Repurposing nucleoside reverse transcriptase inhibitors (NRTIs) to slow aging

Repurposing drugs already approved in the clinic to be used off-label as geroprotectors, compounds that combat mechanisms of aging, are a promising way to rapidly reduce age-related disease incidence in society. Several recent studies have found that a class of drugs — nucleoside reverse transcriptase inhibitors (NRTIs) — originally developed as treatments for cancers and human immunodeficiency virus (HIV) infection, could be repurposed to slow the aging process. Interestingly, these studies propose complementary mechanisms that target multiple hallmarks of aging. At the molecular level, NRTIs repress LINE-1 elements, reducing DNA damage, benefiting the hallmark of aging of ‘Genomic Instability ’ . At the organellar level, NRTIs inhibit mitochondrial translation, activate ATF-4, suppress cytosolic translation, and extend lifespan in worms in a manner related to the ‘Loss of Proteostasis ’ hallmark of aging. Meanwhile, at the cellular level, NRTIs inhibit the P2X7-mediated activation of the inflammasome, reducing inflammation and improving the hallmark of aging of ‘Altered Intercellular Communication ’ . Future development of NRTIs for human aging health will need to balance out toxic side effects with the beneficial effects, which may occur in part through hormesis.


Introduction
Aging has been described as "a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death" (López-Otín et al., 2013).Major human pathologies, including cancers, diabetes, neurodegenerative diseases, and cardiovascular disorders are the direct consequences of this deterioration.Meanwhile, most scientific publications focus on the causes of specific age-related diseases and not the causes of aging itself.However, old age is one of the greatest risk factors for age-related diseases.
Recent events, however, have led to an expanding research field with aging at the forefront.Firstly, major changes in global demography have resulted in a greater proportion of people over the age of 65, while the increase in healthspan has not kept up with the increasing lifespan (Partridge et al., 2018;Niccoli and Partridge, 2012;Hurst et al., 2018).Secondly, despite significant investment, the pharmaceutical industry has not succeeded in developing products able to cure or prevent disease progression of many age-associated disorders, particularly neurogenerative diseases (Hardy and De Strooper, 2017;Tarakad and Jankovic, 2017).Thirdly, intervention studies performed on animal models have established that delaying aging and lifespan extension are both feasible (Harrison et al., 2009;Fontana and Partridge, 2015;Fontana et al., 2010;Partridge et al., 2018), and most of the time are associated with healthspan extension (Bitto et al., 2016;Crimmins, 2015;Justice et al., 2018).Achieving similar results in humans would improve individual quality of life while drastically reducing healthcare costs at the societal level (Goldman, 2016;Olshansky, 2016).
In this context, researchers are working towards repurposing clinically approved drugs for off-label use as 'geroprotectors', or compounds that combat mechanisms of aging.For instance, rapamycin, traditionally used as an immunosuppressive drug through inhibiting the cellular complex mTORC1, also promotes autophagy and increases lifespan in yeast, flies, and mice (Kaeberlein et al., 2005;Bjedov et al., 2010;Harrison et al., 2009).Likewise, metformin, a drug prescribed for type 2 diabetes, interacts with known longevity pathways and extends lifespan in worms and mice (Rena et al., 2017;De Haes et al., 2014;Chen et al., 2017;Martin-Montalvo et al., 2013).In this review, we focus on a drug class recently emerging as geroprotective, namely nucleoside reverse transcriptase inhibitors (NRTIs).NRTIs are a type of reverse transcriptase inhibitor originally developed as a cancer treatment but subsequently used to inhibit the Human Immunodeficiency Virus (HIV) reverse transcriptase enzyme and provide protection against HIV pathogenicity.This is because NRTIs are structural analogues (nucleosides) of DNA nucleotides, but they prevent a new 3' phosphodiesterase bond from forming with incoming nucleotides that are normally incorporated by the reverse transcriptase enzyme into the growing DNA strand (Fig. 1A, B).
Recently, NRTIs have also been found to promote longevity, occurring through multiple mechanisms.In one, described in mice, NRTIs reduce the age-related activity of long interspersed nuclear elements (LINEs), reduce DNA damage and inflammation, and extend the lifespan of Sirt6 -/-mice (Simon et al., 2019a) (Fig. 2).In another, identified by our team using C. elegans, NRTIs function through the longevity-related transcription factor 4 (ATF-4), decrease cytosolic translation, and extend healthspan and lifespan (Fig. 2) (McIntyre et al., 2023).Meanwhile, again in mammals, NRTIs have also been demonstrated to inhibit the P2X7-mediated inflammasome and reduce inflammation, which can also ultimately result in healthy aging benefits (Fowler et al., 2014) (Fig. 2).Taken together, these findings reveal multiple mechanisms and highlight the potential of NRTIs as geroprotective compounds (Fig. 2).

History of NRTIs
NRTIs achieve their antiviral effects on HIV by preventing the HIV reverse transcriptase enzyme from inserting the code for its viral RNA into the human genome.Specifically, upon administration and absorption, NRTIs are phosphorylated inside the cell, becoming active upon diphosphorylation or tri-phosphorylation.This phosphorylation induces the inhibition of the enzymatic action of the reverse transcriptase protein through the incorporation of the nucleotide analog.Due to the fact that di-and tri-phosphorylated NRTIs lack a 3′ -hydroxyl group, the next incoming deoxynucleotide cannot form the following 5′ -, 3′ -phosphodiester bond needed to extend the DNA chain.As a result, the DNA chain is terminated, and DNA synthesis is inhibited.This halts the conversion of (viral) RNA into double-stranded DNA (Pau and George, 2014) (Fig. 1).
This review will specifically discuss several NRTIs that have been studied in the context of longevity and aging, including zidovudine, didanosine, zalcitabine, stavudine, lamivudine, and alovudine (Fig. 3), though others also exist.Notably, some of these, and many others, have been introduced as treatments for AIDS and subsequently removed from the market due to side effects and the emergence of drug-induced resistance, a pattern that has occurred in various waves in NRTI development.In what follows, we briefly describe the historic use of the main NRTIs studied in the context of longevity and aging.
Zidovudine (AZT) was the first HIV NRTI to be clinically approved by the FDA in 1987 (Rathbun et al., 2006).However, because of its toxicity, which can include myopathy (Rachlis and Fanning, 1993), zidovudine is no longer recommended as a first-line drug for HIV treatment.
Didanosine was researched in the mid-1980 s (Mitsuya and Broder, 1986;Mitsuya and Broder, 1987) and approved by the FDA in 1991.Interestingly, didanosine undergoes several modifications before it becomes a potent reverse transcriptase inhibitor.After initial phosphorylation, an adenylosuccinate synthetase and lyase modify the compound and another phosphorylation occurs for its activation (Amblard et al., 2022).Didanosine also induces severe side effects (Faulds and Brogden, 1992), which contributed to its removal from the market in 2020.
Zalcitabine was approved by the FDA in 1992 as an anti-HIV treatment.It was first identified as an anti-cancer compound in 1966 (Horwitz et al., 1966), before being evaluated as a potent inhibitor of HIV reverse transcriptase (Mitsuya and Broder, 1986).Due to the side effects of zalcitabine, including peripheral neuropathy (Adkins et al., 1997), the drug was removed from the market in 2006.
Stavudine, another NRTI, was initially designed as a potential anticancer compound in the 1960 s (Horwitz et al., 1964).Decades later, a collaboration between research groups (Tai-Shun et al., 1987;Baba et al., 1987;Hamamoto et al., 1987) documented the anti-HIV activity of stavudine, leading to its approval by the FDA as a treatment in 1994.Unfortunately, the use of stavudine also resulted in major side effects at the prescription dose, including pancreatitis (Lea and Faulds, 1996), lactic acidosis (Shah et al., 2003), lipoatrophy (Saint-Marc et al., 1999), and peripheral neuropathy (Subbaraman et al., 2007), and the drug was ultimately removed from the market in 2020.
Lamivudine was first synthesized in 1989 and was approved by the FDA in 1995 as an anti-HIV treatment (Diederich and Steuber, 2015).While its efficacy against HIV has been established during multiple clinical trials either alone or in cocktails with other HIV treatments, and the compound is still in use medically to treat HIV, toxic side effects exist including lactic acidosis, liver disease, and more (Cahn et al., 2020;Johnson et al., 2019;Rial-Crestelo et al., 2021;Drugs.com, 2022).
Alovudine, another NRTI, has never been approved by a medical agency due to its potential bone marrow toxicity (Mansuri et al., 1990).
However, later studies demonstrated that alovudine was ten times more potent than zidovudine in vitro (Kong et al., 1992).Therefore, investigation to use this compound as a therapy is still ongoing (Ghosn et al., 2007;Flexner et al., 1994;Yehudai et al., 2019).
All in all, NRTIs are vital drugs against HIV but come with severe side effects.These side effects, which may take diverse forms, are thought to stem from the inherent toxicity NRTIs cause in human mitochondrial DNA since they also inhibit mitochondrial DNA polymerase gamma (Nolan and Mallal, 2004;Lewis et al., 2003).Fortunately, newer NRTI drugs, like abacavir, tenofovir, lamivudine, and emtricitabine have a weaker inhibitory effect on mitochondrial DNA polymerase gamma, generating much less mitochondrial-associated toxicity (Moyle, 2005).Interestingly, despite these side effects, NRTIs may nonetheless slow aging, as has been specifically shown for lamivudine and stavudine in mice (Simon et al., 2019a) and zidovudine and didanosine in C. elegans worms (McIntyre et al., 2023).

NRTIs inhibit age-driving effects of retrotransposon L1 elements in mice
Genomic instability can be summarized as "the accumulation of genetic damage throughout life" (Moskalev et al., 2013).Additionally, many diseases related to premature aging are mainly caused by increasing DNA damage accumulation (Burtner and Kennedy, 2010).The integrity and stability of genomic DNA are repeatedly challenged by numerous agents, both endogenous (DNA replication errors, reactive oxygen species, and spontaneous hydrolytic reactions) and exogenous (physical, biological, and chemical agents) (Hoeijmakers, 2009).Additionally, long interspersed nuclear element 1 (LINE 1; L1) retrotransposons also induce genomic instability due to their insertion into the genome: L1 retrotransposition either disrupts coding exons or introns, leading to mis-splicing or exon skipping, which ultimately generates null or hypomorphic expression alleles (Beck et al., 2011;Hulme et al., 2006).As a result, L1 retrotransposition is involved in several Fig. 2. NRTI longevity mechanisms.Based on findings of NRTI's influences on aging and longevity related pathways in mammals (Simon et al., 2019a;Fowler et al., 2014) and C. elegans worms (McIntyre et al., 2023).In mammals, two possible longevity routes have been described.NRTIs can inhibit the P2X7-mediated inflammasome, resulting in inflammation reduction (Fowler et al., 2014) that can theoretically result in longevity.Independently, it has been demonstrated that NRTIs inhibit LINE-1 cDNA formation in mammals, which reduces inflammation and DNA damage, together ultimately contributing to longevity (Simon et al., 2019a).In C. elegans worms, mitochondrial translation inhibition activates ATF-4 that suppresses cytosolic translation, also ultimately contributing to longevity.A side effect of mitochondrial translation inhibition however is mitochondrial toxicity, which, while causing side-effects, may also produce benefitical mitohormetic effects that extend lifespan (as in C. elegans worms).
L1 elements represent up to 20% of the human and mouse genomes (Lander et al., 2001;Anon, 2002).Fully functional retrotransposons can replicate themselves and expand within their host genome.Given that their insertion requires DNA breakage, L1 activity has been linked to genome instability, including DNA damage and mutagenesis (Iskow et al., 2010;Gasior et al., 2006;Gilbert et al., 2002).In mammalian aging, de-repression of L1 elements occurs, which seems to be caused by an impaired heterochromatin reorganization and redistribution, usually occurring during the mammalian aging process (López-Otín et al., 2023).
Recently, a landmark study used the Sirtuin 6 gene knock-out (Sirt6 KO) mouse model to address the role of L1s in age-related pathology (Simon et al., 2019a).SIRT6 expression induces the ribosylation of KAP1, promoting its complex formation with Histone Protein 1 (HP1) thereby packaging the L1 DNA into heterochromatin, preventing the transcription of retrotransposons (Van Meter et al., 2014).Sirt6 KO mice provide an ideal model to study L1 elements, as Sirt6 KO results in a strong activation of L1 elements, genomic instability, and DNA damage (Mostoslavsky et al., 2006;Van Meter et al., 2014).
Given the dependence of L1 elements on the reverse transcriptase enzyme, Sirt6 KO mice were treated with two different NRTIs, namely stavudine and lamivudine, to inhibit L1 retrotransposons (Simon et al., 2019a).Indeed, L1 activity causes an increase of L1 DNA in the cytoplasm, building a type I interferon response through DNA sensing of cyclic GMP-AMP synthase (cGAS), thus promoting inflammation.Remarkably, inhibition of the L1 retrotransposon activity in Sirt6 KO mice by exposure to NRTIs prevented these deleterious molecular changes, alleviated the cellular and physiological dysfunctions, and increased both the healthspan and lifespan of these mice.Consequently, their results support the hypothesis that L1 elements negatively influence the aging process, through genomic instability and inflammation, and that NRTIs may serve as a therapy for this (Fig. 2).
A milestone study published in the same year used a human cellular senescence model to demonstrate that L1 elements become activated during senescence and aging in humans, leading to accumulation of L1 DNA in the cytoplasm (De Cecco et al., 2019).Treatment with stavudine and lamivudine inhibited formation of L1 cDNA and alleviated type I interferon response and senescence-associated inflammatory markers (De Cecco et al., 2019).
Taken together, L1 elements are actors of genomic instability in humans, due to altered chromatin reorganization during aging.L1 elements then trigger an inflammatory response in the cytosol through the cGAS sensor, leading up to a type I interferon response and subsequent inflammation damage.NRTIs can intervene in this mechanism, inhibiting the ability of L1 elements to spread.Therefore, NRTIs may slow down the aging process due to their initial inhibition of LINE-1 retrotransposons (Fig. 2).Fig. 3. Specific NRTIs described in this review.These include zidovudine (also known as azidothymidine and AZT), didanosine (also known as ddl, DDl, and videx), zalcitabine (also known as ddC and dideoxycytidine), stavudine (also known as d4T and zerit), lamivudine (also known as 3TC) and alovudine (also known as fluorothymidine).

NRTIs extend C. elegans lifespan in an ATF-4-dependent manner
Activating Transcription Factor 4 (Atf4 in mice, and initially named as atf-5 in C. elegans worms, though recently renamed to atf-4) is activated during mild stresses at the cellular level.Under certain stressful conditions (including nutrient deprivation, and oxidative-, thermal-, and endoplasmic reticulum stress), cells protect themselves by inhibiting protein synthesis (Costa-Mattioli and Walter, 2020;Harding et al., 2003).Subsequently, the Integrated Stress Response (ISR) is induced, culminating in the phosphorylation of the translation initiation factor eIF2α.This subunit can be phosphorylated by four different kinases: GCN2 (amino acid starvation), HRI (reduced heme levels), PKR (double-stranded RNA), and PERK (ER stress) (Balachandran et al., 2000;Harding et al., 2000;Han, 2001).ATF4 becomes active by integrating the signals from those eIF2α kinases.This leads up to a significant reduction in cap-dependent mRNA translation (Costa-Mattioli and Walter, 2020; Harding et al., 2003).Consequently, ATF4 is induced in response to the suppression of translation, resulting in the mobilization of stress defense mechanisms to maintain homeostasis (Costa-Mattioli and Walter, 2020;Harding et al., 2003).
Lower cytosolic protein synthesis has been associated with healthier phenotypes (López-Otín et al., 2013).Indeed, overexpression of ATF-4 alone extended lifespan in C. elegans (Statzer et al., 2022).In line with the role of ATF4 in longevity, long-lived mouse models including the Snell Dwarf and PAPP-A knock-out mouse, or those that underwent rapamycin treatment and nutrient reduction, exhibited a higher level and activity of ATF4 (Li et al., 2014;Li and Miller, 2015).ATF4 expression and that of its transcriptional targets were all elevated in the livers of mice in lifespan-extending conditions including treatment with acarbose, treatment with rapamycin, or diets low in calories or low in methionine (Li et al., 2014).These findings were in line with previous work, where long-lived mice with mutations that increase lifespan by alteration of endocrine pathways related to Growth Hormone (GH) and/or Interleukin Growth Factor-1 (IGF-1) had a similar increase in ATF4 protein in liver cells (Li and Miller, 2015), suggesting that an elevation of ATF4 function probably contributed to a slower aging process.Finally, our team recently described that inhibiting mitochondrial translation initiates an atf-4/ATF4-dependent cascade leading to coordinated repression of cytosolic translation and lifespan extension (Molenaars et al., 2020).Altogether, it seems clear that ATF4/ATF-4 has a conserved function in longevity promotion.
Our group identified NRTIs as geroprotective while computationally screening for small molecules producing a similar transcriptional profile to overexpression of the FOXO3 longevity transcription factor (McIntyre et al., 2021;McIntyre et al., 2023).Our initial screen identified zidovudine as a candidate longevity drug and subsequent experiments showed zidovudine extended lifespan, though this was not dependent on FOXO3 (daf-16 in worms) (McIntyre et al., 2021).RNA sequencing of zidovudine-treated worms demonstrated, amongst other changes, that treatment reduced mitochondrial ribosomal gene expression and cytosolic ribosomal gene expression (McIntyre et al., 2023).This finding was in line with previous work, identifying an Atf4/atf-4 dependent communication between mitochondrial and cytosolic translation longevity pathways (Molenaars et al., 2020).We then hypothesized that ATF-4 was required for lifespan extension and tested this hypothesis along with several other candidate longevity mechanisms.Of the candidate regulators we tested, atf-4 was the only gene required for zidovudine's lifespan extension.We further demonstrated that ATF-4 was not only necessary for lifespan extension but also improvements in healthspan and suppression of cytosolic translation in the worms (McIntyre et al., 2023).
Following our characterization of zidovudine as ATF-4-dependent and a life-and healthspan extending drug, we proceeded to test other NRTIs for their longevity effects in worms.Interestingly, most other NRTIs, including stavudine, zalcitabine, and alovudine, did not influence C. elegans lifespan at an equivalent dose to zidovudine (50 μM) (McIntyre et al., 2023).Only didanosine was able to increase lifespan at a dose 4-fold higher (200 μM) than zidovudine, and also did so in a manner dependent on atf-4 (McIntyre et al., 2023).These findings suggest that NRTIs affect worm lifespan through a process known as mitohormesis.In mitohormesis, treatment with a sub-lethal dose of a mitochondrial toxin would result in a mitochondrial and cellular defense response from the cell, ultimately providing net benefit to the cell and organism.Indeed, it is likely not a coincidence that both zidovudine and didanosine were early-developed NRTIs with high side-effect profiles due to mitochondrial toxicity.This would suggest that, upon treatment with zidovudine or didanosine (or equivalently mitochondrially toxic doses of other NRTIs), the mitochondrial function becomes partially impaired, subsequently triggering ATF-4 activation (McIntyre et al., 2023).This ATF-4 activation and suppressed translation then likely further results in the preferential translation of protective proteins (Rogers et al., 2011) and increased transcription of stress-related genes (Robida-Stubbs et al., 2012;Wang et al., 2010).Taken together, NRTIs like zidovudine and didanosine in C. elegans are able to achieve their lifespan-benefiting effects through a signaling cascade originating at the mitochondria, mediated by ATF-4, and culminated in the suppression of cytosolic protein translation (Fig. 2).

NRTIs reduce inflammaging
As seen from the inhibition of retrotransposon L1 elements in mice, reduced inflammaging is a major component contributing to NRTI benefits.Inflammaging is a term that describes the chronic and systemic inflammation that accompanies the natural aging process (Ferrucci and Fabbri, 2018).This inflammation is different from acute inflammation and is characterized by low-grade, persistent inflammatory signals.It arises in part from the accumulation of cellular damage over time, including oxidative stress and DNA damage, and is further compounded by dysregulation of the aging immune system (Ferrucci and Fabbri, 2018;López-Otín et al., 2023).Inflammaging is associated with numerous age-related diseases, including cardiovascular disease, diabetes, neurodegenerative conditions, and cancer, and can also contribute to frailty in the elderly (Ferrucci and Fabbri, 2018).
Remarkably, NRTIs have been described to prevent the activation of caspase-1, the crucial mediator of the NLRP3 inflammasome (Youm et al., 2013;Fowler et al., 2014).More specifically, NRTIs do not reduce the expression of NLRP3 but inhibit several cell permeabilization pathways activated by the P2X purinoceptor 7 (P2X7) protein (Baroja-Mazo et al., 2013), an LPS/ATP (Qu et al., 2011) and Alu RNA (Kerur et al., 2013) receptor capable of triggering the NLRP3 inflammasome.This results in the inhibition of caspase-1 activation, and the subsequent suppression of NLRP3 and NLRP3-mediated inflammatory cytokine production.
Following these discoveries, numerous other studies have repurposed NRTIs to specifically target the NLRP3 inflammasome in various age-related diseases, such as Alzheimer's Disease (Magagnoli et al., 2023), atrophic macular degeneration (Fukuda, Narendran et al., 2021), amyloid induced retinal degeneration (Narendran et al., 2021), and diabetes (Ambati et al., 2020), as well as non-aging related diseases including rhegmatogenous retinal detachment (Huang et al., 2023).In relation to anti-inflammatory therapy, it has been suggested that modified NRTIs, which are methoxylated and no longer act as chain terminators in reverse transcription, may avoid undesirable side effects on polymerase toxicity while retaining anti-inflammatory properties (Fowler et al., 2014).These developments may serve to improve NRTI repurposing in inflammaging.In summary, NRTIs have emerged as promising candidates to counteract NLRP3 inflammasome activation, offering potential treatments for multiple age-related diseases.

NRTIs and mitohormesis
It has been noted that NRTIs induce mitochondrial toxicity via inhibition of mitochondrial polymerase γ (Nolan and Mallal, 2004;Lewis et al., 2003).It may, therefore, seem paradoxical that NRTIs also have the capacity to extend lifespan and healthspan in C. elegans worms (McIntyre et al., 2023).In fact, these compounds may be acting through mitochondrial hormesis (mitohormesis).Hormesis is the term used to describe effects of stressors that are beneficial at small doses due to the activation of protective stress response pathways, yet become toxic at higher doses (Gems and Partridge, 2008).Hormesis has been designated a "hallmark of health" (López-Otín and Kroemer, 2021), as a growing body of research demonstrates this phenomenon contributes to physiological maintenance and prevention of pathologies.
Therefore, as NRTIs induce mitochondrial DNA (mtDNA) damage, yet confer a longevity benefit, it is likely they are acting in part through a mitohormetic mechanism, at least in C. elegans worms.Though polymerase γ was not studied specifically in relation to the NRTI longevity phenotype observed in C. elegans, the disruption of cytosolic translation via ATF-4 suggests impaired mitochondrial translation or other mitochondrial stress (McIntyre et al., 2023).Additionally, in mammals, damage to mtDNA has been connected to the mitochondrial integrated stress response (ISR), which regulates one carbon metabolism, nucleotide synthesis and dNTP pools via Atf4 (Chung et al., 2022).It is therefore plausible that activation of ATF-4 in worms treated with zidovudine is a protective stress response to the damage incurred upon mtDNA and associated mitochondrial stress.
Treatment of C. elegans with zidovudine also led to significant alterations in pyrimidine metabolism (McIntyre et al., 2023).This could also be explained by mitohormetic effects induced by mtDNA damage.Specifically, zidovudine caused a depletion of orotic acid, which could be explained by effects on dihydro-orotate dehydrogenase (DHODH/DHOD-1).DHODH is localized in the mitochondria and is connected to the respiratory chain via the pool of coenzyme Q (Boukalova et al., 2020).It is therefore likely that both zidovudine itself as a thymidine analog, and mitochondrial stress induced by damage to mtDNA, influence the observed alterations to pyrimidine metabolism.
Despite the robust lifespan extension observed with zidovudinetreated worms, didanosine-treated worms required four times the dosing of zidovudine to extend longevity in worms, and to a reduced degree (McIntyre et al., 2023).Moreover, stavudine, zalcitabine and alovudine did not noticeably extend longevity at either concentration tested (McIntyre et al., 2023).Since all these compounds have the potential to interfere with polymerase γ, it is surprising not to observe longevity benefits with all compounds.Yet, one of the hallmarks of hormesis is that specific doses are required to create beneficial outcomes (Gems and Partridge, 2008).Therefore, it is possible that testing a wider range of doses of these other NRTIs-both larger and smaller than those already studied-could lead to similar longevity benefits to those seen with zidovudine.Taken together, mito-hormesis may be a likely explanation for the longevity benefits of NRTIs observed in C. elegans worms.

Geroprotective use of NRTIs in humans
The NRTI-aging studies discussed in this review suggest a potential geroprotective effect for NRTIs.However, the toxic side effects observed clinically at therapeutic doses (including peripheral neuropathy, myopathy, lipoatrophy, hepatic steatosis with lactic acidosis, and more (Margolis et al., 2014)) present a major downside for these drugs.
Therefore, two questions emerge: is there a dose of NRTIs that could confer the geroprotective benefits seen in preclinical models in humans, while minimizing the existing detrimental side effects?And could new NRTI-related compounds be developed with minimal side effects?
While future studies will be needed to address this question, at least two clinical trials have studied administration of NRTIs as treatments for age-related diseases in humans.One of these is a clinical trial with the NRTI Lamivudine, where 12 subjects in the age range of 50-80 years with a clinical diagnosis of early Alzheimer's diseases receive 300 mg lamivudine, daily for 24 weeks (Clinical Trial ID NCT04552795).In the second study, the NRTI emtricitabine, is also being tested as a treatment for Alzheimer's disease (Clinical Trials ID NCT04500847).In this second study, individuals aged 50-85 years showing signs of either Alzheimer's disease or mild cognitive impairment are given a 200 mg daily oral dose.Excitingly, the results of these trials may lead to a novel therapy for Alzheimer's that slows aging at the same time.Notably, these doses are within the ranges of HIV treatments, and similar side effects may therefore be expected.It would be of future interest to titrate lower doses of NRTIs in an attempt to minimize the mitochondrial toxic effects of NRTIs while maximizing the protective effects of ATF4-activation, DNA protection, and inflammation-reduction demonstrated in aging models.
Nonetheless, these clinical trials highlight that dementia, including Alzheimer's disease, may be the most obvious first candidate for NRTI treatment for aging in humans.Indeed, retrotransposons have been linked to dementia (Ochoa Thomas et al., 2020;Gorbunova et al., 2021) and it has also been demonstrated in C. elegans worms and mammals that targeting mitochondrial translation can reduce amyloid aggregations (Sorrentino et al., 2017).Therefore, both mechanisms of NRTIs to slow aging are relevant for dementia (Fig. 2) and make Alzheimer's disease and dementia, in general, interesting first candidate age-related diseases for NRTIs in humans.

Conclusion
NRTIs have been a core component of HIV medications since 1987 and have provided a dramatic survival benefit for infected people (Fischl et al., 1987).In an effort to identify FDA-approved pharmaceuticals that extend lifespan, NRTIs have emerged as efficient geroprotective compounds (McIntyre et al., 2023;Simon et al., 2019a;De Cecco et al., 2019;Fowler et al., 2014).While the studies that recently described NRTIs as geroprotective modify longevity pathways that produce the same outcome-health and lifespan extension-their results point to alternative mechanisms in different studies (Fig. 2) (Simon et al., 2019a;McIntyre et al., 2023;De Cecco et al., 2019;Fowler et al., 2014).Together, this points to NRTIs interacting directly upon multiple hallmarks of aging (López-Otín et al., 2023).At the molecular level, inhibition of reverse transcriptase represses LINE-1 elements, reducing DNA damage and acting on the hallmark of aging of 'Genomic Instability' (Simon et al., 2019b;De Cecco et al., 2019;López-Otín et al., 2023).At the organellar level, NRTIs inhibit mitochondrial translation, activating ATF-4 and suppressing cytosolic translation, improving the hallmark of aging of 'Loss of Proteostasis ' (McIntyre et al., 2023;López-Otín et al., 2023).Finally, at the cellular level, NRTIs inhibit the P2X7-mediated activation of the inflammasome, reducing inflammation and improving the hallmark of aging of 'Altered Intercellular Communication' (Fowler et al., 2014;López-Otín et al., 2023).
One important consideration in the NRTI-longevity model we describe (Fig. 2) is that both proposed longevity mechanisms stemming from mammalian data (LINE-1-driven and P2X7-driven) involve a reduction of inflammation.Therefore, it remains to be seen if LINE-1 inhibition is upstream of the P2X7-driven reduction of inflammation from NRTI treatment.It may be that these observations are in fact in the same pathway, with NRTI benefits ultimately stemming initially from LINE-1 inhibition.Furthermore, additional benefits of NRTIs in aging requires more exploration.For example, NRTIs may prevent the reverse transcription of other retroelements in the genome that play a role in age-related diseases, as has been observed with Alu retroelements in recent studies (Fukuda et al., 2021;Fukuda, Narendran et al., 2021).It will be interesting to see what further developments emerge, which will add yet more dimentions to the mechanisms and hallmarks of aging through which NRTIs slow aging.Interestingly, it is not the first time geroprotective molecules are proposed to have multiple complementary mechanisms to slow aging.For example, HSP90 inhibitors may benefit health in mammals by clearing senescence cells (Fuhrmann-Stroissnigg et al., 2017), while in C. elegans, HSP90 inhibitors extend lifespan by activating the Heat Shock Factor 1 (HSF1) longevity-related transcription factor, eliciting improved proteostasis and health and lifespan extension (Janssens et al., 2019).In this case, and the case of NRTIs, these mechanisms are not necessarily mutually exclusive and could both ultimately contribute to benefits when working to translate findings to humans.

Contributions
All authors contributed to the writing of this review.

Declaration of Competing Interest
Authors declare no competing interests.

Fig. 1 .
Fig. 1.NRTIs inhibit reverse transcription.A, the reverse transcriptase of the human immunodeficiency virus (HIV) normally functions to incorporate nucleotides based on its viral RNA template creating a growing DNA strand.B, with nucleoside reverse transcriptase inhibitors (NRTIs), the nucleosides are used by the reverse transcriptase enzyme, but block the reverse transcription occurring by preventing additional nucleotides from being further incorporated.Figure is adapted from ViralZone 2014 SIB Swiss Institute of Bioinformatics.