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Kidney ischemia/reperfusion injury emerges in various clinical settings as a great problem complicating the course and outcome. Ischemia/reperfusion injury is still an unsolved puzzle with a great diversity of investigational approaches, putting the focus on oxidative stress and mitochondria. Mitochondria are both sources and targets of ROS. They participate in initiation and progression of kidney ischemia/reperfusion injury linking oxidative stress, inflammation, and cell death. The dependence of kidney proximal tubule cells on oxidative mitochondrial metabolism makes them particularly prone to harmful effects of mitochondrial damage. The administration of antioxidants has been used as a way to prevent and treat kidney ischemia/reperfusion injury for a long time. Recently a new method based on mitochondria-targeted antioxidants has become the focus of interest. Here we review the current status of results achieved in numerous studies investigating these novel compounds in ischemia/reperfusion injury which specifically target mitochondria such as MitoQ, Szeto-Schiller (SS) peptides (Bendavia), SkQ1 and SkQR1, and superoxide dismutase mimics. Based on the favorable results obtained in the studies that have examined myocardial ischemia/reperfusion injury, ongoing clinical trials investigate the efficacy of some novel therapeutics in preventing myocardial infarct. This also implies future strategies in preventing kidney ischemia/reperfusion injury.

Point mutations of the NADP⁺-dependent isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) occur early in the pathogenesis of gliomas. When mutated, IDH1 and IDH2 gain the ability to produce the metabolite (R)-2-hydroxyglutarate (2HG), but the downstream effects of mutant IDH1 and IDH2 proteins or of 2HG on cellular metabolism are unknown. We profiled >200 metabolites in human oligodendroglioma (HOG) cells to determine the effects of expression of IDH1 and IDH2 mutants. Levels of amino acids, glutathione metabolites, choline derivatives, and tricarboxylic acid (TCA) cycle intermediates were altered in mutant IDH1- and IDH2-expressing cells. These changes were similar to those identified after treatment of the cells with 2HG. Remarkably, N-acetyl-aspartyl-glutamate (NAAG), a common dipeptide in brain, was 50-fold reduced in cells expressing IDH1 mutants and 8.3-fold reduced in cells expressing IDH2 mutants. NAAG also was significantly lower in human glioma tissues containing IDH mutations than in gliomas without such mutations. These metabolic changes provide clues to the pathogenesis of tumors associated with IDH gene mutations.

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) targeting HER2, exhibiting significant clinical efficacy in breast cancer (BC) with varying HER2 expression, including HER2-low and HER2-ultralow. However, the precise mechanism underlying its efficacy and the contribution of immune activation in these settings remain unclear. Here, we demonstrate that T-DXd efficacy in HER2-low and HER2-negative BC is independent of HER2 engagement and ADC internalization. Instead, its activity relies on extracellular proteases, such as cathepsin L (CTSL), within the tumor microenvironment. Irrespective of their HER2 status, tumor and stromal compartments of invasive BC abundantly express CTSL, which efficiently cleaves the specialized linker of T-DXd, facilitating payload release and inducing cytotoxicity against HER2-low/negative tumors. In HER2-positive BC, the antibody backbone of T-DXd engages Fcγ-receptors and drives antibody-dependent cellular phagocytosis (ADCP). Concurrently, its cytotoxic payload (DXd) induces immunogenic cell death, further activating myeloid cells via TLR4 and STING pathways to enhance tumor antigen presentation to CD8+ T cells. Notably, T-DXd cytotoxicity also upregulates tumor CD47 expression, dampening immune activation. Combining T-DXd with CD47 checkpoint blockade significantly enhances anti-tumor immune responses in a HER2-transgenic BC mouse model, while also inducing durable CD8+ T cell memory to prevent tumor recurrence after therapy cessation. Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate targeting HER2 but paradoxically efficient even in breast cancers expressing HER2 at very low levels. Here authors show that invasive breast cancers, even if their HER2 expression is negligible, secrete extracellular proteases, such as cathepsin L, which cleave the specialized linker of T-DXd, releasing the drug in the tumour microenvironment, while in HER2 positive breast cancers, T-DXd engages Fcγ receptors to promote phagocytosis of HER2-expressing cells and triggers payload-induced immunogenic cell death.

Background: Manganese porphyrin, MnTnBuOE-2-PyP5+ (BMX-001), improves neurologic deficits in experimental ischemic stroke and has the potential to serve as an adjunct with thrombolysis or thrombectomy in stroke patients. In 10–30% of stroke patients following thrombolysis, the hemorrhagic transformation, associated with iron release, occurs. This study aimed to examine the neurologic outcome following the BMX-001 treatment in a mouse intracerebral hemorrhage (ICH) model with relevance to prospective ischemic stroke clinical trials. Methods: Twenty C57Bl6 mice were randomly assigned to groups after surgery and received vehicle or BMX-001 treatment immediately following stereotaxic left striatum collagenase injection. Post-ICH body weight, the Corner test, neurological deficit score, and Rotarod test were examined. Six sham surgery mice serve as a control group. At 72 h, the brain histological evaluation was performed, including hemorrhage size, Prussian blue staining, and the activation of macrophages. Data were collected by a researcher who was blind to groups. Results: No significant difference in body weight, neurological deficits, and hemorrhage size was found between groups. However, BMX-001 reduced the number of macrophages in the hemorrhagic area (48 ± 10 in vehicle, 33 ± 8 in BMX-001, p = 0.008) and the number of cells stained with Prussian blue—an indicator of iron released during hemorrhage (65 ± 22 in vehicle and 41 ± 15 in BMX-001, p = 0.027). Conclusions: The results support the safe use of BMX-001 in stroke patients in combination with thrombolysis or thrombectomy and, moreover, indicate the beneficial anti-inflammatory effect of BMX-001, alike to that previously reported in stroke studies of analogous, similarly redox-active, Mn porphyrins.

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX , defining molecular alterations in IDH -mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX-deficient glioma models in the presence and absence of the IDH1 R132H mutation. ATRX-deficient glioma cells are sensitive to dsRNA-based innate immune agonism and exhibit impaired lethality and increased T-cell infiltration in vivo. However, the presence of IDH1 R132H dampens baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1 R132H inhibition. IDH1 R132H co-expression does not interfere with the ATRX deficiency-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1 R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytomas. ATRX inactivation occurs often in IDH -mutant gliomas and has been associated with immune dysfunction. Here, using preclinical models of glioma, the authors show that ATRX inactivation promotes innate immune signalling in response to double stranded RNA-based innate immune agonists, an effect that is masked in IDH -mutant tumours, presenting a therapeutic vulnerability.

Diffuse midline glioma (DMG) is an incurable brain cancer without a single FDA-approved drug that prolongs survival. CDK4/6 inhibitors have been evaluated in children with DMG with limited efficacy. Since MAPK pathway activation is upstream of cell proliferation, we hypothesized that MEK inhibitors may increase the anti-tumor effects of CDK4/6 inhibitors. Here, we evaluated the efficacy of the CDK4/6 inhibitor ribociclib and the MEK inhibitor trametinib in human and murine DMG models to investigate combinational effects. We conducted in vitro and in vivo assays using DMG cell lines from human patient-derived xenografts (PDX) and genetically engineered mouse (GEM) models. In vitro, we assessed synergy across human DMG lines. In vivo, we evaluated therapeutic effects with histological examinations, survival analysis, pharmacokinetic measurements, and RNA-sequencing analysis. In vitro, ribociclib and trametinib had variable synergistic effects against human DMG cell lines. In vivo, a five-day treatment with combination therapy in the GEM DMG model significantly decreased cell proliferation and increased apoptosis compared with the vehicle, with trametinib having mostly cytotoxic effects and ribociclib having primarily cytostatic effects. In addition, a 21-day treatment with combination therapy significantly prolonged mice survival compared with the vehicle in the GEM DMG model (median survival: 112 days vs. 71.5 days, log rank test p = 0.0195). In an orthotopic PDX model, combination therapy did not prolong mice survival compared with vehicle, ribociclib, and trametinib. LC/MS analysis showed adequate drug delivery across the blood-brain-barrier (BBB) into tumor tissue in both GEM and PDX models. Transcriptomic analysis in the GEM model suggests that combination therapy inhibited the MAPK pathway and inflammation. Combination therapy with ribociclib and trametinib significantly prolonged survival in the GEM model but not in the PDX model, highlighting the importance of testing novel therapies in diverse models.

The current opioid crisis urgently calls for developing non-addictive pain medications. Progress has been slow, highlighting the need to uncover targets with unique mechanisms of action. Extracellular adenosine alleviates pain by activating the adenosine A1 receptor (A1R). However, efforts to develop A1R agonists have faced obstacles. The equilibrative nucleoside transporter subtype 1 (ENT1) plays a crucial role in regulating adenosine levels across cell membranes. We postulate that ENT1 inhibition may enhance extracellular adenosine levels, potentiating endogenous adenosine action at A1R and leading to analgesic effects. Here, we modify the ENT1 inhibitor dilazep based on its complex X-ray structure and show that this modified inhibitor reduces neuropathic and inflammatory pain in animal models while dilazep does not. Notably, our ENT1 inhibitor surpasses gabapentin in analgesic efficacy in a neuropathic pain model. Additionally, our inhibitor exhibits less cardiac side effect than dilazep via systemic administration and shows no side effects via local/intrathecal administration. ENT1 is colocalized with A1R in mouse and human dorsal root ganglia, and the analgesic effect of our inhibitor is linked to A1R. Our studies reveal ENT1 as a therapeutic target for analgesia, highlighting the promise of rationally designed ENT1 inhibitors for non-opioid pain medications. In this study, Wrigth et al. use structural, medicinal chemistry, and behavioral approaches to examine how equilibrative nucleoside transporter subtype 1 (ENT1) exerts its functions; and their findings reveal ENT1 as a potential target for analgesia.

TRPV4 ion channels represent osmo-mechano-TRP channels with pleiotropic function and wide-spread expression. One of the critical functions of TRPV4 in this spectrum is its involvement in pain and inflammation. However, few small-molecule inhibitors of TRPV4 are available. Here we developed TRPV4-inhibitory molecules based on modifications of a known TRPV4-selective tool-compound, GSK205. We not only increased TRPV4-inhibitory potency, but surprisingly also generated two compounds that potently co-inhibit TRPA1, known to function as chemical sensor of noxious and irritant signaling. We demonstrate TRPV4 inhibition by these compounds in primary cells with known TRPV4 expression - articular chondrocytes and astrocytes. Importantly, our novel compounds attenuate pain behavior in a trigeminal irritant pain model that is known to rely on TRPV4 and TRPA1. Furthermore, our novel dual-channel blocker inhibited inflammation and pain-associated behavior in a model of acute pancreatitis – known to also rely on TRPV4 and TRPA1. Our results illustrate proof of a novel concept inherent in our prototype compounds of a drug that targets two functionally-related TRP channels, and thus can be used to combat isoforms of pain and inflammation in-vivo that involve more than one TRP channel. This approach could provide a novel paradigm for treating other relevant health conditions.

Oxidative status of maternal blood represents an important parameter of pregnancy that is involved in both, regulation of physiological processes and (if significantly altered) development of different pregnancy complications. Inherited thrombophilias represent genetic disorders that increase the risk of thromboembolism in pregnancy. Little is known about the impact of thrombophilia on the oxidative status of maternal blood. In this study, we analyzed oxidative status of blood of 56 women with pregnancies burdened by inherited thrombophilias. The status was established at three different trimesters using biochemical assays and electrochemical measurements, and it was compared to 10 age- and trimester-matching controls. Activities of superoxide dismutase, catalase, and glutathione reductase in the 1.sup.st and the 2.sup.nd trimester of thrombophilic pregnancy were lower than controls. Also, there was less oxidation in the plasma, according to higher concentration of reduced thiols and lower oxidation-reduction potential. Therefore, it appears that thrombophilic mothers do not experience oxidative stress in the circulation in the first two trimesters. However, the rise in GPx, GR and SOD activities in the 3.sup.rd trimester of thrombophilic pregnancy implies that the risk of oxidative stress is increased during the late pregnancy. These results are important for developing antioxidative treatment that could tackle thrombophilia-related pregnancy complications.

The interactions of drugs with iron are of interest in relation to the potential effects of iron-rich foods and iron supplements on sorption and bioavailability. Doxycycline (DOX), a member of the tetracycline class of broad-spectrum antibiotics, is frequently administered by oral route. In the digestive tract, DOX can be exposed to iron at different pH values (stomach pH 1.5–4, duodenum pH 5–6, distal jejunum and ileum pH 7–8). In relation to this, we analyzed the impact of pH on Fe 3+ -DOX complex formation. The optimal conditions for Fe 3+ -DOX complex formation are pH = 4 and [Fe 3+ ]/[DOX] = 6 molar ratio. HESI-MS showed that Fe 3+ -DOX complex has 1:1 stoichiometry. Raman spectra of Fe 3+ -DOX complex indicate the presence of two Fe 3+ -binding sites in DOX structure: tricarbonylamide group of ring A and phenolic-diketone oxygens of BCD rings. The Fe 3+ -DOX complex formed at pH = 4 is less susceptible to oxidation than DOX at this pH. The increase of pH induces the decomposition of Fe 3+ -DOX complex without oxidative degradation of DOX. The pH dependence of Fe 3+ -DOX complex formation may promote unwanted effects of DOX, impeding the absorption that mainly takes place in duodenum. This could further result in higher concentrations in the digestive tract and to pronounced impact on gut microbiota. Graphical abstract