Intergraded Stress Response Inhibitor: ISRIB
Overview produce by Michael Slattery, Age-Regression.com 3/21/2021
Introduction
ISRIB is a powerful new tool with the potential to address so many diseases that when appropriately implemented will effectively appear to be antiaging intervention. To truncate a great deal of research into a few words, cellular stress is a precursor to disease, and the ability to downregulate protein expression in cells that are experiencing stress, constitutes a safety check the body utilizes to prevent those diseases. That cellular response is called the integrated stress response or ISR. Learning how to safely modulate releasing this safety system will entail a learning curve, adapting both dose, interval, timing and environmental factors. Because of the target and mechanism of action there are already 30 disease indications potentially treatable utilizing ISRIB or analogs. It is a safe bet that this list will grow dramatically.
“Just a few doses of an experimental drug can reverse age-related declines in memory and mental flexibility in mice, according to a new study by UC San Francisco scientists. The drug, called ISRIB, (pronounced IzRib) has already been shown in laboratory studies to restore memory function months after traumatic brain injury (TBI), reverse cognitive impairments in Down Syndrome, prevent noise-related hearing loss, fight certain types of prostate cancer, and even enhance cognition in healthy animals.” Additionally ISRIB has demonstrated its ability to improve mood, sleep quality, cognition, appetite and ameliorate pain. Improvements in neuron and immune cell functions have also been noted. [EXT-LINK] [EXT-LINK]
ISRIB was discovered at UCSF in a screening process designed to identify drugs that are ISR inhibitors by Carmela Sidrauski Ph.D., in the lab of Peter Walter, Ph.D. Carmela identified a potent small molecule that activates the translation initiation factor eIF2B and antagonizes the integrated stress response (ISR). The drugs derived from that first lead compound are currently in development by Calico Life Sciences, (Press Release) a subsidiary of Alphabet; Google (GOOG), in collaboration with AbbVie (NYSE: ABBV), (Abbvie’s Drugs in Development). Dr. Sidrauski is now the principal investigator of the development program at Calico, the Google life science division heavily involved in developing antiaging drugs. She was also the individual that identified the specific molecule now referred to as ISRIB as a lead therapeutic candidate. Although no information has recently been released to the public, it is likely that several analogs including ABBV-CLS-7262, were produced to improved the pharmacokinetics of the lead molecule, primary improving solubility.
ISRIB is currently available in both trans and cis forms from more than 800 sources online.
MOA Overview
During translation, regulation of protein synthesis by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is a common consequence of diverse stress stimuli, which leads to reprogramming of gene expression. This process, known as the integrated stress response, is one of the most fundamental mechanisms of translational control conserved throughout eukaryotes. It is also a promising therapeutic target in neurodegenerative diseases and traumatic brain injury including neurocognitive impairment secondary to HIV.
Pharmacological modulation of this central pathway has many potential therapeutic avenues that Carmela’s lab is exploring. In her graduate work, Carmela studied the unfolded protein response and discovered that this inter-organelle signaling pathway is mediated by an unconventional cytosolic splicing event catalyzed by a unique kinase/RNAse IRE1 and tRNA ligase.
The lead Calico neurodegeneration molecule (ABBV-CLS-7262) is an eIF2B activator which targets a key regulator of the highly conserved integrated stress response pathway. Inhibition of this pathway has therapeutic potential in a number of neurodegenerative diseases, such as ALS, Parkinson’s disease and traumatic brain injury. ABBV-CLS-7262 is currently in Phase I studies with plans to begin a study later this year in patients with ALS. ABBV-CLS-7262 is based on novel technology licensed from the lab of Peter Walter, Professor of Biochemistry and Biophysics at the University of California, San Francisco. Source Calico.
Background
The Integration Stress Response
Cellular stress comes from both inside and outside of the cell. These threats to the cells health and functionality take many forms; oxidative stress, toxins, nutrient deprivation, diseases, including cancer and viral infections, starvation or calorie restriction. There are of course many others including age-related toxicities produced by the cells internal originals as healthy processes deteriorate because of age. This is exemplified by the Unfolded Protein Response or UPR. All proteins produced in the cell must be correctly folded into functional and conformational appropriate proteins. If the proteins produced by the endoplasmic reticulum (ER), don’t get folded into the prescribed bioactive and appropriate shapes and these defective proteins start accumulating within the cell in an unfolded state, that triggers the UPR.
The integrated stress response is a cellular stress response conserved in eukaryotic cells that downregulates protein synthesis and upregulates specific genes in response to internal or environmental stresses
The integrated stress response can be triggered within a cell due to either extrinsic or intrinsic conditions. Extrinsic factors include hypoxia, amino acid deprivation, glucose deprivation, viral infection and presence of oxidants. The main intrinsic factor is endoplasmic reticulum stress due to the accumulation of unfolded proteins. It has also been observed that the integrated stress response may trigger due to oncogene activation. The integrated stress response will either cause the expression of genes that fix the damage in the cell due to the stressful conditions, or it will cause a cascade of events leading to apoptosis, which occurs when the cell cannot be brought back into homeostasis. [44]
The Endoplasmic Reticulum (ER)
▶︎The ER is an essential organelle of the cell that plays important role in protein folding and quality control [91, 92], lipid synthesis [93] and Ca2+ homeostasis [94]. During the life of the cell, different factors may perturb these functions, leading to a cellular state referred to as ‘ER stress’. These stressors may be intrinsic, i.e., cancer [95–98], neurodegenerative disease [99, 100], or diabetes [101, 102], or extrinsic, i.e., micro-environmental stress [103], exposure to ER stressors [104], temperature [105] or reactive oxygen species production [106, 107]. Nevertheless, every time the ER is stressed, it triggers an adaptive response. This adaptive response is called the unfolded protein response (UPR). This UPR will help the cells to counter the stress by attenuating protein synthesis, clearing the unfolded proteins and enhancing the ability of the ER to fold proteins. The UPR is an intracellular signal transduction mechanism that protects cells from ER stress. Three ER-resident transmembrane proteins function as stress sensors: RNA-activated protein kinase (PKR)-like endoplasmic reticular kinase (PERK); activating transcription factor 6 (ATF6); and IRE1. In basal state, these three transmembrane proteins are bound to BiP, an ER resident chaperone and are inactive [108, 109]. Upon a stress, the folding capacity of the ER is surpassed, leading to the dissociation of BiP from PERK, ATF6 and IRE1. This dissociation allows the activation of the three sensors [110]. Their activations transduce the unfolded protein stress signal across ER membrane and lead to UPR activation [111]. PERK is transmembrane ER resident protein of 1116 amino acids with two functional domains, a luminal and a cytosolic Ser/Thr kinase domain [112]. The dissociation of BiP from the luminal domain leads to oligomerization [108] and trans-autophosphorylation [113]. Activation of the PERK pathway leads to attenuation of general protein translation by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) [114]. Phosphorylated eIF2α inhibits eukaryotic translation initiation 2B activity, thus leading to a decrease of protein synthesis [115]. The blockage of the translation during ER stress diminishes the protein load on the ER folding machinery and is a prerequisite to a reestablishment of the ER homeostasis. In contrast to its attenuation of translation, eIF2α phosphorylation can selectively enhance the translation of mRNAs containing inhibitory upstream open reading frames in their 5_ untranslated region, such as activating transcription factor 4 (ATF4) [116]. The production of ATF4 induces the expression of a plethora of adaptive genes involved in amino acid transport, metabolism, protection from oxidative stress, protein homeostasis and autophagy [117]. Finally, ATF4 favors the expression of CAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), which will result in the expression of genes that are involved in protein synthesis and the UPR. If the expression of CHOP is sustained, the increased protein synthesis will lead to oxidative stress and cell death [118]. ◀︎[23]
Protein homeostasis (proteostasis) refers to an extensive network of components that acts to maintain proteins in the correct concentration, conformation, and subcellular location, to cooperatively achieve the stability and functional features of the proteome.
ISRIB & ABBV-CLS-7262
A phase one study of ABBV-CLS-7262 has been started by Abbie. Link is to a Clinical Trials.Gov overview of the clinical design and recruiting status.
ISRIB inhibits the Integrated Stress Response (ISR) in eukaryotic cells. When the ISR is activated by cell stress, the production of proteins by cells are downregulated. ISRIB turns this production back on restoring cellular function. ISRIB reverses the protein production block by allosterically antagonizing the inhibitory effect of phosphorylated eIF2 on eIF2B, a dedicated guanine nucleotide exchange factor, enhancing its activity independently of phosphorylation.
Aging activates the integrated stress response, contributing to changes in brain function and memory. In particular, aging leads to diminished protein synthesis within the brain related to protein folding defects. The effects of the drug were rapid, occurring after a few doses. “ISRIB’s extremely rapid effects show for the first time that a significant component of age-related cognitive losses may be caused by a kind of reversible physiological “blockage” rather than more permanent degradation,” explained Susanna Rosi, a UCSF professor in prepared remarks. [42]
The ISR, together with other cellular adaptation pathways, functions as an important part of the cellular defense strategy in response to stress. It does this mainly through altering / inhibiting global protein synthesis and through the regulation of genes that promote pro‐survival signaling such as through the activation of autophagy, or that counteract pathways that lead to cell death such as apoptosis or proteotoxicity (impairment of cell function due to the effects of misfolded proteins). Notably, there is also a cross‐talk between the ISR and other pro‐survival pathways such as the UPR, phosphatidylinositol‐3 kinase (PI3K) signaling, autophagy, and the ubiquitin‐proteasome system. (See graphic above right.)
The accumulation of unfolded proteins in the ER induces a condition of ER stress, which is relieved by the reduced level of incoming proteins when global protein synthesis is inhibited 1. PKR activation of the ISR during viral infection helps to reduce the translation of viral mRNAs, thus protecting the cells 8, 42. Under conditions of amino acid depletion, activation of the ISR by GCN2 reduces the need for amino acids for protein synthesis, thus alleviating this stress 34. Activation of the ISR by HRI under conditions of low heme lessens the need for heme by attenuating the translation of globin mRNAs, thus reducing the stress and promoting survival 52. It is important, however, to note that timely termination of the ISR also plays a key role in promoting long‐term cell survival, by re‐starting synthesis of essential proteins. This is achieved through dephosphorylation of eIF2α by the phosphatase GADD34, which is induced by ATF4 and its downstream targets CHOP and ATF3 15, 78, 79, 166. [23]
ISRIB Pre-Clinical Research
Testing in 2017 indicated an experimental drug (ISRIB) improved the ability of brain-injured mice to learn and form memories on memory tests, thus appearing to reverse impairments from a wide variety of disease and aging related cognitive impairments, including traumatic brain injury. Little clinical information exists outside of the initial mouse studies with the exception of a large number of antidotal reports of individuals self-medicating. [29]
ISRIB treatment also corrects spatial memory deficits and improves working memory in aged mice [10] Just a few doses of an experimental drug can reverse age-related declines in memory and mental flexibility in mice, according to a new study by UC San Francisco scientists. The drug, called ISRIB, has already been shown in laboratory studies to restore memory function months after traumatic brain injury (TBI), reverse cognitive impairments in Down Syndrome , prevent noise-related hearing loss, fight certain types of prostate cancer , and even enhance cognition in healthy animals.
In the new study, researchers led by Rosi lab postdoc Karen Krukowski , Ph.D., trained aged animals to escape from a watery maze by finding a hidden platform, a task that is typically hard for older animals to learn. But animals who received small daily doses of ISRIB during the three-day training process were able to accomplish the task as well as youthful mice, much better than animals of the same age who didn't receive the drug.
The researchers then tested how long this cognitive rejuvenation lasted and whether it could generalize to other cognitive skills. Several weeks after the initial ISRIB treatment, they trained the same mice to find their way out of a maze whose exit changed daily—a test of mental flexibility for aged mice who, like humans, tend to get increasingly stuck in their ways. The mice who had received brief ISRIB treatment three weeks before still performed at youthful levels, while untreated mice continued to struggle.
Current Target List of Potentially Treatable Disease Indications Utilizing eIF2B activators like ISRIB
Age-Related Cognitive Decline
Age-Related Immune Suppression
Allergic Rhinitis
Alzheimer's Disease Alector Different Drug
Amyotrophic Lateral Sclerosis (ALS)
Anti-Anxiety
Anti-Psychotic
Arteriosclerosis
Appetite Stimulation
Bipolar Disorder,
Bipolar Depression
Chronic Rhinosinusitis
Demyelinating Diseases
Down Syndrome
Frontotemporal Dementia
Hearing Loss
HIV Cognitive Impairment
HIV Kick & Kill
Kidney Disease
Major depression disorder
Migraine
Mood Elevation
Multiple Sclerosis
Neurodegenerative disease
Obstructive Sleep Apnea
Parkinson's Disease
Peripheral Neuropathy
Post-Traumatic Stress Syndrome
Prion Disease
Prostate Cancer
Sarcopenia
Schizophrenia
Social Anxiety Disorder
Stroke, TIAs
T-Cell Regulated Immune suppression
Traumatic Brain Injury & Recurring TBI
Vanishing White Matter Disorder
Wasting Syndrome
HIV, UPR and ISR
Many types of viruses actively regulate the ISR in their host cells, showing a great variety in the manners and mechanisms by which they do so, preventing components of ISR activity harmful to virus survival and replication and exploiting ISR components beneficial to the virus, and even showing cell‐type specificity. Importantly, while the primary purpose of the ISR is to promote cellular survival in the short term, chronic ISR activation in response to sustained injury/insult or in cases of extreme insult can also lead to orchestrated cell death 15, 36, 38, 41, 42. Thus, chronic ISR activation may provide a link between the inflammatory response to HIV infection in the CNS and neuronal dysfunction, damage and death in HAND. [3] One strategy to address quiescent HIV infected cells is to activate those cells utilizing a process euphemistically referred to as Kick and Kill. Partially releasing the virally induced protein suppression via ISR may have a part in improving this strategy. This is also an immunological stranglehold the virus induces to provide a cellular sanctuary.
Reovirus and the Host Integrated Stress Response: On the Frontlines of the Battle to Survive
One potential solution to this problem is if the virus encodes or induces an ISR antagonist that can reverse the effects of P-eIF2α inhibition on eIF2B function. Recently, a small molecule ISR inhibitor (ISRIB) was identified that acts to stabilize assembled eIF2B [87,88]. As mentioned previously, eIF2B is composed of two copies of five subunits—α, β, δ, γ, and ε—that assemble to form the fully function eIF2B. ISRIB is able to bind to and stabilize the interaction between the two eIF2Bδ subunits that are part of the regulatory unit composed of eIF2Bα, eIF2Bβ, and eIF2Bδ [88,89]. An increased assembly of eIF2B results in increased GEF activity, allowing for increased resistance to P-eIF2α [90]. If one of the MRV proteins mimics ISRIB, we would expect that even with virus-induced P-eIF2α, the function of eIF2B would be enhanced, and translation could continue. Furthermore, since most of the MRV proteins localize to VF, we might expect this increased eIF2B assembly and function to also localize around the VFs. Another possible solution to this problem would be if MRV encoded a protein that is functionally similar to the AcP10 protein of beluga whale coronavirus, which is a P-eIF2α/eIF2B inhibitor [91]. AcP10 specifically binds to eIF2B and inhibits P-eIF2α binding, thereby allowing non-phosphorylated eIF2α to bind and GEF function to continue, effectively reversing the impact of P-eIF2α even when induced by high levels of stress via sodium arsenite [91]. Further investigation into the function of MRV proteins will identify if the virus encodes proteins involved in altering eIF2B function or has other mechanisms to overcome the eIF2B problem.
Conversely, elvitegravir but not lopinavir led to increased eIF2α phosphorylation, indicating the activation of a common adaptive pathway termed the integrated stress response (ISR), and elvitegravir-mediated neurotoxicity was partially alleviated by the ISR inhibitor trans-ISRIB, suggesting ISR as a promoter of elvitegravir-associated neurotoxicity.
HIV infection and antiretroviral therapy lead to unfolded protein response activation
HIV promotes translational attenuation through eIF2α activation
The expression of the phosphorylated form of eIF2α (P-eIF2α) was analyzed under both in vitro and in vivo conditions by Western blotting. Phosphorylation of eIF2α represents its activation, leading to translational inhibition inside the cell. Under these conditions only, certain spe- cific genes related to the ER stress response do not have their expression attenuated. Furthermore, eIF2α phos- phorylation is a consequence of previous PERK activation, suggesting activation of this UPR sensor. In the present study, no difference in P-eIF2α levels was observed in non-infected PBMCs, regardless of the ARV treatment. However, HIV-infected PBMCs that were submitted to ARV treatment showed significantly higher expression of P-eIF2α compared with non-infected cells (Fig. 2a). Inter- estingly, the highest expression of P-eIF2α was found in cells treated only with ritonavir (p < 0.001) compared with the other treatments (p < 0.05).
ATF6 is cleaved in HIV-infected cells
ATF6 is one of the three major sensors of the UPR path- way, and it is activated after cleavage that occurs in the Golgi apparatus. Cleaved ATF6 is a transcription factor that leads to increased expression of UPR-related genes, including the gene encoding the chaperone BiP. In the current study, Western blotting assays were performed with an antibody capable of detecting the cleaved and non-cleaved forms of ATF6. In both in vitro and in vivo assays, a larger amount of ATF6 was cleaved when cells were infected with HIV, and ARV therapy did not significantly affect the expression profile of this protein. In particular, the expression of cleaved ATF6 in HIV- infected PBMCs was significantly higher (p < 0.05) com- pared with expression in non-infected PBMCs (Fig. 4a). Monocytes from HIV-infected patients showed higher, but not statistically significantly higher, expression of the cleaved form of ATF6 compared with monocytes from healthy individuals (Fig. 4b). A similar phenotype was observed in CD4+ T lymphocytes, although the expres- sion of cleaved ATF6 was significantly higher in cells from HIV-infected patients. Moreover, treatment-naïve patients showed slightly higher expression of this protein (p < 0.001) compared with patients under ARV therapy without a protease inhibitor (p < 0.05) or with a protease inhibitor (p < 0.01). These ATF6 cleavage results from both the in vitro and the in vivo assays suggest that HIV is responsible for the activation of this UPR pathway sensor.
Conclusions: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus, two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs’ pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented.
Several studies have shown that some of the soluble factors that are released as part of the HIV‐induced inflammatory response, as well as HIV viral proteins themselves, are able to induce the integrated stress response (ISR) [endoplasmic reticulum (ER) stress response, unfolded protein response], suggesting that the ISR may play a role in HAND 10. The ISR orchestrates a multitude of cellular responses against a wide range of extra‐ and intracellular stresses by regulating intracellular Ca levels; ER chaperone genes; protein synthesis, folding and degradation; and the endogenous antioxidant response 10-18. A variety of insults can trigger the ISR, such as oxidative stress, hypoxia, toxins, nutrient deprivation and viral infection. In fact, many types of viruses actively regulate the ISR in their host cells, showing a great variety in the manners and mechanisms by which they do so, preventing components of ISR activity harmful to virus survival and replication and exploiting ISR components beneficial to the virus, and even showing cell‐type specificity
ISRIB Mechanism of Action
Model for ISRIB’s mechanism of action: ISRIB staples together tetrameric eIF2B(βγδε) subcomplexes, building a more active eIF2B(βγδε)2 octamer. In turn, the ISRIB-stabilized octamer binds eIF2B(α2) with greater affinity, enhancing the formation of a fully-active, decameric holoenzyme.
Abbvie and Calico Collaboration
Abbvie has not identified the molecular structure of their current drug candidate; ABBV-CLS-7262. How close an analog to the structure of ISRIB is unknown at this time. It is very likely that there is very strong strong sequence homology, but it is unlikely that this lead compound is identical to ISRIB. A phase one clinical trails has been initiated. Oncology Pipeline, All Other Indications.
A press release put out by Calico indicates that the first clinical trial will begin later this year. On February 3, 2021– Calico Life Sciences and AbbVie (NYSE: ABBV) announced clinical-stage programs in two areas – immuno-oncology and neurodegeneration, currently in Phase I studies.
The lead Calico neurodegeneration molecule (ABBV-CLS-7262) is an eIF2B activator which targets a key regulator of the highly conserved integrated stress response pathway. Inhibition of this pathway has therapeutic potential in a number of neurodegenerative diseases, such as ALS, Parkinson’s disease and traumatic brain injury. ABBV-CLS-7262 is currently in Phase I studies with plans to begin a study later this year in patients with ALS. ABBV-CLS-7262 is based on novel technology licensed from the lab of Peter Walter, Professor of Biochemistry and Biophysics at the University of California, San Francisco.
Similar Drug Candidates
Abbott Pharmaceuticals becomes Abbvie in 2013: https://en.m.wikipedia.org/wiki/AbbVie
Denali Therapeutics:
https://www.denalitherapeutics.com
A similar drug (DNL343) is being developed by Denali Therapeutics. DNL343 is a brain-penetrant activator of the eukaryotic initiation factor EIF2b. It inhibits the cell's unfolded protein response in an attempt to restore protein synthesis. Normally, EIF2b, with its EIF2a subunit, functions to initiate mRNA translation. Under conditions of cellular stress, phosphorylation of the EIF2a subunit by the PERK kinase leads to inhibition of EIF2b, and pauses protein synthesis as part of the cell’s integrated stress response (ISR). Loss-of-function mutations in EIF2b that impair protein translation cause a progressive neurodegenerative syndrome affecting the brain and spinal cord, called vanishing white matter disease (see OMIM). DNL343 is the most advanced product candidate in our EIF2B program, and is a brain penetrant small molecule designed to activate EIF2B function in ALS / FTD.
Chemical Syntheses
General methods: Commercially available reagents and solvents were used as received. Compounds ISRIB-A1 and ISRIB-A2 were prepared as previously reported (Sidrauski et al., 2013b). Compound ISRIB-A7 was available commercially from Specs (The Netherlands). 1H NMR spectra were recorded on a Varian INOVA-400 400 MHz spectrometer and a Bruker Avance 300 300 MHz spectrometer. Chemical shifts are reported in δ units (ppm) relative to residual solvent peak. Coupling constants (J) are reported in hertz (Hz). LC-MS analyses were carried out using Waters 2795 separations module equipped with Waters 2996 photodiode array detector, Waters 2424 ELS detector, Waters micromass ZQ single quadropole mass detector, and an XBridge C18 column (5 μm, 4.6 Å~ 50 mm). Microwave reactions were carried out in a CEM Discover microwave reactor.General procedure A for amide coupling To a solution of the carboxylic acid (1 equiv.) in N,N-dimethylformamide, were sequentially added 1-hydroxybenzotriazole hydrate (1.2 equiv.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 equiv.), 2-(4-chlorophenoxy)-N-[(1r,4r)-4-aminocyclohexyl]acetamide trifluoroacetic acid (1.0 equiv., prepared as described in the synthesis of ISRIB-A8, below) and N,N-diisopropylethylamine (1.5 equiv). The reaction mixture was stirred at room temperature until judged complete by LC-MS and then diluted with water (2 ml). The mixture was vigorously vortexed, centrifuged and the water was decanted. This washing protocol was repeated with water (2 ml) and then with diethyl ether (2 ml). The wet solid was dissolved in dichloromethane (10 ml) 47 and dried over anhydrous magnesium sulfate. The solids were removed by filtration and the filtrate was concentrated by rotary evaporation to obtain the product.
Online Sellers of ISRIB:
https://en.isrib.shop 874 hits for “ISRIB” on Alibaba
There are also many chemist within the biohacking community know to those on the respective blog sites who will fill orders from individuals within those sites.
Formulation
ISRIB 1.0 or ISRIB 1.5 were prepared the day of administration and dissolved into 5 mg/ml in either DMSO or NMP by vortexing, heating, and sonication. Immediately prior to PO dosing nine volumes of HPMT was slowly added to 5 mg/ml solvent dissolved ISRIB to generate a milky suspension of 0.5 mg/ml. Mice were subsequently dosed at 10 ul/g. Immediately prior to IP dosing, solvent dissolved ISRIB 1.0 or ISRIB 1.5 was diluted to 4 mg/ml and combined 1:1 with PEG400. Mice were then dosed at 2.5 ul/g.
Sublingual Oral Formulation
The drugs that are administered sublingually generally have low solubility. Therefore, to enhance dissolution, it is crucial to reduce and control the particle size of the API. This attribute is important in the case of all drugs with low solubility. However, a tighter control on particle size of API is desirable in sublingual drug products to maintain the reproducible quality and performance of the drug product in view of the limited window of dissolution and absorption time.
The directly compressible sublingual tablet formulation contains directly compressible soluble excipients, a super disintegrant, and lubricant. It may also contain microcrystalline cellulose, dry binder, buffers, surface-active agents, sweeteners, and flavors. Sugar-based excipients are widely used as bulking agents because of their high aqueous solubility, sweetness, pleasant feeling in the mouth, and good taste-masking. Nearly all sublingual formulations incorporate some saccharide-based material (60). The choice of a suitable disintegrant and its amount are critical for achieving a fast disintegration and dissolution rate. Sometimes effervescent agents are used to increase disintegration and dissolution of sublingual tablets.
Test of TBI injury (Proof of Concept in an Animal Model)
Rosi and her team first used mechanical pistons to hit anesthetized mice in precise parts of their surgically exposed brains, resulting in contusive injuries, focused blows that can also result from car accidents or being hit with a heavy object. After 4 weeks of rest, Rosi trained the mice to swim through a water maze, where they used cues to remember the location of a hidden resting platform. Healthy mice got better with practice, but the injured ones didn’t improve. However, when the injured mice were given ISRIB 3 days in a row, they were able to solve the maze just as quickly as healthy mice up to a week later. “We kept replicating experiments, thinking maybe something went wrong,” Rosi says. So the team decided to study ISRIB in a second model of traumatic brain injury known as a closed head injury, which resembles a concussion from a fall. They again used a mechanical piston, but this time landed a broad blow to the back of the skull. Two weeks later, the mice were trained on a tougher maze, full of bright lights and loud noise. They had to scurry around a tabletop with 40 holes, looking for the one with an escape hatch. Again, while the uninjured mice improved at the task, the concussed mice never got the hang of it. But after four daily doses of ISRIB, the concussed mice performed as well as their healthy counterparts. “This is the most exciting piece of work I’ve ever done, no doubt,” Rosi says. [42] Check out this ref for dup text prior to this paragraph.
Review of antidotal human experience
The biohacking community has been ingesting ISRIB via various routes for several years. Although the observations and self-reporting from these individuals are uncontrolled the fact that multiple individuals have reported similar responses implies that the antidotal reports are not just a placebo effects. Multiple, uncontrolled first-person reports of their experiences can be found on the following sites: Discord-ISRIB, Reddit-r/ISRIB, Reddit-r/ISRIB, Phoenix Rising, (1, LongeCity-ISRIB Group, Those results are cataloged by type below. A review of ISRIB results are difficult to accurately ascertain because you are assuming that all respondents are: 1) actually taking an active, structurally identical molecule it ISRIB; 2) are administering pharmacologically active doses within a suitable biologically active dose range; 3) are incorporating a route of administration that delivers active PK levels of the drug; 4) are being straight forward and have no ulterior motives; 5) are not reporting placebo effects; 6) are not taking other concomitant psychoactive medications or recreational drugs; 7) do not have ongoing active disease processes causing cellular stress like viral infections, calorie restriction, cancer, or diabetes, resulting in the acute activation of the ISR response; 8) have not recently experienced some form of traumatic stress or physical concussions and finally 9) there are no controls to contrast and compare between these observational reports.
The majority of the sites where individuals are self-reporting, are self-identified as nootropic blogs. That presupposes most of the individuals on these sites have or are currently taking some kind of psychoactive smart drugs or cognitive enhancing compounds. Some of the sites are longevity or anti-aging sites. Many of the individuals on these sites are taking multiple nutritional supplements (referred to as a stack) and untested compounds reported in the literature, but not currently approved as a drug by the FDA. One such site providing untested drugs and compounds can be reviewed here.
The drug appears to cross the blood brain barrier in multiple doses ranges and routes of administration including snorting the pure ISRIB compound, rectal insertion, oral ingestion with multiple carriers, IV IM and IP, dissolved in DMSO and/or PEG400.
A consensus appears to be emerging on the best route of administration from several of these sites. The following process is utilized: Dissolve ISRIB in DMSO in a small sterilized glass container. A shot glass will accomplish this, dipping it in boiling/hot water to sterilize it. A bath of hot water will also facilitate getting it to dissolve faster. The syringe can also be utilized to surge the ISRIB/DMSO back and forth between the syringe and the shot glass to assist in dissolving the ISRIB in the DSMO solution. An ultrasonic vibrator can also be utilized to facilitate the process. Then you use a syringe to pierce and extract the fish oil from a soft gel capsule. . You refill the gel-cap capsule with the ISRIB/DMSO solution. utilizing the same syringe then swallow the pill. To increase the bioavailability some individuals are also adding propylene glycol or PEG 400 to this formula.
From a pharmacokinetic perspective this is likely successfully being administered via all of these routes in spite of its very low solubility factor. This low solubility is likely being overcome by a very high therapeutic index, requiring only .005ng /mol to activate eIF2B.
YouTube: ISRIB in DMSO into Gel Capsules
ISRIB appears to be safe. Indeed Dr. Walker can be viewed in his YouTube video categorically stating that the drug is “Safe.” Very few individuals reported self-limiting toxic side-effects but those reports are suspect. Several individuals have reported “Brain Fog,” without providing a detailed description of what exactly that means. They also indicated that it was resolved with food. From these first person reports ISRIB looks very promising, but also not without some potential problems. It appears that there are counter-indications. You would not want to take this with any recreational and/or addictive drugs as it could increase the likelihood of reinforcing memories that would facilitate a chemical dependence. You would also want to avoid ISRIB if you have recently experienced some kind of traumatic event as it would likely reinforce those memories, potentially leading to PTSD. Because the ISR is a cellular protein production safety-break, triggered by oxidative stress, viral infections, amino acid starvation, and UPR-ER stress, it would seem likely that long term constant steady-state-drug-levels of ISRIB could be detrimental. Ideally short intervals of drug administration, limiting both dosage and time of administration would be the safest strategy until dose ranging human clinical trials have been conducted. In the complete absence of controlled human clinical trials pulsing the drug at the lowest does that improves memory or cognition would be prudent. Additionally, any individual dosing ISRIB would want to stop well in advance of events that could be described as periods of vulnerability (POV). POVs would be any event that is likely to induce a high level of stress or anxiety. Again, you don’t want to indelibly imprint negative memories onto your long term memory. Human anecdotal experience has reported the occurrence of a single cardiac related adverse events. These cardiac events where not first person and unattributed to a source. There was one additional reports of an individual who experienced heart arrhythmias, accompanied by fainting spells and that is detailed below. Cardiac events reported in dog, where an adverse event seen in a clinical analog of ISRIB, not ISRIB. That compound is not being moved forward by Abbvie.
The following are the reported effects experienced and grouped by type. Where many similar effects where reported on a few anecdotal experiences where cataloged here.
APPETITE
Dramatic increase in appetite
“my appetite increased twice”
“However, and this part concerns me the most, throughout the day I sometimes experienced brain fog if I didn't eat enough. I was eating a whole lot more than usual (compared to my pre-ISRIB state) yet I did not seem to be gaining weight... I was over-eating to counteract the effects of what I'd considered brain fog from ISRIB — and I assumed that this was because my brain now simply required more "energy"/calorie consumption because of my perceived increase in cognition”
“I find myself needing to consume more food or else I will feel unusually groggy (brain fog); this used to not be the case. I'm not sure of the mechanism of action behind this (perhaps glucose metabolism, but that's purely speculation), but it could be that ISRIB is taxing my brain more.”
ANXIETY
Resolution of some social anxiety
15mg Oral Nasal: “it made me less anxious. Now a am able to speak to people, not having any bad feelings, not thinking they will definitely deceive me, what is a new thing to me.”
CARDIOVASCULAR
No conclusion to draw from the reported and conflicting events
Mike - “I was the one that went to the ER. Tbh I am pretty frail and suffer from some undiagnosed, wierd shit, It was atrial fibrillation and I had a very irregular heartbeat. I may be predisposed to it plus I was taking a large variety of various nootropics/supplements over the past several months”
Evan- “I had some problems with my heart before isrib. Now all is ok, I feel better with my cardiovascular system.”
DREAMS / NIGHTMARES
Increased vivid dreams
15mg Oral Nasal: “I consider such things like taking a pill and becoming a genius being a nonsense but can say that dreams (while sleeping, don’t know how its called in English) become very strange, strong and awful in some way.”
ENERYG / ACTIVITIES OF DAILY LIVING
Several individuals described, Brain Fog,” increases of energy and better willingness to attend to activities of daily living.
“I find myself needing to consume more food or else I will feel unusually groggy (brain fog); this used to not be the case. I'm not sure of the mechanism of action behind this (perhaps glucose metabolism, but that's purely speculation)”
“My typical dosage is about 15mg orally without DMSO. After 5 minutes I feel a strong surge of energy.”
“ridiculous amounts of energy”
EMOTIONAL STATE
“I experienced a kind of emotionlessness rather quickly after taking ISRIB which never really bothered me. It was a kind of contentment - emotional apathy, even though I felt euphoric for most of the time.”
MEMORY LONG TERM
Improved long term memory
“I also felt as if I had regained long-lost memories of events that had vanished”
MEMORY SHORT TERM
“My memory and ability to perceive information significantly improved,”
“My brain started to work in another way, my memory and learning patterns are so good.”
“It mens practicaly, that while reading a text, it is way much easier to remember the words, expression, and contexts as well (after 1-2 days).”
PERIPHERAL NEUROPATHY
Resolution of multiple neuropathy pain.
Several individuals described the resolution of pain in their flank , thighs, fingers and toes.
PSYCHIATRIC
Resolution of severe depression.
15mg Oral Nasal: “Throughout this time, perhaps the most severe depressive episode of my life that had finally ended”
15mg Oral Nasal: “10 minutes i felt weak euphoria,”
15mg Oral Nasal: “Music and food felt like i'm on acid”
15mg Oral Nasal: “After a few days my serious mania and craziness weaked a bit so i could controll them (yeah, on the second day of this trial i took a knife(not willing to kill anyone of course) and went running in forest at night, feeling like superhero)”
SOCIALIZATION
Dramatic increase in socialization
“I'm now noticing profound changes in social awareness - the body language processing that was absent previously is now really apparent.”
“near constant state of extroverted excitement and euphoria”
“My confidence in pretty much every conceivable aspect accelerated”
“socialized to new heights that I had previously considered impossible,”
SLEEP
Improved sleep quality, reduced need for normal night’s sleep duration
“My energy levels felt absolutely phenomenal; I couldn't seem to be able to get more than six or seven hours of sleep because as soon as I woke up I instantly felt "wide awake" and I wanted to make the most of the day. I was happy as hell to just be alive. Note: I was also sleeping regular hours, so these benefits definitely did not arise from sleep deprivation. This is important for to emphasise, because before taking ISRIB I felt so utterly drained of energy regardless of whether I slept 7-12 hours.”
“On another note, each time I have taken ISRIB, for the first and second night, I am getting the longest "Deep Sleep" cycles I have had in a long time. After taking ISRIB, the Deep Sleep cycles have ranged from ~30 mins to ~60 mins. I typically only get a deep sleep cycle every few days and it rarely extends beyond ~15 mins.”
“I'm finding it extremely difficult to get more than 6 hours of sleep per night. And, when I do sleep, it is always disturbed. Always. I will wake up from terrifically vivid dreams (just had one - no lucid dreaming for me yet, though) and be only able to get 30-60 minutes before awakening again.”
TINNITUS
Transient resolution
“Initially after taking ISRIB, I did notice the ringing was reduced, but its back now. Tinnitus comes and goes on its own, so I don't think ISRIB had any affect on it.”
VISION
Improvement in vision were reported by one individual.
Drug Profile
ISRIB has poor solubility. So poor that it was initially rejected in a preliminary drug screen. ISRIB has an extremely high cellular potency [trans-ISRIB’s IC50 is about 5 nM], which equates to equal concentrations of ISRIB is 1000x more effective than most other primary compounds. Accordingly, one could use a thousand times less of it and still get the desired effect. It turns out that solubility is not a limiting factor for ISRIB. After pharmacokinetic profiling, it became clear that ISRIB would reach sufficient bioavailability for in vivo studies, even more importantly, it could cross the blood-brain barrier. They discovered that even a single injection of ISRIB was sufficient to deliver ISRIB to the brain several folds higher than its required IC50.
Companies with ISR Inhibitors in Development
Calico Patents
In collaboration with Abbvie
ABBV-CLS-7262
Quick Search Links
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YouTube videos on ISRIB
https://www.youtube.com/watch?v=bqB1sCivIXY
https://www.youtube.com/watch?v=PqHZhG6e4Vg
https://www.youtube.com/watch?v=Ge3V9W1qO7I
Wikipedia
ISRIB — Integrated Stress Response Inhibitor
Articles in Press
In stressed cells, phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation and gene expression known as the integrated stress response. We show here that DCs are characterized by high eIF2α phosphorylation, mostly caused by the activation of the ER kinase PERK (EIF2AK3). Despite high p-eIF2α levels, DCs display active protein synthesis and no signs of a chronic integrated stress response. This biochemical specificity prevents translation arrest and expression of the transcription factor ATF4 during ER-stress induction by the subtilase cytotoxin (SubAB). PERK inactivation, increases globally protein synthesis levels and regulates IFN-β expression, while impairing LPS-stimulated DC migration. Although the loss of PERK activity does not impact DC development, the cross talk existing between actin cytoskeleton dynamics; PERK and eIF2α phosphorylation is likely important to adapt DC homeostasis to the variations imposed by the immune contexts.
Down syndrome (DS) is the most common genomic disorder characterized by the increased incidence of developing early Alzheimer’s disease (AD). In DS, the triplication of genes on chromosome 21 is intimately associated with the increase of AD pathological hallmarks and with the development of brain redox imbalance and aberrant proteostasis. Increasing evidence has recently shown that oxidative stress (OS), associated with mitochondrial dysfunction and with the failure of antioxidant responses (e.g., SOD1 and Nrf2), is an early signature of DS, promoting protein oxidation and the formation of toxic protein aggregates. In turn, systems involved in the surveillance of protein synthesis/folding/degradation mechanisms, such as the integrated stress response (ISR), the unfolded stress response (UPR), and autophagy, are impaired in DS, thus exacerbating brain damage. A number of pre-clinical and clinical studies have been applied to the context of DS with the aim of rescuing redox balance and proteostasis by boosting the antioxidant response and/or inducing the mechanisms of protein re-folding and clearance, and at final of reducing cognitive decline. So far, such therapeutic approaches demonstrated their efficacy in reverting several aspects of DS phenotype in murine models, however, additional studies aimed to translate these approaches in clinical practice are still needed.
[3] Viruses Jan 2021 Reovirus and the Host Integrated Stress Response: On the Frontlines of the Battle to Survive
Cells are continually exposed to stressful events, which are overcome by the activation of a number of genetic pathways. The integrated stress response (ISR) is a large component of the overall cellular response to stress, which ultimately functions through the phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF2α) to inhibit the energy-taxing process of translation. This response is instrumental in the inhibition of viral infection and contributes to evolution in viruses. Mammalian orthoreovirus (MRV), an oncolytic virus that has shown promise in over 30 phase I–III clinical trials, has been shown to induce multiple arms within the ISR pathway, but it successfully evades, modulates, or subverts each cellular attempt to inhibit viral translation. MRV has not yet received Food and Drug Administration (FDA) approval for general use in the clinic; therefore, researchers continue to study virus interactions with host cells to identify circumstances where MRV effectiveness in tumor killing can be improved. In this review, we will discuss the ISR, MRV modulation of the ISR, and discuss ways in which MRV interaction with the ISR may increase the effectiveness of cancer therapeutics whose modes of action are altered by the ISR.
The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B’s nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other’s binding to eIF2B. These findings point to antagonistic allosteric in ISRIB action on eIF2B, culminating in inhibition of the ISR.
• Exposure to cholesterol or oxidized phospholipids triggers phenotypic switching of vascular smooth muscle cells to a macrophage/fibroblast–like cell and is associated with an unfolded protein response (UPR), involving activation of all 3 arms of endoplasmic reticulum stress: Perk (protein kinase RNA-like endoplasmic reticulum kinase), Ire (inositol-requiring enzyme) 1α, and Atf (activating transcription factor) 6.
• Induction of endoplasmic reticulum stress and UPR is necessary for phenotypic switching of smooth muscle cells to macrophage/fibroblast-like cells.
• Cholesterol-induced phenotypic switching is mediated primarily by the Perk-eIF2α (α-subunit of the eukaryotic elongation factor 2)-Atf4 axis of UPR.
• Chemically induced UPR without cholesterol exposure is sufficient to cause phenotypic switching of smooth muscle cells to a macrophage/fibroblast-like cell.
Reanalysis of previously published single-cell RNA sequencing data shows preliminary evidence of UPR activation in modulated smooth muscle cells in vivo.Treatment with 200 nmol/L ISRIB (integrated stress response inhibitor) reverses cholesterol-induced upregulation of macrophage marker genes (D) and proteins (E). F, Exposure to ISRIB prevents the cholesterol-induced increase in Klf4 transcriptional activity. G, Quantitative polymerase chain reaction analysis demonstrates successful downregulation of protein kinase RNA-like ER kinase (Perk) mRNA by shRNA (short hairpin RNA) treatment. H,
Expectedly, ISRIB treatment blocks the Perk-eIF2α-Atf4 sensor, as indicated by the lack of cholesterol-driven increases of Atf4, but has no effect on the Ire1α or Atf6 pathways (Figure IXB through IXG in the Data Supplement).
With exposure to free cholesterol, SMCs decrease expression of contractile markers, activate Klf4, and upregulate a subset of macrophage and fibroblast markers characteristic of modulated SMCs that appear with atherosclerotic plaque formation. These phenotypic changes are associated with activation of all 3 pathways of the endoplasmic reticulum unfolded protein response (UPR), Perk (protein kinase RNA-like endoplasmic reticulum kinase), Ire (inositol-requiring enzyme) 1α, and Atf (activating transcription factor) 6. Blocking the movement of cholesterol from the plasma membrane to the endoplasmic reticulum prevents free cholesterol–induced UPR, Klf4 activation, and upregulation of the majority of macrophage and fibroblast markers. Cholesterol-induced phenotypic switching is also prevented by global UPR inhibition or specific inhibition of Perk signaling. Exposure to chemical UPR inducers, tunicamycin and thapsigargin, is sufficient to induce these same phenotypic transitions. Finally, analysis of published single-cell RNA sequencing data during atherosclerotic plaque formation in hyperlipidemic mice provides preliminary in vivo evidence of a role of UPR activation in modulated SMCs.
[6] Current Eye Research. Jan 2021 Targeting the integrated stress response in ophthalmology
Purpose: To summarize the Integrated Stress Response (ISR) in the context of ophthalmology, with special interest on the cornea and anterior segment.
Results: The ISR is a powerful and conserved signaling pathway that allows for cells to respond to a diverse array of both intracellular and extracellular stressors. The pathway is classically responsible for coordination of the cellular response to amino acid starvation, ultraviolet light, heme dysregulation, viral infection, and unfolded protein. Under normal circumstances, it is considered pro-survival and a necessary mechanism through which protein translation is controlled. However, in cases of severe or prolonged stress the pathway can promote apoptosis, and loss of normal cellular phenotype. The activation of this pathway culminates in the global inhibition of cap-dependent protein translation and the canonical expression of the activating transcription factor 4 (ATF4).
Conclusion: The eye is uniquely exposed to ISR responsive stressors due to its environmental exposure and relative isolation from the circulatory system which are necessary for its function. We will discuss how this pathway is critical for the proper function of the tissue, its role in development, as well as how targeting of the pathway could alleviate key aspects of diverse ophthalmic diseases.
[7] Medical Press Dec 2020 Drug reverses age-related cognitive decline within days (Lay Press)
Just a few doses of an experimental drug can reverse age-related declines in memory and mental flexibility in mice, according to a new study by UC San Francisco scientists. The drug, called ISRIB, has already been shown in laboratory studies to restore memory function months after traumatic brain injury (TBI), reverse cognitive impairments in Down Syndrome , prevent noise-related hearing loss, fight certain types of prostate cancer , and even enhance cognition in healthy animals
In the new study, published December 1, 2020 in the open-access journal eLife , researchers showed rapid restoration of youthful cognitive abilities in aged mice, accompanied by a rejuvenation of brain and immune cells that could help explain improvements in brain function
[8] Dec 2020. eIF2B Conformation and Assembly State Regulate the Integrated Stress Response
The integrated stress response (ISR) is activated by phosphorylation of the translationinitiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2’s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B’s α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.
[9] New Atlas. Dec 2020. Drug reboots protein production to reverse age-related cognitive decline [Lay Press]
Scientists studying the mechanisms behind cellular stress and its effects on aging have found success in reversing cognitive decline in mice using an experimental drug. The findings not only bode well for further study of aging and its effects in human subjects, but sheds light on a growing list of conditions thought to be spurred on by chronic stress of this type.
[10] BioRxiv Nov 2020: BioRxiv Nov 2020: Small molecule cognitive enhancer reverses age-related memory decline in mice.
With increased life expectancy, age-associated cognitive decline becomes a growing concern. The integrated stress response (ISR) is activated during aging and contributes to age-related brain phenotypes. We demonstrate that treatment with the drug-like small-molecule ISR inhibitor ISRIB reverses ISR activation in the brain, as indicated by decreased activating transcription factor 4 (ATF4) protein levels. Furthermore, ISRIB treatment reverses spatial memory deficits and ameliorates working memory in old mice. At the cellular level in the hippocampus, ISR inhibition i) rescues intrinsic neuronal electrophysiological properties, ii) restores spine density and iii) reduces immune profiles, specifically interferon and T cell-mediated responses. Thus, pharmacological interference with the ISR emerges as a promising intervention strategy for combating age-related cognitive decline.
ONE SENTENCE SUMMARY Inhibition of the integrated stress response restores neuronal and immune dysfunction and alleviates memory deficits in aged mice
35 Neuronal protein synthesis is essential for long-term memory consolidation. Conversely, 36 dysregulation of protein synthesis has been implicated in a number of neurodegenerative 37 disorders, including Alzheimer’s disease (AD). Several types of cellular stress trigger the 38 activation of protein kinases that converge on the phosphorylation of eukaryotic 39 translation initiation factor 2b (eIF2b-P). This leads to attenuation of cap-dependent 40 mRNA translation, a component of the integrated stress response (ISR). We show that 41 AD brains exhibit increased eIF2b-P and reduced eIF2B, key components of the eIF2 42 translation initiation complex. We further demonstrate that attenuating the ISR with the 43 small molecule compound ISRIB (ISR Inhibitor) rescues hippocampal protein synthesis 44 and corrects impaired synaptic plasticity and memory in mouse models of AD. Our 45 findings suggest that attenuating eIF2b-P-mediated translational inhibition may comprise 46 an effective approach to alleviate cognitive decline in AD.
[12] ReserchGate Oct 2020. ISRIB prevents synaptic plasticity disruption and cognitive deficits in live rat model of Alzheimer’s disease
Growing evidence shows that targeting the integrated stress response (ISR) through the inhibition of phosphorylation of eIF2α provides beneficial effects in animal models of neurodegenerative diseases including Alzheimer’s disease (AD). However, those results are inconsistent and somehow conflicting likely due to the important role of ISR in both cell death and survival. Aβ-triggered pathologies including microvascular hypoxia, neuronal hyperactivation, neuroinflammation are common inducers of the ISR. The small-molecule inhibitor of the ISR called ISRIB, which only partially restores protein synthesis and confers neuroprotection without adverse effects on the pancreas most probably due to its state-dependent action, remarkably enhances cognition in animals. Methods To elucidate the roles of ISR in AD pathogenesis, we systemically treated exogenous Aβ-injected animals with ISRIB. Both Aβ-facilitated long-term depression (LTD) and Morris water maze were used to characterize Aβ-induced dysfunction. Results Acute treatment with ISRIB prevented Aβ-facilitated LTD and repeated treatment abrogated the spatial learning and memory deficits in exogenous Aβ-injected animals. Moreover, ISRIB restored aberrant high level of ATF4 to normal but did not affect the aberrant high level of phosphorylated eIF2α in the hippocampus of exogenous Aβ-injected rats. Conclusions Targeting the ISR by suppressing the eIF2α cascade with ISRIB may provide protective effects against the synaptic and cognitive disruptive effects of Aβ which likely mediate the early stage of sporadic AD.
[13] Nature Oct 2020 eIF2α controls memory consolidation via excitatory and somatostatin neurons
An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory1,2,3,4. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories5,6,7,8,9,10. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF211. Phosphorylation of the α-subunit of eIF2 (p-eIF2α), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds11,12,13. By contrast, inhibiting the ISR by mutating the eIF2α phosphorylation site, genetically11 and pharmacologically inhibiting the ISR kinases14,15,16,17, or mimicking reduced p-eIF2α with the ISR inhibitor ISRIB11, enhances long-term memory in health and disease18. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2α phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2α in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2α-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons.
Currently, thirty-seven million people are infected with human immunodeficiency virus-1 (HIV-1) worldwide. Thankfully, the development of combined antiretroviral therapy (cART) regimens has decreased mortality and significantly improved the overall quality of life for these patients. However, approximately half of all patients clinically manifest with HIV-associated neurocognitive disorder (HAND), a spectrum of cognitive, motor, and behavioral abnormalities which histologically present as non-specific gliosis, synaptodendritc damage and loss of white matter and myelin. Furthermore, the severity of white matter damage correlates with the length of ART duration. However, almost no studies have been performed to determine how the myelin sheath or the oligodendrocytes that synthesize the sheath are damaged. Thus, we hypothesized that the administration of ART contributed in part to the myelin loss in the CNS of HIV-positive patients. Previously, we have reported that the protease inhibitor class of ART drugs hampered the in vitro differentiation of oligodendrocytes. Given that the new US guidelines for treating HIV patients recommends anew class of drugs, the integrase strand transfer inhibitors (INSTIs) as front-line therapy, we examined if two specific INSTIs, Elvitegravir (EVG) and raltegravir (RAL), altered the survival and/or maturation of developing oligodendrocytes in vitro and in vivo. We found that treatment of oligodendrocyte precursor cells (OPCs) with EVG, but not RAL, during differentiation reduced the number of cells positive for immature oligodendrocyte marker galactosylceramide (GalC) and mature oligodendrocyte marker myelin basic protein (MBP) in vitro, as well as the synthesis of myelin proteins. However, neither EVG or RAL induced cell loss or apoptosis, as determined by cell counts and TUNEL assays, suggesting that EVG does not affect OPC viability but instead, inhibits differentiation. EVG-induced oligodendrocyte differentiation deficits could be reversed by pre-treating the cells with a drug that pharmacologically inhibits the phosphorylation of eukaryotic initiation factor 2α(eIF2α) through the cellular integrated stress response (ISR). Finally, in vivo, mice receiving EVG/COBI failed to remyelinate the corpus callosum during the three week recovery period following demyelination, after cuprizone treatment. Although EVG/COBI treatment by itself did not cause overt white matter loss in this brain region. Our study demonstrates that EVG, but not RAL, inhibits oligodendrocyte precursor cell differentiation both in vitro and in vivo. Furthermore, EVG may be inhibiting oligodendrocyte precursor cell differentiation though activation of the ISR. Also, we found that the effects of EVG on oligodendrocyte differentiation could be attenuated in vitro by inhibiting the ISR. These studies suggest that ART may contribute to cognitive impairment by inhibiting renewal and replacement of oligodendrocytes in adults or development of oligodendrocytes in children. Further, our results suggest an ISR inhibitor might attenuate the negative effect of EVG on the maturation of oligodendrocytes. Our findings also suggest that development of less toxic ART compounds and adjunctive therapies are needed to minimize the side effects of ART on the CNS.
[15] Front Pharmacol Sept 2020 Metformin: A Prospective Alternative for the Treatment of Chronic Pain
Metformin (biguanide) is a drug widely used for the treatment of type 2 diabetes. This drug has been used for 60 years as a highly effective antihyperglycemic agent. The search for the mechanism of action of metformin has produced an enormous amount of research to explain its effects on gluconeogenesis, protein metabolism, fatty acid oxidation, oxidative stress, glucose uptake, autophagy and pain, among others. It was only up the end of the 1990s and beginning of this century that some of its mechanisms were revealed. Metformin induces its beneficial effects in diabetes through the activation of a master switch kinase named AMP-activated protein kinase (AMPK). Two upstream kinases account for the physiological activation of AMPK: liver kinase B1 and calcium/calmodulin-dependent protein kinase kinase 2. Once activated, AMPK inhibits the mechanistic target of rapamycin complex 1 (mTORC1), which in turn avoids the phosphorylation of p70 ribosomal protein S6 kinase 1 and phosphatidylinositol 3-kinase/protein kinase B signaling pathways and reduces cap-dependent translation initiation. Since metformin is a disease-modifying drug in type 2 diabetes, which reduces the mTORC1 signaling to induce its effects on neuronal plasticity, it was proposed that these mechanisms could also explain the antinociceptive effect of this drug in several models of chronic pain. These studies have highlighted the efficacy of this drug in chronic pain, such as that from neuropathy, insulin resistance, diabetic neuropathy, and fibromyalgia-type pain. Mounting evidence indicates that chronic pain may induce anxiety, depression and cognitive impairment in rodents and humans. Interestingly, metformin is able to reverse some of these consequences of pathological pain in rodents. The purpose of this review was to analyze the current evidence about the effects of metformin in chronic pain and three of its comorbidities (anxiety, depression and cognitive impairment).
In support of this, the specific inhibitor (ISRIB) of the ISR diminishes eIF2αSer51 phosphorylation and reduces and reverts methylglyoxal-induced nociception and also reduces type 1 diabetes-induced neuropathic pain in mice and rats. Interestingly, the AMPK activator drug metformin also lessens eIF2αSer51 phosphorylation (Barragán-Iglesias et al., 2019). These data imply that metformin can decrease the effects of methylglyoxal on pain by reducing eIF2αSer51phosphorylation in an AMPK-dependent manner (see Table 1). Taken together, data suggest that metformin behaves as a disease-modifying drug in insulin resistance- and experimental diabetes-induced pathological pain.
Stress granules (SGs) are assemblies of mRNA and proteins that form from mRNAs stalled in translation initiation in response to stress. Chronic stress might even induce formation of cytotoxic pathological SGs. SGs participate in various biological functions including response to apoptosis, inflammation, immune modulation, and signalling pathways; moreover, SGs are involved in pathogenesis of neurodegenerative diseases, viral infection, aging, cancers and many other diseases. Emerging evidence has shown that small molecules can affect SG dynamics, including assembly, disassembly, maintenance and clearance. Thus, targeting SGs is a potential therapeutic strategy for the treatment of human diseases and the promotion of health. The established methods for detecting SGs provided ready tools for large-scale screening of agents that alter the dynamics of SGs. Here, we describe the effects of small molecules on SG assembly, disassembly, and their roles in the disease. Moreover, we provide perspective for the possible application of small molecules targeting SGs in the treatment of human diseases.
Major depression disorder is one of the most common psychiatric disorders that greatly threaten the mental health of a large population worldwide. Previous studies have shown that endoplasmic reticulum (ER) stress plays an important role in the pathophysiology of depression, and current research suggests that brain-derived neurotrophic factor precursor (proBDNF) is involved in the development of depression. However, the relationship between ER and proBDNF in the pathophysiology of depression is not well elucidated. Here, we treated primary hippocampal neurons of mice with corticosterone (CORT) and evaluated the relationship between proBDNF and ERS. Our results showed that CORT induced ERS and upregulated the expression of proBDNF and its receptor, Follistatin-like protein 4 (FSTL4), which contributed to significantly decreased neuronal viability and expression of synaptic-related proteins including NR2A, PSD95, and SYN. Anti-proBDNF neutralization and ISRIB (an inhibitor of the ERS) treatment, respectively, protected neuronal viabilities and increased the expression of synaptic-related proteins in corticosterone-exposed neurons. ISRIB treatment reduced the expression of proBDNF and FSTL4, whereas anti-proBDNF treatment did not affect ERS markers (Grp78, p-PERK, ATF4) expression. Our study presented evidence that CORT-induced ERS negatively regulated the neuronal viability and the level of synaptic-related protein of primary neurons via the proBDNF/FSTL4 pathway.
The endoplasmic reticulum (ER) is an important organelle for normal cellular function and homeostasis in most living things. ER stress, which impairs ER function, occurs when the ER is overwhelmed by newly introduced immature proteins or when calcium in the ER is depleted. A number of diseases are associated with ER stress, including otorhinolaryngological diseases. The relationship between ER stress and otorhinolaryngologic conditions has been the subject of investigation over the last decade. Among otologic diseases associated with ER stress are otitis media and hearing loss. In rhinologic diseases, chronic rhinosinusitis, allergic rhinitis, and obstructive sleep apnea are also significantly associated with ER stress. In this review, we provide a comprehensive overview of the relationship between ER stress and otorhinolaryngological diseases, focusing on the current state of knowledge and mechanisms that link ER stress and otorhinolaryngologic diseases.
Keywords: endoplasmic reticulum stress; otorhinolaryngologic disease; otitis media; hearing loss; chronic rhinosinusitis; allergic rhinitis; obstructive sleep apnea; intermittent hypoxia
Mild repetitive traumatic brain injury (rTBI) induces chronic behavioral and cognitive alterations and increases the risk for dementia. Currently, there are no therapeutic strategies to prevent or mitigate chronic deficits associated with rTBI. Previously we developed an animal model of rTBI that recapitulates the cognitive and behavioral deficits observed in humans. We now report that rTBI results in an increase in risk-taking behavior in male but not female mice. This behavioral phenotype is associated with chronic activation of the integrated stress response and cell-specific synaptic alterations in the type A subtype of layer V pyramidal neurons in the medial prefrontal cortex. Strikingly, by briefly treating animals weeks after injury with ISRIB, a selective inhibitor of the integrated stress response (ISR), we (1) relieve ISR activation, (2) reverse the increased risk-taking behavioral phenotype and maintain this reversal, and (3) restore cell-specific synaptic function in the affected mice. Our results indicate that targeting the ISR even at late time points after injury can permanently reverse behavioral changes. As such, pharmacological inhibition of the ISR emerges as a promising avenue to combat rTBI-induced behavioral dysfunction.
Loss of protein folding homeostasis features many of the most prevalent neurodegenerative disorders. As coping mechanism to folding stress within the endoplasmic reticulum (ER), the unfolded protein response (UPR) comprises a set of signaling mechanisms that initiate a gene expression program to restore proteostasis, or when stress is chronic or overwhelming promote neuronal death. This fate-defining capacity of the UPR has been proposed to play a key role in amyotrophic lateral sclerosis (ALS). However, the several genetic or pharmacological attempts to explore the therapeutic potential of UPR modulation have produced conflicting observations. In order to establish the precise relationship between UPR signaling and neuronal death in ALS, we have developed a neuronal model where the toxicity of a familial ALS-causing allele (mutant G93A SOD1) and UPR activation can be longitudinally monitored in single neurons over the process of neurodegeneration by automated microscopy. Using fluorescent UPR reporters we established the temporal and causal relationship between UPR and neuronal death by Cox regression models. Pharmacological inhibition of discrete UPR processes allowed us to establish the contribution of PERK (PKR-like ER kinase) and IRE1 (inositol-requiring enzyme-1) mechanisms to neuronal fate. Importantly, inhibition of PERK signaling with its downstream inhibitor ISRIB, but not with the direct PERK kinase inhibitor GSK2606414, significantly enhanced the survival of G93A SOD1-expressing neurons. Characterization of the inhibitory properties of both drugs under ER stress revealed that in neurons (but not in glial cells) ISRIB overruled only part of the translational program imposed by PERK, relieving the general inhibition of translation, but maintaining the privileged translation of ATF4 (activating transcription factor 4) messenger RNA. Surprisingly, the fine-tuning of the PERK output in G93A SOD1-expressing neurons led to a reduction of IRE1-dependent signaling. Together, our findings identify ISRIB-mediated translational reprogramming as a new potential ALS therapy.
The integrated stress response (ISR) regulates protein synthesis under conditions of stress. Phosphorylation of translation initiation factor eIF2 by stress‐sensing kinases converts eIF2 from substrate to competitive inhibitor of its dedicated nucleotide exchange factor, eIF2B, arresting translation. A drug‐like molecule called integrated stress response inhibitor (ISRIB) reverses the effects of eIF2 phosphorylation and restores translation by targeting eIF2B. When administered to mice, ISRIB enhances cognition and limits cognitive decline due to brain injury. To determine ISRIB’s mechanism of action, we solved an atomic structure of ISRIB bound to the human eIF2B decamer. We found that ISRIB acts as a molecular staple, pinning together tetrameric subcomplexes of eIF2B along the assembly path to a fully active, decameric enzyme. In this Structural Snapshot, we discuss ISRIB’s mechanism, its ability to rescue disease mutations in eIF2B and conservation of the enzyme and ISRIB‐binding pocket.
Methylglyoxal (MGO) is a reactive glycolytic metabolite associated with painful diabetic neuropathy at plasma concentration is between 500 nM and 5 μM. The mechanisms through which MGO causes neuropathic pain at these pathological concentrations are not known. Because MGO has been linked to diabetic neuropathic pain, which is prevalent and poorly treated, insight into this unsolved biomedical problem could lead to much needed therapeutics. Our experiments provide compelling evidence that ~1-μM concentrations of MGO activate the integrated stress response (ISR) in IB4-positive nociceptors in the dorsal root ganglion (DRG) of mice in vivo and in vitro. Blocking the integrated stress response with a specific inhibitor (ISRIB) strongly attenuates and reverses MGO-evoked pain. Moreover, ISRIB reduces neuropathic pain induced by diabetes in both mice and rats. Our work elucidates the mechanism of action of MGO in the production of pain at pathophysiologically relevant concentrations and suggests a new pharmacological avenue for the treatment of diabetic and other types of MGO-driven neuropathic pain.
Blocking the integrated stress response with a specific inhibitor (ISRIB) strongly attenuates and reverses MGO-evoked pain. Moreover, ISRIB reduces neuropathic pain induced by diabetes in both mice and rats. Our work elucidates the mechanism of action of MGO in the production of pain at pathophysiologically relevant concentrations and suggests a new pharmacological avenue for the treatment of diabetic and other types of MGO-driven neuropathic pain.
[23] AEMB Jan 2020 At the Crossing of ER Stress and MAMs: A Key Role of Sigma-1 Receptor?
Calcium exchanges and homeostasis are finely regulated between cellular organelles and in response to physiological signals. Besides ionophores, including voltage-gated Ca²⁺ channels, ionotropic neurotransmitter receptors, or Store-operated Ca²⁺ entry, activity of regulatory intracellular proteins finely tune Calcium homeostasis. One of the most intriguing, by its unique nature but also most promising by the therapeutic opportunities it bears, is the sigma-1 receptor (Sig-1R). The Sig-1R is a chaperone protein residing at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), where it interacts with several partners involved in ER stress response, or in Ca²⁺ exchange between the ER and mitochondria. Small molecules have been identified that specifically and selectively activate Sig-1R (Sig-1R agonists or positive modulators) at the cellular level and that also allow effective pharmacological actions in several pre-clinical models of pathologies. The present review will summarize the recent data on the mechanism of action of Sig-1R in regulating Ca²⁺ exchanges and protein interactions at MAMs and the ER. As MAMs alterations and ER stress now appear as a common track in most neurodegenerative diseases, the intracellular action of Sig-1R will be discussed in the context of the recently reported efficacy of Sig-1R drugs in pathologies like Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis.
[24] USPTO Dec 2019 Modulators of the eif2alpha pathway [U.S. Patent 9708247]
Provided herein, inter alia, are compounds and methods useful for modulating the translational effects of eIF2α phosphorylation, the Integrated Stress Response (ISR), and the unfolded protein response (UPR); for treating diseases; for increasing protein production, and for improving long-term memory.
Down syndrome (DS) is the most common genetic cause of intellectual disability. Protein homeostasis is essential for normal brain function, but little is known about its role in DS pathophysiology. In this study, we found that the integrated stress response (ISR)–a signaling network that maintains proteostasis–was activated in the brains of DS mice and individuals with DS, reprogramming translation. Genetic and pharmacological suppression of the ISR, by inhibiting the ISR-inducing double-stranded RNA-activated protein kinase or boosting the function of the eukaryotic translation initiation factor eIF2-eIF2B complex, reversed the changes in translation and inhibitory synaptic transmission and rescued the synaptic plasticity and long-term memory deficits in DS mice. Thus, the ISR plays a crucial role in DS, which suggests that tuning of the ISR may provide a promising therapeutic intervention.
The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a guanosine triphosphatase that becomes activated by eIF2B, a two-fold symmetric and heterodecameric complex that functions as eIF2’s dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from a substrate into an inhibitor of eIF2B. We report cryo–electron microscopy structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B’s bipartite catalytic centers to catalyze nucleotide exchange. Phosphorylation refolds eIF2α, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequestering it into a nonproductive complex.
Currently, thirty-seven million people are infected with human immunodeficiency virus-1 (HIV-1) worldwide. Thankfully, the development of combined antiretroviral therapy (cART) regimens has decreased mortality and significantly improved the overall quality of life for these patients. However, approximately half of all patients clinically manifest with HIV-associated neurocognitive disorder (HAND), a spectrum of cognitive, motor, and behavioral abnormalities which histologically present as non-specific gliosis, synaptodendritc damage and loss of white matter and myelin. Furthermore, the severity of white matter damage correlates with the length of ART duration. However, almost no studies have been performed to determine how the myelin sheath or the oligodendrocytes that synthesize the sheath are damaged. Thus, we hypothesized that the administration of ART contributed in part to the myelin loss in the CNS of HIV-positive patients. Previously, we have reported that the protease inhibitor class of ART drugs hampered the in vitro differentiation of oligodendrocytes. Given that the new US guidelines for treating HIV patients recommends a new class of drugs, the integrase strand transfer inhibitors (INSTIs)as front-line therapy, we examined if two specific INSTIs, Elvitegravir (EVG) and raltegravir (RAL), altered the survival and/or maturation of developing oligodendrocytes in vitroand in vivo. We found that treatment of oligodendrocyte precursor cells (OPCs) with EVG, but not RAL, during differentiation reduced the number of cells positive for immature oligodendrocyte marker galactosylceramide (GalC) and mature oligodendrocyte marker myelin basic protein (MBP) in vitro, as well as the synthesis of myelin proteins. However, neither EVG or RAL induced cell loss or apoptosis, as determined by cell counts and TUNEL assays, suggesting that EVG does not affect OPC viability but instead, inhibits differentiation. EVG-induced oligodendrocyte differentiation deficits could be reversed by pre-treating the cells with a drug that pharmacologically inhibits the phosphorylation of eukaryotic initiation factor 2α(eIF2α) through the cellular integrated stress response (ISR). Finally, in vivo,mice receiving EVG/COBI failed to remyelinate the corpus callosum during the three week recovery period following demyelination, after cuprizone treatment. Although EVG/COBI treatment by itself did not cause overt white matter loss in this brain region. Our study demonstrates that EVG, but not RAL, inhibits oligodendrocyte precursor cell differentiation both in vitroand in vivo. Furthermore, EVG may be inhibiting oligodendrocyte precursor cell differentiation though activation of the ISR. Also, we found thatthe effects of EVG on oligodendrocyte differentiation could be attenuated in vitroby inhibiting the ISR. These studies suggest that ART may contribute to cognitive impairment by inhibiting renewal and replacement of oligodendrocytes in adults or development of oligodendrocytes in children. Further, our results suggest an ISR inhibitor might attenuate the negative effect of EVG on the maturation of oligodendrocytes. Our findings also suggest that development of less toxic ART compounds and adjunctive therapies are needed to minimize the side effects of ART on the CNS.
The integrated stress response (ISR) protects cells from a variety of harmful stressors by temporarily halting protein synthesis. However, chronic ISR activation has pathological consequences and is linked to several neurological disorders. Pharmacological inhibition of chronic ISR activity emerges as a powerful strategy to treat ISR-mediated neurodegeneration but is typically linked to adverse effects due to the ISR’s importance for normal cellular function. Paradoxically, the small-molecule ISR inhibitor ISRIB has promising therapeutic potential in vivo without overt side effects. We demonstrate here that ISRIB inhibits low-level ISR activity, but does not affect strong ISR signaling. We thereby provide a plausible mechanism of how ISRIB counteracts toxic chronic ISR activity, without disturbing the cytoprotective effects of a strong acute ISR.
The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.
Hearing loss is a significant public health concern, affecting over 250 million people worldwide. Both genetic and environmental etiologies are linked to hearing loss, but in many cases the underlying cellular pathophysiology is not well understood, highlighting the importance of further discovery. We found that inactivation of the gene Tmtc4 (transmembrane and tetratricopeptide repeat 4), which was broadly expressed in the mouse cochlea, caused acquired hearing loss in mice. Our data showed Tmtc4 enriched in the endoplasmic reticulum, and that it functioned by regulating Ca2+ dynamics and the unfolded protein response (UPR). Given this genetic linkage of the UPR to hearing loss, we demonstrated a direct link between the more common noise-induced hearing loss (NIHL) and the UPR. These experiments suggested a novel approach to treatment. We demonstrated that the small-molecule UPR and stress response modulator ISRIB (integrated stress r modulator ISRIB (integrated stress response inhibitor), which activates eIF2B, prevented NIHL in a mouse model. Moreover, in an inverse genetic complementation approach, we demonstrated that mice with homozygous inactivation of both Tmtc4 and Chop had less hearing loss than knockout of Tmtc4 alone. This study implicated a novel mechanism for hearing impairment, highlighting a potential treatment approach for a broad range of human hearing loss disorders. inhibitor), which activates eIF2B, prevented NIHL in a mouse model. Moreover, in an inverse genetic complementation approach, we demonstrated that mice with homozygous inactivation of both Tmtc4 and Chop had less hearing loss than knockout of Tmtc4 alone. This study implicated a novel mechanism for hearing impairment, highlighting a potential treatment approach for a broad range of human hearing loss disorders.
Hearing loss is a significant public health concern, affecting over 250 million people worldwide. Both genetic and environmental etiologies are linked to hearing loss, but in many cases the underlying cellular pathophysiology is not well understood, highlighting the importance of further discovery. We found that inactivation of the gene Tmtc4 (transmembrane and tetratricopeptide repeat 4), which was broadly expressed in the mouse cochlea, caused acquired hearing loss in mice. Our data showed Tmtc4 enriched in the endoplasmic reticulum, and that it functioned by regulating Ca2+ dynamics and the unfolded protein response (UPR). Given this genetic linkage of the UPR to hearing loss, we demonstrated a direct link between the more common noise-induced hearing loss (NIHL) and the UPR. These experiments suggested a novel approach to treatment. We demonstrated that the small-molecule UPR and stress response modulator ISRIB (integrated stress response inhibitor), which activates eIF2B, prevented NIHL in a mouse model. Moreover, in an inverse genetic complementation approach, we demonstrated that mice with homozygous inactivation of both Tmtc4 and Chop had less hearing loss than knockout ofTmtc4 alone. This study implicated a novel mechanism for hearing impairment, highlighting a potential treatment approach for a broad range of human hearing loss disorders.
Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic pathways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism by which these adaptive responses are established, their functional consequences for tumor development, and their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively activated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor 2–α (P-eIF2α) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models and an inhibitor of P-eIF2α activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.
[33] Neuroscience March 2018 Detailed structure illuminates brain-enhancing drug's action (Lay Press)
Researchers have shown that the drug, nicknamed ISRIB, completely eliminates learning and memory deficits in mice with concussions. Now scientists have a clearer view of how ISRIB works—by pinning together parts of a protein involved in cellular stress.
Mounting evidence suggests that antiretroviral drugs may contribute to the persistence of HIV-associated neurocognitive disorders (HAND), which impact 30%-50% of HIV-infected patients in the post-antiretroviral era. We previously reported that two first generation HIV protease inhibitors, ritonavir and saquinavir, induced oxidative stress, with subsequent neuronal death in vitro, which was reversed by augmentation of the endogenous antioxidant response by monomethyl fumarate. We herein determined whether two newer-generation PIs, darunavir and lopinavir, were deleterious to neurons in vitro. Further, we expanded our assessment to include three integrase strand transfer inhibitors, raltegravir, dolutegravir, and elvitegravir. We found that only lopinavir and elvitegravir were neurotoxic to primary rat neuroglial cultures as determined by the loss of microtubule-associated protein 2 (MAP2). Intriguingly, lopinavir but not elvitegravir led to oxidative stress and induced the endogenous antioxidant response (EAR). Furthermore, neurotoxicity of lopinavir was blocked by pharmacological augmentation of the endogenous antioxidant heme oxygenase-1 (HO-1), expanding our previous finding that protease inhibitor-induced neurotoxicity was mediated by oxidative stress. Conversely, elvitegravir but not lopinavir led to increased eIF2α phosphorylation, indicating the activation of a common adaptive pathway termed the integrated stress response (ISR), and elvitegravir-mediated neurotoxicity was partially alleviated by the ISR inhibitor trans-ISRIB, suggesting ISR as a promoter of elvitegravir-associated neurotoxicity. Overall, we found that neurotoxicity was induced only by a subset of protease inhibitors and integrase strand transfer inhibitors, providing evidence for class- and drug-specific neurotoxic effects of antiretroviral drugs. Future in vivo studies will be critical to confirm the neurotoxicity profiles of these drugs for incorporation of these findings into patient management. The EAR and ISR pathways are potential access points for the development of adjunctive therapies to complement antiretroviral therapies and limit their contribution to HAND persistence.
[35] Science March 2018. ISRIB Memory-Enhancing Drug Acts as Molecular ‘Staple' [LAY PRESS]
New research utilizing UC San Francisco’s cutting-edge cryo-electron microscopy facility has revealed atomic-scale details about the workings of an experimental drug that boosts learning and memory and restores function following concussion in mice.
In a study published March 29, 2018 in Science, UCSF biochemist Peter Walter, PhD, and structural biologist Adam Frost, MD, PhD, showed that the drug, ISRIB (pronounced “iz-rib”), acts like a molecular staple, pinning together parts of a much larger protein involved in cellular stress
This 3-D rendering shows the propeller-shaped molecule ISRIB (red and white) tucked into the center of eIF2B, stapling two halves of the protein complex together. Image credit: J. Tsai et al./Science 2018
eIF2B is a dedicated guanine nucleotide exchange factor for eIF2, the GTPase that is essential to initiate mRNA translation. The integrated stress response (ISR) signaling pathway inhibits eIF2B activity, attenuates global protein synthesis and upregulates a set of stress-response proteins. Partial loss-of-function mutations in eIF2B cause a neurodegenerative disorder called Vanishing White Matter Disease (VWMD). Previously, we showed that the small molecule ISRIB is a specific activator of eIF2B (Sidrauski et al., 2015). Here, we report that various VWMD mutations destabilize the decameric eIF2B holoenzyme and impair its enzymatic activity. ISRIB stabilizes VWMD mutant eIF2B in the decameric form and restores the residual catalytic activity to wild-type levels. Moreover, ISRIB blocks activation of the ISR in cells carrying these mutations. As such, ISRIB promises to be an invaluable tool in proof-of-concept studies aiming to ameliorate defects resulting from inappropriate or pathological activation of the ISR.
[37] Medium Jan 2018 A tale of serendipitous discovery [Lay Press]
Having discovered ISRIB, Carmela was due to some surprises once she started further characterizing the compound. First off, she realized that ISRIB impairs the PERK signaling pathway but has no effect on PERK itself or PERK phosphorylation. In effect, PERK phosphorylation seemed to be even increased.
Progress has been made in our understanding of the mechanisms of endoplasmic reticulum (ER) proteostasis, ER stress and the unfolded protein response (UPR), as well as ER stress-induced autophagy, in the kidney. Experimental models have revealed that disruption of the UPR, including a protein that senses misfolded proteins (namely, inositol-requiring enzyme 1α) in mouse podocytes causes podocyte injury and albuminuria as mice age. Protein misfolding and ER stress are evident in various renal diseases, including primary glomerulonephritides, glomerulopathies associated with genetic mutations, diabetic nephropathy, acute kidney injury, chronic kidney disease and renal fibrosis. The induction of ER stress may be cytoprotective, or it may be cytotoxic by activating apoptosis. The UPR may interact in a coordinated manner with autophagy to alleviate protein misfolding and its consequences. Monitoring the excretion of ER chaperones into the urine can potentially serve as a biomarker of renal ER stress. In specific kidney diseases, the treatment of experimental animals with chemical chaperones that improve protein folding or with chaperone inducers has alleviated kidney injury. Given the limited availability of mechanism-based therapies for kidney diseases, normalization of ER stress using pharmacological agents represents a promising therapeutic approach towards preventing or arresting the progression of kidney disease.
[39] Medical Press July 2017. Drug reverses memory failure caused by traumatic brain injury (Lay Press)
In an unprecedented finding, UC San Francisco scientists used an experimental drug to completely reverse severe learning and memory impairments caused by traumatic brain injury (TBI) in mice. Surprisingly, the drug fully restored the ability to learn and remember in the brain-injured mice even when the animals were first treated as much as a month after injury.
[40] Science July 2017 Memory-enhancing drug reverses effects of traumatic brain injury in mice [Lay Press]
Traumatic brain injuries, which cause cell death and inflammation in the brain, affect 2 million Americans each year. But the condition is difficult to study, in part because every fall, concussion, or blow to the head is different. Some result in bleeding and swelling, which must be treated immediately by drilling into the skull to relieve pressure. But under the microscope, even less severe cases appear to trigger an “integrated stress response,” which throws protein synthesis in neurons out of whack and may make long-term memory formation difficult.
CACNA1C, encoding the Cav1.2 subunit of L-type Ca2+ channels, has emerged as one of the most prominent and highly replicable susceptibility genes for several neuropsychiatric disorders. Cav1.2 channels play a crucial role in calcium-mediated processes involved in brain development and neuronal function. Within the CACNA1C gene, disease-associated single-nucleotide polymorphisms have been associated with impaired social and cognitive processing and altered prefrontal cortical (PFC) structure and activity. These findings suggest that aberrant Cav1.2 signaling may contribute to neuropsychiatric-related disease symptoms via impaired PFC function. Here, we show that mice harboring loss of cacna1c in excitatory glutamatergic neurons of the forebrain (fbKO) that we have previously reported to exhibit anxiety-like behavior, displayed a social behavioral deficit and impaired learning and memory. Furthermore, focal knockdown of cacna1c in the adult PFC recapitulated the social deficit and elevated anxiety-like behavior, but not the deficits in learning and memory. Electrophysiological and molecular studies in the PFC of cacna1c fbKO mice revealed higher E/I ratio in layer 5 pyramidal neurons and lower general protein synthesis. This was concurrent with reduced activity of mTORC1 and its downstream mRNA translation initiation factors eIF4B and 4EBP1, as well as elevated phosphorylation of eIF2α, an inhibitor of mRNA translation. Remarkably, systemic treatment with ISRIB, a small molecule inhibitor that suppresses the effects of phosphorylated eIF2α on mRNA translation, was sufficient to reverse the social deficit and elevated anxiety-like behavior in adult cacna1c fbKO mice. ISRIB additionally normalized the lower protein synthesis and higher E/I ratio in the PFC. Thus this study identifies a novel Cav1.2 mechanism in neuropsychiatric-related endophenotypes and a potential future therapeutic target to explore.
Traumatic brain injury (TBI) is a leading cause of long-term neuro- logical disability, yet the mechanisms underlying the chronic cognitive deficits associated with TBI remain unknown. Consequently, there are no effective treatments for patients suffering from the long-lasting symptoms of TBI. Here, we show that TBI persistently activates the integrated stress response (ISR), a universal intracellular signaling pathway that responds to a variety of cellular conditions and regulates protein translation via phosphorylation of the translation initiation factor eIF2α. Treatment with ISRIB, a potent drug-like small-molecule inhibitor of the ISR, reversed the hippocampal- dependent cognitive deficits induced by TBI in two different injury mouse models—focal contusion and diffuse concussive injury. Surprisingly, ISRIB corrected TBI-induced memory deficits when administered weeks after the initial injury and maintained cognitive improvement after treatment was terminated. At the physiological level, TBI suppressed long-term potentiation in the hippocampus, which was fully restored with ISRIB treatment. Our results indicate that ISR inhibition at time points late after injury can reverse memory deficits associated with TBI. As such, pharmacological inhibition of the ISR emerges as a promising avenue to combat head trauma- induced chronic cognitive deficits.
CACNA1C, encoding the Cav1.2 subunit of L-type Ca2+ channels, has emerged as one of the most prominent and highly replicable susceptibility genes for several neuropsychiatric disorders. Cav1.2 channels play a crucial role in calcium-mediated processes involved in brain development and neuronal function. Within the CACNA1C gene, disease-associated single-nucleotide polymorphisms have been associated with impaired social and cognitive processing and altered prefrontal cortical (PFC) structure and activity. These findings suggest that aberrant Cav1.2 signaling may contribute to neuropsychiatric-related disease symptoms via impaired PFC function. Here, we show that mice harboring loss of cacna1c in excitatory glutamatergic neurons of the forebrain (fbKO) that we have previously reported to exhibit anxiety-like behavior, displayed a social behavioral deficit and impaired learning and memory. Furthermore, focal knockdown of cacna1c in the adult PFC recapitulated the social deficit and elevated anxiety-like behavior, but not the deficits in learning and memory. Electrophysiological and molecular studies in the PFC of cacna1c fbKO mice revealed higher E/I ratio in layer 5 pyramidal neurons and lower general protein synthesis. This was concurrent with reduced activity of mTORC1 and its downstream mRNA translation initiation factors eIF4B and 4EBP1, as well as elevated phosphorylation of eIF2α, an inhibitor of mRNA translation. Remarkably, systemic treatment with ISRIB, a small molecule inhibitor that suppresses the effects of phosphorylated eIF2α on mRNA translation, was sufficient to reverse the social deficit and elevated anxiety-like behavior in adult cacna1c fbKO mice. ISRIB additionally normalized the lower protein synthesis and higher E/I ratio in the PFC. Thus this study identifies a novel Cav1.2 mechanism in neuropsychiatric-related endophenotypes and a potential future therapeutic target to explore.
[44] EMBO Reports Oct 2016. The integrated stress response
In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four members of the eIF2α kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro‐survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.
A pharmacological approach to ameliorate Alzheimer's disease (AD) has not yet been established. In the present study, we investigated the pharmacological characteristics of the recently identified memory enhancing compound, ISRIB for the amelioration of AD. ISRIB potently attenuated amyloid b-induced neuronal cell death at concentrations of 12.5e25 nM, but did not inhibit amyloid b production in the HEK293T cell line expressing the amyloid precursor protein (APP). These results suggest that ISRIB possesses the unique pharmacological property of attenuating amyloid b-induced neuronal cell death without affecting amyloid b production.
Background: The unfolded protein response (UPR) is one of the pathways triggered to ensure quality control of the proteins assembled in the endoplasmic reticulum (ER) when cell homeostasis is compromised. This mechanism is primarily composed of three transmembrane proteins serving as stress sensors: PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1). These three proteins’ synergic action elicits translation and transcriptional downstream pathways, leading to less protein production and activating genes that encode important proteins in folding processes, including chaperones. Previous reports showed that viruses have evolved mechanisms to curtail or customize this UPR signaling for their own benefit. However, HIV infection’s effect on the UPR has scarcely been investigated.
Conclusions: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus, two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs’ pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented.
Keywords: HIV, Aids, UPR pathway, Endoplasmic reticulum stress, Antiretroviral therapy, Cell stress
[47] Nature: Cell Death and Disease March 2015 Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity
• ISRIB prevents neurodegeneration without toxicity
Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative disorders that are currently incurable.
Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.
[49] eLife Digest May 2013 Pharmacological brake-release of mRNA translation enhances cognitive memory
Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders.
Several studies have shown that some of the soluble factors that are released as part of the HIV‐induced inflammatory response, as well as HIV viral proteins themselves, are able to induce the integrated stress response (ISR) [endoplasmic reticulum (ER) stress response, unfolded protein response], suggesting that the ISR may play a role in HAND 10. The ISR orchestrates a multitude of cellular responses against a wide range of extra‐ and intracellular stresses by regulating intracellular Ca levels; ER chaperone genes; protein synthesis, folding and degradation; and the endogenous antioxidant response 10-18. A variety of insults can trigger the ISR, such as oxidative stress, hypoxia, toxins, nutrient deprivation and viral infection. In fact, many types of viruses actively regulate the ISR in their host cells, showing a great variety in the manners and mechanisms by which they do so, preventing components of ISR activity harmful to virus survival and replication and exploiting ISR components bene#cial to the virus, and even showing cell‐type specificity
[51] Molecules Sept 2009: Nasal Delivery of High Molecular Weight Drugs
Nasal drug delivery may be used for either local or systemic effects. Low molecular weight drugs with are rapidly absorbed through nasal mucosa. The main reasons for this are the high permeability, fairly wide absorption area, porous and thin endothelial basement membrane of the nasal epithelium. Despite the many advantages of the nasal route, limitations such as the high molecular weight (HMW) of drugs may impede drug absorption through the nasal mucosa. Recent studies have focused particularly on the nasal application of HMW therapeutic agents such as peptide-protein drugs and vaccines intended for systemic effects. Due to their hydrophilic structure, the nasal bioavailability of peptide and protein drugs is normally less than 1%. Besides their weak mucosal membrane permeability and enzymatic degradation in nasal mucosa, these drugs are rapidly cleared from the nasal cavity after administration because of mucociliary clearance. There are many approaches for increasing the residence time of drug formulations in the nasal cavity resulting in enhanced drug absorption. In this review article, nasal route and transport mechanisms across the nasal mucosa will be briefly presented. In the second part, current studies regarding the nasal application of macromolecular drugs and vaccines with nano- and micro-particulate carrier systems will be summarized.
The endoplasmic reticulum (ER) communicates with the nucleus through the unfolded protein response(UPR), which senses accumulation of unfolded proteins in the ER lumen and leads to increased transcription of genes encoding ER-resident chaperones. As a key regulatory step in this signaling pathway, the mRNA encoding the UPR-specific transcription factor Hac1p becomes spliced by a unique mechanism that requires tRNA ligase but not the spliceosome. Splicing is initiated upon activation of Ire1p, a transmembrane kinase that lies in the ER and/or inner nuclear membrane. We show that Ire1p is a bifunctional enzyme: in addition to being a kinase, it is a site-specific endoribonuclease that cleaves HAC1mRNA specifically at both splice junctions. The addition of purified tRNA ligase completes splicing; we therefore have reconstituted HAC1 mRNA splicing in vitro from purified components.