mTORC1; mTORC2; Natural product; secondary metabolite

(+)-Usnic acid (1) is a common bioactive lichen-derived secondary metabolite with a characteristic dibenzofuran scaffold. It displayed low micromolar antiproliferative activity levels and, notably, induced autophagy in a panel of diverse breast cancer cell lines, suggesting the mechanistic (formerly "mammalian") target of rapamycin (mTOR) as a potential macromolecular target. The cellular autophagic markers were significantly upregulated due to the inhibition of mTOR downstream effectors. Additionally, 1 showed an optimal binding pose at the mTOR kinase pocket aided by multiple interactions to critical amino acids. Rationally designed benzylidene analogues of 1 displayed excellent fitting into a targeted deep hydrophobic pocket at the core of the kinase cleft, through stacking with the phenolic side chain of the Tyr2225 residue. Several potent analogues were generated, including 52, that exhibited potent (nM concentrations) antiproliferative, antimigratory, and anti-invasive activities against cells from multiple breast cancer clonal lines, without affecting the nontumorigenic MCF-10A mammary epithelial cells. Analogue 52 also exhibited potent mTOR inhibition and autophagy induction. Furthermore, 52 showed potent in vivo antitumor activity in two athymic nude mice breast cancer xenograft models. Collectively, usnic acid and analogues are potential lead mTOR inhibitors appropriate for future use to control breast malignancies.[1]
We loaded (+)-usnic acid into modified polyurethane and quantitatively assessed the capacity of (+)-usnic acid to control biofilm formation by either S. aureus or Pseudomonas aeruginosa under laminar flow conditions by using image analysis. (+)-Usnic acid-loaded polymers did not inhibit the initial attachment of S. aureus cells, but killing the attached cells resulted in the inhibition of biofilm. Interestingly, although P. aeruginosa biofilms did form on the surface of (+)-usnic acid-loaded polymer, the morphology of the biofilm was altered, possibly indicating that (+)-usnic acid interfered with signaling pathways.[2]

In Vivo Activity of (+)-usnic acid Analogue 52 against Breast Cancer in Nude Mice Xenograft Models [1]
The antitumor efficacy of 52 was assessed using mTOR-dysregulated orthotopic xenograft breast cancer models, generated by inoculating nude mice with MCF-7 or MDA-MB-231/GFP cells. Intraperitoneal administration of 52 at a dose regimen of 10 mg/kg, 3×/week, significantly attenuated tumor growth in both models by the end of the study (Figure 14). In the MDA-MB-231/GFP model, the mean tumor volume at the sacrifice time (34 days post-inoculation) in vehicle-treated control group animals was 1835 ± 251 mm3 (±SE). Meanwhile, the mean tumor volume in treated mice reached 692 ± 146 mm3, representing a nearly 62.3% tumor growth inhibition in the treatment group (Figure 14A). In the MCF-7 model, vehicle-treated control group mice showed mean tumor volumes of 1093 ± 185 mm3 by the end of the study (75 days postinoculation). On the other hand, mice receiving treatment with 52 harbored tumors with a mean volume of only 385 ± 96 mm3 (Figure 14), reflecting a 64.8% mean tumor growth inhibition. Collectively, these results clearly demonstrated the robust efficacy of 52 in attenuating MCF-7 and MDA-MB-231/GFP breast tumor growth, cells of both lines having dysregulated function convergent on mTOR-governed pathways. Moreover, the body weights of the animals were monitored for the duration of the study and results taken as a general indicator of toxicity. Analogue 52 did not induce any gross toxicity signs or significant reduction of the mean body weights of animals in treatment groups as compared to the vehicle-treated control groups (Figure S57, Supporting Information).

Loading of the antimicrobial agent in the polymer. [2]
The loading of (+)-usnic acid in PEUADED was performed by preparing an acetone solution containing either (+)-usnic acid (2% [wt/vol]) or PEUADED (5% [wt/vol]). (+)-Usnic acid-loaded PEUADED disks (treated polymer) were obtained by casting of the solution described above on Teflon plates, followed by solvent evaporation under a vacuum at 30°C. A high-affinity antibiotic-polymer interaction was established by combining polyurethane provided with basic tertiary amino groups in the side chain and an antimicrobial agent, (+)-usnic acid, displaying acidic groups. 1H-nuclear magnetic resonance analysis and acidic-alkaline titration of the amidation reaction efficacy revealed a content of 75% of amino groups present in the polymer side chain (data not shown).

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(+)-usnic acidrelease from the polymer. [2]
The kinetics of the release of antibiotic from polymer disks in water was determined by measuring the concentration of (+)-usnic acid in the water the disks were immersed in by spectrometry, looking at the absorbance at 270 nm every 24 h for 6 days (the planned experimental period). Because of the limited solubility of usnic acid in water, standard solutions were prepared in a solution of 95% water and 5% acetone. No leaching of (+)-usnic acid was detected over this period (data not shown).

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Determination of the MIC of (+)-usnic acid. [2]
The MICs of (+)-usnic acid for P. aeruginosa and S. aureus were determined by the microdilution method (26). Because of the limited solubility of (+)-usnic acid in water, acetone was used as the solvent mediator for the antimicrobial agent, after ruling out any intrinsic activity of acetone by plating viability. A 0.2% (wt/vol) solution of (+)-usnic acid was prepared and then diluted to the desired concentrations with LB broth for P. aeruginosa and with tryptic soy broth for S. aureus. An inoculum of 5 × 105 CFU/ml was used for both species. The MICs of (+)-usnic acid were 32 μg/ml for S. aureus 1945GFPuvr and 256 μg/ml for P. aeruginosa strain pMF230.

After 30-min and 24-h exposure without shear and in the presence of nutrients, S. aureus adhered to the surface of the (+)-usnic acid-treated polymer (Fig. 6) but did not grow to form a mature biofilm. Viability staining showed that the relative proportion of attached live cells decreased from approximately 80% after 30 min to less than 1% after 24 h.[2]
that (+)-usnic acid may be used in the development of antimicrobial catheters to resist biofilm formation by S. aureus and possibly other gram-positive organisms. The modified polymer successfully inhibited the formation of S. aureus biofilm for a period of up to 6 days under flow conditions with multiple challenges of high concentrations of bacteria.[2]

In Vivo Nude Mice Xenograft Models [1]
Female athymic nude mice (5–6 weeks old) were housed at the Animal Facility and maintained under clean conditions in sterile filter-top cages, at a temperature of 24 ± 2 °C, 50 ± 10% relative humidity, and 12:12 h artificial light–dark cycle. Mice received mouse chow and water ad libitum.
MDA-MB-231 green florescent protein-tagged (MDA-MB-231/GFP) cells were harvested, washed with PBS, and resuspended in RPMI-1640 medium. Cells (2 × 106 cells/25 μL) were injected into the mammary fat pad of each nude mouse, using a 29G hypodermic needle. Animals were observed daily for the growth of palpable tumors at the site of injection. Ten days postimplantation, tumors became visible with an approximate average volume of 50 mm3. Mice were randomized and allocated to control and treatment groups (5 mice/group). In the MCF-7 xenografted animals, a 17β-estradiol pellet delivering controlled release over 90 days was implanted beneath the skin of each nude mouse. After recovery from surgery (5 days), animals were injected with 2 × 106 cells suspended in 25 μL of serum-free media into the mammary fat pad of each nude mouse. It took approximately 20 days for generated tumors to reach the average volume of 50 mm3. Mice were randomized and allocated to control and treatment groups (5 mice/group). (+)-usnic acid analogue 52 was prepared as a stock solution in sterile DMSO (1 mg/20 μL), diluted with sterile PBS containing 0.1% Tween 80, and injected intraperitoneally at a dose regimen of 10 mg/kg body weight, three times per week, for the indicated times in each set of experiments. Animals in the control groups received the same volume of vehicle, following the same treatment protocol. Tumor dimensions were measured using a digital caliper. Tumor volume was calculated using the well-established formula: tumor volume (mm3) = [(length × width2)/2]. Animals were monitored daily for any signs of treatment- or vehicle-associated toxicity. Animals were sacrificed at the indicated times, unless they appeared to be moribund or a tumor showed signs of necrosis. At termination, tumors were excised from the connective tissues and snap-frozen for subsequent analysis.

Hepatotoxicity
Several cases of clinically apparent acute liver injury have been attributed to commercial dietary supplements that contain usnic acid. “LipoKinetix” was one such supplement advertised as a weight loss and body building supplement. Each tablet contained sodium usniate (100 mg), norephedrine (25 mg), diiodothyronine (100 mg), yohimbine (3 mg) and caffeine (100 mg). The product has been linked to multiple instances of acute liver injury. The time to onset was 2 to 12 weeks and the clinical presentation resembled acute viral hepatitis with onset of fatigue and nausea, followed by jaundice. The pattern of serum enzyme elevations was hepatocellular, with marked elevations in serum ALT and minimal increases in alkaline phosphatase levels. Liver biopsy demonstrated acute hepatocellular necrosis and inflammation. Immunoallergic features (fever, rash and eosinophilia) were not common and autoantibodies were usually not present. Recovery was rapid with stopping the dietary supplement, but some cases were severe and led to acute liver failure and either death or need for emergency liver transplantation. Instances of acute hepatitis have also been reported with other multi-ingredient dietary supplements that contain usnic acid, but much more rarely than with LipoKinetix which was withdrawn from distribution after a FDA warning letter. Rare instances of hepatotoxicity have also been reported with use of lichen based teas known as Kombucha tea, but whether these were due to usnic acid or another contaminant of the tea was not shown.
Likelihood score (usnic acid): B (highly likely cause of clinically apparent liver injury).
Likelihood score (Kombucha tea): C (probable cause of clinically apparent liver injury).

[1]. Usnic Acid Benzylidene Analogues as Potent Mechanistic Target of Rapamycin Inhibitors for the Control of Breast Malignancies. J Nat Prod. 2017 Apr 28;80(4):932-952.

[2]. Usnic acid, a natural antimicrobial agent able to inhibit bacterial biofilm formation on polymer surfaces. Antimicrob Agents Chemother. 2004;48(11):4360-4365

(-)-usnic acid is the (-)-enantiomer of usnic acid. It has a role as an EC 1.13.11.27 (4-hydroxyphenylpyruvate dioxygenase) inhibitor. It is a conjugate acid of a (-)-usnic acid(2-). It is an enantiomer of a (+)-usnic acid.
Usnic acid is a furandione found uniquely in lichen that is used widely in cosmetics, deodorants, toothpaste and medicinal creams as well as some herbal products. Taken orally, usnic acid can be toxic and has been linked to instances of clinically apparent, acute liver injury.
(-)-Usnic acid has been reported in Ramalina hierrensis, Stereocaulon alpinum, and other organisms with data available.

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(+)-Usnic acid (1) is a common bioactive lichen-derived secondary metabolite with a characteristic dibenzofuran scaffold. It displayed low micromolar antiproliferative activity levels and, notably, induced autophagy in a panel of diverse breast cancer cell lines, suggesting the mechanistic (formerly "mammalian") target of rapamycin (mTOR) as a potential macromolecular target. The cellular autophagic markers were significantly upregulated due to the inhibition of mTOR downstream effectors. Additionally, 1 showed an optimal binding pose at the mTOR kinase pocket aided by multiple interactions to critical amino acids. Rationally designed benzylidene analogues of 1 displayed excellent fitting into a targeted deep hydrophobic pocket at the core of the kinase cleft, through stacking with the phenolic side chain of the Tyr2225 residue. Several potent analogues were generated, including 52, that exhibited potent (nM concentrations) antiproliferative, antimigratory, and anti-invasive activities against cells from multiple breast cancer clonal lines, without affecting the nontumorigenic MCF-10A mammary epithelial cells. Analogue 52 also exhibited potent mTOR inhibition and autophagy induction. Furthermore, 52 showed potent in vivo antitumor activity in two athymic nude mice breast cancer xenograft models. Collectively, usnic acid and analogues are potential lead mTOR inhibitors appropriate for future use to control breast malignancies.[1]

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In modern medicine, artificial devices are used for repair or replacement of damaged parts of the body, delivery of drugs, and monitoring the status of critically ill patients. However, artificial surfaces are often susceptible to colonization by bacteria and fungi. Once microorganisms have adhered to the surface, they can form biofilms, resulting in highly resistant local or systemic infections. At this time, the evidence suggests that (+)-usnic acid, a secondary lichen metabolite, possesses antimicrobial activity against a number of planktonic gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. Since lichens are surface-attached communities that produce antibiotics, including usnic acid, to protect themselves from colonization by other bacteria, we hypothesized that the mode of action of usnic acid may be utilized in the control of medical biofilms. We loaded (+)-usnic acid into modified polyurethane and quantitatively assessed the capacity of (+)-usnic acid to control biofilm formation by either S. aureus or Pseudomonas aeruginosa under laminar flow conditions by using image analysis. (+)-Usnic acid-loaded polymers did not inhibit the initial attachment of S. aureus cells, but killing the attached cells resulted in the inhibition of biofilm. Interestingly, although P. aeruginosa biofilms did form on the surface of (+)-usnic acid-loaded polymer, the morphology of the biofilm was altered, possibly indicating that (+)-usnic acid interfered with signaling pathways.[2]

C18H16O7

344.32

344.089

C, 62.79; H, 4.68; O, 32.53

7562-61-0

Usnic acid;125-46-2

442614

Light yellow to yellow solid

1.5±0.1 g/cm3

638.2±55.0 °C at 760 mmHg

201-203 °C(lit.)

236.0±25.0 °C

0.0±2.0 mmHg at 25°C

1.679

2.3

3

7

2

25

734

1

O1C2=C(C(C([H])([H])[H])=O)C(=C(C([H])([H])[H])C(=C2[C@]2(C([H])([H])[H])C(C(C(C([H])([H])[H])=O)=C(C([H])=C12)O[H])=O)O[H])O[H]

WEYVVCKOOFYHRW-GOSISDBHSA-N

InChI=1S/C18H16O7/c1-6-14(22)12(8(3)20)16-13(15(6)23)18(4)10(25-16)5-9(21)11(7(2)19)17(18)24/h5,21-23H,1-4H3/t18-/m1/s1

(9bS)-2,6-diacetyl-3,7,9-trihydroxy-8,9b-dimethyldibenzofuran-1-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: ~4 mg/mL warmed (~11.6 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 0.63 mg/mL (1.83 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 6.3 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.)