AlphaFold is an artificial intelligence approach for predicting the three-dimensional (3D) structures of proteins with atomic accuracy. One challenge that limits the use of AlphaFold models for drug discovery is the correct prediction of folding in the absence of ligands and cofactors, which compromises their direct use. We have previously described the optimization and use of the histone deacetylase 11 (HDAC11) AlphaFold model for the docking of selective inhibitors such as FT895 and SIS17. Based on the predicted binding mode of FT895 in the optimized HDAC11 AlphaFold model, a new scaffold for HDAC11 inhibitors was designed, and the resulting compounds were tested in vitro against various HDAC isoforms. Compound 5a proved to be the most active compound with an IC50 of 365 nM and was able to selectively inhibit HDAC11. Furthermore, docking of 5a showed a binding mode comparable to FT895 but could not adopt any reasonable poses in other HDAC isoforms. We further supported the docking results with molecular dynamics simulations that confirmed the predicted binding mode. 5a also showed promising activity with an EC50 of 3.6 μM on neuroblastoma cells.
- MeSH
- antitumorózní látky * farmakologie chemie chemická syntéza MeSH
- histondeacetylasy * metabolismus MeSH
- inhibitory histondeacetylas * farmakologie chemie chemická syntéza MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- neuroblastom * farmakoterapie patologie MeSH
- racionální návrh léčiv * MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- umělá inteligence MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.
- MeSH
- anaplastická lymfomová kináza * genetika metabolismus antagonisté a inhibitory MeSH
- apoptóza účinky léků genetika MeSH
- chemorezistence genetika účinky léků MeSH
- dibenzocyklohepteny * MeSH
- farnesyltranstransferasa * antagonisté a inhibitory metabolismus MeSH
- GTP-fosfohydrolasy * genetika metabolismus MeSH
- inhibitory proteinkinas * farmakologie terapeutické užití MeSH
- lidé MeSH
- membránové proteiny metabolismus genetika MeSH
- mikro RNA * genetika metabolismus MeSH
- mutace MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- neuroblastom * farmakoterapie genetika patologie metabolismus MeSH
- piperidiny * farmakologie terapeutické užití MeSH
- pyridiny * farmakologie terapeutické užití MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- synergismus léků MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitochondria are central for cancer responses to therapy-induced stress signals. Refractory tumors often show attenuated sensitivity to apoptotic signaling, yet clinically relevant molecular actors to target mitochondria-mediated resistance remain elusive. Here, we show that MYC-driven neuroblastoma cells rely on intact mitochondrial ribosome (mitoribosome) processivity and undergo cell death following pharmacological inhibition of mitochondrial translation, regardless of their multidrug/mitochondrial resistance and stem-like phenotypes. Mechanistically, inhibiting mitoribosomes induced the mitochondrial stress-activated integrated stress response (ISR), leading to downregulation of c-MYC/N-MYC proteins prior to neuroblastoma cell death, which could be both rescued by the ISR inhibitor ISRIB. The ISR blocks global protein synthesis and shifted the c-MYC/N-MYC turnover toward proteasomal degradation. Comparing models of various neuroectodermal tumors and normal fibroblasts revealed overexpression of MYC proteins phosphorylated at the degradation-promoting site T58 as a factor that predetermines vulnerability of MYC-driven neuroblastoma to mitoribosome inhibition. Reducing N-MYC levels in a neuroblastoma model with tunable MYCN expression mitigated cell death induction upon inhibition of mitochondrial translation and functionally validated the propensity of neuroblastoma cells for MYC-dependent cell death in response to the mitochondrial ISR. Notably, neuroblastoma cells failed to develop significant resistance to the mitoribosomal inhibitor doxycycline over a long-term repeated (pulsed) selection. Collectively, we identify mitochondrial translation machinery as a novel synthetic lethality target for multidrug-resistant MYC-driven tumors.
- MeSH
- apoptóza MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- neuroblastom * farmakoterapie genetika metabolismus MeSH
- protoonkogen n-myc genetika metabolismus MeSH
- protoonkogenní proteiny c-myc genetika metabolismus MeSH
- signální transdukce MeSH
- umělé letální mutace * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alzheimer's disease (AD) is a complex disease with an unknown etiology. Available treatments, limited to cholinesterase inhibitors and N-methyl-d-aspartate receptor (NMDAR) antagonists, provide symptomatic relief only. As single-target therapies have not proven effective, rational specific-targeted combination into a single molecule represents a more promising approach for treating AD, and is expected to yield greater benefits in alleviating symptoms and slowing disease progression. In the present study, we designed, synthesized, and biologically evaluated 24 novel N-methylpropargylamino-quinazoline derivatives. Initially, compounds were thoroughly inspected by in silico techniques determining their oral and CNS availabilities. We tested, in vitro, the compounds' effects on cholinesterases and monoamine oxidase A/B (MAO-A/B), as well as their impacts on NMDAR antagonism, dehydrogenase activity, and glutathione levels. In addition, we inspected selected compounds for their cytotoxicity on undifferentiated and differentiated neuroblastoma SH-SY5Y cells. We collectively highlighted II-6h as the best candidate endowed with a selective MAO-B inhibition profile, NMDAR antagonism, an acceptable cytotoxicity profile, and the potential to permeate through BBB. The structure-guided drug design strategy applied in this study imposed a novel concept for rational drug discovery and enhances our understanding on the development of novel therapeutic agents for treating AD.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- Alzheimerova nemoc * farmakoterapie MeSH
- cholinesterasové inhibitory terapeutické užití MeSH
- inhibitory MAO terapeutické užití MeSH
- lidé MeSH
- monoaminoxidasa metabolismus MeSH
- neuroblastom * farmakoterapie MeSH
- racionální návrh léčiv MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Butyrylcholinesterase (BChE) is one of the most frequently implicated enzymes in the advanced stage of Alzheimer's disease (AD). As part of our endeavors to develop new drug candidates for AD, we have focused on natural template structures, namely the Amaryllidaceae alkaloids carltonine A and B endowed with high BChE selectivity. Herein, we report the design, synthesis, and in vitro evaluation of 57 novel highly selective human BChE (hBChE) inhibitors. Most synthesized compounds showed hBChE inhibition potency ranging from micromolar to low nanomolar scale. Compounds that revealed BChE inhibition below 100 nM were selected for detailed biological investigation. The CNS-targeted profile of the presented compounds was confirmed theoretically by calculating the BBB score algorithm, these data were corroborated by determining the permeability in vitro using PAMPA-assay for the most active derivatives. The study highlighted compounds 87 (hBChE IC50 = 3.8 ± 0.2 nM) and 88 (hBChE IC50 = 5.7 ± 1.5 nM) as the top-ranked BChE inhibitors. Compounds revealed negligible cytotoxicity for the human neuroblastoma (SH-SY5Y) and hepatocellular carcinoma (HepG2) cell lines compared to BChE inhibitory potential. A crystallographic study was performed to inspect the binding mode of compound 87, revealing essential interactions between 87 and hBChE active site. In addition, multidimensional QSAR analyses were applied to determine the relationship between chemical structures and biological activity in a dataset of designed agents. Compound 87 is a promising lead compound with potential implications for treating the late stages of AD.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- alkaloidy amarylkovitých * farmakologie MeSH
- Alzheimerova nemoc * farmakoterapie MeSH
- butyrylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory chemie MeSH
- lidé MeSH
- neuroblastom * farmakoterapie MeSH
- simulace molekulového dockingu MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Melanins belong to a group of pigments of different structure and origin. They can be produced synthetically or isolated from living organisms. A number of studies have reported testing of various melanins in neurological studies providing different outcomes. Because the structure of melanins can have an effect on obtained results in cell toxicity studies, we present here our original study which aimed to compare the biological effects of bacterial melanin (biotechnologically obtained from B. thuringiensis) with that of synthetic melanin in neuroblastoma cells. Both melanins were structurally characterized in detail. After melanin treatment (0-200 μg/mL), cell viability, glutathione levels, cell morphology and respiration were assessed in SH-SY5Y cells. The structural analysis showed that bacterial melanin is more hydrophilic according to the presence of larger number of -OH moieties. After melanin treatment, we found that synthetic melanin at similar dosage caused always larger cell impairment compared to bacterial melanin. In addition, more severe toxic effect of synthetic melanin was found in mitochondria. In general, we conclude that more hydrophilic, bacterial melanin induced lower toxicity in neuroblastoma cells in comparison to synthetic melanin. Our findings can be useable for neuroscientific studies estimating the potential use for study of neuroprotection, neuromodulation or neurotoxicity.
- MeSH
- Bacteria MeSH
- glutathion MeSH
- lidé MeSH
- melaniny * MeSH
- mitochondrie MeSH
- neuroblastom * farmakoterapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug-resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient-matched tumor cells obtained at diagnosis. Electron microscopy identified reduced ER-mitochondria-associated membranes (MAMs; ER-mitochondria contacts, ERMCs) in therapy-resistant cells, and genetically or biochemically reducing MAMs in therapy-sensitive tumors phenocopied resistance. MAMs serve as platforms to transfer Ca2+ and bioactive lipids to mitochondria. Reduced Ca2+ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ER-mitochondria-associated membranes as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Neuroblastoma is the most frequent extra-cranial solid tumor in early childhood. Intensive combined therapy of high-risk neuroblastoma consists of surgery followed by chemotherapy and autologous hematopoietic stem cell transplantation, as well as radiotherapy. Such intensive therapeutic approach generates negative burden for patients’ immune system. Particularly radiotherapy and treatment using 13-cis-retinoid acid or topotecan are initiating processes that may result in accelerated ageing and immunosenescence. This project aims to address the impact of persistent chronic inflammation, as it occurs in neuroblastoma patients, to the progress of immunosenescence. We hypothesize that long-term inflammation caused by intense therapy changes the properties of subsets of myeloid cells and their precursors circulating in peripheral blood and negatively impact patient’s immunity. This project aims to develop new prognostic markers allowing to assess the progress of immunosenescence in order to prevent further serious complications, which occur long-term after the successful therapy.
Neuroblastom je nejčastější extrakraniální solidní nádor dětského věku. Intensivní kombinovaná terapie vysoce rizikové formy neuroblastomu zahrnuje chirurgické odstranění nádoru následované chemoterapií a autologní transplantací krvetvorné tkáně spolu s radioterapií. Takto intensivní terapeutický přístup má značný negativní vliv na imunitu. Především radioterapie a léčba pomocí 13-cis-retinové kyseliny a topotecanu mohou iniciovat procesy, které jsou zodpovědné za rozvoj předčasného stárnutí a imunosenescence. Tento projekt se zaměřuje na vliv chronického zánětu, který vzniká následkem léčby neuroblastomu, na rozvoj imunosenescence. Předpokládáme, že dlouhodobý zánět způsobený intenzivní léčbou mění vlastnosti myeloidních buněk a jejich prekurzorů v periferní krvi a to může mít negativní vliv na pacientovu imunitu. Cílem projektu je vyvinout prognostické znaky, které pomohou k časné detekci případných závažných následků léčby, které se vyskytují po úspěšné terapii pacientů s neuroblastomem.
- MeSH
- antitumorózní látky škodlivé účinky MeSH
- biologické markery analýza MeSH
- chronická nemoc MeSH
- dítě MeSH
- imunosenescence MeSH
- lidé MeSH
- myeloidní buňky účinky léků MeSH
- neuroblastom farmakoterapie komplikace MeSH
- nežádoucí účinky léčiv diagnóza MeSH
- prognóza MeSH
- stárnutí buněk MeSH
- telomery MeSH
- zánět MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- alergologie a imunologie
- onkologie
- pediatrie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Alzheimer's disease (AD) is a multifactorial neurodegenerative condition of the central nervous system (CNS) that is currently treated by cholinesterase inhibitors and the N-methyl-d-aspartate receptor antagonist, memantine. Emerging evidence strongly supports the relevance of targeting butyrylcholinesterase (BuChE) in the more advanced stages of AD. Within this study, we have generated a pilot series of compounds (1-20) structurally inspired from belladine-type Amaryllidaceae alkaloids, namely carltonine A and B, and evaluated their acetylcholinesterase (AChE) and BuChE inhibition properties. Some of the compounds exhibited intriguing inhibition activity for human BuChE (hBuChE), with a preference for BuChE over AChE. Seven compounds were found to possess a hBuChE inhibition profile, with IC50 values below 1 μM. The most potent one, compound 6, showed nanomolar range activity with an IC50 value of 72 nM and an excellent selectivity pattern over AChE, reaching a selectivity index of almost 1400. Compound 6 was further studied by enzyme kinetics, along with in-silico techniques, to reveal the mode of inhibition. The prediction of CNS availability estimates that all the compounds in this survey can pass through the blood-brain barrier (BBB), as disclosed by the BBB score.
- MeSH
- acetylcholinesterasa chemie MeSH
- alkaloidy amarylkovitých chemie MeSH
- butyrylcholinesterasa chemie MeSH
- cholinesterasové inhibitory chemie farmakologie MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- neuroblastom farmakoterapie patologie MeSH
- počítačová simulace MeSH
- proliferace buněk MeSH
- simulace molekulového dockingu * MeSH
- tyramin analogy a deriváty chemie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
Východiská: Neuroblastóm je najčastejší solídny extrakraniálny nádor detského veku s extrémne heterogénnym biologickým a klinickým správaním. Napriek pokrokom v liečbe je dlhodobá prognóza pacientov s vysokorizikovým neuroblastómom a s relapsom ochorenia nepriaznivá. Potenciál zlepšiť ju prináša implementácia imunoterapie do liečebných protokolov. Dinutuximab, chimerická monoklonálna protilátka, vedie k apoptóze nádorových buniek prostredníctvom väzby na GD2 receptor. Cieľom článku je prezentácia prvých skúseností nášho centra s liečbou dinutuximabom. Súbor a metódy: V rokoch 2018–2019 sme podali 7 pacientom 31 cyklov dinutuximabu. Piatim pacientom s vysokorizikovým neuroblastómom bol dinutuximab podávaný v 1. línii, u dvoch pacientov s relapsom ochorenia bol dinutuximab podávaný v rámci 2. línie liečby. Na vyhodnotenie toxicity liečby boli retrospektívne analyzované ošetrovateľské záznamy pacientov počas podávania imunoterapie. Výsledky: U dvoch pacientov liečených dinutuximabom v 1. línii pretrváva po ukončení imunoterapie kompletná remisia, traja pacienti dosiahli parciálnu odpoveď. Obom pacientom s relapsom neuroblastómu bol po ukončení imunoterapie diagnostikovaný druhý relaps a exitovali na progresiu ochorenia. Tolerancia liečby bola u väčšiny pacientov primeraná – u 6 pacientov boli nežiaduce účinky zvládnuté podpornou liečbou. Išlo najmä o prejavy capillary leak syndrómu, bolesti a hypersenzitívne reakcie. U jedného pacienta bola liečba prerušená pre závažnú neurotoxicitu. Záver: Dinutuximab má preukázateľný benefit v eradikácii minimálnej reziduálnej choroby pri liečbe neuroblastómu. Imunoterapia je v súčasnosti štandardom prvolíniovej liečby pacientov s vysokorizikovým neuroblastómom. Jej úloha v liečbe pacientov s relapsom ochorenia je predmetom viacerých prebiehajúcich štúdií, rovnako ako aj optimalizácia terapeutických schém v oboch indikáciách. Liečba dinutuximabom je spojená s rizikom rozvoja širokej palety nežiaducich účinkov, preto jeho podávanie patrí do rúk skúseného centra detskej onkológie.
Background: Neuroblastoma is the most common extracranial solid tumour of childhood with extremely heterogeneous biological and clinical behaviour. Despite advances in its treatment, the long-term prognosis of patients with a high-risk and relapsed neuroblastoma remains poor. The implementation of immunotherapy into the treatment protocols has the potential to improve it. Dinutuximab, a chimeric monoclonal antibody, leads to the apoptosis of tumour cells through binding to the GD2 receptor. The article aim is to present the first experience of our centre with dinutuximab treatment. Patients and methods: In 2018–2019, we administered 31 cycles of dinutuximab to seven patients. Five patients with high-risk neuroblastoma received dinutuximab in the first line, in two patients with relapse, dinutuximab was administered in the second line of treatment. To evaluate the toxicity of the treatment, the nursing records of patients during immunotherapy were retrospectively analysed. Results: Two patients treated with dinutuximab in the first line are in complete remission, three patients achieved a partial response. Both patients with relapsed neuroblastoma were diagnosed with a second relapse after immunotherapy and died of disease progression. The treatment tolerance was acceptable in most patients – in six patients adverse events were managed with adequate supportive care. These were mainly symptoms of capillary leak syndrome, pain and hypersensitivity reactions. In one patient, the treatment was discontinued due to severe neurotoxicity. onclusion: Dinutuximab has a proven benefit in the eradication of the minimal residual disease in the treatment of neuroblastoma. Immunotherapy is currently the standard for first-line treatment of high-risk neuroblastoma. Its role in the treatment of relapsed neuroblastoma is a subject of several ongoing studies as well as the optimization of therapeutic regimens. Dinutuximab administration is associated with a considerable risk of severe adverse reactions, so the treatment belongs to the hands of an experienced paediatric oncology centre.
