Mechanism of allosteric inhibition of human p97/VCP ATPase and its disease mutant by triazole inhibitors.

Human p97 ATPase is crucial in various cellular processes, making it a target for inhibitors to treat cancers, neurological, and infectious diseases. Triazole allosteric p97 inhibitors have been demonstrated to match the efficacy of CB-5083, an ATP-competitive inhibitor, in cellular models. However, the mechanism is not well understood. This study systematically investigates the structures of new triazole inhibitors bound to  both wild-type and disease mutant forms of p97 and measures their effects on function. These inhibitors bind at the interface of the D1 and D2 domains of each p97 subunit, shifting surrounding helices and altering the loop structures near the C-terminal α2 G helix to modulate domain-domain communications. A key structural moiety of the inhibitor affects the rotameric conformations of interacting side chains, indirectly modulating the N-terminal domain conformation in p97 R155H mutant. The differential effects of inhibitor binding to wild-type and mutant p97 provide insights into drug design with enhanced specificity, particularly for oncology applications.

Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor. Our observations suggest a multi-layered genetic response to drive tumor recurrence and implicate PTP4A2 (protein tyrosine phosphatase 4A2) as a modulator of self-renewal, proliferation and tumorigenicity in recurrent GBM. Genetic perturbation or small-molecule inhibition of PTP4A2 acts through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and its downstream molecular players, exploiting a functional dependency on ROBO signaling. Because a pan-PTP4A inhibitor was limited by poor penetrance across the blood-brain barrier in vivo, we engineered a second-generation chimeric antigen receptor (CAR) T cell therapy against ROBO1, a cell surface receptor enriched across recurrent GBM specimens. A single dose of ROBO1-targeted CAR T cells doubled median survival in cell-line-derived xenograft (CDX) models of recurrent GBM. Moreover, in CDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma, ROBO1 CAR T cells eradicated tumors in 50-100% of mice. Our study identifies a promising multi-targetable PTP4A-ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors.

Mitigation of Fetal Radiation Injury from Mid-Gestation Total-body Irradiation by Maternal Administration of Mitochondrial-Targeted GS-Nitroxide JP4-039.

Victims of a radiation terrorist event will include pregnant women and unborn fetuses. Mitochondrial dysfunction and oxidative stress are key pathogenic factors of fetal radiation injury. The goal of this preclinical study is to investigate the efficacy of mitigating fetal radiation injury by maternal administration of the mitochondrial-targeted gramicidin S (GS)-nitroxide radiation mitigator JP4-039. Pregnant female C57BL/6NTac mice received 3 Gy total-body irradiation (TBI) at mid-gestation embryonic day 13.5 (E13.5). Using novel time-and-motion-resolved 4D in utero magnetic resonance imaging (4D-uMRI), we found TBI caused extensive injury to the fetal brain that included cerebral hemorrhage, loss of cerebral tissue, and hydrocephalus with excessive accumulation of cerebrospinal fluid (CSF). Histopathology of the fetal mouse brain showed broken cerebral vessels and elevated apoptosis. Further use of novel 4D Oxy-wavelet MRI capable of probing in vivo mitochondrial function in intact brain revealed a significant reduction of mitochondrial function in the fetal brain after 3 Gy TBI. This was validated by ex vivo Oroboros mitochondrial respirometry. One day after TBI (E14.5) maternal administration of JP4-039, which passes through the placenta, significantly reduced fetal brain radiation injury and improved fetal brain mitochondrial respiration. Treatment also preserved cerebral brain tissue integrity and reduced cerebral hemorrhage and cell death. JP4-039 administration following irradiation resulted in increased survival of pups. These findings indicate that JP4-039 can be deployed as a safe and effective mitigator of fetal radiation injury from mid-gestational in utero ionizing radiation exposure.

Synthesis and Biological Analysis of Iso-dimethyltryptamines in a Model of Light-Induced Retinal Degeneration.

Iso-dimethyltryptamine (isoDMT) analogues with heterocyclic substitutions at the indole C(3) were prepared in a hydrogen autotransfer alkylation and tested in combination with natural and unnatural clavine alkaloids in a model of light-induced retinal degeneration for protection against retinal degeneration. On the basis of measurements with optical coherence tomography and electroretinography, three compounds showed better efficacy than the positive control bromocriptine at equivalent systemically administered doses. These studies provide further insights into the role of serotonin receptors and their potential therapeutic applications in ocular diseases.

JP4-039, a Mitochondria-Targeted Nitroxide, Mitigates the Effect of Apoptosis and Inflammatory Cell Migration in the Irradiated Mouse Retina.

We hypothesize that the injection of JP4-039, a mitochondria-targeted nitroxide, prior to irradiation of the mouse retina may decrease apoptosis and reduce neutrophil and macrophage migration into the retina. In our study, we aimed to examine the effects of JP4-039 in the mouse retina using fluorescent microscopy, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and flow cytometry. Forty-five mice and one eye per mouse were used. In Group 1, fluorescent microscopy was used to determine retinal uptake of 10 µL (0.004 mg/µL) of intravitreally injected BODIPY-labeled JP4-039 at 0, 15, and 60 min after injection. In Group 2, the TUNEL assay was performed to investigate the rate of apoptosis after irradiation in addition to JP4-039 injection, compared to controls. In Group 3, flow cytometry was used to determine the extent of inflammatory cell migration into the retina after irradiation in addition to JP4-039 injection, compared to controls. Maximal retinal uptake of JP4-039 was 15 min after intravitreal injection (p < 0.0001). JP4-039-treated eyes had lower levels of retinal apoptosis (35.8 ± 2.5%) than irradiated controls (49.0 ± 2.7%; p = 0.0066) and demonstrated reduced migration of N1 cells (30.7 ± 11.7% vs. 77.7 ± 5.3% controls; p = 0.004) and M1 cells (76.6 ± 4.2 vs. 88.1 ± 3.7% controls, p = 0.04). Pretreatment with intravitreally injected JP4-039 reduced apoptosis and inflammatory cell migration in the irradiated mouse retina, marking the first confirmed effect of this molecule in retinal tissue. Further studies may allow for safety profiling and potential use for patients with radiation retinopathy.

SARS-CoV-2 Spike Protein Induces Oxidative Stress and Senescence in Mouse and Human Lung.

BACKGROUND/AIM: There is concern that people who had COVID-19 will develop pulmonary fibrosis. Using mouse models, we compared pulmonary inflammation following injection of the spike protein of SARS-CoV-2 (COVID-19) to radiation-induced inflammation to demonstrate similarities between the two models. SARS-CoV-2 (COVID-19) induces inflammatory cytokines and stress responses, which are also common to ionizing irradiation-induced acute pulmonary damage. Cellular senescence, which is a late effect following exposure to SARS-CoV-2 as well as radiation, was investigated. MATERIALS AND METHODS: We evaluated the effect of SARS-CoV-2 spike protein compared to ionizing irradiation in K18-hACE2 mouse lung, human lung cell lines, and in freshly explanted human lung. We measured reactive oxygen species, DNA double-strand breaks, stimulation of transforming growth factor-beta pathways, and cellular senescence following exposure to SARS-CoV-2 spike protein, irradiation or SARS-COV-2 and irradiation. We also measured the effects of the antioxidant radiation mitigator MMS350 following irradiation or exposure to SARS-CoV-2. RESULTS: SARS-CoV-2 spike protein induced reactive oxygen species, DNA double-strand breaks, transforming growth factor-ß signaling pathways, and senescence, which were exacerbated by prior or subsequent ionizing irradiation. The water-soluble radiation countermeasure, MMS350, reduced spike protein-induced changes. CONCLUSION: In both the SARS-Co-2 and the irradiation mouse models, similar responses were seen indicating that irradiation or exposure to SARS-CoV-2 virus may lead to similar lung diseases such as pulmonary fibrosis. Combination of irradiation and SARS-CoV-2 may result in a more severe case of pulmonary fibrosis. Cellular senescence may explain some of the late effects of exposure to SARS-CoV-2 spike protein and to ionizing irradiation.

Rhodium(I)-Catalyzed Annulation of Bicyclo[1.1.0]butyl-Substituted Dihydroquinolines and Dihydropyridines.

Bicyclo[1.1.0]butane-containing compounds feature a unique chemical reactivity, trigger "strain-release" reaction cascades, and provide novel scaffolds with considerable utility in the drug discovery field. We report the synthesis of new bicyclo[1.1.0]butane-linked heterocycles by a nucleophilic addition of bicyclo[1.1.0]butyl anions to 8-isocyanatoquinoline, or, alternatively, iminium cations derived from quinolines and pyridines. The resulting bicyclo[1.1.0]butanes are converted with high regioselectivity to unprecedented bridged heterocycles in a rhodium(I)-catalyzed annulative rearrangement. The addition/rearrangement process tolerates a surprisingly large range of functional groups. Subsequent chemo- and stereoselective synthetic transformations of urea, alkene, cyclopropane, and aniline moieties of the 1-methylene-5-azacyclopropa[cd]indene scaffolds provide several additional new heterocyclic building blocks. X-ray structure-validated quantum mechanical DFT calculations of the reaction pathway indicate the intermediacy of rhodium carbenoid and metallocyclobutane species.

Mitigation of Fetal Irradiation Injury from Mid-Gestation Total Body Radiation with Mitochondrial-Targeted GS-Nitroxide JP4-039.

Victims of a radiation terrorist event will include pregnant women and unborn fetuses. Mitochondrial dysfunction and oxidative stress are key pathogenic factors of fetal irradiation injury. The goal of this preclinical study is to investigate the efficacy of mitigating fetal irradiation injury by maternal administration of the mitochondrial-targeted gramicidin S (GS)- nitroxide radiation mitigator, JP4-039. Pregnant female C57BL/6NTac mice received 3 Gy total body ionizing irradiation (TBI) at mid-gestation embryonic day 13.5 (E13.5). Using novel time- and-motion-resolved 4D in utero magnetic resonance imaging (4D-uMRI), we found TBI caused extensive injury to the fetal brain that included cerebral hemorrhage, loss of cerebral tissue, and hydrocephalus with excessive accumulation of cerebrospinal fluid (CSF). Histopathology of the fetal mouse brain showed broken cerebral vessels and elevated apoptosis. Further use of novel 4D Oxy-wavelet MRI capable of probing in vivo mitochondrial function in intact brain revealed significant reduction of mitochondrial function in the fetal brain after 3Gy TBI. This was validated by ex vivo Oroboros mitochondrial respirometry. Maternal administration JP4-039 one day after TBI (E14.5), which can pass through the placental barrier, significantly reduced fetal brain radiation injury and improved fetal brain mitochondrial respiration. This also preserved cerebral brain tissue integrity and reduced cerebral hemorrhage and cell death. As JP4-039 administration did not change litter sizes or fetus viability, together these findings indicate JP4-039 can be deployed as a safe and effective mitigator of fetal radiation injury from mid-gestational in utero ionizing radiation exposure. One Sentence Summary: Mitochondrial-targeted gramicidin S (GS)-nitroxide JP4-039 is safe and effective radiation mitigator for mid-gestational fetal irradiation injury.

Biological studies of clavine alkaloids targeting CNS receptors.

In contrast to well established psychedelics such as lysergic acid diethylamide (LSD) and psilocybin, ergot alkaloids of the clavine subclass have not been thoroughly investigated, in spite of their broad occurrence in nature and their well-established potent physiological effects. This study presents the current knowledge on the biological properties of clavine alkaloids, draws comparisons to the pharmacology of ergolines and related psychedelics, and demonstrates opportunities to develop novel structure-activity relationship (SAR) profiles. The latter could usher in a new stage of medicinal chemistry studies that enable an expansion of the currently structurally limited portfolio of psychedelic therapeutics.

Synthesis of 13C-methyl-labeled amino acids and their incorporation into proteins in mammalian cells.

Isotopic labeling of methyl-substituted proteinogenic amino acids with 13C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with 15N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of 13C-methyl labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups. Recently, similar methodology has been applied to enable 13C-methyl labeling of Ile, Val, and Leu in yeast, extending the approach to proteins that do not readily fold when produced in bacteria. Mammalian or insect cells are nonetheless preferable for production of many human proteins, yet 13C-methyl labeling using similar metabolic precursors is not feasible as these cells lack the requisite biosynthetic machinery. Herein, we report versatile and high-yielding synthetic routes to 13C methyl-labeled amino acids based on palladium-catalyzed C(sp3)-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, 13C-γ2-Ile and 13C-γ1,γ2-Val, into human receptor extracellular domains with multiple disulfides using suspension-cultured HEK293 cells. Production costs are reasonable, even at moderate expression levels of 2-3 mg purified protein per liter of medium, and the method can be extended to label other methyl groups, such as 13C-δ1-Ile and 13C-δ1,δ2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of 13C methyl-labeled amino acids generated de novo by a versatile synthetic route.

C3-Functionalization of indoles with α-heteroaryl-substituted methyl alcohols.

The transition metal-free Cs2CO3/Oxone®-mediated C3-alkylation of indoles proceeds in moderate to high yields with a variety of C4-C7 functionalized indoles and is applicable to 2-, 3- and 4-hydroxymethyl pyridines and related electron-deficient heterocycles, permitting novel late-stage drug functionalizations. Preliminary mechanistic studies support a hydrogen autotransfer-type chain process starting with an initial oxidation of the alcohol to the corresponding aldehyde, followed by a subsequent condensation onto indole and reduction/hydride delivery from another equivalent of the primary alcohol.

Optimization of 1,2,4-Triazole-Based p97 Inhibitors for the Treatment of Cancer.

The AAA+ ATPase p97 (valosin-containing protein, VCP) is a master regulator of protein homeostasis and therefore represents a novel target for cancer therapy. Starting from a known allosteric inhibitor, NMS-873, we systematically optimized this scaffold, in particular, by applying a benzene-to-acetylene isosteric replacement strategy, specific incorporation of F, and eutomer/distomer identification, which led to compounds that exhibited nanomolar biochemical and cell-based potency. In cellular pharmacodynamic assays, robust effects on biomarkers of p97 inhibition and apoptosis, including increased levels of ubiquitinated proteins, CHOP and cleaved caspase 3, were observed. Compound (R)-29 (UPCDC-30766) represents the most potent allosteric inhibitor of p97 reported to date.

Metabolism-guided development of Ko143 analogs as ABCG2 inhibitors.

ATP-binding cassette subfamily G member 2 (ABCG2), an efflux transporter, is involved in multiple pathological processes. Ko143 is a potent ABCG2 inhibitor; however, it is quickly metabolized through carboxylesterase 1-mediated hydrolysis of its t-butyl ester moiety. The current work aimed to develop more metabolically stable ABCG2 inhibitors. Novel Ko143 analogs were designed and synthesized by replacing the unstable t-butyl ester moiety in Ko143 with an amide group. The synthesized Ko143 analogs were evaluated for their ABCG2 inhibitory activity, binding mode with ABCG2, cytotoxicity, and metabolic stability. We found that the amide modification of Ko143 led to metabolically stable ABCG2 inhibitors. Among these Ko143 analogs, K2 and K34 are promising candidates with favorable oral pharmacokinetic profiles in mice. In summary, we synthesized novel Ko143 analogs with improved metabolic stability, which can potentially be used as lead compounds for the future development of ABCG2 inhibitors.

Inhibition of tyrosine kinase Fgr prevents radiation-induced pulmonary fibrosis (RIPF).

Cellular senescence is involved in the development of pulmonary fibrosis as well as in lung tissue repair and regeneration. Therefore, a strategy of removal of senescent cells by senolytic drugs may not produce the desired therapeutic result. Previously we reported that tyrosine kinase Fgr is upregulated in ionizing irradiation-induced senescent cells. Inhibition of Fgr reduces the production of profibrotic proteins by radiation-induced senescent cells in vitro; however, a mechanistic relationship between senescent cells and radiation-induced pulmonary fibrosis (RIPF) has not been established. We now report that senescent cells from the lungs of mice with RIPF, release profibrotic proteins for target cells and secrete chemotactic proteins for marrow cells. The Fgr inhibitor TL02-59, reduces this release of profibrotic chemokines from the lungs of RIPF mice, without reducing numbers of senescent cells. In vitro studies demonstrated that TL02-59 abrogates the upregulation of profibrotic genes in target cells in transwell cultures. Also, protein arrays using lung fibroblasts demonstrated that TL02-59 inhibits the production of chemokines involved in the migration of macrophages to the lung. In thoracic-irradiated mice, TL02-59 prevents RIPF, significantly reduces levels of expression of fibrotic gene products, and significantly reduces the recruitment of CD11b+ macrophages to the lungs. Bronchoalveolar lavage (BAL) cells from RIPF mice show increased Fgr and other senescent cell markers including p16. In human idiopathic pulmonary fibrosis (IPF) and in RIPF, Fgr, and other senescent cell biomarkers are increased. In both mouse and human RIPF, there is an accumulation of Fgr-positive proinflammatory CD11b+ macrophages in the lungs. Thus, elevated levels of Fgr in lung senescent cells upregulate profibrotic gene products, and chemokines that might be responsible for macrophage infiltration into lungs. The detection of Fgr in senescent cells that are obtained from BAL during the development of RIPF may help predict the onset and facilitate the delivery of medical countermeasures.

Synthesis of Veliparib Prodrugs and Determination of Drug-Release-Dependent PARP-1 Inhibition.

Poly(ADP-ribose) polymerase (PARP) plays a key role in repairing DNA damage, and several PARP inhibitors have been approved as treatments in BRCA1/2 mutated breast and ovarian cancers. Mounting evidence also supports their application as neuroprotective agents since PARP overactivation compromises the mitochondrial homeostasis by consumption of NAD+ reserves, leading to an increase in reactive oxygen and nitrogen species and a spike in intracellular Ca2+ levels. Herein, we present the synthesis and preliminary evaluation of new mitochondria-targeting PARP inhibitor prodrugs of (±)-veliparib, with the goal to advance potential neuroprotective properties without impairing the repair of damaged DNA in the nucleus.

OGG1 and MUTYH repair activities promote telomeric 8-oxoguanine induced cellular senescence.

Telomeres are prone to formation of the common oxidative lesion 8-oxoguanine (8oxoG), and the acute production of 8oxoG damage at telomeres is sufficient to drive rapid cellular senescence. OGG1 and MUTYH glycosylases initiate base excision repair (BER) at 8oxoG sites to remove the lesion or prevent mutation. Here, we show OGG1 loss or inhibition, or MUTYH loss, partially rescues telomeric 8oxoG-induced senescence, and loss of both glycosylases results in a near complete rescue. Loss of these glycosylases also suppresses 8oxoG-induced telomere fragility and dysfunction, indicating that single-stranded break (SSB) intermediates arising downstream of glycosylase activity impair telomere replication. The failure to initiate BER in glycosylase-deficient cells suppresses PARylation at SSB intermediates and confers resistance to the synergistic effects of PARP inhibitors on damage-induced senescence. Our studies reveal that inefficient completion of 8oxoG BER at telomeres triggers cellular senescence via SSB intermediates which impair telomere replication and stability.

Mitochondrial ROS Triggers KIN Pathogenesis in FAN1-Deficient Kidneys.

Karyomegalic interstitial nephritis (KIN) is a genetic adult-onset chronic kidney disease (CKD) characterized by genomic instability and mitotic abnormalities in the tubular epithelial cells. KIN is caused by recessive mutations in the FAN1 DNA repair enzyme. However, the endogenous source of DNA damage in FAN1/KIN kidneys has not been identified. Here we show, using FAN1-deficient human renal tubular epithelial cells (hRTECs) and FAN1-null mice as a model of KIN, that FAN1 kidney pathophysiology is triggered by hypersensitivity to endogenous reactive oxygen species (ROS), which cause chronic oxidative and double-strand DNA damage in the kidney tubular epithelial cells, accompanied by an intrinsic failure to repair DNA damage. Furthermore, persistent oxidative stress in FAN1-deficient RTECs and FAN1 kidneys caused mitochondrial deficiencies in oxidative phosphorylation and fatty acid oxidation. The administration of subclinical, low-dose cisplatin increased oxidative stress and aggravated mitochondrial dysfunction in FAN1-deficient kidneys, thereby exacerbating KIN pathophysiology. In contrast, treatment of FAN1 mice with a mitochondria-targeted ROS scavenger, JP4-039, attenuated oxidative stress and accumulation of DNA damage, mitigated tubular injury, and preserved kidney function in cisplatin-treated FAN1-null mice, demonstrating that endogenous oxygen stress is an important source of DNA damage in FAN1-deficient kidneys and a driver of KIN pathogenesis. Our findings indicate that therapeutic modulation of kidney oxidative stress may be a promising avenue to mitigate FAN1/KIN kidney pathophysiology and disease progression in patients.

Quassinoid analogs exert potent antitumor activity via reversible protein biosynthesis inhibition in human colorectal cancer.

Cellular protein synthesis is accelerated in human colorectal cancer (CRC), and high expression of protein synthesis regulators in CRC patients is associated with poor prognosis. Thus, inhibition of protein synthesis may be an effective therapeutic strategy for CRC. We previously demonstrated that the quassinoid bruceantinol (BOL) had antitumor activity against CRC. Herein, potent tumor growth suppression (>80%) and STAT3 inhibition was observed in two different mouse models following BOL administration. Loss of body and spleen weight was observed but was eliminated upon nanoparticle encapsulation while maintaining strong antitumor activity. STAT3 siRNA knockdown exhibited modest suppression of cell proliferation. Surprisingly, STAT3 inhibition using a PROTAC degrader (SD-36) had little effect on cancer cell proliferation suggesting the possibility of additional mechanism(s) of action for quassinoids. BOL-resistant (BR) cell lines, HCT116BR and HCA7BR, were equally sensitive to standard CRC therapeutic agents and known STAT3 inhibitors but resistant to homoharringtonine (HHT), a known protein synthesis inhibitor. The ability of quassinoids to inhibit protein synthesis was dependent on the structure of the C15 sidechain. Of note, BOL did not inhibit protein synthesis in normal human colon epithelial cells whereas HHT and napabucasin remained effective in these normal cells. Novel quassinoids were designed, synthesized, and evaluated in pre-clinical CRC models. Treatment with the most potent analog, 5c, resulted in significant inhibition of cell proliferation and protein synthesis at nanomolar concentrations. These quassinoid analogs may represent a novel class of protein synthesis inhibitors for the treatment of human CRC.

Non-Essential Amino Acid Availability Influences Proteostasis and Breast Cancer Cell Survival During Proteotoxic Stress.

Protein homeostasis (proteostasis) regulates tumor growth and proliferation when cells are exposed to proteotoxic stress, such as during treatment with certain chemotherapeutics. Consequently, cancer cells depend to a greater extent on stress signaling, and require the integrated stress response (ISR), amino acid metabolism, and efficient protein folding and degradation pathways to survive. To define how these interconnected pathways are wired when cancer cells are challenged with proteotoxic stress, we investigated how amino acid abundance influences cell survival when Hsp70, a master proteostasis regulator, is inhibited. We previously demonstrated that cancer cells exposed to a specific Hsp70 inhibitor induce the ISR via the action of two sensors, GCN2 and PERK, in stress-resistant and sensitive cells, respectively. In resistant cells, the induction of GCN2 and autophagy supported resistant cell survival, yet the mechanism by which these events were induced remained unclear. We now report that amino acid availability reconfigures the proteostasis network. Amino acid supplementation, and in particular arginine addition, triggered cancer cell death by blocking autophagy. Consistent with the importance of amino acid availability, which when limited activates GCN2, resistant cancer cells succumbed when challenged with a potentiator for another amino acid sensor, mTORC1, in conjunction with Hsp70 inhibition. IMPLICATIONS: These data position amino acid abundance, GCN2, mTORC1, and autophagy as integrated therapeutic targets whose coordinated modulation regulates the survival of proteotoxic-resistant breast cancer cells.

Potentiation of neuromuscular transmission by a small molecule calcium channel gating modifier improves motor function in a severe spinal muscular atrophy mouse model.

Spinal muscular atrophy (SMA) is a monogenic disease that clinically manifests as severe muscle weakness owing to neurotransmission defects and motoneuron degeneration. Individuals affected by SMA experience neuromuscular weakness that impacts functional activities of daily living. We have used a mouse model of severe SMA (SMNΔ7) to test whether a calcium channel gating modifier (GV-58), alone or in combination with a potassium channel antagonist (3,4-diaminopyridine; 3,4-DAP), can improve neuromuscular function in this mouse model. Bath application of GV-58 alone or in combination with 3,4-DAP significantly restored neuromuscular transmission to control levels in both a mildly vulnerable forearm muscle and a strongly vulnerable trunk muscle in SMNΔ7 mice at postnatal days 10-12. Similarly, acute subcutaneous administration of GV-58 to postnatal day 10 SMNΔ7 mice, alone or in combination with 3,4-DAP, significantly increased a behavioral measure of muscle strength. These data suggest that GV-58 may be a promising treatment candidate that could address deficits in neuromuscular function and strength and that the addition of 3,4-DAP to GV-58 treatment could aid in restoring function in SMA.