Peumus boldus, and its capacity to block some dysfunctional processes caused by AβO. We examined the protective effect of boldine (1–10 μM) in primary hippocampal neurons and HT22 hippocampal-derived cell line treated with AβO (24–48 h). We found that boldine interacts with Aβ in silico affecting its aggregation and protecting hippocampal neurons from synaptic failure induced by AβO. Boldine also normalized changes in intracellular Ca2+ levels associated to mitochondria or endoplasmic reticulum in HT22 cells treated with AβO. In addition, boldine completely rescued the decrease in mitochondrial membrane potential (ΔΨm) and the increase in mitochondrial reactive oxygen species, and attenuated AβO-induced decrease in mitochondrial respiration in HT22 hippocampal cells. We conclude that boldine provides neuroprotection in AD models by both direct interactions with Aβ and by preventing oxidative stress and mitochondrial dysfunction. Additional studies are required to evaluate the effect of boldine on cognitive and behavioral deficits induced by Aβ in vivo.Alzheimer’s disease (AD) is the most common cause of senile dementia worldwide, characterized by both cognitive and behavioral deficits. Amyloid beta peptide (Aβ) oligomers (AβO) have been found to be responsible for several pathological mechanisms during the development of AD, including altered cellular homeostasis and synaptic function, inevitably leading to cell death. Such AβO deleterious effects provide a way for identifying new molecules with potential anti-AD properties. Available treatments minimally improve AD symptoms and do not extensively target intracellular pathways affected by AβO. Naturally-derived compounds have been proposed as potential modifiers of Aβ-induced neurodysfunction and cytotoxicity based on their availability and chemical diversity. Thus, the aim of this study was to evaluate boldine, an alkaloid derived from the bark and leaves of the Chilean tree  Alzheimer’s Disease (AD) is the most prevalent neurodegenerative disorder characterized by cognitive and behavioral deficits  that is N-methyl-D-aspartate (NMDA) receptors (NMDARs) caused by AβO, ultimately leading to early cognitive deficits (2+ (Ca2+i). Additionally, AβO per se induces an immediate Ca2+i rise involving GluN2B-containing NMDARs in cortical neurons   triggered by mitochondrial dysfunction and ER stress culminates in the oxidative damage observed in AD cells and animal models, as well as in AD patient’s biological fluids and peripheral blood mononuclear cells (PBMCs)  , highligsurvival . In addival  .Cholinesterase inhibitors and memantine (uncompetitive NMDAR antagonist) are the only drugs approved by the Food and Drug Administration (FDA) to slowdown the cognitive symptoms in the early and late stages of AD, respectively, however, neither of them inhibit the disease progression. Therefore, the development and testing of new drugs, such as natural compounds, is imperative as a primary prevention of cognitive decline associated with AD .Peumus boldus (commonly known as boldo) and traditionally consumed in South America  and reconstituted following the instructions of the manufacturer. Aβ1–42 oligomers used in HT22 cells (referred as AβO) were prepared as previously described  and stored in aliquots at −20°C. For AβO preparation (80 μM), aliquots of 5 mL were added to 137.5 mL ultrapure water in an Eppendorf tube. After 15 min incubation at room temperature, the samples were centrifuged at 14,000 g for another 15 min, and the supernatant fraction transferred to a new tube. The samples were stirred at 500 rpm using a Teflon-coated microstir bar for 24–48 h at room temperature (approximately 22°C) and stored at 4°C until required. Fura2-AM and TMRM+ were purchased from Molecular Probes-Invitrogen . Poly-L-lysine, MitoPY1, oligomycin, FCCP and 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. . Thapsigargin was purchased from Tocris Bioscience . The monoclonal mouse anti-synaptic vesicle protein 2 (SV2) antibody was from Developmental Studies  and rabbit anti-MAP2 was from Santa Cruz Biotechnology . Anti-rabbit IgG conjugated with Cy5 and anti-mouse IgG conjugated with Cy3 were from Jackson Immuno Research Laboratories . DAKO mounting medium was purchased from Agilent, United States. All other reagents were of analytical grade. The final concentration of DMSO in assays was not higher than 1% v/v.Boldine was purchased from Sigma (Germany) and a 50 mM stock solution was prepared in DMSO and stored at −20°C according to manufacturer indications. Synthetic Aβescribed . For eleescribed . For insin silico interaction between boldine and Aβ1–42, a protein-ligand docking was performed. First, the interaction grids (15 Å) for the C-terminal, central regions and full peptide were generated using the structure of monomeric Aβ1–42 (PDB ID: 1IYT) with Glide . Boldine structure was obtained from PubChem (CID: 10154). A protein-ligand docking was performed using Glide with a high precision (XP) configuration. The complexes were analyzed using the docking score provided by Glide and the calculation of the MM-GBSA ΔGbind using Prime . All images presented were created with PyMOL .In order to study the 1–42 peptide obtained from Genemed Syn-thesis, Inc. (United States). Oligomeric species were prepared by measuring absorbance of Aβ samples at 23°C (1–42 (40 μM) aggregation with GBP for 24 h under a stirring condition of 500 rpm. Absorbance at 405 nm was measured for 9 h with readings every 20 min using a Novostar microplate reader . Protein absorbance was measured at 482 nm and all the experiments were performed in triplicates. Briefly, 40 μM Aβ was added to a 96 well plate in the presence and absence of 10 and 100 μM boldine.For the aggregation and experiments in hippocampal neurons, we used a synthetic Aβ at 23°C . We stud2 and subsequently euthanized by cervical dislocation. The embryos were removed and rapidly decapitated. Brains were removed and the hippocampi were dissected from the cortices free of meninges. The hippocampus was mechanically and enzymatically dissociated  were treated with 1 μM AβO in the presence or absence of 10 μM boldine for 24 h.Hippocampal neurons were obtained from C57BL/J6 mice or Sprague-100 Dawley rat embryos as previously described e.g., . Animal sociated . After b2, 1 mM MgCl2, 10 mM glucose and 10 mM HEPES, pH 7.4/NaOH; 320 mOsm/L). Cells were stabilized at room temperature for 20 min before experiments. The internal solution used to record spontaneous post-synaptic currents (sPSCs) contained 120 mM KCl, 2.0 mM MgCl2, 2 mM Na2ATP, 10 mM BAPTA, 0.5 mM NaGTP and 10 mM HEPES, pH 7.4/KOH; 310 mOsm/L). All currents were recorded by adjusting the membrane potential to −60 mV using an Axopatch-200B amplifier  and an inverted microscope . The acquisition was made using a computer connected to the recording system with a Digidata 1440A acquisition card  and the pClamp10 software . Electrodes with a resistance of 4–5 MΩ were pulled from borosilicate capillaries  in a horizontal puller . A 5 mV pulse was used to monitor series resistance throughout the recording period and only cells with a stable access resistance (less than 15 MΩ and that did not change more than 20%) were included for data analysis. After acquiring the synaptic recording, the area under the current trace was integrated (pA⋅ms) and expressed as charge transferred (nC) during the whole recording period (2 min) using Clampfit 10.5 .For voltage-clamp experiments in the whole cell mode, culture medium was replaced with a normal external solution (NES) containing: 150 mM NaCl, 5.4 mM KCl, 2 mM CaClHippocampal neurons were fixed for 15 min with 4% paraformaldehyde in PBS. Thereafter, cells were incubated with permeabilization and blocking solution with 0.1% Triton X-100 in horse serum (HS):PBS 1:10 for 20 min. Cells were then incubated with primary antibodies [monoclonal mouse anti-synaptic vesicle 2 protein (SV2) antibody (1:200) and rabbit anti-MAP2 (1:300)] overnight at 4°C followed by incubation with secondary antibodies [anti-rabbit IgG conjugated with Cy5 (1:500) and anti-mouse IgG conjugated with Cy3 (1:500)] for 2 h at room temperature. All antibodies were diluted with HS (10%) in PBS. Coverslips were mounted in DAKO mounting medium and cells observed under a spectral confocal laser scanning microscope  using a 63 × 1.4 numerical aperture oil immersion objective . Images of 16-bit were collected using a pixel time of 1.58 μs and a pixel size of 110 nm.per condition were counted.In fluorescence microscopy, light undergoes diffraction while traveling in an imaging system leading to image blurring and limiting visual access to details. The blurring is characterized by a point-spread function (PSF) that along with the original image can be used in a deconvolution algorithm to restore microscopic details. Therefore, using the Richardson−Lucy algorithm provided by DeconvolutionLab2 plugin  in Image2 culture flasks in high-glucose Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated (HI) fetal bovine serum (FBS), 12 mM NaHCO3, 5 mM HEPES and 100 μg/mL penicillin-streptomycin, pH 7.3 in a humidified incubator with 5% CO2 and 95% air at 37°C. When 80% confluency was reached, cells were detached using a Ca2+-Mg2+-free dissociation medium containing 140 mM NaCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, 1.47 mM KCl, 0.55 mM EDTA, pH 7.3 and then centrifuged at 800 rpm for 5 min and subsequently sub-cultured or plated at a density of 0.005 × 106 cell/well in 96-multiwell plates. Twenty four hour after plating, cells were treated with 1 μM AβO in the presence or absence of 1, 10 or 100 μM boldine in culture conditioned medium (medium where the cells were plated) for 24 h at 37°C in a humidified culture chamber containing 95% O2 and 5% CO2. The effect of boldine alone was also tested.HT22 cells, a mouse hippocampal cell line obtained from the immortalization of primary hippocampal neurons using a temperature sensitive SV40 T-antigen and subcloned from HT4 cells based on sensitivity to glutamate , were ob2, 0.4 mM KH2PO4, 1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES, pH 7.4 for 2 h at 37°C in the dark. Krebs medium was removed and formazan crystals were dissolved in 0.04 M HCl in isopropanol. The absorbance at 570 nm was then measured spectrophotometrically using a SpectraMax plus microplate reader .Cell viability was determined using the colorimetric MTT assay based on the reduction of MTT into an insoluble formazan product via nicotinamide adenine dinucleotide phosphate (NADH)-dependent dehydrogenases by viable cells. HT22 cells were incubated with 0.5 g/L MTT in Krebs medium containing 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2+ and ΔΨm measurements, HT22 cells were rinsed with Krebs medium and co-loaded with the high affinity Ca2+ probe Fura2-AM (5 μM) that accumulates in the cytoplasm plus the ΔΨm sensing dye TMRM+  that accumulates in polarized mitochondria, for 40 min at 37°C. Cells were then rinsed with Krebs medium and fluorescence was measured in the presence of TMRM+ (in order to prevent leakage) using a Spectrofluorometer Gemini EM . The fluorescence was recorded for both Fura2 at 340/380 nm excitation and 510 emission, and for TMRM+ at 540 nm excitation and 590 nm emission. After the establishment of a 2-min baseline, cells were stimulated with 2 μM FCCP to induce maximal mitochondrial depolarization in the presence of 2 μg/mL oligomycin, to prevent ATP hydrolysis. The cytoplasmic Ca2+ increase upon mitochondrial Ca2+ release was recorded as an increase in the Fura2 fluorescence, whereas ΔΨm was evaluated by the increase in TMRM+ fluorescence signal in response to mitochondrial full depolarization. For ER-Ca2+ measurements, cells were incubated in Fura2-containing Krebs medium for 30 min at 37°C. Following a washing step, fluorescence was measured in Ca2+-free Krebs medium. After the baseline establishment, cells were stimulated with 1 μM thapsigargin, a selective non-competitive inhibitor of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), that leads to ER-Ca2+ depletion. The increase in Fura2 fluorescence signal was recorded and analyzed as relative levels of ER-Ca2+. Fluorescence was measured using a Spectrofluorometer Gemini EM . Ca2+ levels and ΔΨm presented in bar graphs were calculated by subtracting the last baseline value (before stimuli) to the highest fluorescence value after the stimuli.For mitochondrial-Ca2O2. Cells were incubated with the probe during 20 min at 37°C in a humidified culture chamber containing 95% O2 and 5% CO2. Then, cells were rinsed with Krebs medium and fluorescence was read at 489 excitation and 540 emission in a Spectrofluorometer Gemini EM .HT22 cells were rinsed with Krebs medium and loaded with 10 μM mitochondrial peroxy yellow 1 (MitoPY1), a fluorescent probe that selectively accumulates in the mitochondria and reacts with H2 consumption by HT22 cells, cultured as described before, was measured by using the XF24 flux analyzer according to 4 cells per well 48 h before the experiments. On the following day, cells were incubated with or without 1 μM AβO in the absence or in the presence of 1 μM boldine and cultured for an additional 24 h. The effect of boldine alone was also tested. On the day of experiments, cell culture medium was carefully removed and cells were washed two times with 1 mL DMEM5030 medium, pH 7.4, supplemented with 2 g/L glucose and 0.3 g/L glutamine to fully remove the previous medium. Then, the cells were incubated in 450 μL DMEM at 37°C in a CO2-free incubator for 1 h. Cellular bioenergetics was determined using the extracellular flux analyzer (Seahorse Bioscience). This system allows for real time, non-invasive measurements of oxygen consumption rate (OCR), which can be correlated with mitochondrial function/oxidative burst. Sequential injection of mitochondrial inhibitors oligomycin (2.5 μg/mL), FCCP (4 μM), and antimycin A  plus rotenone (2 μM) were added to evaluate basal respiratory capacity, maximal respiration in the presence of FCCP, oligomycin-sensitive O2 consumption coupled to ATP synthesis, proton leak, spare respiratory capacity and non-mitochondrial respiration as previously described (2 per minute (pmol O2/min).Oescribed . Resultspost hoc test. Statistical differences were represented as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.Data were analyzed by using Excel and GraphPad Prism 8  software and results are expressed as the mean ± SEM. Comparison among groups was performed using the Kruskal-Wallis test followed by uncorrected Dunn’s multiple comparisons test or one-way ANOVA followed by Tukey’s bind (MM-GBSA), one of which was boldine (1–42 peptide (1IYT) docking interactions was performed considering 3 grids: central region   in the presence of two concentrations of boldine (10 and 100 μM) under constant agitation. Results in ctively) . No signp < 0.05) and this effect was reduced when the neurons were co-incubated with AβO and 10 μM boldine (p < 0.05)  , suggest < 0.05) .h) of −60 mV in neurons treated with AβO in the absence or presence of boldine using the whole-cell technique. Under voltage clamp conditions, bursts of synaptic activity were recorded reflected by synaptic events (p < 0.01) in the number and amplitude of the synaptic currents. Co-incubation of AβO with 10 μM boldine significantly recovered the presence of postsynaptic currents and synaptic transmission. Boldine alone had no effect on the functional activity.To independently confirm the effect of boldine in recovering the SV2 protein labeling, and functionally preventing synaptic impairment, spontaneous synaptic currents were recorded at a holding potential , as evidenced by the increase in cytosolic Ca2+ rise following complete mitochondrial depolarization (with FCCP plus oligomycin) (2+ levels (p < 0.05) as observed after ER-Ca2+ depletion induced by 1 μM thapsigargin (selective SERCA inhibitor) in HT22 cells   . Moreove22 cells . Boldinectively) . Neverthrganelle . These r+, a fluorescent probe that predominantly accumulates in polarized mitochondria. Our results showed that AβO-treated HT22 cells exhibited a significant decrease in ΔΨm (p < 0.05), which was largely prevented by 1 μM boldine (p < 0.001)  . Treatme< 0.001) . These d2O2 produced by mitochondria. Our results indicate that Aβ enhanced mitochondrial ROS levels (p < 0.05) and this effect was largely prevented by co-treatment with boldine (p < 0.001) (p < 0.05)  . Boldine< 0.001) . HT22 ne < 0.05) . Data sh < 0.05) .2+ levels is relevant for mitochondrial overall activity, we analyzed mitochondrial respiration profile and ATP synthesis in cells exposed to AβO and/or boldine. Mitochondrial respiration was analyzed in HT22 cells incubated with AβO in the absence or presence of boldine (1 μM) by using a Seahorse XF24 flux analyzer  and oxidative stress  was shown to restore ΔΨm and ROS levels in a murine model of renal dysfunction . In addi2O2 levels induced by AβO, which is in agreement with previous studies in several models indicating that the neuroprotective effects of boldine are due to its antioxidant effects . Therefore, the neuroprotective effect of boldine may result from: (1) Interaction with residues implicated in the association of Aβ to the membrane and the formation of channels permeable to Ca2+; (2) Interaction with residues implicated in ion permeation (mainly Ca2+) through the cellular membrane ; (3) Direct interaction with Aβ, preventing ER and mitochondria deregulation due to Ca2+ rise, thus ameliorating mitochondrial function. In conclusion, low concentrations of boldine interfere with Aβ aggregation, prevent synaptic failure and organelle dysfunction, centering on mitochondria as one of the relevant players relevant for cell survival, and thus constituting a potential neuroprotective drug in AD.Our data indicate that boldine interacts with the Aβpairment . FurtherThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Ethics Committee at the University of Concepcion.in vitro experiments, and discussed the results. CB designed and performed in silico studies and analysis. JT, EF-P, NR-L, BP-C, LP-P, and DM carried out material preparation, data collection and analysis of in vitro studies. All authors read and approved the final manuscript.JT, EF-P, IF, ACR, and LA wrote the manuscript, designed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A reliable assay that assesses the biological consequences of any radionuclide is much needed. Here, we evaluated the cell-free plasmid DNA assay to determine the relative biological effects of radionuclides such as Auger electron-emitting [67Ga]GaCl3 or [111In]InCl3 for up to 73 h or was exposed to EBRT. The induction of relaxed and linear plasmid DNA, representing single and double strand breaks, respectively, was assessed by gel electrophoresis. Chelated forms of 67Ga were also investigated using DOTA and THP. Topological conversion rates for supercoiled-to-relaxed Supercoiled pBR322 plasmid DNA (1.25 or 5 ng/μL) was incubated with 0.5 or 1 MBq [67Ga]GaCl3 and [111In]InCl3. Damage caused by [67Ga]GaCl3 decreased when chelated. [67Ga]GaCl3 proved more damaging than [111In]InCl3; 1.25 ng/μL DNA incubated with 0.5 MBq [67Ga]GaCl3 for 2 h led to a 70% decrease of intact plasmid DNA as opposed to only a 19% decrease for [111In]InCl3. For both EBRT and radionuclides, conversion rates were slower for 5 ng/μL than 1.25 ng/μL plasmid DNA. DNA damage caused by 1 Gy EBRT was the equivalent to damage caused by 0.5 MBq unchelated [67Ga]GaCl3 and [111In]InCl3 after 2.05 ± 0.36 and 9.3 ± 0.77 h of incubation, respectively.DNA damage increased both with EBRT dose and incubation time for [This work has highlighted the power of the plasmid DNA assay for a rapid determination of the relative biological effects of radionuclides compared to external beam radiotherapy. It is envisaged this approach will enable the systematic assessment of imaging and therapeutic radionuclides, including Auger electron-emitters, to further inform radiopharmaceutical design and application. However, dose limits between isotopes are not comparable and by taking [131I]-NaI as the guide might be setting it too low I [111In]In-Octreotide and [111In]In-EGF have come closest to clinical translation [123I]I-MAPi and [125I]I-DCIBzL intended to treat brain tumours and prostate cancer, respectively, are proving exciting avenues to explore the use of Auger electron-emitters in the clinic [MRT has mostly focussed on radionuclides emitting alpha or beta particles. Auger electron MRT has recently come to the fore due to its therapeutic potential for micrometastases or single circulating tumour cells in cancer . The ranolecules . It was  125I]I ,20–25, aII ,20–25nslation ,27. Moree clinic –30.Redefining dose limits for radionuclides emitting Auger electrons is intrinsically linked to recent work, which accurately recalculates their S values by including the contribution of these low energy electrons ,32. In iIn this study, gel electrophoresis of plasmid pBR322 was used to systematically relate the DNA damage caused by different radionuclides. This included both co-incubation with the DNA or use as an external source of radiation separate to and relatively distant from the DNA. DNA damage was compared to that caused by EBRT to better understand the RBE of Auger electron-emitters and other radionuclides in a simplified context without the complicating features of living cells and tissues.22.167Ga]Ga-citrate  was obtained in 5.5 mL citrate solution  and converted to [67Ga]GaCl3 as described previously [67Ga]Ga-citrate was converted with a SEP-PAK® Silica Plus Light cartridge . After pre-conditioning the cartridge with 10 mL milliQ water at 1 mL/min, [67Ga]Ga-citrate was passed through the cartridge up to three times at 0.5-1 mL/min to maximise retention on the cartridge, which was then washed three times with 5 mL milliQ water. Finally, [67Ga]GaCl3 was eluted with 600 μL 0.1 MHCl  and collected in 50 μL fractions. Fractions with the highest activity  were used for chelator labelling. [111In]InCl3  was supplied in 0.5 mL 0.05 M hydrochloric acid . Radionuclide radiation spectra have been previously published [[eviously . In shorublished .2.267Ga]GaCl3 or [111In]InCl3 for up to 25 h to determine the effect of plasmid concentration, activity and radionuclide on induction of DNA damage. For up to 73 h, plasmid DNA was also incubated with gallium citrate or chelated [67Ga]GaCl3, i.e. [67Ga]Ga-DOTA or [67Ga]Ga-THP, to ascertain any protective effects against DNA damage from the chelation of [67Ga]GaCl3 (0.4-0.5 MBq). [67Ga]GaCl3 was also used as an external source of radiation by placing a 0.5 mL Eppendorf tube containing plasmid inside a 15 mL centrifuge tube containing 0.5 MBq [67Ga]GaCl3. Exposure times were chosen to enable us to kinetically model the DNA damage caused by radionuclides. During incubation, samples were kept in the fridge. In the final reaction, the pH was neutral for all conditions.Supercoiled plasmid DNA, pBR322, was obtained from New England Biolabs at 250 μg/mL, in 10 mM Tris-HCl, 1 mM EDTA and pH 8.0. Plasmid DNA, used at 1.25 ng/μL and 5 ng/μL in 20 μL PBS, was irradiated at 0-40 Gy by external beam radiotherapy (EBRT) using a 0.6 MeV gamma-emitting Caesium-137 source  at 5 Gy/min. Separately, plasmid DNA  was incubated with 0.5-1 MBq GaCl3 was chelated with either S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid  or (tris)hydroxypyridinone  and heated to 95 °C for 15min. Separately, THP  in 0.5M ammonium acetate was mixed with [67Ga]GaCl3  at room temperature for 10 min. In the final reaction, the pH was neutral for all conditions. Radiochemical purity was assessed by instant thin layer chromatography utilising iTLC-SG  giving good separation between [67Ga]GaCl3 (Rf = 0) and [67Ga] Ga-DOTA or [67Ga]Ga-THP (Rf = 1) with a mobile phase of 1 M ammonium acetate in water/methanol  or h−1 (radionuclides) ± standard error. There is a possibility of an additional transition from S directly to L due to the creation of double strand breaks (DSBs), however, this was deemed unlikely, and we opted for the simplest model that could explain the observations.Topological conversion rates for supercoiled-to-relaxed, x is either a given dose, D (Gy), or time, t (h). The data for all three species proportions were fit globally with the above equations using ‘nlinfit’ in custom MATLAB code  weighted by the standard deviation. This code is publicly available at github.com/danielburnham/DNA-Damage-Kinetics. With the successful quantitative model in place, it is possible to form the basis of a quantitative comparison between DNA damage created by EBRT and MRT. Briefly, employing the proportion of a specific DNA state as a proxy for DNA damage, we can equate EBRT and MRT-produced damage. Taking the proportion of supercoiled DNA remaining (EBRT(D) and SMRT(t) respectively, and equating them we can determine the equivalent dose from EBRT, D, required to achieve the same damage as t hours of MRT to be Solving the master equations describing this scheme, the proportions of the respective species, as a function of either time or dose, are: emaining  after EBt, required for a radionuclide at a given activity to achieve the same damage as an EBRT dose, D, was Equivalently, the incubation time, The same principle was applied to both relaxed and linear states as given in the Supplementary Methods.2.567Ga]GaCl3 and [111In]InCl3. Here, slight alterations to the method are described in the Supplementary Methods.Studies with PET isotopes largely followed the same methods as described above for GaCl3 also decreased the relative amount of supercoiled DNA from 92 ± 3% (0 h incubation) to 31 ± 7% to 0.14 ± 0.05% at 2 and 25 h, respectively  at 25 h. There was no significant difference found between values obtained for [67Ga]Ga-DOTA or [67Ga]Ga-THP (P = 0.09).The effect on plasmid DNA damage induction by chelated [iled DNA . Chelati67Ga]Ga-cit-rate as well as when [67Ga]GaCl3 was used as an external source of radiation, i.e. not in the same tube as the plasmid (P < 0.0001).Separately, a preventative effect was also observed for [3.467Ga]GaCl3 and [111ln]lnCl3 Auger electron-emitters reduced the level of supercoiled plasmid DNA over time (67Ga]GaCl3 proved highly damaging with little difference in damage induced when plasmid DNA was incubated with either 0.5 or 1 MBq (P = 0.8026). On the other hand, increasing the activity from 0.5 to 1 MBq for [111In]InCl3 did decrease the level of supercoiled DNA from 81 ± 8% to 50 ± 24% at 2 h (P = 0.0299). Furthermore, [67Ga]GaCl3 proved to be more damaging than [111In]InCl3 (P < 0.0001 at 0.5 MBq and P = 0.0140 at 1 MBq). For example, supercoiled DNA levels were at 31 ± 7% and 81 ± 8% for [67Ga]GaCl3 and [111In]InCl3, respectively at 0.5 MBq at 2 h.Both [ver time . [67Ga]G3.5−1; −1) compared to 5 ng/μL  (67Ga]GaCl3 at a concentration of 1.25 ng/μL (−1) compared to 5 ng/μL (−1)  . Topolog02 Gy−1) . Similar009 h−1) . Through67Ga]GaCl3 (0.59 ± 0.10 h−1 at 0.5 MBq) than for [111In]InCl3 (0.13 ± 0.01 h−1 at 0.5 MBq). This was also the case for the rate of conversion of relaxed to linear DNA with values of 0.003 ± 0.001 h−1 for [67Ga] Gacl3 and 0.0010 ± 0.0002 h−1 for [111In]InCl3 at 1.25 ng/μL , we , we 3), 67Ga]GaCl3 for 1 h, the equivalent dose is found to be DGa = (0.585 ± 0.1 h−1 / 1.21 ± 0.04 Gy−1) × 1 h = 0.48 Gy ± 0.08 Gy, and for [111In]InCl3 under the same conditions DIn = (0.131 ± 0.01 h−1 / 1.21 ± 0.04 Gy−1) × 1 h = 0.11 Gy ± 0.01 Gy. Similarly, incubating plasmid DNA with 1 MBq [67Ga]GaCl3 or [111In]InCl3 for 1 h induces that same amount of DNA damage as 0.5 ± 0.3 Gy and 0.224 ± 0.009 Gy of EBRT, respectively.To elucidate the principle, we now consider some examples using 67Ga]GaCl3 is found to be, tGa = (1.21 ± 0.04 Gy−1 / 0.585 ± 0.1 h−1) × 1 Gy = 2.05 ± 0.36 h and for [111In]InCl3 under the same conditions tIn = (1.21 ± 0.04 Gy−1 / 0.131 ± 0.01 h−1) × 1 Gy = 9.3 ± 0.77 h , was calculated to be 3.1 ± 0.1 Gy and 2.8 ± 0.1 Gy for an external 137Cs source (+DTPA) and DTPA-chelated [111In]InCl3, respectively [111In]InCl3 was most effective in the absence of DTPA (i.e. when up to 15% was bound to DNA) with a D0 value of 15.3 + 0.7 × 1010disintegrations/cm3 iodoacridine, and a non-intercalating equivalent, 4-[125I]iodoacridine, were 22.4 ± 0.6 × 1011 disintegrations and 4.7 ± 0.4 × 1011 disintegrations, respectively GaCl3  and demo67Ga]GaCl3. These data thus show that the plasmid assay could provide useful mechanistic insights into DNA damage caused by radionuclides. Given the similarities between the effects of increasing plasmid concentration for both EBRT and MRT, we propose that there is a common mechanism at work. Also, it highlights the need for a systematic approach to maximise output and validity of results as values such as D0 Ga-THP. The change in charge likely results in reduced, or even repulsive (for ([67Ga]Ga-DOTA)1−) forces between the radioactive species and the negatively charged DNA backbone. The use of [67Ga]Ga-citrate, which has a charge of−5, or [67Ga]GaCl3 as an external source of radiation proved to completely protect the DNA against damage from [67Ga] Ga3+, as seen previously MnCl2, [89Zr]Zr-oxalate, and [68Ga]GaCl3 could induce damage . Given the observables of our experiments, we are unable to distinguish between these two models and a resolution is for future studies. Also, as seen in Fitting a kinetic model has not previously been presented alongside biological pBR322 study data and yet it is a potentially powerful tool. Here, we assume an irreversible, three state model to explain the observations, however, it is likely an additional irreversible step exists from supercoiled to linear DNA due to single strand breaks occurring sufficiently close to linearise the DNA. This additional kinetic rate, thus model free parameter, does not change the general solution to the master equations of the kinetic scheme, rather only including the addition of a constant to k67Ga]GaCl3 or [111In]InCl3 to induce the same amount of plasmid DNA damage as a specific amount of absorbed radiation dose by EBRT (and vice versa). Traditionally, in vitro or preclinical RBE values are based on clonogenic survival data or studies looking at spermatogenesis in mouse testes [In this study, kinetic modelling has enabled us to determine the duration of time required to incubate with [e testes –51. These testes . This ree testes ,53. The As such, it remains to be seen whether this cell-free pBR322 assay will inform RBE and, in turn, affect radiation weighting doses in the clinic. However, this straightforward, cell-free assay with which we can determine cell-free RBE does hold promise and merit in preclinical nuclear imaging/MRT-focussed research groups, in order to better inform choices of ‘safe’ imaging radionuclides and effective therapeutic radionuclides. These data also highlight the gross gaps in understanding that is attainable by conventional dosimetry and weighting factors and how this assay could provide a way to improve that understanding.5This work demonstrated the utility of the plasmid DNA assay for rapid assessment of the relative effects of medical radionuclides on DNA integrity. This is important for the evaluation of the safety and therapeutic efficacy of radioisotopes used in radionuclide imaging and/or MRT research and to compare them relative to one another and in relation to EBRT. This assay is of particular importance for assessing Auger electron-emitting radioisotopes and to further inform radiopharmaceutical design and in vitro and in vivo therapeutic studies.Appendix A. Supplementary datahttps://doi.org/10.1016/j.nucmedbio.2021.06.004.Supplementary data to this article can be found online at Supplementary data


Many studies already identified appropriate NTCP inhibitors. However, most of them interfere with NTCP’s physiological function as a hepatic bile acid transporter. To overcome this drawback, the present study aimed to find compounds that specifically block HBV/HDV binding to NTCP without affecting its transporter function. A novel assay was conceptualized to screen for both in parallel; virus binding to NTCP (measured via binding of a preS1-derived peptide of the large HBV/HDV envelope protein) and bile acid transport via NTCP. Hits were subsequently validated by in vitro HDV infection studies using NTCP-HepG2 cells. Derivatives of the birch-derived pentacyclic lupane-type triterpenoid betulin revealed clear NTCP inhibitory potency and selectivity for the virus receptor function of NTCP. Best performing compounds in both aspects were 2, 6, 19, and 25. In conclusion, betulin derivatives show clear structure–activity relationships for potent and selective inhibition of the HBV/HDV virus receptor function of NTCP without tackling its physiological bile acid transport function and therefore are promising drug candidates.Current treatment options against hepatitis B and D virus (HBV/HDV) infections have only limited curative effects. Identification of Na The genome of the enveloped DNA virus HBV consists of 3.2 kb which encode for three envelope proteins, referred to as small (SHBs), middle (MHBs), and large (LHBs)2. The 2–48 N-terminal amino acids of the myristoylated preS1 domain (myr-preS12-48 lipopeptide) of the LHBs are essential for virus binding to hepatocytes4. As HDV is a satellite virus of HBV and bears identical envelope proteins, it also shares this myr-preS12-48 lipopeptide5. About 5% of all chronic HBV carriers are additionally infected with HDV1. This co-infection causes more rapid disease progression, increased mortality rates, and increased incidence of HCC and liver cirrhosis6. To date, there are only few treatment options for HBV and HDV infections available. Chronic hepatitis can usually be controlled by antiviral therapy with nucleoside reverse transcriptase inhibitors and interferon. This is, however, rarely curative. Furthermore, interferon therapy is highly prone to adverse drug reactions and nucleoside reverse transcriptase inhibitors have to be given life-long8.Infections with the Hepatitis B (HBV) and D (HDV) viruses are the main cause of hepatocellular carcinoma (HCC) and liver cirrhosis as consequences of chronic hepatitis. Although vaccination is possible, more than 250 million people worldwide suffer from chronic hepatitis based on HBV/HDV infections, and 887,000 deaths can be traced back to this every year+/taurocholate co-transporting polypeptide NTCP (gene symbol SLC10A1), which has been identified as the bona fide hepatic receptor for HBV/HDV10. It is generally accepted that HBV/HDV entry into hepatocytes essentially involves attachment of the virus particles to heparan sulfate proteoglycans11, followed by high-affinity binding of the myr-preS1 domain of the large envelope protein to certain domains of NTCP that finally triggers subsequent steps such as endocytosis of the virus-receptor complex12.A promising novel drug target to block HBV/HDV virus entry into hepatocytes is represented by the Na2-48 lipopeptide itself can block in vitro HBV/HDV infection in a dose-dependent manner with inhibitory constants (IC50) in the low nanomolar range4. Based on this, a synthetic analogue of the myr-preS12-48 lipopeptide, called bulevirtide , was developed as a therapeutic agent against HBV and HDV infections. Several clinical studies have been successfully completed and very recently, bulevirtide was approved as a new drug to treat hepatitis D in Europe and Russia and will be marketed under the brand name Hepcludex13. Furthermore, it was demonstrated that viral DNA (HBV) and RNA (HDV) levels can be reduced significantly in chronically infected patients with a combination of bulevirtide and pegylated interferon alpha (PEG-IFNα)14. These data clearly indicate that NTCP is an appropriate drug target to control hepatic HBV/HDV levels. In addition, an HBV/HDV entry inhibitor addressing NTCP might be beneficial to prevent new infections of hepatocytes during chronic infection with HBV/HDV12.It is known for long that an artificial myr-preS117. Thus, NTCP has a dual function as bile salt transporter and virus receptor. It is already known that both functions interfere with each other. The myr-preS12-48 lipopeptide can block the bile salt transport in primary human hepatocytes and NTCP-overexpressing HepG2 hepatoma cells in a concentration-dependent manner. Vice versa, bile salts can prevent myr-preS12-48 lipopeptide binding to NTCP as well as in vitro HBV infection18. Based on this, it was expected that in clinical trials using bulevirtide as a therapeutic agent, the total plasma bile salt levels would increase significantly. In a current study on healthy volunteers who received 10 mg bulevirtide, which is above the therapeutic dose, total plasma bile salt levels indeed increased 19.2-fold, and single bile salts even higher 19. This data can be interpreted as dysregulation of the bile acid homeostasis under bulevirtide treatment.Physiologically, NTCP is expressed dominantly at the basolateral membrane of hepatocytes and acts as a sodium-dependent uptake carrier of bile salts to enable their enterohepatic recirculation20 or by screening for inhibitors of myr-preS12-48 lipopeptide attachment and/or in vitro HBV/HDV infection21 in appropriate cell culture models overexpressing NTCP. However, none of these studies addressed and fulfilled all of the criteria outlined above. In contrast, the present study aimed to find small molecular HBV/HDV entry inhibitors that specifically block myr-preS12-48 lipopeptide binding to NTCP without affecting its bile salt transport function. We analyzed a set of derivatives of the pentacyclic lupane-type triterpenoid betulin, and indeed found individual compounds that were quite potent and selective for myr-preS12-48 lipopeptide binding inhibition and significantly blocked in vitro HDV infection of NTCP-expressing HepG2 cells. These compounds now can be further optimized for increased potency and then can be forwarded for in vivo infection experiments.On the basis of the therapeutically used effects of bulevirtide, a promising second strategy for the development of HBV/HDV entry inhibitors targeting NTCP would be to (I) investigate small molecules with oral bioavailability which (II) block virus binding to NTCP, but (III) without tackling its physiologically important bile salt transporter activity at relevant therapeutic drug concentrations. Currently, several studies identified novel chemical entry inhibitors for HBV and HDV by screening for bile salt transport inhibitors2-48 lipopeptide binding to NTCP in a cell culture system. This was achieved by plating NTCP-HEK293 cells onto 96-well plates and by using tritium-labelled analytes, being [3H]taurocholic acid ([3H]TC) and [3H]myr-preS12-48 lipopeptide ([3H]preS1), which were analyzed in a microplate scintillation counter. For validation of this assay, transport of 1 µM [3H]TC first was inhibited by increasing concentrations of TC  itself and by myr-preS12-48 lipopeptide (concentrations ranging from 0.001 to 10 µM). In addition, binding of 5 nM [3H]preS1 was inhibited by myr-preS12-48 lipopeptide itself (concentrations ranging from 0.001 to 10 µM) and by TC . As shown in Fig. 3H]TC transport with IC50 of 30 µM and of the [3H]preS1 binding with IC50 of 42 µM. The same was true for myr-preS12-48 lipopeptide as the inhibitor that revealed IC50 of 0.35 µM for [3H]TC transport inhibition and IC50 of 0.55 µM for [3H]preS1 binding inhibition  in both assays compared to the physiological transport substrate TC (IC50 = 30 µM / 42 µM). Four reference compounds were analyzed  which had shown inhibition of the transporter and receptor function of NTCP before, namely ciglitazone22, troglitazone22, cyclosporine A25, and rapamycin26. Interestingly, all compounds showed comparable inhibition of [3H]TC uptake and [3H]preS1 binding with IC50 values of 1 µM and 1.3 µM for ciglitazone  up to values > 1,000 µM with very low, if any transport inhibition . This clearly indicates structure–activity relationships over three orders of magnitude. Most of the betulin derivatives showed quite potent inhibition of [3H]preS1 binding with IC50 values ranging from 2–3 µM  up to 199 µM (24), also demonstrating clear structure–activity relationships over a broad inhibitor concentration range.Next, 30 derivatives of betulin were tested in both assays and IC50 values for [3H]TC transport inhibition and the IC50 values for [3H]preS1 binding inhibition were calculated and are further referred to as selectivity index. The higher this index, the more selective the respective compound for myr-preS12-48 lipopeptide inhibition. Therefore, the data listed in Table 2-48 lipopeptide binding inhibitors but also to assess their selectivity. These values ranged from an index of 1, including more potent (e.g. 4) and less potent (e.g. 12) non-selective inhibitors, up to an index of > 60, including the highly selective and potent inhibitors 2 and 25. Figure 3H]TC transport inhibition (in blue) and [3H]preS1 binding inhibition (in red). This panel shows compounds with different characteristics. Within the group of potent [3H]preS1 binding inhibitors (IC50 < 10 µM), there were (I) non-selective inhibitors , (II) poorly selective inhibitors (e.g. 21), and highly selective inhibitors, being 2 and 25. Also, from the group of less potent [3H]preS1 binding inhibitors (IC50 = 11–50 µM), some were poorly selective (e.g. 18) and others were highly selective . Taken together, these data clearly indicate structure–activity relationships of the betulin derivatives for both, bile acid transport inhibition and myr-preS12-48 lipopeptide binding inhibition.In addition, ratios between the IC1) itself showed relatively low inhibitory potency for bile acid transport inhibition (IC50 = 748 µM) and [3H]preS1 binding inhibition (IC50 = 109 µM) resulting in a selectivity index of 7. However, relatively moderate modifications at the betulin core structure revealed significantly improved potency and selectivity index of the individual compounds. Some of these modifications are depicted in more detail in Fig. O-acetylbetulin (24) itself has a relatively low potency for [3H]preS1 binding inhibition (IC50 = 199 µM) and poor selectivity (selectivity index: 5)  improved the potency for [3H]preS1 binding inhibition to IC50 = 8 µM and increased the selectivity index to 125  conjugation of betulin (1) also reduced the IC50 value for [3H]preS1 binding inhibition to 6 µM and increased the selectivity index to 22  increased the inhibitory potency for [3H]preS1 binding by one order of magnitude (109 µM vs. 11 µM) and also improved the selectivity index (7 vs. 48)   and HpreS1 bi µM) and HpreS1 bi µM) and HpreS1 bi µM) and HpreS1 bi µM) and HpreS1 bi µM) and HpreS1 bi50 preS1 binding < 10 µM) and non-selective (selectivity index < 10) NTCP inhibitors from the group of betulin derivatives bear at least one acidic group. This applies to compounds 4, 8—12, 20, and 21 (Tab. 4 (pKa = 4.8) and 8 (pKa = 4.1) were tested for [3H]TC uptake inhibition and [3H]preS1 binding inhibition at pH 7.4 and pH 5.5. Betulin (1) was included as a non-acidic compound for control. In the case of compound 4 a shift of the IC50 value for [3H]TC uptake inhibition from 0.8 to 103 µM was observed  compared to the [3H]preS1 binding assay (15 min). While NTCP-HepG2 cells without inhibitor were used as control (set to 100% infection rate), co-incubation with 0.5 µM of the myr-preS12-48 lipopeptide completely abolished infection of the cells (set to 0% infection rate). As inhibitors for in vitro HDV infection, betulinic acid (4) was used as a potent but non-selective inhibitor, 20,29-dihydrobetulin (2) as a potent and highly selective inhibitor, as well as lupenone (17) and betulonoyl dimethyl-L-aspartate (19) as less potent but still selective inhibitors. All four compounds significantly blocked in vitro HDV infection of the NTCP-HepG2 cells in a concentration-dependent manner  uptake and [3H]preS1 (5 nM) binding were analyzed for 10 min at 37 °C. Cells pretreated with solvent (DMEM) alone served as 100% controls. Data from HepG2 cells without NTCP expression were set to 0%. Even after three intense washing steps, [3H]TC uptake was significantly reduced after short (15 min) and long (6 h) term preincubation with compound 4. In contrast, [3H]preS1 binding did not change, clearly confirming plasma membrane localization of NTCP even after long term preincubation with high inhibitor concentrations of 600 µM. In the case of compound 17, no differences were observed after short term (15 min) preincubation, while [3H]TC uptake and [3H]preS1 binding slightly changed in opposite directions after long term (6 h) preincubation. These data confirm that also after preincubation with compound 17, NTCP remained at the plasma membrane.Compounds 9. Since then, many studies have identified compounds that are capable to inhibit the virus receptor function of NTCP: Hepcludex13, cyclosporine A and other cyclosporine derivatives25, ezetimibe10, irbesartan27, ritonavir28, (-)-epigallocatechin-3-gallate29, vanitaracin A30, Ro41-525331, proanthocyanidin32, zafirlukast33, sulfasalazine33, and Chicago Sky Blue 6B (an azo dye)33. Among them, Hepcludex is a peptide-based drug and cannot be administered orally13. Furthermore, it has correspondingly complex manufacturing, storage, and distribution requirements. Therefore, it is still desirable to develop a small molecular drug with HBV/HDV cell-entry inhibitor activity that can be orally applied and would be easier to handle.The identification of the hepatic bile acid transporter NTCP as the high-affinity receptor for HBV and HDV was the basis for the development of a novel class of anti-HBV/anti-HDV drugs, being cell-entry inhibitors targeting NTCP17. Others were identified as novel HBV/HDV inhibitors and later revealed their interference with the physiological bile acid transport function of NTCP. Overall, this interference indicates that NTCP domains relevant for substrate binding and transport overlap with the domains mediating myr-preS12-48 lipopeptide / virus binding to NTCP. However, there are also some hints that both functions can be separated. In a previous study, we identified amino acid G158 of the human NTCP as absolutely essential for myr-preS12-48 lipopeptide binding and in vitro HBV and HDV infection. This amino acid is located at a domain (amino acids 157–165) that previously was shown to be involved in myr-preS12-48 binding to human NTCP18. Accordingly, G158R NTCP mutants were completely insusceptible for in vitro HBV/HDV infection, but still supported transport of bile acids. It was discussed that the larger amino acid side chain of arginine compared to glycine might sterically preclude myr-preS1 peptide binding, while bile acids can still bind to their respective binding pocket34. Furthermore, Shimura et al.25 showed for the cyclosporine derivative SCY995 higher potency against in vitro HBV infection (IC50 < 8 µM) than for bile acid transport inhibition (IC50 > 25 µM).Many of the other mentioned compounds, e.g. cyclosporine A, were previously known to inhibit the bile acid transport function of NTCP3H]preS1 binding to NTCP as a surrogate parameter for HBV/HDV infection and inhibition of [3H]TC transport via NTCP. In the present study, a novel chemical class of promising HBV/HDV cell-entry inhibitors was identified, represented by derivatives of the natural pentacyclic lupane-type triterpenoid betulin. Betulin can be isolated from various plant species, belonging to a variety of families such as Betulaceae, Platanaceae, Dilleniaceae, Rhamnaceae, Rosaceae, and Fagaceae36. Characteristics of the lupane skeleton of betulin are the five-membered E ring and the isopropylidene group37. Interestingly, nearly all betulin derivatives tested in the present study significantly inhibited [3H]TC transport and [3H]preS1 binding. However, the ratios between the IC50 values for transport inhibition and for [3H]preS1 binding inhibition were quite different and the resulting selectivity indices ranged from 1 up to 65. The latter was the case for compound 2 with an IC50 of 4 µM for [3H]preS1 binding inhibition and an IC50 of 260 µM for bile acid transport inhibition. Some other compounds showed no transport inhibition at all, being 24, 25, 28, and 30, but at least in the case of 25, potent inhibition of [3H]preS1 peptide binding with IC50 of 8 µM. This data clearly indicate structure–activity relationships for both NTCP functions. Of note, only small changes in the molecular structure of the betulin derivatives significantly changed their potency and selectivity index. Moreover, hydroxylation of compound 24 to compound 25, as an example, improved both factors in parallel: potency for [3H]preS1 binding inhibition and the selectivity index. Also other modifications of the betulin molecule (1) such as 3-O-caffeoly conjugation (compound 27) or C20-C30 saturation (compound 2) significantly improved both factors simultaneously.Based on this, the present study was designed to enable the identification of a selective HBV/HDV cell-entry inhibitor from the group of small molecules. Therefore, all test compounds were a priori tested for both, inhibition of TC uptake inhibitory potency when the pH was reduced to 5.5 and, thereby reducing the relative charge of the molecules. In contrast, the [3H]preS1 binding inhibitory potency was insensitive to this pH shift and only showed little differences between pH 7.4 and pH 5.5 conditions. This again indicates that the transporter and the receptor function of NTCP can be separated under certain experimental conditions and that blocking of the preS1 peptide binding site of NTCP does not require negative charge of the inhibitor. In contrast to compounds 4 and 8, betulin itself (compound 1) does not bear any acidic group. Consequently, betulin could be less active at the bile acid binding site, what would explain the relatively low potency for TC uptake inhibition (IC50 = 748 µM) at neutral and acidic pH values. However, no uniform legality could be established for the other chemical modifications of betulin, which would precisely predict the potency and selectivity pattern of the respective betulin derivative. Nevertheless, based on the structure–activity relationships described in the present study it is most likely that even more potent betulin derivatives can be identified, while sparing the cross-reactivity on the bile acid transport function of NTCP.Inhibition studies at pH 5.5 revealed additional structure–activity relationships regarding the charge of the tested compounds. All potent but non-selective inhibitors Fig. b bear at4) particularly underwent intensive toxicological assessment43. In vitro and in vivo model systems consistently categorized betulinic acid (4) as a safe compound with low toxicity. More precisely, no toxic effects were observed in rats that obtained single i.p. application of 400 mg/kg or in mice receiving 100 mg/kg i.p. every 3–4 days for a total of six treatments, or 500 mg/kg i.p. once43. Unfortunately, betulinic acid (4), although potent in [3H]preS1 binding inhibition (IC50 = 2 µM), is not selective and showed equipotent inhibition of the bile acid transport function of NTCP at least at physiological pH. Therefore, betulinic acid (4) is not regarded as the optimal HBV/HDV entry inhibitor candidate from the betulin class of compounds. Apart from betulinic acid (4), betulin (1) also has a good safety profile. Betulin (1) is approved for medical use in humans and is currently available as a betulin containing gel . It was authorized in 2016 by the European Medicines Agency (EMA) for treatment of partial thickness wounds in adults. This gel contains an extract from birch bark of Betula pendula Roth, Betula pubescens Ehrh., and hybrids of both species, obtained with n-heptane as the extraction solvent. The resulting betulin (1) concentrations are in the range of 72–88 mg betulin (1) per 100 mg extract. The gel itself is composed of 10% birch bark extract in 90% sunflower oil. Moreover, Episalvan is currently undergoing a phase III efficacy and safety study in patients with inherited epidermolysis bullosa (EB) that started in 2017 with an estimated enrollment of 250 participants (NCT03068780). Based on this good safety profile and the established use in human medicine, betulin (1) is an attractive early lead candidate for selective HBV/HDV cell-entry inhibition. However, despite its superior selectivity in comparison to betulinic acid (4), the potency for [3H]preS1 binding inhibition is relatively low (IC50 = 109 µM). Nevertheless, the present study clearly demonstrates that even small modifications of the lupane skeleton of betulin can improve the potency and selectivity of the respective compounds significantly. Therefore, it seems likely that advanced rational drug design can further improve this compound class to end up with a potent and selective HBV/HDV cell-entry inhibitor.Among the betulin derivatives, betulinic acid (1) and betulin derivatives have a broad spectrum of pharmacological effects, including anti-cancer, anti-viral, anti-microbial, anti-inflammatory, and anti-fibrotic effects48. Interestingly, betulinic acid (4) was also used in HBV infection studies before, which showed inhibition of HBV replication by suppression of manganese superoxide dismutase (SOD2) expression with subsequent mitochondrial reactive oxygen species (ROS) over-generation49. Based on this and the results from the present study, betulinic acid (4) might have additive HBV/HDV antiviral properties by addressing different cellular targets involved in cell-entry (NTCP) and replication (SOD2). Therefore, further studies with betulin derivatives should take selective NTCP inhibition and potential suppression of SOD2 into account, which was beyond the scope of the present study.Apart from the above-mentioned clinical application, several previous studies showed that betulin  and in vitro HDV infection experiments (hours). Another factor might be the use of different cell lines and incubation media. Whereas, the [3H]preS1 binding assays were performed in NTCP-HEK293 cells in pure DMEM, the infection experiments needed to be performed with NTCP-HepG2 cells in HGM containing significant amounts of bovine serum albumin (BSA) as well as polyethylene glycol (PEG). The latter medium conditions potentially alter free drug concentrations due to protein binding. Lowering of the inhibitory potency of betulinic acid (4) due to protein binding has already been identified in a previous study, where this compound was used as inhibitor for the organic anion transporting polypeptide OATP1B350. A further factor could be the different stoichiometry of the single preS1 peptide molecule and the multitude of preS1 domains of the large envelope proteins of HBV/HDV virions. The latter one could need a higher degree of blocked NTCP receptors for successful virus binding and cell-entry inhibition. On this background, it could be beneficial if an NTCP inhibitor would not only be selective, but would also irreversibly block the preS1/virus binding sites at NTCP. However, this was not achieved with the betulin derivatives analyzed in the present study. The data obtained on compounds 4 and 17 more suggest a reversible mode of inhibition for myr-preS12-48 peptide binding to NTCP. Their inhibitory effect on [3H]preS1 binding was completely abrogated by washing the cell surface with inhibitor-free medium  belongs to the group of acidic betulin derivatives one can speculate that betulinic acid is transported by NTCP, accumulates inside the cell during preincubation and so by trans-inhibiton blocks substrate binding but not preS1 peptide binding to the outer surface of the NTCP protein. This again would support the idea that bile acid and preS1 peptide binding to NTCP occur at separate, but overlapping domains. In contrast, compound 17 showed a paradoxical effect after long term preincubation and increased the capacity for [3H]preS1 peptide binding to approximately 130%, while the [3H]TC uptake rate was slightly reduced. This could be explained by longer lasting allosteric effects of this compound that differently affects the transport and receptor function of NTCP. The mechanism behind these effects could not be clarified in the present study and needs further investigation, including LC–MS/MS supported direct transport experiments with key betulin derivatives.Of interest are also the long-term effects of compounds , betulin derivatives show clear structure–activity relationships for potent and selective inhibition of the HBV/HDV virus receptor function of NTCP without tackling its physiological bile acid transport function. Therefore, betulin derivatives are promising candidates for further development of HBV/HDV cell-entry inhibitors. Further studies with this compound class should additionally focus on the mode of inhibition (reversible vs. irreversible) and should be extended to all relevant HBV genotypes.In conclusion51. Cells were maintained at 37 °C, 5% CO2 and 95% humidity in DMEM/F-12 medium  supplemented with 10% fetal calf serum , 4 mM L-glutamine  and penicillin/streptomycin (PAA). HepG2 cells stably transfected with NTCP-FLAG (further referred to as NTCP-HepG210) were cultured under the same conditions in DMEM with all supplements listed above, except for L-glutamine. For induction of the transgene, the medium was supplemented with 1 µg/ml tetracycline  in the case of the NTCP-HEK293 cells or with 2 µg/ml doxycycline (Sigma-Aldrich) in the case of the NTCP-HepG2 cells.Human embryonic kidney (HEK293) cells were stably transfected with human NTCP, C-terminally tagged with the FLAG epitope (further referred to as NTCP-HEK293 cells) as reported before3H]TC  as reported before51. In parallel, [3H]preS1 peptide binding experiments were performed with a tritium-labelled myr-preS12-48 lipopeptide -HBV subgenotype D3- that was purchased from Pharmaron . Briefly, cells were seeded onto polylysine-coated 96-well plates, induced with 1 µg tetracycline per ml, and grown to confluence over 72 h at 37 °C. Then, cells were washed once with tempered phosphate buffered saline  at 37 °C and preincubated with 80 µl DMEM for 5 min at 37 °C. The medium was replaced by 80 µl DMEM containing the respective betulin derivative  or solvent alone (100% uptake/binding control), and cells were further incubated for 5 min at 37 °C. After this preincubation, bile acid transport experiments were started by adding 20 µl DMEM containing 5 µM [3H]TC . Binding of [3H]preS1 was initiated by adding 20 µl DMEM containing 25 nM [3H]preS1 . Experiments were stopped after 10 min by two-times washing with ice-cold PBS. For 0% uptake/binding control, the NTCP-HEK293 cells were not induced with tetracycline (-tet). Cell-associated radioactivity of [3H]TC or [3H]preS1 was quantified by liquid scintillation counting in a Packard Microplate Scintillation Counter TopCount NXT . Transport rates and [3H]preS1 binding were determined in counts per minute (cpm). The mean of the 0% control was subtracted and the net [3H]TC transport rates and net [3H]preS1 binding rates, respectively, were expressed as % of control. Experiments at pH 5.5 were performed the same way, using DMEM acidified with hydrochloric acid . A set of betulin derivatives was purchased from Adipogen AG , including the compounds betulin (1), betulinic acid (4), lupeol (3), betulonaldehyde (18), lupenone (17), 3-O-caffeoylbetulin (27), 3,28-di-O-acetylbetulin (24), 28-O-betulin (10), 3,28-di-O-betulin (12), 28-O-succinoylbetulin (8), 3,28-di-O-succinoylbetulin (9), 3-O-betulinic acid (11), and 3-O-acetylbetulinic acid (21) (TargoSet). All other betulin derivatives were synthesized as outlined elsewhere54. IC50 values were calculated from quadruplicate determinations by GraphPad Prism 6 .Bile acid transport measurements were performed in the NTCP-HEK293 cells with TC uptake and [3H]preS1 binding experiments were started by adding 300 µl of 1 µM [3H]TC or 5 nM [3H]preS1, respectively. After 10 min, experiments were stopped by washing the cells 5 times with ice-cold PBS. Cell-associated radioactivity was quantified by liquid scintillation counting as described above. [3H]TC uptake and [3H]preS1 binding rates were determined in pmol/mg protein/10 min. Means of the 0% controls were subtracted and the net [3H]TC uptake and net [3H]preS1 binding rates, respectively, were expressed as % of control. Data were calculated from two independent experiments, each with quadruplicate determinations by GraphPad Prism 6 .NTCP-expressing HepG2 hepatoma cells were seeded onto rat tail collagen coated 24-well plates, induced with 2 µg doxycycline per ml, and grown to confluence over 72 h at 37 °C. For experiments, cells were preincubated with a fix concentration (600 µM) of compound Pierce lactate dehydrogenase (LDH) cytotoxicity assay (Thermo Fisher Scientific) was performed according to the manufacturer’s protocol. Briefly, NTCP-HepG2 cells were incubated with 300 µM or 600 µM of the respective betulin derivatives over 6 h at 37 °C. 45 min before the end of incubation, lysis buffer was added to the maximal LDH control. After 6 h, 50 µl of each sample and control medium were transferred to a new well plate, 50 µl of the reaction mixture were added and incubation was performed over 30 min at room temperature. Finally, the reaction was stopped by adding 50 µl of stop solution and samples were measured by ELISA reader .4 and 8.2D compound structures were generated using the MAESTRO 12.2 Molecular Modeling Interface of SCHRÖDINGER, Inc. . The Jaguar pKa prediction module of the SCHRÖDINGER software was used to calculate pKa values of compounds 50 values was done by nonlinear regression analysis using the equation log(inhibitor) vs. response settings of the GraphPad Prism 6.0 software (GraphPad). Data of bile acid transport and [3H]preS1 binding are expressed as means ± SD from quadruplicate determinations. IC50 values are listed with their 95% confidence intervals in Table 50 values were calculated for each experiment. Infection studies show data from three independent experiments, each with triplicate determinations represented as means ± SEM. Statistical analyses of the HDV infection experiments and NTCP-HepG2 preincubation studies were performed by two-way ANOVA, followed by Dunnett's multiple comparison test by GraphPad Prism 6.0, considering p < 0.01 as statistically significant.Determination of IC


Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the  The faithful replication of the genome by the DNA replication machinery is hindered by a range of exogenous and endogenous factors that are capable of disrupting replication forks. Such cellular events are a prominent feature of the early stages of cancer and have been collectively referred to as replication stress (RS) . RS occuOn encountering RS, the replisome is thought to be stabilised at the replication fork until the impeding context is removed or bypassed and replication restarted . A failuSaccharomyces cerevisiae) are activated by the accumulation of excessive RPA-coated single stranded DNA (Sc Mrc1) (Sc Tof1)   and Tof1Sc Tof1) .Sc Csm3) are required to stimulate DNA replication in vivo and in vitro (in vivo (tof1Δ and csm3Δ (but not mrc1Δ) cells are acutely sensitive to the chemotherapeutic drug camptothecin (CPT), which stabilises the covalently DNA-bound intermediate of Top1  and are listed in Table All yeast strains in this study were generated in the W303 background    by NotI Saccharomyces cerevisiae S288C Chromosome XII: 459799–460920. This was assembled with PfoI-linearized pRS426 using the NEBuilder® HiFi DNA Assembly Cloning Kit .Plasmid pRS426-RFB was generated by a two-fragment Gibson assembly. Specifically, a single RFB site was PCR-amplified from plasmid 4xRFB (gift from Luis Aragon) corresponding to the sequence from top2–4 pRS316-containing strains, cells were grown to mid-log phase in synthetic complete media without uracil +2% glucose, before being re-suspended in YP 2% Glucose (YPD). Cells were arrested in G1 by addition of 10 μg/ml alpha factor peptide (Genscript) for 1.5 h, after which a second dose of alpha factor (5 μg/ml) was added. When >90% of cells were unbudded, cultures were shifted to the restrictive temperature for top2–4 (37°C) 1 h before release into S-phase achieved by washing three times with YPD at 37°C. Time 0 indicates the time from addition of the first wash. 50 μg/ml nocodazole was added to cultures at 45 min to prevent mitotic entry, and at 80 min from release 10 ml samples for 2D gel and Southern blotting analysis were collected by centrifugation and snap-freezing the pellets in liquid nitrogen.For plasmid catenation experiments in For fork pausing experiments cells containing pRS426-RFB were grown to mid-log phase in synthetic complete media without uracil +2% glucose. 10 ml cultures were collected by centrifugation and snap-freezing the pellets in liquid nitrogen.For experiments involving treatment with HU, cells were grown in YPD to mid-log phase before either HU being added to 200 mM for 2 h  for 45 min at 25°C. 125 mM glycine was then added followed by a 5 min incubation at 25°C. Cells were washed with PBS before being pelleted and snap-frozen in liquid nitrogen.10 ml of mid-log yeast cultures were centrifuged at 3500 rpm for 5 min and the resulting cell pellets were snap-frozen in liquid nitrogen before storing at −80°C. All further steps were carried out on ice, and centrifugation and homogenisation steps at 4°C. 200 μl of 20% TCA was added to thawed cell pellets and the cell suspension was transferred to screw-cap tubes containing 500 μl of 0.5 mm zirconia/silica beads (BioSpec Products). Cells were homogenised using a FastPrep-24  on max speed (6.5 m/s) for 4 × 1 min pulses, with 1 min on ice in between pulses. Beads were separated from the lysate by piercing the tubes and centrifugation of the mixtures into fresh tubes at 3000 rpm for 2 min. Beads were washed once with 600 μl 5% TCA and centrifuged again into tubes containing the cell lysate. Cell extract/TCA mixtures were then centrifuged at 13 000 rpm for 5 min before removing all TCA from the resulting pellets. This step was repeated once to ensure complete removal of all TCA. To the pellets 200 μl of 1× sample buffer was added before boiling samples for 5 min. Samples were spun at 13 000 rpm for 5 min and the resulting supernatants were collected and stored at −20°C for SDS-PAGE and western blotting analysis.Protein extracts prepared by TCA extraction were run on either 8%, 10% or 12% SDS-PAGE gels before being wet-transferred to nitrocellulose membranes at 50V for 90 min, 4°C. Proteins were visualised by staining in Ponceau for 30 seconds before membranes were blocked in 5% milk (Marvel) PBS 0.2% Tween-20 (PBS-T). All primary antibodies were diluted in 5% milk PBS-T and incubated overnight at 4°C. In between primary and secondary antibody incubations membranes were washed 3 times with PBS-T for 15 min. Secondary antibodies were diluted in 5% milk PBS-T and incubated with membranes for 1 h at room temperature. Proteins were detected using Western Lightning Plus-ECL  and images were acquired on an ImageQuant LAS4000 system . Densitometry analysis was carried out using ImageQuant TL software.Primary antibodies used for immunoblotting in this study: OctA-Probe Antibody 1:1000 (for detecting FLAG-tagged proteins) (sc-166355), anti-Csm3 , anti-PA® Chromatography Columns (Bio-Rad #7311550) and the bound fraction was eluted in 0.5 ml of 0.2 M glycine pH 3.0. For Top1 pulldowns in 200 ml of mid-log yeast cultures were centrifuged for 5 min at 3500 rpm and washed once in ice-cold PBS. The resulting cell pellet was snap-frozen in liquid nitrogen and stored at −80°C before proceeding. Frozen pellets were re-suspended in 0.5 ml of ice-cold lysis buffer  and 1 phosphatase inhibitor tablet  per 50ml of lysis buffer) in screw-cap tubes. 500 μl of zirconia/silica beads (BioSpec Products) were added and the cell suspensions were homogenised using the FastPrep-24  on max speed (6.5 m/s) for 4 pulses of 1 min each, with 3 min on ice in between each pulse. Beads were separated from the lysate by piercing the tubes and centrifugation into fresh tubes at 3000 rpm for 2 min. For Csm3 pulldowns in Figure Frozen pellets were re-suspended in 0.4 ml of DNA Extraction Buffer  along with 40 Units of lyticase  and 5 μl 2-mercaptoethanol . Samples were incubated at 37°C for 5 min before addition of 450 μl phenol/chloroform/isoamylalcohol  and mixing by rotation. Phase lock tubes  were used to collect the aqueous layer, by centrifugation for 5 min at 12 000 rpm. DNA was ethanol precipitated by addition of roughly 2× volume of 100% EtOH and washed once in 70% EtOH before air-drying and solubilisation in 10 mM Tris pH 8.0.For plasmid DNA catenation analysis of pRS316, purified DNA was nicked with Nb.Bsm1  according to manufacturer's instructions.For fork pausing analysis of pRS426-RFB, purified DNA was digested with BamHI-HF  and SnaBI  or SnaBI alone according to manufacturer's instructions.After nicking/digestion the DNA was precipitated, washed and solubilised as above with the addition of 300 mM Sodium Acetate pH 5.2 at the first ethanol addition.Purified DNA was separated in the first dimension by electrophoresis in 0.4% MegaSieve/MegaBase Agarose , 1× TBE .top2–4 cells, first dimension gels were run at room temperature for 16–18 h at 30 V. A lane of the gel containing a small amount of each DNA sample was excised and stained in 0.5 μg/ml Ethidium Bromide 1× TBE to reveal extent of genomic DNA mobility. The remaining non-stained gel slices containing the plasmid were excised and embedded in 1.2% MegaSieve/MegaBase Agarose 1X TBE and run in the second dimension in 1× TBE at 4°C for 16–17 h, 120 V.For DNA catenation analysis of plasmid pRS316 from For analysis of paused replication intermediates from plasmid pRS426-RFB, first dimension gels were run at room temperature for 15.5 h at 30 V. First dimension gels were stained in 0.5 μg/ml Ethidium Bromide in 1× TBE and gel slices containing the replication intermediates were excised and embedded in 1% MegaSieve/MegaBase Agarose 1× TBE 0.3 μg/ml Ethidium bromide gels. Second dimension gels were run in 1× TBE at 4°C for 8 h, 120 V with re-circulation of the running buffer.2O in-between each buffer. DNA was transferred onto Hybond-N+ membrane  by capillary action in 20× SCC . Membranes were cross-linked using a UV Stratalinker 1800 (Stratagene) at 1200 J/m and subsequently blocked in hybridisation buffer  0.2% Tropix I-Block , 0.1% SDS) for at least 1 h at 60°C.Following 2D electrophoresis, gels were washed sequentially in depurination buffer (0.125 M HCl), denaturation buffer  and neutralisation buffer  with washes in ddHURA3 gene). Labelling and detection used random prime labelling incorporating fluorescein tagged dUTP (Roche). Following probing, hybridized fluorescein tagged dUTP was detected with alkaline phosphatase tagged anti fluorescein Fab fragments (Roche), revealed with CDP-Star  and non-saturating exposures acquired on an ImageQuant LAS4000 system . Densitometry analysis was carried out using ImageQuant TL software.Catenated pRS316 plasmids or replication intermediates from pRS426-RFB were probed with DNA amplified from pRS316  was added. Cells were incubated overnight at 37°C, pelleted and re-suspended in freshly made 5 mg/ml pepsin (Sigma) in 5 mM HCl and incubated again at 37°C for 30 min. Fixed cells were washed once more in 50 mM Tris pH 8.0 and re-suspended in 0.5 mg/ml propidium iodide in 50 mM Tris pH 8.0. Samples were sonicated for 5 s each on low power to reduce clumping before analysis using the BD Accuri™ C6 Plus (BD Biosciences).Yeast cells were grown to mid-log phase before being serially diluted 10-fold in YPD. 5 μl of each dilution was spotted onto YPD plates containing the indicated dose of drug or control reagent and incubated for 48 h at 25°C or 30°C before imaging.Yeast cells were grown to mid-log phase before being arrested in G1 by the addition of 10 μg/ml alpha factor peptide. When cells were >90% unbudded they were released into the cell cycle in the presence of 200 mM HU for 1 h. Following the HU treatment cells were counted, diluted in YPD medium and plated onto YPD plates. Colonies were counted 48 h after plating and the viability was calculated as the percentage of plated cells able to form colonies. Statistical significance was calculated using a two-tailed unpaired Students t-test.Pellets from 75 ml cultures were resuspended in 500 μl SDS buffer , PhosSTOP (Roche)). 200 μl of 0.5 mm zirconia/silica beads were added to samples and cells were lysed using the FastPrep-24  on max speed (6.5 m/s), with five rounds of 1 min each. Lysate was spun out and IP buffer , cOmplete Tablets Mini EDTA-free EASYpack (Roche), PhosSTOP (Roche)) was added to a final volume of 1 ml. Samples were sonicated using the Focused-Ultrasonicator (Covaris) . The sample was centrifuged for 20 min at 13 000 rpm at 4°C. Supernatant was then diluted to 7.5 ml with IP buffer. 75 μl protein A Dynabeads (Invitrogen) and 75 μl protein G Dynabeads (Invitrogen), were washed 3 times in IP buffer before adding to the sample and incubating for 2 h at 4°C. 2 ml of the supernatant was taken to 15 ml falcon tubes, and the rest was kept at −20°C as an input sample. To the 2 ml sample RPA1 antibody  was added followed by overnight incubation on a rotating wheel at 4°C.3) for 30 min at room temperature. At the same time 50 μl from the input sample was added to 150 μl of elution buffer. 20 μl of 5 M NaCl and 10 μl of 10 mg/ml proteinase K (Invitrogen) was then added to the input, and 40 μl and 20 μl to the IP samples respectively. These were incubated at 65°C overnight. Then 10 μl of DNase-free RNase (Roche) was added to the input and 20 μl to the IP samples, and they were left at 37°C for 30 min. All DNA was purified with a Qiagen PCR purification kit and eluted in 40 μl H2O. 34 μl from the RPA1 samples and 1 ng DNA in 34 μl water from the input were used for library preparation. 5 μl 10× NEB2.1 buffer and 5 μl of random primers  were added and the samples were boiled at 95°C for 5 min and immediately placed on ice for 5 min. 5 μl 10× dNTPs with dUTP instead of dTTP (2 mM each) and 1 μl T4 polymerase (NEB) were added and the mixture was incubated at 37°C in a thermal cycler for 20 min, and 5 μl 0.5 M EDTA (pH 8) was immediately added to stop the reaction. The resulting dsDNA was used to create libraries using the Ultra II library kit (NEB) as per the manufacturer's instructions with 13 cycles at the amplification step.A mix of Dynabeads, Protein A (30 μl) and Protein G (30 μl), was washed 3 times in IP buffer. This was added to each sample and incubated at 4°C for 4 h. Supernatant was removed and beads were washed at 4°C for 6 min in TSE-150 , 150 mM NaCl), followed by TSE-500 , 500 mM NaCl), followed by LiCl wash ) and finally Tris-EDTA (TE pH8). Elution was carried out in 400 μl elution buffer  or NextSeq 500 (42 bp reads from each side) systems to result >2 million reads.https://basespace.illumina.com/home/index). The resulting sequences were aligned to a reference genome  using Bowtie 2 generating a SAM output file for each sample (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Reads from MySeq were trimmed 25 bp from 3′ and 1 bp from the 5′ end, while reads from NextSeq were not trimmed.FASTQ files were generated by Illumina basespace (Command for MySeq reads:bowtie2 -p 14 -x [path to index folder] –trim3 25 –trim5 1 -1 [Path and name of R1 fastq file] -2 [Path and name of R2 fastq file] -S [name of the resulting .sam file]Command for NextSeq reads:bowtie2 -p 14 -x [path to index folder] –trim3 0 –trim5 0 -1 [Path and name of R1 fastq file] -2 [Path and name of R2 fastq file] -S [name of the resulting .sam file]http://samtools.sourceforge.net/):SAM files were then converted into sorted BAM files by using SAMtools (samtools sort [name of the .sam file generated with bowtie2] -o [name for the resulting .bam file] -O bam -T [name for resulting .bam file wo .bam]https://broadinstitute.github.io/picard)Duplicates were then removed using picard . We used the ‘call peak’ function which also generates genome wide score data. These were used to generate fold enrichment tracks. Example command:macs2 callpeak -t [sorted BAM file from yh2a data]-c [sorted BAM file from h2a data]-f BAMPE -g 12100000 -n [name for output file] -B -q 0.01 –SPMRThe data then was sorted into 50 bp bins, normalized to have a mean value of 1, and used for meta data analysis using custom-made R programs.Regions within 10 kb of LTR-retrotransposons, transposable element genes, rRNA genes and long terminal repeats were removed from the meta analysis as these regions have high RPA enrichment.2O. 50 ng of DNA in 50 ul water was used for library preparation using the Ultra II library kit (NEB) as per the manufacturer's instructions with 6 cycles at the amplification step.Pellets from 2 ml cultures were resuspended in 500 μl SDS buffer , PhosSTOP (Roche)). 200 μl of 0.5 mm zirconia/silica beads were added to samples and cells were lysed using the FastPrep-24  on max speed (6.5 m/s), with five rounds of 1 min each. Lysate was spun out and IP buffer , cOmplete Tablets Mini EDTA-free EASYpack (Roche), PhosSTOP (Roche)) was added to a final volume of 1 ml. Samples were sonicated using the Focused-Ultrasonicator (Covaris) . 200 μl of sample was removed and 10 μl of DNase-free RNase was added and incubated at 37°C for 30 min. DNA was then purified with a Qiagen PCR purification kit and eluted in 50 μl HPaired end sequencing was performed using NextSeq 500 (42 bp reads from each side) systems to result >2 million reads.https://github.com/DNAReplicationLab/.Sync-seq analysis  was carrlocalMapper.sh -g [path to index folder] [Path and name of R1 fastq file] -2 [Path and name of R2 fastq file] -s [name of the output files] -w 3000 -c 14The resulting .bed files were then read in to R:repBed ← loadBed(file name for the replicating sample)nrepBed ← loadBed(file name for the non-replicating sample)Outliers were removed:repBed←rmOutliersrepBed←rmOutliersnrepBed←rmOutliersnrepBed←rmOutliersRatio between non-replicated and replicated samples were calculated:ratio ← makeRatioAnd normalised:ratio ← normaliseRatiowhere rFactor was empirically determined to fit the lowest replicating regions to 1.The resulting ratios were smoothed by a moving average of 7 and plotted using custom-made R programs. TOF1 ORF sequence optimised for budding yeast expression  long Tof1 protein are required for each of its characterised functions we generated a series of premature stop codons in a pression  and the 1 Figure . We noten Figure . The sizpression . We onlytof1Δ cells, as visualised by Southern blotting of replication products  (corresponding to Chromosome XII sequence: 459799–460920) into the multicopy yeast episomal plasmid pRS426 . We. WeS. ce6 Figure , whereasn Figure . Expressg Figure . However Figures . TogetheTOF1 results in hypersensitivity to CPT, an agent which stabilises the Topoisomerase 1 covalent complex (Top1cc) to DNA, forming a DNA protein cross-link (DPC). Deletion of CSM3 results in similar sensitivity to CPT treatment while mrc1Δ cells display only mild sensitivity to this agent  have normal levels of resistance to CPT. Conversely, tof1 truncations that do not support replication fork pausing (tof1 627 and tof1 762) cannot suppress CPT sensitivity. We conclude that CPT sensitivity is closely associated with the ability of the Tof1-Csm3 complex to support pausing of the fork at a protein block to replication.Deletion of is agent . Therefond break ,32. Altend break , potentiT Figure . Therefotof1 997 expressing cells. Addition of a C terminal TAP tag did not adversely affect all regions of the protein, as the tagged forms of TOF1 or tof1 627 did not appear to be more sensitive to CPT than their untagged forms  also requires Tof1 function. Following acute treatment of cells with HU both Tof1 and Mrc1 are required to maintain the coupling of helicase and polymerase activities at the fork . In the e Figure . Expresss Figure . Thus, es Figure , indicatS. cerevisiae sustained activation of the effector kinase Rad53 in S phase following HU treatment can either be through the DRC, mediated by Mrc1 and Tof1, or through the Rad9-mediated DDC . We conclude that Csm3 is not essential for DRC activation but that its presence likely stabilises the DRC proficient conformation of the replisome.Our phenotypic analysis of the s Figure . We did Defining how the Timeless protein functions to maintain genome stability, both generally and following RS has been complicated by its multiple functions. Here we have shown that distinct regions of Tof1 are required for its roles during DNA replication.in vivo interaction of Tof1 and Top1  of Tof1 is required to suppress excessive fork rotation in cells. This region is also sufficient to mediate a two-hybrid interaction with the type 1B topoisomerase Top1  and is rand Top1   of Tof1 is sufficient for intra-S-checkpoint signalling in response to HU as assayed by the Rad53 phospho-shift assay. Both Mrc1 and Tof1 are required for efficient checkpoint signalling in HU ,10. In m of Csm3  whereas  of Csm3 ,44. Duri of Csm3 , and Tof of Csm3 ,16, we f of Csm3 . Since ctof1Δ cells also suggests that the replisome structure formed in the absence of Tof1 may not be proficient in replication following restart after RS. The phenotype of the separation of function mutation tof1 627, which is proficient for checkpoint signalling but not proficient for replisome coupling or fork pausing, indicates that Tof1 has important roles in completing DNA replication following RS beyond purely mediating DRC activation. Although TOF1 and tof1 627 cells appear to have equivalent levels of Rad53 mobility shift, we cannot exclude the possibility that the cellular activity of the DRC may be different in the two conditions. Nevertheless, our data argues that Tof1 functions separate from DRC activation are required to stabilise replication forks following resumption of DNA replication after RS. The reason for fork failure following resumption of DNA replication is unclear but we speculate that tof1 mutants deficient in coupling helicase and polymerase activity in HU are also defective in efficient recoupling during recovery from HU treatment, leading to frequent fork failure during elongation. Notably, the sensitivity of tof1 mutants to HU is highly dependent on the activity of Rad9, consistent with Rad9 dependent repair pathways being required to resolve tof1 linked DNA lesions. Although we show that Tof1 has roles in recovering from RS beyond mediation of the DRC, we cannot rule out a role for Tof1 downstream of DRC activation. The phosphorylation state of Tof1 is partially dependent on Mec1/Tel1 activity All raw and processed data are available at the Gene Expression Omnibus (GEO) under the accession number: GSE144321 (The data on UCSC can be reached on this link:https://genome.ucsc.edu/s/a.keszhelyi/Tof1_Westhorpe.gkaa963_Supplemental_FileClick here for additional data file.