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Artykuły w czasopismach na temat "Protein targeted lead molecule"
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Hyun, Soonsil, i Dongyun Shin. "Chemical-Mediated Targeted Protein Degradation in Neurodegenerative Diseases". Life 11, nr 7 (24.06.2021): 607. http://dx.doi.org/10.3390/life11070607.
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Neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease, are a class of diseases that lead to dysfunction of cognition and mobility. Aggregates of misfolded proteins such as β-amyloid, tau, α-synuclein, and polyglutamates are known to be among the main causes of neurodegenerative diseases; however, they are considered to be some of the most challenging drug targets because they cannot be modulated by conventional small-molecule agents. Recently, the degradation of target proteins by small molecules has emerged as a new therapeutic modality and has garnered the interest of the researchers in the pharmaceutical industry. Bifunctional molecules that recruit target proteins to a cellular protein degradation machinery, such as the ubiquitin–proteasome system and autophagy–lysosome pathway, have been designed. The representative targeted protein degradation technologies include molecular glues, proteolysis-targeting chimeras, hydrophobic tagging, autophagy-targeting chimeras, and autophagosome-tethering compounds. Although these modalities have been shown to degrade many disease-related proteins, such technologies are expected to be potentially important for neurogenerative diseases caused by protein aggregation. Herein, we review the recent progress in chemical-mediated targeted protein degradation toward the discovery of drugs for neurogenerative diseases.
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Giardina, Sarah F., Elena Valdambrini, Michael Peel, Manny D. Bacolod, Mace L. Rothenberg, Richard B. Lanman, J. David Warren i Francis Barany. "Cure-PROs: Next-generation targeted protein degraders." Journal of Clinical Oncology 41, nr 16_suppl (1.06.2023): e15101-e15101. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.e15101.
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e15101 Background: Many proteins, including transcription factors and scaffolding proteins, are not amenable to targeting by traditional small molecule inhibitors due to the lack of a well-defined binding pocket or active site. Proteolysis-Targeting Chimeras (PROTACs) are a new class of hetero-bifunctional molecules that bind both a target protein and an E3 ubiquitin ligase, bringing the two into proximity for appending ubiquitin, and subsequently marking the target protein for proteasomal degradation. Currently, thirteen PROTACs are in clinical trials for oncology indications. However, the clinical utility of PROTACs is challenged by their large size and long development timelines. Also, resistance mutations in the E3 ligase or transporter overexpression inevitably evolve. Thus, a new platform for small-molecule degraders that enables ultra-rapid drug development timelines, efficient cellular uptake, and can be developed to overcome innate and acquired drug resistance is needed. Methods: Coferons, developed in our laboratory, are small molecules that self-assemble upon binding to a target, where they form reversible covalent dimers through bio-orthogonal linker chemistries. We have combined features of the Coferon platform and PROTACs to generate CURE-PROs (Combinatorial Ubiquitination REal-time PROteolysis), consisting of one target protein ligand and one E3 ligase ligand that form reversible heterodimers that lead to targeted protein degradation within cells. By modifying known ligands for BRD4, and the E3 ubiquitin ligases Cereblon, VHL, and MDM2, with linkers able to reversibly join the BRD4 to the ligase ligands, we synthesized libraries of CURE-PRO monomers that can be combined to create thousands of CURE-PRO dimer combinations. We explored whether this platform could yield meaningful BRD4 degradation in vitro and in vivo. Results: Rapid combinatorial cell-based screening identified several BRD4-E3 ligase CURE-PRO combinations that induced greater than 50% BRD4 degradation, with the most promising CURE-PRO pairs achieving more than 95% protein degradation. Consistent with a PROTAC mechanism-of-action, successful CURE-PRO combinations confirmed significant protein degradation which was inhibited by proteasome inhibitors or competition with parent ligands. Significant BRD4 degradation was also observed in mice bearing bilateral human xenograft tumors, confirming CURE-PRO proof-of-mechanism in vivo. Conclusions: The combinatorial nature of our platform has the potential to significantly reduce synthesis time and effort to identify the optimal linker length and E3 ligase for each target protein. The CURE-PRO platform consists of expanding libraries of monomers for both additional oncoprotein targets as well as E3 ligases, which can be redeployed to shorten lead development timelines.
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Musielak, Bogdan, Weronika Janczyk, Ismael Rodriguez, Jacek Plewka, Dominik Sala, Katarzyna Magiera-Mularz i Tad Holak. "Competition NMR for Detection of Hit/Lead Inhibitors of Protein–Protein Interactions". Molecules 25, nr 13 (1.07.2020): 3017. http://dx.doi.org/10.3390/molecules25133017.
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Screening for small-molecule fragments that can lead to potent inhibitors of protein–protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low μM–mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak μM–mM ligand–protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex’s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.
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Thomas, Bedwyr ab Ion, H. Lois Lewis, D. Heulyn Jones i Simon E. Ward. "Central Nervous System Targeted Protein Degraders". Biomolecules 13, nr 8 (25.07.2023): 1164. http://dx.doi.org/10.3390/biom13081164.
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Diseases of the central nervous system, which once occupied a large component of the pharmaceutical industry research and development portfolio, have for many years played a smaller part in major pharma pipelines—primarily due to the well cited challenges in target validation, valid translational models, and clinical trial design. Unfortunately, this decline in research and development interest has occurred in tandem with an increase in the medical need—in part driven by the success in treating other chronic diseases, which then results in a greater overall longevity along with a higher prevalence of diseases associated with ageing. The lead modality for drug agents targeting the brain remains the traditionally small molecule, despite potential in gene-based therapies and antibodies, particularly in the hugely anticipated anti-amyloid field, clearly driven by the additional challenge of effective distribution to the relevant brain compartments. However, in recognition of the growing disease burden, advanced therapies are being developed in tandem with improved delivery options. Hence, methodologies which were initially restricted to systemic indications are now being actively explored for a range of CNS diseases—an important class of which include the protein degradation technologies.
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Das, Debanu, Matthew Duncton, Patricia Pellicena, Ashley Deacon, David Wilson i Millie Georgiadis. "Development of a DNA damage response (DDR) therapeutics platform for oncology." Journal of Clinical Oncology 39, nr 15_suppl (20.05.2021): e15036-e15036. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e15036.
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e15036 Background: Cancer cells respond to increases in DNA damage by deploying their DNA damage response (DDR) pathways. We are building a platform for the discovery and development of target-specific DDR therapeutics, including small molecule inhibitors and targeted protein degradation warheads, founded on fragment- and structure-based drug discovery. Methods: Our DDR platform, which includes hit-to-lead, lead optimization and candidate selection, starts with hit generation from a new technology that uses high-throughput protein X-ray crystallography to directly screen compound libraries. Our hit generation process produces empirical evidence of direct target engagement. The elucidation of high-quality ligand-bound 3D structures reveals the location and pose of the ligand and details of the protein-ligand interactions. Thus we can predict the structure-function consequences of the hit molecule engagement, which sets the stage for rapid assessment of synthetic tractability and intellectual property. After hit identification, we apply a multi-pronged approach in hit-to-lead conversion and lead optimization using iterative biophysical and biochemical assays, coupled with crystallography. We are applying our approach to several new targets in DDR and will present some early progress in this space. Results: Apurinic/apyrimidinic endonuclease 1 (APE1) is the major repair enzyme for abasic sites in DNA and contributes to DNA strand break processing. Many studies have associated increased APE1 levels with enhanced growth, migration, and drug resistance in human tumor cells, and with decreased patient survival. APE1 has been implicated in over 20 human cancers, including glioblastoma, making the protein an attractive target for the development of anticancer therapeutics. Despite intensive effort, there are no clinical endonuclease inhibitors of APE1. We have identified 25 diverse small molecule fragments that bind to APE1 at two distinct sites, including the endonuclease site. Pol eta (or PolH) is a DNA polymerase implicated, among other things, in the development of cisplatin resistance in a subset of ovarian cancers. In our quest to develop PolH inhibitors, we have identified 5 diverse fragments that bind to two distinct sites in the polymerase including a new potential allosteric site. Our results on APE1 and PolH represent the first known cases of crystal structures of small molecules bound to these proteins. Flap endonuclease (FEN1) is implicated in several cancers including for example ER/tamoxifen-resistant breast cancer. We are developing a targeted protein degradation approach using PROTACs (Proteolysis-Targeting Chimeras) toward the development of novel therapeutics against FEN1. Conclusions: Our results will help us develop small molecule inhibitors and targeted protein degradation against DDR targets that may be effective as single therapies or be used to make existing therapies more effective.
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Milosevic, Ivana. "Resistance to targeted therapy in chronic lymphocytic leukemia". Medical review 75, Suppl. 1 (2022): 57–61. http://dx.doi.org/10.2298/mpns22s1057m.
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Targeted therapy with inhibitors of cell signaling pathways and inhibitors of anti-apoptotic molecules significantly improved treatment of chronic lymphocytic leukemia. Inhibitors of Bruton?s tyrosine kinase and inhibitors of bcl2 protein showed significant efficacy in either treatment-na?ve or relapsed/refractory patients and in patients with poor risk factors. The majority of patients respond to treatment and have durable remissions, but some of them develop resistance, which leads to clinical relapse. The most frequent cause of resistance is mutations on the binding site of targeted molecules, such as Bruton?s tyrosine kinase mutations in patients treated with ibrutinib or acalabrutinib, or bcl2 mutations in patients treated with venetoclax. There are also alternative mechanisms that can lead to resistance, such as mutations of another molecule in Bruton?s tyrosine kinase signaling pathway, PLCG2, or overexpression of bcl2 protein. These mutations have been detected several months before clinical sings of relapse, and therefore could serve as predictive markers of treatment failure. When resistance to inhibitors of Bruton?s tyrosine kinase occurs, treatment with bcl2 inhibitors will be effective in most cases, and vice versa. Other strategies for overcoming resistance to inhibitors of Bruton?s tyrosine kinase or bcl2 protein are treatment with PI3K inhibitors, second and third generation Bruton?s tyrosine kinase inhibitors, bispecific antiCD3/CD19 antibodies, chimeric antigen receptor T-cells and allogenic stem cell transplantation. Several molecules, which can inhibit or degrade different signaling targets in chronic lymphocytic leukemia cells, are currently under investigation, and they could be effective in patients resistant to inhibitors of Bruton?s tyrosine kinase and bcl2 protein.
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Qokoyi, Ndibonani Kebonang, Priscilla Masamba i Abidemi Paul Kappo. "Proteins as Targets in Anti-Schistosomal Drug Discovery and Vaccine Development". Vaccines 9, nr 7 (8.07.2021): 762. http://dx.doi.org/10.3390/vaccines9070762.
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Proteins hardly function in isolation; they form complexes with other proteins or molecules to mediate cell signaling and control cellular processes in various organisms. Protein interactions control mechanisms that lead to normal and/or disease states. The use of competitive small molecule inhibitors to disrupt disease-relevant protein–protein interactions (PPIs) holds great promise for the development of new drugs. Schistosome invasion of the human host involves a variety of cross-species protein interactions. The pathogen expresses specific proteins that not only facilitate the breach of physical and biochemical barriers present in skin, but also evade the immune system and digestion of human hemoglobin, allowing for survival in the host for years. However, only a small number of specific protein interactions between the host and parasite have been functionally characterized; thus, in-depth understanding of the molecular mechanisms of these interactions is a key component in the development of new treatment methods. Efforts are now focused on developing a schistosomiasis vaccine, as a proposed better strategy used either alone or in combination with Praziquantel to control and eliminate this disease. This review will highlight protein interactions in schistosomes that can be targeted by specific PPI inhibitors for the design of an alternative treatment to Praziquantel.
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Olsen, Sarah Naomi, Laura Godfrey, James P. Healy, Charles Hatton i Scott A. Armstrong. "Abstract 681: Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition". Cancer Research 82, nr 12_Supplement (15.06.2022): 681. http://dx.doi.org/10.1158/1538-7445.am2022-681.
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Abstract MLL1 (KMT2A) translocations lead to aberrant expression of stem cell associated gene programs in hematopoietic cells producing a particularly aggressive subtype of leukemia, namely MLL-rearranged acute myeloid and lymphoblastic leukemia (AML and ALL, respectively). In pediatric leukemia, the most common rearrangement is the t(9; 11) (p22; q23) reciprocal translocation, which results in the expression of the MLL-AF9 fusion gene. Since the MLL-AF9 rearrangement does not lead to direct activation of an enzyme, small molecule drug discovery remains challenging and despite decades of research on MLL-rearranged leukemia, we have been limited by the inability of acutely assessing the consequences of direct MLL-AF9 inactivation in relevant model systems. By coupling rapid PROTAC-mediated degradation of the oncogenic MLL-AF9 fusion protein with genome-wide gene expression and chromatin analyses we have now been able to identify the immediate transcriptional and chromatin state consequences of MLL-AF9 degradation. Specifically, we have established a core set of MLL-AF9 target genes whose expression changes within minutes of MLL-AF9 degradation. These rapid expression changes are mediated by changes in productive RNA Polymerase II elongation and increased RNA Polymerase II pausing. At later timepoints, degradation of the fusion protein also induces loss of an active chromatin landscape at MLL-AF9 target genes, characterized by loss of MLL-AF9 associated proteins and activating histone modifications. These insights improve our mechanistic understanding of how MLL-AF9 mediates chromatin remodeling and leukemogenesis, while also helping us evaluate small molecule inhibitors of epigenetic mechanisms that target the MLL-AF9 protein complex. Specifically, we investigated how degradation of MLL-AF9 compares to enzymatic inhibition of DOT1L or inhibition of the MENIN-MLL interaction. Interestingly, both DOT1L and MENIN inhibitors fell short when comparing their effects to MLL-AF9 degradation and only combined DOT1L/MENIN inhibition induced global MLL-AF9 destabilization on chromatin, thus disrupting the full MLL-AF9 directed epigenetic and transcriptional program. Ultimately, we were able to confirm these findings in a patient-derived xenograft model in vivo, where only the combination was able to significantly prolong leukemia free survival. In summary, our studies establish the cellular and molecular consequences of targeted degradation of the MLL-AF9 fusion oncoprotein and help improve our understanding of small molecule inhibitors of epigenetic mechanisms as cancer therapies. Citation Format: Sarah Naomi Olsen, Laura Godfrey, James P. Healy, Charles Hatton, Scott A. Armstrong. Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 681.
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Khattri, Ram B., Daniel L. Morris, Stephanie M. Bilinovich, Erendra Manandhar, Kahlilah R. Napper, Jacob W. Sweet, David A. Modarelli i Thomas C. Leeper. "Identifying Ortholog Selective Fragment Molecules for Bacterial Glutaredoxins by NMR and Affinity Enhancement by Modification with an Acrylamide Warhead". Molecules 25, nr 1 (30.12.2019): 147. http://dx.doi.org/10.3390/molecules25010147.
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Illustrated here is the development of a new class of antibiotic lead molecules targeted at Pseudomonas aeruginosa glutaredoxin (PaGRX). This lead was produced to (a) circumvent efflux-mediated resistance mechanisms via covalent inhibition while (b) taking advantage of species selectivity to target a fundamental metabolic pathway. This work involved four components: a novel workflow for generating protein specific fragment hits via independent nuclear magnetic resonance (NMR) measurements, NMR-based modeling of the target protein structure, NMR guided docking of hits, and synthetic modification of the fragment hit with a vinyl cysteine trap moiety, i.e., acrylamide warhead, to generate the chimeric lead. Reactivity of the top warhead-fragment lead suggests that the ortholog selectivity observed for a fragment hit can translate into a substantial kinetic advantage in the mature warhead lead, which bodes well for future work to identify potent, species specific drug molecules targeted against proteins heretofore deemed undruggable.
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Yang, Kylie, Jacek L. Kolanowski i Elizabeth J. New. "Mitochondrially targeted fluorescent redox sensors". Interface Focus 7, nr 2 (6.04.2017): 20160105. http://dx.doi.org/10.1098/rsfs.2016.0105.
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The balance of oxidants and antioxidants within the cell is crucial for maintaining health, and regulating physiological processes such as signalling. Consequently, imbalances between oxidants and antioxidants are now understood to lead to oxidative stress, a physiological feature that underlies many diseases. These processes have spurred the field of chemical biology to develop a plethora of sensors, both small-molecule and fluorescent protein-based, for the detection of specific oxidizing species and general redox balances within cells. The mitochondrion, in particular, is the site of many vital redox reactions. There is therefore a need to target redox sensors to this particular organelle. It has been well established that targeting mitochondria can be achieved by the use of a lipophilic cation-targeting group, or by utilizing natural peptidic mitochondrial localization sequences. Here, we review how these two approaches have been used by a number of researchers to develop mitochondrially localized fluorescent redox sensors that are already proving useful in providing insights into the roles of reactive oxygen species in the mitochondria.
Rozprawy doktorskie na temat "Protein targeted lead molecule"
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Nilsson, Jonas. "Design, Synthesis and Characterization of Small Molecule Inhibitors and Small Molecule : Peptide Conjugates as Protein Actors". Doctoral thesis, Linköpings universitet, Organisk Kemi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-3943.
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This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.
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Nacheva, Katya Pavlova. "Development of a Bio-Molecular Fluorescent Probe Used in Kinetic Target-Guided Synthesis for the Identification of Inhibitors of Enzymatic and Protein-Protein Interaction Targets". Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4376.
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Abstract
Fluorescent molecules used as detection probes and sensors provide vital information about the chemical events in living cells. Despite the large variety of available fluorescent dyes, new improved fluorogenic systems are of continued interest. The Diaryl-substituted Maleimides (DMs) exhibit excellent photophysical properties but have remained unexplored in bioscience applications. Herein we present the identification and full spectroscopic characterization of 3,4-bis(2,4-difluorophenyl)-maleimide and its first reported use as a donor component in Forster resonance energy transfer (FRET) systems. The FRET technique is often used to visualize proteins and to investigate protein-protein interactions in vitro as well as in vivo. The analysis of the photophysical properties of 3,4-bis(2,4-difluorophenyl)-maleimide revealed a large Stokes shift of 140 nm in MeOH, a very good fluorescence quantum yield in DCM (Ffl 0.61), and a high extinction coefficient ε(340) 48,400 M-1cm-1, thus ranking this molecule as superior over other reported moieties from this class. In addition, 3,4-bis(2,4-difluorophenyl)-maleimide was utilized as a donor component in two FRET systems wherein different molecules were chosen as suitable acceptor components - a fluorescent quencher (DABCYL) and another compatible fluorophore, tetraphenylporphyrin (TPP). It has been demonstrated that by designing a FRET peptide which contains the DM donor moiety and the acceptor (quencher) motif, a depopulation of the donor excited state occurred via intermolecular FRET mechanism, provided that the pairs were in close proximity. The Forster-Radius (R0) calculated for this FRET system was 36 % and a Forster-Radius (R0) of 26 % was determined for the second FRET system which contained TPP as an acceptor. The excellent photophysical properties of this fluorophore reveal a great potential for further bioscience applications. The 3,4-bis(2,4-difluorophenyl)-maleimide fluorescent moiety was also implemented in an alternative application targeting the enzyme carbonic anhydrase (CAs) are metalloenzymes that regulate essential physiologic and physio-pathological processes in different tissues and cells, and modulation of their activities is an efficient path to treating a wide range of human diseases. Developing more selective CA fluorescent probes as imaging tools is of significant importance for the diagnosis and treatment of cancer related disorders. The kinetic TGS approach is an efficient and reliable lead discovery strategy in which the biological target of interest is directly involved in the selection and assembly of the fragments together to generate its own inhibitors. Herein, we investigated whether the in situ click chemistry approach can be implemented in the design of novel CA inhibitors from a library of non-sulfonamide containing scaffolds, which has not been reported in the literature. In addition, we exploit the incorporation of the (recently reported by us) fluorescent moiety 3,4-bis(2,4-difluorophenyl)-maleimide) as a potential biomarker with affinity to CA, as well as two coumaine derivatives representing a newly discovered class of inhibitors. The screening of a set of library with eight structurally diverse azides AZ1-AZ8 and fifteen functionalized alkynes AK1-AK12 led to the identification of 8 hit combinations among which the most prominent ones were those containing the coumarine and fluorescent maleimide scaffolds. The syn- and anti-tirazole hit combinations, AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3 were synthesized, and in a regioisomer-assignment co-injection test it was determined that the enzyme favored the formation of the anti-triazoles for all identified combinations. The mechanism of inhibition of these triazoles was validated by incubating the alkyne/azide scaffolds in the presence of Apo-CA (non-Zn containing) enzyme. It was demonstrated that the Zn-bound water/hydroxide was needed in order to hydrolyze the coumarins which generated the actual inhibitor, the corresponding hydroxycinnamic acid. The time dependent nature of the inhibition activity typical for all coumarine-based inhibitors was also observed for the triazole compounds whose inhibition constants (Ki) were determined in two independent experiments with pre-incubation times of 3 and 25 minutes, respectively. It was observed that the lower Ki values were determined, the longer the pre-incubations lasted. Thus, a novel type of coumarin-containing triazoles were presented as in situ generated hits which have the potential to be used as fluorescent bio-markers or other drug discovery applications.
The proteins from the Bcl-2 family proteins play a central role in the regualtion of normal cellular homeostasis and have been validated as a target for the development of anticancer agents. Herein, in a proof-of-concept study based on a previous kinetic TGS study targeting Bcl-XL, it was demonstrated that a multi-fragment kinetic TGS approach coupled with TQMS technology was successfully implemented in the identification of known protein-protein modulators. Optimized screening conditions utilizing a triple quadruple mass spectrometer in the Multiple Reaction Monitoring (MRM) mode was demonstrated to be very efficient in kinetic TGS hit identification increasing both the throughput and sensitivity of this approach. The multi-fragment incubation approach was studied in detail and it was concluded that 200 fragment combinations in one well is an optimal and practical number permitting good acylsulfonamide detectability. Subsequently, a structurally diverse liberty of forty five thio acids and thirty eight sulfonyl azides was screened in parallel against Mcl-1 and Bcl-XL, and several potential hit combinations were identified. A control testing was carried out by substituting Bcl-XL with a mutant R139ABcl-XL, used to confirm that the potential kinetic TGS hit combinations were actually forming at the protein's hot spot and not elsewhere on the protein surface. Although, the synthesis of all these kinetic TGS hit compounds is currently ongoing, preliminary testing of several acylsulfonamides indicate that they disrupt the Bcl-XL/Bim or Mcl-1/Bim interaction.
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Verow, Mark. "The development and validation of small molecule inhibitors targeted against the HIV-l Nef protein". Thesis, University of Leeds, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.659183.
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It is now more than 30 years since the Human Immunodeficiency Virus (HIV) became a pandemic, and now it is still a major global pathogen. Although several classes of antiretovirals are currently available for therapy, resistance and toxicity remain the biggest threats to the successful control and treatment of HIV. Therefore it is important to increase the range of targets to help reduce the chance of treatment failure. Nef serves as a pathogencity factor, and is closely associated with disease progression to Acquired ImmunoDeficiency Syndrome (AIDS) . Using structure based drug design (SBDD) methods, a range of compounds were identified to bind Nef and prevent CD4 down-modulation by Nef. Both cellular and in vitro assays were utilised to screen molecules for inhibitory activity. A set of 11 compounds were selected for the 1st generation screen. This approach identified two 'hits' with 50% inhibition of Nef function at 10 IlM. The lead molecule was selected for structural refinement, which lead to the identification of 9 analogues for use in the 2nd generation screenings. This screen identified 3 'hits' and activity against Nef function was titrated to nanomolar concentrations. Each subsequent generation utilised a re-iterative improvement/modification process, and resulted in 3 distinct compound groups, the 3rd being used to study ' differences in activity amongst similar structures.
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Omar, Hany Ahmed Mostafa Mohamed. "Molecular Pharmacology and Preclinical Studies of Novel Small-molecule Targeted Agents for The Treatment of Hepatocellular Carcinoma". The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1290565602.
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Farrington, Caroline Cain. "TARGETED DEGRADATION OF THE MYC ONCOGENE USING PP2AB56ALPHASELECTIVE SMALL MOLECULE MODULATORS OF PROTEINPHOSPHATASE 2A AS A THERAPEUTIC STRATEGY FOR TREATING MYCDRIVENCANCERS". Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1579905487094187.
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Lu, Felice. "Protein inhibition by targeted small molecule libraries /". 2005. http://wwwlib.umi.com/dissertations/search.
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Matos, Ana Marta de Jesus Gomes de. "From a multitarget antidiabetic glycosyl isoflavone towards new molecular entities against diabetes and Alzheimer’s disease : generation of lead series and target assessment". Doctoral thesis, 2018. http://hdl.handle.net/10451/42532.
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Type 2 diabetes and Alzheimer’s disease are closely related amyloid diseases globally affecting millions of people. However, the pathophysiological mechanisms connecting both diseases still require further investigation. In this work, we compile the existing evidence in the literature to allow the establishment of etiological links needed for drug discovery against diabetes-induced dementia. Furthermore, we provide an extensive revision of bioactive lead molecules that encourage further studies, particularly focusing on polyphenol sugar conjugates endowed with antidiabetic and neuroprotective activities.
The state-of-the-art synthetic approaches for the generation of these types of molecules are also covered, thus setting the organic chemistry background for the original research work here developed.
The use of carbohydrate-based molecules in drug research and development has multiple recognized benefits. In addition to enhanced solubility, bioavailability, and antidiabetic effects as previously reported, in this work we show, for the first time, that C-glucosylation is able to reverse the membrane dipole potential decrease induced by planar lipophilic polyphenols, elsewhere described as Pan-Assay Interference Compounds. This is a relevant discovery for drug development, particularly in the context of this thesis due to the polyphenolic nature of the compounds here presented.
One of these compounds, 8-β-D-glucosylgenistein, was investigated in a diet-induced obese mouse of type 2 diabetes and found to exert a multitarget antidiabetic mechanism of action that goes beyond prior conjectures. Indeed, this antihyperglycemic glucosyl isoflavone reduces the renal threshold for glucose reabsorption, ameliorates diabetes-associated non-alcoholic fatty liver disease and hypercholesterolemia, normalizes insulin-degrading enzyme expression, and increases glucosestimulated insulin secretion. However, the detected inability of this polyphenol to permeate the blood brain barrier and to exert neuroprotective effects encouraged the pursuit of new scaffolds with therapeutic potential against diabetes-induced dementia.
The role of amyloid β in the neurodegenerative processes occurring in Alzheimer’s disease and diabetes-induced dementia is, nowadays, unquestionable. Yet, targeted therapies aimed at inhibiting amyloid secretion or aggregation have, so far, failed clinical trials. In the past decade, the role of the cellular prion protein (PrPC) – a high-affinity ligand of amyloid β oligomers (Aβo) – has, in fact, been regarded as the limiting step in the cascade of events leading to neurodegeneration. Fyn kinase is one of the key players in this cascade, which culminates with the formation of neurofibrillary tangles composed by hyperphosphorylated tau, eventually leading to cell death. In this perspective, we have identified innovative N-methylpiperazinyl flavones and their glucosyl derivatives as Aβo-binders and non-toxic disruptors of Aβo-PrPC interactions. Furthermore, easily accessed glucosyl polyphenols with improved pharmacokinetic properties were also investigated and revealed to inhibit Aβ-induced Fyn activation with concomitant decrease in tau phosphorylation. Fyn kinase inhibition is considered a novel therapeutic strategy for Alzheimer’s disease, and these compounds are the first to accomplish this goal, with proven downstream effects. These molecules thus share the potential for further development against Alzheimer’s disease and diabetes-induced dementia.
The work presented in this thesis elucidates the therapeutic relevance of natural and nature-inspired C-glucosyl polyphenols in the studied biological context, and highlights the usefulness of carbohydrate-based molecules for medicinal chemistry applications.
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Bhowmick, Tuhin. "Crystal Structure Of Mycobacterium Tuberculosis Histone Like Protein HU And Structure Based Design Of Molecules To Inhibit MtbHU-DNA Interaction : Leads For A New Target. Structure Aided Computational Analysis Of Metal Coordinated Complexes Containing Amino Acids And Organic Moieties Designed For Photo Induced DNA Cleavage". Thesis, 2012. http://hdl.handle.net/2005/2469.
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In bacteria, nucleoid associated proteins (NAPs) represent a prominent group of global regulators that perform the tasks of genome compaction, establishing chromosomal architecture and regulation of various DNA transactions like replication, transcription, recombination and repair. HU, a basic histone like protein, is one of the most important NAPs in Eubacteria. Mycobacterium tuberculosis produces a homodimeric HU (MtbHU), which interacts with DNA non-specifically through minor groove binding. Exploration for essential genes in Mtb (H37Rv) through transposon insertion has identified HU coding gene [Rv2986c, hupB; Gene Id: 15610123; Swiss-Prot ID: P95109)] to be vital for the survival and growth of this pathogen.
MtbHU contains two domains, the N-terminal domain which is considerably conserved among the HU proteins of the prokaryotic world, and a C–terminal domain consisting of Lys-Ala rich multiple repeat degenerate motifs. Sequence analysis carried out by the thesis candidate showed that MtbHU exhibits 86 to 100 percent identity within the N-term region among all the mycobacterium species and some of the members of actinobacteria, including important pathogens like M. tuberculosis, M. leprae, M. ulcerans, M. bovis, Nocardia; while C term repeat region varies relatively more. This strikingly high cross species identity establishes the MtbHU N-terminal domain (MtbHUN) as an important representative structural model for the above mentioned group of pathogens.
The thesis candidate has solved the X-ray crystal structure of MtbHUN, crystallized in two different forms, P2 and P21. The crystal structures in combination with computational analyses elucidate the structural details of MtbHU interaction with DNA. Moreover, the similar mode of self assembly of MtbHUN observed in two different crystal forms reveals that the same DNA binding interface of the protein can also be utilized to form higher order oligomers, that HU is known to form at higher concentrations. Though the bifunctional interface involved in both DNA binding and self assembly is not akin to a typical enzyme active site, the structural analysis identified key interacting residues involved in macromolecular interactions, allowing us to develop a rationale for inhibitor design. Further, the candidate has performed virtual screening against a vast library of compounds, and design of small molecules to target MtbHU and disrupt its binding to DNA. Various biochemical, mutational and biological studies were performed in the laboratory of our collaborator Prof. V. Nagaraja, MCBL, IISc., to investigate these aspects. After a series of iterations including design, synthesis and validation, we have identified novel candidate molecules, which bind to MtbHU, disrupt chromosomal architecture and arrest M. tuberculosis growth. Thus, the study suggests that, these molecules can serve as leads for a new class of DNA-interaction inhibitors and HU as a druggable target, more so because HU is essential to Mtb, but absent in human. Our study proposes that, targeting the nucleoid associated protein HU in Mtb can strategize design of new anti-mycobacterial therapeutics. Perturbation of MtbHU-DNA binding through the identified compounds provides the first instance of medium to small molecular inhibitors of NAP, and augurs well for the development of chemical probe(s) to perturb HU functions, and can be used as a fundamental chemical tool for the system level studies of HU-interactome.
Section I: “Crystal structure of Mycobacterium tuberculosis histone like protein HU and structure based design of molecules to inhibit MtbHU-DNA interaction: Leads for a new target.” of this thesis presents an elaborate elucidation of the above mentioned work.
The candidate has additionally carried out structure based computational and theoretical work to elucidate the interaction of amino acid based metal complexes which efficiently bind to DNA via minor-groove, major-groove or base intercalation interaction and display DNA cleavage activity on photo-irradiation. This understanding is crucial for the design of molecules towards Photodynamic Therapy (PDT). PDT is an emerging method of non-invasive treatment of cancer in which drugs like Photofrin show localized toxicity on photoactivation at the tumor cells leaving the healthy cells unaffected.
The work carried out in our group in close collaboration with Prof. A.R. Chakravarty of Inorganic and Physical Chemistry Department elaborates the structure based design of Amino acid complexes containing single Cu (II), such as [Cu(L-trp)(dpq)(H2O)]+ , [Cu (L-arg) 2](NO3)2 , Amino acid complexes containing oxobridged diiron Fe(III), such as [{Fe(L-his)(bpy)}2(μ-O)](ClO4)2 , [{Fe(L-his)(phen)}2(μ-O)](ClO4)2 , and Complexes containing Binuclear Cu(II) coordinated organic moiety, such as [{(dpq) CuII}2(μ-dtdp)2], which bind to DNA through minor groove/major groove/base intercalation interactions. Docking analysis was performed with the X-ray crystallographic structure of DNA as receptor and the metal complexes as ligands, to study the mode of binding to DNA and to understand the possible mode of DNA cleavage (single/double strand) when activated with laser.
Section II: “Structure based computational and theoretical analysis of metal coordinated complexes containing amino acids and organic moieties designed for photo induced DNA cleavage” of this thesis presents a detailed presentation of the above mentioned work.
Książki na temat "Protein targeted lead molecule"
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Cui, Zhao, Neil Turner i Ming-hui Zhao. Alport post-transplant antiglomerular basement membrane disease. Redaktor Neil Turner. Oxford University Press, 2015. http://dx.doi.org/10.1093/med/9780199592548.003.0075.
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Alport antiglomerular basement membrane (anti-GBM) disease is a rare example of disease caused by allo-sensitization after renal transplantation, first described in 1992. Because the recipient lacks a specific glomerular basement membrane (GBM) protein, they can become sensitized to the normal molecule present in the GBM of the donor kidney. The disease is restricted to the allograft. Interestingly severe disease arises from this only arises rarely, certainly less than 1 in 20, probably closer to 1 in 50. It characteristically causes late graft loss in a first transplant with accelerated tempo in later allografts, and in its most extreme form recurs within days. However, inexplicably some subsequent transplants do not provoke aggressive recurrence. Treatment of the most aggressive disease is difficult and in most cases has been ultimately unsuccessful. Lower levels of immune response, marked by linear binding of immunoglobulin-G to GBM without glomerular disease, are not uncommon in Alport patients after transplantation and should not lead to altered treatment. Immunoassays for anti-GBM antibodies can be misleading as in most cases the target of antibodies is the α5 chain of type IV collagen, rather than the α3 chain which is the target in spontaneous anti-GBM disease. Overall the outcome of transplantation in Alport syndrome is better than average. This complication is more likely in patients with partial or total gene deletion rather than point mutations, but no other predictive features have been identified.
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Colbert, Robert A., i Paul Bowness. Immune mechanisms: HLA-B27. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780198734444.003.0006.
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HLA-B27 is present in the majority of patients with ankylosing spondylitis (AS). Although we have learned a considerable amount about the natural immunologic function of HLA class I proteins, this has not provided a definitive mechanism of AS pathogenesis. While HLA-B27 is adept at presenting antigenic peptides to CD8+ T cells, ‘arthritogenic’ peptides targeted by a cross-reactive T or natural killer cell response have not been described, nor have autoreactive T cells been found. Newer concepts have evolved based on the propensity of HLA-B27 to ‘misbehave’, both inside cells and on the cell surface. Misfolded HLA-B27 molecules may stimulate an endoplasmic reticulum stress response, promoting production of IL-23 and then IL-17 and related cytokines. Aberrant cell-surface HLA-B27 molecules are ligands for natural killer and related immunoreceptors, and recognition can lead to IL-17 proinflammatory responses. There is growing evidence to suggest that these aberrant behaviours contribute to AS pathogenesis.
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Malcangio, Marzia. Glia. Redaktorzy Paul Farquhar-Smith, Pierre Beaulieu i Sian Jagger. Oxford University Press, 2018. http://dx.doi.org/10.1093/med/9780198834359.003.0035.
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The landmark review discussed in this chapter, published in 2003 by Watkins and Maier, showed how glia have a major role in the modulation of pain mechanisms in the spinal cord and act remotely from peripheral nerve injury. This review led the way to a substantial body of literature demonstrating the pivotal role played by both microglia and astrocytes in chronic pain mechanisms. Since 2003 the modalities underlying neuron–microglia communication (e.g. chemokines, proteases, the translocator protein TSPO) have been dissected, and novel pathways of interactions delineated. Concrete molecular targets expressed by spinal microglia in response to a remote injury have been identified and they hold promise for future analgesic therapies for chronic pain.
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Beattie, R. Mark, Anil Dhawan i John W.L. Puntis. The pancreas. Oxford University Press, 2011. http://dx.doi.org/10.1093/med/9780198569862.003.0046.
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Congenital anomalies 326Pancreatitis 328Pancreatic insufficiency 335These are rare. Most anomalies are sporadic. Only the gene coding for the homeodomain protein PDX1 is clearly demonstrated to be causal of pancreatic agenesis. Inactivation or inhibition of signalling molecule sonic hedgehog (Shh) could potentially lead to annular pancreas, pancreatic divisum, and pancreatic ectopia....
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Gropman, Andrea L., Belen Pappa i Nicholas Ah Mew. The Urea Cycle Disorders. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780199937837.003.0063.
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The urea cycle is the primary nitrogen disposal pathway in humans. The urea cycle requires the coordinated function of six enzymes and two mitochondrial transporters to catalyze the conversion of a molecule of ammonia, the α-nitrogen of aspartate and bicarbonate into urea. Whereas ammonia is toxic, urea is relatively inert, soluble in water, and readily excreted by the kidney in the urine. The accumulation of ammonia and other toxic intermediates of the cycle lead to predominantly neurological sequelae. All of the genes have been identified. The disorders may present at any age from the neonatal period to adulthood, with the more severe patients presenting earlier in life. Patients are at risk for metabolic decompensation throughout life, often triggered by illness, fasting, surgery and postoperative states, peripartum, stress, and increased exogenous protein load. This chapter addresses common somatic and neurological presentation, differential diagnosis, laboratory testing, and treatments.
Części książek na temat "Protein targeted lead molecule"
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Weber, L. "Discovery of New MCRs, Chemical Evolution and Lead Optimization". W Small Molecule — Protein Interactions, 189–99. Berlin, Heidelberg: Springer Berlin Heidelberg, 2003. http://dx.doi.org/10.1007/978-3-662-05314-0_12.
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Whitty, Adrian. "Small-Molecule Inhibitors of Protein-Protein Interactions: Challenges and Prospects". W Gene Family Targeted Molecular Design, 199–233. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2008. http://dx.doi.org/10.1002/9780470423936.ch7.
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Ursu, Andrei, Matthew G. Costales, Jessica L. Childs-Disney i Matthew D. Disney. "Chapter 15. Small-molecule Targeted Degradation of RNA". W Protein Degradation with New Chemical Modalities, 317–36. Cambridge: Royal Society of Chemistry, 2020. http://dx.doi.org/10.1039/9781839160691-00317.
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Prakash, Om, i Feroz Khan. "CoSSDb: A Database of Co-crystallized Ligand Sub-structures for Anticancer Lead Designing & Optimization". W Proceedings of the Conference BioSangam 2022: Emerging Trends in Biotechnology (BIOSANGAM 2022), 133–41. Dordrecht: Atlantis Press International BV, 2022. http://dx.doi.org/10.2991/978-94-6463-020-6_14.
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AbstractThe Discovery of the novel optimized structures of small molecules for selective targeting is one of the challenging tasks in drug designing. Bioisosteres are the key components of the lead compound, which provide hidden power to the compound scaffold for selective targeting. We are presenting a database, named CoSSDb which stands for Co-crystallized Sub-Structure Database. The CoSSDb contains ligand sub-structures as possible bioisosteres. extracted from PDB files, available in Protein Data Bank. Sub-structures were extracted through an algorithm, which utilizes the location of atoms in the 3D domain of the complex ligand & protein. It processes the relative positioning of atoms for demarcation of the influential part of the ligand, which interacts with macromolecule and provides potency to that ligand for binding with a specific binding pocket of the protein. The algorithm was used to extract sub-structures from the ligands co-crystallized with proteins involved in cancer. About 7721 x-ray crystallography PDB files were processed, and 654 non-redundant substructures were identified. These sub-structures will be useful during designing & optimization of novel ligands for selective targets. The database is freely accessible at ‘https://opticket49.wixsite.com/substructdb’.
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Sharma, Sahil, i Cynthia M. Sharma. "Identification of RNA Binding Partners of CRISPR-Cas Proteins in Prokaryotes Using RIP-Seq". W Methods in Molecular Biology, 111–33. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1851-6_6.
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AbstractCRISPR-Cas systems consist of a complex ribonucleoprotein (RNP) machinery encoded in prokaryotic genomes to confer adaptive immunity against foreign mobile genetic elements. Of these, especially the class 2, Type II CRISPR-Cas9 RNA-guided systems with single protein effector modules have recently received much attention for their application as programmable DNA scissors that can be used for genome editing in eukaryotes. While many studies have concentrated their efforts on improving RNA-mediated DNA targeting with these Type II systems, little is known about the factors that modulate processing or binding of the CRISPR RNA (crRNA) guides and the trans-activating tracrRNA to the nuclease protein Cas9, and whether Cas9 can also potentially interact with other endogenous RNAs encoded within the host genome. Here, we describe RIP-seq as a method to globally identify the direct RNA binding partners of CRISPR-Cas RNPs using the Cas9 nuclease as an example. RIP-seq combines co-immunoprecipitation (coIP) of an epitope-tagged Cas9 followed by isolation and deep sequencing analysis of its co-purified bound RNAs. This method can not only be used to study interactions of Cas9 with its known interaction partners, crRNAs and tracrRNA in native systems, but also to reveal potential additional RNA substrates of Cas9. For example, in RIP-seq analysis of Cas9 from the foodborne pathogen Campylobacter jejuni (CjeCas9), we recently identified several endogenous RNAs bound to CjeCas9 RNP in a crRNA-dependent manner, leading to the discovery of PAM-independent RNA cleavage activity of CjeCas9 as well as non-canonical crRNAs. RIP-seq can be easily adapted to any other effector RNP of choice from other CRISPR-Cas systems, allowing for the identification of target RNAs. Deciphering novel RNA-protein interactions for CRISPR-Cas proteins within host bacterial genomes will lead to a better understanding of the molecular mechanisms and functions of these systems and enable us to use the in vivo identified interaction rules as design principles for nucleic acid-targeting applications, fitted to each nuclease of interest.
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Jayaraj, Abhilash, Ruchika Bhat, Amita Pathak, Manpreet Singh i B. Jayaram. "Development of a Web-Server for Identification of Common Lead Molecules for Multiple Protein Targets". W Methods in Pharmacology and Toxicology, 487–504. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/7653_2018_9.
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Takaoka, Yousuke, i Minoru Ueda. "Enantiodifferential Approach for the Target Protein Detection of the Jasmonate Glucoside That Controls the Leaf Closure of Samanea saman". W Methods in Molecular Biology, 149–58. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7874-8_13.
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Ramos, Kenneth S., Stefano Guerra i Randa El-Zein. "Precision Medicine Approaches for Stratification and Development of Novel Therapies of Latin(x) Patients at Risk of Lung Malignancy". W Advancing the Science of Cancer in Latinos, 89–98. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-14436-3_8.
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AbstractPrecision medicine has emerged as an optimal health-care delivery platform, which emphasizes integration of individual patient characteristics into patient care. For lung cancer, precision approaches have focused mostly on targeted therapies directed at tyrosine kinases and immunotherapy. It is proposed that refinements should focus on improved risk stratification of patients at heightened risk of lung malignancy, namely patients with chronic obstructive pulmonary disease (COPD). African ancestry is associated with worsened clinical outcomes in COPD and lung cancer, which is relevant for Latinx populations given that varying degrees of African ancestry exist among several Latinx subgroups. The work reviewed here focuses on ORF1p, a protein encoded by Long Interspersed Element-1 (LINE-1) and associated with genetic instability. Because high expression of ORF1p is associated with poor prognosis in patients with non-small-cell lung cancer (NSCLC), it is hypothesized that circulating ORF1p can be monitored as a proxy of genetic instability in patients with COPD and lung cancer. Circulating ORF1p levels correlate with FEV1 deficits and airflow limitation (the hallmark of COPD) in former smokers, and tissue expression of ORF1p is increased in TP53 mutant NSCLC compared to wildtype. Understanding the role of ORF1p in COPD and lung cancer and its utility as a biomarker of genetic instability may lead to advances in lung cancer care and development of novel targeted therapies.
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Das, Amit, i Simanti Bhattacharya. "Different Types of Molecular Docking Based on Variations of Interacting Molecules". W Pharmaceutical Sciences, 795–819. IGI Global, 2017. http://dx.doi.org/10.4018/978-1-5225-1762-7.ch031.
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Molecular docking plays an important role in drug discovery research by facilitating target identification, target validation, virtual screening for lead identification and lead optimization. Depending upon the nature of the disease of interest, targets can be either protein or DNA while drugs are mostly organic small molecules. Different types of molecular docking techniques like protein-protein or protein-DNA or protein-small molecule or DNA-small molecule are employed for achieving the above mentioned objectives. This chapter provides a clear idea of the position of molecular docking in drug discovery with detailed discussion on different types of molecular docking based on the varieties of interacting partners. Subsequently the authors provide a detailed list of tools that can be used for docking in drug discovery and discus some examples of molecular docking in drug discovery before concluding with a remark on future areas of improvement in molecular docking related to drug discovery.
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Das, Amit, i Simanti Bhattacharya. "Different Types of Molecular Docking Based on Variations of Interacting Molecules". W Methods and Algorithms for Molecular Docking-Based Drug Design and Discovery, 148–72. IGI Global, 2016. http://dx.doi.org/10.4018/978-1-5225-0115-2.ch006.
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Molecular docking plays an important role in drug discovery research by facilitating target identification, target validation, virtual screening for lead identification and lead optimization. Depending upon the nature of the disease of interest, targets can be either protein or DNA while drugs are mostly organic small molecules. Different types of molecular docking techniques like protein-protein or protein-DNA or protein-small molecule or DNA-small molecule are employed for achieving the above mentioned objectives. This chapter provides a clear idea of the position of molecular docking in drug discovery with detailed discussion on different types of molecular docking based on the varieties of interacting partners. Subsequently the authors provide a detailed list of tools that can be used for docking in drug discovery and discus some examples of molecular docking in drug discovery before concluding with a remark on future areas of improvement in molecular docking related to drug discovery.
Streszczenia konferencji na temat "Protein targeted lead molecule"
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Quertermous, T., J. M. Schnee, M. S. Runge, G. R. Matsueda, N. W. Hudson, J. G. Seidman i E. Haber. "EXPRESSION OF A RECOMBINANT ANTIBODY-TARGETED THROMBOLYTIC MOLECULE". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644616.
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We have recently shown that targeting tissue-type plasminogen activator (t-PA) by covalent linkage to a fibrin-specific monoclonal antibody (59D8) produces a more potent thrombolytic agent which also induces less fibrinogenolysis. A recombinant molecule encoding a t-PA-59D8 fusion protein was constructed to provide a ready source of this agent for further study, and to allow tailoring of the active moities for maximal activity. DNA sequence coding for the 59D8 heavy chain (HC) antigen combining site was cloned from a lambdaphage library by selection with a joining region probe. Gene segments coding for this cloned HC rearrangement, the amino portion of the mouse gamma 2b HC constant region, and the catalytic B chain of t-PA were joined in the pSV2-gpt expression vector. The desired coding sequence was confirmed by nucleotide sequence analysis. The construct was transfected by electroporation into 59D8 hybridoma HC loss variants. Transfectants were screened for antifibrin antibody activity. Positive clones were shown to produce mRNA which hybridized to the human t-PA gene in Northern blot analysis. Supernatants from 5 of these clones were subjected to affinity chromatography on a synthetic fibrin-like peptide-Sepharose column followed by a benzamidine-Sepharose column. Western blots of SDS polyacrylamide gels run under reducing conditions revealed binding to a 60 kd band by a monoclonal antihuman t-PA antibody, consistent with a 59D8 HC-t-PA fusion protein. Also, binding to a 25 kd band by goat anti-mouse Fab indicated the presence of 59D8 light chain. Affinity purified protein was shown to have amidolytic activity of similar potency to t-PA in a chromogenic substrate assay utilizing S-2288. Bifunctionality of the purified protein was demonstrated first by an assay which requires the protein to bind to immobilized fibrin and simultaneously exhibit activity in the S2288 assay, and second by simultaneous fibrin and iodinated anti-t-PA binding.
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Turchi, John, Katherine S. Pawelczak, Navnath Gavande i Pamela S. VanderVere-Carozza. "Abstract A095: Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku". W Abstracts: AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; October 26-30, 2019; Boston, MA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1535-7163.targ-19-a095.
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Chen, Kok Hao, i Jong Hyun Choi. "DNA Oligonucleotide-Templated Nanocrystals: Synthesis and Novel Label-Free Protein Detection". W ASME 2009 International Mechanical Engineering Congress and Exposition. ASMEDC, 2009. http://dx.doi.org/10.1115/imece2009-11958.
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Semiconductor and magnetic nanoparticles hold unique optical and magnetic properties, and great promise for bio-imaging and therapeutic applications. As part of their stable synthesis, the nanocrystal surfaces are usually capped by long chain organic moieties such as trioctylphosphine oxide. This capping serves two purposes: it saturates dangling bonds at the exposed crystalline lattice, and it prevents irreversible aggregation by stabilizing the colloid through entropic repulsion. These nanocrystals can be rendered water-soluble by either ligand exchange or overcoating, which hampers their widespread use in biological imaging and biomedical therapeutics. Here, we report a novel scheme of synthesizing fluorescent PbS and magnetic Fe3O4 nanoparticles using DNA oligonucleotides. Our method of PbS synthesis includes addition of Na2S to the mixture solution of DNA sequence and Pb acetate (at a fixed molar ratio of DNA/S2−/Pb2+ of 1:2:4) in a standard TAE buffer at room temperature in the open air. In the case of Fe3O4 particle synthesis, ferric and ferrous chloride were mixed with DNA in DI water at a molar ratio of DNA/Fe2+/Fe3+ = 1:4:8 and the particles were formed via reductive precipitation, induced by increasing pH to ∼11 with addition of ammonium hydroxide. These nanocrystals are highly stable and water-soluble immediately after the synthesis, due to DNA termination. We examined the surface chemistry between oligonucleotides and nanocrystals using FTIR spectroscopy, and found that the different chemical moieties of nucleobases passivate the particle surface. Strong coordination of primary amine and carbonyl groups provides the chemical and colloidal stabilities, leading to high particle yields (Figure 1). The resulting PbS nanocrystals have a distribution of 3–6 nm in diameter, while a broader size distribution is observed with Fe3O4 nanoparticles as shown in Figure 1b and c, respectively. A similar observation was reported with the pH change-induced Fe3O4 particles of a bimodal size distribution where superparamagnetic and ferrimagnetic magnetites co-exist. In spite of the differences, FTIR measurements suggest that the chemical nature of the oligonucleotide stabilization in this case is identical to the PbS system. As a particular application, we demonstrate that aptamer-capped PbS QD can detect a target protein based on selective charge transfer, since the oligonucleotide-templated synthesis can also serve the additional purpose of providing selective binding to a molecular target. Here, we use thrombin and a thrombin-binding aptamer as a model system. These QD have diameters of 3∼6 nm and fluoresce around 1050 nm. We find that a DNA aptamer can passivate near IR fluorescent PbS nanocrystals, rendering them water-soluble and stable against aggregation, and retain the secondary conformation needed to selectively bind to its target, thrombin, as shown in Figure 2. Importantly, we find that when the aptamer-functionalized nanoparticles binds to its target (only the target), there is a highly systematic and selective quenching of the PL, even in high concentrations of interfering proteins as shown in Figure 3a and b. Thrombin is detected within one minute with a detection limit of ∼1 nM. This PL quenching is attributed to charge transfer from functional groups on the protein to the nanocrystals. A charge transfer can suppress optical transition mechanisms as we observe a significant decrease in QD absorption with target addition (Figure 3c). Here, we rule out other possibilities including Forster resonance energy transfer (FRET) and particle aggregation, because thrombin absorb only in the UV, and we did not observe any significant change in the diffusion coefficient of the particles with the target analyte, respectively. The charge transfer-induced photobleaching of QD and carbon nanotubes was observed with amine groups, Ru-based complexes, and azobenzene compounds. This selective detection of an unlabeled protein is distinct from previously reported schemes utilizing electrochemistry, absorption, and FRET. In this scheme, the target detection by a unique, direct PL transduction is observed even in the presence of high background concentrations of interfering negatively or positively charged proteins. This mechanism is the first to selectively modulate the QD PL directly, enabling new types of label free assays and detection schemes. This direct optical transduction is possible due to oligonucleotidetemplated surface passivation and molecular recognition. This chemistry may lead to more nanoparticle-based optical and magnetic probes that can be activated in a highly chemoselective manner.
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Sarkar, Saugata, i Marissa Nichole Rylander. "Treatment Planning Model for Nanotube-Mediated Laser Cancer Therapy". W ASME 2008 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2008. http://dx.doi.org/10.1115/sbc2008-192997.
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The goal of the project is to develop an effective treatment planning computational tool for nanotube-mediated laser therapy that maximizes tumor destruction and minimizes tumor recurrence. Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, the effectiveness of these therapies is limited due to nonspecific heating of target tissue and diffusion limited thermal deposition which often leads to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to induction of molecular chaperones called heat shock protein (HSP) in tumor regions where non-lethal temperature elevation occurs causing enhanced tumor cell viability and imparting resistance to chemotherapy and radiation treatments which are generally employed in conjunction with hyperthermia.
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Tavousi, Pouya, Morad Behandish, Kazem Kazerounian i Horea T. Ilieş. "An Improved Free Energy Formulation and Implementation for Kinetostatic Protein Folding Simulation". W ASME 2013 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/detc2013-12671.
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Protein structure prediction remains one of the significant challenges in computational biology. We have previously shown that our kinetostatic compliance method can overcome some of the key difficulties faced by other de novo structural prediction methods, such as the very small time steps required by the molecular dynamics approaches, or the very large number of samples required by the sampling based techniques. In this paper we extend the previous free energy formulation by adding the solvent effects, which contribute predominantly to the folding phenomena. We show that the addition of the solvation effects, which complement the existing Coulombic and van der Waals interactions, lead to a physically effective energy function. Furthermore, we achieve significant computational speed-up by employing efficient algorithms and data structures that effectively reduce the time complexity from O(n2) to O(n), n being the number of atoms. Our simulations are consistent with the general behavior observed in protein folding, and show that the hydrophobic atoms tend to pack inside the core of the molecule in an aqueous solvent, while a vacuum environment produces no such effect.
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Hussein, Ola, Feras Alali, Ala‐Eddin Al Mustafa i Ashraf Khalil. "Development of Novel Chalcone Analogs as Potential Multi-Targeted Therapies for Castration-Resistant Prostate Cancer". W Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2021. http://dx.doi.org/10.29117/quarfe.2021.0114.
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Prostate cancer (PCa) is the second most frequently diagnosed malignancy, as well as a leading cause of cancer-related mortality in men globally. Despite the initial response to hormonal targeted therapy, the majority of patients ultimately progress to a lethal form of the disease, castration-resistant prostate cancer (CRPC). Therefore, the objective of this study was to discover and develop novel treatment modalities for CRPC. Chalcones are among the highly attractive scaffolds being investigated for their antitumor activities. A library of 26 chalcone analogs were designed, synthesized and evaluated as potential therapies for CRPC. The design was guided by in-silico ADMET prediction in which analogs with favorable drug-likeness properties were prioritized. The new compounds were synthesized, purified and characterized by extensive structural elucidation studies. The compounds in vitro cytotoxicity was evaluated against two androgen receptor (AR)-negative prostate cancer cell lines (PC3 and DU145). Among the tested compounds, pyridine containing analogs (13, 15 and 16) showed potent antiproliferative activities with IC50 values ranging between 4.32-6.47 µM against PC3 and DU145 cell lines. Detailed biological studies of the lead molecule 16 revealed that it can significantly induce apoptosis through upregulation of Bax and downregulation of Bcl-2. In addition, compound 16 potently inhibited colony formation and reduced cell migration of AR-negative PCa cell lines (PC3 and DU145). The molecular pathway analysis showed that the anticancer activity of compound 16 is associated with blocking of ERK1/2 and Akt activities. Furthermore, compound 16 inhibited angiogenesis in the chick chorioallantoic membrane (CAM) model as compared to control. Structure-activity relationship study revealed that the cytotoxicity could dramatically improve via changing the methoxylation pattern by more than 2-folds (IC50 << 2.5 μM). These results indicate that pyridine-based chalcones could serve as promising lead molecules for the treatment of CRPC; thus, further in vitro and in vivo studies are warranted.
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Vieira, Daniella Serafin Couto, Laura Otto Walter, Ana Carolina Rabello de Moraes, João Péricles da Silva Jr i Maria Cláudia Santos Silva. "CROSS-SECTIONAL ANALYSIS OF CLINICAL AND MORPHOLOGICAL FACTORS OF BREAST CANCER IMMUNOPHENOTYPES: A COMPARATIVE STUDY OF TWO DIFFERENT METHODOLOGIES OVER A 24‑YEAR HISTORICAL SERIES". W Scientifc papers of XXIII Brazilian Breast Congress - 2021. Mastology, 2021. http://dx.doi.org/10.29289/259453942021v31s1043.
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Introduction: Breast cancer (BC) is the most incident form of cancer in women worldwide. The widespread use of breast screening programs, as well advances in molecular biology, and new drugs in chemotherapy have contributed to the recent survival rate improvement in high income countries. Furthermore, the study of cancer genome led to the elucidation of the intrinsic subtypes of invasive breast cancer (IBC), consequently, the success rate of targeted therapies improved the outcome in patients. However, considering that immunohistochemistry (IHC) is one of the main methods to determine the profile of protein expression in surgical pathology, most antibodies used have a presumed or already established role and represent proteins whose transcription has been previously described in genetic profile studies. Objectives: Describe the prevalence of IBC in women admitted to a public hospital in Brazil from 1994 to 2018, to establish a correlation between two models of immunohistochemical analysis, the 13th St. Gallen Conference classification and the biomarkerdefined subtypes based on HER2 and estrogen receptor (ER) status, and to investigate the profile of these cases. Methods: Retrospective database analysis was performed. 1,335 women with histologic diagnosis of IBC were included in the study from a public hospital in Brazil between 1994 and 2018. Frequencies and univariate associations were estimated by using chi-square tests. Agreement between the immunohistochemical groups were tested by using Cohen’s kappa coefficients. Results: The mean age was 56.1 years. The most prevalent subtype was luminal B/HER2 and the frequency of tumors with worse prognosis was 62.7%. An association was found between histological grade 3 (G3) and the worst prognostic subtypes: non-luminal A (OR=31.18; 95%CI 13.76–70.64), TNBC (OR=8.77; 95%CI 6.20–12.41), non-ER+/HER2- (OR=5.37; 95%CI 4.11–7.04) and ER-/HER2- (OR=8.50; 95%CI 6.10–11.85). A similar association was found for nuclear G3: non-luminal A (OR=6.3; 95%CI 4.29–9.47), TNBC (OR=5.14; 95%CI 3.64–7.31), non-ER+/HER2- (OR=4.83; 95%CI 3.80–6.15) and ER-/HER2- (OR=5.41; 95%CI 3.92–7.50). When the two models of immunohistochemical analysis were compared, the results showed an agreement rate of 99.48% to 100%. Conclusions: Our results show that most cases had worse outcomes, and there was absolute agreement between the two models of immunohistochemical analysis. These results can contribute to institutions that do not have molecular investigation, enabling accessible tools in routine practice.
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Brewer, Bryson M., Yandong Gao, Rebecca M. Sappington i Deyu Li. "Microfluidic Molecular Trap: Probing Extracellular Signaling by Selectively Blocking Exchange of Specific Molecules in Cell-Cell Interactions". W ASME 2013 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/imece2013-64489.
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Communication among cell populations is achieved via a wide variety of soluble, extracellular signaling molecules [1]. In order to investigate the role of specific molecules in a cellular process, researchers often utilize in vitro cell culture techniques in which the molecule under question has been removed from the signaling pathway. Traditionally, this has been accomplished by eliminating the gene in the cell that is responsible for coding the targeted ligand/receptor by using modern DNA technology such as gene knockout; however, this process is expensive, time-consuming, and labor intensive. Previously, we have demonstrated a microfluidic platform that uses a semi-permeable barrier with embedded receptor-coated nanoparticles to selectively remove a specific molecule or ligand from the extracellular signaling pathway in a cell co-culture environment [2]. This initial proof-of-principle was conducted using biotinylated nanoparticles and fluorescently tagged avidin molecules, as the avidin/biotin complex is the strongest known non-covalent interaction between a protein and a ligand (Dissociation constant kd = 10−15 M). Also, the trap was only effective for short time periods (<15 min) because the high concentration of fluorescently tagged avidin molecules required for visualization quickly saturated the barrier. However, nearly all biologically relevant ligand-receptor interactions have lower binding affinities than the avidin-biotin complex, with dissociation constants that are larger by several orders of magnitude. In addition, many in vitro cell culture experiments are conducted over multiple hours or days. Thus, a practically useful molecular trap device must be able to operate in a lower binding affinity regime while also lasting for extended time periods. Here we present results in which a biotinylated-particle barrier was used to successfully block lower concentrations of fluorescently tagged avidin for multiple days, showcasing the applicability of the device for long term experiments. In addition, we introduce a modified molecular trap in which the protein A/goat IgG complex was used to demonstrate the effectiveness of the platform for lower binding affinity protein-ligand interactions. These results indicate the potential usefulness of the microfluidic molecular trap platform for probing extracellular signaling pathways.
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Hussein, Ola, Feras Alali, Ala-Eddin Al Moustafa i Ashraf Khalil. "Design, Synthesis and Biological Evaluation of Novel Chalcone Analogs as Potential Therapeutic Agents for Castration-Resistant Prostate Cancer". W Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0179.
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Prostate cancer (PCa) is the second most frequently diagnosed malignancy, as well as a leading cause of cancer-related mortality in men globally. Despite the initial response to hormonal targeted therapy, the majority of patients ultimately progress to a lethal form of the disease, termed as castration-resistant prostate cancer (CRPC), which currently lacks curative therapeutic options and is associated with poor prognosis. Therefore, the development of novel treatment modalities for PCa is urgently needed. Chalcones, also known as 1,3-diphenyl-2-propen-1-ones, are among the highly attractive scaffolds being investigated for their antitumor activities. Three series of 18 cyclic (tetralone-based) and two acyclic chalcone analogs, in which ring B was either substituted with nitrogen mustard or replaced by pyrrole or pyridine heterocyclic rings, were designed, synthesized and evaluated as potential therapies for CRPC. Compounds were synthesized by Claisen-Schmidt condensation reaction, purified using columnchromatography or recrystallization and characterized by 1H-NMR, 13C-NMR and LC-MS. The compounds' in-vitro cytotoxicity was evaluated against three prostate cancer cell lines (PC3, DU145, and LNCaP). Among the tested compounds, OH14, OH19 and OH22 showed potent antiproliferative activities at low micromolar levels with IC50 values ranging between 4.4 and 10 µM against PC3 and DU145 cell lines. Detailed biological studies of the lead molecule OH19 revealed that it significantly induces apoptosis through upregulation of Bax and downregulation of BCL-2. In addition, OH19 potently inhibits colony formation and reduces cell migration of androgen-independent PCa cell lines (PC3 and DU145). The molecular pathway analysis show that the anticancer activity of OH19 is associated with attenuation in the phosphorylation of Akt and ERK. Furthermore, OH19 inhibits blood vessel formation in the chick chorioallantoic membrane (CAM) model as compared to control. These results indicate that OH19 could serve as a potential promising lead molecule for the treatment of CRPC and thus, further in-vitro and invivo studies are warranted.
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Imai, Yohsuke, Hitoshi Kondo, Young Ho Kang, Takuji Ishikawa, Chwee Teck Lim i Takami Yamaguchi. "A Numerical Model of Adhesion Property of Malaria Infected Red Blood Cells in Micro Scale Blood Flows". W ASME 2009 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2009. http://dx.doi.org/10.1115/sbc2009-206456.
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Infection by malaria parasite changes mechanical properties of red blood cells (RBCs). Infected red blood cells (IRBCs) lose the deformability but also develop the ability to cytoadhere and rosetting. These outcomes can lead to microvascular blockage [1]. The stiffness of IRBCs [2] and its effects on the flow in micro channels [3] were studied with recent experimental techniques. The cytoadherence and rosetting properties of IRBCs have also been studied experimentally. The cytoadherence is mediated by the interaction of the parasite protein PfEMP1 with several endothelial adhesion molecules, such as CD36, intercellular adhesion molecule-1 (ICAM-1), P-selectin, and vascular cell adhesion molecule-1 (VCAM-1) [4]. In particular, the ligand-receptor interaction between PfEMP1 and CD36 shows tight adhesion [5]. Microvascular blockage may be a hemodynamic problem, involving the interactions between IRBCs, healthy RBCs (HRBCs) and endothelial cells (ECs) in flowing blood, but however experimental techniques have several limitations to this topic. First, it is still difficult to observe the RBC behavior interacting with many other cells even with the recent confocal microscopy. Second, the three-dimensional information on flow field is hardly obtained. Third, capillaries in human body are circular channels with complex geometry, but such complex channels cannot be created in micro scale. Instead, numerical modeling can overcome these problems. We presented a two-dimensional hemodynamic model involving adhesive interactions [6]. In this paper, we propose a three-dimensional model of the adhesive interactions for micro scale hemodynamics in malaria infection.
Raporty organizacyjne na temat "Protein targeted lead molecule"
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Gafni, Yedidya, i Vitaly Citovsky. Molecular interactions of TYLCV capsid protein during assembly of viral particles. United States Department of Agriculture, kwiecień 2007. http://dx.doi.org/10.32747/2007.7587233.bard.
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Tomato yellow leaf curl geminivirus (TYLCV) is a major pathogen of cultivated tomato, causing up to 100% crop loss in many parts of the world. The present proposal, a continuation of a BARD-funded project, expanded our understanding of the molecular mechanisms by which CP molecules, as well as its pre-coat partner V2, interact with each other (CP), with the viral genome, and with cellular proteins during assembly and movement of the infectious virions. Specifically, two major objectives were proposed: I. To study in detail the molecular interactions between CP molecules and between CP and ssDNA leading to assembly of infectious TYLCV virions. II. To study the roles of host cell factors in TYLCV assembly. Our research toward these goals has produced the following major achievements: • Characterization of the CP nuclear shuttling interactor, karyopherin alpha 1, its pattern of expression and the putative involvement of auxin in regulation of its expression. (#1 in our list of publication, Mizrachy, Dabush et al. 2004). • Identify a single amino acid in the capsid protein’s sequence that is critical for normal virus life-cycle. (#2 in our list of publications, Yaakov, Levy et al. in preparation). • Development of monoclonal antibodies with high specificity to the capsid protein of TYLCV. (#3 in our list of publications, Solmensky, Zrachya et al. in press). • Generation of Tomato plants resistant to TYLCV by expressing transgene coding for siRNA targeted at the TYLCV CP. (#4 in our list of publications, Zrachya, Kumar et al. in press). •These research findings provided significant insights into (i) the molecular interactions of TYLCV capsid protein with the host cell nuclear shuttling receptor, and (ii) the mechanism by which TYLCV V2 is involved in the silencing of PTGS and contributes to the virus pathogenicity effect. Furthermore, the obtained knowledge helped us to develop specific strategies to attenuate TYLCV infection, for example, by blocking viral entry into and/or exit out of the host cell nucleus via siRNA as we showed in our publication recently (# 4 in our list of publications). Finally, in addition to the study of TYLCV nuclear import and export, our research contributed to our understanding of general mechanisms for nucleocytoplasmic shuttling of proteins and nucleic acids in plant cells. Also integration for stable transformation of ssDNA mediated by our model pathogen Agrobacterium tumefaciens led to identification of plant specific proteins involved.
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Tzfira, Tzvi, Michael Elbaum i Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, grudzień 2005. http://dx.doi.org/10.32747/2005.7695881.bard.
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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.
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Lapidot, Moshe, i Vitaly Citovsky. molecular mechanism for the Tomato yellow leaf curl virus resistance at the ty-5 locus. United States Department of Agriculture, styczeń 2016. http://dx.doi.org/10.32747/2016.7604274.bard.
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Tomato yellow leaf curl virus (TYLCV) is a major pathogen of tomato that causes extensive crop loss worldwide, including the US and Israel. Genetic resistance in the host plant is considered highly effective in the defense against viral infection in the field. Thus, the best way to reduce yield losses due to TYLCV is by breeding tomatoes resistant or tolerant to the virus. To date, only six major TYLCV-resistance loci, termed Ty-1 to Ty-6, have been characterized and mapped to the tomato genome. Among tomato TYLCV-resistant lines containing these loci, we have identified a major recessive quantitative trait locus (QTL) that was mapped to chromosome 4 and designated ty-5. Recently, we identified the gene responsible for the TYLCV resistance at the ty-5 locus as the tomato homolog of the gene encoding messenger RNA surveillance factor Pelota (Pelo). A single amino acid change in the protein is responsible for the resistant phenotype. Pelo is known to participate in the ribosome-recycling phase of protein biosynthesis. Our hypothesis was that the resistant allele of Pelo is a “loss-of-function” mutant, and inhibits or slows-down ribosome recycling. This will negatively affect viral (as well as host-plant) protein synthesis, which may result in slower infection progression. Hence we have proposed the following research objectives: Aim 1: The effect of Pelota on translation of TYLCV proteins: The goal of this objective is to test the effect Pelota may or may not have upon translation of TYLCV proteins following infection of a resistant host. Aim 2: Identify and characterize Pelota cellular localization and interaction with TYLCV proteins: The goal of this objective is to characterize the cellular localization of both Pelota alleles, the TYLCV-resistant and the susceptible allele, to see whether this localization changes following TYLCV infection, and to find out which TYLCV protein interacts with Pelota. Our results demonstrate that upon TYLCV-infection the resistant allele of pelota has a negative effect on viral replication and RNA transcription. It is also shown that pelota interacts with the viral C1 protein, which is the only viral protein essential for TYLCV replication. Following subcellular localization of C1 and Pelota it was found that both protein localize to the same subcellular compartments. This research is innovative and potentially transformative because the role of Peloin plant virus resistance is novel, and understanding its mechanism will lay the foundation for designing new antiviral protection strategies that target translation of viral proteins. BARD Report - Project 4953 Page 2
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Ohad, Nir, i Robert Fischer. Regulation of Fertilization-Independent Endosperm Development by Polycomb Proteins. United States Department of Agriculture, styczeń 2004. http://dx.doi.org/10.32747/2004.7695869.bard.
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Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to identify members of the complex, analyze their interactions, and understand their function. What is the Temporal and Spatial Pattern of Polycomb Proteins Accumulation? The use of transgenic plants expressing tagged FIE and MEA polypeptides as well as specific antibodies were proposed to localize the endogenous polycomb complex. How is Polycomb Protein Activity Controlled? To understand the molecular mechanism controlling the accumulation of FIE protein, transgenic plants as well as molecular approaches were proposed to determine whether FIE is regulated at the translational or posttranslational levels. The objectives of our research program have been accomplished and the results obtained exceeded our expectation. Our results reveal that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms (Publication 1). Moreover our data show that FIE has additional functions besides controlling the development of the female gametophyte. Using transgenic lines in which FIE was not expressed or the protein level was reduced during different developmental stages enabled us for the first time to explore FIE function during sporophyte development (Publication 2 and 3). Our results are consistent with the hypothesis that FIE, a single copy gene in the Arabidopsis genome, represses multiple developmental pathways (i.e., endosperm, embryogenesis, shot formation and flowering). Furthermore, we identified FIE target genes, including key transcription factors known to promote flowering (AG and LFY) as well as shoot and leaf formation (KNAT1) (Publication 2 and 3), thus demonstrating that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Using the Yeast two hybrid system and pull-down assays we demonstrated that FIE protein interact with MEA via the N-terminal region (Publication 1). Moreover, CURLY LEAF protein, an additional member of the SET domain family interacts with FIE as well. The overlapping expression patterns of FIE, with ether MEA or CLF and their common mutant phenotypes, demonstrate the versatility of FIE function. FIE association with different SET domain polycomb proteins, results in differential regulation of gene expression throughout the plant life cycle (Publication 3). In vitro interaction assays we have recently performed demonstrated that FIE interacts with the cell cycle regulatory component Retinobalsoma protein (pRb) (Publication 4). These results illuminate the potential mechanism by which FIE may restrain embryo sac central cell division, at least partly, through interaction with, and suppression of pRb-regulated genes. The results of this program generated new information about the initiation of reproductive development and expanded our understanding of how PcG proteins regulate developmental programs along the plant life cycle. The tools and information obtained in this program will lead to novel strategies which will allow to mange crop plants and to increase crop production.
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Wilson, Thomas E., Avraham A. Levy i Tzvi Tzfira. Controlling Early Stages of DNA Repair for Gene-targeting Enhancement in Plants. United States Department of Agriculture, marzec 2012. http://dx.doi.org/10.32747/2012.7697124.bard.
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Gene targeting (GT) is a much needed technology as a tool for plant research and for the precise engineering of crop species. Recent advances in this field have shown that the presence of a DNA double-strand break (DSB) in a genomic locus is critical for the integration of an exogenous DNA molecule introduced into this locus. This integration can occur via either non-homologous end joining (NHEJ) into the break or homologous recombination (HR) between the broken genomic DNA and the introduced vector. A bottleneck for DNA integration via HR is the machinery responsible for homology search and strand invasion. Important proteins in this pathway are Rad51, Rad52 and Rad54. We proposed to combine our respective expertise: on the US side, in the design of zincfinger nucleases (ZFNs) for the induction of DNA DSBs at any desired genomic locus and in the integration of DNA molecules via NHEJ; and on the Israeli side in the HR events, downstream of the DSB, that lead to homology search and strand invasion. We sought to test three major pathways of targeted DNA integration: (i) integration by NHEJ into DSBs induced at desired sites by specially designed ZFNs; (ii) integration into DSBs induced at desired sites combined with the use of Rad51, Rad52 and Rad54 proteins to maximize the chances for efficient and precise HR-mediated vector insertion; (iii) stimulation of HR by Rad51, Rad52 and Rad54 in the absence of DSB induction. We also proposed to study the formation of dsT-DNA molecules during the transformation of plant cells. dsT-DNA molecules are an important substrate for HR and NHEJ-mediatedGT, yet the mode of their formation from single stranded T-DNA molecules is still obscure. In addition we sought to develop a system for assembly of multi-transgene binary vectors by using ZFNs. The latter may facilitate the production of binary vectors that may be ready for genome editing in transgenic plants. ZFNs were proposed for the induction of DSBs in genomic targets, namely, the FtsH2 gene whose loss of function can easily be identified in somatic tissues as white sectors, and the Cruciferin locus whose targeting by a GFP or RFP reporter vectors can give rise to fluorescent seeds. ZFNs were also proposed for the induction of DSBs in artificial targets and for assembly of multi-gene vectors. We finally sought to address two important cell types in terms of relevance to plant transformation, namely GT of germinal (egg) cells by floral dipping, and GT in somatic cells by root and leave transformation. To be successful, we made use of novel optimized expression cassettes that enable coexpression of all of the genes of interest (ZFNs and Rad genes) in the right tissues (egg or root cells) at the right time, namely when the GT vector is delivered into the cells. Methods were proposed for investigating the complementation of T-strands to dsDNA molecules in living plant cells. During the course of this research, we (i) designed, assembled and tested, in vitro, a pair of new ZFNs capable of targeting the Cruciferin gene, (ii) produced transgenic plants which expresses for ZFN monomers for targeting of the FtsH2 gene. Expression of these enzymes is controlled by constitutive or heat shock induced promoters, (iii) produced a large population of transgenic Arabidopsis lines in which mutated mGUS gene was incorporated into different genomic locations, (iv) designed a system for egg-cell-specific expression of ZFNs and RAD genes and initiate GT experiments, (v) demonstrated that we can achieve NHEJ-mediated gene replacement in plant cells (vi) developed a system for ZFN and homing endonuclease-mediated assembly of multigene plant transformation vectors and (vii) explored the mechanism of dsTDNA formation in plant cells. This work has substantially advanced our understanding of the mechanisms of DNA integration into plants and furthered the development of important new tools for GT in plants.
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Citovsky, Vitaly, i Yedidya Gafni. Suppression of RNA Silencing by TYLCV During Viral Infection. United States Department of Agriculture, grudzień 2009. http://dx.doi.org/10.32747/2009.7592126.bard.
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The Israeli isolate of Tomato yellow leaf curl geminivirus (TYLCV-Is) is a major tomato pathogen, causing extensive (up to 100%) crop losses in Israel and in the south-eastern U.S. (e.g., Georgia, Florida). Surprisingly, however, little is known about the molecular mechanisms of TYLCV-Is interactions with tomato cells. In the current BARD project, we have identified a TYLCV-Is protein, V2, which acts as a suppressor of RNA silencing, and showed that V2 interacts with the tomato (L. esculentum) member of the SGS3 (LeSGS3) protein family known to be involved in RNA silencing. This proposal will use our data as a foundation to study one of the most intriguing, yet poorly understood, aspects of TYLCV-Is interactions with its host plants – possible involvement of the host innate immune system, i.e., RNA silencing, in plant defense against TYLCV-Is and the molecular pathway(s) by which TYLCV-Is may counter this defense. Our project sought two objectives: I. Study of the roles of RNA silencing and its suppression by V2 in TYLCV-Is infection of tomato plants. II. Study of the mechanism by which V2 suppresses RNA silencing. Our research towards these goals has produced the following main achievements: • Identification and characterization of TYLCV V2 protein as a suppressor of RNA silencing. (#1 in the list of publications). • Characterization of the V2 protein as a cytoplasmic protein interacting with the plant protein SlSGS3 and localized mainly in specific, not yet identified, bodies. (#2 in the list of publications). • Development of new tools to study subcellular localization of interacting proteins (#3 in the list of publications). • Characterization of TYLCV V2 as a F-BOX protein and its possible role in target protein(s) degradation. • Characterization of TYLCV V2 interaction with a tomato cystein protease that acts as an anti-viral agent. These research findings provided significant insights into (I) the suppression of RNA silencing executed by the TYLCV V2 protein and (II) characterization some parts of the mechanism(s) involved in this suppression. The obtained knowledge will help to develop specific strategies to attenuate TYLCV infection, for example, by blocking the activity of the viral suppressor of gene silencing thus enabling the host cell silencing machinery combat the virus.
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Meir, Shimon, Michael S. Reid, Cai-Zhong Jiang, Amnon Lers i Sonia Philosoph-Hadas. Molecular Studies of Postharvest Leaf and Flower Senescence. United States Department of Agriculture, styczeń 2011. http://dx.doi.org/10.32747/2011.7592657.bard.
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Original objectives: To understand the regulation of abscission by exploring the nature of changes of auxin-related gene expression in tomato (Lycopersicon esculatumMill) abscission zones (AZs) following organ removal, and by analyzing the function of these genes. Our specific goals were: 1) To complete the microarray analyses in tomato flower and leaf AZs, for identifying genes whose expression changes early in response to auxin depletion; 2) To examine, using virus-induced gene silencing (VIGS), the effect of silencing target genes on ethylene sensitivity and abscission competence of the leaf and flower AZs; 3) To isolate and characterize promoters from AZ-specific genes to be used in functional analysis; 4) To generate stable transgenic tomato plants with selected genes silenced with RNAi, under the control of an AZ-specific promoter, for further characterization of their abscission phenotypes. Background: Abscission, the separation of organs from the parent plant, results in postharvest quality loss in many ornamentals and other fresh produce. The process is initiated by changes in the auxin gradient across the AZ, and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the initiation of the abscission process, as the AZ becomes sensitized to ethylene. The present project was focused on elucidating these early molecular regulatory events, in order to gain a better control of the abscission process for agricultural manipulations. Major conclusions, solutions, achievements: Microarray analyses, using the Affymetrix Tomato GeneChip®, revealed changes in expression, occurring early in abscission, of many genes with possible regulatory functions. These included a range of auxin- and ethylene-related transcription factors (TFs), other TFs that are transiently induced just after flower removal, and a set of novel AZ-specific genes. We also identified four different defense-related genes, including: Cysteine-type endopeptidase, α- DOX1, WIN2, and SDF2, that are newly-associated with the late stage of the abscission process. This supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses. To facilitate functional studies we implemented an efficient VIGS system in tomato, and isolated two abscission-specific promoters (pTAPG1 and pTAPG4) for gene silencing in stable transformation. Using the VIGS system we could demonstrate the importance of TAPGs in abscission of tomato leaf petioles, and evaluated the importance of more than 45 genes in abscission. Among them we identified few critical genes involved in leaf and flower abscission. These included: PTRP-F1, PRP, TKN4, KNOTTED-like homeobox TF, KD1, and KNOX-like homeodomain protein genes, the silencing of which caused a striking retardation of pedicel abscission, and ERF1, ERF4, Clavata-like3 protein, Sucrose transporter protein, and IAA10 genes, the silencing of which delayed petiole abscission. The importance of PRPand KD1 genes in abscission was confirmed also by antisense–silencing using pTAPG4. Experiments testing the effects of RNAi silencing of few other genes are still in progress, The analysis of the microarray results of flower and leaf AZs allowed us to establish a clear sequence of events occurring during acquisition of tissue sensitivity to ethylene, and to confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes in both AZs. Implication, both scientific and agricultural: Our studies had provided new insights into the regulation of the abscission process, and shaded light on the molecular mechanisms that drive the acquisition of abscission competence in the AZ. We pointed out some critical genes involved in regulation of abscission, and further expanded our knowledge of auxin-ethylene cross talk during the abscission process. This permits the development of novel techniques for manipulating abscission, and thereby improving the postharvest performance of ornamentals and other crops.
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Gafni, Yedidya, i Vitaly Citovsky. Inactivation of SGS3 as Molecular Basis for RNA Silencing Suppression by TYLCV V2. United States Department of Agriculture, listopad 2013. http://dx.doi.org/10.32747/2013.7593402.bard.
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The Israeli isolate of Tomato yellow leaf curl geminivirus(TYLCV-Is) is a major tomato pathogen, causing extensive crop losses in Israel and in the south-eastern U.S. Yet, little is known about the molecular mechanisms of its interaction with tomato cells. One of the most interesting aspects of such interaction is how the invading virus counteracts the RNA silencing response of the plant. In the former BARD project, we have shown that TYLCV-Is V2 protein is an RNA silencing suppressor, and that this suppression is carried out via the interaction of V2 with the SGS3 component of the plant RNA silencing machinery. This reported project was meant to use our data as a foundation to elucidate the molecular mechanism by which V2 affects the SGS3 activity. While this research is likely to have an important impact on our understanding of basic biology of virus-plant interactions and suppression of plant immunity, it also will have practical implications, helping to conceive novel strategies for crop resistance to TYLCV-Is. Our preliminary data in regard to V2 activities and our present knowledge of the SGS3 function suggest likely mechanisms for the inhibitory effect of V2 on SGS3. We have shown that V2 possess structural and functional hallmarks of an F-box protein, suggesting that it may target SGS3 for proteasomal degradation. SGS3 contains an RNA-binding domain and likely functions to protect the cleavage produces of the primary transcript for subsequent conversion to double-stranded forms; thus, V2 may simply block the RNA binding activity of SGS3. V2 may also employ a combination of these mechanisms. These and other possibilities were tested in this reported project.
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Tucker, Mark L., Shimon Meir, Amnon Lers, Sonia Philosoph-Hadas i Cai-Zhong Jiang. Elucidation of signaling pathways that regulate ethylene-induced leaf and flower abscission of agriculturally important plants. United States Department of Agriculture, styczeń 2012. http://dx.doi.org/10.32747/2012.7597929.bard.
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The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.
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Gafni, Yedidya, Moshe Lapidot i Vitaly Citovsky. Dual role of the TYLCV protein V2 in suppressing the host plant defense. United States Department of Agriculture, styczeń 2013. http://dx.doi.org/10.32747/2013.7597935.bard.
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TYLCV-Is is a major tomato pathogen, causing extensive crop losses in Israel and the U.S. We have identified a TYLCV-Is protein, V2, which acts as a suppressor of RNA silencing. Intriguingly, the counter-defense function of V2 may not be limited to silencing suppression. Our recent data suggest that V2 interacts with the tomato CYP1 protease. CYP1 belongs to the family of papain-like cysteine proteases which participate in programmed cell death (PCD) involved in plant defense against pathogens. Based on these data we proposed a model for dual action of V2 in suppressing the host antiviral defense: V2 targets SGS3 for degradation and V2 inhibits CYP1 activity. To study this we proposed to tackle three specific objectives. I. Characterize the role of V2 in SGS3 proteasomal degradation ubiquitination, II. Study the effects of V2 on CYP1 maturation, enzymatic activity, and accumulation and, III. Analyze the effects of the CYP1-V2 interaction on TYLCV-Is infection. Here we describe results from our study that support our hypothesis: the involvement of the host's innate immune system—in this case, PCD—in plant defense against TYLCV-Is. Also, we use TYLCV-Is to discover the molecular pathway(s) by which this plant virus counters this defense. Towards the end of our study we discovered an interesting involvement of the C2 protein encoded by TYLCV-Is in inducing Hypersensitive Response in N. benthamianaplants which is not the case when the whole viral genome is introduced. This might lead to a better understanding of the multiple processes involved in the way TYLCV is overcoming the defense mechanisms of the host plant cell. In a parallel research supporting the main goal described, we also investigated Agrobacteriumtumefaciens-encoded F-box protein VirF. It has been proposed that VirF targets a host protein for the UPS-mediated degradation, very much the way TYLCV V2 does. In our study, we identified one such interactor, an Arabidopsistrihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. Another target for VirF is VFP4, a transcription factor that both VirF and its plant functional homolog VBF target to degradation by UPS. Using RNA-seqtranscriptome analysis we showed that VFP4 regulates numerous plant genes involved in disease response, including responses to viral and bacterial infections. Detailed analyses of some of these genes indicated their involvement in plant protection against Agrobacterium infection. Thus, Agrobacterium may facilitate its infection by utilizing the host cell UPS to destabilize transcriptional regulators of the host disease response machinery that limits the infection.