Discovery of MDV6058 (PF-06952229), a selective and potent TGFßR1 inhibitor: Design, synthesis and optimization.

Compound 1 is a potent TGF-ß receptor type-1 (TGFßR1 or ALK5) inhibitor but is metabolically unstable. A solvent-exposed part of this molecule was used to analogue and modulate cell activity, liver microsome stability and mouse pharmacokinetics. The evolution of SAR that led to the selection of 2 (MDV6058 / PF-06952229) as a preclinical lead compound is described.

Dual Inhibition of Bruton's Tyrosine Kinase and Phosphoinositide-3-Kinase p110δ as a Therapeutic Approach to Treat Non-Hodgkin's B Cell Malignancies.

Although new targeted therapies, such as ibrutinib and idelalisib, have made a large impact on non-Hodgkin's lymphoma (NHL) patients, the disease is often fatal because patients are initially resistant to these targeted therapies, or because they eventually develop resistance. New drugs and treatments are necessary for these patients. One attractive approach is to inhibit multiple parallel pathways that drive the growth of these hematologic tumors, possibly prolonging the duration of the response and reducing resistance. Early clinical trials have tested this approach by dosing two drugs in combination in NHL patients. We discovered a single molecule, MDVN1003 (1-(5-amino-2,3-dihydro-1H-inden-2-yl)-3-(8-fluoro-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine), that inhibits Bruton's tyrosine kinase and phosphatidylinositol-3-kinase δ, two proteins regulated by the B cell receptor that drive the growth of many NHLs. In this report, we show that this dual inhibitor prevents the activation of B cells and inhibits the phosphorylation of protein kinase B and extracellular signal-regulated kinase 1/2, two downstream mediators that are important for this process. Additionally, MDVN1003 induces cell death in a B cell lymphoma cell line but not in an irrelevant erythroblast cell line. Importantly, we found that this orally bioavailable dual inhibitor reduced tumor growth in a B cell lymphoma xenograft model more effectively than either ibrutinib or idelalisib. Taken together, these results suggest that dual inhibition of these two key pathways by a single molecule could be a viable approach for treatment of NHL patients.

Novel 3-methylindoline inhibitors of EZH2: Design, synthesis and SAR.

EZH2 (enhancer of zeste homologue 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the methylation of lysine 27 of histone H3 (H3K27). Dysregulation of EZH2 activity is associated with several human cancers and therefore EZH2 inhibition has emerged as a promising therapeutic target. Several small molecule EZH2 inhibitors with different chemotypes have been reported in the literature, many of which use a bicyclic heteroaryl core. Herein, we report the design and synthesis of EZH2 inhibitors containing an indoline core. Partial saturation of an indole to an indoline provided lead compounds with nanomolar activity against EZH2, while also improving solubility and oxidative metabolic stability.

Design, synthesis and optimization of bis-amide derivatives as CSF1R inhibitors.

Signaling via the receptor tyrosine kinase CSF1R is thought to play an important role in recruitment and differentiation of tumor-associated macrophages (TAMs). TAMs play pro-tumorigenic roles, including the suppression of anti-tumor immune response, promotion of angiogenesis and tumor cell metastasis. Because of the role of this signaling pathway in the tumor microenvironment, several small molecule CSF1R kinase inhibitors are undergoing clinical evaluation for cancer therapy, either as a single agent or in combination with other cancer therapies, including immune checkpoint inhibitors. Herein we describe our lead optimization effort that resulted in the identification of a potent, cellular active and orally bioavailable bis-amide CSF1R inhibitor. Docking and biochemical analysis allowed the removal of a metabolically labile and poorly permeable methyl piperazine group from an early lead compound. Optimization led to improved metabolic stability and Caco2 permeability, which in turn resulted in good oral bioavailability in mice.

Temozolomide analogs with improved brain/plasma ratios - Exploring the possibility of enhancing the therapeutic index of temozolomide.

Temozolomide is a chemotherapeutic agent that is used in the treatment of glioblastoma and other malignant gliomas. It acts through DNA alkylation, but treatment is limited by its systemic toxicity and neutralization of DNA alkylation by upregulation of the O6-methylguanine-DNA methyltransferase gene. Both of these limiting factors can be addressed by achieving higher concentrations of TMZ in the brain. Our research has led to the discovery of new analogs of temozolomide with improved brain:plasma ratios when dosed in vivo in rats. These compounds are imidazotetrazine analogs, expected to act through the same mechanism as temozolomide. With reduced systemic exposure, these new agents have the potential to improve efficacy and therapeutic index in the treatment of glioblastoma.

Targeting prostate cancer with compounds possessing dual activity as androgen receptor antagonists and HDAC6 inhibitors.

While enzalutamide and abiraterone are approved for treatment of metastatic castration-resistant prostate cancer (mCRPC), approximately 20-40% of patients have no response to these agents. It has been stipulated that the lack of response and the development of secondary resistance to these drugs may be due to the presence of AR splice variants. HDAC6 has a role in regulating the androgen receptor (AR) by modulating heat shock protein 90 (Hsp90) acetylation, which controls the nuclear localization and activation of the AR in androgen-dependent and independent scenarios. With dual-acting AR-HDAC6 inhibitors it should be possible to target patients who don't respond to enzalutamide. Herein, we describe the design, synthesis and biological evaluation of dual-acting compounds which target AR and are also specific towards HDAC6. Our efforts led to compound 10 which was found to have potent dual activity (HDAC6 IC50=0.0356µM and AR binding IC50=<0.03µM). Compound 10 was further evaluated for antagonist and other cell-based activities, in vitro stability and pharmacokinetics.

Dopamine receptor D3 expressed on CD4+ T cells favors neurodegeneration of dopaminergic neurons during Parkinson's disease.

Emerging evidence has demonstrated that CD4(+) T cells infiltrate into the substantia nigra (SN) in Parkinson's disease (PD) patients and in animal models of PD. SN-infiltrated CD4(+) T cells bearing inflammatory phenotypes promote microglial activation and strongly contribute to neurodegeneration of dopaminergic neurons. Importantly, altered expression of dopamine receptor D3 (D3R) in PBLs from PD patients has been correlated with disease severity. Moreover, pharmacological evidence has suggested that D3R is involved in IFN-γ production by human CD4(+) T cells. In this study, we examined the role of D3R expressed on CD4(+) T cells in neurodegeneration of dopaminergic neurons in the SN using a mouse model of PD. Our results show that D3R-deficient mice are strongly protected against loss of dopaminergic neurons and microglial activation during 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. Notably, D3R-deficient mice become susceptible to MPTP-induced neurodegeneration and microglial activation upon transfer of wild-type (WT) CD4(+) T cells. Furthermore, RAG1 knockout mice, which are devoid of T cells and are resistant to MPTP-induced neurodegeneration, become susceptible to MPTP-induced loss of dopaminergic neurons when reconstituted with WT CD4(+) T cells but not when transferred with D3R-deficient CD4(+) T cells. In agreement, experiments analyzing activation and differentiation of CD4(+) T cells revealed that D3R favors both T cell activation and acquisition of the Th1 inflammatory phenotype. These findings indicate that D3R expressed on CD4(+) T cells plays a fundamental role in the physiopathology of MPTP-induced PD in a mouse model.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Stimulation of dopamine receptor D5 expressed on dendritic cells potentiates Th17-mediated immunity.

Dendritic cells (DCs) are responsible for priming T cells and for promoting their differentiation from naive T cells into appropriate effector cells. Emerging evidence suggests that neurotransmitters can modulate T cell-mediated immunity. However, the involvement of specific neurotransmitters or receptors remains poorly understood. In this study, we analyzed the role of dopamine in the regulation of DC function. We found that DCs express dopamine receptors as well as the machinery necessary to synthesize, store, and degrade dopamine. Notably, the expression of D5R decreased upon LPS-induced DC maturation. Deficiency of D5R on the surface of DCs impaired LPS-induced IL-23 and IL-12 production and consequently attenuated the activation and proliferation of Ag-specific CD4(+) T cells. To determine the relevance of D5R expressed on DCs in vivo, we studied the role of this receptor in the modulation of a CD4(+) T cell-driven autoimmunity model. Importantly, D5R-deficient DCs prophylactically transferred into wild-type recipients were able to reduce the severity of experimental autoimmune encephalomyelitis. Furthermore, mice transferred with D5R-deficient DCs displayed a significant reduction in the percentage of Th17 cells infiltrating the CNS without differences in the percentage of Th1 cells compared with animals transferred with wild-type DCs. Our findings demonstrate that by contributing to CD4(+) T cell activation and differentiation to Th17 phenotype, D5R expressed on DCs is able to modulate the development of an autoimmune response in vivo.

Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer.

BACKGROUND: Enzalutamide (formerly MDV3100 and available commercially as Xtandi), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration-resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: binding of androgens to AR, AR nuclear translocation, and association of AR with DNA. Here, we investigate the effects of enzalutamide on AR signaling, AR-dependent gene expression and cell apoptosis. METHODS: The expression of AR target gene prostate-specific antigen (PSA) was measured in LnCaP and C4-2 cells. AR nuclear translocation was assessed in HEK-293 cells stably transfected with AR-yellow fluorescent protein. The in vivo effects of enzalutamide were determined in a mouse xenograft model of CRPC. Differential gene expression in LNCaP cells was measured using Affymetrix human genome microarray technology. RESULTS: We found that unlike bicalutamide, enzalutamide lacked AR agonistic activity at effective doses and did not induce PSA expression or AR nuclear translocation. Additionally, it is more effective than bicalutamide at inhibiting agonist-induced AR nuclear translocation. Enzalutamide induced the regression of tumor volume in a CRPC xenograft model and apoptosis in AR-over-expressing prostate cancer cells. Finally, gene expression profiling in LNCaP cells indicated that enzalutamide opposes agonist-induced changes in genes involved in processes such as cell adhesion, angiogenesis, and apoptosis. CONCLUSIONS: These results indicate that enzalutamide efficiently inhibits AR signaling, and we suggest that its lack of AR agonist activity may be important for these effects.

Induction of Aryl Hydrocarbon Receptor-Mediated Cancer Cell-Selective Apoptosis in Triple-Negative Breast Cancer Cells by a High-Affinity Benzimidazoisoquinoline.

Triple-negative breast cancer (TNBC) remains a disease with a paucity of targeted treatment opportunities. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in a wide range of physiological processes, including the sensing of xenobiotics, immune function, development, and differentiation. Different small-molecule AhR ligands drive strikingly varied cellular and organismal responses. In certain cancers, AhR activation by select small molecules induces cell cycle arrest or apoptosis via activation of tumor-suppressive transcriptional programs. AhR is expressed in triple-negative breast cancers, presenting a tractable therapeutic opportunity. Here, we identify a novel ligand of the aryl hydrocarbon receptor that potently and selectively induces cell death in triple-negative breast cancer cells and TNBC stem cells via the AhR. Importantly, we found that this compound, Analog 523, exhibits minimal cytotoxicity against multiple normal human primary cells. Analog 523 represents a high-affinity AhR ligand with potential for future clinical translation as an anticancer agent.

Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19).

Rapid on-site diagnosis of emerging pathogens is key for early identification of infected individuals and for prevention of further spreading in a population. Currently available molecular diagnostic tests are instrument-based whereas rapid antibody and antigen tests are often not sufficiently sensitive for detection in pre-symptomatic subjects. There is a need for rapid point of care molecular screening tests that can be easily adapted to emerging pathogens and are selective, sensitive, reliable in different settings around the world. We have developed a simple, rapid (<30 â€‹min), and inexpensive test for SARS-CoV-2 that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers and visual detection with paper-based microfluidics. Our test (CoRapID) is specific for SARS-CoV-2 (alpha to omicron variants) and does not detect other coronaviruses and pathogens by in silico and in vitro analysis. A two-step test protocol was developed with stable lyophilized reagents that reduces handling by using portable and disposable components (droppers, microapplicators/swabs, paper-strips). After optimization of assay components and conditions, we have achieved a limit of detection (LoD) of 1 copy/reaction by adding a blocking primer to the lateral flow assay. Using a set of 138 clinical samples, a sensitivity of 88.1% (P â€‹< â€‹0.05, CI: 78.2-93.8%) and specificity of 93.9% (P â€‹< â€‹0.05, CI: 85.4-97.6%) was determined. The lack of need for instrumentation for our CoRapID makes it an ideal on-site primary screening tool for local hospitals, doctors' offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.

Suppression of Ah Receptor (AhR) increases the aggressiveness of TNBC cells and 11-Cl-BBQ-activated AhR inhibits their growth.

Triple-negative breast cancer (TNBC) represents around 15% of the 2.26 million breast cancers diagnosed worldwide annually and has the worst outcome. Despite recent therapeutic advances, there remains a lack of targeted therapies for this breast cancer subtype. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with biological roles in regulating development, xenobiotic metabolism, cell cycle progression and cell death. AhR activation by select ligands can promote tumor suppression in multiple cancer types. AhR can negatively regulate the activity of different oncogenic signaling pathways and can directly upregulate tumor suppressor genes such as p27Kip1. To determine the role of AhR in TNBC, we generated AhR-deficient cancer cells and investigated the impact of AhR loss on TNBC cell growth phenotypes. We found that AhR-deficient MDA-MB-468 TNBC cells have increased proliferation and formed significantly more colonies compared to AhR expressing cells. These cells without AhR expression grew aggressively in vivo. To determine the molecular targets driving this phenotype, we performed transcriptomic profiling in AhR expressing and AhR knockout MDA-MB-468 cells and identified tyrosine receptor kinases, as well as other genes involved in proliferation, survival and clonogenicity that are repressed by AhR. In order to determine therapeutic targeting of AhR in TNBC, we investigated the anti-cancer effects of the novel AhR ligand 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ), which belongs to a class of high affinity, rapidly metabolized AhR ligands called benzimidazoisoquinolines (BBQs). 11-Cl-BBQ induced AhR-dependent cancer cell-selective growth inhibition and strongly inhibited colony formation in TNBC cells.

Necroptosis inhibition counteracts neurodegeneration, memory decline, and key hallmarks of aging, promoting brain rejuvenation.

Age is the main risk factor for the development of neurodegenerative diseases. In the aged brain, axonal degeneration is an early pathological event, preceding neuronal dysfunction, and cognitive disabilities in humans, primates, rodents, and invertebrates. Necroptosis mediates degeneration of injured axons, but whether necroptosis triggers neurodegeneration and cognitive impairment along aging is unknown. Here, we show that the loss of the necroptotic effector Mlkl was sufficient to delay age-associated axonal degeneration and neuroinflammation, protecting against decreased synaptic transmission and memory decline in aged mice. Moreover, short-term pharmacologic inhibition of necroptosis targeting RIPK3 in aged mice, reverted structural and functional hippocampal impairment, both at the electrophysiological and behavioral level. Finally, a quantitative proteomic analysis revealed that necroptosis inhibition leads to an overall improvement of the aged hippocampal proteome, including a subclass of molecular biofunctions associated with brain rejuvenation, such as long-term potentiation and synaptic plasticity. Our results demonstrate that necroptosis contributes to age-dependent brain degeneration, disturbing hippocampal neuronal connectivity, and cognitive function. Therefore, necroptosis inhibition constitutes a potential geroprotective strategy to treat age-related disabilities associated with memory impairment and cognitive decline.

Eisosomes mark static sites of endocytosis.

Endocytosis functions to recycle plasma membrane components, to regulate cell-surface expression of signalling receptors and to internalize nutrients in all eukaryotic cells. Internalization of proteins, lipids and other cargo can occur by one of several pathways that have different, but often overlapping, molecular requirements. To mediate endocytosis, effectors assemble transiently underneath the plasma membrane, carry out the mechanics of membrane deformation, cargo selection and vesicle internalization, and then disassemble. The mechanism by which endocytosis initiates at particular locations on the plasma membrane has remained unknown. Sites of endocytosis might be formed randomly, induced by stochastic protein and/or lipid clustering. Alternatively, endocytosis might initiate at specific locations. Here we describe large immobile protein assemblies at the plasma membrane in the yeast Saccharomyces cerevisiae that mark endocytic sites. These structures, termed eisosomes (from the Greek 'eis', meaning into or portal, and 'soma', meaning body), are composed primarily of two cytoplasmic proteins, Pil1 and Lsp1. A plasma membrane protein, Sur7, localizes to eisosomes. These structures colocalize with sites of protein and lipid endocytosis, and their components genetically interact with known endocytic effectors. Loss of Pil1 leads to clustering of eisosome remnants and redirects endocytosis and endocytic effector proteins to these clusters.

Correction of eIF2-dependent defects in brain protein synthesis, synaptic plasticity, and memory in mouse models of Alzheimer's disease.

Neuronal protein synthesis is essential for long-term memory consolidation, and its dysregulation is implicated in various neurodegenerative disorders, including Alzheimer's disease (AD). Cellular stress triggers the activation of protein kinases that converge on the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which attenuates mRNA translation. This translational inhibition is one aspect of the integrated stress response (ISR). We found that postmortem brain tissue from AD patients showed increased phosphorylation of eIF2α and reduced abundance of eIF2B, another key component of the translation initiation complex. Systemic administration of the small-molecule compound ISRIB (which blocks the ISR downstream of phosphorylated eIF2α) rescued protein synthesis in the hippocampus, measures of synaptic plasticity, and performance on memory-associated behavior tests in wild-type mice cotreated with salubrinal (which inhibits translation by inducing eIF2α phosphorylation) and in both ß-amyloid-treated and transgenic AD model mice. Thus, attenuating the ISR downstream of phosphorylated eIF2α may restore hippocampal protein synthesis and delay cognitive decline in AD patients.

The unfolded protein response during prostate cancer development.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). The UPR promotes cell survival by adjusting ER protein folding capacity but if homeostasis cannot be re-established, apoptosis is induced. The execution of life/death decisions is regulated by the three UPR branches (IRE1, PERK, ATF6) and their downstream effectors. Events that offset the balance of the UPR branches can have devastating consequences, and UPR misregulation has been correlated with various diseases, including metabolic and neurodegenerative diseases and cancer. In cancer, upregulation of the UPR is thought to provide a growth advantage to tumor cells. In contrast to this prevailing view, we report here an analysis of data obtained by others indicating that all three UPR branches appear selectively down-regulated in mouse models of prostate tumorigenesis.

Cognition-enhancing properties of Dimebon in a rat novel object recognition task are unlikely to be associated with acetylcholinesterase inhibition or N-methyl-D-aspartate receptor antagonism.

Dimebon (dimebolin) treatment enhances cognition in patients with Alzheimer's disease (AD) or Huntington's disease. Although Dimebon was originally thought to improve cognition and memory through inhibition of acetylcholinesterase (AChE) and the N-methyl-d-aspartate (NMDA) receptor, the low in vitro affinity for these targets suggests that these mechanisms may not contribute to its clinical effects. To test this hypothesis, we assessed whether Dimebon enhances cognition in rats and if such an action is related to either mechanism or additional candidate mechanisms. Acute oral administration of Dimebon to rats (0.05, 0.5, and 5 mg/kg) enhanced cognition in a novel object recognition task and produced Dimebon brain concentrations of 1.7 +/- 0.43, 14 +/- 5.1, and 172 +/- 94 nM, respectively. At these concentrations, Dimebon did not alter the activity of recombinant human or rat brain AChE. Unlike the AChE inhibitors donepezil and galantamine, Dimebon did not change acetylcholine levels in the hippocampus or prefrontal cortex of freely moving rats. Dimebon displays affinity for the NMDA receptor (K(i) = 105 +/- 18 microM) that is considerably higher than brain concentrations associated with cognition enhancement in the novel object recognition task and 200-fold weaker than that of memantine (K(i) = 0.54 +/- 0.05 microM). Dimebon did not block NMDA-induced calcium influx in primary neuronal cells (IC(50) > 50 microM), consistent with a lack of significant effect on this pathway. The cognition-enhancing effects of Dimebon are unlikely to be mediated by AChE inhibition or NMDA receptor antagonism, and its mechanism of action appears to be distinct from currently approved medications for AD.

Small molecule cognitive enhancer reverses age-related memory decline in mice.

With increased life expectancy, age-associated cognitive decline becomes a growing concern, even in the absence of recognizable neurodegenerative disease. 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 levels of activating transcription factor 4 (ATF4) and phosphorylated eukaryotic translation initiation factor eIF2. 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 in otherwise healthy individuals.

Integrated Stress Response Inhibitor Reverses Sex-Dependent Behavioral and Cell-Specific Deficits after Mild Repetitive Head Trauma.

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.