Klebsiella pneumoniae (KU726953), Salmonella enterica (KU726954), Enterobacter aerogenes (KU726955), Enterobacter cloaceae (KU726957) showed optimum production of MnP and laccase at 120 and 144 h of growth, respectively. The potential bacterial consortium showed decolourisation of Maillard product up to 70% in presence of glucose (1%), peptone (0.1%) at optimum pH (8.1), temperature (37 °C) and shaking speed (180 rpm) within 192 h of incubation. The reduction of colour of Maillard product correlated with shifting of absorption peaks in UV–Vis spectrophotometry analysis. Further, the changing of functional group in FT-IR data showed appearance of new peaks and GC–MS analysis of degraded sample revealed the depolymerisation of complex MRPs. The toxicity evaluation using seed of Phaseolus mungo L. showed reduction of toxicity of MRPs after bacterial treatment. Hence, this study concluded that developed bacterial consortium have capability for decolourisation of MRPs due to high content of MnP and laccase.Maillard reactions products (MRPs) are a major colorant of distillery effluent. It is major source of environmental pollution due to its complex structure and recalcitrant nature. This study has revealed that sucrose glutamic acid-Maillard reaction products (SGA-MRPs) showed many absorption peaks between 200 and 450 nm. The absorption maximum peak was noted at 250 nm in spectrophotometric detection. This indicated the formation of variable molecular weight Maillard products during the SGA-MRPs formation at high temperature. The identified aerobic bacterial consortium consisting The online version of this article (10.1007/s11274-018-2416-9) contains supplementary material, which is available to authorized users. The majlly AIDA .2O2 treatment , which are predominantly present in sugarcane juice composition which generate the complex of molasses melanoidins due to thermal degradation reaction at subsequent stage Walford . Further−1; glucose 1%, peptone 0.1%, MgSO4·7H2O 0.05%, K2HPO4 0.1%). The flasks were incubated at 37 ± 1 °C in a refrigerated incubator shaker  at 120 rpm for 7 days. The flask containing the sample showing the decolourisation was selected. Subsequently, an aliquot (1.0 mL) was taken serially diluted in order of 10−2, 10−3, 10−4, 10−5, and 10−6, then after diluted sample (0.1 mL) was spread on GPM agar medium plates. Further, the bacterial colonies showing clear zone on agar plates were purified on GPM agar plate by streak plate method.The sludge sample was collected from the dumping site of M/s Unnao Distilleries and Breweries, located in Unnao, Uttar Pradesh , India for isolation of potential bacterial strains. For isolation of bacterial strains, 10 g of sludge sample was transferred to a 250 mL Erlenmeyer flask containing 100 mL sterile GPM broth (g L−1 w/v). Each plate was incubated at 37 ± 1 °C for 48 h and observed for growth of tolerant bacterial colonies. The bacterial strains tolerant to high concentration of melanoidins were selected and screened for MnP and laccase activity by plate assay method. For enzyme screening, the substrate used for MnP (EC 1.11.1.13) was phenol red   dextrose 1%, peptone 0.5%, beef extract 0.3%, NaCl 0.5%, and CuSO4 (1 mM) , MgSO4·7H2O (0.05%) and molasses melanoidins (1000 mg L−1) and incubated for 24 h in continuous shaking condition (125 rpm) at 37 ± 1 °C. After 24 h, the equal volume of each bacterial strains containing cell density 3.1 × 104 cells mL−1 was used for preparation of different bacterial consortium. The bacterial strains IITRCS01, IITRCS06, IITRCS07, and IITRCS11 were taken in the various combinations i.e. in the ratio of 1:1:1:1, 1:2:1:1, 1:2:2:1, 1:2:2:2, 2:1:1:2; 2:2:1:1, 2:2:2:1 and 2:2:2:2 for optimal decolourisation of SGA-MRPs.A loopful pure culture of isolated bacteria strains IITRCS01, IITRCS06, IITRCS07, and IITRCS11 was transferred to a 50 mL modified GPM broth medium supplemented with K2HPO4 0.1%) and MgSO4·7H2O (0.05% g L−1) and 1.0 mL of trace elements solution containing CuSO4·5H2O (0.003%), MnCl2·4H2O (0.001%), ZnSO4·7H2O (0.001%), and CaCl2·2H2O (0.002%), and pH was adjusted to 7.0 with 1 N NaOH  containing 100 mL of previously prepared  were evaluated for the SGA-MRPs decolourisation and degradation. In another experiment, different organic and inorganic nitrogen sources viz. peptone, beef extract, yeast extract, sodium sulphate, ammonium chloride and urea were added into the GPM medium at 0.5% (w/v) concentration. The effect of carbon and nitrogen sources were optimised for decolourisation and degradation SGA-MRPs at different concentration viz. 0.1–1.0 (w/v).To evaluate the effect of different environmental conditions the same experiment was carried out at different temperature (25–50 °C), pH (4–12), and shaking speed (100–220 rpm).g) for 10 min at 4 °C, the pellets were washed thrice with distilled water to remove the attached medium contents. For bacterial biomass determination, the pellet was dried in an hot air oven at 80 °C until getting a constant dried weight reported in the form of dry cell mass (g L−1). For SEM micrograph, the bacterial cells were fixed with 1% (w/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h and washed again with distilled water. The fixed samples were then dehydrated through 25, 50, 75, 95 and 100% ethanol solutions in increasing concentration for 5 min, at each step. The samples were then dried in critical point drier and coated with a thin conductive film of gold in a sputtering coater and observed under SEM .During decolourisation experiment, the optical density was continuously measured till 192 h at 12 h periodic interval by taking absorbance at 620 nm by UV–Vis spectrophotometer as the measurement of the bacterial cell growth. While the bacterial sample for biomass determination and SEM micrograph was collected from the flask after 144 h of incubation. First, the bacterial cells were centrifuged  and guaiacol , respectively as described earlier  in order to reveal the chemical nature of the SGA-MRPs. The separated samples of SGA-MPs were mixed with potassium bromide (KBr) to prepare the pellet for FT-IR analysis.UV–Vis absorption spectra of the treated and untreated SGA-MRPs solution were recorded using a UV–Vis spectrophotometer in the wavelength range between 200 and 700 nm at room temperature. Further, FT-IR analysis of the sample was performed in range of 400–4000 cmThe organic compounds present in treated and untreated SGA-MPs were extracted by ethyl acetate under acidic condition (pH < 2.0) as previously described  by using the gradient of solvent A (Milli-Q water) and solvent B (acetonitrile with 0.1% TFA)  at a flow rate 0.4 mL min−1 for 60. The detection was monitored at wavelength 250 nm (absorption maxima) to assess the decolourisation and degradation of Maillard products. 50 µL of methanol extract was injected into the column by using a glass microsyringe .HPLC analysis was carried out using waters, 515 HPLC instrument equipped with a diode array detector system  and reverse phase C−1. The temperature of GC oven was programmed started from 65 °C (hold for 2 min), increased to 230 °C at a rate of 6 °C min−1 and finally reached to 290 °C (hold for 20 min) at a rate of 10 °C min−1 increased. The transfer line temperature and ion source temperature were kept at 290 and 220 °C, respectively. The mass spectrum (MS) was operated in the positive electron ionization (+ EI) mode at an electron energy of 70 eV with a solvent delay of 7 min. The MS was operated in full-scan mode from m/z, 45–800. The detected organic compounds were identified by matching with the MS library NIST v. 1.0.0.12 available with instrument.In GC–MS analysis, the extracted samples were derivatised with trimethylsilyl (TMS) as described by Chandra and Kumar . An aliqP. mungo L. seed germination using Petri dish method .All the experiment was carried out in triplicate and the results were presented as the mean of three independent observations. To confirm the variability of data obtained and validity of results, the mean concentration of various physico-chemical parameters of untreated and treated SGA-MRPs was calculated by Student’s −1) containing phenol red amended modified GPM agar plates, as well as laccase activity by developing a reddish brown color zone on B&K agar plate during the screening of potential bacterial strains capable for growth on SGA-MRPs amended media. The screened bacterial strains also showed tolerance for growth on melanoidins amended GPM agar plate at 3500 mg L−1 concentration.Twenty-seven morphologically distinct bacterial strains were isolated from distillery sludge. Only four bacterial isolates namely IITRCS01, IITRCS06, IITRCS07, and IITRCS11 could show the MnP activity by changing the deep orange to light yellow color at melanoidins (800 mg LKlebsiella pneumoniae (KU726952), Salmonella enterica (NR044372), Enterobacter aerogenes (NR024643), and Enterobacter cloaceae (KJ437492) found in the GenBank database. Hence, based on the sequence homology, the bacterial strain IITRCS01, IITRCS06, IITRCS07, and IITRCS11 were identified as K. pneumoniae (KU726953), S. enterica (KU726954), E. aerogenes (KU726955), E. cloaceae (KU726957), respectively. These bacterial species belonged to group Gammaproteobacteria. Further, the phylogenetic tree was showed the interrelationship of bacterial species with the most closely related genera inferred from sequences of the 16S rRNA gene as shown in Supplementary Fig. S2.Screened bacterial strains IITRCS01, IITRCS06, IITRCS07, and IITRCS11 showed several morphological and biochemical characteristics are mentioned in Bergey’s manual of Systematic Bacteriology  were found higher as shown in Table −1) was noted as shown in Table The physico-chemical characteristics of untreated SGA-MRPs showed dark brown in color at pH 6.9. In addition, the concentration of BOD 8641), COD , TDS (1050.77), VS (457.83), and phenolics  concentration for 192 h of incubation, and the results are illustrated in Fig. The effect of different organic and inorganic nitrogen sources at a concentration of 0.5% (w/v) along with glucose 1.0% w/v) showed that peptone was most effective organic nitrogen source which enhanced up to 72% decolourisation of Maillard products at 0.1% (w/v) concentration  was optimum for maximum solubility of Maillard products. Further, increase in pH inhibited the decolourisation process  and biomass  at 144 h incubation period during decolourisation process , and laccase activity was found maximum (1.91 U mL−1 min−1) at 144 h of incubation . But, after 120 and 144 h incubation showed gradual decrease of MnP and laccase enzyme activities of bacterial consortium, respectively. The colour reduction of MRPs was significantly initiated at 72 h and achieve maximum decolourisation at 144 h incubation.To observe the function of ligninolytic enzymes in the decolourisation and degradation of SGA-MRPs, enzymes were measured and MnP was found to be the dominating enzymes at the initial stage of decolourisation. MnP activity was noted maximum at 120 h  may correspond to carbonyl groups in primary amide functions, or it could appear because of the existence of C–O stretching of COO− ketonic C–O and aromatic C–C conjugated with COO−. The absorption band at 1411.1 and 1457.8 cm−1 might appear due to aliphatic C–H bending and COO− asymmetric stretching in spectra. The untreated sample showed absorptions peak between 1000 and 1200 cm−1 in untreated and simulated spectra could appear due to C–O bonds  and/or minerals. While a band at 922.8, and 978.0 cm−1 indicated the presence of C–O stretching of polysaccharides or Si–O-asymmetric stretch. The untreated sample also showed an intense peak at 847.7, 778.5, 629.6 and 535.4 cm−1 represent the sulfate, carbohydrate, alkyl halides, nitro group, and C–S bond. The treated sample showed a narrow peak at 3414.0 cm−1 represented the stretching vibration of O–H group. The spectra corresponding to treated sample was displays bands at 2855.4 and 2924.8 cm−1, probably due to stretching vibrations of CH3 and CH2 groups, respectively. The broad stretching adsorption band peak at 2160.0 cm−1 could be assigned to C=C stretching vibrations. The absorption band at 1444.9 cm−1 might appear due to aliphatic C–H bending. The occurrence of aromatic groups is also suggested by absorptions at around 700 cm−1. Moreover, most of the bands were similar between untreated and simulated samples.FT-IR analysis supported the depolymerisation of SGA-MRPs after bacterial treatment by shifting the various functional groups as shown in Supplementary Fig. S5. The FT-IR spectrum of the SGA-MRPs is characterized by an intense absorption between 3200 and 3600 cmThe HPLC analysis report demonstrating the area, height, and retention time (RT), before and after bacterial treatment of Maillard products which confirms the degradation of melanoidins Fig. b. The HPd-lactic acid-DiTMS (RT:8.34), 1-dodecene (RT:8.41), undecane, 2-methyl (RT:8.80), butanoic acid, TMS ester (RT:9.12), dodecane (RT:9.28), silanol, trimethyl, benzoate (RT:12.36), heptadecane, 2,6,10,15-tetramethyl (RT:13.22), benzeneacetic acid, TMS ester (RT:13.44), 1-tetradecanol (RT:13.60), 2-bromo dodecane (RT:13.93), pentadecane (RT:14.03), dodecane, 2,6,11-trimethyl (RT:14.07), heptadecane (RT:14.47), tetradecane (15.77), hexadecane, 2,6,10,14-tetramethyl (RT:17.60), phenol,2,4-bis  (RT:17.95), 1-tetradecanol (RT:18.17), tetradecane, 2,6,10-trimethyl (RT:18.30), hexadecane (RT:19.82), dodecanoic acid, TMS ester (RT:20.77), docosane (RT:21.68), pyrazine, 2,5-dimethyl-3-propyl (RT:21.56), 2,6-diisopropylnaphthalene (RT:22.19), 3,4-tetramethylene-5,5-pentamethyle-2-nepyrazoline (RT:22.66), Hexadecane, 2,6,11,15-tetramethyl (RT:25.64), 7,9-di-tert-butyl-1-oxaspirodeca-6,9-diene-2,8-dione (RT:25.21), heptacosane (RT:29.40), hexadecanoic acid, TMS ester (RT:27.55), 1H-purin-6-amine,[(2-fluorophenyl)]methyl (RT:27.90), pentacosane (RT:29.29), 2,2-bis [(4-trimethylsiloxy)phenyl]propane (RT:30.00), nonacosane (RT:32.30), 1,2-benzenedicarboxylic acid, bis (2-ethylhexyl)ester (RT:33.51), hexadecanoic acid (RT:33.67), octadecane, 3-ethyl-5-(2-ethylbutyl) (RT:33.97), and methylenebis  (RT:43.94). However, the investigation of bacteria treated SGA-MPs sample has showed the existence of various organic compounds such as, 3-hydroxy-2-butanone, TMS ester (RT:9.41), ethanolamine, TBS (RT:9.65), butane, 2,3-bis (TMSoxy) (RT:10.52), silanol, trimethyl, pentadecane (RT:14.03), undecenoic aicd, TMS ester (RT:18.51), cis-5-dodecanoic acid, TMS ester (RT:22.71), tetradecanoic acid, ethyl ester (RT:24.28), 2-hexadecanol acetate (RT:25.52), trans-9-octadecenoic acid 1TMS (25.60), cis-10-heptadecenoic acid, TMS ester (RT:28.79), octadecanoic acid, ethyl ester (RT:29.72), 11-trans-octadecenoic acid, TMS ester (RT:30.10), methyl 19-methyl-eicosanoate (RT:32.16), 1-docosanol, acetate (RT:33.40), docosanoic acid, methyl ester (34.08), 17-pentatriacontene (RT:35.13), 2-monostearin, TMS ether (RT:35.30), octadecanoic acid, 2,3-bis [(TMS)oxy]propyl ester (RT:35.58), and ethyl tetracosanoate (RT:35.72) at different RT. These compound were completely absent in untreated sample, they are the degraded products of SGA-MRPs and few as new metabolites. In bacterial treated sample two organic compounds i.e. docosane (RT:21.68) and dodecanoic acid, TMS ester (RT:20.77) could not be metabolized.In the present study, the GC–MS analysis of ethyl acetate extracted untreated SGA-MRPs showed the existence of different types of organic pollutants, most of which were biotransformed and biodegraded during bacterial treatment Fig. ; Table 2P. mungo L. seeds. It was observed that untreated sample was highly toxic in nature and showed only 75, 70, 60, 15% germination at 2.5, 5, 10 and 15% concentration of SGA-MRPs. But, after treatment with bacterial, the toxicity of SGA-MRPs was reduced extensively and showed 90, 90, 80, 25, and 20% germination at 2.5, 5, 10, 15, and 20% concentration of Maillard products  which is required for the melanoidins degradation , S. enterica (KU726954), E. aerogenes (KU726955), E. cloaceae (KU726957), respectively. This bacterial species belonged to group Gammaproteobacteria. The class Gammaproteobacteria constitutes a very large and diverse group of bacteria that exhibits enormous variety in terms of their phenotype and metabolic capabilities  was noted at pH 8.1 by developed bacterial consortium after 192 h of incubation. Further, increase or decrease in pH inhibited the decolourisation process. This might be due to inhibition of the enzyme activity. For developed aerobic bacterial consortium comprising −1; CFU mL−1 28 × 106) OD620 (2.49) at 144 h of incubation. Further, incubation did not increase the rate of decolourisation. The direct correlation of OD620 with high bacterial biomass indicated the direct involvement in decolourisation and degradation process of MRPs. This indicted high bacterial growth was due to utilisation of MRPs as a carbon source for its growth.For efficient and potential consortium, different inoculums ratio of bacterial strains were also optimized. Consortium having 1:1:1:1 inoculums ratio of bacteria showed maximum (74%) decolourisation of MRPs within biomass (5.15 g L−1 min−1) at 120 h of incubation and laccase activity was found highest (1.91 U mL−1 min−1) at 144 h of incubation. The MnP and laccase have a broad substrate oxidising enzymes reported for detoxification and degradation of phenolic and non-phenolic polymeric compounds  detected in untreated sample were diminished from the treated sample by MnP and laccase enzyme activity of bacteria. Hence, the optimized conditions for developed bacterial consortium can be used for decolourisation of MRPs containing industrial wastewater at large scale.Below is the link to the electronic supplementary material.Supplementary material 1 (DOC 6094 KB)


Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.DOI:http://dx.doi.org/10.7554/eLife.25174.001 These ATPases are essential for many cellular processes, including DNA replication, proteostasis, membrane remodeling, and cytoskeletal organization (The AAA+ (ATPases Associated with diverse cellular Activities) superfamily is comprised of ~100 proteins in humans , are present in metazoan cells and are required for a wide range of cellular processes . TranspoCiliobrevins were recently reported as the first selective, cell-permeable probes of dynein . AlthougThe ciliobrevins are based on a benzoylacrylonitrile-substituted quinazolinone scaffold . This tyDynein is a large, ~4600 amino acid protein that contains six unique AAA ATPase domains . Many AAHere, we characterize the conformation of ciliobrevins and design tricyclic pyrazoloquinazolinone derivatives that are more potent inhibitors of dynein. One derivative, which we name dynapyrazole-A inhibits dyneins 1 and 2 with similar potencies in vitro and in cellular assays. Biochemical analyses of this compound showed that while it inhibits the microtubule-stimulated ATPase activity of dynein, it does not potently block the microtubule-independent basal activity. This mode of activity is unlike that of the ciliobrevins, which inhibit both basal and microtubule-stimulated hydrolysis.1, a derivative with a 2-morpholinoethyl ether substitution, was synthesized using a previously described procedure and was found to readily crystallize . This analysis revealed that the dichlorophenyl D ring of 4 projects from the pyrazoloquinazolinone core such that it is offset from the D ring of ciliobrevin  inhibited dynein-2-dependent microtubule gliding . By contrast, compound 7, with a methoxy group at the 6-position of the A ring, did not substantially inhibit dynein-2-driven microtubule gliding . For the two most active compounds (6 and 8) as well as ciliobrevin D, we performed dose-dependent analyses. We found six- to eightfold increases in potencies of compounds 6 (IC50: 2.9 ± 0.6 µM) and 8 (IC50: 2.6 ± 1.3 µM) relative to ciliobrevin D (IC50 of 20 ± 1.0 µM) in this assay  and compound 9, a 6-CF3-substituted ciliobrevin derivative  ~8 fold more potently than ciliobrevin D  over the 28 hr time course of this experiment. In this assay, we have also observed cell death at high ciliobrevin D concentrations . To stimulate Hedgehog pathway activity, we used the synthetic agonist of smoothened (SAG)  , which ced (SAG) . As dynaed (SAG) .8, 5 µM), retrograde-directed IFT88 punctae were markedly slowed . In contrast, anterograde particle velocities were only reduced by ~18% . After 10 min of treatment, reductions in velocities were similar to those at the 5 min time point  were transferred to solvent-control media with serum  and incubated for an additional 10 min. Both retrograde and anterograde velocities recovered to control levels . Dynapyrazole-A (30 µM) inhibited the microtubule-stimulated ATPase activity to a residual rate of ~0.5 s−1 , ADP-vanadate dependent photocleavage of dynein was reduced ~2.5-fold relative to controls . These data are consistent with dynapyrazole-A binding at the AAA1 site.To better understand how dynapyrazole-A inhibits dynein activity, we monitored its effect on ADP-vanadate-dependent photocleavage of dynein. In this assay, an established read-out of nucleotide binding at dynein's AAA1 site , ultravi−1, n = 5), as has been previously shown for yeast dynein with a similar mutation(−1 (3 µM microtubules) and 1.7 s−1 (11.5 µM microtubules) , a mutation that disrupts nucleotide binding at AAA3 . We foun mutation. The ATPtubules) . The redtubules) .50 of 5.5 ± 1.6 µM, while ciliobrevin D has an IC50 of 38 ± 6 µM  reduced the AAA3 mutant dynein’s ATPase activity by 70–80% . Surpris8 ± 6 µM . Even ins tested . It is nIn this study, we analyzed the structure of the ciliobrevins and designed tricyclic derivatives in which the benzoylacrylonitrile of ciliobrevin was replaced with a cyanopyrazole. These compounds inhibited dynein in vitro and in cells more potently than ciliobrevins and have improved chemical properties. We also find that dynapyrazole-A inhibits dynein by a mechanism that is distinct from that of ciliobrevins.4-8 has not previously been reported in a bioactive compound. The hydrophobicity of the dynapyrazoles remains a limitation that needs to be addressed. We note that the high ClogP value of dynapyrazole-A, which is similar to that of ciliobrevins, may explain why this compound, like ciliobrevin D, has reduced activity in the presence of high protein concentrations (e.g. serum). Likewise, acute reversal of intraflagellar transport inhibition was most effective using cell culture medium with serum. We believe that the dynapyrazole scaffold will be valuable for further optimization of these chemical probes.The pyrazoloquinazolinone scaffold of the dynapyrazoles addresses many of the limitations of the ciliobrevins. First, activated acrylonitriles, such as the benzoylacrylonitrile at the core of the ciliobrevin scaffold, have been shown to be susceptible to attack by intracellular nucleophiles, raising the possibility of unwanted reactivity of these dynein inhibitors . While cIn current models, the motion of membrane-bound organelles requires a balance of motors moving toward microtubule plus- and minus-ends . Studiesm values separated by two orders of magnitude . It is Of the six AAA sites in dynein, only mutations at AAA1 and AAA3 have been found to block motility . In currOur finding that dynapyrazole-A does not potently inhibit ATP hydrolysis in the absence of microtubules suggests that, for dynein 1, the AAA1 site contributes to only a small fraction of this enzyme’s basal activity. We posit that a separate site, likely AAA3, accounts for the majority of dynein’s ATPase activity in the absence of microtubules. This site likely remains active even when ATPase activity at AAA1 is inhibited by dynapyrazoles. Currently, we cannot exclude the possibility that additional ATPase sites, such as AAA4, may also contribute partially to the basal ATPase activity of dynein. The nucleotide state at AAA3 is known to regulate the ATPase cycle of AAA1 when microtubules are present, with the apo- and ATP-bound states of AAA3 slowing overall hydrolysis and the ADP-bound state of AAA3 leading to rapid hydrolysis at AAA1 in wild-type dynein . Our finWhile restricting conformational flexibility of ligands is a well-established strategy for improving potency, its successful application typically depends on structural data that reveal the compound conformation required to bind its target protein . In the 1H NMR spectra were recorded at 500MHz on a Bruker Advance III HD 500 MHz NMR spectrometer equipped with a TCI cryogenic probe with enhanced 1H and 13C detection. Chemical shifts are reported in parts per million (ppm) and coupling constants (J) are expressed in hertz (Hz). All data were collected at 298K, and internally referenced for 1H to the residual chloroform signal at 7.26 ppm; to DMSO signal at 2.50 ppm; or to TMS at 0 ppm. Flash chromatography purifications were performed on CombiFlash Rf (Teledyne ISCO) as the stationary phase. The LC-MS was performed on a Waters ACQUITY H-Class UPLC/MS with a PDA eLambda detector and QDa mass spectrometer. Reactions under microwave irradiation were performed on a CEM Explorer 48 System.General chemical methods: Solvents and reagents were purchased from VWR or Sigma Aldrich. All reactions involving air- or moisture-sensitive compounds were performed under nitrogen atmosphere using dried glassware. 1H NMR  δ 13.36 , 9.88 , 7.95 , 7.66 , 7.50–7.41 , 7.05–6.95 , 6.94–6.86 , 4.50 , 4.09–3.93 , 3.68 , 3.57 , 3.24 . LCMS m/z: 487.1 [M+H]+.To a mixture of (2E)−3-−2-(7-hydroxy-4-oxo-1H-quinazolin-2-ylidene)−3-oxo-propanenitrile   and pota3 (aq.) and extracted with ethyl acetate. The combined organic layer was washed with water and brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (hexane/ethyl acetate) to yield 2-[1-ethylidene]propanedinitrile  as a colorless oil. 1H NMR  δ 7.57 , 7.43 , 7.19 , 2.62 . LCMS m/z: 235.1 [M-H]-.To a solution of 1-ethanone  in toluene (20 mL) - acetic acid (2 mL) were added malononitrile  and ammonium acetate  at room temperature. The mixture was stirred at 100°C for 13 hr. The mixture was neutralized with sat. NaHCO2O3 (aq.) and extracted with dichloromethane. The combined organic layer was washed with sat. NaS2O3 (aq.) and brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (hexane/ethyl acetate) to yield 2-[2-bromo-1-ethylidene]propanedinitrile  as a colorless oil. 1H NMR  δ7.59 , 7.47 , 7.34 , 4.58 . LCMS m/z: 314.856 [M-H]-.To a solution of 2-[1-ethylidene]propanedinitrile  in carbon tetrachloride (4 mL) was added bromine  at room temperature. The mixture was stirred at room temperature for 15 min and at 70°C for 8.5 hr. The starting material remained, so bromine  was added. The mixture was stirred at 70°C for 21 hr. The mixture was diluted with sat. NaS1H NMR  δ 8.81 , 8.40 , 7.84 , 7.66 , 7.57–7.46 , 7.40 , 7.36 . LCMS m/z: 354.1 [M+H]+.To a solution of 2-[2-bromo-1-ethylidene]propanedinitrile  in isopropyl alcohol (30 mL) was added methyl 2-aminobenzoate  at room temperature. The mixture was stirred at 100°C for 20 hr. The mixture was concentrated. The residue was purified by column chromatography (hexane/ethyl acetate) to give 2-−5-oxo-4H-pyrroloquinazoline-3-carbonitrile  as a colorless solid. 1H NMR  δ 12.34 , 8.19 , 7.99 , 7.94 , 7.80 , 7.61–7.53 , 6.42 . LCMS m/z: 329.9 [M+H]+.A mixture of 2-hydrazinobenzoic acid , 3-−3-oxo-propanenitrile  and acetic acid (2 mL) was stirred at 150°C under microwave irradiation for 30 min. The mixture was diluted with water and ethyl acetate, the insoluble material was collected by filtration to give the desired compound as a colorless solid (7.5 mg). The filtrate was extracted with ethyl acetate. The combined organic layer was washed with water and brine, dried over MgSO4, filtered and concentrated in vacuo. The solid was washed with ethyl acetate to give the desired compound (9.4 mg). The combined solid was washed with hexane to yield 2-−4H-pyrazoloquinazolin-5-one  as a colorless solid.4, filtered and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/hexanes) to yield 2-[-hydroxy-methylene]propanedinitrile  as light yellow amorphous powder. This product was subjected to the next reaction. 1H NMR  δ7.55 , 7.46–7.38 . LCMS m/z: 236.992 [M-H]-.To a stirred solution of propanedinitrile  in THF (150 mL), sodium hydride  was added at 0°C. The mixture was stirred at room temperature for 1 hr. The solution of 2,4-dichlorobenzoyl chloride  in THF (50 mL) was added to the mixture at 0°C. The mixture was stirred at room temperature for 4 hr. The mixture was diluted with 1N HCl (aq.) and extracted with ethyl acetate. The combined organic layer was washed with water and brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (hexane/ethyl acetate) to yield 2-[-methoxy-methylene]propanedinitrile  as a brown solid. . 1H NMR  δ 7.62 , 7.49 , 7.34 , 3.85 .A mixture of 2-[-hydroxy-methylene]propanedinitrile , dimethyl sulfate  and N-ethyl-N-isopropyl-propan-2-amine  in dioxane (200 mL) was stirred at 60°C for 23 hr. The reaction was cooled to room temperature and concentrated in vacuo. The residue was dissolved with ethyl acetate and quenched with water. The organic layer was separated, washed with brine, dried over MgSO1H NMR:  δ 12.31 , 7.76 , 7.58–7.41 , 6.49 . LCMS m/z: 253.184 [M+H]+.The mixture of 2-[-methoxy-methylene]propanedinitrile  and hydrazine hydrate  in ethanol (50mL) was stirred at 80°C for 4.5 hr. Additional hydrazine hydrate  was added to the mixture and it was stirred at 80°C for 1.5 hr. The reaction was concentrated in vacuo. The residue was washed with ethanol to yield 5-amino-3-−1H-pyrazole-4-carbonitrile  as an off-white solid. 4 and concentrated in vacuo. The residue was purified by silica-gel column chromatography (hexane/ethyl acetate) to yield 2-−5-oxo-4H-pyrazoloquinazoline-3-carbonitrile  as a white solid. 1H NMR:  δ 8.09 , 7.94 , 7.83 , 7.73–7.56 , 7.40 . LCMS m/z: 353.136 [M-H]-.A mixture of 5-amino-3-−1H-pyrazole-4-carbonitrile , dipotassium carbonate  and methyl 2-fluorobenzoate  in dimethylformamide (1 mL) was stirred at 140°C for 30 min. The mixture was poured into water, and extracted with ethyl acetate. The organic layer was washed with water and brine respectively, dried over MgSO1, 10 g, 50.86 mmol) in THF (100 mL) were added oxalyl chloride  and dimethylformamide . The mixture was stirred at room temperature for 0.5 hr. The mixture was concentrated in vacuo. The mixture was added to a solution of propanedinitrile  and sodium hydride  in THF (100 mL) at 0°C. The mixture was stirred at room temperature for 1 hr. The mixture was diluted with 1N HCl and extracted with ethyl acetate. The combined organic layer was washed with water and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (hexane/ethyl acetate) to yield 2-((1-(4-chlorophenyl)cyclopropyl) (hydroxy)methylene)malononitrile  as a light yellow oil. 1H NMR  δ 7.36–7.30 , 7.22 , 1.22–1.16 , 0.97 . LCMS m/z: not detected.To a solution of 1-(4-chlorophenyl)cyclopropanecarboxylic acid  in dioxane (200 mL) and H2O (20 mL) were added dimethyl sulfate  and NaHCO3 . The mixture was stirred at 100°C for 5 hr. The mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane/ethyl acetate) to yield 2-((1-(4-chlorophenyl)cyclopropyl)(methoxy)methylene)malononitrile  as a yellow oil. 1H NMR  δ 7.55–7.45 , 7.33–7.22 , 4.04 , 1.80–1.71 , 1.68–1.61 . LCMS m/z: not detected.To a solution of 2-[1-(4-chlorophenyl)cyclopropanecarbonyl]propanedinitrile  in ethanol (100 mL) was added hydrazine hydrate . The mixture was stirred at 80°C for 2 hr. The mixture was concentrated in vacuo. The residue was purified by column chromatography (hexane/ethyl acetate) to yield 5-amino-3-[1-(4-chlorophenyl)cyclopropyl]−1H-pyrazole-4-carbonitrile  as a white powder. 1H NMR  δ 11.73 , 7.35 , 7.27–7.16 , 6.28 , 1.43–1.13 . LCMS m/z: 259 [M+H]+.To a solution of 2-[[1-(4-chlorophenyl)cyclopropyl]-methoxy-methylene]propanedinitrile (4 and concentrated in vacuo. The residue was purified by flash chromatography (ethyl acetate/hexane) to yield 2-[1-(4-chlorophenyl)cyclopropyl]−5-oxo-4H-pyrazoloquinazoline-3-carbonitrile  as a white solid. 1H NMR  δ 13.23 , 8.17 , 8.10 , 7.95 , 7.59 , 7.38 , 7.32 , 1.55 , 1.42 . LCMS m/z: 361.233 [M+H]+.A mixture of 5-amino-3-[1-(4-chlorophenyl)cyclopropyl]−1H-pyrazole-4-carbonitrile , dipotassium carbonate  and methyl 2-fluorobenzoate  in dimethylformamide (1 mL) was stirred at 140°C for 30 min. The mixture was poured into water, and extracted with ethyl acetate. The organic layer was washed with water and brine respectively, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/hexane) to give a mixture. The amorphous material was triturated with acetonitrile and the white precipitate was collected to yield 2-[1-(4-chlorophenyl)cyclopropyl]−5-oxo-7-(trifluoromethyl)−4H-pyrazoloquinazoline-3-carbonitrile  as a white solid. 1H NMR  δ 13.49 , 8.36 , 8.25 , 7.42–7.35 , 7.35–7.27 , 1.63–1.52 , 1.48–1.38 . LCMS m/z: 429.2 [M+H]+.A mixture of 5-amino-3-[1-(4-chlorophenyl)cyclopropyl]−1H-pyrazole-4-carbonitrile  , methyl 2-fluoro-5-(trifluoromethyl)benzoate  and dipotassium;carbonate  in dimethylformamide (1 mL) was stirred at 140°C for 30 min under microwave irradiation . The mix4 and concentrated in vacuo. The residue was purified column chromatography (ethyl acetate/hexane) to yield 2-[1-(4-chlorophenyl)cyclopropyl]−7-methoxy-5-oxo-4H-pyrazoloquinazoline-3-carbonitrile  as a white solid. 1H NMR  δ 13.24 , 8.03 , 7.61–7.48 , 7.39–7.23 , 3.89 , 1.52 , 1.39 . LCMS m/z: 391.30 [M+H]+.A mixture of 5-amino-3-[1-(4-chlorophenyl)cyclopropyl]−1H-pyrazole-4-carbonitrile , dipotassium;carbonate  and methyl 2-fluoro-5-methoxy-benzoate  in dimethylformamide (3 mL) was stirred at 140°C for 40 hr. The mixture was poured into water, and extracted with ethyl acetate. The organic layer was washed with water and brine respectively, dried over MgSO1H NMR  δ 9.78 , 8.65 , 8.15 , 7.91 , 7.43–7.32 , 1.64 , 1.41 . LCMS m/z: 487.0 [M+H]+.A mixture of 5-amino-3-[1-(4-chlorophenyl)cyclopropyl]−1H-pyrazole-4-carbonitrile , methyl 2-fluoro-5-iodo-benzoate  and dipotassium carbonate  in dimethylformamide (10 mL) was stirred at 150°C for 1 hr under microwave irradiation. The reaction was cooled to room temperature and poured into water. The white precipitate was collected and washed with ethyl acetate to yield 2-[1-(4-chlorophenyl)cyclopropyl]−7-iodo-5-oxo-4H-pyrazoloquinazoline-3-carbonitrile  as a white solid. 1H NMR  δ 12.98 , 8.38 , 8.17 , 7.90 , 4.61 . LCMS m/z: 263.03 [M+H]+.To a solution of sodium methoxide  was added 2-chloroacetonitrile . The mixture was stirred at 25°C for 30 min. A solution of 2-amino-5-(trifluoromethyl)benzoic acid  in MeOH (7 mL) was added to the mixture. The mixture was stirred at room temperature for 12 hr. The resulting solid was collected by filtration to yield 2-(chloromethyl)−6-(trifluoromethyl)−1H-quinazolin-4-one  as a white solid. 2SO4 and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/hexane) to yield 2-[4-oxo-6-(trifluoromethyl)−1H-quinazolin-2-yl]acetonitrile  as a pale yellow solid. 1H NMR  δ 12.83 , 8.37 , 8.16 , 7.93–7.86 , 4.26 . LCMS m/z: 254.13 [M+H]+.To a solution of 2-(chloromethyl)−6-(trifluoromethyl)−1H-quinazolin-4-one  in DMSO (5 mL) was added sodium cyanide . The mixture was stirred at 25°C for 3 hr. The mixture was poured into aqueous ammonium chloride, and extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The resulting solid was collected by filtration using ethyl acetate to yield (2E)−3-−3-oxo-2-[4-oxo-6-(trifluoromethyl)−1H-quinazolin-2-ylidene]propanenitrile  as a pale yellow solid. 1H NMR  δ 13.66 , 8.23 , 7.91 , 7.67 , 7.56 , 7.47 . LCMS m/z: 428.08 [M+H]+.To a mixture of 2-[4-oxo-6-(trifluoromethyl)−3H-quinazolin-2-yl]acetonitrile in dioxane (4 mL) were added 2,4-dichlorobenzoyl chloride  and triethylamine  at 100°C. The mixture was stirred for 15 hr at 100°C. The mixture was poured into water, and extracted with EtOAc. The organic layer was washed with brine, dried over Na1, measurements were made on a Rigaku R-AXIS RAPID-191R diffractometer using graphite monochromated Cu-K irradiation. The structure was solved by direct methods with SIR2008  with accession numbers CCDC 1510769 (1) and CCDC 1510770 (5), respectively. These data can be obtained free of charge from the CCDC via the internet: http://www.ccdc.cam.ac.uk/Community/Requestastructure/Pages/DataRequest.aspx?.For compound  SIR2008  and was LXL-2013 . All nonLXL-2013 . For com SIR2008  and was L-2014/7 . All nonCrystal data for1: C23H20Cl2N4O4, MW = 487.34; crystal size, 0.21 × 0.10×0.07 mm; colorless, block; monoclinic, space group P21/c, a = 14.2322 (6) Å, b = 6.7917 (3) Å, c = 25.2715 (18) Å, α = γ = 90°, β = 90.494 (6)°, V = 2442.7 (2) Å3, Z = 4, Dx = 1.325 g/cm3, T = 100 K, μ = 2.698 mm−1, λ = 1.54187 Å, R1 = 0.098, wR2 = 0.201.Crystal data for5: C20H13ClN4O, MW = 360.80; crystal size, 0.23 × 0.05×0.03 mm; colourless, needle; monoclinic, space group P21/c, a = 10.32080 (19) Å, b = 4.91042 (7) Å, c = 34.5332 (5) Å, β = 90.6463 (16)°, α = γ = 90°, V = 1750.01 (5) Å3, Z = 4, Dx = 1.369 g/cm3, T = 100 K, μ = 2.067 mm−1, λ = 1.54187 Å, R1 = 0.037, wR2 = 0.096.2, 0.2 mM EGTA, 10% [v/v] glycerol, 300 mM KCl, 0.2 mM ATP, 1 mM DTT, 2 mM PMSF, and protease inhibitor cocktail ) and lysed using a dounce homonizer. The lysate was clarified by centrifugation at 1.2x105 g and then incubated with IgG-sepharose beads beads for 90 min at 4°. The beads were washed with 12 bed volumes of lysis buffer and 12 bed volumes of buffer B . TEV protease was added to the bead slurry and incubated at 4° for 8 hr with rotation. Cleaved soluble protein was collected, concentrated , and loaded onto a gel filtration chromatography column . The protein was eluted in buffer C , peak fractions were collected, concentrated, aliquoted, and then frozen in liquid nitrogen., which bears an N-terminal protein A tag, followed by tobacco etch virus (TEV) protease-cleavable linker, followed by a GFP tag, was expressed using the baculovirus/insect cell expression system and purified using a procedure similar to that described previously . Briefly, which, like the GFP-dynein 2 construct, bears N-terminal protein A tag, followed by a TEV-cleavable linker, followed by a GFP tag, was expressed using the baculovirus/insect cell expression system and purified using a similar procedure to that described for GFP-dynein 2 with the following modifications:TM, Thermo).The lysis buffer was: 30 mM HEPES (pH 7.5 with KOH), 200 mM NaCl, 1 mM DTT, 1 mM PMSF, protease inhibitor cocktail ,150 mM KOAc, 2 mM Mg(OAc)TEV protease was incubated with the bead slurry for 90 min on wet ice without agitation.The protein was not concentrated.5 g and then incubated with Ni-NTA beads for 2 hr at 4°C. The beads were washed with 75 bed volumes of lysis buffer and bound proteins were eluted with buffer containing 30 mM HEPES pH 7.5, 100 mM NaCl, 500 mM imidazole, and 1 mM TCEP. Eluate fractions containing protein were diluted into buffer B 2,1 mM EGTA, 1 mM EDTA, 0.1 mM ATP, 1 mM DTT) and loaded onto a Mono Q anion exchange column . The column was eluted using a salt gradient from 150 to 750 mM KOAc over 20 column volumes. Peak fractions were concentrated using a centrifugal concentrator (Amicon) and subjected to size exclusion chromatography on a Superose 6 column using buffer B. For K1912A and K2601A proteins, the ion exchange chromatography step was omitted and NiNTA-eluate fractions were pooled and subjected directly to size exclusion chromatography. Gel filtration revealed a monodisperse peak at an elution volume of 12.2 mL (wild-type), 12.3 mL (K1912A), and 12.4 mL (K2601A), consistent with the expected molecular weight. Peak fractions were pooled, supplemented with glycerol to a final concentration of 20%, and snap frozen. The protein yield was ~1–2 mg of dynein 1 wild-type or mutants per 1L of Sf9 culture. Cleavage of the TEV-cleavable linker following binding to Ni-NTA beads led to protein precipitation., proteins which contain N-terminal hexahistixine (6x-His), followed by tobacco etch virus (TEV) protease-cleavable linker were expressed using the baculovirus/insect cell expression system and purified as follows. An Sf9 cell pellet was resuspended in a buffer containing, 30 mM HEPES pH 7.6, 200 mM NaCl, 10 mM imidazole, 1 mM TCEP, 2 mM PMSF, and protease inhibitor cocktails  and lysed by the addition of Triton X-100 to a final concentration of 0.2%. The lysate was clarified by centrifugation at 1.2x10Reaction setup: A coverslip-based motility assay was adapted from similar assays reported previously . Anti-GFP antibody  was flowed into the chamber and allowed to adhere non-specifically to the glass for 20 s. The surface was blocked using 0.5 mg/mL α-casein in buffer A. Excess protein was then washed away with buffer A. GFP-dynein motor domain was diluted in buffer A supplemented with 1 mM DTT and 0.1 mM ATP , flowed into the chamber and allowed to bind for 20 s. Excess protein was washed away with buffer A. Reaction mix  was flowed into the chamber using ~4 chamber volumes. For experiments shown in eviously . A flow 8 was observed as above, and upon completion of imaging, the chamber, fresh buffer A supplemented with 1 mM ATP was flowed into the chamber and incubated for 1 min; this wash and incubation cycle was repeated twice. Reaction mix containing DMSO (no compound) was flowed into the chamber using ~4 chamber volumes. The chamber was sealed with valap and returned to the microscope.For reversibility experiments, motility in the presence of 5 µM Microscopy conditions: Following addition of reaction mix, microtubules were allowed to bind for 5 min before imaging. For reversibility experiments, microtubules were allowed to bind for 10 min before imaging. Videos were taken on a Zeiss Axiovert 200M wide-field microscope equipped with a Zeiss 100x/1.45 NA α-Plan-Fluar objective. Data were captured with an EM-CCD camera  with a 0.2 s exposure time and frame rate of 0.25–0.5/second. Four 10–20 s videos were captured per experimental condition. Velocities were measured using the kymograph tool in FIJI ( in FIJI . Data anATPase assays using recombinant Dynein 1 (wild-type and mutants) were performed in a buffer containing 25 mM PIPES pH7.0, 30 mM KCl, 1 mM EGTA, 5 mM MgCl2, 0.01% triton X-100, 1 mM DTT, and 20–30 µM taxol (if microtubules were present). For typical conditions, enzyme was incubated with varying concentrations of inhibitor in buffer containing 2% DMSO for 10 min in a volume of 10 μL. When microtubules were present, they were polymerized by stepwise addition of taxol and high concentrations of DMSO , and re-suspended following centrifugation in ATPase buffer to a concentration up to threefold above the final concentration used in the reaction . For microtubule-stimulated reactions in dose-response format, reactions were assembled by 1:1:1 mixture of 3x enzyme stock, 3x compound stock (in buffer with 6% DMSO), and 3x microtubule stock to each well. For microtubule-stimulated ATPase assays at tubulin concentrations > 2.5 µM, enzyme stock was supplemented with compound stock in pure DMSO (resulting in an intermediate DMSO concentration of 6%), and then the microtubule stock was added. Final enzyme concentrations were 30–100 nM for GFP-dynein 1, wild-type 6x-His-dynein 1, and K2601A 6x-His-dynein 1 and up to 500 nM for K1912A 6x-His-dynein 1. The final DMSO concentration was 2% in all ATPase assays performed.32P ATP  and allowed to proceed for a time predetermined to lie within the linear range of the assay (20–30 min). For typical conditions, ATP was used at a final concentration of 1 mM. Reactions were quenched by the addition of 12 µL 100 mM EDTA, and 2 μL of each quenched reaction was spotted onto PEI-cellulose TLC plates (Millipore). Plates were developed in a glass chamber with a freshly prepared solution of 150 mM formic acid and 150 mM LiCl, dried, exposed to a storage phosphor tray , and scanned on a Typhoon imaging system . The fraction of γ-phosphate hydrolyzed in each condition was quantified using FIJI.Assembled reaction mixtures  were incubated for 10 min before addition of ATP. Each ATPase reaction was initiated by addition of 2 μL of a 6x ATP stock containing trace γ-8  or an equivalent amount of solvent carrier (3.3% DMSO), followed by a 10 min incubation at room temperature. ATP  was added and the mixtures were incubated 5 min at room temperature. NaVO3  was added and the mixtures were again incubated 5 min at room temperature. Vanadate was deliberately omitted from one well. The plate was then transferred to ice and irradiated with 365 nm light for 15 min with the lamp positioned 10 cm above the plate(Spectroline Maxima ML-3500S lamp). Following addition of SDS-PAGE loading buffer and heating , individual reactions were resolved on hand-poured 8% tris-glycine polyacrylamide gels. Gels were stained (Coomassie blue), imaged using a Li-Cor Odyssey, and band intensities were analyzed using FIJI.Thirty-microliter reactions were prepared in 96-well plates as follows. ATPase buffer was supplemented with concentrated stocks of His-dynein 1 , compound CVCL_2721) were maintained in DMEM with 10% bovine calf serum (BCS). Cells were seeded at a density of 30,000 cells/well in 96-well tissue culture-treated plates  in 100 μL in DMEM +10% BCS, and incubated for 48 hr  containing solvent control (0.3% DMSO) or compound. At fixed time points, CellTiter-Glo 2.0 (Promega) reagent was added to the wells, mixed by pipetting, and transferred to a white, opaque 96-well plate. After a 10 min incubation, luminescence for each condition was read using a Synergy Neo plate reader (1 s integration time per well).Murine inner medullary collecting duct (IMCD3) cells stably expressing mNeonGreen-IFT88 (described previously ) were ma5 cells were seeded onto 22-mm coverslips in a six well plate and incubated for 24 hr at 37°C. The media was then replaced with DMEM/F12 +0.2% FBS and cells were incubated 24 hr to promote cilium formation. Immediately prior to imaging, media was replaced with phenol red-free media (Leibovitz's L-15 + 0.2% FBS) containing either carrier solvent control (0.3% DMSO) or compound. IFT88 transport was observed using a TE2000-E spinning disk confocal microscope (Nikon PlanApo 100x/1.45 objective lens) fitted with a Photometrics Cascade II (EMCCD 512) camera and imaged at a frame rate of 2 frames/s. Kymographs were generated in FIJI using KymographClear and the velocities of mNeonGreen-IFT88 foci movement were quantified by KymographDirect (Murine inner medullary collecting duct (IMCD3) cells stably expressing mNeonGreen-IFT88 were maintained in DMEM/F12 supplemented with 10% FBS. Cells were typically passaged fewer than 10 times following thawing of frozen stocks. Cells were described previously . 6 × 105phDirect . The algFor washout experiments, cells were exposed to a 5-min compound treatment as described above. Cells were then washed twice with 5 mL of L-15 supplemented with 10% FBSand 0.3% DMSO. A 5-min incubation followed each wash. Imaging was performed as described above immediately after the second 5-min wash and incubation step had been completed. For washout experiments shown in CVCL_0199) were maintained in DMEM/F12 medium with 10% Fetal Bovine Serum , particle velocities were measured from frame to frame and the total length of trajectories divided by the number of analyzed organelles was calculated for every experimental condition.Microscopy was performed using a Nikon TE-2000 inverted microscope equipped with a LED X-Cite illuminator (Lumen Dynamics) at 37 degrees. Images were acquired with a Hamamatsu ORCA-Flash 4.0 camera driven by MetaMorph software at a rate of 1 frame per second for 1 min. Lysosome motility in neurites was analyzed using Diatrack software (SCR_002285). The angle formed between an edge of a microtubule and the horizontal was measured. This angle, θ, was converted into velocity using the following relationship: velocity = 1/|tangent(θ)| * (unit pixel distance)/.Microtubule velocity measurements: the tracks of microtubules were traced and converted to kymographs  using the kymograph function of FIJI /(1 + 10^((LogIC50-X)*HillSlope)). All variables were allowed to float freely with the exception of the variable denoted ‘bottom’, which was constrained to be >0. The separate IC50 values obtained from fits to each individual experiment's dataset were averaged together and presented as a mean with range  and the respective controls, an un-paired T-test was performed for the difference of means control and compound treatment. This analysis was performed for each direction of motion.Statistical analysis of intraflagellar transport frequencies: Run frequencies output directly from tion see . For eacQuantification of ADP-vanadate dependent photocleavage of dynein was performed as follows: Scanned gels were analyzed with FIJI, and fraction cleaved quantified as follows:Fraction cleaved = (intensity of band at 250 kDa) /(intensity of band at 250 kDa + intensity of band at ~350 kDa) In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Philip Cole (Senior Editor) and three reviewers, one of whom, Wilfred van der Donk, is a member of our Board of Reviewing Editors.Thank you for submitting your article "Chemical structure-guided design of Dynapyazole, a potent cell-permeable dynein inhibitor with a unique mode of action" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This study presents optimization of the chemical properties and potency of ciliobrevins for inhibiting of the microtubule (MT)-associated motor protein, cytoplasmic dynein. Based on conformational analysis of ciliobrevin, the authors covalently locked the preferred conformation in a series of analogs. The final result is an inhibitor, which the authors coin "dynapyrazole", with improved chemical properties and up-to 10-fold higher affinity. It appears to act differently than the parent compound in that it appears to be specific for the microtubule-induced activity and not the basal activity of dyneins. The amount of new biology learned appears modest and inconclusive, but if the essential revisions can be achieved, this work would benefit the field given the steadily increasing demand for understanding dynein function in cells and cilia. With good physiological analysis of dynapyrazole inhibition and reversibility, the paper could be very strong. Unless the questions in point 5 below can be addressed, the reviewers suggest this contribution to be a Tools and Resources paper with omission of the currently insufficiently supported mechanistic data.Essential revisions:1) Physiological analysis is limited to axonal transport of lysosomes. There is much potential information in these experiments, but much of this is currently lacking. The multicolor time projections of lysosomal movement are visually confusing. The reader will understand that the change from multicolored to white implies inhibition, but important details are missing. The lysosomal "displacements" are very long on average, and probably represent a composite of multiple +, –, and stalled behavior. The direction of movement is at minimum important as a test of dynapyrazole specificity – does it affect MT minus end -directed runs alone? Although the challenges to address this are acknowledged, if dynapyrazole is fast acting and physiologically specific, it should show initial dramatic minus end inhibition. At present, bidirectional inhibition is not addressed, but whether it occurs or not, and whether it sets in with increasing time of drug treatment, are important issues to clarify for the cell biological reader/user. Stalls are also important to quantify as evidence for coordinate +/- inhibition, but also as an indicator of cellular toxicity. Kymographs are a much better way to show detailed motile behavior and should be featured. The nature of inhibition can also be better defined for the community that would be using the new compound. Ciliobrevin is said to be a substrate competitor. How was this determined? Similar analysis should be performed for the new compound. Competition analysis between CB, dynapyrazole, and ATP might be useful. In order to show the reversible effect of dynapyrazole, washout experiments should be performed in a physiological setting e. g., lysosome transport.2) At many places in the manuscript the statistical analyses is either not clear or not sufficient. Legends to 3) 4) The authors should address the following experimental concerns: The dynein preparations used for this analysis appear to be less than ideal. Single-headed recombinant dynein-1 and -2 motor domains are each used, though ATPase levels are only reported for dynein 1. However, the measured ATPase activity, especially in the presence of microtubules, is very low compared to native higher eukaryotic cytoplasmic dynein. This implies that a substantial fraction of the dynein may be inactive. The effects of ciliobrevin and dynapyrazole on dynein binding to MTs should also be evaluated and shown. It is possible that these compounds, or contaminants, might serve to oxidize dynein active site residues, and cause formation of dead-heads. MT gliding behavior also needs to be more thoroughly shown. The% of moving MTs and the distribution of velocities (histograms) should be provided.5) The arguments that dynapyrazole targets the AAA1 site specifically are confusing and not well supported. This part of the study should be eliminated, unless it can be performed in a manner that provides conclusive insight in drug and motor mechanisms. If the authors want to keep their mechanistic interpretation, the following questions need to be addressed: Ciliobrevin effects on MT-stimulated ATPase activity should be included for proper comparison with dynpyrazole . The eff6) Based on the reduction in the velocity of dynein-based MT gliding as a result of the reduction in the MT-stimulated ATPase activity of dynein, the application of dynapyrazole is expected to cause a reduction in the velocity of lysosome movement. Why isn't the effect of dynapyrazole on the velocity of lysosome movement reported? It would be highly interesting if differential effects on transport distance and velocity were found. Essential revisions:1) Physiological analysis is limited to axonal transport of lysosomes. There is much potential information in these experiments, but much of this is currently lacking. The multicolor time projections of lysosomal movement are visually confusing. The reader will understand that the change from multicolored to white implies inhibition, but important details are missing. The lysosomal "displacements" are very long on average, and probably represent a composite of multiple +, -, and stalled behavior. The direction of movement is at minimum important as a test of dynapyrazole specificity – does it affect MT minus end -directed runs alone? Although the challenges to address this are acknowledged, if dynapyrazole is fast acting and physiologically specific, it should show initial dramatic minus end inhibition. At present, bidirectional inhibition is not addressed, but whether it occurs or not, and whether it sets in with increasing time of drug treatment, are important issues to clarify for the cell biological reader/user. Stalls are also important to quantify as evidence for coordinate +/- inhibition, but also as an indicator of cellular toxicity. Kymographs are a much better way to show detailed motile behavior and should be featured. The nature of inhibition can also be better defined for the community that would be using the new compound. Ciliobrevin is said to be a substrate competitor. How was this determined? Similar analysis should be performed for the new compound. Competition analysis between CB, dynapyrazole, and ATP might be useful. In order to show the reversible effect of dynapyrazole, washout experiments should be performed in a physiological setting e. g., lysosome transport.We thank the reviewers for raising all these different points. The changes we have made to address these concerns are:A) Physiologic analysis: The physiologic analyses in the original submission included inhibition of lysosome motility and Hedgehog signaling. To address your concern, we have now obtained cell lines from the group of Dr. Max Nachury and with their guidance have done live-cell imaging of intraflagellar transport in living cells. To obtain the level of detailed analysis you requested  we used computer-based tracking algorithms. The findings are presented in a new figure  in the r50 for dynein 2-driven microtubule gliding is very similar to that observed in a previous study of the effects of temperature-sensitive mutants of dynein 2 on intraflagellar transport in Chlamydomonas : 151-167 DOI: 10.1083/jcb.201206068, ; Nicholas et al., rat dynein ~6 s-1 (per dimer), Nat Commun. 2015 Feb 11;6:6206 ; Ori-McKenney et al., mouse dynein, ~50 nmol/mg/min, Nat Cell Biol. 2010 Dec;12(12):1228-34 [Figure 1C]). We note that the ATPase activity observed here is lower than those observed in dyneins from S. cerevisiae and, especially, from D. Discoideum. We observe modest stimulation (~2 fold) at 2.5µM microtubules, and have now added data showing that we observe up to 5-fold stimulation of ATPase activity at higher microtubule concentrations (20µM), which is within the 2- to 6-fold range of stimulation observed at high microtubule concentrations for rat, cow, and mouse dynein 1 constructs . Therefore, we believe, and hope you agree, that the basal and microtubule-stimulated activities we report are in line with the range of activities reported by others for mammalian dyneins.We appreciate the reviewers' concerns regarding the protein activity we report. Respectfully, we believe that the confusion about protein hydrolysis activity may arise from the common practice of presenting the ATPase activity of dynein in units of nmol/min/mg protein. When converted to these units (molecular weight of the construct is ~400kDa), the basal ATPase activity of our single-headed dynein 1 construct is ~90nmol/min/mg protein  The arguments that dynapyrazole targets the AAA1 site specifically are confusing and not well supported. This part of the study should be eliminated, unless it can be performed in a manner that provides conclusive insight in drug and motor mechanisms. If the authors want to keep their mechanistic interpretation, the following questions need to be addressed: Ciliobrevin effects on MT-stimulated ATPase activity should be included for proper comparison with dynpyrazole . The effWe thank the reviewers for their constructive suggestions regarding our mechanistic analysis. We now include analysis of ADP-vanadate-dependent photocleavage of dynein, which sheds light on the mechanism dynein inhibition by dynapyrazole-A. We show that dynein photocleavage, which has been shown to occur at AAA1, is inhibited by dynapyrazole-A treatment, consistent with inhibition of nucleotide binding at AAA1 of dynein. The concentration of dynapyrazole-A necessary to block photocleavage of dynein (4µM) is ~40 times lower than was reported for ciliobrevin A .We have added many of the other analyses requested. We now show the effect of ciliobrevin D on the microtubule-stimulated ATPase activity of dynein, which reveals inhibition to ~40% residual activity at the highest concentration tested (40µM), with no saturation of the inhibitory effect observed. We compare the potencies of inhibition of the basal ATPase activity of the AAA3 mutant of dynein by ciliobrevin D and dynapyrazole-A and show that the new compound is ~8-fold more potent in this setting. We show that the AAA3 mutant of dynein is stimulated by microtubules, but to a lesser degree than observed for the wild-type enzyme, consistent with analyses of comparable mutants by others :11266-74, [Table 1]). We show that dynapyrazole-A suppresses microtubule stimulation of both the wild-type and AAA3 mutant enzymes across a broader range of microtubule concentrations.The new data we present support our previous assertion that dynapyrazole-A primarily blocks the microtubule-stimulated component of dynein’s ATPase activity while ciliobrevin D inhibits the basal and microtubule-stimulated activities of dynein. When taken together with the inhibition of ADP-vanadate-dependent photocleavage at AAA1 of dynein by low concentrations of dynapyrazole-A, we believe that our data now show more clearly that (A) dynapyrazole-A blocks ATP hydrolysis at AAA1, (B) dynapyrazole-A only weakly inhibits basal ATPase activity, (C) dynapyrazole-A strongly inhibits microtubule-stimulated hydrolysis by dynein. We believe these observations are best explained by a model in which AAA1 contributes minimally to the basal ATPase activity of dynein and contributes significantly (and in a microtubule concentration-dependent manner) to the microtubule-stimulated activity of dynein. In this model, inhibition by dynapyrazole-A of AAA1 only would be expected to give results consistent with those we observe. We have attempted to clarify the writing describing our conclusions.stimulation of dynein’s basal rate in constructs with a similar AAA3 walker A lysine-to-alanine mutation. We have added the following statement to the manuscript in the attempt to clarify our reasoning. “Our finding that the AAA3 mutant has elevated basal activity relative to the wild-type is consistent with the protein being in a state that mimics one in which hydrolysis at AAA1 is activated.”The reviewers asked: “if AAA1 contributes only a small fraction of the basal activity, why can AAA1 activity then contribute significantly to the basal activity in the AAA3 K/A mutant?” In current models nucleotide occupancy within one ATPase sites of dynein regulates that at other sites. Thus, in the AAA3 mutant, this site is not only impaired in its own nucleotide binding and hydrolysis, but also its APO-like nucleotide state may be sensed and transmitted to other AAA sites, leading to their activation. We note in the manuscript that others have observed comparable and seemingly counterintuitive We appreciate the reviewers’ suggestion that another site (likely AAA4) might also contribute hydrolysis activity in the setting of the AAA3 mutant. Our newly-added microtubule stimulation data for the AAA3 mutant further support this conclusion, and we have revised the text accordingly.6) Based on the reduction in the velocity of dynein-based MT gliding as a result of the reduction in the MT-stimulated ATPase activity of dynein, the application of dynapyrazole is expected to cause a reduction in the velocity of lysosome movement. Why isn't the effect of dynapyrazole on the velocity of lysosome movement reported? It would be highly interesting if differential effects on transport distance and velocity were found.We appreciate the reviewers' enthusiasm for the potential for dynapyrazole to illuminate the roles of dynein-driven motion in a cellular context. As mentioned above, we have included an expanded analysis of lysosome motility as well as a new analysis of intraflagellar transport. We demonstrate a reduction in the velocities of moving lysosomes in addition to the track shortening previously shown. We also now show that dynapyrazole causes reversible inhibition of intraflagellar transport.


Artemisia argyi H. under liquid-state fermentation by Monascus purpureus (AAFM), which has cellular antioxidant activity and neuronal cell viability, on trimethyltin- (TMT-) induced learning and memory impairment in Institute of Cancer Research (ICR) mice was confirmed. Tests were conducted to determine the neuroprotective effects against H2O2-induced oxidative stress, and the results showed that AAFM has protective effects through the repression of mitochondrial injury and cellular membrane damage against H2O2-induced neurotoxicity. In animal experiments, such as the Y-maze, passive avoidance, and Morris water maze tests, AAFM also showed excellent ameliorating effects on TMT-induced cognitive dysfunction. After behavioral tests, brain tissues were extracted to assess damage to brain tissue. According to the experimental results, AAFM improved the cholinergic system by upregulating acetylcholine (ACh) contents and inhibiting acetylcholinesterase (AChE) activity. AAFM effectively improved the decline of the superoxide dismutase (SOD) level and the increase of the oxidized glutathione (GSH) ratio and lipid peroxidation  production) caused by TMT-induced oxidative stress. The occurrence of mitochondrial dysfunction and apoptosis was also decreased compared with the TMT group. Finally, quinic acid derivatives were identified as the major phenolic compounds in AAFM using ultra-performance liquid chromatography quadrupole-time-of-flight (UPLC-Q-TOF) MS analysis.The cognitive effect of Artemisia argyi H. is a perennial herbaceous plant belonging to the Chrysanthemum family, and it has been used as a drug in traditional medicine for a long time in China, Korea, Japan, and the Russian Far East. Studies on Artemisia argyi H. have revealed its various biological functions, such as antidiabetic, antioxidant, anticancer, and anti-inflammatory  in a 50 mM sodium phosphate buffer (pH 8.0) was added to the above reaction mixture and absorbance was read as 405 nm.The mixed whole brains homogenate (5 3 (in 0.1 N HCl) were added and absorbance was measured at 540 nm.The level of ACh was determined in accordance with the method described by Vincent et al. . The brag (for 10 min at 4°C). 1x Cell Extraction Buffer  is added in pellets and then incubated on ice for 30 min. The mixtures were centrifuged at 10,000 ×g for 10 min at 4°C and the supernatant is used to measure SOD levels using SOD assay kit.Homogenates (brain with PBS) were centrifuged at 400Brain with 5% metaphosphoric acid homogenates centrifuged at 14,000 ×g for 15 min at 4°C. The supernatant was treated to 2 M 4-vinylpyridine and incubated for 1 h at room temperature. And oxidized GSH and GSH were measured using commercial kits.Brain homogenates with PBS were centrifuged (6000 ×g for 10 min at 4°C), and the supernatant was mixed with 0.67% TBA solution with 1% phosphoric acid and then incubated in a water bath (95°C) for 1 h. After cooling, absorbance was measured at 532 nm.According to the procedure of Dragicevic et al., whole brains were homogenized with 5 volumes of isolation buffer containing 215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin , 1 mM EGTA, and 20 mM HEPES (pH 7.2) to separate the mitochondria and centrifuged at 1,300 ×g for 5 min , a solution of 1 μM 5,5′,6,6′-tetrachloro-1,1′3,3′,-tetraehylbenzimi-dazolycarbocyanine iodide (JC-1) in DMSO was reacted, and, then, the mixture was stirred gently at room temperature for 20 min in the dark. It was measured with a fluorescent  microplate reader.In order to measure the membrane potential of isolated mitochondria, after mixing 20 g for 10 min at 4°C. After segregating the protein using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), it was transferred to a polyvinylidene difluoride (PVDF) membrane . The membranes were blocked with 5% nonfat dry milk in Tris-Buffered Saline (TBS) with 0.1% of Tween 20 (TBST) buffer. After 1 h, primary antibodies were diluted (1 : 1000) in a dilute solution (0.1% sodium azide and 0.5% BSA in TBST). After incubating diluted primary antibodies with the membrane under gentle agitation overnight, the membrane was washed 3 times (10 min each time) in TBST. And then, after allowing the secondary antibody solution to react with the membrane for 1 h, it was washed again. Finally, prior to detecting the luminescence with Chemi-doc , the membrane was exposed to an enhanced chemiluminescence reagent. The density of the band was analyzed with Image J Software . The protein was calculated using density of target protein/density of β-actin as a loading control.Brain tissues were added with ProtinEx™ Animal cell/tissue  and 1% protease inhibitor cocktails  and centrifuged at 13,0002) temperature at 35°C, fragmentor voltage at 175 V, nebulizer pressure at 45 psi, drying gas flow at 10 L/min, capillary voltage at 4000 V, and mass range from m/z 100 to 1000.UPLC Q-TOF/MS was used to analyze the quality of main compounds in the AAFM. An electrospray source in negative ion mode was used to obtain MS and MS/MS data. Phenolic compounds were separated on an ACQUITY UPLC BEH C18 with oven temperature at 40°C and a flowing rate of 0.3 mL/min. The following shows how a linear solvent gradient of binary mobile phase during analysis was applied: 99% A/1% B at 0–10 min. The conditions for MS analyses included the drying gas (Np < 0.05) of each group in a high order using Duncan's new multiple-range test of SAS ver. 9.1 .All data were expressed as mean ± SD. The different capital letters from “A” represent statistical difference  compared with that of the control group (100.0 ± 4.7%)  (p < 0.05) had lower cell viability than the control (100.0 ± 6.7%), vitamin C (108.2 ± 2.0%) (p < 0.05), and AAFM  groups (2O2 group (124.6 ± 4.6%) (p < 0.05) was increased compared with that of the control group (100 ± 3.0%)  (p < 0.05) showed decreased LDH release. AAFM also had an excellent inhibitory effect on LDH release into the medium by protecting the neuronal cell membranes. In other words, AAFM protected neuronal cells from H2O2-induced oxidative stress.To measure the protective effect of AAFM against H ± 4.7%) . However) groups . Moreove ± 3.0%) . In addi2O2-induced oxidative stress. In addition, according to a previous study, when Monascus purpureus is fermented with rice, secondary metabolites, such as dimerumic acid and deferricoprogen, are generated, and these secondary metabolites can protect the neuron against oxidative stress through their antioxidant activity [These results showed that the antioxidant activity of the phenolic compounds from AAFM protected the cells against Hactivity . TherefoY-maze and passive avoidance tests were carried out to assess spatial cognition and short-term memory abilities, respectively. The Morris water maze test is a means of evaluating experimental animals' spatial memory learning ability and long-term memory ability , 13.p < 0.05) showed reduced spatial cognition abilities in comparison with the control group (60 ± 8%). On the other hand, the AAFM 5, 10, and 20 mg/kg of body weight groups showed results of 51 ± 5%, 58 ± 8%, and 61 ± 5%, respectively  was reduced compared to that of the control group (264.5 ± 19.3 s). The AAFM groups showed improved memory dysfunction compared to that of the TMT group.The passive avoidance test was conducted to assess short-term memory of stress, as shown in p < 0.05) was decreased in comparison with that of the control group (35.6 ± 4.5%), and those of the AAFM 5, 10, and 20 mg/kg of body weight groups were 30.0 ± 5.1%, 31.5 ± 3.3%, and 36.7 ± 3.3%, respectively  (p < 0.05) was decreased compared with that of the control group (0.93 ± 0.08 mmol/mg of protein). The ACh contents of the AAFM 5, 10, and 20 mg/kg of body weight groups were 0.77 ± 0.2 mmol/mg of protein (p < 0.05), 0.83 ± 0.09 mmol/mg of protein, and 0.93 ± 0.09 mmol/mg of protein (p < 0.05) compared with the control group (100 ± 4.9%), and more effective suppression was found in the AAFM groups ) compared with the TMT group  have AChE inhibitory activity because of phenolic compounds like gallic acid, ellagic acid, and so on [After protein , respectMT group . As a rend so on . TherefoAntioxidant enzymes, such as SOD and GSH, neutralize oxidation phenomena. However, when the amount of free radicals generated exceeds such enzymes' neutralization capacity, neuronal ageing and degenerative deformation occur rapidly .TMT leads to neuronal oxidative damage and the extended elevation of ROS levels . Therefop < 0.05) was higher than that of the control group (70.3 ± 1.3%). Moreover, in the AAFM groups (5 mg/kg of body weight: 82.3 ± 1.1% (p < 0.05), 10 mg/kg of body weight: 79.1 ± 4.4% (p < 0.05), and 20 mg/kg of body weight: 73.8 ± 2.6%), the oxidized GSH/total GSH ratios were lower than that of the TMT group  showed an increase in mitochondrial ROS production in our experimental results. On the other hand, mitochondrial ROS production of the AAFM groups ) was improved from TMT toxicity (p < 0.05) was decreased compared with that of the control group (100.0 ± 9.1%) because of the increased mitochondrial ROS production. Moreover, as predicted, the MMP levels of the AAFM groups (5 mg/kg of body weight: 72.0 ± 7.8% (p < 0.05), 10 mg/kg of body weight: 78.4 ± 8.8% (p < 0.05), and 20 mg/kg of body weight: 91.4 ± 4.7%) were higher than that of the TMT group.In comparison to the control group (100 ± 10.1%), the TMT group (167.0 ± 16.4%) , Bcl-2-associated death promoter (BAD), and caspase 9, by phosphorylating these proteins, and promotes cell longevity pathways [In terms of the improvement of neurodegenerative disorders, Akt plays an important role as a prosurvival kinase. The phosphorylation of Akt as the activated form inactivates the preapoptotic proteins, such as glycogen synthase kinase-3pathways .p < 0.05), and 0.84 ± 0.05, resp.)  stimulates the activation of proapoptotic proteins, and the activation of JNK occurs through the dual phosphorylation of tyrosine (Tyr) and threonine (Thr) residues . Therefo Figures .p < 0.05) and 0.45 ± 0.23, resp.) showed increased expression of BAX and release of Cyto. C in the mitochondria compared with the control group . On the other hand, the AAFM 20 mg/kg of body weight group (relative density of BAX and Cyto. C in mitochondria: 0.93 ± 0.10 (p < 0.05) and 1.24 ± 0.16, resp.) showed better results than the TMT group  protein heterodimer. Bcl-2 and Bcl-xL, which are antiapoptotic proteins, are activated, while BAX/Bak-triggered apoptosis is inactivated. BAX/Bak also leads to a loss in membrane potential by interacting with the mitochondrial voltage-dependent anion channel. Accordingly, Cyto. C is released from the mitochondria to the cytosol . For the Figures .β, a type of tau protein kinase I (TPKI). Tau protein kinases I and II (TPKI and TPKII) are candidate enzymes responsible for the hyperphosphorylation of tau, and TPKII strongly enhances the action of TPKI. The hyperphosphorylation of tau induced by GSK-3β activity leads to neuronal cell death; accordingly, it is closely related to AD [In addition, p-Akt also inactivates GSK-3ed to AD , 31. As To sum up, AAFM effectively inhibited apoptosis through Akt activation and JNK inactivation, and it is thought that the repression of apoptosis in neuronal cells by AAFM could lead to the improvement of cognitive function. In addition, this result may be the result of mitochondrial dysfunction; therefore we confirmed again that AAFA is effective in ameliorating mitochondrial dysfunction.2 scan with mass fragmentation. These phytochemicals in AAFM were identified as seven major peaks . When the results of each MS2 scan were compared with references, the main phytochemicals of AAFM were confirmed to be derivatives of quinic acid   , and 3.4 Artemisia annua L., which also belongs to the genus Artemisia, also has quinic acid derivatives as the major constituents. Moreover, 3.5-Dicaffeoylquinic acid was identified as a major component of Artemisia annua L. in an LC–negative ion ESI-MS total ion current (TIC) profile [ Artemisia argyi H. ethyl acetate fractions [ Monascus purpureus fermentation. However, the summative or synergistic effect of secondary metabolites produced during microbial fermentation on the antiamnesic effect could not be confirmed, and this will be investigated in the near future.Previous research confirmed that profile . In our ractions . In otheIn investigations of the analyzed substances, it has been reported that quinic acid derivatives have neuroprotective effects against oxidative stress through their antioxidant activity . In part2O2-induced oxidative stress in neuronal cells. In an animal behavior test, AAFM intake improved learning and memory abilities in mice with TMT-induced cognitive impairment. According to the results of the brain tissue analysis in the AAFM groups, oxidative stress and the reduction of ACh were ameliorated. AAFM prevented the occurrence of mitochondrial dysfunction caused by TMT-induced oxidative stress, and, thus, apoptosis was inhibited. These physiological activities were assumed based on the quinic acid derivatives in Artemisia argyi H. under liquid-state fermentation by Monascus purpureus. Consequently, the administration of AAFM could lead to the improvement of cognitive function. In addition, we confirmed that phenolic compounds produced by microbial fermentation could be changed by various strains used for fermentation; accordingly, the physiological activity of the fermented product could be affected.The improvement effect of AAFM on TMT-induced cognitive dysfunction in mice was confirmed. AAFM showed a superior protective effect against HMonascus purpureus had the highest polyphenol contents compared to other strains.Supplementary Material 1 shows polyphenol contents of extracts fermented by various strains. In this result, the extract fermented by Monascus purpureus had the best antioxidant activity compared to other strains.Supplementary Material 2 shows antioxidant activity of extracts fermented by various strains. In this result, the extract fermented by Lactobacillus brevis was the highest compared to other strains, but there was no significant differences from the extract fermented by Monascus purpureus. Therefore, in our experiment, the extract fermented by Monascus purpureus was used to investigate.Supplementary Material 3 shows acetylcholinesterase inhibitory activity of extracts fermented by various strains. The AChE inhibition activity of extract fermented by


Cucumis melo L.) SC3-5-1 contained an introgression of the non-climacteric Korean cultivar “Shongwan Charmi” accession PI 161375 (SC) in the genetic background of the non-climacteric cultivar “Piel de Sapo” (PS). The aroma production was monitored during ripening at 21 °C in intact fruit using headspace sorptive bar extraction (HSSE). Bars were composed of polydimethylsiloxane (PDMS) and aromas were desorbed and analyzed by gas-chromatography mass-spectrometry. The aromatic profile was composed of 70 aromatic compounds plus 21 alkanes with a predominance of esters, particularly acetate . Some compounds were severely affected by postharvest time. The acetate esters  decreased with ripening and sulfur-derived compounds (S-methyl butanethioate and S-methyl 3-methylbutanethioate) increased gradually with ripening. A few compounds increased at the senescence phase (propyl ethanoate). Other compounds such as hexadecanoic acid showed a marked decrease after harvest, some decreasing from a relative maximum at harvest .A climacteric aromatic near-isogenic line (NIL) of melon ( The pro et al.  proposedn LG III ,9. et al. [et al. [The climacteric behavior of this NIL is strongly associated with a typical aromatic profile  and softet al.  showed t [et al.  showed aThe goal of this paper is to characterize the main individual changes in volatiles associated with SC3-5-1 climacteric fruit ripening and senescence, particularly those revealing potential ethylene-dependent behavior in intact fruit after harvest.Cucumis melo L.) plants of the NIL SC3-5-1 were harvested in full-ripe stage of maturity in mid July 2009 in Cartagena, Murcia, SE Spain. Harvest indices, experimental design and flesh sampling followed the methodology reported by [Fruits obtained from melon (orted by . The inborted by .n = 4) were 1636 ± 88 g and 992 kg·m−3, respectively. All the fruit samples were sampled for 1 h within hermetic containers of 5283 cm3 at harvest and during ripening.For ripening experiments, fruit were stored at 21 ± 1 °C and 93% ± 3% RH for 10 days. For aroma volatile analysis, four selected fruits (one fruit per replicate) harvested at the end of the season for SC3-5-1 were used. Fruit weight and density . The bars were stacked onto the metallic wall of the container for absorbing aroma headspace. Also, they followed the conditioning process before or after analysis previously reported . The Twi® Desorption Unit (TDU) option and a mass spectrometer 5975 with an hyperbolic quadrupole (Agilent Technologies). For the TDU, the following parameters were used: for the desorption program, 40 to 250 °C (5 min) at 300 °C·min−1; carrier gas (He) flow rate, 45 mL·min−1. The TDU settings were splitless mode with a fixed transfer temperature of 300 °C and standard sample mode. The back inlet (CIS4) worked in solvent vent mode at initial temperature of 250 °C, 8.60 psi (around 59.3 kPa) pressure, a vent flow of 50 mL·min−1, vent pressure of 7.25 psi (around 49.99 kPa), purge flow of 9.2 mL·min−1 and total flow of 13.3 mL·min−1.Volatile analysis was performed as in  on a 689−1. The hold time was 3 min.The PTV was cooled to −100 °C with an equilibration time of 0.5 min using liquid nitrogen, the GC cool down time being 0.1 min with a cryogenic timeout of 30 min (cryogenic cooling parameters). The cryogenic cooling temperature program was as follows: injection temperature 250 °C, reached at 10 °C·s−1. The column operated at constant pressure mode using pentadecane  to block the method at 27.5 min [Capillary GC-MS analyses were performed using an HP-5MS ultra inert (Agilent Technologies) column (0.25 mm × 30 m length × 0.25 µm). Chromatographic conditions were as follows: solvent delay, 0.10 min; split mode 10:1; oven temperature program with an equilibration time of 0.50 min and a ramp from 60 to 240 °C at 3 °C·min27.5 min .The chromatograms and mass spectra were evaluated using ChemStation software . The peaks were registered using a mass spectrometer  coupled to the GC. Volatile compounds were tentatively identified by comparing the experimental spectra with those of the National Institute for Standards and Technology (NIST05a.L) data bank [Raw data or data transformed into their respective logarithm were analyzed by analysis of variance of repeated measurements with ripening time as fixed factor. When time was significant, mean differences were separated by LSD test with type-I error α ≤ 0.05. Only compounds showing a significant effect of the ripening time are reported as time-dependent and for the rest only the mean ± SE is reported. The rest of compounds of the profile with a presence of at least 47% in the samples analyzed were included in −1·s−1 of ethylene, and decreased afterwards (−1·s−1 (data not shown).The NIL SC3-5-1 showed a climacteric behavior from the 3rd day onwards, peaks on the 9th day with levels of 57.1 ± 2.5 pmol·kgterwards . This tr®. This is the reason for the development of new bar coatings able to absorb compounds with different polarities, such as PDMS/polypyrrole, PDMS/metachrylate derivates, PDMS/activated carbon, PDMS-ACB, PDMS/polyvinylalcohol or PDMS/PVA, ethylene glycol (EG)-silicone; polyacrylate (PA) [Twister technology is appropriate for monitoring changes in melon volatiles non-destructively because most of the well-known aromas in the climacteric melon NILs flesh were also recovered here ,4. The bate (PA) ,29.The aroma profile commonly found during ripening of NIL SC3-5-1 was mainly composed of 70 volatile compounds, 39 esters , seven organic acids, five aldehydes, four ketones, one alcohol, four terpenes, another three compounds of other chemical groups, and seven unidentified compounds . Twenty etc.)  . Some coetc.) A, followetc.) E and othethioate . The 2-methioate . It has oleucine . This cooleucine  and is poleucine . Other nThe large amount of esters is also consistent with the strong dependence on ethylene biosynthesis of most of the ester and thioesters catalyzed by several alcohol acetyl transferases ,35,36 anS-methyl 3-methylbutanethioate or S-methyl butanethioate; i.e., ethyl butanoate from 0% to 1.4% after 10 day of ripening), among other compounds, followed the opposite pattern. The data confirmed that all these compounds can be either detected in climacteric NILs either non-destructively  decreased during ripening D,E,G,I. n-hexadecanoic acid (from 4.3% to levels below 0.3% after 1 day of ripening). Other compounds decreased slowly during ripening, such a nonanoic acid (from 0.6% to less than 0.2% after 3 days of ripening) or n-hexanoic acid , or oic acid J. ProbabFinally, other volatiles were typical of melon senescence , though The acetate esters and thioesters, particularly 2-methylbutyl acetate, predominated in the SC3-5-1 profile. Aroma volatiles identified during ripening of the climacteric NIL SC3-5-1 followed different patterns but apparently following an ethylene-dependent pattern due to their biosynthesis or degradation.


Aldama tucumanensis, Ambrosia elatior, Baccharis artemisioides, Baccharis coridifolia, Dimerostemma aspilioides, Gaillardia megapotamica, and Vernonanthura nudiflora presented outstanding antiproliferative activity at 50 μg/mL, with inhibitory values from 93 to 100%, when tested on the acute lymphoblastic leukemia (ALL) cell line CCRF-CEM and the resistant derivative CEM-ADR5000, while 70–90% inhibition was observed against the chronic myelogenous leukemia (CML) cell K562 and its corresponding resistant subline, Lucena 1. Subsequent investigation showed these extracts to possess marked cytotoxicity with IC50 values ranging from 0.37 to 29.44 μg/mL, with most of them being below 7 μg/mL and with ALL cells, including the drug-resistant phenotype, being the most affected. G. megapotamica extract found to be one of the most effective and bioguided fractionation yielded helenalin (1). The sesquiterpene lactone displayed IC50 values of 0.63, 0.19, 0.74, and 0.16 μg/mL against K562, CCRF-CEM, Lucena 1, and CEM/ADR5000, respectively. These results support the potential of these extracts as a source of compounds for treating sensitive and multidrug-resistant leukemia cells and support compound 1 as a lead for developing effective anticancer agents.Plants are a significant reservoir of cytotoxic agents, including compounds with the ability to interfere with multidrug-resistant (MDR) cells. With the aim of finding promising candidates for chemotherapy, 91 native and naturalized plants collected from the central region of Argentina were screened for their cytotoxic effect toward sensitive and MDR P-glycoprotein (P-gp) overexpressing human leukemia cells by means of MTT assays. The ethanol extracts obtained from Leukemia is a malignant disorder with a significant number of deaths annually . AccordiDespite overall improvement in the outcome of conventional leukemia therapies , 4, someVarious mechanisms underlie this type of resistance, the most common being overexpression of the P-glycoprotein (P-gp) transporter (ABCB1/MDR1) . This prA considerable number of plants have been recognized with rich potential as a therapeutic resource, and many of their derived molecules were active on leukemia cells, including resistant phenotypes . Many co Gaillardia megapotamica, was submitted to bioguided fractionation to further isolate the active principle responsible for its cytotoxicity.The present work reports antiproliferative activity on sensitive and P-gp overexpressing leukemia cell lines of a panel of 91 extracts obtained from native and naturalized plants of Central Argentina. One of the most potent extracts,Plants were collected in the hills of Córdoba Province, Argentina, from November to March 2016-2017. Voucher specimens have been deposited in the “Marcelino Sayago” Herbarium of the School of Agricultural Science, Catholic University of Córdoba, and were authenticated by the botanist, G. Ruiz. Plants were selected according to their availability, accessibility, and especially the lack of scientific information about their activity and/or chemical pattern. Crushed, air-dried material (200 g) was extracted by 48 h maceration with 700 mL of 96% ethanol. The yields of each active extract, obtained after exhaustive solvent removal and expressed as percentage weight of air-dried crushed plant material, are shown in  Phaseolus vulgaris (PHA) were purchased from Sigma Aldrich, . Doxorubicin hydrochloride  was obtained from Nanox Release Technology  and was used dissolved in bidistilled water. RPMI-1640 and cell culture reagents Gibco® were purchased from Invitrogen Life Technologies . Sterile plastic material was purchased from Greiner Bio-One . All solvents were HPLC grade. 1H- and 13C-NMR and two-dimensional spectra were recorded with a Bruker AVANCE II 400 spectrometer  with tetramethylsilane (TMS) as the internal reference. HPLC was performed on a Shimadzu LC-10 AS , equipped with a Luna C18, 250 × 4.6 mm reversed-phase column. The mobile phases were ACN/H2O 30 : 70, ACN/H2O 10 : 90, and ACN/H2O 70 : 30 with UV detection at 210 and 280 nm.3--2,5-diphenyl-2H-tetrazolium bromide (MTT) and lectin from G. megapotamica (4 g) was initially subjected to vacuum liquid chromatography on silica gel  eluted with a step gradient of hexane/diethyl ether (Et2O)/methanol (MeOH) to yield 12 fractions, which were combined in 8 groups according to their thin layer chromatography (TLC) profile (F1 to F8). Fractions F1 to F5 and F7 and F8 were not active at the tested concentration (10 μg/mL), while fraction F6, eluted with 100% Et2O, demonstrated cytotoxic effect at this concentration. Therefore, F6 was further processed by radial preparative chromatography using an isocratic mobile phase of hexane/Et2O 30 : 70. The fractions obtained were combined in 10 groups in accordance with the TLC analysis (F6-1 to F6-10). From fraction F6-6, a pure compound 1 was obtained by spontaneous crystallization . This compound, the remaining F6-6, and the rest of the fractions were further tested for their cytotoxic activity at 10 μg/mL. Only 1 exerted a toxic effect at this concentration. According to 1H and 13C NMR spectra , the compound was identified as the sesquiterpene lactone helenalin C15H18O4   × 100.Untreated and DMSO (1%)-treated cells were used as controls, while DOX , the concentrations of the tested samples required to inhibit 50% cell proliferation, were calculated from the mean values of data from wells. Resistance factors (RF) were calculated by dividing the IC50 of resistant cells by the IC50 of the corresponding sensitive cell line [Half-maximal inhibitory concentrations  or 1% DMSO for 48 h. Absorbance (Abs) and percentage of cytotoxicity were determined as described above and the IC50 values were calculated.The cytotoxicity of the extracts on peripheral blood mononuclear cells (PBMC) was evaluated by MTT assay . PBMC wet test using GraphPad Prism software , with p values ≤ 0.05 as statistically significant. IC50s were calculated by GraphPad Prism software, responding to at least five concentrations of each extract at the 95% confidence level with upper and lower confidence limits.The results are expressed as mean ± SE. Data were analyzed with one-way analysis of variance (ANOVA) and one-tailed unpaired μg/mL against a panel of cells consisting of CCRF-CEM and K562 and their respective P-gp overexpressing counterparts, CEM/ADR5000 and Lucena 1. The ethanol extracts obtained from Aldama tucumanensis, Ambrosia elatior, Baccharis artemisioides, Baccharis coridifolia, Dimerostemma aspilioides, Gaillardia megapotamica, and Vernonanthura nudiflora inhibited proliferation by 93–100% in ALL cell lines while 70–90% inhibition was observed in the CML cells  plant screening program for an extract to be considered active following an incubation period of 48 and 72 h [50 values lower than 7 μg/mL. The G. megapotamica and D. aspilioides extracts were the most effective among those with less toxic effect on PBMC.These active extracts were further assessed for their ICand 72 h . As obse A. elatior extract was highly specific, since it showed outstanding proliferation-inhibiting properties against ALL cells but not against CML cells (p < 0.05). The extract obtained from D. aspilioides, although active against all the cell lines, also induced specific cytotoxic activity, being more effective on ALL cell lines than on CML cells (p < 0.05). B. artemisioides and B. coridifolia extracts were highly potent, being equally active (p > 0.05) against almost all the assayed cell lines, with IC50 values from 0.37 to 5.89 μg/mL, and at the same time were the most toxic against PBMC. Most of the extracts showed significantly different cytotoxic effects (p < 0.001–0.05) with respect to DOX, with the same or lower activity in the case of CCRF-CEM and K562, but the same or greater effectiveness in the resistant cells, CEM/ADR5000 and Lucena 1. The latter were >570-fold and 29-fold more resistant to DOX than their parental cell lines, CCRF-CEM and K562, respectively  with CEM/ADR5000 compared to the parental cell line CCRF-CEM  and lack of cross-resistance regarding Lucena 1. Except B. coridifolia, to which both P-gp overexpressing cells showed cross-resistance, the rest of the extracts showed this phenomenon only in one resistant cell. This classification is taken into consideration following Hall et al., [It is remarkable that extracts from et al.,  who defi G. megapotamica was identified as one of the most active species. After submitting this plant to bioguided isolation, helenalin (1) was obtained. This sesquiterpene lactone showed outstanding IC50 values against K562 cells and their MDR counterpart Lucena 1 of 0.63 ± 0.06 and 0.74 ± 0.06 μg/mL, respectively  while 0.19 ± 0.01 and 0.16 ± 0.01 μg/mL were obtained against CCRF-CEM and its respective drug-resistant phenotype CEM/ADR5000 . In coincidence with the results obtained for the complete extract, ALL cells were more sensitive to compound 1 than CML cells.As mentioned, A. tucumanensis, displayed cytotoxic activity against HT-29, PC-3, DU-145, MDA-MB-231, and MCF7 cells, with IC50 values in the range of 0.72 to 2.32 μg/mL, by inducing apoptotic death [μg/mL cutoff established by the NCI to identify good cytotoxic compounds [50 of 0.47 μg/mL against H.Ep.-2 cell line [ A. elatior, has been tested for cytotoxic activity against Jurkat E6-1, GLC4, and COLO320 cells, showing IC50 ranging from 10.21 to 24.62 μg/mL [50 value of 4.89 μg/mL [50 values ranging from 1.8–10.7 μg/mL against HepG2, Hep2, HCT116, RD, and MCF7 [Although none of these most active species have been subjected to bioguided isolation to obtain their cytotoxic principles, there is some related information regarding toxicity on cancer cells of some of their constituents. Leptocarpin, present inic death . The valompounds . Eupatolell line . The fla62 μg/mL , 58. Oth62 μg/mL ; the sam62 μg/mL , while p62 μg/mL  but was and MCF7 . B. coridifolia and most of B. artemisioides studied by Rizzo et al. [50 of 0.0018, 0.0055,0.0012 and 0.0022 μg/mL, respectively against H4TG cells [50 value of 0.002 μg/mL [μg/mL against KB and BC1 cells [ B. coridifolia, demonstrated a toxic effect against KB cells with an IC50 value of 2.7 μg/mL [ D. aspilioides, showed moderate activity against KB cells, with an IC50 of 3.0 μg/mL [All the Argentinian plants ofo et al.  were posTG cells . When te02 μg/mL  while veC1 cells . The com.7 μg/mL . The ger.0 μg/mL . G. megapotamica. Helenalin, with a mean cytotoxic activity of 0.10 μg/mL against EN2 cells [50 of 0.14 μg/mL against EN2 cells [50 ranging from 1.22 to 4.62 μg/mL [50 of 11.70 and 9.30 μg/mL against GLC4 and COLO320 cells, respectively [ V. nudiflora contains many inactive compounds, such as genkwanin, with an IC50 against KB and P388 cells of 8.7 and 10 μg/mL, respectively [50 29.5 and 35.6 μg/mL against HCT 116 and A549 cell lines, respectively [50 = 1.5 μg/mL against KB cells) [50 values from 7.29 to 16.39 μg/mL against Jurkat E6-1, GLC4, and COLO320 cell lines [μg/mL [Some sesquiterpene lactones, among them helenalin and mexicanin, were obtained fromN2 cells  induced N2 cells  and inhiN2 cells , among oN2 cells . The actN2 cells . Dehydro62 μg/mL , and lutectively . V. nudiectively  and germectively , as wellB cells) . In addill lines , 58. A ms [μg/mL . 1, isolated from G. megapotamica, is a promising cytotoxic with outstanding activity against K562 and CCRF-CEM and their respective MDR counterparts, Lucena 1 and CEM/ADR5000. This identifies this sesquiterpene lactone as an important lead for obtaining therapeutics to act against resistant leukemia cells.This study demonstrated that compound D. aspilioides and the scant information about its components and the hypersensitivity of resistant CEM/ADR5000 cells toward its extract merit further investigation to obtain potential candidates for improving leukemia chemotherapies, especially against resistant phenotypes.The outstanding antileukemia effect of