Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-β superfamily. On the other hand, the specific roles of TGF-β and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-β (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-β or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-β inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-β and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-β and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-β subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-β (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.
- MeSH
- buněčná diferenciace genetika MeSH
- dánio pruhované * genetika MeSH
- folikuly stimulující hormon farmakologie metabolismus MeSH
- messenger RNA genetika MeSH
- pyrazoly * MeSH
- spermatogeneze genetika MeSH
- spermatogonie * metabolismus MeSH
- testis metabolismus MeSH
- thiosemikarbazony * MeSH
- transformující růstový faktor beta metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Aim: Discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors using a structure-based drug discovery strategy. Materials & methods: Virtual screening employing covalent and noncovalent docking was performed to discover Mpro inhibitors, which were subsequently evaluated in biochemical and cellular assays. Results: 91 virtual hits were selected for biochemical assays, and four were confirmed as reversible inhibitors of SARS CoV-2 Mpro with IC50 values of 0.4-3 μM. They were also shown to inhibit SARS-CoV-1 Mpro and human cathepsin L. Molecular dynamics simulations indicated the stability of the Mpro inhibitor complexes and the interaction of ligands at the subsites. Conclusion: This approach led to the discovery of novel thiosemicarbazones as potent SARS-CoV-2 Mpro inhibitors.
- MeSH
- antivirové látky farmakologie chemie MeSH
- COVID-19 * MeSH
- inhibitory proteas farmakologie chemie MeSH
- lidé MeSH
- SARS-CoV-2 MeSH
- simulace molekulového dockingu MeSH
- thiosemikarbazony * farmakologie MeSH
- virové nestrukturální proteiny MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A novel fluorescent ligand (H2LCl⋅1.5CH3OH, 1) was synthesized and metal complexes of 1 with Mn(II), Fe(III), Ni(II), Cu(II), and Zn(II) were obtained as Mn(HL)2Cl2 (2), Fe(HL)2Cl3⋅3H2O (3), Ni(L)(HL)Cl⋅8H2O (4), Cu(HL)Cl2⋅4H2O (5), Zn(H2L)Cl3 (6), respectively. These compounds were identified by spectroscopic methods, elemental analysis, molar conductivity, and single-crystal X-ray crystallography. According to the crystal structure of 4 nickel (II), center is surrounded by two ligands in a distorted octahedral geometry. The ligand and its complexes are soluble in water and have excellent stability. In vitro anti-proliferative activity of these compounds was evaluated against human breast adenocarcinoma (MCF-7) and human lipo-sarcoma (SW-872) as cancer cells and human fibroblasts (HFF-2) as normal cells by MTT assay. Interestingly, complex 5 exhibited excellent activity against both cancer cells with low IC50 value 22.18 ± 0.35 μg/mL (35.66 ± 0.56 μM) for SW-872 and 79.41 ± 3.54 μg/mL (127.6 ± 5.69 μM) for MCF-7 among the compounds and in comparison with paclitaxel (PTX) which acts finely. Morphological changes were evaluated by flow cytometry that revealed apoptosis is the main cause of cell death. Likewise, cell cycle studies indicated the cell cycle arrest in the G1 and S phases for complex 5 against MCF-7 and SW-872 cancer cells, while complex 6 could arrest the MCF-7 and SW-872 cells in G2 and G1 phases, respectively. All of the compounds are fluorescent which enabled us to monitor the uptake and intracellular distribution in living human cancer cells by fluorescence microscopy.
- MeSH
- antitumorózní látky * farmakologie chemie MeSH
- komplexní sloučeniny * farmakologie chemie MeSH
- lidé MeSH
- ligandy MeSH
- měď chemie MeSH
- Schiffovy báze farmakologie chemie MeSH
- thiosemikarbazony * chemie MeSH
- železité sloučeniny MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Dysregulation of iron homeostasis is one of the important processes in the development of many oncological diseases, such as pancreatic cancer. Targeting it with specific agents, such as an iron chelator, are promising therapeutic methods. In this study, we tested the cytotoxicity of novel azulene hydrazide-hydrazone-based chelators against pancreatic cancer cell lines (MIA PaCa-2, PANC-1, AsPC-1). All prepared chelators (compounds 4-6) showed strong cytotoxicity against pancreatic cancer cell lines and high selectivity for cancer cell lines compared to the healthy line. Their cytotoxicity is lower than thiosemicarbazone-based chelators Dp44mT and DpC, but significantly higher than hydroxamic acid-based chelator DFO. The chelator tested showed mitochondrial and lysosomal co-localization and its mechanism of action was based on the induction of hypoxia-inducible factor-1-alpha (HIF-1α), N-myc downstream-regulated gene-1 (NDRG1) and transferrin receptor 1 (TfR1). This strongly implies that the cytotoxic effect of tested chelators could be associated with mitophagy induction. Lipinski's rule of five analyses was performed to determine whether the prepared compounds had properties ensuring their bioavailability. In addition, the drug-likeness and drug-score were calculated and discussed.
- MeSH
- azuleny MeSH
- chelátory železa farmakologie MeSH
- hydraziny MeSH
- hydrazony farmakologie MeSH
- kyseliny hydroxamové MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory slinivky břišní * farmakoterapie MeSH
- receptory transferinu MeSH
- thiosemikarbazony * farmakologie MeSH
- železo MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Aminopeptidase N (APN), also known as CD13 antigen or membrane alanyl aminopeptidase, belongs to the M1 family of the MA clan of zinc metallopeptidases. In cancer cells, the inhibition of aminopeptidases including APN causes the phenomenon termed the amino acid deprivation response (AADR), a stress response characterized by the upregulation of amino acid transporters and synthetic enzymes and activation of stress-related pathways such as nuclear factor kB (NFkB) and other pro-apoptotic regulators, which leads to cancer cell death by apoptosis. Recently, APN inhibition has been shown to augment DR4-induced tumor cell death and thus overcome resistance to cancer treatment with DR4-ligand TRAIL, which is available as a recombinant soluble form dulanermin. This implies that APN inhibitors could serve as potential weapons for overcoming cancer treatment resistance. In this study, a series of basically substituted acetamidophenones and the semicarbazones and thiosemicarbazones derived from them were prepared, for which APN inhibitory activity was determined. In addition, a selective anti-proliferative activity against cancer cells expressing APN was demonstrated. Our semicarbazones and thiosemicarbazones are the first compounds of these structural types of Schiff bases that were reported to inhibit not only a zinc-dependent aminopeptidase of the M1 family but also a metalloenzyme.
- MeSH
- aminopeptidasy MeSH
- antigeny CD13 metabolismus MeSH
- lidé MeSH
- nádory * farmakoterapie MeSH
- semikarbazony * MeSH
- thiosemikarbazony * MeSH
- zinek farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Anticancer therapy by anthracyclines often leads to the development of multidrug resistance (MDR), with subsequent treatment failure. Thiosemicarbazones have been previously suggested as suitable anthracycline partners due to their ability to overcome drug resistance through dual Pgp-dependent cytotoxicity-inducing effects. Here, we focused on combining anthracyclines (doxorubicin, daunorubicin, and mitoxantrone) and two thiosemicarbazones (DpC and Dp44mT) for treating cell types derived from the most frequent pediatric solid tumors. Our results showed synergistic effects for all combinations of treatments in all tested cell types. Nevertheless, further experiments revealed that this synergism was independent of Pgp expression but rather resulted from impaired DNA repair control leading to cell death via mitotic catastrophe. The downregulation of checkpoint kinase 1 (CHEK1) expression by thiosemicarbazones and the ability of both types of agents to induce double-strand breaks in DNA may explain the Pgp-independent synergism between anthracyclines and thiosemicarbazones. Moreover, the concomitant application of these agents was found to be the most efficient approach, achieving the strongest synergistic effect with lower concentrations of these drugs. Overall, our study identified a new mechanism that offers an avenue for combining thiosemicarbazones with anthracyclines to treat tumors regardless the Pgp status.
- MeSH
- antibiotika antitumorózní MeSH
- antracykliny * farmakologie MeSH
- checkpoint kinasa 1 metabolismus MeSH
- dítě MeSH
- doxorubicin metabolismus farmakologie MeSH
- inhibitory topoisomerasy II MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- P-glykoprotein metabolismus MeSH
- poškození DNA MeSH
- thiosemikarbazony * farmakologie MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Despite constant advances in the field of pediatric oncology, the survival rate of high-risk neuroblastoma patients remains poor. The molecular and genetic features of neuroblastoma, such as MYCN amplification and stemness status, have established themselves not only as potent prognostic and predictive factors but also as intriguing targets for personalized therapy. Novel thiosemicarbazones target both total level and activity of a number of proteins involved in some of the most important signaling pathways in neuroblastoma. In this study, we found that di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) potently decreases N-MYC in MYCN-amplified and c-MYC in MYCN-nonamplified neuroblastoma cell lines. Furthermore, DpC succeeded in downregulating total EGFR and phosphorylation of its most prominent tyrosine residues through the involvement of NDRG1, a positive prognostic marker in neuroblastoma, which was markedly upregulated after thiosemicarbazone treatment. These findings could provide useful knowledge for the treatment of MYC-driven neuroblastomas that are unresponsive to conventional therapies.
- MeSH
- amplifikace genu účinky léků MeSH
- biologické modely MeSH
- chelátory železa farmakologie MeSH
- down regulace účinky léků MeSH
- erbB receptory metabolismus MeSH
- fosforylace účinky léků MeSH
- fyziologický stres účinky léků MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- neuroblastom metabolismus patologie MeSH
- proteiny buněčného cyklu metabolismus MeSH
- protoonkogen n-myc metabolismus MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- pyridiny farmakologie MeSH
- signální transdukce * MeSH
- thiosemikarbazony farmakologie MeSH
- tvar buňky účinky léků MeSH
- umlčování genů účinky léků MeSH
- upregulace účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Amyotrofická laterální skleróza (ALS) je progresivní neurodegenerativní onemocnění (ND) motoneuronů v mozkové kůře, mozkovém kmeni a míše vedoucí ke ztrátě svalové kontroly a úmrtí vlivem respiračního selhání většinou do 3–5 let od stanovení diagnózy. Většina případů ALS je sporadická (sALS), avšak 5–10 % tvoří případy familiární (fALS). Asi 20 % případů fALS a 2–7 % případů sALS je spojeno s mutací SOD1 genu, který kóduje enzym měď-zinek superoxiddismutázu 1 (SOD1). Nejběžnější volný radikál vznikající v lidském těle je málo reaktivní a tedy nikoliv příliš škodlivý superoxid mající však schopnost spontánní přeměny dismutací na peroxid vodíku. SOD1 tuto dismutaci urychluje a vzniklý peroxid vodíku je odstraňován navazujícími reakcemi. Mutace postihující SOD1 vedou k poruše homeostázy mědi v míše zvířecích (myších) modelů ALS. V současnosti je v Austrálii testována sloučenina Cu2+ diacetyl-di, N4-methylthiosemicarbazon v I/II fázi klinické studie u ALS pacientů. Předpokládá se, že tato molekula by mohla fungovat nejen u případů ALS s mutací SOD1 (SOD1-ALS) jako nosič mědi nebo zinku umožňující jejich interakci se SOD1, a tím správnou funkci enzymu, ale i jako sloučenina vychytávající peroxynitrit. Léčebný potenciál tedy není limitován pouze na SOD1-ALS či ALS obecně, ale jako sloučenina snižující poškození buněk oxidativním a nitrosativním stresem by mohla najít využití i při terapii dalších ND.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of motor neurons in the cerebral cortex, brain stem, and spinal cord leading to loss of muscle control and death from respiratory failure occurring mostly within 3-5 years of the disease diagnosis. The majority of ALS cases are sporadic (sALS); however, 5-10% are familial cases (fALS). Approximately 20% of fALS cases and 2-7% of sALS cases are associated with a mutation in the SOD1 gene that encodes the copper-zinc superoxide dismutase 1 enzyme (SOD1). The most common free radical arising in the human body is a not very reactive, and thus, not a very harmful superoxide which, however, is capable of spontaneous conversion by dismutation to hydrogen peroxide. SOD1 accelerates this dismutation and the produced hydrogen peroxide is eliminated by successive reactions. The mutations affecting SOD1 lead to copper dyshomeostasis in the spinal cord of animal (mice) models of ALS. Currently, the Cu2+ diacetyl-di, N4-methylthiosemicarbazone molecule is being tested in Australia in a phase I/II clinical trial in patients with ALS. It is assumed that this molecule could work not only in cases of ALS with SOD1 mutation (SOD1-ALS) as a copper or zinc carrier allowing their interaction with SOD1, and thus, it's the proper function of the enzyme, but also as a compound for peroxynitrite uptake. As a result, its therapeutic use appears not to be limited only to cases of SOD1-ALS or ALS in general, but it might also have an effect as a compound to reduce cell damage by oxidative and nitrosative stress in other neurodegenerative diseases.
- MeSH
- amyotrofická laterální skleróza * patologie terapie MeSH
- homeostáza MeSH
- lidé MeSH
- měď * metabolismus MeSH
- mutace genetika MeSH
- organokovové sloučeniny MeSH
- riluzol terapeutické užití MeSH
- superoxiddismutasa MeSH
- thiosemikarbazony MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Solid-phase microextraction (SPME) is an alternative method to dialysis and ultrafiltration for the determination of plasma protein binding (PPB) of drugs. It is particularly advantageous for complicated analytes where standard methods are not applicable. Di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a lead compound of novel thiosemicarbazone anti-cancer drugs, which entered clinical trials in 2016. However, this agent exhibited non-specific binding on filtration membranes and had intrinsic chelation activity, which precluded standard PPB methods. In this study, using a simple and fast procedure, we prepared novel SPME fibers for extraction of DpC based on a metal-free, silicon string support, covered with C18 sorbent. Reproducibility of the preparation process was demonstrated by the percent relative standard deviation (RSD) of ≤ 9.2% of the amount of DpC extracted from PBS by several independently prepared fibers. The SPME procedure was optimized by evaluating extraction and desorption time profiles. Suitability of the optimized protocol was verified by examining reproducibility, linearity, and recovery of DpC extracted from PBS or plasma. All samples extracted by SPME were analyzed using an optimized and validated UHPLC-MS/MS method. The developed procedure was applied to the in vitro determination of PPB of DpC at two clinically relevant concentrations (500 and 1000 ng/mL). These studies showed that DpC is highly bound to plasma proteins (PPB ≥ 88%) and this did not differ significantly between both concentrations tested. This investigation provides novel data in the applicability of SPME for the determination of PPB of chelators, as well as useful information for the clinical development of DpC. Graphical abstract.
- MeSH
- adsorpce MeSH
- antitumorózní látky metabolismus MeSH
- design vybavení MeSH
- křemík chemie MeSH
- krevní proteiny metabolismus MeSH
- krysa rodu rattus MeSH
- mikroextrakce na pevné fázi přístrojové vybavení metody MeSH
- pyridiny metabolismus MeSH
- skot MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- thiosemikarbazony metabolismus MeSH
- vazba proteinů MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The possible use of acridines as anticancer agents was first considered in the 1920´s. Since then, a large number of acridine drugs have been tested as antitumour agents, including compounds containing sulphur on the acridine chromophore. In this review, we will discuss recent studies which have investigated the anticancer activity of this class of acridine derivatives. METHODS: We present the results both of our own decade-long research and also of existing research literature into the anticancer activity of acridine derivatives containing sulphur. The evidence of specific tumor-cell killing properties displayed by these compounds suggest the potential of using such molecules as anticancer therapeutics. RESULTS: During the last decade, a number of acridine analogs have been developed by modifying the position and the nature of the substituent on the acridine core. In this paper, we published results on the anticancer activity of acridine derivatives containing sulfur (acridine thioureas, acridine thiazolidine/thiazoidinone, and acridine thiosemicarbazones/ thiosemicarbazides). In cancer chemotherapy, the mechanism of the drugs is complex, although the study of the anticancer activity of acridines has yielded exciting results. CONCLUSION: In this review we have summarized recent literature on the anticancer activity of acridine derivatives containing sulfur. A considerable amount of published data suggests that these compounds exhibit promising anticancer activity against selected cancer cell lines. The obtained results can be helpful in the development of new pharmaceutical agents.
- MeSH
- akridiny farmakologie MeSH
- antitumorózní látky farmakologie MeSH
- léky antitumorózní - screeningové testy MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- thiazolidiny farmakologie MeSH
- thiomočovina analogy a deriváty farmakologie MeSH
- thiosemikarbazony farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
