Disruptors of the Sestrin AMPK/MAPK Complexes

Last Revision: 11/21/2024

DOS QUICK OVERVIEW

Disruptors of Sestrin-MAPK interactions (DOS) target and dismantle a specific protein complex known as the sestrin-MAPK activation complex (sMAC), which is implicated in immune senescence. The key points of their mechanism of action include:

1. Targeting the sMAC Complex: DOS compounds disrupt the interaction between sestrins and MAPK proteins (ERK, JNK, and p38), which are stress-activated protein kinases that collectively drive T cell senescence through metabolic and signal transduction pathways.

2. Ubiquitin-Proteasomal Pathway: By binding to sestrins, DOS compounds facilitate the ubiquitination and proteasomal degradation of the sMAC complex. This prevents the sestrin-mediated activation of MAPKs, effectively reversing age-related immune inhibition.

3. Reactivation of Juvenile Pathways: DOS treatment reactivates youthful cellular pathways in T cells, such as β-catenin signaling, which supports fatty acid oxidation and induces a stem-like state in formerly senescent cells. This rejuvenation is linked to the re-expression of genes involved in antigen-specific TCR rearrangements, enabling the formation of novel immune responses.

4. AMPK Inhibition: DOS inhibits the activation of AMP-activated protein kinase (AMPK) by sestrins, which is a key event in sMAC-driven T cell senescence. The disruption of this AMPK pathway reduces oxidative stress and mitigates the cellular checkpoints associated with energy and genotoxic stress.

5. Selective Penetration: DOS preferentially acts on senescent T cells (e.g., CD27- CD28- subsets) with high sMAC levels, ensuring that their effects are specific to aged or dysfunctional immune cells without broadly affecting other immune populations.

Key Insights:

Rejuvenation and TCR Diversity: DOS compounds enable senescent T cells to regain proliferative capacity and generate new TCR specificities, enhancing immune repertoire diversity.
Potential for Broad Therapeutic Applications: By addressing immune dysfunction at its molecular root, DOS could be leveraged for treating age-related diseases, improving vaccine responses, and combating chronic infections or cancer.
Differentiation from Other Therapies: Unlike traditional approaches targeting telomerase or senolytics, DOS directly modulates the signaling environment, restoring function without inducing off-target effects common in senescence interventions.

DOS POTENTIAL

Disruptors of Sestrin-MAPK interactions (DOS) rejuvenate senescent T cells, leading to enhanced systemic immunity by restoring youthful cellular function, increasing immune repertoire diversity, and potentially mitigating age-related diseases, chronic infections, and cancer through improved immunosurveillance and long-term immune protection. By maintaining robust immune responses and reducing the burden of chronic age-associated conditions, DOS holds the potential to dramatically extend both healthspan and lifespan.

DOS ~ Sestrin MAPK Activation Complex

DOS represents a class of cyclic polypeptides that target sestrins for proteasomal degradation. This results in a diminished signaling cascade between sestrins, AMPK and MAPK complexes, (sMAC) resulting in the rejuvenation of the immune system, via the restoration of senescent T cells and the cascade of benificial downstream effects that this restoration facilitates.

The AMPK and MAPK signaling pathways are involved in a complex interplay that regulates cellular metabolism, proliferation, differentiation, and survival [14]

Sestrins are stress-inducible proteins that play a critical role in regulating cellular protection and metabolism by sensing damage and stress signals [3]. Under normal conditions, sestrins activate AMP-activated protein kinases (AMPK), which suppresses the mTOR (mechanistic target of rapamycin) pathway, a key regulator of cell growth and metabolism [4]. Sestrins also interact with mitogen-activated protein kinases (MAPKs), forming complexes (sMACs) that manage stress responses, including inflammation, cell division, and senescence [7]. In healthy cells, sestrins help maintain cellular balance by promoting autophagy (cellular clean-up) and preventing excessive cell growth during stress. However, as we age, sestrins accumulate in senescent cells, where they contribute to dysfunction and promote chronic inflammation [13].

Structure of SESN-A, SESN-C Sestrin molecule

Senescent Cell Receptors and DOS Response — Click [√] 2 Enlarge

Senescent cells are aged or damaged cells that have permanently stopped dividing but remain biologically active in a harmful way. These cells enter a state of what was previously considered irreversible growth arrest, in response to stress or damage. However, instead of dying, they persist in a dysfunctional state and no longer perform their normal roles [19]. Often referred to as "zombie cells"—they are still alive but stuck in a dysfunctional state, unable to divide or perform their protective functions. Instead, they linger in the body, releasing harmful substances known as senescence-associated secretory phenotypes (SASP). SASP’s refer to a collection of pro-inflammatory cytokines, chemokines, growth factors, and proteases that are secreted by senescent cells, which occurs when cells experience damage, stress, or reach the end of their replicative lifespan, often determined by telomere length. While cellular senescence serves as a tumor-suppressive mechanism by halting cell division, the SASP contributes to inflammaging, a low-grade, systemic inflammation associated with aging. Inflammaging is driven by the persistent release of SASP factors, which alter the tissue microenvironment and influence neighboring cells. This chronic inflammatory state is a major contributor to age-related diseases, such as cardiovascular disorders, cancer, and neurodegenerative conditions. By compounding health risks in elderly individuals, SASP-driven inflammaging accelerates tissue dysfunction and plays a key role in the decline seen during the aging process.

Senescent T cells also exhibit diminished responses when encountering foreign antigens, substances that normally trigger immune reactions. Even when exposed to threats like viruses or tumors, these "zombie" T cells fail to mount an effective or robust immune response. This diminished response is a major contributor to the immune system’s decline with age, leaving the body susceptible to infections, cancer and other health challenges [18].

MAPK signaling controls cellular proliferation, differentiation, and survival, whereas the AMPK signaling regulates cellular metabolism. Hyperactive MAPK signaling is responsible for a large portion of cancers. The interplays between MAPK and AMPK signalings are binary, and the AMPK signaling can regulate MAPK signaling reversely [14]. While age-related accumulation of sestrins are not the primary cause of immune cell senescence, their abnormal accumulation and interaction with MAPKs, forming sestrin-MAPK complexes (sMACs), plays a critical role in maintaining the senescent state. These complexes amplify stress signals and inhibit essential cellular processes, locking cells in a dysfunctional state [24].

The conventional approach to managing senescent cells often involves senolytics; drugs that selectively kill these dysfunctional "zombie" cells to reduce the negative effects, such as chronic inflammation and tissue damage. While effective at clearing out damaging cells, senolytics carry risks like off-target drug effects and the loss of the potential benefits of the immune surveillance cells they are killing off. In contrast to senolytics, DOS compounds do not kill senescent cells; instead, they rejuvenate them, not just restoring their health and functionality but also significantly expanding the immunological potential of these cells. DOS treatment not only brings back lost immune function, these rejuvenated T cells develop new antigen-specific receptors, enabling them to respond to a wider range of infections and diseases even if they have never encountered the threat before and have no immunological memory of them. This process turns previously dysfunctional cells into highly responsive, broadly effective immune cells while simultaneously remodeling the cellular environment into a more youthful environment that promotes appropriate immunological responses by the elimination of SAPS.

Mechanism of Action of DOS46L on Sestrin and Downregulation of the Overactivation of sMAC (Sestrin•MAPK Complex)

DOS compounds work by disrupting the negative effects of sestrin accumulation in aging cells and their interaction with MAPKs. Normally, mitogens like MAPKs stimulate immune cells to divide and grow, a critical process for fighting infections. However, in aging cells, MAPK (ERK, JNK, p38) signaling become overactive, leading to immune cell dysfunction. DOS compounds selectively block the harmful signaling between sestrins and MAPK elements by causing the selective degradation of sestrins, which reactivates immune cells and restores their ability to combat infections and diseases [24].

sMAC ~ Sestrin MAPK Activation Complex

DOS46L is the lead compound developed by Prof. Alessio Lanna, Ph.D. and the founder of SentCell.

Sentcell is an exclusive NCE (New Chemical Entity) company that has developed the first in class human rejuvenating therapies by rational design focused on the sMAC pathway.

DOS46L is a pan-sestrin inhibitor, targeting sestrin 1, sestrin 2, and sestrin 3, although it does not bind directly to sestrin 3 in vitro. After entering senescent T cells (especially CD27⁻ CD28⁻ CD4⁺ T cells with high levels of sMAC), DOS46L binds to a specific site on sestrins, distinct from regions involved in GATOR/mTOR regulation (Not shown right [27].) This binding induces a conformational change in sestrins, exposing regions that lead to their ubiquitination. Once ubiquitinated, the sestrin is recognized by the 26S proteasome, a large proteolytic complex responsible for degrading proteins. The proteasome unfolds the sestrin protein and threads it into its core, where it is broken down into smaller peptides, effectively removing it from the cell and preventing it from performing its biological functions. DOS46L also impedes AMPK's energy sensing by preventing ATP from leaving AMPK's gamma subunit, inactivating AMPK and causing it to lose its affinity for sestrin binding. This leads to the proteasomal degradation of both sestrin and AMPK, dismantling the sestrin-MAPK complex (sMAC).

By disassembling sMAC and halting the pro-senescence MAPK (ERK, JNK, p38) signaling pathways [7], DOS46L reverses immune senescence and rejuvenates immune cell function.

MAPKs Collectively Balance Cellular Responses to Stress, Growth, and Aging Across Various Conditions.

Sestrin MAPK (sMAC) Activation Complex — Click [√] to Enlarge

JNK (c-Jun N-terminal kinase): JNK is primarily activated by stressors like oxidative stress, UV radiation, and inflammatory cytokines, promoting apoptosis and inflammatory responses. In immune cells, JNK suppresses T-cell receptor (TCR) signaling during aging, contributing to immune-senescence.

p38 MAPK: p38 responds to environmental stress and cytokines, managing inflammation, cell cycle arrest, and DNA repair. In aging immune cells, p38 inhibits telomerase activity, leading to telomere shortening and accelerating senescence.

ERK (extracellular signal-regulated kinase): ERK is typically activated by growth factors and mitogens, driving cell proliferation, differentiation, and survival. While mainly growth-associated, ERK can also be triggered by mechanical and oxidative stress, particularly in immune cells during inflammatory responses.

🔷 Rearrangement of T cell Receptors

DOS exhibits a unique capability to reprogram aging T cells, not only restoring their lost function but fundamentally rearranging their T cell receptors (TCRs) to develop entirely new antigen-specific receptors. This reconfiguration allows senescent T cells to recognize and respond to previously unencountered pathogens or tumor cells, effectively transforming "worn-out" cells into highly responsive, functionally rejuvenated immune cells. Such reactivation of TCR diversity is critical to reviving immune surveillance, a process that typically declines with age and leaves the body vulnerable to cancers and age-related diseases. This reactivation of TCR diversity plays a key role in the rejuvenating effects of DOS.

This discovery redefines T cell behavior, challenging the longstanding view that TCR configurations are fixed from birth, selected only stochastically through clonal expansion. DOS prompts a novel mechanism for stem-like behavior in T cells by reprogramming their receptors, suggesting that previous research may have missed this dynamic aspect of T cell immunology, which holds vast potential for extending the immune response against new health threats and achieving broad rejuvenation in the immune system[21, 24].

🔷 Telomeres

Some T cells, especially naïve and memory cells, can extend their telomeres (the protective caps on the ends of DNA) by receiving parts of telomeres from specialized cells called antigen-presenting cells (APCs). This process does not rely on the usual enzyme, telomerase. When APCs come into contact with these T cells, they break down a protective protein called shelterin to release telomeres, which are then cut by another protein (TZAP) and passed to the T cells in tiny packages called vesicles. These telomere pieces attach to the ends of the T cell's DNA, making the telomeres longer by about 3,000 base pairs. This transfer of telomeres helps protect the T cells from aging and ensures they can continue dividing and responding to infections for a long time. Senescent T cells typically lose the ability to efficiently elongate their telomeres or maintain proper telomere length due to immune-aging. Once DOS inhibits the sestrin-MAPK complexes, the targeted T cells regain their ability to receive telomeres from APCs during the immune synapse, leading to telomere elongation and restoration of their proliferative capacity and youth-like function. [17, 24, 26]

🔷 Pharmacokinetics

The pharmacological profile of DOS is particularly impressive. These compounds require only small doses to achieve therapeutic effects, with a single dose being sufficient to initiate long•term immune rejuvenation. In current studies, the identified therapeutic dose for the lead molecule, DOS46L, is 0.1 mg/kg. This dosage has shown significant efficacy in animal models, including aged mice, where a single administration restored immune responsiveness and provided extended protection against infections like H1N1 influenza. The long-lasting benefit, even with minimal dosing, is a major advantage over other immune therapies that require frequent or continuous administration. DOS compounds achieve this sustained effect by promoting the formation of long-lived, stem cell-like memory T cells, which maintain immune readiness for extended periods, potentially lasting for years.

In animal studies, DOS compounds have demonstrated a favorable safety profile. Even at doses of 0.1 mg/kg, no significant toxicities or adverse effects have been observed. Studies in mice revealed that DOS does not induce harmful immune responses, cell damage, or death, as seen with some other therapies that aim to kill senescent cells. Instead, DOS promotes immune cell rejuvenation without causing unwanted inflammation or toxicity. Additionally, there have been no significant off-target effects or organ toxicities, making DOS a promising candidate for long-term, intermittent use in treating age-related immune decline.

Blocking sestrin function entirely could be harmful, disrupting the normal stress response pathways, potentially leading to increased cellular damage or inflammation. DOS compounds selectively disrupt only the harmful, elevated sestrin-MAPK complexes in senescent immune cells. This targeted action ensures that healthy cells continue to benefit from sestrin’s protective roles while allowing senescent immune cells to regain functionality. By focusing on these specific complexes, DOS treatment minimizes toxicity and optimizes immune restoration without the risks associated with broadly inhibiting sestrin pathways.

DOS Technology: A Transformative Approach to Immune Rejuvenation and Systemic Aging

DOS compounds represent a groundbreaking advancement in addressing age-related immune decline by targeting multiple immunological pathways with unprecedented precision. These compounds rejuvenate senescent immune cells, including CD4⁺ and CD8⁺ T cells, B cells, and NK cells, restoring their responsiveness to antigens and cytokine production, such as IFN-γ, IL-4, and IL-10. DOS not only reverses immune-senescence by inhibiting sestrin-MAPK complexes (sMAC) and promoting telomere elongation, but also eliminates the senescence-associated secretory phenotype (SASP), reducing harmful inflammation. By suppressing pro-inflammatory cytokines like IL-6 and TNF-α and enhancing anti-inflammatory pathways, DOS compounds not only rejuvenate the immune system but also improve systemic tissue repair and reduce chronic inflammation. Furthermore, DOS compounds induce de novo T-cell receptor (TCR) rearrangements, significantly expanding the immune repertoire and empowering the body to combat both new and previously encountered infections. Critically, DOS46L doesn't just inhibit sestrin function—it actively promotes its degradation, ensuring a prolonged reduction in sestrin activity even after the drug has dissociated. While the half-life of DOS46L may be measured in minutes to hours, its biological effects, such as the sustained restoration of healthy AMPK/MAPK signaling and reversal of immune cell senescence, continue for weeks to months. This combination of actions positions DOS as a transformative tool capable of preventing a wide range of illnesses, dramatically enhancing healthspan, and potentially extending lifespan. If successful in clinical trials, DOS could revolutionize the entire healthcare ecosystem by redefining the approach to disease prevention, treatment, and aging, making it a pivotal force in the fight against age-related diseases and the effort to extend human longevity.

In comparison to existing therapies like senolytics, exosome transfers and young plasma infusions, DOS compounds offer a more targeted, effective and durable solution. While exosomes and plasma transfers work by introducing youth-associated factors to dilute the pro-inflammatory SASP, DOS technology reprograms senescent cells directly, remodeling the SASP at its source. DOS46L restores to a younger state effective immune survaliance. By suppressing pro-inflammatory cytokines like IL-6 and TNF-α and enhancing anti-inflammatory pathways, DOS compounds not only rejuvenate the immune system but also improve systemic tissue repair and reduce chronic inflammation. This multi-faceted impact on immune cell function and systemic inflammation positions DOS as a highly potent therapy for both immune rejuvenation and the overall mitigation of aging processes.

For an excellent primer on the development and aging of immune cells this highly relevant review article focuses on major changes regarding acquired immunity, especially by T cells. As Professor Alessio’s research illuminates, T cell immunity is an evolving science.

“As humans age, the immune system undergoes dramatic changes, culminating in losing the ability to protect against cancerous cells, infections and foreign invaders. With increasing age, the innate immune system functions continuously progress to create a characteristic pro-inﬂammatory environment, so called “inﬂammaging”, while the adaptive immune response declines, leading to massive T cell dysfunction.” Of piticular note is the majority of the disease causing deficiencies identified in this article and directly associated with aging and are resolved by DOS.

[2020] The MAPK and AMPK signalings: interplay and implication in targeted cancer therapy

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