ISRIB: The ISR InhiBitor

# # # # In December 2020, a research paper was published that drew a lot of attention. It involved a molecule that reversed memory deficits in aged mice – even weeks after treatment has stopped. The treatment involved inhibition of the integrated stress response. In today’s post, we will explore what the integrated stress response is, review the data presented in new report, and consider what might happen next with this line of research (and be warned, there is quite a bit of biology to kick things off). # # # #

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Title: Small molecule cognitive enhancer reverses age-related memory decline in mice.
Authors: Krukowski K, Nolan A, Frias ES, Boone M, Ureta G, Grue K, Paladini MS, Elizarraras E, Delgado L, Bernales S, Walter P, Rosi S.
Journal: Elife. 2020 Dec 1;9:e62048.
PMID: 33258451                  (This report is OPEN ACCESS if you would like to read it)

This report was published in December 2020.

It starts with a quote:

“Of the capacities that people hope will remain intact as they get older, perhaps the most treasured is to stay mentally sharp” (Source)

The report then proceeds to describe an experiment in which a small drug-like molecule reverses memory deficits in aged mice, even weeks after treatment has stopped.

Wow! What was the treatment?

The molecule is an ‘integrated stress response‘ (or ISR) inhibitor that has conveniently been called ISRIB (or ISR InhiBitor).

What is ISR and why would the researchers be inhibiting it?

The integrated stress response (or ISR) is a signaling pathway that is present in eukaryotic cells (cells that have a nucleus). It is activated in response to a selection of internal or external factors.

The external conditions can include nutrient deprivation and viral infection, while internal factors largely focus on endoplasmic reticulum stress as a result of the accumulation of unfolded proteins.

Source: Researchgate

What is endoplasmic reticulum stress?

The endoplasmic reticulum (or ER) is a highly convoluted, net-like mesh structure that extends off the nucleus. It is the assembly line where proteins are produced within a cell.

The endoplasmic reticulum. Source: Britannica

The nucleus is where the blue prints for making and maintaining an organism is kept in the form of DNA. A template of how to produce a particular protein can be generated from this DNA and that template is called messenger RNA (or simply mRNA).

mRNA is used to produce a protein (via a process called translation). A large part of that protein production process is conducted within the endoplasmic reticulum – proteins are produced and start to be folded into their correct confirmation. In times of cellular stress, however, unfolded proteins can start to pile up in the ER and this causes ER stress.

Ok, got it. So ER stress can initiate the integrated stress response?

Yes, it is one of the internal factors involved in initiating the ISR.

The internal or external factors activate four ISR regulators: the stress responsive protein kinases PERK, PKR, HRI, and GCN2.

What is a kinase? 

A kinase is an enzyme – a biological catalyst; a protein that facilitates specific chemical reactions in cells. In particular, kinases catalyze the transfer of phosphate groups from phosphate-donating molecules to specific recipients.

What’s a phosphate group?

A phosphate group is characterised by a phosphorus atom bonded to four oxygen atoms. They are important in activating proteins so that the proteins can perform particular functions in cells.

Ok, so what do the four ISR regulator kinases activate?

All four of the ISR regulator kinases converge on the core of the ISR: a protein called eIF2α.

Source: Embo

What is eIF2α?

Eukaryotic Initiation Factor 2 alpha (or eIF2α) plays a central role in protein synthesis. Specifically, it is involved in the early steps of that process.

But during periods of cellular stress, eIF2α is targeted by the stress responsive protein kinases. These proteins phosphorylate eIF2α which inhibits the ability of it to do it’s function, causing a reduction in global protein production.

Wait – what does phos…phory…late mean?

Phosphorylation of a protein is basically the process of turning it on or off – making it useful or inactivating it. From allowing a protein to fold in a particular manner to actually activating/deactivating the function of a protein, phosphorylation is a critical function in cellular biology.

Phosphorylation of a kinase protein. Source: Nature

Phosphorylation occurs via the addition or removal of phosphates. Their addition or removal determines the state of the protein being phosphorylated.

Phosphorylation of eIF2α by the stress responsive protein kinases inactivates/inhibits it.

This inactivation results in a reduction of protein synthesis, but there is an increase in the translation of a certain number of RNAs that are not affected by eIF2α phosphorylation. One of these gives rise to a protein called ATF4.

And what is ATF4?

Activating transcription factor 4 (or ATF4) is a protein whose activity is associated with its cellular localization. For example, under situations involving ER stress, ATF4 migrates to the nucleus, where it activates genes involved in many cellular pathways focused on restoring normality (or homeostasis) in cells.

Source: Frontiers

Problems start to arise, however, in periods of prolonged (pathological) ER stress. During such times, ATF4 can shift away from activating survival-oriented pathways in neuronal cells to activating pro-cell death (apoptosis) pathways.

Thus, chronic ER stress is not a good thing. And there is evidence that prolonged ER stress maybe occurring in neurodegenerative conditions associated with misfolded protein – like some cases of Parkinson’s.

For an excellent overview of the integrated stress response – click here.

# RECAP #1:  The integrated stress response (or ISR) is a signaling pathway that is activated in cells during times of stress. It results in the inactivation of a protein called eIF2α, which results in a reduction in protein production in the cell. During episodes of mild cellular stress, this inactivation can give the cell an opportunity to recover, but during sustained ISR activation the consequences can be negative (resulting in cell death). #

So what did the researchers investigate in their research report?

The investigators involved in the study had previously presented the discovery of a molecule that could inhibit the integrated stress response.

They called the molecule ISRIB (or ISR InhiBitor).

This is the report about the discovery:

Title: Pharmacological brake-release of mRNA translation enhances cognitive memory
Authors: Sidrauski C, Acosta-Alvear D, Khoutorsky A, Vedantham P, Hearn BR, Li H, Gamache K, Gallagher CM, Ang KK, Wilson C, Okreglak V, Ashkenazi A, Hann B, Nader K, Arkin MR, Renslo AR, Sonenberg N, Walter P.
Journal: Elife. 2013 May 28;2:e00498.
PMID: 23741617          (This report is OPEN ACCESS if you would like to read it)

In this study, the researchers conducted a drug screening experiment that involved evaluating 106,281 compounds for their ability to inhibits the PERK kinase. From that investigation, they identified a molecules that acted downstream of all four of the ISR protein kinases. That molecule potently (IC50 = 5 nM) reversed the effects of eIF2α phosphorylation.

This was the molecule that they called ISRIB.

Alone, ISRIB would be represent an extremely useful investigational tool, but when the researchers injected it into mice, they found a really interesting result: the molecule improved memory .

The investigators trained mice to find a hidden platform in a swimming-based memory test (called the Morris Water Maze). The water is made opaque and during the first training session (“1st trial” in the image below) the mice explore all areas of the pool. After a number of training sessions, the mice remember where the platform is and make a more direct path to it (see the “8th trial” image below).

Source: Brainstuff

The researchers reduced the number of training sessions in this study and found that mice injected with ISRIB were able to locate the hidden platform in the water maze more rapidly than control injected animals (displaying less latency in finding the platform):

Source: PMC

The researchers also reported that ISRIB improved performance in fear conditioning memory tests. These results indicated to the scientists eIF2α phosphorylation has an important role in “modulating higher-order brain function“.

These interesting results were replicated and then further explored in a follow up study, which was published in 2017:

Title: Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury.
Authors: Chou A, Krukowski K, Jopson T, Zhu PJ, Costa-Mattioli M, Walter P, Rosi S.
Journal: Proc Natl Acad Sci U S A. 2017 Aug 1;114(31):E6420-E6426.
PMID: 28696288                 (This report is OPEN ACCESS if you would like to read it)

In this study, the researchers reported that ISRIB rescued the cognitive ability of mice in two models of traumatic brain injury. Remarkably, this protective effect occurred even when ISRIB was “administered weeks after the initial injury and maintained cognitive improvement after treatment was terminated“.

The researchers concluded that their results “indicate that ISR inhibition at time points late after injury can reverse memory deficits associated with traumatic brain injury”.

~ For those interested, the exact mechanism of action for ISRIB has been explored in depth. Un-phosphorylated eIF2α binds with two other proteins, eIEF2β and eIF2γ, and collectively they initiate translation. Once phosphorylated, eIF2α limits the ability of this collection of subunits to do their function, grinding protein synthesis to a halt. Mutation analysis has demonstrated that ISRIB binds to eIF2β, where it stabilizes that protein and stimulates its activity independently of eIF2α phosphorylation. This action favors a restoration of translational activity (Click here and here to read more about this). ~

Interesting. Has ISRIB been tested in models of neurodegenerative conditions?

Yes it has.

Recently ISRIB administration was evaluated in a mouse model of Alzheimer’s and the results indicate a beneficial effect:

Title: Correction of eIF2-dependent defects in brain protein synthesis, synaptic plasticity, and memory in mouse models of Alzheimer’s disease.
Authors: Oliveira MM, Lourenco MV, Longo F, Kasica NP, Yang W, Ureta G, Ferreira DDP, Mendonça PHJ, Bernales S, Ma T, De Felice FG, Klann E, Ferreira ST.
Journal: Sci Signal. 2021 Feb 2;14(668):eabc5429.
PMID: 33531382                  (A preprint manuscript of this report is openly available)

In this report, the researchers evaluated ISR inhibition in multiple models of Alzheimer’s and found that improved performance on memory-associated behavior tests. They also reported that postmortem brain tissue from people who passed away with Alzheimer’s showed increased levels of phosphorylation of eIF2α (compared to unaffected control cases).

It should be noted, however, that this result stands in contrast to two previous studies that found no beneficial effect from ISRIB (Click here and here to read those reports). But the researchers in this new study note a significant difference in the dosing used between the studies: this new study found that low dose ISRIB (0.25 mg/kg) for several days had beneficial effects in the Alzheimer’s mouse model, while not having any noticeable effect in normal control mice. The previous studies used much higher doses (2.5-5 mg/kg).

Researchers have also published research suggesting that ISRIB treatment could have potential applications in motor neurone disease (Click here to read more about this).

Could ISRIB have an effect on age-associate memory issues?

So this is the trillion dollar question – could ISRIB help everyone as we age?

As we get older, there is a significant decline of protein synthesis in the brain. It has been proposed at this reduction correlates with defects in proper protein folding (Click here to read more about this). Inappropriate protein folding is believed to put pressure on the ER, which may result in a gradual increase in ER stress and activation of the ISR.

Recently researchers have looked at this question of whether ISRIB treatment could have any effect on age-associate cognitive issues. They reported their results in December 2020 – which brings us back to the report mentioned at the top of this post:

Title: Small molecule cognitive enhancer reverses age-related memory decline in mice.
Authors: Krukowski K, Nolan A, Frias ES, Boone M, Ureta G, Grue K, Paladini MS, Elizarraras E, Delgado L, Bernales S, Walter P, Rosi S.
Journal: Elife. 2020 Dec 1;9:e62048.
PMID: 33258451                  (This report is OPEN ACCESS if you would like to read it)

In this study, the researchers firstly started by looking at ATF4 levels in the brains of young and old mice. You will recall that we discussed activating transcription factor 4 (or ATF4) above – it is one of the RNAs that are not affected by eIF2α phosphorylation. The investigators looked at ATF4 levels in the brains of young and old mice, and they found that it was significantly higher in the older mice:

Source: PMC

When the researchers treated old mice with ISRIB, ATF4 levels returned to the levels closer to those seen in young mice (see graph above).

Interestingly, these ATF4 levels correlated with the number of errors  in spatial and working memory tests – older mice made more mistakes – but when the researchers treated the older mice with ISRIB, the mice made fewer mistakes:

Source: PMC

The investigators also looked at changes in brain activity that were associated with the ISRIB treatment and found that a single injection of ISRIB the day before testing could reverse certain age-associated changes in neuronal activity.

The researchers concluded that temporary treatment with ISRIB could lead to long lasting reductions of ATF4 (up to 20 days post-treatment). This ‘ISR reset’ was associated with improvements in spatial and working memory, and neuronal activity. They suggest that their data indicates that pharmacological attenuation of the ISR could relieve age-related neuronal changes “potentially resetting age-induced cognitive decline“.

# # RECAP #2: Numerous reports have indicated that a molecule called ISRIB (or ISR InhiBitor) is able to rescue the ISR response and restore protein production in the cell. This action has been reported to have beneficial effects in models of both neurodegenerative conditions and age-associated cognitive issues. # #

Has anyone conducted a clinical trial of ISRIB?

Not yet. But it will not be too far away.

In small print at the bottom of some of the reports discussed in this post, it has been stated that the rights to ISRIB have been licensed by UCSF (the research institute that discovered the molecule) to a biotech company called Calico.

Founded on September, 2013, the “California Life Company” (Calico) is a subsidiary of the Google parent company, Alphabet and it is focused on ‘life extension research and development’. It is being led by Art Levinson, the former chief executive officer of the biotech giant Genentech.

Art Levinson. Source: CSHL

Calico is an extremely secretive company – not disclosing much about what it is working on – but in 2014, it joined forces with the pharmaceutical company AbbVie (Source), and that deal was extended in 2018 (Source).

In February 2021, the companies provided an update about some of their collaborative projects (Click here to read that update), and of particular interest to this post is ABBV-CLS-7262.

What is ABBV-CLS-7262?

ABBV-CLS-7262 is an eIEF2β activator – similar to ISRIB.

The February update states that “ABBV-CLS-7262 is currently in Phase I studies with plans to begin a study later this year in patients with ALS” (Source). Where the Phase I studies are being conducted in unknown to me. But if all goes well, it will be encouraging to see a clinical trial in ALS/motor neurone disease starting later this year.

Is anyone else looking at molecules similar to ISRIB?

This is a really good question.

In the middle of their 2021 outlook earlier this year, Denali Therapeutics mentioned an update on DNL343 (Click here to read the outlook).

DNL343 is a brain-penetrant activator of eIEF2β – similar to ISRIB and ABBV-CLS-7262.

Limited data on DNL343 in preclinical models is available in the public domain (Click here to see a 2020 presentation), but in February 2020 the company began a Phase I clinical trial in the Netherlands evaluating single and multiple ascending oral doses of DNL343 in 88 healthy adults (Click here to read more about this study). And then in December, a second Phase I study was initiated in New Zealand to explore the effects of formulation and food on DNL343’s safety and pharmacokinetics. This second study involves 24 healthy men and women (Click here to read more about this study).

The Denali 2021 outlook suggested that the “results from a Phase 1 study of DNL343 in healthy volunteers are expected to be available in 1H 2021. Denali plans to initiate a Phase 1b study of DNL343 in patients with ALS in 2H 2021​”.

So there appears to be lots of potential clinical activity in the ISR neck of the woods.

Has anyone ever looked at eIF2α or ISRIB in the context of Parkinson’s?

As far as I’m aware, nothing has been published on ISRIB in Parkinson’s models – but folks may be working on this now given the attention that this molecule (and associated pathways) is getting.

And researchers have looked into elF2α levels in Parkinson’s. Back in 2007, this report was published:

Title: Activation of the unfolded protein response in Parkinson’s disease.
Authors: Hoozemans JJ, van Haastert ES, Eikelenboom P, de Vos RA, Rozemuller JM, Scheper W.
Journal: Biochem Biophys Res Commun. 2007 Mar 16;354(3):707-11.
PMID: 17254549

In this study, the researchers reported elevated levels of both phosphorylated PERK and phosphorylated eIF2α in the dopamine neurons on postmortem sections of brain from people who had passed away with Parkinson’s. Dopamine neurons are the population of cells in the brain most severely affected by PD.

Samples of brain from 13 people with Parkinson’s were compared with samples from 7 unaffected control cases, and the researchers found that dopamine neurons with detectable levels of phosphorylated PERK and phosphorylated eIF2α were only present in the PD cases.

Increased levels of phosphorylated PERK and phosphorylated eIF2α have also been detected in the brains of people with progressive supranuclear palsy (a condition similar to PD) and Alzheimer’s (Click here to read more about this).

So there may be broad potential applications for ISRIB-like molecules if they are found to have beneficial effects in other neurodegenerative conditions.

So what does it all mean?

There are an increasing number of researchers and biotech companies who are viewing aging as more of a treatable condition than an inevitable, unavoidable burden of life. What was once viewed as the realm of fantasy and fanatics is gradually being brought into the fold of acceptable research and gaining considerable attention in an aging world. And over the next few years we will start to see the results of clinical evaluations of some of these approaches and whether our understanding of the biology of aging is malleable.

While a lot of Parkinson’s research is focused on ‘personalised medicine’, there may well be opportunities for developing therapies with broader applications based on some of this age-related research (age is a common correlate in many neurodegenerative conditions).

That said, there is still a lot of research to be conducted in this area. All of the reports discussed in this post have come from the last 10 years and the field of ISR is still young. The different results coming from the preclinical Alzheimer’s work suggests that there may be a Goldilocks-like dosing effect that will need to be better understood.

And it will be interesting to see more research in the context of Parkinson’s exploring the biological mechanisms involved with the ISR. If this is conducted in parallel to the early clinical trials, perhaps by the time we have preclinical PD results, we may also have a clinical candidate ready for evaluating in a Phase II study.

Watch this space.

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ADDENDUM: 26th April 2021

Such is the pace of Parkinson’s research that the day after you publish a post on the SoPD, there is already interesting new research on the topic you just wrote about.

This report was published this week:

Title: Cell-type-specific disruption of PERK-eIF2α signaling in dopaminergic neurons alters motor and cognitive function.
Authors: Longo F, Mancini M, Ibraheem PL, Aryal S, Mesini C, Patel JC, Penhos E, Rahman N, Mamcarz M, Santini E, Rice ME, Klann E.
Journal: Mol Psychiatry. 2021 Apr 20. Online ahead of print.
PMID: 33879865

In this study, the researchers were interested in exploring PERK-eIF2α signaling in the normal functioning of dopamine neurons. To do this, they used multiple genetic approaches to selectively deletion of PERK protein in just the dopamine neurons of mice. Curiously, they report that this manipulation results in cognitive and motor complications. Their finding indicates that “proper regulation of PERK-eIF2α signaling in DA neurons is required for normal cognitive and motor function in a non-pathological state“.

The key words in that last sentence are “non-pathological state”. It may be that the PERK-eIF2α signaling pathway is sensitive to aging, but some caution may be required for the current attempts to pharmacologically modulate this pathway in terms of normal aging-associated cognitive issues. Perhaps (as we discussed above) there will need to be a delicate Goldilocks-like balancing act on dosing.

 

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