ◽  
Leukemic Cells ◽  
Type I ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
In Vitro Proliferation ◽  
Adult T Cell Leukemia

Abstract Interleukin 2 (IL-2) receptor/Tac antigen is abnormally expressed on cells of patients with adult T cell leukemia (ATL) caused by infection with human T lymphotropic virus type I (HTLV-I). Twenty-five patients with ATL were examined to determine whether their leukemic cells continued to show IL-2-dependent proliferation. In 21 patients, the in vitro proliferation of HTLV-I-infected nonleukemic T cell clones was found to be dependent on IL-2. However, clonality analysis based on T cell receptor gene rearrangement profiles and the site of HTLV-I provirus integration revealed IL-2-dependent growth in leukemic cells in four patients with ATL. These results provide evidence for the IL-2- dependent proliferation of leukemic cells in some ATL patients.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

IDENTIFICATION OF SELETIVE CLAUDIN CATION CHANNEL BLOCKERS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S25-S26
Author(s):  
Jingjing Ma ◽  
Emma Wu ◽  
Ye Li ◽  
William Seibel ◽  
Le Shen ◽  
...  
Keyword(s):  
Small Molecules ◽  
Homology Model ◽  
Docking Studies ◽  
Channel Blockers ◽  
Binding Modes ◽  
Barrier Dysfunction ◽  
Epithelial Barrier Function ◽  
Dynamics Simulations ◽  
In Silico Models

Abstract Compromised epithelial barrier function is known to be associated with inflammatory bowel disease (IBD) and may contribute to disease development. One mechanism of barrier dysfunction is increased expression of paracellular tight junction ion and water channels formed by claudins. Claudin-2 and -15 are two such channels. We hypothesize that blocking these channels could be a viable therapeutic approach to treat diarrhea. In an effort to develop blockers of these channels, we turn to our previously developed and validated in silico models of claudin-15 (Samanta et al. 2018). We reasoned that compounds that can bind with the interior of claudin pores can limit paracellular water and ion flux. Thus, we used docking algorithms to search for putative small molecules that bind in the claudin-15 pore. AutoDock Vina was initially used to assess rigid docking using small compound databases. The small molecules were analyzed based on binding affinity to the pore and visualized using VMD for their potential blockage of the channel. Clusters of binding modes were identified based on the prominent interacting residues of the protein with the small molecules. We initially screened 10,500 compounds from within the UIC Centre for Drug Discovery and a cross-section of 10,000 compounds from the NCI open compound repository. This initial screen allowed us to identify 2 first-in-class selective claudin-15 blockers with efficacy in MDCK monolayers induced to express claudin-15 and in wildtype duodenum. Next, we screened the entire NCI open compound repository for additional molecules structurally related to our best initially identified molecule and this has allowed us to identify 13 additional molecules that increase TER of claudin-15 expressing MDCK monolayers by 90–160%. Additionally, these molecules possess similar structural components that will be collected in a fragment library and explored through molecular dynamics simulations. We also developed a claudin-2 homology model on which we are performing docking studies and in vitro measurements, which we expect will result in similar candidate ligands for blocking claudin-2. Our study will provide important insight into the role of claudin-dependent cation permeability in fundamental physiology, which we believe will lead to the utility of claudin blockers as a novel and much needed approach to treat diseases such as IBD.

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