- eEF1A2 (eukaryotic translation elongation factor 1A2): Plitidepsin binds to eEF1A2 with a Kd of 80 ± 30 nM (determined by saturation binding with [¹⁴C]-plitidepsin). The dissociation rate constant (koff) is 1.9 ± 0.8 × 10⁻³ s⁻¹, corresponding to a target residence time of approximately 9 minutes. The binding site is at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. [1]
- eEF1A (eukaryotic translation elongation factor 1A) (for antiviral activity): Plitidepsin's anti-SARS-CoV-2 activity is mediated through inhibition of eEF1A. Resistance-conferring mutation A399V in eEF1A reduces antiviral activity by >10-fold. [2]

- Anticancer Activity (Antiproliferative): Plitidepsin shows potent antiproliferative activity against various cancer cell lines. HeLa cells (cervical cancer) have an IC50 of approximately 1 nM. NCI-H460 (lung cancer) and HGC27 (gastric cancer) cells show IC50 values of 0.2 nM and 0.9 nM, respectively, after 72 h exposure. [1]
- eEF1A2 Binding and Protection (DARTS): In HeLa cell protein extracts, plitidepsin (0.1-10 μM) protects eEF1A2 from subtilisin digestion in a concentration-dependent manner, indicating direct binding. [1]
- Signal Transduction: In HeLa cells, plitidepsin (450 nM) induces phosphorylation of p38, ERK, and JNK (MAPK family members). This effect is restored in plitidepsin-resistant HeLa-APL-R cells upon ectopic expression of eEF1A2. [1]
- Antiviral Activity Against SARS-CoV-2: Plitidepsin potently inhibits SARS-CoV-2 replication in vitro. In Vero E6 cells, the IC90 is 1.76 nM. In hACE2-293T human cells, the IC90 is 0.88 nM, which is 27.5-fold more potent than remdesivir (IC90 = 24.2 nM). In human pneumocyte-like cells, the IC90 is 3.14 nM, with a selectivity index of 40.4. The compound shows an additive effect with remdesivir. [2]
- Mechanism of Antiviral Action: Plitidepsin reduces SARS-CoV-2 genomic and subgenomic RNA levels, with a particularly strong and early reduction in subgenomic N RNA (as early as 4 hours post-infection) and viral N protein levels, consistent with inhibition of viral translation. [2]
- Resistance Mechanism: Expression of the eEF1A A399V mutant in 293T cells increases the IC50 of plitidepsin against SARS-CoV-2 by >10-fold (from ~0.1 nM to ~2.2 nM). A CRISPR knock-in 293T cell line (293T-A399V) is also refractory to plitidepsin's antiviral activity (>12-fold increase in IC50). siRNA silencing of eEF1A reduces viral N protein levels. [2]

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In MOLT-4 cells, plitidepsin (20 nM; 1 h) causes VEGF dependency [1]. In normal endothelial cells, which express VEGFR-1 but do not support VEGF, plitidepsin (20 nM; 1 h) does not significantly decrease VEGF-R1 mRNA [1]. SARS-CoV-2 is inhibited by plitidepsin, with an IC90 of 1.76 nM. Plitidepsin exhibits anti-SARS-CoV-2 action in hACE2-293T cells, with an IC90 of 0.88 nM. Plitidepsin suppresses SARS-CoV-2 replication in the established cell model of human lung cell samples, with an IC90 of 3.14 nM and a selectivity index of 40.4 [1].

- Anticancer Efficacy: Not directly tested in vivo in the provided documents; the focus is on target identification and mechanism. [1]
- Antiviral Efficacy (BALB/c-hACE2 Mouse Model): In BALB/c mice transduced with Ad5-hACE2 and infected with SARS-CoV-2 (10⁴ PFU), prophylactic subcutaneous treatment with plitidepsin (0.3 mg/kg, once daily for 3 days) reduced lung viral titers by nearly 2 log units compared to vehicle control. A single dose of 1 mg/kg resulted in a 1.5 log unit reduction. [2]
- Antiviral Efficacy (K18-hACE2 Mouse Model): In K18-hACE2 transgenic mice infected with SARS-CoV-2 (10⁴ PFU), treatment with plitidepsin (0.3 mg/kg, once daily for 3 days starting 2 h pre-infection) reduced lung viral titers by 2 log units at day 3 post-infection, comparable to high-dose remdesivir (50 mg/kg, twice daily). Histopathology analysis showed reduced lung inflammation (score 1/16) compared to vehicle (5.4/16) and remdesivir (2.3/16), with no peribronchiolar inflammation noted. [2]

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In BALB/c samples expressing human ACE2, plitidepsin (ip; 0.3 mg/kg or 1 mg/kg; 2 hours prior to SARS-CoV-2 infection) dramatically lowers SARS-CoV-2 infection. In comparison to the vector desert, the SARS-CoV-2 viral decrease in the lungs was almost two log units lower in the 0.3 mg/kg plitidepsin group and 1.5 log units lower in the 1 mg/kg group [2].

- Saturation Binding Assay: Rabbit muscle eEF1A2 (100 nM) was mixed with varying concentrations of [¹⁴C]-plitidepsin (0.1-4 μM) in buffer (45 mM Hepes-KOH pH 7.5, 5 mM Mg acetate, 75 mM KCl, 1 mM DTT, 5% DMSO, 1 μM Gpp(NH)p). After 1 h incubation at RT, bound radioligand was separated by filtration through GF/C filters and counted. Data were fitted to the Swillens equation to account for ligand depletion, yielding a Kd of 80 ± 30 nM. [1]
- Dissociation Kinetics: eEF1A2 (100 nM) was pre-incubated with 1 μM [¹⁴C]-plitidepsin for 1 h. Unlabeled plitidepsin (10 μM) was then added, and bound radioactivity was measured at various times. Data were fitted to a single exponential decay, yielding a koff of 1.9 ± 0.8 × 10⁻³ s⁻¹. [1]

- Cell Viability/Proliferation Assay (MTT): Cells were seeded in 96-well plates, treated with various concentrations of plitidepsin for 72 h, and then incubated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Formazan crystals were dissolved, and absorbance was measured at 550-570 nm. IC50 values were calculated. [1][2]
- Antiviral Assay (Immunofluorescence): Vero E6 or hACE2-293T cells were pre-treated with plitidepsin for 2 h, then infected with SARS-CoV-2 (MOI 0.05) for 24 h. Cells were fixed, stained with anti-SARS-CoV-2 nucleocapsid antibody and a fluorescent secondary antibody, and imaged. IC50 and IC90 were calculated from percent infection. [2]
- Western Blotting: Cells were lysed in RIPA buffer with protease/phosphatase inhibitors. Proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against eEF1A2, phospho-JNK, phospho-ERK, phospho-p38, SARS-CoV-2 N protein, and loading controls (GAPDH, α-Tubulin). [1][2]
- DARTS Assay (Drug Affinity Responsive Target Stability): HeLa cell lysates were incubated with plitidepsin (0.1-10 μM) for 1 h, then digested with subtilisin for 30 min at RT. Samples were analyzed by Western blot with anti-eEF1A2 antibody. Protection from degradation indicated binding. [1]
- siRNA Knockdown: 293T cells were transfected with siRNA targeting eEF1A. After 48 h, cells were infected with SARS-CoV-2 (MOI 1) for 24 h, and N protein levels were analyzed by Western blot. [2]

- BALB/c-hACE2 Mouse Model: Female BALB/c mice (6-8 weeks) were intranasally transduced with 2.5 × 10⁸ PFU of Ad5-hACE2. Five days later, mice were infected intranasally with 1 × 10⁴ PFU of SARS-CoV-2. Plitidepsin was administered subcutaneously (s.c.) at 0.3 mg/kg (once daily for 3 days, starting 2 h pre-infection) or as a single dose of 1 mg/kg (2 h pre-infection). Remdesivir (50 mg/kg, once daily for 3 days) was used as a comparator. Lungs were harvested at day 3 post-infection for viral titration (TCID50). [2]
- K18-hACE2 Mouse Model: Heterozygous K18-hACE2 transgenic mice were infected intranasally with 1 × 10⁴ PFU of SARS-CoV-2. Plitidepsin (0.3 mg/kg, s.c.) was administered once daily for 3 days, starting 2 h pre-infection. Remdesivir (50 mg/kg, twice daily for 3 days) was used as a comparator. Lungs were harvested at day 3 for viral titration (TCID50) and histopathology (H&E staining, scored 0-16 for inflammation). [2]

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Animal/Disease Models: balb/c (Bagg ALBino) mouse[2]. 1]
Doses: 0.3 mg/kg; 1 mg/kg Dosing: 1 mg/kg QD for 1 day; 0.3 mg/kg daily for 3 days
Experimental Results: Shown in vivo in SARS-CoV-2 infection mouse model Antiviral effect.

In mice, a single s.c. dose of 0.3 mg/kg achieves lung concentrations an order of magnitude greater than the in vitro IC90. Detailed human PK parameters (e.g., half-life, Cmax, AUC) are not provided. [2]

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Metabolism / Metabolites
Liver

- In Vitro Cytotoxicity (Antiproliferative): Plitidepsin shows a cytostatic effect on cell proliferation rather than a cytotoxic one. In Vero E6 cells, the CC50 is approximately 100 nM; in hACE2-293T cells, the CC50 is 6.85 nM; in pneumocyte-like cells, the CC50 is 126.9 nM. [2]
- In Vivo Tolerability (Mouse): In the BALB/c-hACE2 model, mice treated with 0.3 mg/kg plitidepsin daily for 3 days showed slight body weight loss, while a single 1 mg/kg dose did not cause weight loss. In the K18-hACE2 model, no significant body weight loss or other adverse effects were reported at 0.3 mg/kg daily for 3 days. [2]
- Clinical Safety: Plitidepsin has a well-established safety profile from clinical trials for multiple myeloma. It has a good safety profile with minimal side effects at the doses used in COVID-19 clinical trials (max total 7.5 mg over 3 days). Cardiac adverse events are not significantly associated with plitidepsin treatment. [2]

[1]. Translation Elongation Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin. Sci Rep. 2016 Oct 7;6:35100.

[2]. Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A. Science. 2021 Feb 26;371(6532):926-931.

- Mechanism of Action (Anticancer): Plitidepsin binds to eEF1A2, a protein overexpressed in many cancers, with high affinity. This binding disrupts the interaction of eEF1A2 with its partners (e.g., aa-tRNAs, eEF1Bα, actin), leading to potent oxidative stress, activation of MAPK signaling (JNK, p38), and ultimately caspase-dependent apoptosis. The drug causes rapid and sustained activation of JNK and p38. [1]
- Mechanism of Action (Antiviral): Plitidepsin targets the host eEF1A protein, which is required for SARS-CoV-2 replication. Inhibition of eEF1A leads to a specific reduction in viral subgenomic RNA production and viral protein translation, with a greater impact on negative-sense RNA accumulation. This host-targeted approach offers a high barrier to resistance. [2]
- Clinical Status: Plitidepsin has completed a phase III clinical trial for multiple myeloma (NCT01102426). It is also being evaluated in a phase II/III clinical trial for COVID-19. [1][2]
- Comparison to Remdesivir: Plitidepsin is 27.5 times more potent than remdesivir against SARS-CoV-2 in hACE2-293T cells (IC90 0.88 nM vs. 24.2 nM) and shows an additive effect when combined in vitro. [2]

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Plitidepsin is a dipeptide, a derivative of dipeptide B, in which the hydroxyl group of the 1-(2-hydroxypropionyl)-L-prolineamide moiety is oxidized to the corresponding ketone. It was originally isolated from the Mediterranean sea squirt Aplidium albicans. It is a marine metabolite with antitumor and anticoronavirus activities. Apliidine is a peptide found in sea squirts and has shown promising potential in shrinking tumors of pancreatic, gastric, bladder, and prostate cancers. Its source marine organism is the sea squirt Aplidium albicans. Apliidine has also attracted attention as a potential therapy for certain leukemias. Plitidepsin is a cyclic condensate isolated from the marine sea squirt Aplidium albicans. Plitidepsin possesses broad-spectrum antitumor activity and can induce apoptosis by triggering mitochondrial cytochrome c release, initiating the Fas/DC95 and JNK pathways, and activating caspase 3. This drug also inhibits elongation factor 1-α, thereby interfering with protein synthesis and inducing G1 and G2 phase arrest, thus inhibiting tumor cell growth. Drug Indications: For the treatment of various cancers. Treatment of COVID-19: Mechanism of Action: Pritepulsin's antitumor activity stems from its ability to inhibit cell growth and induce apoptosis. Pritepulsin's primary intracellular target is eukaryotic elongation factor 1A2 (eEF1A2). It also inhibits the growth of MOLT-4 cells and induces their apoptosis by inhibiting VEGF secretion, blocking the VEGF/VEGFR-1 autocrine loop required for MOLT-4 cell growth. Furthermore, by inhibiting eEF1A, pritepulsin also exhibits antiviral activity against SARS-CoV-2. Its exact mechanism of action is not fully elucidated, and pritepulsin may have multiple modes of action.

C57H87N7O15

1110.34

1109.63

C, 61.66; H, 7.90; N, 8.83; O, 21.61

137219-37-5

9812534

White to off-white solid powder

1.24g/cm3

1.567

-0.59

4

15

15

79

2200

12

O=C1[C@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([C@@]([H])(C([H])([H])[H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])OC(C([H])([H])[C@@]([H])([C@@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N([H])C([C@]([H])([C@@]([H])(C([H])([H])[H])OC([C@]([H])(C([H])([H])C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])N(C([H])([H])[H])C([C@]2([H])C([H])([H])C([H])([H])C([H])([H])N21)=O)=O)N([H])C([C@@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N(C([H])([H])[H])C([C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C(C(C([H])([H])[H])=O)=O)=O)=O)=O)O[H])=O)=O)=O

UUSZLLQJYRSZIS-LXNNNBEUSA-N

InChI=1S/C57H87N7O15/c1-15-33(8)46-44(66)29-45(67)79-49(32(6)7)48(68)34(9)50(69)58-39(26-30(2)3)54(73)64-25-17-19-41(64)56(75)62(13)43(28-37-20-22-38(77-14)23-21-37)57(76)78-36(11)47(52(71)59-46)60-51(70)42(27-31(4)5)61(12)55(74)40-18-16-24-63(40)53(72)35(10)65/h20-23,30-34,36,39-44,46-47,49,66H,15-19,24-29H2,1-14H3,(H,58,69)(H,59,71)(H,60,70)/t33-,34-,36+,39-,40-,41-,42+,43-,44-,46+,47-,49-/m0/s1

(2S)-N-[(2R)-1-[[(3S,6S,8S,12S,13R,16S,17R,20S,23S)-13-[(2S)-butan-2-yl]-12-hydroxy-20-[(4-methoxyphenyl)methyl]-6,17,21-trimethyl-3-(2-methylpropyl)-2,5,7,10,15,19,22-heptaoxo-8-propan-2-yl-9,18-dioxa-1,4,14,21-tetrazabicyclo[21.3.0]hexacosan-16-yl]amino]-4-methyl-1-oxopentan-2-yl]-N-methyl-1-(2-oxopropanoyl)pyrrolidine-2-carboxamide

dehydrodemnin B; plitidepsin; Aplidine; aplidin; 137219-37-5; Dehydrodidemnin B; aplidine. US brand name: Aplidin.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO: ~100 mg/mL (~90.1 mM)

Solubility in Formulation 1: 2.5 mg/mL (2.25 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.25 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.)