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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., (© 2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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-β 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., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

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.

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.

Isotopic labeling of methyl-substituted proteinogenic amino acids with 13 C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with 15 N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of 13 C-methyl labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups. Recently, similar methodology has been applied to enable 13 C-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 13 C-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 13 C methyl-labeled amino acids based on palladium-catalyzed C(sp 3 )-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, 13 C-γ2-Ile and 13 C-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 13 C-δ1-Ile and 13 C-δ1,δ2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of 13 C methyl-labeled amino acids generated de novo by a versatile synthetic route.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tasker, Pazur and Wipf.)

The transition metal-free Cs 2 CO 3 /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.

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., Competing Interests: Declaration of competing interest X.M., J.Z., and J.L. are inventors on a patent (WO2020236901) and hold equity in Portal Therapeutics, Inc. The authors declare no other competing interests., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

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., (© 2023. The Author(s).)

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., (©2023 American Association for Cancer Research.)

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., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

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, HCT116 BR and HCA7 BR , 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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 Ca 2+ 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., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

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.

Protein tyrosine phosphatase type IVA member 3 (PTP4A3 or PRL-3) is a nonreceptor, oncogenic, dual-specificity phosphatase that is highly expressed in many human tumors, including ovarian cancer, and is associated with a poor patient prognosis. Recent studies suggest that PTP4A3 directly dephosphorylates SHP-2 phosphatase as part of a STAT3-PTP4A3 feedforward loop and directly dephosphorylates p38 kinase. The goal of the current study was to examine the effect of a PTP4A phosphatase inhibitor, 7-imino-2-phenylthieno[3,2- c ]pyridine-4,6(5 H ,7 H )-dione (JMS-053), on ovarian cancer STAT3, SHP-2, and p38 kinase phosphorylation. JMS-053 caused a concentration- and time-dependent decrease in the activated form of STAT3, Y 705 phospho-STAT3, in ovarian cancer cells treated in vitro. In contrast, the phosphorylation status of two previously described direct PTP4A3 substrates, SHP-2 phosphatase and p38 kinase, were rapidly increased with JMS-053 treatment. We generated A2780 and OVCAR4 ovarian cancer cells resistant to JMS-053, and the resulting cells were not crossresistant to paclitaxel, cisplatin, or teniposide. JMS-053-resistant A2780 and OVCAR4 cells exhibited a 95% and 50% decrease in basal Y 705 phospho-STAT3, respectively. JMS-053-resistant OVCAR4 cells had an attenuated phosphorylation and migratory response to acute exposure to JMS-053. These results support a regulatory role for PTP4A phosphatase in ovarian cancer cell STAT3 and p38 signaling circuits. SIGNIFICANCE STATEMENT: This study demonstrates that chemical inhibition of PTP4A phosphatase activity with JMS-053 decreases STAT3 activation and increases SHP-2 phosphatase and p38 kinase phosphorylation activation in ovarian cancer cells. The newly developed JMS-053-resistant ovarian cancer cells should provide useful tools to further probe the role of PTP4A phosphatase in ovarian cancer cell survival and cell signaling., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Now entering its fourth decade, research on the biological function, small molecule inhibition, and disease relevance of the three known isoforms of protein kinase D, PKD1, PKD2, and PKD3, has entered a mature development stage. This mini-perspective focuses on the medicinal chemistry that provided a structurally diverse set of mainly active site inhibitors, which, for a brief time period, moved through preclinical development stages but have yet to be tested in clinical trials. In particular, between 2006 and 2012, a rapid expansion of synthetic efforts led to several moderately to highly PKD-selective chemotypes but did not yet achieve PKD subtype selectivity or resolve general toxicity and pharmacokinetic challenges. In addition to cancer, other unresolved medical needs in cardiovascular, inflammatory, and metabolic diseases would, however, benefit from a renewed focus on potent and selective PKD modulators.

Alterations in phospholipids have long been associated with spinal cord injury (SCI). However, their specific roles and signaling cascades in mediating cell death and tissue repair remain unclear. Here we investigated whether alterations of cardiolipin (CL), a family of mitochondrion-specific phospholipids, play a crucial role in mitochondrial dysfunction and neuronal death following SCI. Lipidomic analysis was used to determine the profile of CL alteration in the adult rat spinal cord following a moderate contusive SCI at the 10th thoracic (T10) level. Cellular, molecular, and genetic assessments were performed to determine whether CL alterations mediate mitochondrial dysfunction and neuronal death after SCI, and, if so, whether reversing CL alteration leads to neuroprotection after SCI. Using lipidomic analysis, we uncovered CL alterations at an early stage of SCI. Over 50 distinct CL species were identified, of which 50% showed significantly decreased abundance after SCI. The decreased CL species contained mainly polyunsaturated fatty acids that are highly susceptible to peroxidation. In parallel, 4-HNE, a lipid peroxidation marker, significantly increased after SCI. We found that mitochondrial oxidative stress not only induced CL oxidation, but also resulted in CL loss by activating cPLA 2 to hydrolyze CL. CL alterations induced mitochondrial dysfunction and neuronal death. Remarkably, pharmacologic inhibition of CL alterations with XJB-5-131, a novel mitochondria-targeted electron and reactive oxygen species scavenger, reduced cell death, tissue damage and ameliorated motor deficits after SCI in adult rats. These findings suggest that CL alteration could be a novel mechanism that mediates injury-induced neuronal death, and a potential therapeutic target for ameliorating secondary SCI., (© 2022. The Author(s).)

Rh(I)-catalyzed cycloisomerizations of bicyclo[1.1.0]butanes provide a fruitful approach to cyclopropane-fused heterocycles. Products and stereochemical outcome are highly dependent on catalyst. The triphenylphosphine (PPh 3 ) ligand provides pyrrolidines, placing substituents anti to the cyclopropyl group. The 1,2-bis(diphenylphosphino)ethane (dppe) ligand yields azepanes with substituents syn to the cyclopropyl group. In this work, quantum mechanical DFT calculations pinpoint a reversal of regio- and diastereoselectivity, suggesting a concerted (double) C-C bond cleavage and rhodium carbenoid formation, driven by strain-release. The ligand-influenced cleavage step determines the regioselectivity of carbometalation and product formation, and suggests new applications of bicyclobutanes., (© 2022. The Author(s).)

Key transformations in a four-step synthesis of the ergot alkaloid scaffold include a novel cesium carbonate-mediated hydrogen autotransfer alkylation to generate the C(3)-C(4) bond and an intramolecular Heck reaction that directly establishes the C(9)-C(10) alkene of methyl lysergate. An ester reduction and a streamlined experimental procedure establish a readily scalable, expedient total synthesis of all four stereoisomers of lysergol and isolysergol, including the previously unknown (-)-lysergol, for pharmacological evaluation at 5-HT 1A and 5HT 2A,B,C receptors. A bicyclic scaffold is also characterized for the first time in the intramolecular Heck coupling.

To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV 50 ) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain., (Copyright © 2022. Published by Elsevier Ltd.)

Quassinoid natural products have gained considerable recognition for their diverse biological properties and their synthetically challenging, highly oxygenated polycyclic structures. Herein, we discuss strategies and tactics in the total synthesis of quassinoids that have been evolving over the past 15 years. Additionally, recent structure-activity relationships and potential biological mechanisms of actions are briefly summarized.

Identification of novel androgen receptor (AR) antagonists may lead to urgently needed new treatments for patients with prostate cancer resistant to current AR antagonists. AR is presently the main target for treating prostate cancer. Clinically approved AR antagonists compete with dihydrotestosterone (DHT) for binding to the ligand-binding domain (LBD) of AR, and patients eventually develop resistance to these treatments. One approach to overcoming resistance is to discover compounds that inhibit AR in alternative ways. Our lab previously identified a small molecule, JJ-450, that is capable of inhibiting AR lacking LBD. To optimize the efficacy of this class of inhibitors, we developed structural analogues of JJ-450 and identified (+)-JJ-74-138 as a promising candidate. Here, we show that (+)-JJ-74-138 is more potent than JJ-450 in the inhibition of androgen-independent AR activity in enzalutamide-resistant LN95 cells. Further studies showed (+)-JJ-74-138 inhibition of castration-resistant PSA expression in all tested castration-resistant prostate cancer (CRPC) cells. (+)-JJ-74-138 inhibited mRNA expression of AR and ARv7 target genes and reduced AR level in the nucleus in the absence of androgens. Also, this analogue noncompetitively inhibited androgen-stimulated AR activity in C4-2, LN95, and 22Rv1 CRPC cells. At low dosages, (+)-JJ-74-138 inhibited the proliferation of enzalutamide-resistant AR-positive LN95 and 22Rv1 cells, but not AR-negative PC3 and DU145 cells. A surface plasmon resonance assay detected (+)-JJ-74-138 binding to AR and a chromatin immunoprecipitation assay indicated (+)-JJ-74-138 inhibited AR binding to androgen response elements. In addition, (+)-JJ-74-138 inhibited 22Rv1 xenograft tumor growth. Our observations suggest that (+)-JJ-74-138 is a novel noncompetitive AR antagonist capable of inhibiting enzalutamide-resistant CRPC., (©2022 American Association for Cancer Research.)

Purpose or the Research: Nearly 300,000 people are affected by spinal cord injury (SCI) with approximately 18,000 new cases annually, according to the National SCI Statistics Center. SCI affects physical mobility and impairs the function of multiple internal organs to cause lower urinary tract (LUT) dysfunctions manifesting as detrusor sphincter dyssynergia (DSD) and neurogenic detrusor overactivity (NDO) with detrimental consequences to the quality of life and increased morbidity. Multiple lines of evidence now support time dependent evolution of the complex SCI pathology which requires a multipronged treatment approach of immediate, specialized care after spinal cord trauma bookended by physical rehabilitation to improve the clinical outcomes. Instead of one size fits all treatment approach, we propose adaptive drug treatment to counter the time dependent evolution of SCI pathology, with three small molecule drugs with distinctive sites of action for the recovery of multiple functions., Principal Results: Our findings demonstrate the improvement in the recovery of hindlimb mobility and bladder function of spinal cord contused mice following administration of small molecules targeting neurotrophin receptors, LM11A-31 and LM22B-10. While LM11A-31 reduced the cell death in the spinal cord, LM22B-10 promoted cell survival and axonal growth. Moreover, the soluble guanylate cyclase (sGC) activator, cinaciguat, enhanced the revascularization of the SCI injury site to promote vessel formation, dilation, and increased perfusion., Major Conclusions: Our adaptive three drug cocktail targets different stages of SCI and LUTD pathology: neuroprotective effect of LM11A-31 retards the cell death that occurs in the early stages of SCI; and LM22B-10 and cinaciguat promote neural remodeling and reperfusion at later stages to repair spinal cord scarring, DSD and NDO. LM11A-31 and cinaciguat have passed phase I and IIa clinical trials and possess significant potential for accelerated clinical testing in SCI/LUTD patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Polysubstituted tetrahydroquinolines were obtained in moderate to high yields (28% to 92%) and enantiomeric ratios (er 89:11 to 99:1) by a three-component Povarov reaction using a chiral phosphoric acid catalyst. Significantly, post-Povarov functional group interconversions allowed a rapid access to a library of 36 enantioenriched 4-aminotetrahydroquinoline derivatives featuring five points of diversity. Selected analogs were assayed for their ability to function as glucagon-like peptide-1 (GLP-1) secretagogues., (© 2021 Wiley Periodicals LLC.)

JP4-039 is an alkene peptide isostere that acts as a low-micromolar inhibitor of erastin- and RSL-3-induced ferroptotic cell death in the HT-1080 cell line. In this work, we have developed new synthetic strategies that allow access to analogues of this lead structure. Enantioselective vinylogous Mannich or cross-metathesis reactions were key to the preparation of a series of analogues that culminated in the preparation of the ca. 30-fold more potent analogue ( S )- 6c . Structure-activity relationship analyses used both HT-1080 cells and a luminescence-based ferroptosis assay in RAW 264.7 macrophages. In particular, α,α-disubstituted alkene peptide isosteres (R α ≠ H) were found to exceed the potency of the corresponding glycine (R α = H) derivatives., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Ferroptosis is a novel iron-dependent regulated cell death mechanism that affects cell metabolism; however, a detailed metabolomic analysis of ferroptotic cells is not yet available. Here, we elucidated the metabolome of H9c2 cardioblasts by gas chromatography-mass spectrometry during ferroptosis induced by RSL3, a GPX4 inhibitor, in the presence of ferrostatin-1 (a ferroptosis inhibitor), XJB-5-131 (a mitochondrial-targeted ROS scavenger), or TSM-1005-44 (a newly developed cellular ROS scavenger). Results demonstrated that RSL3 decreased the levels of amino acids involved in glutathione synthesis more than two-fold. In contrast, saturated fatty acids levels were markedly increased in RSL3-challenged cells, with no effects on unsaturated fatty acids. RSL3 significantly altered the levels of mitochondrial tricarboxylic acid cycle intermediates; isocitrate and 2-oxoglutarate were found to increase, whereas succinate was significantly decreased in RSL3-challenged cells. Ferrostatin-1, XJB-5-131, and TSM-1005-44 prevented RSL3-induced cell death and conserved the metabolomic profile of the cells. Since 2-oxoglutarate is involved in the regulation of ferroptosis, particularly through glutamine metabolism, we further assessed the role of glutaminolysis in ferroptosis in H9c2 cardioblasts. Genetic silencing of GLS1, which encodes the K-type mitochondrial glutaminase (glutaminase C), protected against ferroptosis in the early stage. In conclusion, our study demonstrates that RSL3-induced ferroptosis impairs the metabolome of H9c2 cardioblasts.

Chiral sp 3 -rich bicyclo[3.3.1]nonane scaffolds 10-12 were synthesized as single diastereomers from aldehyde 9, which was prepared from 4,4-dimethoxycyclohexa-2,5-dienone through a copper-catalyzed enantioselective reduction. Three different types of intramolecular addition reactions were studied: SmI 2 -mediated reductive cyclization, base-promoted aldol reaction, and one-pot Mannich reaction. We succeeded in introducing three side-chains to scaffold 11 and construct an sp 3 -rich compound library in both enantiomeric variants by simply changing the chirality of the ligands. The biological evaluation revealed that all synthesized compounds exhibited a concentration-dependent inhibition of hypoxia-inducible factor-1 (HIF-1) transcriptional activity, with IC 50 values in the range of 17.2-31.7 µM, whereas their effects on cell viability were varied (IC 50  = 3.5 to > 100 µM). The most active compound 16f inhibits the accumulation of HIF-1α protein and mRNA in hypoxia, indicating that it has a mechanism of action distinctly different from other known compounds bearing the common bicyclo[3.3.1]nonane skeleton., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Mitochondria (MT) are energy "powerhouses" of the cell and the decline in their function from oxidative damage is strongly correlated in many diseases. To suppress oxygen damage, we have developed and applied XJB-5-131 as a targeted platform for neutralizing reactive oxygen species (ROS) directly in MT. Although the beneficial activity of XJB-5-131 is well documented, the mechanism of its protective effects is not yet fully understood., Objective: Here, we elucidate the mechanism of protection for XJB-5-131, a mitochondrial targeted antioxidant and electron scavenger., Methods: The Seahorse Flux Analyzer was used to probe the respiratory states of isolated mouse brain mitochondria treated with XJB-5-131 compared to controls., Results: Surprisingly, there is no direct impact of XJB-5-131 radical scavenger on the electron flow through the electron transport chain. Rather, XJB-5-131 is a mild uncoupler of oxidative phosphorylation. The nitroxide moiety in XJB-5-131 acts as a superoxide dismutase mimic, which both extracts or donates electrons during redox reactions. The electron scavenging activity of XJB-5-131 prevents the leakage of electrons and reduces formation of superoxide anion, thereby reducing ROS., Conclusion: We show here that XJB-5-131 is a mild uncoupler of oxidative phosphorylation in MT. The mild uncoupling property of XJB-5-131 arises from its redox properties, which exert a protective effect by reducing ROS-induced damage without sacrificing energy production. Because mitochondrial decline is a common and central feature of toxicity, the favorable properties of XJB-5-131 are likely to be useful in treating Huntington's disease and a wide spectrum of neurodegenerative diseases for which oxidative damage is a key component. The mild uncoupling properties of XJB-5-131 suggest a valuable mechanism of action for the design of clinically effective antioxidants.

A radiation combined injury is defined as an injury that occurs in the setting of irradiation, such as those expected after a nuclear accident, radiation dispersal device release (a 'dirty bomb'), or a nuclear weapon detonation. There is much research on irradiation-associated burns and their healing, but there is less known about other injuries sustained in the context of irradiation. Animal models are limited in their correlations to clinical situations but can support research on specific questions about injuries and their healing. Mouse models of irradiation with skin or bone wounds are validated as highly reproducible and quantitative. They show dose-dependent impairment of wound healing, with later recovery. Irradiation-induced delay of bone wound healing was mitigated to different extents by single doses of gramicidin S-nitroxide JP4-039, a plasmid expressing manganese superoxide dismutase, amifostine/WR2721, or the bifunctional sulfoxide MMS-350. These models should be useful for research on mechanisms of radiation dermal and osseous damage and for further development of new radioprotectors. They also provide information of potential relevance to the effects of clinical radiation therapies., (© 2021 Society for Radiological Protection. Published on behalf of SRP by IOP Publishing Limited. All rights reserved.)

The role of molecular chaperones, such as heat shock protein 70 (Hsp70), is not typically studied as a function of biological sex, but by addressing this gap we might improve our understanding of proteinopathic disorders that predominate in one sex. Therefore, we exposed male or female primary hippocampal cultures to preformed α-synuclein fibrils in a model of early-stage Lewy pathology. We first discovered that two mechanistically distinct inhibitors of Hsp70 function increased phospho-α-synuclein + inclusions more robustly in male-derived neurons. Because Hsp70 is released into extracellular compartments and may restrict cell-to-cell transmission/amplification of α-synucleinopathy, we then tested the effects of low-endotoxin, exogenous Hsp70 (eHsp70) in primary hippocampal cultures. eHsp70 was taken up by and reduced α-synuclein + inclusions in cells of both sexes, but pharmacological suppression of Hsp70 function attenuated the inhibitory effect of eHsp70 on perinuclear inclusions only in male neurons. In 20-month-old male mice infused with α-synuclein fibrils in the olfactory bulb, daily intranasal eHsp70 delivery also reduced inclusion numbers and the time to locate buried food. eHsp70 penetrated the limbic system and spinal cord of male mice within 3 h but was cleared within 72 h. Unexpectedly, no evidence of eHsp70 uptake from nose into brain was observed in females. A trend towards higher expression of inducible Hsp70-but not constitutive Hsp70 or Hsp40-was observed in amygdala tissues from male subjects with Lewy body disorders compared to unaffected male controls, supporting the importance of this chaperone in human disease. Women expressed higher amygdalar Hsp70 levels compared to men, regardless of disease status. Together, these data provide a new link between biological sex and a key chaperone that orchestrates proteostasis., (© 2021. The American Society for Experimental NeuroTherapeutics, Inc.)

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants, whose pathogenesis remains incompletely understood, although activation of the Gram-negative bacterial receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium plays a critical role. Patients with NEC typically display gastrointestinal dysmotility before systemic disease is manifest, suggesting that dysmotility could drive NEC development. Both intestinal motility and inflammation are governed by the enteric nervous system, a network of enteric neurons and glia. We hypothesized here that enteric glia loss in the premature intestine could lead to dysmotility, exaggerated TLR4 signaling, and NEC development. We found that intestinal motility is reduced early in NEC in mice, preceding the onset of intestinal inflammation, whereas pharmacologic restoration of intestinal motility reduced NEC severity. Ileal samples from mouse, piglet, and human NEC revealed enteric glia depletion, and glia-deficient mice ( Plp1 ΔDTR , Sox10 ΔDTR , and Bdnf ΔDTR ) showed increased NEC severity compared with wild-type mice. Mice lacking TLR4 on enteric glia ( Sox10-Tlr4 ko ) did not show NEC-induced enteric glia depletion and were protected from NEC. Mechanistically, brain-derived neurotrophic factor (BDNF) from enteric glia restrained TLR4 signaling on the intestine to prevent NEC. BDNF was reduced in mouse and human NEC, and BDNF administration reduced both TLR4 signaling and NEC severity in enteric glia–deficient mice. Last, we identified an agent (J11) that enhanced enteric glial BDNF release, inhibited intestinal TLR4, restored motility, and prevented NEC in mice. Thus, enteric glia loss might contribute to NEC through intestinal dysmotility and increased TLR4 activation, suggesting enteric glia therapies for this disorder.

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We developed JMS-053, a potent inhibitor of the dual specificity phosphatase PTP4A3 that is potentially suitable for cancer therapy. Due to the emerging role of the unfolded protein response (UPR) in cancer pathology, we sought to identify derivatives that combine PTP4A3 inhibition with induction of endoplasmatic reticulum (ER) stress, with the goal to generate more potent anticancer agents. We have now generated bifunctional analogs that link the JMS-053 pharmacophore to an adamantyl moiety and act in concert with the phosphatase inhibitor to induce ER stress and cell death. The most potent compound in this series, 7a, demonstrated a ca. 5-fold increase in cytotoxicity in a breast cancer cell line and strong activation of UPR and ER stress response genes in spite of a ca. 13-fold decrease in PTP4A3 inhibition. These results demonstrate that the combination of phosphatase inhibition with UPR/ER-stress upregulation potentiates efficacy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

High grade serous ovarian cancer (OvCa) frequently becomes drug resistant and often recurs. Consequently, new drug targets and therapies are needed. Bioinformatics-based studies uncovered a relationship between high Protein Tyrosine Phosphatase of Regenerating Liver-3 (PRL3 also known as PTP4A3) expression and poor patient survival in both early and late stage OvCa. PTP4A3 mRNA levels were 5-20 fold higher in drug resistant or high grade serous OvCa cell lines compared to nonmalignant cells. JMS-053 is a potent allosteric small molecule PTP4A3 inhibitor and to explore further the role of PTP4A3 in OvCa, we synthesized and interrogated a series of JMS-053-based analogs in OvCa cell line-based phenotypic assays. While the JMS-053 analogs inhibit in vitro PTP4A3 enzyme activity, none were superior to JMS-053 in reducing high grade serous OvCa cell survival. Because PTP4A3 controls cell migration, we interrogated the effect of JMS-053 on this cancer-relevant process. Both JMS-053 and CRISPR/Cas9 PTP4A3 depletion blocked cell migration. The inhibition caused by JMS-053 required the presence of PTP4A3. JMS-053 caused additive or synergistic in vitro cytotoxicity when combined with paclitaxel and reduced in vivo OvCa dissemination. These results indicate the importance of PTP4A3 in OvCa and support further investigations of the lead inhibitor, JMS-053.

Molecular chaperones, such as Hsp70, prevent proteotoxicity and maintain homeostasis. This is perhaps most evident in cancer cells, which overexpress Hsp70 and thrive even when harboring high levels of misfolded proteins. To define the response to proteotoxic challenges, we examined adaptive responses in breast cancer cells in the presence of an Hsp70 inhibitor. We discovered that the cells bin into distinct classes based on inhibitor sensitivity. Strikingly, the most resistant cells have higher autophagy levels, and autophagy was maximally activated only in resistant cells upon Hsp70 inhibition. In turn, resistance to compromised Hsp70 function required the integrated stress response transducer, GCN2, which is commonly associated with amino acid starvation. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating PERK. These data reveal an unexpected route through which breast cancer cells adapt to proteotoxic insults and position GCN2 and autophagy as complementary mechanisms to ensure survival when proteostasis is compromised., Competing Interests: SS, MY, MS, SO, AL, PW, JB No competing interests declared, (© 2021, Sannino et al.)

The addition of carbamoyl anions to azirines affords synthetically useful 2-aziridinyl amide building blocks. The reaction scope was explored with respect to both formamide and azirine, and the addition was found to be highly diastereoselective. A one-pot conversion of a ketoxime to an aziridinyl amide was demonstrated. The method was employed to incorporate an aziridine residue into a dipeptide segment.

Recent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized antibody capture, or nonselective immobilization via electrostatic interactions or Schiff base formation. We report a powerful and flexible method for the affinity capture of target proteins for cryo-EM analysis that utilizes small-molecule ligands as bait for concentrating human target proteins directly onto the grid surface for single-particle reconstruction. This approach is demonstrated for human p97, captured using two different small-molecule high-affinity ligands of this AAA+ ATPase. Four electron density maps are revealed, each representing a p97 conformational state captured from solution, including a double-hexamer structure resolved to 3.6 Å. These results demonstrate that the noncovalent capture of protein targets on EM grids modified with high-affinity ligands can enable the structure elucidation of multiple configurational states of the target and potentially inform structure-based drug design campaigns.

Selective lithiation of arylsulfonylbicyclo[1.1.0]butanes at the bridgehead methine and addition to carbonyl compounds yield tertiary bicyclobutyl alcohols that form spiro[3.4]octanes and related heteroatom-containing spirocycles via an acid- or halogen-mediated semipinacol rearrangement. Further synthetic transformations at the carbonyl or arylsulfone positions, in general in high yield and good chemoselectivity, allow access to acetals, difluorides, amides, and methylenecyclobutene building blocks.

Exposure to loud noise can cause hearing loss and tinnitus in mice and humans. In mice, one major underlying mechanism of noise-induced tinnitus is hyperactivity of auditory brainstem neurons, due at least in part, to decreased Kv7.2/3 (KCNQ2/3) potassium channel activity. In our previous studies, we used a reflex-based mouse model of tinnitus and showed that administration of a non-specific KCNQ channel activator, immediately after noise trauma, prevented the development of noise-induced tinnitus, assessed 1 week after trauma. Subsequently, we developed RL-81, a very potent and highly specific activator of KCNQ2/3 channels. Here, to test the timing window within which RL-81 prevents tinnitus in mice, we modified and employed an operant animal model of tinnitus, where mice are trained to move in response to sound but not move in silence. Mice with behavioral evidence of tinnitus are expected to move in silence. We validated this mouse model by testing the effect of salicylate, which is known to induce tinnitus. We found that transient administration of RL-81 1 week after noise exposure did not affect hearing loss but reduced significantly the percentage of mice with behavioral evidence of tinnitus, assessed 2 weeks after noise exposure. Our results indicate that RL-81 is a promising drug candidate for further development for the treatment of noise-induced tinnitus.

Sulfite oxidase (SO) deficiency is a disorder caused either by isolated deficiency of SO or by defects in the synthesis of its molybdenum cofactor. It is characterized biochemically by tissue sulfite accumulation. Patients present with seizures, progressive neurological damage, and basal ganglia abnormalities, the pathogenesis of which is not fully established. Treatment is supportive and largely ineffective. To address the pathophysiology of sulfite toxicity, we examined the effects of intrastriatal administration of sulfite in rats on antioxidant defenses, energy transfer, and mitogen-activated protein kinases (MAPK) and apoptosis pathways in rat striatum. Sulfite administration decreased glutathione (GSH) concentration and glutathione peroxidase, glucose-6-phosphate dehydrogenase, glutathione S-transferase, and glutathione reductase activities in striatal tissue. Creatine kinase (CK) activity, a crucial enzyme for cell energy transfer, was also decreased by sulfite. Superoxide dismutase-1 (SOD1) and catalase (CAT) proteins were increased, while heme oxygenase-1 (HO-1) was decreased. Additionally, sulfite altered phosphorylation of MAPK by decreasing of p38 and increasing of ERK. Sulfite further augmented the content of GSK-3β, Bok, and cleaved caspase-3, indicating increased apoptosis. JP4-039 is a mitochondrial-targeted antioxidant that reaches higher intramitochondrial levels than other traditional antioxidants. Intraperitoneal injection of JP4-039 before sulfite administration preserved activity of antioxidant enzymes and CK. It also prevented or attenuated alterations in SOD1, CAT, and HO-1 protein content, as well as changes in p38, ERK, and apoptosis markers. In sum, oxidative stress and apoptosis induced by sulfite injection are prevented by JP4-039, identifying this molecule as a promising candidate for pharmacological treatment of SO-deficient patients., (© 2020 SSIEM.)

The objective of this study was to investigate the molecular response to damage at the blood brain barrier (BBB) and to elucidate critical pathways that might lead to effective treatment in central nervous system (CNS) pathologies in which the BBB is compromised. We have used a human, stem-cell derived in-vitro BBB injury model to gain a better understanding of the mechanisms controlling BBB integrity. Chemical injury induced by exposure to an organophosphate resulted in rapid lipid peroxidation, initiating a ferroptosis-like process. Additionally, mitochondrial ROS formation (MRF) and increase in mitochondrial membrane permeability were induced, leading to apoptotic cell death. Yet, these processes did not directly result in damage to barrier functionality, since blocking them did not reverse the increased permeability. We found that the iron chelator, Desferal© significantly decreased MRF and apoptosis subsequent to barrier insult, while also rescuing barrier integrity by inhibiting the labile iron pool increase, inducing HIF2α expression and preventing the degradation of Ve-cadherin specifically on the endothelial cell surface. Moreover, the novel nitroxide JP4-039 significantly rescued both injury-induced endothelium cell toxicity and barrier functionality. Elucidating a regulatory pathway that maintains BBB integrity illuminates a potential therapeutic approach to protect the BBB degradation that is evident in many neurological diseases.

Necrotizing enterocolitis (NEC) is a disease of premature infants characterized by acute intestinal necrosis. Current dogma suggests that NEC develops in response to post-natal dietary and bacterial factors, and so a potential role for in utero factors in NEC remains unexplored. We now show that during pregnancy, administration of a diet rich in the aryl hydrocarbon receptor (AHR) ligand indole-3-carbinole (I3C), or of breast milk, activates AHR and prevents NEC in newborn mice by reducing Toll-like receptor 4 (TLR4) signaling in the newborn gut. Protection from NEC requires activation of AHR in the intestinal epithelium which is reduced in mouse and human NEC, and is independent of leukocyte activation. Finally, we identify an AHR ligand ("A18") that limits TLR4 signaling in mouse and human intestine, and prevents NEC in mice when administered during pregnancy. In summary, AHR signaling is critical in NEC development, and maternally-delivered, AHR-based therapies may alleviate NEC.

Nuclear localization of the androgen receptor (AR) is necessary for its activation as a transcription factor. Defining the mechanisms regulating AR nuclear localization in androgen-sensitive cells and how these mechanisms are dysregulated in castration-resistant prostate cancer (CRPC) cells is fundamentally important and clinically relevant. According to the classical model of AR intracellular trafficking, androgens induce AR nuclear import and androgen withdrawal causes AR nuclear export. The present study has led to an updated model that AR could be imported in the absence of androgens, ubiquitinated, and degraded in the nucleus. Androgen withdrawal caused nuclear AR degradation, but not export. In comparison with their parental androgen-sensitive LNCaP prostate cancer cells, castration-resistant C4-2 cells exhibited reduced nuclear AR polyubiquitination and increased nuclear AR level. We previously identified 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) in a high-throughput screen for its inhibition of androgen-independent AR nuclear localization in CRPC cells. The current study shows that CPPI is a competitive AR antagonist capable of enhancing AR interaction with its E3 ligase MDM2 and degradation of AR in the nuclei of CRPC cells. Also, CPPI blocked androgen-independent AR nuclear import. Overall, these findings suggest the feasibility of targeting androgen-independent AR nuclear import and stabilization, two necessary steps leading to AR nuclear localization and activation in CRPC cells, with small molecule inhibitors.

The rising interest in Kv7 modulators originates from their ability to evoke fundamental electrophysiological perturbations in a tissue-specific manner. A large number of therapeutic applications are, in part, based on the clinical experience with two broad-spectrum Kv7 agonists, flupirtine and retigabine . Since precise molecular structures of human Kv7 channel subtypes in closed and open states have only very recently started to emerge, computational studies have traditionally been used to analyze binding modes and direct the development of more potent and selective Kv7 modulators with improved safety profiles. Herein, the synthetic and medicinal chemistry of small molecule modulators and the representative biological properties are summarized. Furthermore, new therapeutic applications supported by in vitro and in vivo assay data are suggested., Competing Interests: One of the authors (P. W.) is a co-inventor of patents on the composition of matter and use of RL-81 and related compounds, filed and assigned to the University of Pittsburgh. M. B., P. M. and R. L. have no conflicts to declare., (This journal is © The Royal Society of Chemistry.)