FOXO4-DRI – a Promising Anti-aging Peptide
The human body comprised of up to 1600 transcription factor proteins, also known as sequence specific DNA binding factor, which primarily regulates the rate of genetic information transcription from DNA to RNA (1). These proteins bind to the specific sequences of the DNA and produce their effects by controlling the gene expression and direct cell cycle including cell growth and cell death.
One such class of transcription factor proteins is the FOXO group, or forkhead family of transcription factor-O (2), which includes four members namely FOXO1, FOXO3, FOXO4, and FOXO6. The FOXO4 factor regulates various cellway pathways including insulin signaling, and cell cycle progression, and other functions that regulate growth and differentiation.
A synthetic version of the FOXO4 protein is available namely FOXO4-DRI, which is identical to the endogenously available FOXO4 protein, except for the alteration in its amino acid structure. The alteration and further benefits of the peptide are discussed in detail below.
FOXO4-DRI peptide, also called Proxofim, is an acronym for Forkhead box O transcription factor 4-D-Retro-Inverso peptide (3).
FOXO4-DRI peptide is the same as that of FOXO4 protein, except that the L amino acids in its structure are replaced by D amino acids. As a result of this, FOXO4-DRI peptide is less susceptible towards the normal clearance mechanism as compared to FOXO4 and hence remains in the body for longer periods of time.
Overview of “DRI” peptides
The retro inverso peptides (DRI peptides) are linear chains of amino acid sequence that is “reversed”, hence also reversing the chirality of the structure (i.e., L amino acid structure altered to D amino acid and vice versa). The primary benefit of this alteration is that it provides resistance towards peptide degradation and potentially elongates their half lives.
D amino acids represent the mirror image of the naturally occurring L amino acids in the biological proteins. The main advantage of exchanging L amino acid with D amino acid is that the latter is resistant towards degradation, making the protein structure more durable.
FOXO4-DRI Peptide Working Mechanism
The main benefit of the FOXO4-DRI peptide is its ability to prevent the binding of the FOXO4 protein in the body with the other p53 protein (4).
The p53 protein is an endogenous regulator protein, which regulates the progression of the cell cycle, including cell death. Primarily, when FOXO4 protein binds with p53, it does not allow p53 to bind with the DNA and thereby prevent apoptosis and cell death. In the presence of FOXO4-DRI peptide, this usual process is inhibited and p53 goes on to bind with DNA and thereby help the cell cycle to continue and die.
One may wonder how is this beneficial if it eventually leads to cell death? Interestingly, FOXO4-DRI peptide is selective in nature and only exert this effect on cells that have become dysfunctional over time due to aging, known as the senescent cells (4).
As a result of this biological pathway, the functioning of the tissues is improved, which further helps in promoting cell growth and differentiation. This is how the peptide leads to better biological functions and decrease in aging effects.
Biological Uses of FOXO4-DRI
This peptide has demonstrated various advantages in the therapeutic field, including:
- Helps address senescence (process of cell deterioration associated with age)
- Anti aging effects
- Assistance with insulin signal pathway
- Prevents heart damage
- Potential benefits in combating neurological disorders
Research and Clinical Studies
Aging and Senescence Studies
As mentioned earlier, the natural FOXO4 functioning is that the protein protects the senescent cells by not allowing p53 to bind with DNA. With the peptide, it blocks the natural protein, helps p53 to bind with DNA and cause the senescent cells to die. Researchers refer to this process as the “rejuvenation” process by biological elimination of the aged cells (5).
This is because once the damaged (senescent) cells are out from the body, all the energy produced is redirected towards healthy cells, which leads to better growth, development and functioning of the body.
While FOXO4-DRI peptide does not completely stop the cell senescence process, it does help to slow down the process by preventing the FOXO4 mediated senescence.
The below image represents which factors lead to cell senescence and what are the outcomes due to this occurrence:
Over time, there may be irreparable cell damage i.e., beyond the body’s ability to combat this, which leads to reduced health span. It should be noted that healthspan is the duration of healthy cells during which the organism remains healthy – which is different from lifespan.
By preventing senescence and cell damage, this peptide increases the healthspan of the cells, which may not inadvertently increase the number of years lived, but results in better, healthier living of the organism.
This theory was also proven by a 2017 study (6) where an experiment was conducted on aged mouse models that were administered with either the protein compound or the placebo. The peptide administered mice showed improved fitness, better renal functioning and increased density of the fur. While the mice did not show increased longevity in their lifespan, the results depict that the peptide leads to better cellular and tissue health, resulting in fewer ailments and disabilities due to age.
Cardiovascular Disorders Studies
It is a known fact that with increasing age, there are higher chances of cardiovascular disorders. Research from 2002 (7) has shown that as age increases, the levels of proteasome enzymes decrease. These enzymes played a primary role in removing any cells that were identified as damaged or dysfunctional. Since their levels decrease, the number of damaged cells in the body eventually increases with age.
FOXO4 protein regulates the levels of proteasome enzymes in the body. This interpretes as FOXO4 protein leading to increased levels of proteasomes in the body, but it does not necessarily help in reducing damaged cells in aged beings.
It is understood that with the help of FOXO4-DRI peptide, it will boost the body’s natural process and eliminate all the dysfunctional cells, which in turn may help prevent the occurrence of age-related heart diseases (8). Clinical trials are yet to be conducted to prove this theory is correct.
Insulin Signaling Studies
Research from 2017 (9) has shown that FOXO proteins mediate the insulin signaling pathway, where they regulate the inhibitory functions of the insulin most involved in cell metabolism, cell cycle, oxidative stress, senescence, and aging.
Any alterations in the levels of FOXO proteins lead to serious disorders including cancer, metabolic disorders and altered lifespan of the organisms. It is of major concern especially in diabetics, as altered FOXO levels lead to hyperlipidemia and hyperglycemia, which may potentially lead to stroke, kidney damage and more.
While studies are yet to be conducted to clearly demonstrate if and how FOXO4-DRI peptide works, it is understood that the peptide may improve downstream effects of insulin, which will lead to reducing excessive blood sugar levels. This may further help in avoiding more complications pertaining to altered FOXO levels and insulin pathways.
Studies Demonstrating Peptide Role in Neurodegenerative Disorders
It is known that with increased age, the cognitive functions impair. While the pathophysiology of certain neurological diseases, such as Alzheimer’s, remains unclear, it is presumed that there are changes in the proteasome enzyme activity with increased age, which causes cognitive impairment.
Research (10) has shown that the proteasome activities were downregulated in disorders like Parkinson, Alzeihmer and Prion disease. It is not known whether this downregulation is the primary cause of these diseases or not, but it sure is a contributing factor.
Continued research has shown that the levels of FOXO proteins in the central nervous system are altered in the patients suffering from neurodegenerative disorders (NDDs)(11). This has led to the belief that exogenous FOXO protein, such as FOXO4-DRI peptide, may help in regulating optimal levels of FOXO proteins and thereby prevent or alleviate the progression of any NDDs associated with this mechanism.
Age-related Male Hypogonadism Studies
One of the common age associated ailments in men is late-onset hypogonadism (LOH) which is characterized by decreased serum testosterone levels, low sexual desire, erectile dysfunction, and also obesity and depression. This is primarily due to impaired functioning of the senescent Leydig cells (cells that are adjacent to the tubules in the testicles)(12).
A study (12) was conducted with the purpose of establishing the use of FOXO4-DRI peptide in treating age-related male LOH. An in vitro model composed of senescent Leydig cells was used. These Leydig cells were previously isolated from male mice and treated with hydrogen peroxide chemical which caused the senescence.
Helps Combat Male Hypogonadism
On analysis of these damaged cells, researchers noted that FOXO4 cells helped to maintain the viability of these cells and prevent its apoptosis. When these isolated senescence cells were treated with FOXO4-DRI peptide, it blocked FOXO4 protein and allowed p53 to bind with DNA, which led to the apoptosis of the senescent Leydig cells.
Another study (12) in naturally aged mice showed that FOXO4-DRI peptide improved the functioning and overall health of Leydig cells, which contributed towards improved testicular functioning and increased testosterone secretion.
These results depict that the peptide may also be clinically useful in combating male late onset hypogonadism.
Side Effects With FOXO4-DRI
Clinical trials of the peptide are yet to be conducted to fully demonstrate any serious adverse effects associated with peptide use.
However, as with other peptides, some of the common side effects may be:
- Pain, redness, swelling at the site of the injection
- Dry mouth
- Flu like symptoms
- Pain in joints
FOXO4-DRI peptide (Proxofim) was first discovered in 2017 by Biologist Peter de Keizer and his team. While the recent studies available are only on mice, there are developments in progress to obtain approval to conduct clinical trials in humans.
Currently, only preliminary studies have been conducted with the Proxofim compound, which so far has yielded promising results. FDA approval is yet to be obtained for the peptide. This compound is not approved to be used on humans and animals of any kind, and is available for research purposes only.
FOXO4-DRI, or Proxofim peptide, is an identical amino acid sequence as the endogenously occurring FOXO4 transcription protein, with the only difference being that the L amino acids are replaced with D amino acids. Since both D- and L- amino acid structures are mirror images of each other, this peptide is called FOXO4-D-Retro-Inverso peptide.
Unlike other analogous compounds, FOXO4-DRI acts by preventing (or reversing) the normal functioning of the FOXO4 protein. The compound blocks the protein from binding with p53 protein, allowing p53 to bind with DNA instead and leading to cell death.
Owing to its highly selective nature, the peptide exerts this effect on senescent or damaged cells only. This fact promotes healthier and better functioning cells and tissues in the body. Preliminary research shows promising results that the peptide may be used to combat several age related ailments and senescence in humans.
(1) Babu MM, Luscombe NM, Aravind L, Gerstein M, Teichmann SA. Structure and evolution of transcriptional regulatory networks. Curr Opin Struct Biol. 2004 Jun;14(3):283-91. https://pubmed.ncbi.nlm.nih.gov/15193307/
(2) Sun, Yan et al. “FOXO4 Inhibits the Migration and Metastasis of Colorectal Cancer by Regulating the APC2/β-Catenin Axis.” Frontiers in cell and developmental biology vol. 9 659731. 23 Sep. 2021. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8495124/
(3) Huang, Yuzhao et al. “Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro Expanded Human Chondrocytes.” Frontiers in bioengineering and biotechnology vol. 9 677576. 29 Apr. 2021, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116695/
(4) Zhang, C., Xie, Y., Chen, H., Lv, L., Yao, J., Zhang, M., Xia, K., Feng, X., Li, Y., Liang, X., Sun, X., Deng, C., & Liu, G. (2020). FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice. Aging, 12(2), 1272–1284. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053614/
(5) Krimpenfort P, Berns A. Rejuvenation by Therapeutic Elimination of Senescent Cells. Cell. 2017 Mar 23;169(1):3-5. https://pubmed.ncbi.nlm.nih.gov/28340347/
(6) Marjolein P. Baar et al, Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. Vol 169 Issue 1, https://doi.org/10.1016/j.cell.2017.02.031
(7) Anne-Laure Bulteau, Luke I. Szweda, Bertrand Friguet, Age-Dependent Declines in Proteasome Activity in the Heart, Archives of Biochemistry and Biophysics, Volume 397, Issue 2, 2002, Pages 298-304, ISSN 0003-9861, https://doi.org/10.1006/abbi.2001.2663.
(8) Murtaza G, Khan AK, Rashid R, Muneer S, Hasan SMF, Chen J. FOXO Transcriptional Factors and Long-Term Living. Oxid Med Cell Longev. 2017;2017:3494289. doi: 10.1155/2017/3494289. Epub 2017 Aug 15. https://pubmed.ncbi.nlm.nih.gov/28894507
(9) Lee, S., & Dong, H. H. (2017). FoxO integration of insulin signaling with glucose and lipid metabolism. The Journal of endocrinology, 233(2), R67–R79. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480241/
(10) Ciechanover A, Brundin P. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron. 2003 Oct 9;40(2):427-46. https://pubmed.ncbi.nlm.nih.gov/14556719/
(11) Wei Hu, Zhi Yang, Wenwen Yang, Mengzhen Han, Baoping Xu, Zihao Yu, Mingzhi Shen, Yang Yang, Roles of forkhead box O (FoxO) transcription factors in neurodegenerative diseases: A panoramic view, Progress in Neurobiology, Volume 181, 2019, 101645, ISSN 0301-0082, https://doi.org/10.1016/j.pneurobio.2019.101645.
(12) Zhang, C., Xie, Y., Chen, H., Lv, L., Yao, J., Zhang, M., Xia, K., Feng, X., Li, Y., Liang, X., Sun, X., Deng, C., & Liu, G. (2020). FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice. Aging, 12(2), 1272–1284. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053614/
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