Dynamin-dependent transferrin endocytosis (IC50 = 5.7 μM)

Large GTPase dynein cleaves clathrin-coated vesicles that are membrane-bound. In cell physiology, endocytosis—the internalization of extracellular material and portions of the cytoplasmic membrane—is essential. With IC50 values of 2.7 μM and 0.38 μM, Hydroxy Dynasore inhibits dynamin I (Dyn I) activity in the GTPase assay [1], whether or not 0.06% Tween-80 is present. In U2OS cells, Hydroxy Dynasore inhibited Tfn-A594 uptake with an IC50 of 5.7 μM in clathrin-mediated endocytosis (CME) [1]. In the absence of Tween -80, Hydroxy Dynasore had IC50 values of 0.38 μM and 1.1 μM, while in the presence of Tween -80, IC50 values of 4.9 μM and 30.0 μM, respectively. Hydroxy Dynasore exhibits 2.1 times greater selectivity for DynI in this GTPase experiment compared to DynII of Sf21 cells and DynII (a recombinant protein derived from Sf21 cells) [1]. In motor nerve terminals and cultured hippocampus neurons, Hydroxy Dynasore inhibits the absorption of BoNT/A-Hc [2]. Hippocampal neurons depolarize when exposed to Hydroxy Dynasore (1-100 μM; 20 minutes before Alexa Fluor 488-BoNT/A-Hc addition); this inhibitory effect is dose-dependent and has an IC50 of 16.0 μM[2].

In the phrenic nerve-diaphragm twitch model in CD-1 mice, Hydroxy Dynasore (ip; 30 mg/kg; 1.5–2 hours before BoNT/A injection) once more offered protection against BoNT/A-induced paralysis [2].

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Dyngo-4a Disrupts BoNT/A-induced Paralysis and Delays the Onset of Botulism[2]
Researchers determined whether Dyngo-4a could be used to prevent the muscle paralysis induced by purified BoNT/A. We investigated this using a rat hemidiaphragm twitch model. The muscles were stimulated at 0.2 Hz, and the contractile force was recorded over 6–8 h. Representative traces of the twitch recordings for each condition are shown in Fig. 7A. Neither the untreated nor the Dyngo-4a-treated control muscles showed any sign of decline in contractions for up to 8 h. Following addition of BoNT/A, the amplitude of contractions decreased, consistent with BoNT-induced paralysis. Muscles pretreated with Dyngo-4a prior to addition of BoNT/A showed significantly less decline in contractile strength (Fig. 7A and Table 2). The contractile force, graphed as percent decline in contractions, fitted well to a four-parameter logistic curve (R2 = 0.999 and 0.994) (Table 2). Both the t½ and the Hill slope were significantly different (p = 0.0007 and 0.0018, respectively). These results show that both the decline in contraction and the time to reach 50% of decline were significantly increased in the Dyngo-4a-treated muscles compared with the BoNT/A control group. Together, these results demonstrate that Dyngo-4a provides significant protection against BoNT/A-induced muscle paralysis.

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Finally, researchers investigated whether Dyngo-4a could prevent the onset of botulism in an in vivo murine model. CD-1 mice were given an intraperitoneal injection of Dyngo-4a (1 mg) or vehicle followed by a booster 4.5–8 h later. BoNT/A (2 LD50) was injected via the tail vein 1.5–2 h following the initial injection. Mice were scored based on their appearance, behavior, and breathing and were euthanized upon reaching acute respiratory distress, indicative of botulism. Mice injected with Dyngo-4a took significantly longer to exhibit clear signs of botulism, 860 ± 65 min compared with 656 ± 55 min. When the data were fitted to a survival curve, a Mantel-Cox test revealed a significant difference (p = 0.0022) (Fig. 7B) between the two treatments. Mice treated with Dyngo-4a alone or vehicle alone showed no signs of toxicity. Overall, this indicates that Dyngo-4a pretreatment provides significant protection against botulism.

Dynamin GTPase assay[1]
The Malachite Green colorimetric GTPase assay was as described 10. Dynamin I activity was measured in its SAI activity state or was stimulated by three different methods. As each stimulus activates dynamin to different extents, each assay required different dynamin concentrations. First, maximal dynamin activity was stimulated by sonicated PS liposomes 10. Purified dynamin I (10–20 nM, diluted in: 6 mM Tris–HCl, 20 mM NaCl and 0.01% Tween 80, pH 7.4) was incubated in 96‐well plates in GTPase buffer (5 mM Tris–HCl, 10 mM NaCl, 2 mM Mg2+, 0.05% Tween 80, pH 7.4, 1 µg/mL leupeptin and 0.1 mM PMSF) and GTP 0.3 mM in the presence of test compound for 30 min at 37°C in a final assay volume of 150 μL. Reactions were terminated with 10 μL of 0.5 M ethylenediaminetetraacetic acid (EDTA) pH 7.4 and Malachite Green solution (40 μL: 2% w/v ammonium molybdate tetrahydrate, 0.15% w/v malachite green and 4 M HCl) was added for 5 min. Second, dynamin (20 nM) was stimulated by 10 µg/mL of taxol‐stabilized preformed bovine brain microtubules (Cytoskeleton, Inc) using the same protocol. Third, dynamin I (50 nM) was stimulated by 1 μM of recombinant growth factor receptor‐bound protein 2 (grb2), a SH3 (Src homology)‐containing protein that stimulates dynamin about 5–10 times less efficiently than liposomes or microtubules 54. The assay conditions were as described above. Finally, dynamin (500 nM) SAI activity was measured using high concentrations of dynamin, which promote its cooperative self‐assembly into rings (but not helices) 26. The final DMSO concentration in the GTPase or endocytosis assays was at most 3.3 or 1%, respectively, but typically was at 1%. The GTPase assay for dynamin I was unaffected by DMSO up to 3.3%. Compounds were dissolved as 30 mM stocks in 100% DMSO. These stock solutions can be stored at −20°C for several months. Compounds were subsequently diluted into solutions of 50% DMSO made up in 20 mM Tris–HCl pH 7.4 and diluted again into the final assay. For analysis of the kinetics of Dyngo-4a inhibition, dynamin I at a final concentration of 17 nM was incubated with GTPase buffer containing PS (2 µg/mL) and varying amounts of GTP (50–250 μM) in the presence of Dyngo-4a at a concentration range between 0.5 and 6 μM. The reaction was stopped after 30 min by addition of EDTA (0.5 mM, pH 7.4).

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Glutamate release from synaptosomes[1]
Release of glutamate from synaptosomes was performed as described previously 60, with minor changes. The assay was carried out at 37°C in a Perkin Elmer LS50 fluorimeter. Control (1% DMSO) and Dyngo-4a‐treated samples were incubated in the presence of the compound for 30 min prior to stimulation of glutamate release.

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Fluorescence imaging of SV turnover using FM1‐43[1]
Neuronal cultures were removed from culture medium and left for 10 min in incubation medium [170 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4, 20 mM TES (N‐tris[hydroxy‐methyl]‐methyl‐2‐aminoethane‐sulfonic acid), 5 mM NaHCO3, 5 mM glucose, 1.2 mM Na2SO4, 1.2 mM MgCl2 and 1.3 mM CaCl2, pH 7.4]. Cultures were then mounted in a Warner imaging chamber (RC‐21BRFS). Invaginating membrane was loaded with FM1‐43 (10 μM) by evoking SV turnover with a brief train of action potentials (80 Hz for 10 seconds, 100 mA and 1‐millisecond pulse width, delivered using platinum wires embedded in the imaging chamber). Dye kept present for 1 min after stimulation to ensure all retrieving membrane was labeled (S1 loading). After a 10‐min rest period, accumulated dye was unloaded from nerve terminals using two consecutive maximal stimuli with incubation media supplemented with 50 mM KCl (50 mM NaCl removed to maintain osmolarity). The fluorescence decrease due to dye loss provides an estimate of the total number of synaptic vesicles turned over during stimulation (S1). After a 20‐min rest period the S1 protocol was repeated (S2 loading and unloading). Thus, for any selected nerve terminal, the S2 response has a matched individual internal control (S1). Dyngo compound Dyngo-4a (30 μM) was present for 15 min prior to and including either, S2 loading (to monitor effects on endocytosis) or S2 unloading (exocytosis).

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Labeling of endocytosis pathways by horseradish peroxidase[1]
Granule neurons were processed for electron microscopy as previously described 40. Briefly, cells were transferred to incubation medium for 10 min and subsequently incubated with or without 30 μM Dyngo-4a for 15 min. Cultures were next stimulated with 800 action potentials (80 Hz) in incubation medium supplemented with HRP (10 mg/mL) in the presence or absence of Dyngo-4a.

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Dynamin II GTPase assay[1]
Assay conditions were based on the dynamin I assay but contained modifications. Recombinant dynamin II was used at 50 nM, stimulated by 10 µg/mL PS. The GTPase reaction was allowed to occur for 90 min at 37°C before termination.

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Plasma protein binding[1]
Plasma protein binding was estimated using an immobilized human serum albumin column (ChromTech Chiral‐HSA, 50 × 3.0 mm, 5 µm) with gradient elution based on a previously published method 56.

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Fluorescence imaging of dextran uptake[1]
Uptake of tetramethyrhodamine‐dextran (40 kDa) into nerve terminals of CGNs was monitored as described previously 40. Briefly, cells were left for 10 min in incubation medium and then stimulated with a train of 800 action potentials (80 Hz for 10 seconds) in the presence of tetramethyrhodamine‐dextran (50 μM). Dyngo compound Dyngo-4a (30 μM) was present for 15 min prior to and including action potential stimulation.

Cell‐based endocytosis[1]
Quantitative analysis of the inhibition of Alexa 594‐Tfn endocytosis in U2OS cells was performed on large numbers of serum‐starved cells as described 10. Synaptic vesicle recycling in cultured CGNs was monitored using FM1‐43 as described 57 (see Supporting Information). Dynamin‐independent endocytosis was measured using internalization of CT in NIH3T3 cells (using Tfn as a control) as described previously 58 with minor changes (see Supporting Information). The average number of cells for each data point was ˜1200. IC50 values were calculated using GraphPad Prism 5 and data were expressed as mean ± 95% confidence interval (CI) for three wells and ˜1200 cells.

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Internalization Studies[2]
Cultured hippocampal neurons were prepared from embryonic age 18 C57BL/6 embryos and co-cultured with astroglia as described previously. The neurons were allowed to mature for at least 14 days in vitro before use. Neurons were removed from the co-culture and incubated for 5 min at 37 °C with 100 nm Alexa Fluor 488-BoNT/A-Hc in a low K+ buffer (15 mm HEPES, 145 mm NaCl, 5.6 mm KCl, 2.2 mm CaCl2, 0.5 mm MgCl2, 5.6 mm d-glucose, 0.5 mm ascorbic acid, 0.1% bovine serum albumin (BSA), pH 7.4) or high K+ buffer (modified to contain 95 mm NaCl and 56 mm KCl), with or without Dyngo-4a or Dynasore as indicated. The cells were fixed with 4% paraformaldehyde, processed for immunocytochemistry, imaged, and analyzed using Zen software or LaserPix.

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SNAP25 Cleavage Assay[2]
Cultured hippocampal neurons were removed from co-culture and washed once with low K+ buffer. They were then treated with either DMSO or Dyngo-4a (30 μm) for 20 min. Neurons were stimulated with high K+ buffer with and without BoNT/A (100 pm) in the continuing presence of DMSO or Dyngo-4a for 5 min. The cells were washed five times with low K+ buffer containing DMSO or Dyngo-4a and left for 90 min before being transferred back to co-culture with astroglia and conditioned medium for a further 24 h. The neurons were then removed from co-culture and processed for Western blotting as follows: the cells were washed twice with ice-cold PBS before scraping in 20 mm HEPES, 150 mm NaCl, pH 7.5, containing protease inhibitors. The cell membranes were collected and resuspended in Laemmli sample buffer containing 10% β-mercaptoethanol. Samples were run on an SDS-PAGE and then transferred to a PVDF membrane. The membrane was probed for cleaved SNAP25 using an antibody designed against the SNAP25-cleaved product, which does not recognize full-length SNAP25. The intensity of the bands was normalized to β-actin, and the integrated intensity was used to determine the amount of cleavage relative to the control (ImageJ).

Animal/Disease Models: CD-1 mice[2].
Doses: 30 mg/kg
Route of Administration: intraperitoneal (ip)injection; 1.5–2 h before BoNT/A injection
Experimental Results: Protected BoNT/A-induced paralysis in vivo.

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Rat Phrenic Nerve-Hemidiaphragm Twitch Experiments[2]
The hemidiaphragm and innervating phrenic nerve were dissected from 5-week-old male Wistar rats. The nerve muscle preparation was suspended in an organ bath containing carbogen-bubbled Tyrode's solution (136.7 mm NaCl, 2.68 mm KCl, 1.75 mm NaH2PO4, 16.3 mm NaHCO3, 1 mm MgCl2, 1 mm CaCl2, 7.8 mm d-glucose). The nerve was stimulated with 0.1-ms square pulses of 10 V at 0.2 Hz and the force of contractions (mN) was recorded through Powerlab and Bridge Amp Systems with Chart software. Upon reaching stable contractions, 30 μm Dyngo-4a or vehicle was added for 1 h prior to the addition of BoNT/A (100 pm). Control preparations were as indicated. Contractions were recorded over 6–8 h and analyzed by converting contractile strength to percentage decline.

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In Vivo Assay[2]
BoNT/A was diluted in 0.9% saline containing 0.1 mg/ml BSA, immediately prior to use. Female CD-1 mice (30–40 g) were injected intraperitoneally with 1 mg of Dyngo-4a (which is 30 mg/kg body weight) or vehicle (1/9 NMP/PEG300 (1 part NMP to 9 parts PEG300) in PBS). 1.5–2 h later, mice were injected with 2LD50 BoNT/A via the tail vein. A top-up of 1 mg of Dyngo-4a or vehicle was administered 4.5–8 h after the initial intraperitoneal injection. Mice were constantly monitored for signs of botulism and euthanized upon development of acute respiratory distress.

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Dyngo-4a was made up in DMSO (30 mm) for in vitro experiments and dissolved in a formulation containing 1-methyl-2-pyrrolidione (NMP) and polyethylene glycol 300 (PEG300) (1 part NMP to 9 parts PEG300), then diluted 1/9 in phosphate-buffered saline (PBS) for in vivo experiments.

[1]. Building a Better Dynasore: The Dyngo Compounds Potently Inhibit Dynamin and Endocytosis. Traffic. 2013 Dec;14(12):1272-89.

[2]. Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism. J Biol Chem. 2011 Oct 14;286(41):35966-76.

Dyngo-4a is dynamin inhibitor. It has a role as an EC 3.6.5.5 (dynamin GTPase) inhibitor.

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Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC₅₀ ~ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC₅₀ = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC₅₀ of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.[1]

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The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.[2]

C18H14N2O5

338.31

338.09

C, 63.90; H, 4.17; N, 8.28; O, 23.64

1256493-34-1

136227923

Light brown to brown solid powder

1.5±0.1 g/cm3

1.683

5.12

5

6

3

25

500

0

C1=CC=C2C=C(C(=CC2=C1)C(=O)N/N=C/C3=CC(=C(C=C3O)O)O)O

UAXHPUSKEWEOAP-DJKKODMXSA-N

InChI=1S/C18H14N2O5/c21-14-8-17(24)16(23)7-12(14)9-19-20-18(25)13-5-10-3-1-2-4-11(10)6-15(13)22/h1-9,21-24H,(H,20,25)/b19-9+

3-Hydroxynaphthalene-2-carboxylic acid 2-[(2,4,5-trihydroxyphenyl)methylene]hydrazide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)