Human peptide deformylase (HsPDF) with IC50 = 0.043 μM (as determined in the PDF assay, Table 3). [1]

In different human tumor cell lines, actinonin inhibits cell proliferation. The compounds had IC50 values of 4, 6.9, 12.8, 16.6, 27.4, 15.7, and 49.3 μM for Raji cells, MDA-MB-468 cells, PC3 cells, SK-LC-19 cells, Hela cells, HT-1080 cells, and AL67 cells, in that order[1]. Actinonin's primary target, HsPDF, is inhibited in the mitochondria by this protein, which causes tumor cells to die. In a dose- and time-dependent manner, actinonin administration of cells resulted in ATP depletion and tumor-specific mitochondrial membrane depolarization[1]. All three types (Zn-, Ni-, and Fe-) of peptide deformylases from S. aureus and E. coli bacteria are strongly inhibited by actinonin. The IC50 values for actinonin under test conditions are, respectively, 90, 3, 0.8, and 11 nM for Zn-PDF (E. coli), Ni-PDF (E. coli), Fe-PDF (E. coli), and Ni-PDF (S. aureus[2]. Actinonin exhibits antibacterial activity against Gram-positive bacteria, such as S. aureus (MIC value: 8–16 μg/mL), Streptococcus pyogenes (MIC value: 8 μg/mL), and Streptococcus epidermidis (MIC value: 2-4 μg/mL). Actinonin exhibits antibacterial activity against fastidious Gram-negative bacteria, including H. influenzae (MIC value of 1-2 μg/mL), Moraxella catarrhalis (MIC value of 0.5 μg/mL), and Neisseria gonorrhoeae (MIC value of 1-4 μg/mL). Actinonin exhibits high activity against the efflux pump mutants of H. influenzae acr (MIC value of 0.13 μg/mL) and E. coli acr (MIC value of 0.25 μg/mL)[2].

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Actinonin exhibited antiproliferative activity against 16 out of 17 human tumor cell lines representing 9 different lineages, with IC50 values ranging from 4.0 μM to 60.0 μM (e.g., Daudi 5.2±2.2 μM, HL60 9.3±2.9 μM, Raji 4.0±1.0 μM, RL 5.9±0.6 μM, MDA-MB-468 6.9±0.3 μM, SK-BR-3 14.0±5.1 μM, CWR22 32.3±2.3 μM, TSU-PRI 60.0±7.1 μM, DU145 28.5±3.7 μM, PC3 12.8±3.5 μM, SK-LC-8 20.6±0.2 μM, SK-LC-16 16.6±0.4 μM, A2780 12.5±0.0 μM, HeLa 27.4±4.6 μM, HT-1080 15.7±0.2 μM). HEK293 renal cells were resistant (>250 μM). Normal cell lines (WI-38, NIH-3T3, hPBMCs) were resistant with IC50 >500 μM, while ras-transformed mouse fibroblast AL67 had IC50 49.3±29.9 μM. Assays used were thymidine incorporation, XTT metabolism, or trypan blue exclusion. [1]
- Thirty-three analogs of actinonin were synthesized; all compounds with potent HsPDF inhibition (IC50 <0.1 μM) also showed potent antiproliferative activity (IC50 <50 μM) on at least one cell line (CWR22Rv1, TSU-PRI, Daudi, HL60). Compounds with no measurable HsPDF inhibition exhibited no cell growth inhibition. This correlation confirms target specificity. (Table 3 and Figure 4) [1]
- Small interfering RNA (siRNA) targeting HsPDF (siRNA HsPDF 581-601 and 659-679) significantly reduced HsPDF mRNA and protein expression in HeLa cells. At doses as low as 10 nM, these siRNAs significantly inhibited cell proliferation as measured by tritiated thymidine incorporation, with maximal effect observed 48 hours after transfection. Control siRNAs had no effect. [1]
- Actinonin treatment of RL human B lymphoma cells caused a time- and dose-dependent depolarization of the mitochondrial membrane, measured by JC-1 dye (red/green fluorescence ratio). At 100 μg/ml, depolarization equaled that of CCCP (positive control). Depolarization was reversible upon actinonin removal (recovery after ~8 hours). Similar depolarization was seen in HeLa and HL60 cells, but not in normal peripheral blood lymphocytes at the same doses. [1]
- Actinonin depleted ATP levels in Daudi cells in a time- and dose-dependent manner. At 20 μg/ml, ATP depletion was 56.0% at 12 hours, 67.3% at 24 hours, and 95.8% at 36 hours. [1]
- Actinonin induced a low level of apoptosis (approximately 7% annexin V-positive, propidium iodide-negative) in Daudi cells, as determined by annexin V staining. [1]

Mice have been given actinonin as an antibiotic at levels up to 400 mg/kg without risk. Actinonin exhibits anticancer action in vitro, however it does not seem to be very harmful to normal tissues. It is noteworthy that actinonin, administered i.p. or orally, significantly reduces tumor growth in a CWR22 human prostate tumor xenograft model in naked mice. The animals exhibit no symptoms of toxicity while receiving treatment[1].

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In athymic nude mice bearing CWR22 human prostate tumor xenografts, actinonin administered i.p. at 250 mg/kg twice daily for 2 weeks (excluding weekends) significantly inhibited tumor growth (P<0.005). Oral administration at 500 mg/kg twice daily was similarly effective, indicating oral bioavailability. No signs of clinical toxicity were observed in animals during treatment. [1]
- In athymic nude mice bearing A549 human non-small cell lung cancer xenografts, actinonin administered i.p. at 150 mg/kg once daily for 2 weeks (excluding weekends) significantly inhibited tumor growth (P<0.01). Similar antitumor activity was observed in PC3 human prostate cancer xenografts. [1]

Peptide deformylase (PDF) activity was measured using a formate dehydrogenase-coupled spectrophotometric assay. The reaction mixture contained 50 mM HEPES (pH 7.4), 10 mM NaCl, 0.2 mg/ml BSA, 2.4 mM NAD+, 1 U formate dehydrogenase, and 0–32 mM N-formylated peptide substrates (formyl-Met-Ala-Ser, formyl-Met-Ala-His-Ala, or formyl-Met-Thr-Met-His). The reaction was initiated by adding 20–100 μg of HsPDF enzyme (cobalt-substituted N-terminal truncation mutant with C-terminal 6x-histidine tag). The rate of NADH production was measured by monitoring the increase in absorbance at 340 nm. Kinetic parameters (Vmax, Km, Kcat, Kcat/Km) for each substrate were determined. Actinonin and its analogs were tested for inhibition of HsPDF activity, and IC50 values were calculated as the concentration inhibiting 50% of enzymatic activity compared to controls. [1]

Cell proliferation was assessed using tritiated thymidine incorporation assay. Cells (10,000 per well) were plated in 96-well plates with or without actinonin (serial dilutions). After 1–5 days of incubation, 50 μl of 10 μCi/ml tritiated thymidine was added to each well and incubated for 5 hours. Plates were frozen overnight, cells harvested onto filtermats, and counted in a liquid scintillation counter. [1]
- XTT assay: Cells in log-phase growth were plated in 96-well plates. Actinonin was added at various concentrations. When control wells reached confluence, 50 μl of XTT (1 mg/ml in serum-free medium) with phenazine methosulphate was added to each well, incubated for 2–4 hours at 37°C, and absorbance read at dual wavelength 450/630 nm. [1]
- Mitochondrial membrane depolarization was measured using JC-1 dye. Cells were incubated with varying concentrations of actinonin (0–100 μg/ml) at 1×10^6 cells/ml. CCCP (100 nM for 10 minutes) was used as positive control for maximal dissipation. Cells were incubated with JC-1 for 5 minutes at 37°C, washed three times in PBS, and resuspended in media with actinonin. Flow cytometry was performed, measuring green fluorescence (FL-1, 525 nm) for mitochondrial mass and red fluorescence (FL-2, 590 nm) for membrane potential. Mitochondrial membrane potential was expressed as red/green ratio. [1]
- ATP levels were measured using an ATP assay kit. Daudi cells were treated with actinonin (5, 10, 20 μg/ml) for 1–36 hours. At each time point, cells were counted and resuspended to 100,000 cells/ml. ATP was quantitated in 1,000 cells using a luminometer with an ATP standard curve. Results expressed as grams ATP per 1,000 cells. [1]
- Apoptosis was assessed by annexin V staining. Daudi cells were treated with actinonin (5, 10, 20 μg/ml) for 1–96 hours. At each time point, 1×10^6 cells were stained with annexin V and propidium iodide, and analyzed by flow cytometry for percentage of annexin V-positive, propidium iodide-negative cells. [1]
- siRNA transfection: HeLa cells were transfected with siRNA duplexes targeting HsPDF (positions 581-601 or 659-679 relative to start codon) using Oligofectamine. Control siRNA (nonspecific duplex pool) was used. Cell proliferation was determined by tritiated thymidine incorporation on days 1, 2, and 3 after transfection. HsPDF protein expression was analyzed in HeLa cells stably expressing full-length HsPDF-YFP fusion protein by flow cytometry (mean peak fluorescence) 24 and 48 hours after transfection. [1]

For xenograft models, 8- to 10-week-old athymic NCr-nu mice were inoculated subcutaneously in the flank with minced CWR22 (prostate), A549 (lung), or PC3 (prostate) tumor cells mixed with Matrigel. Once tumors became palpable, mice were randomized into control and treatment groups (3-5 animals per group). Actinonin was administered intraperitoneally (i.p.) or orally daily for 2 weeks except weekends. Doses: CWR22 model – 250 mg/kg i.p. twice daily or 500 mg/kg orally twice daily; A549 model – 150 mg/kg i.p. once daily. Control group received vehicle alone. Tumors were measured every 3-5 days with calipers. Tumor volumes calculated by formula 4/3 × π × [(larger diameter + smaller diameter)/4]^3. [1]
- A preliminary toxicity study in mice was conducted to determine maximal tolerated dose, with weight loss as the limiting criterion. [1]

Actinonin is orally bioavailable, as evidenced by similar antitumor efficacy in the CWR22 xenograft model when administered orally (500 mg/kg) compared to intraperitoneal (250 mg/kg) administration. [1]

Actinonin was well tolerated in mice at doses up to 400 mg/kg as an antibiotic. In the xenograft studies, animals showed no signs of clinical toxicity during treatment with i.p. (150 or 250 mg/kg) or oral (500 mg/kg) regimens. [1]
- In vitro, normal cell lines (WI-38 human fibroblasts, NIH-3T3 mouse fibroblasts, normal human PBMCs) were resistant to actinonin with IC50 >500 μM, more than 5-fold higher than the IC50 for tumorigenic hematopoietic cell lines. However, actinonin decreased colony formation of human bone marrow cells (myelosuppressive activity) at doses >5-fold the IC50 for tumor cells. [1]
- Actinonin did not cause mitochondrial membrane depolarization in normal peripheral blood lymphocytes at doses up to 100 μg/ml, whereas tumor cells (RL, HeLa, HL60) showed significant depolarization. [1]

[1]. Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics. J Clin Invest. 2004 Oct;114(8):1107-16.

[2]. Actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor. Biochemistry. 2000 Feb 15;39(6):1256-62.

[3]. Hydroxamate inhibitors of human gelatinase B (92 kDa). Bioorganic & Medicinal Chemistry Letters 5(4), 349-352 (1995).

[4]. Modes of action of microbially-produced phytotoxins. Toxins (Basel). 2011 Aug;3(8):1038-64.

[5]. Human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors. Biochem J. 2004 Mar 1;378(Pt 2):383-9.

There have been reports that streptomyces contains actinomycin, and relevant data is available for reference.

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Mechanism of action: Actinonin inhibits HsPDF, a human mitochondrial enzyme that removes formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins. This inhibition leads to accumulation of unprocessed proteins, disruption of electron transport chain complexes, reduction of proton gradient, mitochondrial membrane depolarization, and ATP depletion. Additionally, accumulation of unfolded proteins may induce a mitochondrial-specific stress response, upregulating transcription factor CHOP, which is implicated in programmed cell death. The reversible nature of membrane depolarization upon actinonin withdrawal suggests indirect effects on the electron transport chain. [1]
- Selectivity: Tumor cells appear more sensitive to actinonin-induced mitochondrial insult than normal cells. The reasons for tumor selectivity are under investigation, but may relate to differences in tumor mitochondria. [1]
- Anticancer activity: Actinonin inhibited growth of human prostate cancer (CWR22, PC3) and lung cancer (A549) xenografts in mice. An analog (SKI-AC-111111) showed approximately 3-fold more potent in vivo antitumor activity in a prostate tumor xenograft model compared to actinonin (preliminary results). [1]

C19H35N3O5

385.4983

385.257

13434-13-4

443600

White to off-white solid powder

1.1±0.1 g/cm3

137-139ºC

1.513

0.59

4

5

11

27

498

3

CCCCC[C@H](CC(=O)NO)C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@H]1CO

XJLATMLVMSFZBN-VYDXJSESSA-N

InChI=1S/C19H35N3O5/c1-4-5-6-8-14(11-16(24)21-27)18(25)20-17(13(2)3)19(26)22-10-7-9-15(22)12-23/h13-15,17,23,27H,4-12H2,1-3H3,(H,20,25)(H,21,24)/t14-,15+,17+/m1/s1

(2R)-N'-hydroxy-N-[(2S)-1-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]-2-pentylbutanediamide

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 : ~50 mg/mL (~129.70 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (12.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 50.0 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: 5 mg/mL (12.97 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (12.97 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 50.0 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.)