Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. Kinesins are a superfamily of microtubule-dependent ATPases that play important roles in the parasite replicative machinery, which is a potential target for antiparasite drugs. Kinesin-5, a molecular motor that cross-links microtubules, is an established antimitotic target in other disease contexts, but its mechanism in Plasmodium falciparum is unclear. Here, we characterized P. falciparum kinesin-5 (PfK5) using cryo-EM to determine the motor's nucleotide-dependent microtubule-bound structure and introduced 3D classification of individual motors into our microtubule image processing pipeline to maximize our structural insights. Despite sequence divergence in PfK5, the motor exhibits classical kinesin mechanochemistry, including ATP-induced subdomain rearrangement and cover neck bundle formation, consistent with its plus-ended directed motility. We also observed that an insertion in loop5 of the PfK5 motor domain creates a different environment in the well-characterized human kinesin-5 drug-binding site. Our data reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as antiparasite targets. With resistance to current frontline therapeutics rapidly rising (https://apps.who.int/iris/handle/10665/274362)  inhibitors have been characterized that block motor ATPase activity and bind to allosteric sites in the motor domain. The best studied of these allosteric binding pockets is defined by kinesin-5-specific sequences in a key structural region of the motor domain, loop5 —the deadliest form of human malaria—could be a selective antimalarial therapeutic target, we characterized the biochemical properties and MT-bound structure of the PfK5 motor domain. We show that the PfK5 motor domain is an ATPase with MT plus-end directed motility, as demonstrated in MT gliding experiments. MT-bound structures of the PfK5 motor domain were determined using cryo-electron microscopy (cryo-EM), and processed in our RELION-based MT image processing pipeline  contains a 105 amino acid asparagine and lysine-rich insertion in loop6 that is characteristic of malaria proteins , but which is poorly conserved (16–30% sequence identity) among Plasmodium kinesin-5s. We engineered loop6 out of our construct, an approach previously taken by another group .To characterize proteins  , but addition of MTs stimulated PfK5ΔL6-MD ATPase activity (C). Changes in pH or ionic strength showed no or minimal impact respectively on PfK5ΔL6-MD ATPase rate (A and B). From these data, the motor kcat and km of MTs (kMT) for PfK5ΔL6-MD were calculated. The km of ATP (KATP) was also determined (D). PfK5ΔL6-MD has a MT-stimulated ATP hydrolysis rate of 0.13 ATP s−1, which is slow compared with kinesin-5 from Sus pombe (Sus cerevisiae (H. sapiens (−1 respectively). However, PfK5ΔL6-MD has similar kMT (5.4 μM) and KATP (9.5 μM) values compared with previously characterized kinesin-5s , purified to 92% purity (A), covalently labeled with biotin, and attached to a neutravidin-coated surface. The velocity of fluorescently labeled MTs driven by PfK5ΔL6-MD-SNAP activity was measured. PfK5ΔL6-MD-driven MTs moved at an average velocity of 5.4 nm/s  (B and S1C). This is slow compared with 23–92 nm/s reported for HsK5-MD (PfK5ΔL6-MD is a plus-end directed motor (B).To determine the motile properties of % purity A, covale= 5–5.9) B and S1C HsK5-MD , 24, 25.ed motor B.Figure PfK5ΔL6-MD, we analyzed the interactions of single molecules of fluorescently labeled PfK5ΔL6-MD-SNAP with MTs. We made single-molecule measurements in different nucleotide conditions, to investigate how MT affinity changes with nucleotide state (C and S1D), using the nonhydrolyzable ATP analogue AMPPNP to mimic the ATP bound state. From these data, we calculated the MT association rate, or kMTlanding (D), and MT dissociation rate, or koff of PfK5ΔL6-MD (E). This demonstrated that in saturating ADP conditions, PfK5ΔL6-MD-SNAP had a comparatively low kMTlanding and a high koff, indicating that PfK5ΔL6-MD-SNAP has low MT affinity when bound to ADP (F). In the absence of nucleotide or in saturating AMPPNP conditions, PfK5ΔL6-MD-SNAP had a comparatively high kMTlanding and a low koff, showing that when no nucleotide is present, or in its ATP-bound state, PfK5ΔL6-MD-SNAP has high MT affinity.To provide some context for the slow ATPase and MT gliding activity we observed for de state C and S1DTlanding D, and MTK5ΔL6-MD E. This dd to ADP F. In theMTlanding was higher compared with the ADP state; however, koff was similar, suggesting that PfK5ΔL6-MD-SNAP also has relatively low MT affinity in the presence of ATP. Overall, we observe that PfK5ΔL6-MD-SNAP has high MT affinity in the no nucleotide and AMPPNP states compared with ADP and ATP saturating conditions, which is comparable to HsK5 , our previously developed pipeline for image processing of MTs with RELION (https://github.com/moores-lab/MiRPv2).To gain molecular insight into the behavior of g AMPPNP . To do tg AMPPNP , A and Bh RELION , 27. As PfK5ΔL6-MD binds MTs every 8 nm (one PfK5ΔL6-MD per αβ-tubulin) on the ridge of MT protofilaments (A), a binding site shared by all other kinesins characterized to date (B and S2C) and AMPPNP states (C and S2C) at overall resolutions of 4.8 Å and 4.5 Å, respectively. The resolution in the MT region of both reconstructions is approximately 4.5–5.5 Å—in contrast, the resolution of PfK5ΔL6-MD exhibits a marked falloff as a function of distance from the MT surface (C). This is typical of kinesin-MT reconstructions (PfK5ΔL6-MD protein itself. We therefore used symmetry expansion and 3D classification focused on PfK5ΔL6-MD from one PfK5ΔL6-MD:αβ-tubulin dimer asymmetric unit to select data with the best PfK5ΔL6-MD occupancy (A). We rejected classes that had a clear absence of PfK5ΔL6-MD density, as well as classes that did not contain continuous motor density (B). The resulting 3D structures  were substantially improved, as shown by reduced resolution decay in the motor domain density (C and D). The average resolution of these reconstructions was 4.4 and 4 Å for the no nucleotide and AMPPNP states, respectively, with PfK5ΔL6-MD having a resolution range of 4.5–7 Å.ilaments A, a bind to date , 29. MiR to date B and S2CP states C and S2C surface C. This iructions , 31 and ccupancy A. We rej density B. The re density , C and DA). However, to facilitate interpretation of the differences, we also calculated a PfK5ΔL6-MD homology model and performed flexible fitting of this model within the cryo-EM density, in which individual secondary structure elements were well resolved (B). Local cross-correlation scoring showed overall improvement of model fit to density as a result of flexible fitting (C) and produced molecular models of each of the PfK5ΔL6-MD:αβ-tubulin dimer complexes  and a β-hairpin (β-lobe2) (F and G). Using these models, we analyzed conformational differences between the no nucleotide and AMPPNP states.Alignment and superposition of our reconstructions directly reveal nucleotide-dependent differences in these structures A. Howeveresolved B. Local  fitting C and proomplexes . These pβ-lobe2) , F and GPfK5ΔL6-MD nucleotide-binding site (NBS) is located away from the MT surface, and despite the overall low sequence conservation of PfK5ΔL6-MD compared with HsK5 . In addition, density corresponding to the C-terminal end of loop11, which is approximately 19 Å from the NBS, and contains a two residue Plasmodium-conserved insertion, is not visible. In the no nucleotide state, density corresponding to loop9 and the P-loop is well separated, while density is observed connecting loop9 and 11 (A). These configurations create an “open” NBS primed for ATP binding formed by a cavity between these three loops. Docking of a model of no nucleotide MT-bound kinesin-1 . In contrast to the no nucleotide state, in the AMPPNP state, there is clear density corresponding to the bound nucleotide in the PfK5ΔL6-MD NBS (A and S5B). In addition, the nucleotide is surrounded by loop9 and 11, which have closed around the bound nucleotide to form an NBS that supports ATP hydrolysis (A) .Fi.FiPfK5ΔLPfK5ΔL6-MD no nucleotide and AMPPNP state models by alignment on αβ-tubulin allows visualization of the structural response of the PfK5ΔL6-MD NBS to AMPPNP binding (B), while docking of the AMPPNP state model into nucleotide density and vice versa confirms the structural differences between the two states (C). Even while the resolution is not sufficient to determine the exact conformation of each of these loops, it is very clear that in the presence of AMPPNP, all three NBS loops move, with loop9 and the P-loop coming closer together, thereby burying the nucleotide. In summary, AMPPNP binding to PfK5ΔL6-MD causes a conformational rearrangement that forms a closed, catalytically competent NBS.Superimposing the  binding B, while o states C. Even wPfK5ΔL6-MD? The structure of PfK5ΔL6-MD can be subdivided into three distinct subdomains (C). The tubulin-binding subdomain  consists of MT-binding elements in which helixα4 binds a shallow cavity at the intratubulin dimer interface. In addition, helixα5 and β-lobe2 contact β-tubulin. The remaining two subdomains, the P-loop subdomain  and switch-I/II subdomain , contain approximately half of the central β-sheet each, along with adjacent secondary structure elements. The NBS is located at the junction of these subdomains .What are the consequences of these NBS rearrangements on the structure of bdomains , which abdomains C. The tuD). The P-loop subdomain pivots such that helixα0 moves toward the MT surface, while helixα6 and the majority of the subdomain move away from the MT surface. The switch-I/II subdomain rotates such that its constituent secondary structure elements move toward β-tubulin.We measured the relative rotation of each helix  in the tFig. 4PfK5ΔL6-MD? Approximately 18 Å away from the MT surface, and 27 Å away from the NBS, is the neck linker. The neck linker is a C-terminal peptide extending from helixα6, which links the motor domain to the kinesin stalk, and which is relatively conserved in the kinesin superfamily (A). In the PfK5ΔL6-MD no nucleotide reconstruction, no density is observed extending from helixα6, indicating that the neck linker is disordered in this nucleotide state (A and S6B). At equivalent thresholds in the AMPPNP state, however, there is clear density corresponding to the neck linker at the C-terminus of helixα6 that extends along the motor domain in the direction of the MT plus-end (B). Docking of the AMPPNP state model into no nucleotide density confirms a lack of density to accommodate the neck linker, while density is unaccounted for when the no nucleotide model is docked in AMPPNP density (C). In addition, density corresponding to the PfK5ΔL6-MD N-terminus is also visualized in the AMPPNP state; this is consistent with formation of backbone interactions between this region and the neck linker, to form short β-strands known as the CNB, which is characteristic of plus-end directed kinesins . No such CNB density is observed in the no nucleotide reconstruction (B). Neck linker docking is enabled by the above-described rotation of the P-loop subdomain, which moves the N-terminus and the central β-sheet away from helixα5/loop13 in the static MT-binding subdomain (D)  , 40. Thimotility .Figure 5PfK5ΔL6-MD binds to one αβ-tubulin dimer, with helixα4 centered at the intradimer interface (A and B). At the PfK5ΔL6-MD-binding site, the sequence of the Sus scrofa αβ-tubulin used in our reconstruction is identical to that of P. falciparum αβ-tubulin . This analysis shows that helixα4 interacts with both α- and β-tubulin, while βlobe1, loop11, and helixα6 interact with helices 4, 5, and 12 of α-tubulin, and loop7, βlobe2, and helixα5 interact with similar secondary structure elements in β-tubulin.nterface , A and B-tubulin , facilit-tubulin C. This aPfK5ΔL6-MD-MT interface is similar between the no nucleotide and AMPPNP states  and is longer compared with HsK5 (A). In the no nucleotide state of PfK5ΔL6-MD, some very poorly defined density corresponding to loop5 can be seen at a low threshold (B), suggesting that this region is largely disordered in this state.Loop5 plays an important role in the mechanochemistry of HsK5 , 16, andhibitors . It is aith HsK5 A. In thehreshold B, suggesC). This region is at lower resolution than other parts of the reconstruction, possibly owing to intrinsic flexibility, but also possibly because of the residual resolution gradient toward the outer surface of PfK5ΔL6-MD (C). There is no secondary structure-like density in this region of the motor, consistent with sequence-based predictions . This also suggests that, although residues outside of loop5 involved in interactions with STLC are largely conserved between HsK5 and PfK5 (E), loop5 of PfK5ΔL6-MD might alter the environment of this putative drug-binding site. To test this idea, we measured whether the ATPase activity of PfK5ΔL6-MD was susceptible to inhibition by STLC . Thus, despite the conserved aspects of PfK5ΔL6-MD mechanochemistry uncovered by our data, evolutionary divergence between PfK5ΔL6-MD and HsK5 mediates differential inhibition of these kinesin-5 motors.Strikingly, however, the density corresponding to loop5 in the AMPPNP state does not protrude away from the surface of the motor but appears to cover the site between helicesα2 and 3, equivalent to the well-described inhibitor-binding site in  density D. This aand PfK5 E, loop5  by STLC . ConsistK5ΔL6-MD F. Thus, P. falciparum kinesin-5 PfK5ΔL6-MD construct shares with HsK5 a comparatively slow MT-stimulated ATPase, plus-end directed MT gliding activity, and nucleotide-dependent conformational changes that support plus-end directed motility. Significantly, however, our structures revealed a different configuration of the well characterized loop5-defined drug-binding pocket. Further, we also showed that PfK5ΔL6-MD exhibits no sensitivity to the classical HsK5 loop5-binding drug STLC.We have determined the biochemical properties and MT-bound cryo-EM structures of a spindle-associated kinesin-5 motor from the malaria parasite. Despite considerable divergence from the human host kinesin-5 sequence, our PfK5ΔL6-MD is ∼340 times slower than H. sapiens kinesin-1  was expressed and purified, but with low yield due to precipitation during purification. Therefore, PfK5MD was engineered such that 105 amino acids of the asparagine/lysine-rich insertion in loop6 (residues 175–269) were removed. The resulting construct, which we refer to as PfK5ΔL6-MD, was cloned in a pET-151D-TOPO vector (Invitrogen) with an N-terminal His6-tag and TEV protease cleavage site, and each preparation was expressed in 12 L BL21 Star DE3 Escherichia coli cells (Invitrogen) grown in LB media. Cells were grown at 37 °C until they reached an optical density of 0.8–1.0 and were then induced with 0.1 mM IPTG for 3 h at 26 °C. Cells were harvested  and stored at –80 °C.The 2, 2 mM DTT, 1 mM ATP, 10 mM imidazole pH 8), which was supplemented with 2× cOmplete EDTA-free protease inhibitor tablets (Roche), 15 μg/ml DNase I (Roche), 0.5 mg/ml Lysozyme, 10% v/v glycerol. The lysate was then clarified by centrifugation . PfK5ΔL6-MD was purified from the clarified lysate at 4 °C using an ÄKTA Pure  in 1 day, which reduced loss of protein due to aggregation. First, nickel affinity chromatography was performed where the lysate was loaded onto a 5 ml HisTrap Excel column , followed by a wash with 20 column volumes (CV) of IMAC W, then reverse elution with 10 CV of IMAC E buffer . The eluate from this step was concentrated to 10–15 ml with a Vivaspin concentrator (Sartorius). Concentrated sample was then exchanged into IEX W buffer  using a HiPrep 26/10 desalting column . Next, anion exchange chromatography (1 ml HiTrap Q HP) was performed, where the flow-through and wash fractions containing PfK5ΔL6-MD were pooled. The His6-tag was then cleaved by incubation with 100 μg/ml TEV protease for 2–4 h at 4 °C. TEV-protease and any remaining contaminants were removed using nickel affinity chromatography (1 ml HisTrap HP), where flow-through and wash fractions containing PfK5ΔL6-MD were pooled. PfK5ΔL6-MD was concentrated to 100–150 μl then, using 0.5 ml Zeba 7K MWCO spin columns, was exchanged into T50K20 buffer . This purified PfK5ΔL6-MD was snap frozen in liquid nitrogen and stored at –80 °C. PfK5ΔL6-MD purity was measured using Coomassie-stained SDS-PAGE band intensity measurement in Fiji (Cells were lysed with three passages through a C3 homogenizer (Avestin) in 100 ml IMAC W buffer  for use in total internal reflection microscopy (TIRFM) experiments. Expression and purification of PfK5ΔL6-MD-SNAP were performed as for PfK5ΔL6-MD, except the anion exchange chromatography step was altered as follows: concentrated and desalted sample was added to a 1 ml HiTrap Q HP column, washed with 10 CV IEX W buffer, and eluted with a 20 CV gradient elution to 500 mM NaCl. Eluted fractions containing PfK5ΔL6-MD-SNAP were pooled. PfK5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to PfK5ΔL6-MD-SNAP. Free SNAP-ligand was removed by two repeats of buffer exchange in T50K20 buffer (0.5 ml Zeba 7K MWCO spin columns).Gibson assembly  was usedS. scrofa brain tubulin—except X-rhodamine, which was from Bos taurus— was reconstituted to 10 mg/ml in BRB80 , centrifuged at 611,453g for 10 min at 4 °C, and the supernatant snap frozen in liquid nitrogen and stored at –80 °C. Double cycle GMPCPP polymerization was performed as follows: reconstituted tubulin was supplemented with 1 mM GMPCPP (Jena Biosciences) and incubated for 5 min on ice. Tubulin was then polymerized at 4–5 mg/ml for 20 min at 37 °C. MTs were then pelleted at 611,453g for 10 min at 23 °C, washed twice, then resuspended, both with BRB80. MTs were then depolymerized on ice for 15 min, and a second round of polymerization performed as above. MTs for the ATPase assay were pelleted on a 50% v/v sucrose/BRB80 cushion after the second polymerization, to aid separation of MTs from unpolymerized tubulin.Purified and lyophilized unlabeled, X-rhodamine labeled, or biotin labeled g, room temperature), the pellet resuspended with bright MT caps, incubated 37 °C for 15 min, then 40 μM paclitaxel added.For labeled MTs, X-rhodamine and unlabeled tubulin were mixed in a 1:9 ratio, or X-rhodamine, biotinylated, and unlabeled tubulin were mixed in a 1:1:8 ratio and polymerized at approximately 4 mg/ml total tubulin with 1 mM GTP at 37 °C for 20 min 40 μM Paclitaxel (Merck) dissolved in DMSO was then added, followed by a further incubation at 37 °C for 15 min, and then incubation at room temperature for at least 1 day before use. To prepare polarity marked MTs , long, dPfK5ΔL6-MD ATPase rates. Reactions were performed in T50K40 buffer  with 100–200 nM PfK5ΔL6-MD. A340 readings were taken on a SpectraMax 384 (Molecular Devices) from 100 μl reactions at 26 °C, with automatic path length correction to 1 cm, taking readings every 10 s for 30 min. Background readings containing no PfK5ΔL6-MD were subtracted from all readings. ATP hydrolysis per second per site was derived with the Beer–Lambert equation, and kcat calculated using the Michaelis–Menten equation with the term K0 (rate at substrate concentration of 0).An NADH-coupled ATPase assay  containiPfK5ΔL6-MD-SNAP was added for 2 min, then washed twice with T50TC. (4) Finally, T50TC supplemented with 20 mM glucose, 300 μM glucose oxidase, 60 μg/ml catalase, 71 mM ß-mercaptoethanol, and 0.5 μM X-rhodamine MTs was added. For single-molecule experiments, the same preparations were performed with the following exceptions. (3) 0.5 μM X-rhodamine/biotin MTs was added for 2 min, then washed twice with T50TC. (4) As above, except 1–100 nM PfK5ΔL6-MD-SNAP-alexa647 was added instead of MTs.TIRFM assays with fluorescently labeled protein were performed in a flow-chamber, created by adhering biotin-PEG coverslips (MicroSurfaces Inc) to glass slides with double-sided tape. To prepare flow chambers for the MT gliding assay, the following treatments were made. (1) 0.75% Pluronic acid +5 mg/ml casein was added for 5 min, and the chamber was washed with T50K20 buffer + 20 μM paclitaxel (T50T). (2) 0.5 mg/ml neutravidin was added for 2 min, then the chamber was washed twice with T50T + 1 mg/ml casein (T50TC). (3) 1–2.5 μM PfK5ΔL6-MD-SNAP MT binding were calculated from kymographs generated in FIJI  . Images  in FIJI . For MT PfK5ΔL6-MD was prepared by incubation with 10 U/ml apyrase (Sigma) for at least 15 min at 4 °C, to remove any nucleotides still present following purification. UltrAuFoil 1.2/1.3 gold (Quantifoil) electron microscopy grids were glow discharged in air for 1 min at 0.3 mpa using a PELCO easiGlow . Using a Vitrobot Mark IV (ThermoFisher), with the chamber set to 23 °C and 95% humidity, 4 μl of 3.5 μM GMPCPP MTs was added to grids and incubated for 30 s, then blotted. Four microliter of 50 μM no nucleotide PfK5ΔL6-MD was then immediately added, incubated for 30 s, then blotted. A second round of PfK5ΔL6-MD was added before blotting and plunge freezing into an ethane slush. AMPPNP PfK5ΔL6-MD was prepared by incubation with 5 mM AMPPNP (R&D Systems) for at least 5 min at 4 °C. Vitrification was performed as above, except using C-flat 2/2 carbon (Protochips) holey grids with 5 μM GMPCPP MTs.Nucleotide-free −/Å2 for 18 s and 60 frames collected with a pixel size at the sample of 1.39 Å.Imaging was manually or automatically performed on a G2 Polara (FEI/ThermoFisher) operating at 300 kV using SerialEM . Images via supervised 3D classification was then performed, and MTs with less than 50% confidence in seam class assignment were removed. C1 reconstructions were obtained with a 3D auto-refine run (using the parameters for X/Y refine in https://github.com/moores-lab/MiRPv2).All image processing steps were performed with RELION v3.0 and 3.1 , 61 excePfK5ΔL6-MD heterogeneity, 3D classification was performed at the level of a PfK5ΔL6-MD:αβ-tubulin asymmetric unit. For this, symmetry expansion was applied to all particles, then 3D classification  without alignment and a mask around one PfK5ΔL6-MD site opposite the seam was applied. This resulted in one class with clear PfK5ΔL6-MD decoration, which was selected and subjected to a 3D auto-refine procedure. The MT-bound nucleotide-free and AMPPNP-bound PfK5ΔL6-MD reconstructions are deposited with the Electron Microscopy Data Bank, deposition number 12257 and 12258, respectively.Symmetrized maps were obtained by first performing 2D classification without alignment  and selecting well-aligned classes with many particles and an estimated resolution better than 6 Å. A 3D auto-refine run was then performed where 14-fold local symmetry was applied, as previously described . To addrTo obtain a kinesin-5 family sequence alignment, the motor domains of all kinesin-5 family members in the Swiss-Prot database were aligned with MAFFT . A hiddePfK5ΔL6-MD, using the human kinesin-5 AMPPNP bound crystal structure  was defined as a rigid body, while loop regions were treated as “all atoms.” Models of the N-terminus, loops 2, 8, 9, 10, 11, and 12, and the neck linker were predicted using Rosetta, firstly using a coarse method  as a teID: 3HQD , then scID: 3HQD  and the  Flex-EM  (cap shi Chimera . During ing step ), then ting step ). A locaity term . Finally HADDOCK  as descr HADDOCK , using tCK (3JAT  as αβ-tuCK (3JAT , 84. ThePlotting was performed with GraphPad Prism 8. Cryo-EM density and model analysis was done in Chimera  and Chimhttps://github.com/moores-lab/MiRPv2. The MT-bound nucleotide-free and AMPPNP-bound PfK5ΔL6-MD reconstructions are deposited with the Electron Microscopy Data Bank, deposition number 12257 and 12258, respectively. The molecular models of MT-bound nucleotide-free and AMPPNP-bound PfK5ΔL6-MD are deposited with the Worldwide Protein Data Bank, deposition number 7NB8 and 7NBA, respectively. The kinesin-5 family motor domain sequence alignment is provided in Updated MiRP scripts and instructions are available on GitHub: This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Accumulation of β-amyloid (Aβ) causes oxidative stress, which is the major pathological mechanism in Alzheimer’s disease (AD). β-asarone could reduce Aβ-induced oxidative stress and neuronal damage, but the molecular mechanism remains elusive. In this study, we used an Aβ-stimulated PC12 cell model to explore the neuroprotective effects and potential mechanisms of β-asarone. The results showed that β-asarone could improve cell viability and weaken cell damage and apoptosis. β-asarone could also decrease the level of ROS and MDA; increase the level of SOD, CAT, and GSH-PX; and ameliorate the mitochondrial membrane potential. Furthermore, β-asarone could promote the expression of Nrf2 and HO-1 by upregulating the level of PI3K/Akt phosphorylation. In conclusion, β-asarone could exert neuroprotective effects by modulating the P13K/Akt/Nrf2 signaling pathway. β-asarone might be a promising therapy for AD. Alzheimer’s disease (AD) is the most common neurodegenerative disease and the primary cause of dementia in elderly people. The neuropathology of AD includes accumulation of β-amyloid (Aβ), neurofibrillary tangles, and loss of neurons . NumerouNuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor that plays an important role in regulating oxidative stress. Accumulating studies showed that the activation of Nrf2 exerted neuroprotective activity via reducing oxidative stress injury in AD . MoreoveAcorus tatarinowii Schott could ameliorate cognitive deficit via controlling oxidative stress damage and was widely used in the treatment of AD  were obtained from Sigma-Aldrich Inc. . Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin were obtained from Hyclone . Phosphate-buffered saline (PBS) was obtained from Nanjing SunShine Biotechnology Co., Ltd. . A total superoxide dismutase (SOD) assay kit, lipid peroxidation malondialdehyde (MDA) assay kit, catalase (CAT) assay kit, total glutathione peroxidase (GSH-PX) assay kit, lactate dehydrogenase (LDH) cytotoxicity assay kit, and 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) probe were purchased from Beyotime Biotechnology . An apoptosis detection kit was purchased from Thermo Fisher Scientific . MitoSOX Red Mitochondrial Superoxide Indicator was purchased from Yeasen Biotech Co., Ltd. . A mitochondrial membrane potential assay kit with JC-1 was obtained from Solarbio . Antibodies against HO-1 were obtained from Gene Tex Inc. . Antibodies against Nrf2, Bax, Bcl-2, cleaved caspase-3, P13K, P-P13K, P-Akt, Akt, β-actin, and Lamin A were obtained from Abcam .β-asarone, Aβ2. Aβ1–42 was dissolved in PBS and incubated at 37°C for 7 days to induce aggregation (1–42 was diluted to the desired concentration. β-asarone was dissolved to a concentration of 10 mM in dimethyl sulfoxide (DMSO). Working solutions were diluted from the stock solutions at a suitable concentration for further use.Rat pheochromocytoma (PC12) cells were obtained from the Chinese Academy of Sciences, cultured in Dulbecco’s modified Eagle’s medium (DMEM), and supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere at 37°C with 5% COregation . Then, t4 cells per well, and cultured for 24 h. After the cells were treated with β-asarone or Aβ1–42 or coprocessing, MTT  was added in each well and incubated for 4 h at 37°C. Then, the medium was removed and replaced with 150 μl/well of DMSO. The formazan crystals were dissolved, and the optical density was measured at 570 nm by using a microplate reader .Cell viability was tested by using MTT assay. The PC12 cells were plated in 96-well plates, with 1 × 104 cells/well). The LDH level was measured according to the manufacturer’s protocol. The absorbance was determined using a microplate reader at 450 nm.Cell damage was determined by using an LDH cytotoxicity assay kit. The PC12 cells were seeded in 96-well plates (1 × 106 cells/well) for 24 h. After exposure to 10 μM Aβ1–42 for 12 h and treatment with β-asarone  for another 12 h, the cells were washed and suspended in 100 μl binding buffer. Then, Annexin V-FITC and propidium iodide (PI) were added and incubated for 15 min at room temperature. The cells were analyzed by flow cytometry .Cell apoptosis was detected by using an apoptosis detection kit according to the manufacturer-recommended protocol. In brief, PC12 cells were cultured in six-well plates . After pretreatment with Aβ1–42 for 12 h, the cells were treated with various concentrations of β-asarone for another 12 h. Then, the cells were washed with PBS and stained with 1 μM DCFH-DA for 20 min. Then, the cells were washed three times with PBS, and the fluorescence intensity was detected with a fluorescence microscope .The accumulation of ROS was monitored by using a fluorescent probe, 2’, 7’-dichlorodihydrofluorescin diacetate (DCFH-DA). In brief, the PC12 cells were cultured in six-well plates .The PC12 cells were lysed with RIPA buffer after treatment. Then, protein concentration was quantified by using a BCA kit. Proteins were separated by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies against HO-1, P13K, P-P13K, P-Akt, Akt, Bax, Bcl-2, cleaved caspase-3, Nrf 2, and β-actin (1:1000) at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies (1:5000) at 37°C for 2 h. The bands were visualized with enhanced chemiluminescence reagent (ECL) under a Tanon-5200 Chemiluminescent Imaging System .p < 0.05 was considered statistically significant.The data were analyzed with SPSS 22.0 statistic program, and the results were expressed as means ± SD by one-way ANOVA and Turkey’s post hoc test. The value of 1–42 alone on PC12 cell proliferation. As shown in 1–42 significantly inhibited PC12 cell viability at a concentration range of 1–20 μM (p < 0.01), and the 50% inhibitory concentration was about 10 μM. β-asarone (0–60 μM) showed no significant effect on the proliferation of PC12 cells. When the concentration was increased to 120 μM, the cell viability decreased (p < 0.05). We selected 10 μM Aβ1–42 and 0–60 μM β-asarone for subsequent experiments. Further study revealed that the cell viability was significantly increased  by β-asarone treatment. The better protective effect was shown at a concentration of 60 μM . When cells were treated with β-asarone, the LDH release decreased (p < 0.05). The results indicated that β-asarone could reduce Aβ1–42-induced cell cytotoxicity in a concentration-dependent manner . However, compared with treatment with Aβ alone, treatment with β-asarone could decrease apoptosis in a dose-dependent manner (p < 0.05). Furthermore, the expressions of Bcl-2, Bax, and cleaved caspase-3 were tested. The results showed that treatment with Aβ1–42 alone could decrease the ratio of Bcl-2/Bax slightly and increase the level of cleaved caspase-3. However, β-asarone could reverse these markers markedly (p < 0.05).To explore the effect of β-asarone on Aβ1–42-induced mitochondrial membrane potential, JC-1 assay was used. When cells were treated with Aβ alone, the green fluorescence increased. The results implied that Aβ induced the loss of the mitochondrial membrane potential. After β-asarone treatment, the fluorescence shifted from green to red in a concentration-dependent manner. Hence, the reduction of the mitochondrial membrane potential could be reversed by β-asarone treatment (p < 0.05).To assess the effect of β-asarone on Aβ1–42-induced oxidative stress, the levels of MDA, SOD, CAT, and GSH-PX were measured. After exposure to 10 µM Aβ1–42, the MDA level was increased (p < 0.05) and the levels of SOD, CAT, and GSH-PX were suppressed compared with the control group (p < 0.05). Nevertheless, the levels of MDA, SOD, CAT, and GSH-PX were noticeably reversed by different concentrations of β-asarone treatment (p < 0.05).To observe the effect of β-asarone on Aβ1–42-induced ROS production, DCFH-DA fluorescein-labeled dye was used. After Aβ1–42 treatment, the green fluorescence was enhanced. The results confirmed that the intracellular ROS level was increased by Aβ treatment (p < 0.05). However, β-asarone could inhibit Aβ1–42-induced ROS accumulation. Next, mitochondrial ROS generation was measured using MitoSOX specific red dye. As shown in 1–42-alone treatment group (p < 0.05). Thus, β-asarone could alleviate Aβ1–42-induced mitochondrial superoxide generation.To examine the effect of β-asarone on Aβp < 0.05). HO-1 is a key enzyme regulated by Nrf2, and the expression of HO-1 was reduced after Aβ treatment (p < 0.05). Compared with the Aβ1–42-alone treatment group, the expressions of Nrf2 and HO-1 were increased by β-asarone treatment (p < 0.05). Moreover, there was significant difference among different doses of β-asarone (p < 0.05). However, the expression of Nrf2 in the cytoplasm was not apparently changed after β-asarone treatment (p > 0.05).We examined Nrf2 and HO-1 expression levels in PC12 cells by Western blot. As shown in 1–42-stimulated oxidative stress by the P13K/Akt signaling pathway. Compared with the control group, the ratios of phosphorylated to total P13K (p-P13K/t-P13K) and phosphorylated to total Akt (p-Akt/t-Akt) were significantly reduced (p < 0.05). However, the p-P13K/t-P13K and p-Akt/t-Akt ratios were upregulated after β-asarone treatment (p < 0.05). Thus, we speculated that β-asarone might regulate Aβ1–42-induced oxidative stress by activating the P13K/Akt signaling pathway.Next, we investigated whether β-asarone regulates Aβin vitro model to evaluate neurotoxicity in AD. Moreover, the cells are particularly sensitive to Aβ-induced injury , translocation of Nrf2 into the nucleus, and the combination of antioxidant response elements (AREs). These events ultimately resulted in upregulation of antioxidant enzymes, such as HO-1 . HO-1 isIn summary, our study demonstrated that β-asarone could protect Aβ-induced PC12 cells against oxidative stress, mitochondria dysfunction, and cell apoptosis. We describe a possible molecular mechanism by which β-asarone exerts its neuroprotective effects in PC12 cells in


Background and Purpose:Mitragyna speciosa extract and kratom alkaloids decrease alcohol consumption in mice at least in part through actions at the δ-opioid receptor (δOR). However, the most potent opioidergic kratom alkaloid, 7-hydroxymitragynine, exhibits rewarding properties and hyperlocomotion presumably due to preferred affinity for the mu opioid receptor (µOR). We hypothesized that opioidergic kratom alkaloids like paynantheine and speciogynine with reduced µOR potency could provide a starting point for developing opioids with an improved therapeutic window to treat alcohol use disorder.Experimental Approach: We characterized paynantheine, speciociliatine, and four novel kratom-derived analogs for their ability to bind and activate δOR, µOR, and κOR. Select opioids were assessed in behavioral assays in male C57BL/6N WT and δOR knockout mice.Key Results: Paynantheine  produced aversion in a limited conditioned place preference (CPP) paradigm but did not produce CPP with additional conditioning sessions. Paynantheine did not produce robust antinociception but did block morphine-induced antinociception and hyperlocomotion. Yet, at 10 and 30 mg∙kg−1 doses (i.p.), paynantheine did not counteract morphine CPP. 7-hydroxypaynantheine and 7-hydroxyspeciogynine displayed potency at δOR but limited µOR potency relative to 7-hydroxymitragynine in vitro, and dose-dependently decreased voluntary alcohol consumption in WT but not δOR in KO mice. 7-hydroxyspeciogynine has a maximally tolerated dose of at least 10 mg∙kg−1 (s.c.) at which it did not produce significant CPP neither alter general locomotion nor induce noticeable seizures.Conclusion and Implications: Derivatizing kratom alkaloids with the goal of enhancing δOR potency and reducing off-target effects could provide a pathway to develop novel lead compounds to treat alcohol use disorder with an improved therapeutic window. Mitragyna speciosa, commonly known as kratom, is growing increasingly popular in the United States, with nearly 1% of the population aged 12 and older using kratom in 2019 . Corynoxine and corynoxine B were purchased from BOC Sciences . Leu-enkephalin, forskolin, and morphine sulfate pentahydrate were purchased from Sigma-Aldrich . (2S)-2-[[2-[[(2R)-2-[[(2S)-2-Amino-3-(4-hydroxyphenyl)propanoyl] amino] propanoyl] amino]acetyl]-methylamino]-N-(2-hydroxyethyl)-3-phenylpropanamide (DAMGO), 2--N-methyl-N-[-2-pyrrolidin-1-ylcyclohexyl]acetamide , and naloxone hydrochloride were purchased from Tocris Bioscience . [3H]DAMGO , [3H]U69,593 , and [3H]DPDPE  were purchased from PerkinElmer . For 31H: 7.26, 13C: 77.3). Peak multiplicity is reported as follows: s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. Coupling constants (J) are expressed in Hz. High resolution mass spectra were obtained on a Bruker Daltonics 10 Tesla Apex Qe Fourier-Transform Ion Cyclotron Resonance–Mass Spectrometer by electrospray ionization (ESI). Accurate masses are reported for the molecular ion [M + Na]+.All chemicals were purchased from Sigma-Aldrich Chemicals and used without further purification. Reactions were carried out in flame-dried reaction flasks under Argon. Reaction mixtures were purified by silica flash chromatography on E. Merck 230–400 mesh silica gel 60 using a Teledyne ISCO CombiFlash Rf instrument with UV detection at 280 and 254 nm. RediSep Rf silica gel normal phase columns were used. The yields reported are isolated yields. NMR spectra were recorded on a Varian 400/500 MHz NMR spectrometer. NMR spectra were processed with MestReNova software. The chemical shifts were reported as δ ppm relative to TMS using residual solvent peak as the reference unless otherwise noted . Then this crude kratom extract was subjected to silica gel column chromatography, using 0–15% MeOH in dichloromethane to isolate mitragynine (4.7 g), paynantheine (568 mg), speciogynine (343 mg), and speciociliatine (754 mg), along with some minor alkaloids.Mitragynine was extracted from the powdered leaves by following our previously reported methods . Kratom 3 solution was added, and the mixture extracted with EtOAc (3 × 15 ml). The organic phase was washed with brine (20 ml) and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure. The residue was purified on a silica column using 10–75% EtOAc in hexanes as eluent. The fractions containing the product were evaporated to yield 42 mg (40%) of 9 as a light magenta amorphous powder. 1H δ : 7.31 ; 7.29 ; 7.19 ; 6.74 ; 5.57 ; 4.99 ; 4.94 ; 3.86 ; 3.79 ; 3.68 ; 3.46 ; 3.23 ; 3.03 ; 3.01 ; 2.85 ; 2.73 ; 2.72 ; 2.66 ; 2.39 ; 2.30 ; 2.05 ; 1.70 . 13C δ : 183.5 (2); 168.8 (16-CO); 159.8 (17); 155.9 (9); 154.9 (13); 139.3 (19); 131.0 (11); 126.4 (8); 115.4 (18); 114.3 (12); 111.4 (16); 109.1 (10); 81.0 (7); 61.6 (21); 61.5 (17-OMe); 60.2 (3); 55.5 (9-OMe); 51.2 (16-COOMe); 49.8 (5); 42.8 (20); 38.2 (15); 35.9 (6); 30.4 (14). Relative configuration was determined based on the NOE cross peaks between the following 1H nuclei: 3 – 5/2; 3 – 14/2; 3 – 21/2; 3 – 5/2; 15 – 19; 19 – 21/2 . HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C23H28N2NaO5 435.189043; found. 435.189116.Paynantheine  was dissolved in acetonitrile (7 ml), and then water (2 ml) was added. The resulting suspension was cooled to 0°C, and PIFA  dissolved in acetonitrile (1.1 ml) was added slowly over the course of several minutes. The reaction mixture was stirred at 0°C for 45 min. Then saturated aqueous NaHCO9, 40 mg, 0.1 mmol) was dissolved in dry toluene (1.5 ml), and Zn(OTf)2  was added. The reaction mixture was stirred in a sealed tube for 30 min at 115°C. To the cooled mixture were added 2 ml sat. aqueous NaHCO3 solution and water (5 ml) and the organics were extracted with EtOAc (10 ml). The organic layer was rinsed with brine (10 ml) and dried over anhydrous Na2SO4. After evaporation of the solvent under reduced pressure, the residue was purified by flash column chromatography on silica (gradient: 40–75% EtOAc in hexanes) to yield 15 mg (38%) of product as a light yellow gum. 1H δ : 7.32 ; 7.18 ; 6.37 ; 6.13 ; 5.49 ; 5.25 ; 4.95 ; 4.9 ; 3.89 ; 3.73 ; 3.62 ; 3.23 ; 3.11 ; 2.87 ; 2.49 ; 2.39 ; 2.39 ; 2.34 ; 1.98 ; 1.94 ; 1.79 ; 1.26 . 13C δ : 199.8 (7); 168.2 (16-C=O); 162.1 (13); 159.7 (17); 158.7 (9); 139.5 (19); 139 (11); 115.6 (18); 111.9 (16); 109.5 (8); 104 (12); 99.2 (10); 74.7 (2); 72.4 (3); 61.5 (17-O-CH3); 58.8 (21); 55.8 (9-OCH3); 53.2 (5); 51.1 (COO-CH3); 42.3 (20); 36.9 (15); 35.3 (6); 28.3 (14). Relative configuration was determined based on the NOE cross peaks between the following 1H nuclei: 1 – 6/1; 3 – 14/2; 1 – 14/1; 14/1 – 20; 15 – 19; 19 – 21/2. HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C23H28N2NaO5 435.189043; found. 435.189219.7-Hydroxypaynantheine . The organic phase was washed with brine (30 ml) and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure. The residue was redissolved in DCM and was purified using silica column chromatography 10–75% EtOAc in hexanes. The fractions containing the product were evaporated to yield 107 mg (57%) of 9 as a light brown amorphous powder. 1H NMR  δ 7.36–7.29 , 7.26 , 7.17 , 6.71 , 3.84 , 3.75 , 3.66 , 3.21 – 3.08 , 2.82 , 2.77–2.69 , 2.64 , 2.54 , 2.30 , 2.17 , 2.06 , 1.80 , 1.69 , 1.40 , 1.02 , 0.82 . 13C NMR  δ 183.9, 169.61, 160.10, 156.07, 155.15, 131.15, 126.52, 114.42, 111.44, 109.18, 81.16, 61.98, 61.49, 61.52, 55.66, 51.64, 50.21, 39.54, 38.87, 36.13, 24.49, 11.56, and 11.29. HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C23H30N2NaO5 437.204693; found. 437.204951.Speciogynine  was dissolved in acetonitrile (15 ml), and then water (5 ml) was added. The resulting suspension was cooled to 0°C, and PIFA  dissolved in acetonitrile (2.2 ml) was added slowly over the course of several minutes. The reaction mixture was stirred at 0°C for 1 h. Then saturated aqueous NaHCO9, 200 mg, 0.48 mmol) was dissolved in dry toluene (6 ml), and Zn(OTf)2  was added. The reaction was stirred in a sealed tube for 2 h at 100°C. To the cooled mixture were added 10 ml sat. aqueous NaHCO3 solution and water (20 ml), and extracted with EtOAc (30 ml). The organic layer was rinsed with brine (20 ml) and dried over anhydrous Na2SO4. After evaporation of the solvent under reduced pressure, the residue was redissolved in DCM and purified by flash column chromatography (gradient: 40–75% EtOAc in hexanes) to yield 78 mg (39%) of 10 as a light yellow amorphous powder. 1H NMR  7.31 , 7.23 , 6.36 , 6.12 , 5.34 , 3.89 , 3.72 , 3.62 , 3.25 – 3.23 , 3.22 – 3.21 , 2.37 – 2.35 , 2.33 – 2.31 , 2.29 – 2.28 , 2.08 – 2.04 , 1.94 – 1.90 , 1.81 – 1.77 , 1.75 – 1.73 , 1.34 –1.30 , 1.18–1.15 , 0.95–0.92 , and 0.79 . 13C NMR  200.18 (7), 168.02 (16-CO), 162.25 (13), 160.27 (17), 158.83, (9), 139.17 (11), 112.22 (16), 109.5 (8), 104.26 (12), 99.17 (10), 74.94 (2), 72.94 (3), 61.51 (17-OMe), 58.42 (21), 55.99 (9-OMe), 53.57 (5), 51.07 (16-COOMe), 38.15 (20), 37.50 (15), 35.48 (6), 28.95 (4), 24.46 (9), and 11.35 (18). Relative configuration was determined based on the NOE cross peaks between the following 1H nuclei: 1 – 6/1; 1 – 14/1; 15 – 19; 19 – 21/2 . HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C23H30N2NaO5 437.204693; found. 437.204760.7-hydroxyspeciogynine  or U2OS cells (κOR) (DiscoverX), and using OR specific radiolabels U69,593 .cAMP inhibition assays were performed in HEK cells and transiently transfected with pGloSensor22F, and either expressing FLAG-mouse δOR, HA-mouse µOR, or FLAG-mouse κOR, as previously described .β-Arrestin recruitment assays were performed in PathHunter cells stably expressing the μOR, δOR, or κOR and β-arrestin-2, as previously described .Data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology . The dathttps://www.purdue.edu/research/regulatory-affairs/animal-research/staff.php). Animal studies were carried out in accordance with the ARRIVE guidelines /12-h dark cycle. All experiments were conducted between 10:30 and 15:00, and all mice were habituated to the test room at least 30 min prior to experimentation. Rotarod, nociception, and seizure experiments were conducted in well-lit rooms, whereas conditioned place preference, two-bottle choice, and locomotor experiments were conducted in the dark.The animal protocols (#1305000864 and #1605001408) describing the care and use of experimental animals was approved by the Purdue University Institutional Animal Care and Use Committee (idelines  and reco−1 (i.p.) paynantheine. For the paynantheine antagonist nociception assays, a separate group of 10 mice were exposed to 6 mg∙kg−1 morphine (s.c.) by itself, and then again after treatment with 10 and 30 mg∙kg−1 paynantheine (i.p.). For agonist and antagonist antinociception assays with 7-hydroxyspeciogynine, a total of 11 wild-type male mice were used; all received 7-hydroxyspeciogynine for the agonist mode, and then for antagonist mode, n = 6 received morphine plus 7-hydroxyspeciogynine and n = 5 received vehicle plus 7-hydroxyspeciogynine. For specifics on drug administration timing in the nociception assays, see the Methods section titled n = 8 males for 7-hydroxypaynantheine, n = 12 males and n = 10 females for 7-hydroxyspeciogynine). For the two-bottle choice experiments with δOR KO mice, a group of mice (n = 9) was repeatedly tested once per week with different drug treatments . Similarly, five wild-type mice from the naloxone-block locomotor experiment were reused to assess seizure activity of 30 mg∙kg−1 paynantheine (i.p.) following a week of drug washout. In the rotarod assay, n = 8 wild-type male and n = 8 δOR KO male mice were used to assess motor incoordination effects following treatment with speciociliatine. Note that one δOR KO mouse died after experiencing severe level 5–6 seizures following i.p. administration of 30 mg/kg speciociliatine in the rotarod assay, leading to an overall n = 7 instead of n = 8 for this genotype. For the CPP paradigms, independent groups of wild-type male mice were used to examine paynantheine by itself , paynantheine with morphine , and 7-hydroxyspeciogynine (n = 8).For the locomotor assays with 7-hydroxymitragynine, a group of 10 male mice was used. For the paynantheine agonist nociception assays, 10 male mice were treated on different days with 10 and 30 mg∙kgt test was used. For paynantheine locomotor data in via a one-way ANOVA with Dunnett’s multiple comparisons to morphine (MOR). For 7-hydroxyspeciogynine locomotor data in t test was used; one mouse was removed from this analysis after being identified as an outlier with Grubb’s test.To assess drug-induced effects on ambulation for 7-hydroxymitragynine, locomotor activity was assessed in a 2-day protocol as previously described . To asseMice were conditioned to drugs and vehicle as described previously in two-chamber conditioned place preference (CPP) boxes in a counterbalanced, unbiased approach for either two drug conditioning sessions over 2 days (brief) or four drug conditioning sessions over 8 days (extended) . For briiSpyConnect.com). A recording time of 90 min was chosen for the tested compounds based on previous observations of seizures time lengths in experiments with 30 mg∙kg−1 paynantheine. If animals were not presenting with seizure activity after 30 min, the recording time was shortened accordingly. Seizure severity was scored based on the modified Racine scale  in bins of 3–5 min. Onset to first seizure symptom, onset to highest Racine score, and highest Racine score were also assessed. A summary of all statistical analyses for the seizure data can be found in t test with Welch’s correction on area under the curve data generated from graphing the highest Racine score per time bin over 90 min for each mouse.To assess drug-induced seizurogenic activity, mice were placed in a clear plastic cylinder  immediately following drug injection and their activity was recorded in a well-lit, quiet room using iSpy camera software (via the tail flick assay was measured as previously described (−1 morphine (s.c.) 10 min later. Tail flick responses were collected 20 min after morphine administration. Data are represented as percent maximal possible effect (%MPE) and is calculated as %MPE = (treatment response time − baseline response time)/(cutoff time − baseline response time) * 100. Data are normalized to vehicle treatment: drug treatment %MPE − saline treatment %MPE. A summary of all statistical analyses for the antinociceptive data can be found in via a two-tailed, paired t test to compare vehicle and drug treatment. For antagonist antinociception assays with three treatment groups in the same group of mice (via repeated measures (RM) one-way ANOVA with Dunnett’s multiple comparisons to the morphine-only treatment group. For antagonist antinociception assays with two treatment groups in two different groups of mice . A summary of all statistical analyses for the drinking data can be found in post hoc analyses were used for water consumption and ethanol preference data. For the change in alcohol consumption, change in water consumption, and change in ethanol preference data for 7-hydroxyspeciogynine where male and female data were analyzed together, a mixed-effects model was used  with the Geisser–Greenhouse correction for main effects, followed by Dunnett’s MC between alcohol consumption baseline vs. treatment day consumption. Sex differences between baseline data were evaluated using RM two-way ANOVA for main effects of sex, treatment baseline, and sex × treatment baseline; Sidak’s multiple comparisons (MC) between male and female mice were then used as the post hoc test for each treatment week tested.Mice were subject to drinking in the dark (DID), limited access (4 h per day), two-bottle choice (10% ethanol vs. water) paradigm in which they were trained to consume alcohol voluntarily as previously described . Mice re−1 speciociliatine (i.p.) and immediately tested for performance on the apparatus , and then tested again at 15, 30, 60, and 120 min post-injection. Each mouse’s performance was normalized to its own baseline and reported as a percentage. A summary of all statistical analyses for the rotarod data can be found in t test vs. a hypothetical mean of 100 (baseline was 100%). Rotarod results between WT and δOR KO genotypes were compared with a mixed-effects model with fixed effects for timepoint, genotype, and timepoint × genotype.Mice were trained to walk on a rotarod apparatus  with 1.25” diameter drums 2 days prior to drug testing. The rotarod started at 3 rpm and was increased to 30 rpm over 300 s. A trial for a mouse ended when it fell and tripped the sensor, when it rode the rotarod for two consecutive revolutions, or after 300 s . Mice rehttp://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY   .−1, but unlike 7-hydroxymitragynine does not produce hyperlocomotion at its effective dose   . In the GloSensor assay of cAMP inhibition, paynantheine displayed partial to full agonism at the ORs , neither the 10 nor 30 mg∙kg−1 dose displayed meaningful antinociceptive effects  = 12.38, p = 0.0005, with Dunnett’s MC to 6 mg∙kg−1 morphine: p = 0.6330 for 10 mg∙kg−1 dose, p = 0.0019 for 30 mg∙kg−1 dose)   = 39.25, p < 0.0001, with Dunnett’s MC to 6 mg∙kg−1 morphine: p = 0.0004 for 10 mg∙kg−1 dose, p < 0.0001 for 30 mg∙kg−1 dose)  . However 0.0612) . Additio . However−1 dose) .2 in refluxing toluene. We adopted the same strategy to synthesize 7-hydroxyspeciogynine (9) and speciogynine pseudoindoxyl (10) as shown in In order to produce better lead candidates to treat alcohol use disorder that lack adverse locomotor and rewarding effects, we next aimed to discover kratom alkaloids or alkaloid derivatives with increased δOR affinity and potency but with limited µOR potency. To this end, we extracted paynantheine (2), speciogynine (3), and speciociliatine (4) from dry kratom powder using a modified protocol reported by Affinity wise, we noted that the paynantheine analogs, especially the 7-hydroxyl analog, showed weak µOR affinity, whereas 7-hydroxyspeciogynine displayed the strongest µOR affinity . At the In terms of cAMP inhibition, we noted clear signs of partial agonism for the analogs at the µOR, with paynantheine pseudoindoxyl, 7-hydroxypaynantheine, and 7-hydroxyspeciogynine displaying the lowest potency at the µOR . 7-hydroin vitro as a partial agonist for μOR and κOR but antagonist at δOR  = 36.48, p < 0.0001, time: F  = 50.17, p < 0.0001, dose × time: F  = 13.30, p < 0.0001, with Sidak’s MC (T-R vs F), p < 0.0001 for the 30 mg∙kg−1 dose)  = 25.36, p = 0.0007, time: F  = 61.69, p < 0.0001, dose × time: F  = 83.26, p < 0.0001, with Sidak’s MC (T-R vs F), p < 0.0001 for the 30 mg∙kg−1 dose)  = 17.10, p = 0.002, dose × time: F  = 7.521, p = 0.0007, with Sidak’s MC (T-R vs F), p < 0.0001 for the 30 mg∙kg−1 dose) and δOR KO mice  = 23.26, p = 0.0009, dose × time: F  = 64.72, p < 0.0001, with Sidak’s MC (T-R vs F), p < 0.0001 for the 30 mg∙kg−1 dose). The 30 mg∙kg−1 dose also significantly reduced the ability of treated wild-type mice to perform in the rotarod assessment , with the peak effect occurring between 15 and 30 min , and the mice fully recovering at 120 min . The same effect was observed in δOR KO mice  = 1.930, p = 0.1906).Based on our hypothesis that G-protein–biased δOR agonism drives decreased alcohol intake following kratom alkaloid injection, we did not expect speciociliatine to decrease alcohol intake as it behaves t at δOR . However−1 dose)  and with−1 dose) . Treatmeype mice   = 13.79, p = 0.0006, dose × time: F  = 8.675, p = 0.0017, with Sidak’s MC (T-R vs F), p = 0.0802 for the 3 mg∙kg−1 dose, p < 0.0001 for the 10 mg∙kg−1 dose). This decrease in ethanol consumption at the 10 mg∙kg−1 dose was accompanied by a concomitant increase in water consumption during the time course of the voluntary alcohol consumption paradigm  = 4.161, p = 0.0661, dose × time: F  = 3.489, p = 0.0483, with Sidak’s MC (T-R vs F), p = 0.0112) as well as a corresponding decrease in ethanol preference  = 8.284, p = 0.0150, dose × time: F  = 4.140, p = 0.0298, with Sidak’s MC (T-R vs F), p = 0.0036). We found that 7-hydroxypaynantheine was able to significantly reduce alcohol intake at a 10 and 30 mg∙kg−1 dose  = 13.79, p = 0.0075, dose × time: F  = 5.515, p = 0.0171, with Sidak’s MC (T-R vs F), p = 0.0219 for the 10 mg∙kg−1 dose, p < 0.0001 for the 30 mg∙kg−1 dose). Similarly, the decrease in ethanol consumption at the 30 mg∙kg−1 dose of 7-hydroxypaynantheine was accompanied by a concomitant increase in water consumption during the time course of the voluntary alcohol consumption paradigm  = 4.920, p = 0.0621, dose x time: F  = 4.149, p = 0.0385, with Sidak’s MC (T-R vs F), p = 0.0015) and a corresponding decrease in ethanol preference  = 5.193, p = 0.0567, dose × time: F  = 3.980, p = 0.0428, with Sidak’s MC (T-R vs F), p = 0.0036). In δOR KO mice subject to the same voluntary alcohol consumption paradigm, 10 mg∙kg−1 7-hydroxyspeciogynine significantly decreased ethanol consumption  = 6.407, p = 0.0007, time: F  = 16.46, p = 0.0036, dose × time: F  = 1.851, p = 0.1435, with Sidak’s MC (T-R vs F), p = 0.0269) but not the 3 mg∙kg−1dose of 7-hydroxyspeciogynine or the 30 mg∙kg−1dose of 7-hydroxypaynantheine  = 6.295, p = 0.0208, dose × time: F  = 0.1027, p = 0.7520, with Sidak’s MC , p < 0.0001 for the vehicle treatment baseline, p < 0.0001 for the 3 mg∙kg−1 7-hydroxyspeciogynine treatment baseline). However, no sex difference was apparent in the Δ ethanol intake  = 7.758, p = 0.0114, sex × dose: F  = 0.2487, p = 0.6234, with Sidak’s MC , p = 0.9993 for the Δ ethanol consumption following vehicle treatment, p = 0.7635 for the Δ ethanol consumption following 3 mg∙kg−1 7-hydroxyspeciogynine treatment). Combining the Δ ethanol intake for males and females, we found that there was a significant ethanol modulation effect at the 3 mg∙kg−1 dose when collectively analyzing male and female responses , p = 0.0165 for the 3 mg∙kg−1 dose, p = 0.0064 for the 10 mg∙kg−1 dose). After finding similar sex differences in water consumption and ethanol preference but not in the Δ of these parameters , p = 0.1804 for the 3 mg∙kg−1 dose, p = 0.0342 for the 10 mg∙kg−1 dose). Accordingly, in the pooled data, a significant decrease in ethanol preference was noted at the 10 mg∙kg−1 dose , p = 0.1644 for the 3 mg∙kg−1 dose, p = 0.0255 for the 10 mg∙kg−1 dose).In female mice exposed to the voluntary alcohol consumption paradigm, 7-hydroxyspeciogynine did not significantly modulate ethanol consumption, water consumption, or ethanol preference at the 3 mg∙kg−1 dose , female in vivo potency as 7-hydroxymitragynine at the δOR but lower µOR potency. Next, we assessed whether 7-hydroxyspeciogynine exhibited a better side effect profile than 7-hydroxymitragynine due to its limited potency at the µOR. Additionally, to determine if 10 mg∙kg−1 7-hydroxyspeciogynine was the maximum tolerated dose (MTD), we assessed the side effect profile for the 10 mg∙kg−1 dose. We found that mice treated with 10 mg∙kg−1 7-hydroxyspeciogynine did not develop conditioned place preference in our “extended” conditioned place preference protocol, which involves four conditioning sessions each for drug and vehicle    or block morphine analgesia   . The sam 0.4787)  or induc 0.4787) . Akin to 0.7991) .in vivo pharmacology and in search of novel treatment options for alcohol use disorder. We report 7-hydroxyspeciogynine as an effective lead compound to reduce alcohol with an MTD of at least 10 mg∙kg−1.Over the past decade, kratom has been reported as a source for naturally occurring, G-protein–biased opioidergic alkaloids, and has been investigated for its effects on pain management , opioid −1 dose, which was the minimal effective dose to reduce alcohol intake . Kratom use can lead to seizures  = 16.46, p = 0.0036, dose × time: F  = 1.851, p = 0.1435, with Sidak’s MC (T-R vs F), p < 0.0001). This analysis provides further evidence that many of paynantheine’s in vivo effects are not mediated by δOR.We previously demonstrated that 7-hydroxymitragynine as well as paynantheine could decrease alcohol consumption . Howeverl intake . It has l intake  as well l intake . Here, wl intake . Since tl intake , μOR potcomotion . In lineseizures  and we nseizures ; howeverseizures . As suchseizures ; however−1 doses of paynantheine failed to produce meaningful antinociception in the tail flick paradigm. In contrast, paynantheine blocks morphine analgesia at a 30 mg∙kg−1 dose but not at 10 mg∙kg−1, yet neither dose blocks morphine CPP. Additionally, paynantheine both at 10 and 30 mg∙kg−1 doses can block morphine hyper-ambulation. Paynatheine, at a 10 mg∙kg−1 dose, only blocks morphine hyper-ambulation within the first 15–20 min of the 40-min conditioning session. Detailed pharmacokinetic data for paynantheine have yet to be reported, but a recent study has shown that following oral administration in rats, a 1.1 mg∙kg−1 dose of paynantheine had a Tmax of 10 min in plasma and was undetectable after an hour , the activity at this receptor is still less potent than 7-hydroxymitragynine (pIC50 = 7.8 ± 0.1). The G-protein–biased μOR activity of 7-hydroxyspeciogynine likely does not contribute to decreased alcohol use because of the lack of effect in δOR KO mice at the 3 mg∙kg−1 dose and because we have previously shown that selective activation of μOR G-protein signaling using Oliceridine/TRV130 did not decrease alcohol consumption , yet speciociliatine acts as a δOR antagonist in the cAMP assay. When tested in mice, speciociliatine did cause a significant and sharp decrease in alcohol consumption at a relatively high 30 mg∙kg−1 dose  for 10 mg∙kg−1 7-hydroxyspeciogynine, and 1.1 ± 0.3 g∙kg−1 ethanol (40 ± 11%) for 30 mg∙kg−1 7-hydroxypaynantheine), which indicates an off-target effect. In support of this explanation, a 30 mg∙kg−1 dose of speciociliatine similarly decreases ethanol consumption in δOR KO mice and significantly impairs motor incoordination in wild-type and δOR KO mice, which likely contributes to the effects we see in the alcohol consumption paradigm. We did not test the kratom analogs or alkaloids in conjunction with δOR antagonists because the role of δOR antagonists in these behaviors is not well defined. For example, we have previously found that δOR-selective antagonist naltrindole does not decrease alcohol intake at a 10 mg∙kg−1 dose in this alcohol model, whereas another δOR-selective antagonist, naltriben, dose-dependently decreases alcohol consumption at 6 and 10 mg∙kg−1 doses  will be insightful. Further behavioral analysis, including modulation of respiratory depression and anxiety-like behavior (Given that agonist-bound structures of both the μOR and δOR are available , it may behavior  would esbehavior  that conin vitro and in vivo but with limited μOR in vivo agonist activity, leading to a broadened therapeutic window as evident from a lack of rewarding, locomotive, and seizurogenic effects and a MTD of at least 10 mg∙kg−1. Our findings support the utility of targeting the δOR to reduce volitional alcohol consumption, and further demonstrate the effectiveness of using the kratom alkaloids as lead scaffolds for developing G-protein–biased δOR agonists for treatment of AUD.In summary, our current and past pharmacological characterization of kratom analogs suggest that alkaloids with sub-micromolar δOR potency, micromolar potency at the μOR, and G-protein bias provide the strongest opportunity to reduce alcohol use in mice with limited side effects. We discovered 7-hydroxyspeciogynine as a novel kratom-derived analog that decreases alcohol intake by activating δORs


In the crystal, inversion-related C—H⋯O hydrogen bonds form chains extending parallel to the  21H20N2O3S, the styryl and ester substituents are displaced to opposite sides of the plane of the pyridine ring. In the crystal, C—H⋯O hydrogen bonds form chains extending parallel to the a-axis direction, which pack with partial intercalation of the styryl and ester substituents. A Hirshfeld surface analysis indicates that the most significant contributions to the crystal packing are from H⋯H (43.6%), C⋯H/H⋯C (15.6%), O⋯H/H⋯O (14.9%) and N⋯H/H⋯N (11.2%) contacts.In the title molecule, C Other interactions contributing less to the crystal packing are S⋯H/H⋯S (5.9%), C⋯C (4.4%), N⋯C/C⋯N (1.5%), S⋯O/O⋯S (1.1%), O⋯C/C⋯O (1.0%), O⋯O (0.3%), N⋯N (0.2%) and S⋯C/C⋯S (0.2%).To quantify the intermolecular interactions in the title compound, a Hirshfeld surface analysis was performed and two-dimensional fingerprint plots were generated using et al., 2016et al., 2007et al., 2007et al., 2018et al., 2018et al., 2018A search of the Cambridge Structural Database -thione , ethyl chloroacetate  and sodium acetate trihydrate  in ethanol (40 ml) was heated under reflux for 30 min. The solid that formed after dilution with water (20 ml) was filtered off and recrystallized from methanol in the form of fine colourless crystals of the title compound, yield 85%; m.p. 343–344 K. Its IR spectrum showed characteristic absorption bands at 2219 cm−1 for (C≡N), at 1748 cm−1 for  and at 1724 cm−1 for . The 1H NMR spectrum  displayed a multiplet at δ = 6.60–7.63 ppm (7H: CH=CH and Ar-Hs), a multiplet at δ = 4.16–4.37 ppm (6H: two OCH2 and SCH2), a singlet at δ = 2.52 ppm  and a multiplet at δ = 1.21–1.27 ppm (6H: two CH3 of ester groups).A mixture of 5-acetyl-3-cyano-6-methyl-4-styrylpyridine-2(1Uiso(H) = 1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021005600/wm5610sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989021005600/wm5610Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021005600/wm5610Isup3.cmlSupporting information file. DOI: 2087180CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Abnormal accumulation of TDP43-related mutant proteins in the cytoplasm causes amyotrophic lateral sclerosis (ALS). Herein, unbiased drug screening approaches showed that SC75741, a multi-target inhibitor, inhibited inflammation-induced aggregation by inhibiting NF-κB and also degraded already aggregated proteins by inhibiting c-Abl mediated autophagy-lysosomal pathway. We delineate the mechanism that SC75741 could markedly enhance TFEB nuclear translocation by an mTORC1-independent TFEB regulatory pathway. In addition, SC75741 enhanced the interaction between p62 with TDP25 and LC3C, thus promoting TDP25 degradation. Taken together, these findings show that SC75741 has beneficial neuroprotective effects in ALS. Our study elucidates that dual-targeted inhibition of c-Abl and NF-κB may be a potential treatment for TDP43 proteinopathies and ALS. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with progressive muscle denervation, weakness, atrophy, spasticity, paralysis, and eventually death . About 9TDP43, a DNA-RNA binding protein encoded by the TARDBP gene, was first identified in 2006 as the key aggregate component found in the brains of ALS patients . NotablyC-Abl, a non-receptor tyrosine kinase, was originally discovered as an oncogene, which can be fused with B cell receptors to cause chronic myeloid leukemia . Since ivia the UPS pathway. However, ubiquitinated protein aggregates cause UPS impairment which prevents their recognition and degradation in several neurodegenerative diseases  and the ubiquitin-proteasome system (UPS) are the protein degradation pathways . Over 80diseases . AutophaIn this study, 2110 FDA-approved drugs and drug candidates were screened and identified SC75741 as a novel TDP25 degradation agent. We demonstrate that SC75741 promoted the degradation of TDP-43-related protein aggregation and reduced the inflammation-induced aggregation by inhibiting NF-κB pathway. SC75741, as a new c-Abl inhibitor, enhance autophagy activity in ATG5-dependent manner and activate TFEB nuclear translocation in mTORC1-independent manner. SC75741 also enhanced the interaction between p62 with TDP25 and LC3C, promoted mitigation of TDP25 aggregation by activation of ALP.−/−, HEK293ATG5−/−, HelaWT, Hela ATG8−/− and N2A cells were grown in DMEM medium ; H4 cells were grown in DMEM medium ; SH-SY5Y cells were grown in MEM medium ; These media were supplemented with 10% FBS (Gibco TM), 1% Penicillin/Streptomycin (Gibco TM). Doxycycline-inducible H4GT25, SH-SY5YGT25 and SH-SY5YKT25 stable cell lines were generated by co-transfecting HP138-GFP-TDP25 (HP138-Keima-TDP25) and HP216 plasmids (a gift from Dr. Hui Yang) into H4 and SH-SY5Y cells using Lipofectamine TM 2000 (Invitrogen TM) and selected with 10 μg/ml puromycin (Sangon® Biotech). All cells were cultured at 37°C with 5% CO2.HEK293WT, HEK293p62The chemicals and their sources are as follows: Doxycycline (#A603456), Puromycin (#A606719), NH4Cl (#A501569) from Sangon® Biotech; MG132 (#S2619), Bafilomycin A1 (#S1413), E-64D (#S7393), Leupeptin hemisulfate (#S7380) from SelleckChem; Arsenite (#S7400) from Sigma; SC75741 (#T6661), Ibudilast (#T2137), Ouabain (#T1318), Imatinib (#T6230), PP-121 (#T2415), JSH-23 (#T1930), CAPE (#T6429) from Topscience, Inc. ; Anti-Flag (DYKDDDDK) Affinity Gel (#B23102) and Anti-HA magnetic beads (#B26202) were purchased from Bimake; Anti-GFP beads (#SM03801) was purchased from Smartlifesciences; Lipofectamine TM 2000 (#1901433) and Lipofectamine TM 3000 (#2067450) were from Invitrogen. The following antibodies were used in this study: β-tubulin (#M1305-2), Flag-Tag (#M1403-2), HA-Tag (#0906-1), GFP-Tag (#ET1607-31) from HuaAn Biotechnology; c-Abl (#2862), phospho-c-Abl (Tyr245) (#2861), TFEB (#37785), NF-κB (#8242), phospho-NF-κB (#5733) from Cell Signaling Technology®; LC3C (#18726-1-AP), p62 (#18420-1-AP) from Proteintech TM; LC3C (#A8295), Lamp1 (#A16894), TFEB (#A7311) from ABclonal; The secondary antibodies for western blotting were used: Goat anti-Mouse IgG (H + L) , Goat anti-Rabbit IgG (H + L) ; The fluorescent secondary antibodies for immunofluorescence were used: goat anti-rabbit Alexa Fluor 546 (#A-11010), goat anti-mouse Alexa Fluor 405 (#A-31553) from Thermo Fisher Scientific.5 H4GT25 cells were pretreated with 1 μg/ml DOX (doxycycline) for 24 h, incubated with 2110 FDA approved drugs and bioactive compounds for 24 h. MG132 (1 μM) treatment was used as negative control to induce aggregate formation, ibudilast (10 μM) was used as a positive control to promote aggregate degradation. The GFP-TDP25 fluorescence was analyzed by fluorescence microscopy and compared with fluorescence of cells treated with DMSO. A total of 178 small molecules with fluorescence intensity below 50% were obtained as candidate compounds. In the second screening, 1 × 105 H4GT25 cells were pretreated with 1 μg/ml DOX for 24h, treated with aresnite (1 h) or MG132 (12 h), incubated with 178 hits for 24h, and the GFP-TDP25 fluorescence was analyzed by fluorescence microscopy and compared with fluorescence of cells treated with DMSO. A total of 2 small molecules with fluorescence intensity below 50% were obtained as candidate compounds. In the third screening, 1 × 105 H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with aresnite (1 h) or MG132 (12 h), and treated with Ibudilast (10 μM), SC75741 (10 μM) or ouabain (10 μM) for another 24 h, analyzed by fluorescence microscopy and cell viability. Cell viability was detected using CellTiter-Glo® Luminescent Cell Viability Assay (#G7573 Promega) according to manufacturer’s instructions. The average fluorescence intensity of GFP-TDP25 aggregates was used to analyze the statistics of the three screening rounds. The aggregates with an average fluorescence intensity >5,000 were counted. Statistical analysis was performed with Gen5™ 3.02 software.All FDA approved drugs and bioactive compounds were commercially purchased from Topscience, Inc. . Drug in the high-throughput screening was 10 μg/ml. MG132 (1 μM) treatment was used as positive control to induce aggregate formation, ibudilast (10 μM) was used as a negative control to promote aggregate degradation. High-throughput screening was done using Biotek Cytation® 3 system excitation at 488 nm and emission at 510 nm. Every plate has negative control (MG132) and positive control (ibudilast), every plate has three replicates, and the screen Z-factor of every plate was >0.5. There are three screenings. In the first screening, 1 × 10Cell lysates or immunoprecipitation samples were added SDS-PAGE 2Х loading buffer and heated at 100°C for 10 min, subjected to 10–15% SDS-PAGE and then transferred onto PVDF membranes for 0.5–1 h at 0.2 A with semi-dry transfer system of BioRad. Membranes were blocked in PBST buffer containing 10% (w/v) skimmed milk for 1 h and probed with the indicated antibodies in PBST containing 5% (w/v) BSA at 4°C overnight. Detection was performed using HRP-conjugated secondary antibodies and chemiluminescence reagents .H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with aresnite (1 h) or MG132 (12 h), and treated with ibudilast (10 μM), SC75741 (10 μM) or ouabain (10 μM) for another 24 h, cell viability was detected using CellTiter-Glo® Luminescent Cell Viability Assay (#G7573 Promega) according to manufacturer’s instructions.H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with or wihtout 5 μM SC75741 for another 24 h, cells were washed twice with phosphate-buffered saline, lysed in cold RIPA buffer ), and sonicated. Cell lysates were cleared by centrifugation at 100,000 g for 30 min at 4°C to generate the RIPA-soluble samples. The supernatants collected from the first centrifugation was analyzed. Then, the resulting pellets were washed twice with RIPA buffer. RIPA-insoluble pellets were then extracted with urea buffer , sonicated, and centrifuged at 100,000 g for 30 min at 22°C.H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with or wihtout 5 μM SC75741 for another 24 h, cells were washed twice with phosphate-buffered saline, Lyse cells with 0.5 ml of 0.5% Triton X-100 lysis buffer (50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 137.5 mM NaCl, 10% Glycerol, 5 mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitor cocktail ) for 15 min in ice, shaking gently. Cell lysates were cleared by centrifugation at 13,000 rpm for 15 min at 4°C to generate the cytosolic and membrane fractions. The supernatants collected from the first centrifugation were analyzed. Then, the resulting pellets were washed twice with lysis buffer. Resuspend the pellet in 0.1 ml of lysis buffer, supplemented with 0.5% sodium dodecyl sulfate (SDS), and sonicate in cold room three times for 3 s at low output to shear genomic DNA. Cell lysates were cleared by centrifugation at 13,000 rpm for 15 min at 4°C to generate the nuclear fractions.Total RNA from H4GT25 cells was extracted using an RNA isolation Kit (Vazyme), and reverse-transcribed to cDNA using the HiScript III first Strand cDNA Synthesis Kit (Vazyme). Real-time PCR assays were performed using B818-480II (Rocher Applied Science). The oligonucleotides used were as follows: GAPDH; fw: 5′-attgttcgtcatgggtgtgaa-3′, rev: 5′-aggggtgctaagcagttggt-3′, DPP7; fw: 5′-gattcggaggaacctgagtg-3′, rev: 5′-cggaagcaggatcttctgg-3′, CTSD; fw: 5′-cttcgacaacctgatgcagc-3′, rev: 5′-tacttggagtctgtgccacc-3′, MCOLN1; fw: 5′-gagtccctgcgacaagtttc-3′, rev: 5′-tgttctcttcccggaatgtc-3′, LAMP1; fw: 5′-ccaacttctctgctgccttc-3′, rev: 5′-agcaatcaacgagactgggg-3’. CTSF; fw: 5′-acagaggaggagttccgcacta-3′, rev: 5′-gcttgcttcatcttgttgcca-3′, ATP6V0E1; fw: 5′-cattgtgatgagcgtgttctgg-3′, rev: 5′-aactccccggttaggaccctta-3′, SQSTM1; fw: 5′-caggtcttccaaggttgagg-3′, rev: 5′-ataaaaacacggccatttgc-3′, Map1LC3; fw: 5′- agcgctacaagggtgagaa-3′, rev: 5′-gttcaccagcaggaagaag-3’.Cells were cultured in 12-well plates on circular glass coverslips and washed twice with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were blocked with blocking buffer  at room temperature for 1 h, and incubated with primary antibodies overnight at 4°C. After washing with PBS, secondary antibodies were incubated at room temperature for 1 h. All samples were examined with an inverted FV3000 confocal microscope system using a 60 ×1.42-NA oil objective. All images were taken at room temperature and analyzed with FV10-ASW 4.0a Viewer.H4GT25 cells were grown in confocal dishes, pretreated with 1 μg/ml DOX for 24 h, treated with 5 μM SC75741 and using Nanolive Fluo-3D Cell Explorer® (Nanolive) microscope to explore aggregates change for a time-lapse of 720 min every 30s (60× magnification and depth of field 30 μm). images were exported using Steve software v.2.6® (Nanolive).2.SH-SY5YKT25 cells were transfected with HP138-Keima-TDP25, treated with 1 μg/ml DOX for 24 h, treated with or without SC75741 (5 μM) for another 24 h. Imaging was done using Biotek Cytation® 3 system excitation at 469 nm/586 nm and emission at 620 nm. Autophagy levels were estimated by the fraction of cells that were fluorescent upon excitation at 469 and 586 nm using Gen5™ software. Subsequently, the Keima-green/Keima-red ratio and Keima-red/Keima-green ratio were calculated. For living-cell colocalization imaging, SH-SY5YKT25 stable cells were grown in confocal dishes and stained with LysoTracker™ Green DND-26  and imaged with FV3000 confocal microscope system at 37°C with 5% COHigh throughput screening analysis: The average fluorescence intensity of GFP-TDP25 aggregates was used to analyze the statistics of the three screening rounds. The aggregates with an average fluorescence intensity >5,000 were counted. Statistical analysis was performed with Gen5™ 3.02 software.Keima-TDP25 assay analysis: The average fluorescence intensity of Keima-TDP25 aggregates was used to analyze the statistics in the Keima-TDP25 assay. Keima-red with an average fluorescence intensity >1,000 and Keima-green with an average fluorescence intensity >3,000 were counted. Statistical analysis was performed with Gen5™ 3.02 software.Co-localization analysis: ImageJ was used to analysis the co-localization of fluorescently labeled proteins.All experiments western blot, micrographs assay, and qPCR assay were carried out at least three independent times with the similar results. Statistical analyses were performed with GraphPad Prism 8. Data were analyzed with a Student’s t-test. Data points are shown as Mean ± SD.H4 cell line expressing GFP-TDP25 in a doxycycline (DOX)-inducible manner (H4GT25) was generated to establish a reporter system suitable for high-throughput screening. Ibudilast-treated cells showed a decreased aggregation and were used as positive controls, while MG132-treated cells showed an increased aggregation and were used as negative controls . The scrIn the past few decades, over 50 TDP43-related pathological mutations have been identified in ALS patients. The previous experiments have suggested that SC75741 effectively clears TDP25, indicating that SC75741 may have a similar effect on ALS-related TDP43 pathological mutations. In this study, wild-type TDP43, truncated mutation , and point mutation TDP43 A315T in SH-SY5Y cells were overexpressed to confirm the role of SC75741 on ALS-related TDP43 pathological mutations. The protein levels and GFP fluorescence levels of TDP43-related mutations were partially decreased after SC75741 treatment .SOD1, Cu/Zn superoxide dismutase 1, was the first ALS gene identified in 1993 . SOD1 muOverall, these results indicate that SC75741 reduces the accumulation of various pathological mutations of TDP43 and SOD1.Inflammation has been increasingly recognized as an important factor underlying the cell-specific neurodegeneration observed in neurodegenerative diseases, including ALS . NF-κB, in vitro kinase assay was conducted to obtain an inhibitory concentration (IC50) of 263 nM for further analysis  at acidic pH and a green fluorescent signal (438 nm) at neutral pH. We generated a stable cell line, which overexpressed Keima-TDP25 in SH-SY5Y . The Kei−/− cell lines overexpressing Keima-TDP25 showed that the decreased ratio of Keima-green/Keima-red under SC75741 treatment was ATG5 dependent , frontotemporal dementia (FTD), and Alzheimer’s disease (AD). TDP25, a C-terminal fragment of TDP43, is a key aggregate component found in the brains of ALS patients. Our study showed that SC75741, as a novel multi-target drug, promotes TDP25 degradation or misfolded TDP43-related proteins through the autophagy pathway. C-Abl, as a new target of SC75741, is essential in enhancing TFEB nuclear translocation by an mTORC1-independent TFEB regulatory pathway to promote TDP25 degradation. SC75741, as an intermediate, promotes the interaction between p62 with LC3 and TDP25, facilitating TDP25 degradation.Our study focused on TDP25, which has the highest degree of pathological aggregation, and a cell-based image high-throughput screening assay was conducted. HSP90, PI3K, Akt, mTOR, and MAPK inhibitors reduced the TDP25 levels in the first screening, consistent with the previous reports , showingIn vitro studies show that c-Abl directly interacts with α-synuclein and catalyzes its phosphorylation at tyrosine 39 (pY39) (Increasing evidence suggests that c-Abl plays important roles in neurodegenerative diseases. 9 (pY39) . Further9 (pY39) . Our stuvia the inhibition of c-Abl and the NF-κB pathway. Ibudilast, an anti-inflammatory and neuroprotective oral agent, is currently at clinical trials to treat progressive multiple sclerosis and ALS. These findings suggest that inflammation also promotes ALS occurrence. However, how inflammation affects the formation of TDP25 aggregates and how the formation of TDP25 aggregates promotes inflammation is unclear. SC75741, as a multi-target drug, could be a promising strategy for treating ALS by inhibiting inflammation and promoting aggregate degradation.Past studies have found that the mRNA and protein levels of NF-κB are higher in the spinal cord of ALS patients than in normal patients . FurtherIn conclusion, our study demonstrates for the first time that SC75741 as a novel autophagy enhancer can inhibit c-Abl pathway, activate autophagy, and trigger TDP43-related aggregates clearance by the ALP. Our findings will facilitate the development of dual-targeted inhibition of c-Abl and NF-κB as a promising drug target for the treatment of ALS.


Corynebacterium glutamicum as a host to produce 3-hydroxypropionic acid (3-HP) from acetate was explored. First, the expression level of malonyl-CoA reductase from Chloroflexus aurantiacus was optimized through several strategies, strain Cgz2/sod-N-C* showed an MCR enzyme activity of 63 nmol/mg/min and a 3-HP titer of 0.66 g/L in flasks. Next, the expression of citrate synthase in Cgz2/sod-N-C* was weakened to reduce the acetyl-CoA consumption in the TCA cycle, and the resulting strain Cgz12/sod-N-C* produced 2.39 g/L 3-HP from 9.32 g/L acetate. However, the subsequent deregulation of the expression of acetyl-CoA carboxylase genes in Cgz12/sod-N-C* resulted in an increased accumulation of intracellular fatty acids, instead of 3-HP. Accordingly, cerulenin was used to inhibit fatty acid synthesis in Cgz14/sod-N-C*, and its 3-HP titer was further increased to 4.26 g/L, with a yield of 0.50 g 3-HP/g-acetate. Finally, the engineered strain accumulated 17.1 g/L 3-HP in a bioreactor without cerulenin addition, representing the highest titer achieved using acetate as substrate. The results demonstrated that Corynebacterium glutamicum is a promising host for 3-HP production from acetate.Acetate is an economical and environmental-friendly alternative carbon source. Herein, the potential of harnessing  Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae  with strong robustness (3-Hydroxypropionic acid (3-HP) is an important chemical raw material. It has been broadly used in agriculture, food, and materials , which crevisiae , and a vrevisiae . So far,ubstrate . Corynebbustness , which ibustness . Meanwhibustness .C. glutamicum ATCC 13032, when more than 6-fold of acetyl-CoA would be accumulated compared to glucose , and 15.8 g/L 3-HP was produced with a yield of 0.71 g-3-HP/g-acetate  was synthesized by GENEWIZ , and three reported point mutations  were introduced to increase enzyme activity  to induce expression.The original strain was activity . To consC. glutamicum was achieved via a two-step homologous recombination using suicide plasmid pD-sacB. For replacing the native promoter of gltA, the vector pD-sacB-P1-gltA was constructed as follows: Both the upstream and downstream sequences of gltA promoter were amplified by PCR and fused with promoter P1, followed by digesting and ligating to corresponding sites of vector pD-sacB. The plasmids pD-sacB-P5-gltA and pD-sacB-P7-gltA were constructed analogously.Genome editing of fasO sites of accBC and accD1, the vector pD-sacB-fasO(M)-accBC was constructed as follows: the flanking regions of the fasO site of accBC with relevant modifications were amplified and fused using PCR, after which the fused fragment was digested and ligated to corresponding sites of vector pD-sacB. The plasmid pD-sacB-fasO(M)-accD1 was constructed analogously.To introduce mutations into E. coli DH5α was incubated at 37°C and 220 rpm in test tubes containing 5 ml LB medium; 40 μg/ml kanamycin was added to the medium if needed.For the cultivation of the plasmid host, C. glutamicum strains, a single colony was used to inoculate 5 ml BHI broth (74 g/L) in a test tube for overnight pre-cultivation, after which 1 ml of the seed was used to inoculate 50 ml CGIII medium  with 20 g/L glucose in a 250-ml flask. When OD600 reached 15–20, the culture was used to inoculate 50 ml CGXII-YA medium 2SO4, 5 g/L urea, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgSO4·7H2O, 21 g/L MOPS, 10 mg/L CaCl2, 10 mg/L FeSO4·7H2O, 0.1 mg/L ZnSO4·7H2O, 0.2 mg/L CuSO4·5H2O, 20 μg/L NiCl2·H2O, 0.2 mg/L biotin, pH 7.0) in a 250-ml flask to an initial OD600 of 0.5. Then 25 μg/ml kanamycin and 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 0 h were added to the medium if needed. And 15 μM cerulenin was added to the medium at 12 h if needed. All fermentations were performed at 30°C and 220 rpm.For the shake flask cultivation of various recombinant 4)2SO4, 5 g/L urea, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgSO4·7H2O, 10 mg/L CaCl2, 10 mg/L FeSO4·7H2O, 0.1 mg/L ZnSO4·7H2O, 0.2 mg/L CuSO4·5H2O, 20 μg/L NiCl2·H2O, 0.2 mg/L biotin, pH 7.0) to a working volume of 2 L in a 5-L bioreactor . Temperature was set at 30 °C, and air flow rate was set at 1 vvm. The initial agitation speed was 300 rpm, which was set to adjust automatically to maintain dissolved oxygen above 30% of saturation. Pure acetate was added automatically into the broth to maintain pH at 7.0, and an adequate amount of sodium acetate was added externally when needed.The seed used for fed-batch cultivation was prepared in the same way, and 200 ml seed cultured in CGIII medium was used to inoculate CGXII-YB medium . Organic acids were quantified using a high-performance liquid chromatography (HPLC) system  equipped with a cation-exchange column , as described previously  in negative electrospray ionization (ESI) mode. Most of the metabolites were identified with LC equipped with a SeQuant ZIC-HILIC column . Then 10 mM ammonium acetate and 100% acetonitrile were used as mobile phases A and B, respectively, and the flow rate was set at 0.2 ml/min with a gradient as follows: 0–3 min, 90% B; 3–6 min, 90–60% B; 6–25 min, 60–50% B; 25–30 min, 50% B; 30–30.5 min, 50–90% B; and 30.5–38 min, 90% B. The relative content of metabolites was normalized to cell density.Procedures for extraction of intracellular metabolites are listed in the C. glutamicum strains were cultured in CGXII-YA medium till the exponential growth phase. Procedures of total RNA extraction and qRT-PCR were followed as previously described  producer due to the lack of the malonyl-CoA reductase (MCR). Therefore, the mcr* gene encoding a bifunctional malonyl-CoA reductase mutant   reductase (MCR-N) activity and the C-terminal half with malonyl-CoA reductase (MCR-C) activity   from Chll of MCR : the seql of MCR , were inactivity  were sepity (H36  and Psod strains . An opti strains . Both MCmcr showed neglectable MCR activity (2 nmol/mg/min), which explained the undetectable 3-HP in the medium. MCR activity of Cgz2/mcr* was increased to 33 nmol/mg/min, which was 15.5-fold higher than Cgz2/mcr, and 0.14 g/L 3-HP , and 3-HP titer was also increased to 0.22 g/L. It was reported that C-terminal half was the rate-limiting part  from -36.15 kJ/mol to -27.24 kJ/mol and -24.8 kJ/mol, respectively . Therefore, Cgz2/sod-N-C* was selected for further modification.The mbp-tag and his-tag were shown to improve the enzyme activity of MCR and the overall 3-HP production under the control of promoter Pectively , which csod-N-C* ; howeversod-N-C* . It was C. glutamicum was increased by about 5-fold, and the majority of which was depleted through the TCA cycle .Acetyl-CoA is the indirect key precursor for 3-HP synthesis, whose availability would extensively affect malonyl-CoA pool and 3-HP production. It was reported that when glucose was replaced with acetate as the sole carbon source, acetyl-CoA concentration in CA cycle . In orde1, P5, and P7 with different strengths  were selected from a promoter library constructed in our previous work  exhibited the highest biomass reduction (25.1%) compared with Cgz2/sod-N-C* —a heterodimer comprising subunits AccBC and AccD1 . It was nd accD1 . Since FfasO sequences of accBC and accD1 in Cgz2/sod-N-C* were substituted, as previously reported . Moreover, intracellular levels of cofactors NADH and NADPH were also influenced by the decreased flux through the TCA cycle as the intracellular NADH content and NADH/NAD+ ratio were decreased by 10 and 22%, respectively; meanwhile, the NADPH content and NADPH/NADP+ ratio were decreased by 59 and 69%, respectively  led to different metabolic responses, and the exact reason for which was undetermined. It was reported that the Km value for NADPH of MCR from Chloroflexus aurantiacus was 25 μM  in the two hosts, and malonyl-CoA might tend to be metabolized by fatty acid synthesis pathway under a lower intracellular NADPH level (acc genes further reduced the amount of acetyl-CoA metabolized in TCA cycle. The NADPH level in Cgz14/sod-N-C* was further decreased by 18%, indicating the NADPH availability in Cgz14/sod-N-C* needs to be improved to increase 3-HP synthesis.The detailed intracellular metabolites in Cgz14/sod-N-C* were also compared with Cgz12/sod-N-C* to analyze the metabolic response of deregulating as 25 μM , which wutamicum . TherefoPH level . MoreoveIn view of the fact that the increased supply of malonyl-CoA in Cgz14/sod-N-C* was mainly directed to fatty acids synthesis, cerulenin was used to inhibit fatty acid synthesis to explore its potential in 3-HP production with Cgz12/sod-N-C* as control. As shown in −1 till 120 h, reaching an OD600 of 132.6. The yield of 3-HP in bioreactor decreased by 61.5% compared with that of the same strain in shake flasks, which might be caused by increased carbon flow toward cell growth in the bioreactor, indicating the conditions for fed-batch fermentation could be further optimized.To further evaluate the ability of 3-HP production from acetate, Cgz14/sod-N-C* was cultured in a 5-L bioreactor without cerulenin addition. As shown in C. glutamicum strain proved its potential in 3-HP production from acetate.Strain Cgz14/sod-N-C* produced 4.26 g/L 3-HP from 8.52 g/L acetate with cerulenin addition in shake flasks, and 17.1 g/L 3-HP using acetate as substrate without addition of cerulenin in bioreactor, which is the highest titer achieved using acetate as substrate. The performance of the engineered C. glutamicum to sufficiently assimilate acetate as the sole substrate to produce 3-HP. With optimization of MCR expression and weakening of CS expression, 3-HP titer of strain Cgz12/sod-N-C* was increased to 2.39 g/L. With the subsequent deregulation of ACC and addition of cerulenin, strain Cgz14/sod-N-C* produced 4.26 g/L 3-HP with a yield of 0.50 g/g-acetate. Furthermore, Cgz14/sod-N-C* accumulated 17.1 g/L 3-HP without addition of cerulenin in a 5 L bioreactor. The results demonstrate that C. glutamicum is a promising host for 3-HP production from acetate.This is the first report on engineering