Neuronal Wiskott-Aldrich syndrome protein (N-WASP)-mediated actin polymerization
Wiskostatin targets neuronal Wiskott-Aldrich syndrome protein (N-WASP), a key regulator of actin polymerization. N-WASP activates the Arp2/3 complex, which nucleates new actin filaments, driving processes such as cell migration, endocytosis, and filopodia formation. Wiskostatin binds to N-WASP and stabilizes its closed, autoinhibited conformation, preventing its activation by upstream signals. This inhibition of N-WASP prevents the activation of the Arp2/3 complex and subsequent actin polymerization.

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In the current studies with this drug, researchers observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, researchers found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. The above data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo[1].

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In vitro, wiskostatin is a potent inhibitor of N-WASP-mediated actin polymerization. Its activity is typically assessed in actin polymerization assays, where it inhibits the formation of actin filaments. It is selective for N-WASP over other members of the WASP family, such as WASP. Its reversible inhibition allows for controlled studies of actin dynamics.

In vivo, wiskostatin is used to study the role of N-WASP in cell motility and actin dynamics. Its ability to inhibit N-WASP-mediated actin polymerization makes it a valuable tool for investigating the role of actin in various cellular processes, including cell migration, invasion, and vesicle trafficking. Specific in vivo studies, including dosing and administration routes, are not detailed in the provided sources but would be described in the primary literature.

Wiskostatin (1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol) (1) is a carbazole-based compound reported as a specific and relatively potent inhibitor of the N-WASP actin remodelling complex (S-isomer EC50 = 4.35 μM; R-isomer EC50 = 3.44 μM). An NMR solution structure showed that wiskostatin interacts with a cleft in the regulatory GTPase binding domain of N-WASP. However, numerous studies have reported wiskostatin's actions on membrane transport and cytokinesis that are independent of the N-WASP-Arp2/3 complex pathway, but offer limited alternative explanation. The large GTPase, dynamin has established functional roles in these pathways. This study reveals that wiskostatin and its analogues, as well as other carbazole-based compounds, are inhibitors of helical dynamin GTPase activity and endocytosis. We characterise the effects of wiskostatin on in vitro dynamin GTPase activity, in-cell endocytosis, and determine the importance of wiskostatin functional groups on these activities through design and synthesis of libraries of wiskostatin analogues. We also examine whether other carbazole-based scaffolds frequently used in research or the clinic also modulate dynamin and endocytosis. Understanding off-targets for compounds used as research tools is important to be able to confidently interpret their action on biological systems, particularly when the target and off-targets affect overlapping mechanisms (e.g. cytokinesis and endocytosis). Herein we demonstrate that wiskostatin is a dynamin inhibitor (IC50 20.7 ± 1.2 μM) and a potent inhibitor of clathrin mediated endocytosis (IC50 = 6.9 ± 0.3 μM). Synthesis of wiskostatin analogues gave rise to 1-(9H-carbazol-9-yl)-3-((4-methylbenzyl)amino)propan-2-ol (35) and 1-(9H-carbazol-9-yl)-3-((4-chlorobenzyl)amino)propan-2-ol (43) as potent dynamin inhibitors (IC50 = 1.0 ± 0.2 μM), and (S)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8a) and (R)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8b) that are amongst the most potent inhibitors of clathrin mediated endocytosis yet reported (IC50 = 2.3 ± 3.3 and 2.1 ± 1.7 μM, respectively).https://pubmed.ncbi.nlm.nih.gov/36577213/

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The inhibitory activity of wiskostatin is assessed using in vitro actin polymerization assays. Purified actin, the Arp2/3 complex, and the VCA domain of N-WASP are incubated with varying concentrations of the compound. The polymerization of actin is monitored by measuring the increase in fluorescence of pyrene-labeled actin. The IC50 is calculated from the dose-response curve.

The cellular activity of wiskostatin is evaluated in cell lines where N-WASP plays a role, such as neurons or cancer cells. Cells are treated with the compound, and its effect on actin polymerization, cell morphology, and cell motility is assessed. The formation of filopodia and lamellipodia can be visualized by fluorescence microscopy. Its effect on cell migration can be measured in wound healing or transwell assays.

In animal studies, wiskostatin would typically be administered to mice via intraperitoneal (i.p.) or intravenous (i.v.) injection. In models of cancer metastasis, its effect on tumor cell invasion and metastasis would be assessed. In models of neurological disorders, its effect on neuronal morphology and function would be studied.

Wiskostatin is a small molecule with a molecular weight of 426.15 g/mol and a chemical formula of C17H18Br2N2O. It is soluble in DMSO and is typically stored as a powder at -20°C. It has a purity of ≥95%. Its stability and solubility are important for its use in cell-based assays.

Formal toxicology data for wiskostatin is not extensively documented in the provided sources, as it is a research compound. Its safety profile has not been established in comprehensive toxicology studies. However, its use in cell-based assays at effective concentrations suggests it is tolerated in those contexts. Its use is limited to research applications.

[1]. N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels. Am J Physiol Cell Physiol. 2007;292(4):C1562-C1566.

1-(3,6-Dibromocarbazole-9-yl)-3-(dimethylamino)prop-2-ol is a carbazole compound with two bromine substituents attached to the 3 and 6 positions of the carbazole ring. It belongs to the carbazole class, organobromine compounds, secondary alcohols, and tertiary amines.

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Wiskostatin (CAS: 253449-04-6) is a valuable research tool for studying actin dynamics and cell motility. Its selective inhibition of N-WASP-mediated actin polymerization makes it a key compound for investigating the role of N-WASP in various cellular processes, including cell migration, invasion, and neuronal function. It is available from various commercial suppliers for research purposes.

C17H18BR2N2O

426.15

423.978

C, 47.91; H, 4.26; Br, 37.50; N, 6.57; O, 3.75

253449-04-6

2775510

White to off-white solid powder

1.6±0.1 g/cm3

320.2±22.0 °C at 760 mmHg

153-155ºC

147.4±22.3 °C

0.0±0.7 mmHg at 25°C

1.657

5.65

1

2

4

22

356

0

XUBJEDZHBUPBKL-UHFFFAOYSA-N

InChI=1S/C17H18Br2N2O/c1-20(2)9-13(22)10-21-16-5-3-11(18)7-14(16)15-8-12(19)4-6-17(15)21/h3-8,13,22H,9-10H2,1-2H3

1-(3,6-dibromocarbazol-9-yl)-3-(dimethylamino)propan-2-ol

Wiskostatin; 253449-04-6; 1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol; 1-(3,6-dibromocarbazol-9-yl)-3-(dimethylamino)propan-2-ol; CHEBI:78012; C17H18Br2N2O; MFCD00218393; Maybridge1_002006;

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)

DMSO : ~25 mg/mL (~58.66 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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