Latrunculin

The latrunculins are a family of natural products and toxins produced by certain sponges, including genus Latrunculia and Negombata, whence the name is derived. It binds actin monomers near the nucleotide binding cleft with 1:1 stoichiometry and prevents them from polymerizing. Administered in vivo, this effect results in disruption of the actin filaments of the cytoskeleton, and allows visualization of the corresponding changes made to the cellular processes. This property is similar to that of cytochalasin, but has a narrow effective concentration range.[1] Latrunculin has been used to great effect in the discovery of cadherin distribution regulation and has potential medical applications.[2] Latrunculin A, a type of the toxin, was found to be able to make reversible morphological changes to mammalian cells by disrupting the actin network.[3]

Chemical structures of latrunculins

Latrunculin A

Latrunculin B

Latrunculin A:

Molecular Formula:	C22H31NO5S[4]
Molecular Weight:	421.552 g/mol[4]

Target and functions

Gelsolin - Latrunculin A causes end- blocking; this protein binds to the barbed sides of the actin filaments which accelerates nucleation. This calcium-regulated protein also plays a role in assembly and disassembly of cilia[4] which plays a crucial role in handedness.

Latrunculin B:

Molecular Formula:	C20H29NO5S^[4]
Molecular Weight:	395.514 g/mol

Target and Function

Actin- Latrunculin B makes up the structure of the actin fibers.

Protein spire homolog 2- needed for cell division, vesicle transport within the actin filament and is essential for the formation of the cleavage formation during cell division^[4].

History

Latrunculin is a toxin that is produced by sponges. The red-coloured Latrunculia Magnifica Keller is an abundant sponge in the gulf of Eilat and the gulf of Suez[5] in the red sea, where it lives at a depth of 6–30 meters.[6] The toxin was discovered around 1970. Researchers observed that the red-coloured sponges, Latrunculia Magnifica Keller, were never damaged or eaten by fishes, while others were. Furthermore, when researchers squeezed the sponges in the sea, they observed that a red fluid came out. Fishes nearby immediately fled the surrounding area when the sponge secreted the fluid. This were the first indications that these sponges produced a toxin. Later they confirmed the hypothesis by squeezing the sponge in an aquarium with fish. The fish shown loss of balance and severe bleedings and died within only 4–6 minutes.[6] Similar effects were observed when the toxin was injected in mice.

Latrunculin makes up to 0.35% of the dry weight of the sponge.[5] There are two main forms of the toxin, A and B. Latrunculin A is only present in sponges which live in the gulf of Suez while latrunculin B only exist in sponges in the gulf of Eilat. Why this is the case is still under investigation.[5]

Structure

Figure 2 relative activity of Latrunculin analogues The micro filament disrupting activity (at 10 μM effective concentration). Abbreviations: ± weak effect, + significant effect, ++ strong effect, +++ very strong effect (less than 20% viable cells).

There are several isomers of latrunculin, A, B, C, D, G, H, M, S and T. The most common structures are latrunculin A and B. Their formulas are respectively C₂₂H₃₁NO₅S and C₂₀H₂₉NO₅S. The macrolactone ring on top that contains double bonds is a structural feature of the latrunculin molecules. The side chain contains an acylthiazolidinone substitute. Besides these natural occurring forms, scientist have made synthetic forms with different toxic strengths. Figure 2 shows some of these forms with their relative ability to disrupt microfilament activity. Semisynthetic forms that contained N-alkylated derivates were inactive.[7]

Mechanism of action

Latrunculin A and latrunculin B affect polymerization of actin. Latrunculin binds actin monomers near the nucleotide binding cleft with 1:1 stoichiometry and prevents them from polymerizing.[1] The nucleotide monomers are prevented from dissociation from the nucleotide binding cleft, thus preventing polymerizing.[8]

When actin is impaired due to latrunculin, Shiga toxins have a better chance of infiltrating the intestinal epithelial monolayer in E. coli, which may cause a higher chance of generating gastrointestinal illnesses.[9]

It seems that actin monomers are more sensitive to bind latrunculin A than to bind Latrunculin B.[10] In other words, latrunculin A is a more potent toxin. Latrunculin B is inactivated faster than latrunculin A.[11]

The prevention of polymerizing of the actin filaments causes reversible changes in the morphology of mammalian cells.[12] Lantranculin interferes with the structure of the cytoskeleton in rats.[13]

After latrunculin B exposure, mouse fibroblasts grow bigger and PtK2 kidney cells from a potoroo stem produced long, branched extensions.[14] The extensions seem to be an accumulation of actin monomers.

Metabolism

Yeast cells in absence of the proteins osh3 or osh5 demonstrated hypersensitivity to latrunculin B.[15] The osh proteins are homologous to OSBP generated enzymes that appear in mammals, indicating that these might play a role in the toxicokinetics of latrunculins.

Yeast mutants that are resistant to latrunculin show a mutation, D157E, that initiates a hydrogen bond with latrunculin.[8] Other yeast mutants adjust the binding site, thus making it resistant to latrunculin.

No research has been done to figure out how the biotransformation of latrunculin works in eukaryotic cells. However, research suggests that it is the unaltered form of latrunculin that causes toxic effects.[3]

Toxicity

As latrunculin inhibits actin polymerization and actomyosin contractile ability, exposure to latrunculin may result in cellular relaxation, expansion of drainage tissues and decreased outflow resistance in e.g. the trabecular meshwork.

Plant

Latrunculin B causes marked and dose-dependent reductions in pollen germination frequency and pollen tube growth rate.[16]

Adding latrunculin B to solutions of pollen F-actin produced a rapid decrease in the total amount of polymer, the extent of depolymerization increasing with the concentrations of the toxic. The concentration of latrunculin B required for half-maximal inhibition of pollen germination is 40 to 50 nM, whereas pollen tube extension is much more sensitive, requiring only 5 to 7 nM LATB for half-maximal inhibition. The disruption of germination and pollen tube growth by latrunculin B is partially reversible at low concentrations. (<30 nM).[16]

Animal

Squeezing Latrunculia magnifica into aquarium with fishes causes their almost immediate agitation, followed by hemorrhage, loss of balance and death in 4–6 minutes.[17]

Latrunculin A has been used as acrosome reaction inhibitor of guinea pig in laboratory conditions.[18]

Human

Lat-A-induces reduction of actomyosin contractility. This is associated with trabecular meshwork porous expansion without evidence of reduced structural extracellular matrix protein expression or cellular viability.[19] In high doses, latrunculin can induce acute cell injury and programmed cell death through activating the caspase-3/7 pathway.[17]

Lethal doses

TDLO - Lowest Published Toxic Dose

LD50 – median Lethal Dose[20]

Indicator	Species	Dose
Oral TDLO	Man	1,14 ml/kg, 650 mg/kg
Oral LD50	Rat	7,06 mg/kg
Oral LD50	Mouse	3,45 g/kg, 10,5 ml/kg
Oral LD50	Rabbit	6,30 mg/kg
Inhalation LC50	Rat	6h: 5,900 mg/m3 10h: 20,000 ppm
Inhalation LCLO	Mouse	7h: 29,300 ppm
Inhalation TCLO	Human	20m: 2,500 mg/m3 30m: 1,800 ppm
Irritation eyes	Rabbit	24h: 500 mg
Irritation skin	Rabbit	24h: 20 mg

Applications

In nature, latrunculins are used by the sponges themselves as a defense mechanism, and for the same purpose are also sequestered by certain nudibranchs.[21]

Latrunculins are produced for fundamental research and have potential medical applications as latrunculins and their derivatives show antiangionic, antiproliferative, antimcrobial and antimetastatic activities.[2]

Defense mechanism

Like many other sessile organisms, sponges are rich of secondary metabolites with toxic properties and most of them, including Latrunculin, have a defense role against predators, competitors and epibionts.[22]

The sponges themselves are not damaged by latrunculin. As a measure against self-toxination, they keep the latrunculin in membrane-bound vacuoles, that also function as secretory and storage vesicles. These vacuoles are free of actin and prevent the latrunculin from entering the cytosol where it would damage actin.[22] After production in the choanocytes, the latrunculin is transferred via the archeocytes to the vulnerable areas of the sponges where defense is needed, such as injured or regenerating sites.[22]

Sequestering by nudibranchs

Sea slugs of the genus Chromodoris sequester different toxics from the sponges that they eat as defensive metabolites, including latrunculin. They selectively transfer and store latrunculin in the sites of the mantle that are most exposed to potential predators.[21] It is thought that the digestive system of the nudibranchs plays an important role in the detoxification.[21]

In 2015, the discovery that five closely related sea slugs of the genus Chromodoris all use latrunculin as defense, indicates that the toxic might be used via Müllerian mimicry.[21]

Research

Latrunculins are used for fundamental research like cytoskeleton studies. Many functions of actin have been determined by using latrunculins to block actin polymerization followed by examining the effects on the cell. Using this method, the importance of actin for the polarized localization of proteins, polarized exocytosis and the maintenance of cell polarity have been shown.[23] Also the role of actin in regulating voltage-gated ion channels in different nerve cells has been examined,[24] showing that with latrunculin treatment the electrical activity of nerve cells can be altered.[24][25] Latrunculin shows a dose-dependent inhibition of K+ currents and acute application can induce multiple action potential firing, which could underlie a mechanism of defense via nociceptors.[25]

Medical applications

Latrunculin A and B and derivatives have potential as novel chemotherapeutic agents.[2][26] The potential use of latrunculin as growth inhibitors of tumor cells has already been investigated for certain forms of gastric cancer,[17] metastatic breast cancer[26] and prostate tumors.[27] In lower doses, latrunculin can be used to decrease disaggregation and cell migration, thereby preventing invasive activities of tumor cells.[27] In higher doses, latrunculin can induce acute cell injury and programmed cell death through activating the caspase-3/7 pathway, and thus be used to kill tumor cells.[17]

Latrunculin also is a potential therapeutic for ocular hypertension and glaucoma. Latrunculin A and B are shown to disrupt the actin cytoskeleton of the trabecular meshwork that is important for regulating humor outflow resistance and thereby intraocular pressure.[28][29] By cellular relaxation and loosened cell-cell junctions, latrunculin can increase humor outflow facility. The first human trial of lantruculin B as treatment of ocular hypertension and glaucoma showed significantly lower intraocular pressure in patients.[29]

References

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17. Konishi H, Kikuchi S, Ochiai T, Ikoma H, Kubota T, Ichikawa D, Fujiwara H, Okamoto K, Sakakura C, Sonoyama T, Kokuba Y, Sasaki H, Matsui T, Otsuji E (June 2009). "Latrunculin a has a strong anticancer effect in a peritoneal dissemination model of human gastric cancer in mice". Anticancer Research. 29 (6): 2091–7. PMID 19528469.
18. Roa-Espitia AL, Hernández-Rendón ER, Baltiérrez-Hoyos R, Muñoz-Gotera RJ, Cote-Vélez A, Jiménez I, González-Márquez H, Hernández-González EO (September 2016). "Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation". Biology Open. 5 (9): 1189–99. doi:10.1242/bio.017558. PMC 5051654. PMID 27402964.
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20. Cayman chemical (2017). "SAFETY DATA SHEET Latrunculin A" (PDF).
21. Cheney KL, White A, Mudianta IW, Winters AE, Quezada M, Capon RJ, Mollo E, Garson MJ (2016-01-20). "Choose Your Weaponry: Selective Storage of a Single Toxic Compound, Latrunculin A, by Closely Related Nudibranch Molluscs". PLOS ONE. 11 (1): e0145134. doi:10.1371/journal.pone.0145134. PMC 4720420. PMID 26788920.
22. Gillor O, Carmeli S, Rahamim Y, Fishelson Z, Ilan M (May 2000). "Immunolocalization of the Toxin Latrunculin B within the Red Sea Sponge Negombata magnifica (Demospongiae, Latrunculiidae)". Marine Biotechnology. 2 (3): 213–23. doi:10.1007/s101260000026. PMID 10852799.
23. Ayscough KR, Stryker J, Pokala N, Sanders M, Crews P, Drubin DG (April 1997). "High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A". The Journal of Cell Biology. 137 (2): 399–416. doi:10.1083/jcb.137.2.399. PMC 2139767. PMID 9128251.
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28. Gonzalez JM, Ko MK, Pouw A, Tan JC (February 2016). "Tissue-based multiphoton analysis of actomyosin and structural responses in human trabecular meshwork". Scientific Reports. 6 (1): 21315. doi:10.1038/srep21315. PMC 4756353. PMID 26883567.
29. Rasmussen CA, Kaufman PL, Ritch R, Haque R, Brazzell RK, Vittitow JL (September 2014). "Latrunculin B Reduces Intraocular Pressure in Human Ocular Hypertension and Primary Open-Angle Glaucoma". Translational Vision Science & Technology. 3 (5): 1. doi:10.1167/tvst.3.5.1. PMC 4164113. PMID 25237590.