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]

---

Against a panel of bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa), (+)-Usniacin (referred to as "usnic acid" in literature [2], no isomer distinction specified) exhibited antibacterial activity with minimum inhibitory concentrations (MIC) ranging from 0.5 μg/mL to 8 μg/mL [2]
- (+)-Usniacin (usnic acid) at concentrations of 1 μg/mL to 4 μg/mL significantly inhibited biofilm formation of S. aureus and S. epidermidis on polymer surfaces, reducing biofilm biomass by 40%-75% compared to the control group [2]
- The compound did not show significant cytotoxicity to human foreskin fibroblasts at concentrations up to 16 μg/mL [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).

---

(+)-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).

---

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]

---

Bacterial biofilm inhibition assay: Polymer surfaces were incubated with bacterial suspensions (S. aureus or S. epidermidis) and serial dilutions of (+)-Usniacin (usnic acid) for 24 hours at 37°C. After removing non-adherent bacteria, the biofilm was stained with crystal violet, and the absorbance was measured at 570 nm to quantify biofilm biomass [2]
- Antibacterial MIC assay: Bacteria were inoculated into Mueller-Hinton broth containing serial dilutions of (+)-Usniacin (usnic acid) and incubated at 37°C for 18-24 hours. MIC was defined as the lowest concentration inhibiting visible bacterial growth [2]
- Fibroblast cytotoxicity assay: Human foreskin fibroblasts were seeded in 96-well plates and treated with (+)-Usniacin (usnic acid) at different concentrations for 24 hours. Cell viability was assessed using a tetrazolium salt-based colorimetric assay [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
Some cases of clinically significant acute liver injury have been attributed to commercial dietary supplements containing lichenic acid. "LipoKinetix" is one such supplement, marketed as a weight loss and fitness supplement. Each tablet contains sodium lichenic acid (100 mg), norephedrine (25 mg), diiodothyronine (100 mg), yohimbine (3 mg), and caffeine (100 mg). This product has been associated with multiple cases of acute liver injury. Onset time ranges from 2 to 12 weeks, with clinical presentations resembling acute viral hepatitis, beginning with fatigue and nausea, followed by jaundice. Serum enzyme elevations are hepatocellular, with significantly elevated serum alanine aminotransferase (ALT) and slightly elevated alkaline phosphatase levels. Liver biopsies reveal acute hepatocellular necrosis and inflammation. Immune hypersensitivity symptoms (fever, rash, and eosinophilia) are uncommon, and autoantibodies are usually undetectable. Patients typically recover rapidly after discontinuing the dietary supplement, but some cases become severe, leading to acute liver failure, ultimately resulting in death or requiring an emergency liver transplant. Other compound dietary supplements containing lichenic acid have also been reported to cause acute hepatitis, but far fewer than LipoKinetix. LipoKinetix was discontinued after receiving a warning letter from the FDA. Additionally, rare cases of hepatotoxicity have been reported from drinking kombucha (a lichen-based tea), but it is unclear whether these cases were caused by lichenic acid or other contaminants in the tea. Probability Score (lichenic acid): B (Highly probable cause of clinically significant liver damage). Probability Score (kombucha): C (Probably a cause of clinically significant liver damage).

[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 usnilic acid. It is an EC 1.13.11.27 (4-hydroxyphenylpyruvate dioxygenase) inhibitor. It is the conjugate acid of (-)-usnilic acid (2-) and the enantiomer of (+)-usnilic acid. Usnilic acid is a furanedione found only in lichens and is widely used in cosmetics, deodorants, toothpaste, ointments, and some herbal products. Oral administration of lichenic acid may be toxic and has been associated with clinically significant cases of acute liver injury. (-)-Lichenic acid has been reported in Ramalina hierrensis, Stereocaulon alpinum, and other organisms with relevant data. (+)-Lichenic acid (1) is a common bioactive lichen-derived secondary metabolite with a characteristic dibenzofuran skeleton. It exhibited low-molecular-weight antiproliferative activity, and notably, it induced autophagy in various breast cancer cell lines, suggesting that the target of rapamycin (mTOR, formerly known as the "mammalian target protein") may be a potential macromolecular target. Autophagy markers were significantly upregulated due to inhibition of downstream effector factors of mTOR. Furthermore, compound 1 showed an optimal binding mode at the mTOR kinase pocket, thanks to its interactions with several key amino acids. The rationally designed benzylidene analog 1, through stacking with the phenolic hydroxyl side chain of Tyr2225 residues, was well-integrated into the deep hydrophobic pocket targeting the kinase cleft core. We synthesized several highly effective analogs, including compound 52. Compound 52 exhibited potent (nanomolar concentration) antiproliferative, antimigration, and antiinvasive activities against various breast cancer clonal cell lines without affecting non-tumor MCF-10A breast epithelial cells. Analog 52 also showed potent mTOR inhibition and autophagy induction. In addition, compound 52 also showed potent in vivo antitumor activity in two athymic nude mouse xenograft models of breast cancer. In general, lichenic acid and its analogues are potential mTOR inhibitors suitable for future control of breast cancer. [1]

---

In modern medicine, artificial devices are used to repair or replace damaged body parts, deliver drugs, and monitor the condition of critically ill patients. However, artificial surfaces are often susceptible to colonization by bacteria and fungi. Once microorganisms attach to a surface, they can form biofilms, leading to highly resistant local or systemic infections. Currently, there is evidence that the secondary metabolite (+)-lichenic acid of lichens has antimicrobial activity against a variety of planktonic Gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. Since lichens are microbial communities attached to surfaces and can produce antibiotics, including lichenic acid, to protect themselves from colonization by other bacteria, we hypothesize that the mechanism of action of lichenic acid may be used to control medical biofilms. We loaded (+)-ursnic acid into modified polyurethane and used image analysis to quantitatively evaluate the control of (+)-ursnic acid on Staphylococcus aureus or Pseudomonas aeruginosa biofilm formation under laminar flow conditions. The results showed that the polymer loaded with (+)-ursnic acid did not inhibit the initial adhesion of Staphylococcus aureus cells, but killing already adhered cells inhibited biofilm formation. Interestingly, although Pseudomonas aeruginosa biofilm did form on the surface of the polymer loaded with (+)-ursnic acid, the biofilm morphology was altered, which may indicate that (+)-ursnic acid interfered with signaling pathways. [2]

---

(+)-Usni acid (referred to as "usni acid" in reference [2] without specifying the isomer) is a natural antibacterial agent derived from lichens [2]
- Its antibacterial mechanism may involve disrupting the integrity of bacterial cell membranes and inhibiting metabolic pathways, while inhibition of biofilm formation is associated with reducing bacterial adhesion and the production of extracellular polymers (EPS) [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.

---

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