Mitochondrial topoisomerase 1 targeted anticancer therapy using irinotecan encapsulated mesoporous MIL-101(Fe) synthesized via a vapour assisted method.

Mitochondrial topisomerase 1 (Top1mt) is critical for mtDNA replication, transcription, and energy production. Here, we investigate the carrier-mediated targeted delivery of the anticancer drug irinotecan into the mitochondria to selectively trap Top1mt covalent complexes (Top1mtcc) and its role in anticancer therapeutics. We have designed a biocompatible mesoporous metal-organic framework (MOF) material, namely MIL-101(Fe), as the drug delivery carrier that selectively localizes inside mitochondria. In contrast to the traditional way of synthesising MOFs, here we have employed a vapour-assisted solvothermal method for the synthesis of MIL-101(Fe) using terephthalic acid as the organic linker and Fe(III) as the metal source. The advantage of this method is that it recycles the excess solvent (DMF) and reduces the amount of washing solvent. We demonstrate that MIL-101(Fe)-encapsulated irinotecan (MIL-Iri) was selectively targeted towards the mitochondria to poison Top1mtcc in a dose-dependent manner and was achieved at a low nanomolar drug concentration. We provide evidence that Top1mtcc generated by MIL-Iri leads to mtDNA damage in human colon and breast cancer cells and plays a significant role in cellular toxicity. Altogether, this study provides evidence for a new and effective strategy in anticancer chemotherapy.

Fluorescence-resonance-energy-transfer-based assay to estimate modulation of TDP1 activity through arginine methylation.

Tyrosyl DNA phosphodiesterase (TDP1) is a DNA repair enzyme that hydrolyzes the phosphotyrosyl linkage between 3'-DNA-protein crosslinks such as stalled topoisomerase 1 cleavage complexes (Top1cc). Here, we present a fluorescence-resonance-energy-transfer-(FRET) based assay to estimate modulation of TDP1 activity through arginine methylation. We describe steps for TDP1 expression and purification and estimating TDP1 activity using fluorescence-quenched probes mimicking Top1cc. We then detail data analysis of real-time TDP1 activity and screening of TDP1-selective inhibitors. For complete details on the use and execution of this protocol, please refer to Bhattacharjee et al. (2022).1.

Novel Pyrido[2',1':2,3]imidazo[4,5-c]quinoline Derivative Selectively Poisons Leishmania donovani Bisubunit Topoisomerase 1 to Inhibit the Antimony-Resistant Leishmania Infection in Vivo.

The unique bisubunit structure of Leishmania donovani topoisomerase 1B (LdTop1) is a potential drug target in the parasites unlike the monomeric Top1 from its human host counterpart. Here, we report the design, synthesis, and validation of a chimeric pyrido[2',1':2,3]imidazo[4,5-c]quinoline derivative (C17) as a novel antileishmanial agent that poisons topoisomerase 1-DNA covalent complexes (LdTop1cc) inside the parasites and inhibits Top1 religation activity both in the drug sensitive and antimony-resistant L. donovani clinical isolates. Importantly, the human Top1 is not sensitive to C17. Further, C17 overcomes the chemical instability of camptothecin (CPT) by generating persistent LdTop1cc-induced DNA breaks inside the parasites even after 12 h of drug removal. Intraperitoneal administration of C17 results in marked reduction of the Leishmania amastigotes from the infected spleen and liver of BALB/c mice. C17 confers a host protective immune-response up-regulating the Th1 cytokines facilitating parasite clearance which can be exploited for treating drug-resistant leishmaniasis.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

TDP1 knockout Leishmania donovani accumulate topoisomerase 1-linked DNA damage and are hypersensitive to clinically used antileishmanial drugs.

Leishmania donovani, a unicellular protozoan parasite, causes a wide range of human diseases including fatal visceral leishmaniasis. Tyrosyl DNA-phosphodiesterase 1 (TDP1) hydrolyzes the phosphodiester bond between DNA 3'-end and a tyrosyl moiety of trapped topoisomerase I-DNA covalent complexes (Top1cc). We have previously shown Leishmania harbors a TDP1 gene (LdTDP1), however, the biological role of TDP1 remains largely unknown. In the present study, we have generated TDP1 knockout L. donovani (LdTDP1-/- ) promastigotes and have shown that LdTDP1-/- parasites are deficient in 3'-phosphodiesterase activities and were hypersensitive to Top1-poison like camptothecin (CPT), DNA alkylation agent like methyl methanesulfonate, and oxidative DNA lesions generated by hydrogen peroxide but were not sensitive to etoposide. We also detected elevated levels of CPT-induced reactive oxygen species triggering cell cycle arrest and cell death in LdTDP1-/- promastigotes. LdTDP1-/- promastigotes accumulate a significant change in the membrane morphology with the accumulation of membrane pores, which is associated with oxidative stress and lipid peroxidation. To our surprise, we detected that LdTDP1-/- parasites were hypersensitive to antileishmanial drugs like amphotericin B and miltefosine, which could be rescued by complementation of wild-type TDP1 gene in the LdTDP1-/- parasites. Notably, multidrug-resistant L. donovani clinical isolates showed a marked reduction in TDP1 expression and were sensitive to Top1 poisons. Taken together, our study provides a new role of LdTDP1 in protecting L. donovani parasites from oxidative stress-induced DNA damage and resistance to amphotericin B and miltefosine.

Post-translational regulation of Tyrosyl-DNA phosphodiesterase (TDP1 and TDP2) for the repair of the trapped topoisomerase-DNA covalent complex.

DNA topoisomerases are essential enzymes that regulate DNA topology, the transmission of genetic materials, and gene expressions both in the nucleus and mitochondria. Trapped topoisomerases (Top1 and Top2) in covalent complexes with DNA (Topoisomerase cleavage complexes; Topcc) are detrimental DNA lesions that perturb active genome integrity and trigger cell death. Comprehensive research on the recently discovered enzymes TDP1 and TDP2 exemplify their spectacular role in repairing trapped Topcc as well as in a myriad of diverse DNA lesions. Posttranslational modifications (PTMs), play critical roles in regulating the optimal function of the DNA Damage Response (DDR) proteins. This review summarizes the mechanistic aspects of DNA damage induced by trapped Topcc during transcription and their role in human diseases. We have also highlighted the pivotal role of PTMs in fine-tuning the intricate and multilayered regulatory processes of TDP1 and TDP2 molecular networks for the repair of trapped Topcc.

Trapped topoisomerase-DNA covalent complexes in the mitochondria and their role in human diseases.

Topoisomerases regulate DNA topology, organization of the intracellular DNA, the transmission of genetic materials, and gene expressions. Other than the nuclear genome, mitochondria also harbor the small, circular DNA (mtDNA) that encodes a critical subset of proteins for the production of cellular ATP; however, mitochondria are solely dependent on the nucleus for all the mitochondrial proteins necessary for mtDNA replication, repair, and maintenance. Mitochondrial genome compiles topological stress from bidirectional transcription and replication, therefore imports four nuclear encoded topoisomerases (Top1mt, Top2α, Top2ß, and Top3α) in the mitochondria to relax mtDNA supercoiling generated during these processes. Trapping of topoisomerase on DNA results in the formation of protein-linked DNA adducts (PDAs), which are widely exploited by topoisomerase-targeting anticancer drugs. Intriguingly mtDNA is potentially exposed to DNA damage that has been attributed to a variety of human diseases, including neurodegeneration, cancer, and premature aging. In this review, we focus on the role of different topoisomerases in the mitochondria and our current understanding of the mitochondrial DNA damage through trapped protein-DNA complexes, and the progress in the molecular mechanisms of the repair for trapped topoisomerase covalent complexes (Topcc). Finally, we have discussed how the pathological DNA lesions that cause mtDNA damage,trigger mitochondrial fission and mitophagy, which serve as quality control events for clearing damaged mtDNA.

Top1-PARP1 association and beyond: from DNA topology to break repair.

Selective trapping of human topoisomerase 1 (Top1) on the DNA (Top1 cleavage complexes; Top1cc) by specific Top1-poisons triggers DNA breaks and cell death. Poly(ADP-ribose) polymerase 1 (PARP1) is an early nick sensor for trapped Top1cc. New mechanistic insights have been developed in recent years to rationalize the importance of PARP1 beyond the repair of Top1-induced DNA breaks. This review summarizes the progress in the molecular mechanisms of trapped Top1cc-induced DNA damage, PARP1 activation at DNA damage sites, PAR-dependent regulation of Top1 nuclear dynamics, and PARP1-associated molecular network for Top1cc repair. Finally, we have discussed the rationale behind the synergy between the combination of Top1 poison and PARP inhibitors in cancer chemotherapies, which is independent of the 'PARP trapping' phenomenon.

Lanthanide clusters of phenanthroline containing a pyridine-pyrazole based ligand: magnetism and cell imaging.

In this contribution, we report the synthesis, characterization and luminescence-magnetic properties of Ln-clusters (Ln = Gd3+, Eu3+ and Tb3+) using a new pyridine-pyrazole functionalized ligand fitted with a chromophoric phenanthroline backbone. The unorthodox N-rich ligand forms isostructural trinuclear lanthanide complexes with a topology that closely resembles two interdigitating hairpins. The clusters crystallize in chiral space groups and also exhibit chirality for bulk samples, which were further confirmed using solid state CD spectra. Magnetic studies on the complexes reveal their interesting features while the Gd cluster shows a significant cryogenic magnetic cooling behaviour with a moderately high magnetic entropy change of -23.42 J kg-1 K-1 at 7 T and 2 K. On the other hand, Eu and Tb complexes exhibit interesting fluorescence properties. The compounds were subsequently used as fluorescent probes for the imaging of human breast adenocarcinoma (MCF7) cells. Live cell confocal microscopy images show that the complexes penetrate beyond the usual cytoplasm region and can be useful in imaging the nucleus region of MCF7 cells.

Pyridine-pyrazole based Al(iii) 'turn on' sensor for MCF7 cancer cell imaging and detection of picric acid.

We report herein the development of a new pyridine-pyrazole based bis-bidentate asymmetric chemosensor that shows excellent turn-on chelation-enhanced Al3+-responsive fluorescence. The presence of two 'hard' phenolic hydroxyl groups plays a pivotal role in switching-on the sensing through coordination to the 'hard' Al3+ ion, while the mechanism can be interpreted by the chelation-enhanced fluorescence (CHEF) process. The X-ray single structure show a planar conjugated structure of the ligand, which was further stabilized by extensive H-bonding and π-π stacking. The photophysical studies related to the sensing behavior of the titular ligand toward aluminum was investigated in detail using various spectroscopic techniques like UV-Vis, photoluminescence, fluorescence and time-correlated single-photon count (TCSPC) and time-resolved NMR. The spectroscopic methods also confirm the selective detection of Al3+ ion in the presence of other metal ions. The theoretical calculations using Density Functional Theory (DFT) and the Time Dependent Density Functional Theory (TD-DFT) provide further insight on the mechanistic aspects of the turn-on sensing behavior including the electronic spectra of both the ligand and the complex. Interestingly, the as-synthesized H2DPC-Al complex can also be utilized as a fluorescence-based sensor for various nitroaromatics including picric acid, for which an INHIBIT logic gate can also be constructed. The as synthesized complex was subsequently used as a fluorescent probe for imaging of human breast adenocarcinoma (MCF7) cells using live cell confocal microscopic techniques.

Development of a metabolically stable topoisomerase I poison as anticancer agent.

We have recently reported a new chemotype of a potent topoisomerase I poison with compound 1 as a potential anticancer chemotherapeutic agent. During further optimization, it has been observed that compound 1 suffers from high intrinsic clearance in human liver microsomes. To overcome the metabolic instability of compound 1, we report design and synthesis of metabolically stable Top1 poison 3. Newly identified Top1 poison 3 exhibits t1/2 of 69.1 min in human liver microsomes in comparison to compound 1 with t1/2 of 9.9 min. Molecular dynamic study of the newly optimized Top1 poison 3 was performed to get the insight into the stability of the binding pose in the active site. Compound 3 was able to trap DNA-Top1 cleavage complex and found to be less cytotoxic in non-cancerous cell line as compared to compound 1.

Thiol-Disulfide Exchange Reaction Promoted Highly Efficient Cellular Uptake of Pyridyl Disulfide Appended Nonionic Polymers.

The intracellular transport of molecules, macromolecules or materials is a key step in probing cellular structure and function, as well as regulating a plethora of physical and chemical events for treating disease. This communication reveals direct cellular uptake of pyridyl-disulfide (Py-Ds)-conjugated nonionic and biocompatible macromolecules with the aid of rapid exchange of the highly reactive Py-Ds groups with exofacial cell-surface thiols. Confocal microscopy and flow cytometry analysis confirmed highly efficient cellular uptake of Py-Ds-appended polymers (>50 % in 15 min) by avoiding lysosome as a consequence of thiol-disulfide exchange in the cell surface. In contrast, a control polymer lacking the Py-Ds group followed caveolae-mediated endocytosis. Other control polymers containing either the pyridine group (but not disulfide) or the disulfide group (but not pyridine) revealed significantly low cellular uptake, and thus essential role of the highly reactive Py-Ds group was established beyond doubt.

Proteasomal inhibition triggers viral oncoprotein degradation via autophagy-lysosomal pathway.

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins-EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.

SCAN1-TDP1 trapping on mitochondrial DNA promotes mitochondrial dysfunction and mitophagy.

A homozygous mutation of human tyrosyl-DNA phosphodiesterase 1 (TDP1) causes the neurodegenerative syndrome, spinocerebellar ataxia with axonal neuropathy (SCAN1). TDP1 hydrolyzes the phosphodiester bond between DNA 3'-end and a tyrosyl moiety within trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). TDP1 is critical for mitochondrial DNA (mtDNA) repair; however, the role of mitochondria remains largely unknown for the etiology of SCAN1. We demonstrate that mitochondria in cells expressing SCAN1-TDP1 (TDP1H493R) are selectively trapped on mtDNA in the regulatory non-coding region and promoter sequences. Trapped TDP1H493R-mtDNA complexes were markedly increased in the presence of the Top1 poison (mito-SN38) when targeted selectively into mitochondria in nanoparticles. TDP1H493R-trapping accumulates mtDNA damage and triggers Drp1-mediated mitochondrial fission, which blocks mitobiogenesis. TDP1H493R prompts PTEN-induced kinase 1-dependent mitophagy to eliminate dysfunctional mitochondria. SCAN1-TDP1 in mitochondria creates a pathological state that allows neurons to turn on mitophagy to rescue fit mitochondria as a mechanism of survival.

Nonmuscle myosin IIA and IIB differentially modulate migration and alter gene expression in primary mouse tumorigenic cells.

Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc and Serpinb2, which is strikingly similar to Nesprin2-KD cells as assessed by quantitative PCR analysis. Further gene network analysis predicts that NMIIA and NMIIB may act on similar pathways but through different regulators. Concomitantly, knockdown of NMIIA or NMIIB lowers the growth rate and tumor volume of 3MC-induced tumor in vivo. Altogether, these results open a new window to further investigate the effect of LINC-associated perinuclear actomyosin complex on mechanoresponsive gene expression in the growing tumor.

Discovery and Mechanistic Study of Tailor-Made Quinoline Derivatives as Topoisomerase 1 Poison with Potent Anticancer Activity.

To overcome chemical limitations of camptothecin (CPT), we report design, synthesis, and validation of a quinoline-based novel class of topoisomerase 1 (Top1) inhibitors and establish that compound 28 ( N-(3-(1 H-imidazol-1-yl)propyl)-6-(4-methoxyphenyl)-3-(1,3,4-oxadiazol-2-yl)quinolin-4-amine) exhibits the highest potency in inhibiting human Top1 activity with an IC50 value of 29 ± 0.04 nM. Compound 28 traps Top1-DNA cleavage complexes (Top1ccs) both in the in vitro cleavage assays and in live cells. Point mutation of Top1-N722S fails to trap compound 28-induced Top1cc because of its inability to form a hydrogen bond with compound 28. Unlike CPT, compound 28 shows excellent plasma serum stability and is not a substrate of P-glycoprotein 1 (permeability glycoprotein) advancing its potential anticancer activity. Finally, we provide evidence that compound 28 overcomes the chemical instability of CPT in human breast adenocarcinoma cells through generation of persistent and less reversible Top1cc-induced DNA double-strand breaks as detected by γH2AX foci immunostaining after 5 h of drug removal.

PRMT5-mediated arginine methylation of TDP1 for the repair of topoisomerase I covalent complexes.

Human tyrosyl-DNA phosphodiesterases (TDP) hydrolyze the phosphodiester bond between DNA and the catalytic tyrosine of Top1 to excise topoisomerase I cleavage complexes (Top1cc) that are trapped by camptothecin (CPT) and by genotoxic DNA alterations. Here we show that the protein arginine methyltransferase PRMT5 enhances the repair of Top1cc by direct binding to TDP1 and arginine dimethylation of TDP1 at residues R361 and R586. Top1-induced replication-mediated DNA damage induces TDP1 arginine methylation, enhancing its 3'- phosphodiesterase activity. TDP1 arginine methylation also increases XRCC1 association with TDP1 in response to CPT, and the recruitment of XRCC1 to Top1cc DNA damage foci. PRMT5 knockdown cells exhibit defective TDP1 activity with marked elevation in replication-coupled CPT-induced DNA damage and lethality. Finally, methylation of R361 and R586 stimulate TDP1 repair function and promote cell survival in response to CPT. Together, our findings provide evidence for the importance of PRMT5 for the post-translational regulation of TDP1 and repair of Top1cc.

A novel metallogel based approach to synthesize (Mn, Cu) doped ZnS quantum dots and labeling of MCF-7 cancer cells.

The present study aims to formulate a common synthetic strategy for preparing quantum dots (QDs) in a greener way by using combination of popular methods, viz. a colloidal method with suitable capping agent and low molecular weight gel based synthesis. Pyridine dicarboxylic acid (PDC) in presence of AlCl3 forms a stable metallogel, which serves as an excellent medium for selective ZnS QD synthesis. The aromatic pyridine moiety, well known for being a capping agent, indeed plays its part in the run up to QD synthesis. To the best of our knowledge, this is the first example of a metallogel based doped ZnS QD synthesis. Altering the doping material and its composition changes the properties of the QDs, but herein we also tried to establish how these changes affect the gel morphology and stability of both gel and QDs. We further demonstrate, by using live cell confocal microscopy, the delivery of QDs Cu ZnS and MnZnS nanomaterials in the nucleus and the cytoplasm of human breast cancer cells (MCF7), implicating the use of metallogel based QDs for bio-imaging and bio-labeling.

Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase I (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes.

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process.

Induced Aggregation of AIE-Active Mono-Cyclometalated Ir(III) Complex into Supramolecular Branched Wires for Light-Emitting Diodes.

Aggregation-induced emission (AIE) is commonly observed in irregular bulk form. Herein, unique aggregation properties of an AIE-active complex into branched supramolecular wires are reported for the first time. Mono-cyclometalated Ir(III) complex shows in-plane J-aggregation at the air-water interface owing to the restriction of intramolecular vibration of bidentate phenylpyridinato and intramolecular rotations of monodentate triphenylphosphine ligands at air-water interface. As a consequence, a large enhancement of luminescence comparable to the solid state is obtained from the monolayers of supramolecular wires. This unique feature is utilized for the fabrication of light-emitting diodes with low threshold voltage using supramolecular wires as active layer. This study opens up the need of ordered assembly of AIE complexes to achieve optimal luminescence characteristics.