What is YK11?

(17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester, more commonly known as YK11, is a potent non-steroidal selective androgen receptor modulator (SARM) that shows potential in improving anabolic activity in muscles and bones while decreasing overall fat mass. Initial research has found that YK11 increases levels of follistatin, resulting in the inhibition of myostatin. This mechanism of action is important to mention considering that high levels of myostatin have been shown to suppress excessive muscle growth. Additionally, YK11 is able to activate pathways that respond in a manner similar to testosterone and DHT. For example, administration of the SARM activates protein kinase B and ultimately results in enhanced osteoblast activity and increased bone density.

 

Main Research Findings

1) YK11 prevents cases of severe bacterial sepsis due to its ability to inhibit the actions of myostatin.

2) YK11 is capable of acting as a partial agonist to androgen receptors.

 

Selected Data

1) The study conducted by researchers Lee et. Al utilized 8-week-old male BALB/c mice obtained from Orient Bio as their test subjects. The experimental groups of mice were compared to a wild-type (WT) control group. All test subjects were administered a daily treatment of 350 or 700 mg/kg of YK11 over a 10 day experimental period. After 10 days of treatment researchers administered the E. coli strain, RS218, to some of the WT mice in order to induce sepsis. Body weight, food intake, and thigh muscle weight were continuously monitored throughout experimentation [1].

Figure 1: Visual representation of experimental procedures

In order to analyze the survival rate of the test subjects, the mice were administered YK11 first followed by varying doses of different E. coli strains as well as Carbapenem-resistant Acinetobacter baumannii (CRAB), in order to induce sepsis. The researchers observed a 72 hour survival rate after administration of the septic compounds. The lungs, liver, and kidneys were all removed from the mice for different analyses to take place. The lungs were extracted and fixed in a 4% paraformaldehyde (PFA) solution prior to dehydration and proper preparation of the samples. After the necessary organs were extracted they were placed in 1 mL sterile PBs solution and then homogenized on ice. All homogenates were further diluted with PBS and samples were prepared accordingly so bacterial cultures could be accurately assessed [1].

The research team was also able to identify the presence of various proteins and endotoxins in the samples gathered from the test subjects. Follistatin protein levels and pro-inflammatory cytokines in tissue lysates from total muscle extracts were recorded through the use of an ELISA assay kit. The protein samples obtained were separated by SDS-PAGE and transferred to fluoride membranes blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T). After separation the samples were incubated with the respective antibodies, washed with TBS-T, and incubated again with the secondary antibody. After preparing the samples, researchers recorded any changes through the use of Western blot analysis and an enhanced chemiluminescence kit. Additionally, endotoxin levels in the mice were recorded using a limulus amebocyte lysate (LAL) chromogenic end-point assay. After the mouse sera were diluted with an endotoxin-free PBS the assay was completed in order to reveal the relative amounts of endotoxin in each sample [1].

The researchers noted that all of the experiments were conducted at least three times in order to ensure consistent results. Tukey’s multiple comparison test and the Student’s t-tests were used to compare the experimental and control groups, while the survival rates were analyzed by the Kaplan-Meier curves and completion of a log-rank test.

2) The research team of Kanno et. Al began their experimentation regarding YK11’s agonistic properties by preparing a 100 mL two-necked round-bottomed flask. Added to the flask was a magnetic stirring bar, 6.5 mg, 0.025 mmol of (CH3CN2)PdCl2, 108.1 mg, 1.0 mmol of p-benzoquinone, and 7 mL of MeOH. The flask was then fitted with a rubber septum and three-way stopcock connected to a CO-filled balloon. A 170.3 mg/ 0.5 mmol solution of substrate 1 in 35 mL of MeOH was added dropwisely to the stirred mixture. After 70 hours of stirring the mixture was further diluted with 70 mL of CH2Cl2, washed with 50 mL of 5% aq NaOH, dried over MgSO4, and concentrated under reduced pressure. The product of the process was purified through the use of column chromatography while the resulting fraction was eluted with hexane/ethyl acetate. The purification process yielded YK11 (93 mg, 43%) as a mixture of inseparable diastereomers [2].

From there, various tests were performed in order to assess how YK11 acts an agonist to androgen receptors (AR). First, cell culturing of the human embryonic kidney cell in, HEK293, and the human breast cancer cell line, MDA-MB 453 expressing wild-type AR, took place. Culturing took place in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS) and penicillin-streptomycin in a humidified climate containing 5% CO2. The HEK293 cells were transfected with pcDNA5/TO-AR; the next day the medium containing 100 mcg/mL of hygromycin was replaced. After 2 weeks the researchers collected and sub-cultured all single colonies. All ARs that expressed the HEK293 cells were marked as 293AR cells [2].

Following the cell culture, the researchers conducted a luciferase reporter analysis. Cells were seeded in 48-well plates and transfected with different expression plasmids. The internal standards of the study were the ARE-luciferase reporter plasmid and a Renilla pGL4.74 [hRluc/TK]; this was determined through the reverse-transfection method using a transfection reagent. The cells were incubated overnight in phenol DMEM containing 5% charcoal-stripped FBS, followed by 24 hours of treatment with various AR ligands. Luciferase activity was then measured through the use of Dual-Luciferase Reporter Assay System and a Turner Designs Luminometer.

The next step of the study was to complete mammalian two-hybrid assays. Similar to the luciferase assay, the cells were seeded in 48 well plates and transfected. However, these cells were specifically transfected with the expression plasmids encoding the GAL4 and VP16 fusion proteins, the pG5-Luc reporter plasmid, and pGL4.74. Transfection occurred using the reverse transfection method using the GeneJuice transfection reagent. The cells were incubated overnight in phenol red[free DMEM containing %5 csBS, followed by AR ligand treatment for 24 hours. Additionally, the GeneJuice transfection agent was used in combination with 500 ng of Yellow fluorescent protein (YFP)-tagged AR in the transfection process of HEK293 cells. This process was performed on 4-well chambered coverglasses, followed by overnight incubation and a 6 hour treatment with either YK11 or DHT. A Zeiss LSM 510 confocal laser scanning microscope was used in order to observe the localization of YFP-tagged proteins [2].

The researchers then completed a real time reverse transcription (RT)-PCR in order to detect the genetic makeup of the cell cultures included in the study. The cells were first seeded in 6-well plates in a medium of phenol red-free DMEM containing 5% csFBS. The cell plates were left overnight and treated the next day with either DHT of YK11, for 24 hours. Following treatment, total RNA was isolated through the use of the TaKaRa RNAiso Reagent and digested with DNase1. cDNA was then synthesized with the Rever Tra Ace qPCR RT Kit, while the RT-PCR was performed with the SYBR Green Real-time PCR Master Mix-plus, as well as PCR primers composed of different oligonucleotide sequences.

The final test conducted by the research team was a Western blot analysis. Sodium dodecyl sulfate (SDS) was used to extract the total protein, which was further supplemented with a potent protease inhibitor cocktail. The protein concentration was then measured using a 2-D Quant kit obtained from GE Healthcare in England, while protein samples were separated through the use of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was then performed using rabbit anti-AR antibody as the primary antibody, and horseradish peroxidase-conjugated anti-rabbit immunoglobulin as the secondary antibody. Researchers were able to effectively observe and record the protein bands with the assistance of the Immobilon Western Detection Reagent [2].

 

Discussion

1) The purpose of this study was to examine the relationship between muscle atrophy and its relation to myostatin and how these variables change in response to bacterial sepsis. YK11 was administered at the same time for 10 days and on the 10th day, sepsis was induced via intraperitoneal injection of E. coli or CRAB. Results of the study reported that YK11 decreased muscle atrophy in a dose-dependent manner, meaning the higher the dose of YK11 administered, the more effective the compound was. While total body weight increased the ratio of fats as a percentage of total body weight decreased in the mice treated with YK11. These findings are consistent with prior research that states myostatin inhibition leads to decreased fat mass and increased muscle mass. YK11 was also shown to recover muscle mass that was lost in the back and thighs of the animals as a result of the K1 E. coli strain. Staining of the cell samples revealed reduced atrophy in muscle cells as well. The SARM was also effective against the resistant bacteria strand, CRAB; the results reported reverted muscle mass loss and increased total body. This indicates that YK11 is capable of treating muscle atrophy triggered by standard and resistant gram-negative bacteria strains [1].

Figure 2: Changes in body weight, muscle weight, and fat weight in response to different experimental treatments.

In addition to the effects of the SARM on muscle atrophy, the research team sought out to observe how YK11 responds to the pathophysiology of sepsis. After treatment with YK11 and an injection of E. coli or CARB, the survival rate of the mice was estimated to be 20% for the 350 mg/kg dose of YK11 and 40% for the 700 mg/kg dose. Serum endotoxin levels were also shown to be significantly reduced in the YK11 treated animals, as well as the levels of various inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-10. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) were reduced after administration of YK11; the researcher thought it was important to include these results considering that plasma levels of ALT, AST, and BUN act as biomarkers for organ damage. Treatment with YK11 was also successful in promoting the clearance of bacteria out of the organs of septic mice. These results indicate that YK11 prevents occurrences of septic shock induced by gram-negative bacteria by regulating levels of inflammatory cytokines [1].

Figure 3: Visual representation of changes observed in A) survival rate, B) endotoxin levels, C) inflammatory cytokines, and D) biomarkers for muscle damage.

When examining the mechanism through which YK11 prevents cases of septic shock, the researchers focused on bacterial LPS-induced NF-KB activation and TGF-beta signaling. Results revealed that in the mice injected with the K1 strain of E. coli, YK11 was able to successfully inhibit myostatin and improve muscle protein metabolism. These inhibitory effects were also seen in correlation with the expression of follistatin; the same animal model demonstrated the natural inhibitory effect follistatin elicits on myostatin. The researchers thought it was interesting to note that the E. coli K1 group experienced diminished levels of follistatin, however, these levels were recovered in the experimental groups treated with YK11 [1].

Figure 4: Follistatin levels in response to different treatments

In addition to myostatin, levels of myogenin, and MyoD were increased by induction of the E. coli K1 strain of bacteria, however, these proteins were repressed following oral administration of YK11. Furthermore, several compounds belonging to the TGF-beta category are capable of signaling through pathways similar to myostatin. However, this typically leads to altered phosphorylation of FOXO3a and Sma2 transcription factors downstream; both of these factors were reduced when the mice were treated with YK11. THe research team of Lee et. Al were able to conclude that administration of YK11 can alleviate bacteria-induced sepsis by inhibiting the activity of myostatin in order to combat muscle atrophy [1].

Figure 5: Variations in the expression of myostatin, myogenin, and MyoD as well as different transcription factors

2) As it was outlined in the selected data portion, the research team of Kanno et. Al used the AR-overexpressing HEK193 cell line, named AR293, in order to determine the effects of YK11 on AR expressions. AR expression was observed in 293AR cells, but not the original HEK293 parent cell line. That being said, the 293AR cell line was treated with DHT and YK11 due to the ability of these compounds to activate the transcription process of the ARE-luciferase reporter gene. Results of the study reported that ARE-luciferase reporter activity was elicited in the sub-micromolar range by both DHT and YK11. However, it is important to note that the maximal activity of YK11 only reached approximately 10-20% of the maximal activity induced by DHT. However, when examining the effects of YK11 in the presence of DHT, YK11 was shown to repress DHT-induced reported activity at concentrations of 5 and 10 uM. Since DHT-induced activity was blocked by the expression of Yk11, the researchers were able to conclude that YK11 is capable of acting as a partial agonist of the androgen receptors [2].

Figure 6: Effects of YK11 versus DHT on total ARE-luciferase activity

Furthermore, the research team examined how treatment with YK11 affects the expression of AR-target genes in 293AR cells. Following 24 hour treatment with 0.1 uM concentration of DHT of doses ranging from 0.1 to 10 uM of YK11, the RT-PCR found that FKBP51 mRNA expression was found to be significantly enhanced in all cases. It was revealed that the improvement in FKBP51 mRNA expression was induced by all treatment doses of YK11 to the same extent as 0.1 uM DHT. However, the mRNA expression did not exhibit any significant improvements in the parent HEK293 cells. The next step of the study was to examine the effects of YK11 in AR-target cells, specifically, the endogenous AR-expressing MD-MB 453 cells. Improvement in FKBP51 and FGF18 mRNA expression was induced by both YK11 and DHT to the same levels.

In contrast, specifically androgen-regulated genes (SARG) mRNA expression was only induced by DHT and not YK1, while induction of HSD11B2 mRNA expression elicited by YK11 was weaker than mRNA expression elicited by DHT. It was also reported that the AR antagonist, bicalutamide (BIC), inhibited the induction of FKBP51 mRNA expression by YK11. This indicates that the ability of YK11 to affect gene expression relative to the androgen agonist is dependent on the context of the gene [2].

Figure 7: Changes in mRNA expression in response to different treatments.

Results of the study determined that the AR is localized in the cytoplasm when other ligands are absent. That being said, the first step of ligand-dependent activation of the AR is nuclear translocation. HEK293 were transfected with YFP-AR expressing plasmid; the plasmid localized in the cytoplasmic region due to the absence of the ligands. Additionally, YFP-AR began to accumulate in the nucleus when both DHT and YK11 were present, indicating that YK11 is capable of accelerating nuclear translocation of the androgen receptor.

In comparison to other nuclear receptors, androgen receptors are maximally active when a ligand is able to promote the interaction between the amino- and carboxy-terminal domains; this is referred to as an N/C interaction. The effects of YK11 on the N/C interactions were observed through the completion of a mammalian two-hybrid assay. Results of the assay found that in the presence of DHT, simultaneous expression of fusion proteins GAL4-AR LBD and VP16-AR NTD only resulted in a strong transactivation of the GAL4-reporter gene. However, YK11 did not support transactivation of the reporter gene, but rather inhibit DHT-dependent transactivation of the reporter gene. The recorded results suggest that YK11 prevents DHT-induced N/C interactions between the LBD/AF-2 and NTD of AR in a similar manner to the DHT antagonist, hydroxyflutamide. Additionally, the researchers determined that YK11 is able to activate the androgen receptor without causing an N/C interaction, thus leading to its agonistic nature [2].

 

Overall, the researchers were able to conclude that in the presence of YK11, the two transactivation domains of the androgen receptor are able to work independently since there is no occurrence of an N/C interaction. However, the effects of YK11 on the transactivation domains were assessed individually. Expression plasmids were coded for two AR mutants truncated at both ends: NTD-DBD and DBD-LBD. HEK293 cells were then transfected with the expression plasmids in combination with the ARE-luciferase reporter plasmid. Results of transfection found that ligand-induced transactivation of the reporter gene was only observed in the presence of the full length AR [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Lee SJ, Gharbi A, Shin JE, Jung ID, Park YM. Myostatin inhibitor YK11 as a preventative health supplement for bacterial sepsis. Biochem Biophys Res Commun. 2021 Mar 5;543:1-7. doi: 10.1016/j.bbrc.2021.01.030. Epub 2021 Feb 12. PMID: 33588136.

[2] Kanno Y, Hikosaka R, Zhang SY, Inoue Y, Nakahama T, Kato K, Yamaguchi A, Tominaga N, Kohra S, Arizono K, Inouye Y. (17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11) is a partial agonist of the androgen receptor. Biol Pharm Bull. 2011;34(3):318-23. doi: 10.1248/bpb.34.318. PMID: 21372378.

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