Specific Inhibitor Research Articles

Epigenetic mechanisms differentially regulate blood pressure and renal dysfunction in male and female Npr1 haplotype mice.

We determined the epigenetic mechanisms regulating mean arterial pressure (MAP) and renal dysfunction in guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene-targeted mice. The Npr1 (encoding NPRA) gene-targeted mice were treated with class 1 specific histone deacetylase inhibitor (HDACi) mocetinostat (MGCD) to determine the epigenetic changes in a sex-specific manner. Adult male and female Npr1 haplotype (1-copy; Npr1+/-), wild-type (2-copy; Npr1+/+), and gene-duplicated heterozygous (3-copy; Npr1++/+) mice were intraperitoneally injected with MGCD (2 mg/kg) for 14 days. BP, renal function, histopathology, and epigenetic changes were measured. One-copy male mice showed significantly increased MAP, renal dysfunction, and fibrosis than 2-copy and 3-copy mice. Furthermore, HDAC1/2, collagen1alpha-2 (Col1α-2), and alpha smooth muscle actin (α-SMA) were significantly increased in 1-copy mice compared with 2-copy controls. The expression of antifibrotic microRNA-133a was attenuated in 1-copy mice but to a greater extent in males than females. NF-κB was localized at significantly lower levels in cytoplasm than in the nucleus with stronger DNA binding activity in 1-copy mice. MGCD significantly lowered BP, improved creatinine clearance, and repaired renal histopathology. The inhibition of class I HDACs led to a sex-dependent distinctive stimulation of acetylated positive histone marks and inhibition of methylated repressive histone marks in Npr1 1-copy mice; however, it epigenetically lowered MAP, repaired renal fibrosis, and proteinuria and suppressed NF-kB differentially in males versus females. Our results suggest a role for epigenetic targets affecting hypertension and renal dysfunction in a sex-specific manner.

The silent information regulator 1 agonist SRT1720 reduces experimental intracerebral hemorrhagic brain injury by regulating the blood-brain barrier integrity.

Intracerebral hemorrhage (ICH) is a significant public health matter that has no effective treatment. ICH-induced destruction of the blood-brain barrier (BBB) leads to neurological deterioration. Astrocytic sonic hedgehog (SHH) alleviates brain injury by maintaining the integrity of the BBB after ICH. Silent information regulator 1 (SIRT1) is neuroprotective in several central nervous system diseases via BBB regulation. It is also a possible influential factor of the SHH signaling pathway. Nevertheless, the role of SIRT1 on BBB and the underlying pathological process associated with the SHH signaling pathway after ICH remain unclear. We established an intracerebral hemorrhagic mouse model by collagenase injection. SRT1720 (a selective agonist of SIRT1) was used to evaluate the effect of SIRT1 on BBB integrity after ICH. SIRT1 expression was reduced in the mouse brain after ICH. SRT1720 attenuated neurobehavioral impairments and brain edema of ICH mouse. After ICH induction, SRT1720 improved BBB integrity and tight junction expressions in the mouse brain. The SHH signaling pathway-related factors smoothened and glioma-associated oncogene homolog-1 were increased with the intervention of SRT1720, while cyclopamine (a specific inhibitor of the SHH signaling pathway) reversed these effects. These findings suggest that SIRT1 protects from ICH by altering BBB permeability and tight junction expression levels. This process is associated with the SHH signaling pathway, suggesting that SIRT1 may be a potential therapeutic target for ICH.

Efficacy of ivermectin and its metabolites against Plasmodium falciparum liver stages in primary human hepatocytes.

Ivermectin, a broad-spectrum anti-parasitic drug, has been proposed as a novel vector control tool to reduce malaria transmission by mass drug administration. Ivermectin and some metabolites have mosquito-lethal effect, reducing Anopheles mosquito survival. Ivermectin inhibits liver stage development in a rodent malaria model, but no inhibition was observed in a primate malaria model or in a human malaria challenge trial. In the liver, cytochrome P450 3A4 and 3A5 enzymes metabolize ivermectin, which may impact drug efficacy. Thus, understanding ivermectin metabolism and assessing this impact on Plasmodium liver stage development is critical. Using primary human hepatocytes (PHHs), we characterized ivermectin metabolism and evaluated the efficacy of ivermectin and its primary metabolites M1 (3″-O-demethyl ivermectin) and M3 (4-hydroxymethyl ivermectin) against Plasmodium falciparum liver stages. Two different modes of ivermectin exposure were evaluated: prophylactic mode (days 0-3 post-infection) and curative mode (days 3-5 post-infection). We used two different PHH donors and modes to determine the inhibitory concentration (IC50) of ivermectin, M1, M3, and the known anti-malarial drug pyrimethamine, with IC50 values ranging from 1.391 to 14.44, 9.95-23.71, 4.767-8.384, and 0.9073-5.416 µM, respectively. In our PHH model, ivermectin and metabolites M1 and M3 demonstrated inhibitory activity against P. falciparum liver stages in curative treatment mode (days 3-5) and marginal activity in prophylactic treatment mode (days 0-3). Ivermectin had improved efficacy when co-administered with ketoconazole, a specific inhibitor of cytochrome P450 3A4 enzyme. Further studies should be performed to examine ivermectin liver stage efficacy when co-administered with CYP3A4 inhibitors and anti-malarial drugs to understand the pharmacokinetic and pharmacodynamic drug-drug interactions that enhance efficacy against human malaria parasites in vitro.

Microglia through MFG-E8 signaling decrease the density of degenerating neurons and protect the brain from the development of cortical infarction after stroke.

Neuronal loss is a hallmark of stroke and other neurodegenerative diseases, and as such, neuronal loss caused by microglia has been thought to be a contributing factor to disease progression. Here, we show that microglia indeed contribute significantly to neuronal loss in a mouse model of stroke, but this microglial-dependent process of neuronal clearance specifically targets stressed and degenerating neurons in the ischemic cortical region and not healthy non-ischemic neurons. Nonspecific stimulation of microglia decreased the density of neurons in the ischemic cortical region, whereas specific inhibition of MFG-E8 signaling, which is required for microglial phagocytosis of neurons, had the opposite effect. In both scenarios, the effects were microglia specific, as the same treatments had no effect in mice whose microglia were depleted prior to stroke. Finally, even though the inhibition of MFG-E8 signaling increased neuronal density in the ischemic brain region, it substantially exacerbated the development of cortical infarction. In conclusion, microglia through MFG-E8 signaling contribute to the loss of ischemic neurons and, in doing so, minimize the development of cortical infarction after stroke.

KIF11 promotes vascular smooth muscle cell proliferation by regulating cell cycle progression and accelerates neointimal formation after arterial injury in mice.

Background and aims: One of the primary causes of lumen narrowing is vascular injury induced during medical procedures. Vascular injury disrupts the integrity of the endothelium, triggering platelet deposition, leukocyte recruitment, and the release of inflammatory factors. This, in turn, induces the proliferation of vascular smooth muscle cells (VSMCs), leading to neointima formation. However, the molecular mechanism underlying VSMC proliferation following injury remains unknown. KIF11 is critical in regulating the cell cycle by forming bipolar spindles during mitotic metaphase. This process may contribute to VSMCs proliferation and neointima formation following vascular injury. Yet, the function of KIF11 in VSMCs has not been elucidated. This study aims to investigate the role and mechanisms of KIF11 in regulating VSMCs cycle progression and proliferation. Methods: After conducting biological analysis of the transcriptome sequencing data from the mouse carotid artery injury model and the cell transcriptome data of PDGF-BB-induced VSMCs, we identified a potential target gene, KIF11, which may play a crucial role in vascular injury. Then we established a vascular injury model to investigate how changes in KIF11 expression and activity influence in vivo VSMCs proliferation and neointimal formation. In addition, we employed siRNA and specific inhibitors to suppress KIF11 expression and activity in VSMCs cultured in vitro to study the mechanisms underlying VSMCs cycle progression and proliferation. Results: The results of immunohistochemistry and immunofluorescence indicate a significant upregulation of KIF11 expression in the injured vascular. The intraperitoneal injection of the KIF11 specific inhibitor, K858, partially inhibits intimal hyperplasia in the vascular injury model. In vitro experiments further demonstrate that PDGF-BB upregulates KIF11 expression through the PI3K/AKT pathway, and enhances KIF11 activity. Inhibition of both KIF11 expression and activity partially reverses the pro-cycle progression and pro-proliferation effects of PDGF-BB on VSMCs. Additionally, KIF11 overexpression partially counteracts the proliferation arrest and cell cycle arrest induced by inhibiting the PI3K/AKT pathway in VSMCs. Conclusion: Our study highlights the crucial role of KIF11 in regulating the cycle progression and proliferation of VSMCs after vascular injury. A comprehensive understanding of these mechanisms could pave the way for potential therapeutic interventions in treating vascular stenosis.

Enhanced glycolysis causes extracellular acidification and activates acid-sensing ion channel 1a in hypoxic pulmonary hypertension.

In pulmonary hypertension (PHTN), a metabolic shift to aerobic glycolysis promotes a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs). Enhanced glycolysis induces extracellular acidosis, which can activate proton-sensing membrane receptors and ion channels. We previously reported that activation of the proton-gated cation channel acid-sensing ion channel 1a (ASIC1a) contributes to the development of hypoxic PHTN. Therefore, we hypothesize that enhanced glycolysis and subsequent acidification of the PASMC extracellular microenvironment activate ASIC1a in hypoxic PHTN. We observed decreased oxygen consumption rate and increased extracellular acidification rate in PASMCs from chronic hypoxia (CH)-induced PHTN rats, indicating a shift to aerobic glycolysis. In addition, we found that intracellular alkalization and extracellular acidification occur in PASMCs following CH and in vitro hypoxia, which were prevented by the inhibition of glycolysis with 2-deoxy-d-glucose (2-DG). Inhibiting H+ transport/secretion through carbonic anhydrases, Na+/H+ exchanger 1, or vacuolar-type H+-ATPase did not prevent this pH shift following hypoxia. Although the putative monocarboxylate transporter 1 (MCT1) and -4 (MCT4) inhibitor syrosingopine prevented the pH shift, the specific MCT1 inhibitor AZD3965 and/or the MCT4 inhibitor VB124 were without effect, suggesting that syrosingopine targets the glycolytic pathway independent of H+ export. Furthermore, 2-DG and syrosingopine prevented enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMCs from CH rats. These data suggest that multiple H+ transport mechanisms contribute to extracellular acidosis and that inhibiting glycolysis-rather than specific H+ transporters-more effectively prevents extracellular acidification and ASIC1a activation. Together, these data reveal a novel pathological relationship between glycolysis and ASIC1a activation in hypoxic PHTN.NEW & NOTEWORTHY In pulmonary hypertension, a metabolic shift to aerobic glycolysis drives a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells. We demonstrate that this enhanced glycolysis induces extracellular acidosis and activates the proton-gated ion channel, acid-sensing ion channel 1a (ASIC1a). Although multiple H+ transport/secretion mechanisms are upregulated in PHTN and likely contribute to extracellular acidosis, inhibiting glycolysis with 2-deoxy-d-glucose or syrosingopine effectively prevents extracellular acidification and ASIC1a activation, revealing a promising therapeutic avenue.

Inhibition of Cutaneous TRPV3 Channels by Natural Caffeic Acid for the Alleviation of Skin Inflammation.

Natural caffeic acid (CA) and its analogues have been studied for their potential applications in the treatment of various inflammatory and infectious skin diseases. However, the molecular mechanism underlying the effects of the CA remains largely unknown. Here, we report that CA and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), inhibit TRPV3 currents in their concentration- and structure-dependent manners with IC50 values ranging from 102 to 410 μM. At the single-channel level, CA reduces the channel open probability and open frequency without alteration of unitary conductance. CA selectively inhibits TRPV3 relative to other subtypes of thermo-TRPs, such as TRPA1, TRPV1, TRPV4, and TRPM8. Molecular docking combined with site-specific mutagenesis reveals that a residue T636 in the Pore-loop is critical for CA binding to TRPV3. Further in vivo evaluation shows that CA significantly reverses TRPV3-mediated skin inflammation induced by skin sensitizer carvacrol. Altogether, our findings demonstrate that CA exerts its anti-inflammatory effects by selectively inhibiting TRPV3 through binding to the pocket formed by the Pore-loop and the S6. CA may serve as a lead for further modification and identification of specific TRPV3 channel inhibitors.

Capsaicin-insensitivity of TRPV1-R575D mutant located at the lipid-water-interface region can be rescued by either extracellular Ca2+-chelation or cholesterol reduction

TRPV1 acts as a unique polymodal ion channel having distinct structure and gating properties. In this context, TRPV1-R575D represents a special mutant located at the inner lipid-water-interface (LWI) region that has less possibility of interaction with membrane cholesterol. In control conditions, this lab-generated mutant of TRPV1 shows no “ligand-sensitivity”, reduced surface expression, reduced localization in the lipid rafts, yet induces high cellular lethality. Notably, the cellular lethality induced by TRPV1-R575D expression can be rescued by adding 5′I-RTX (a specific inhibitor of TRPV1) or by introducing another mutation in the next position, i.e. in TRPV1-R575D/D576R. In this work we characterized TRPV1-R575D and TRPV1-R575D/D576R mutants in different cellular conditions and compared with the TRPV1-WT. We report that the “ligand-insensitivity” of TRPV1-R575D can be rescued in certain conditions, such as by chelation of extracellular Ca2+, or by reduction of the membrane cholesterol. Here we show that Ca2+ plays an important role in the channel gating of TRPV1-WT as well as LWI mutants (TRPV1-R575D, TRPV1-R575D/D576R). However, chelation of intracellular Ca2+ or depletion of ER Ca2+ did not have a significant effect on the TRPV1-R575D. Certain properties related to channel gating of mutant TRPV1-R575D/D576R can be rescued partially or fully in a context -dependent manner. Cholesterol depletion also alters these properties. Our data suggests that lower intracellular basal Ca2+ acts as a pre-requisite for further opening of TRPV1-R575D. These findings enable better understanding of the structure-function relationship of TRPV1 and may be critical in comprehending the channelopathies induced by other homologous thermosensitive TRPVs.

Correlation between Molecular Docking and the Stabilizing Interaction of HOMO-LUMO: Spirostans in CHK1 and CHK2, an In Silico Cancer Approach.

Checkpoint kinases 1 and 2 (CHK1 and CHK2) are enzymes that are involved in the control of DNA damage. At the present time, these enzymes are some of the most important targets in the fight against cancer since their inhibition produces cytotoxic effects in carcinogenic cells. This paper proposes the use of spirostans (Sp), natural compounds, as possible inhibitors of the enzymes CHK1 and CHK2 from an in silico analysis of a database of 155 molecules (S5). Bioinformatics studies of molecular docking were able to discriminate between 13 possible CHK1 inhibitors, 13 CHK2 inhibitors and 1 dual inhibitor for both enzymes. The administration, distribution, metabolism, excretion and toxicity (ADMETx) studies allowed a prediction of the distribution and metabolism of the potential inhibitors in the body, as well as determining the excretion routes and the appropriate administration route. The best inhibition candidates were discriminated by comparing the enzyme-substrate interactions from 2D diagrams and molecular docking. Specific inhibition candidates were obtained, in addition to studying the dual inhibitor candidate and observing their stability in dynamic molecular studies. In addition, Highest Occupied Molecular Orbital-Lowest Unoccupied Molecular Orbital (HOMO-LUMO) interactions were analyzed to study the stability of interactions between the selected enzymes and spirostans resulting in the predominant gaps from HOMOCHKs to LUMOSp (Highest Occupied Molecular Orbital of CHKs-Lowest Unoccupied Molecular Orbital of spirostan). In brief, this study presents the selection inhibitors of CHK1 and CHK2 as a potential treatment for cancer using a combination of molecular docking and dynamics, ADMETx predictons, and HOMO-LUMO calculation for selection.

21 Unconventional mechanisms of action and resistance to rapalogs in renal cancer

Abstract Background Clear cell renal cell carcinoma (ccRCC) is a prevalent cancer type in the United States driven by inactivation of the von Hippel-Lindau (VHL) gene and with frequent hyperactivation of mammalian target of rapamycin complex 1 (mTORC1). Multiple drugs have been developed to target VEGF/VEGFR2 and mTORC1 for advanced RCC treatment. Two specific mTORC1 inhibitors, temsirolimus and everolimus (known as rapalogs), are FDA-approved for the treatment of RCC but their clinical efficacy is hindered by the emergence of resistance. Methods To study the development of rapalog resistance in RCC, we utilized patient-derived tumorgrafts (TGs) implanted in immunocompromised mice and treated the mice with rapamycin for prolonged periods to generate resistance. Resistance was studied by transplanting resistant tumors into additional cohorts of mice and establishing primary cultures. The impact of rapamycin on tumor growth and mTORC1 activity in both tumor and stromal cells was assessed using molecular techniques. To dissect the role of the tumor microenvironment (TME), we generated genetically engineered immunocompromised recipient mice with an mTOR inhibitor resistance mutation (S2035T). Coculture experiments were performed to further explore the interplay. Results Prolonged rapamycin treatment resulted in the development of resistance in TGs. Notably, this occurred despite persistent inhibition of mTORC1 in tumor cells. Resistance was transient and lost with subsequent transplantation and in cell culture. Surprisingly, resistance was accompanied by mTORC1 reactivation in the TME, specifically in cancer associated fibroblasts. Introducing an mTOR resistance mutation (S2035T) into recipient mice was sufficient to cause resistance. Co-culture experiments with fibroblasts further demonstrated that rapamycin-resistant mutant fibroblasts conferred resistance in tumor cells even in the absence of mTORC1 activity. Conclusions This study uncovers a critical role of the TME, in mediating the response and resistance to rapalogs in RCC. Our data show that inhibition of mTORC1 in non-tumor cells is essential for the anti-tumor activity of rapalogs. These findings shed light on why mTOR mutations are rarely observed in resistant RCC patients and highlight the significance of the TME in modulating drug response and resistance. Moreover, our study emphasizes the potential of targeting TME cells as a therapeutic strategy in RCC and possibly other cancers. DOD CDMRP Funding: yes

Medicinal chemistry aspects of fat mass and obesity associated protein: structure, function and inhibitors.

Adiposity and obesity-related proteins (FTO), the earliest identified mRNA N6-methyladenosine (m6A) demethylases, are known to play crucial roles in several biological processes. Therefore, FTO is a promising target for anticancer treatment. Understanding the biological functions and regulatory mechanisms of FTO targets can serve as guidelines for drug development. Despite significant efforts to develop FTO inhibitors, no specific small-molecule inhibitors have entered clinical trials so far. In this manuscript, we review the relationship between FTO and various cancers, the small-molecule inhibitors developed against FTO targets from the perspective of medicinal chemistry and other fields, and describe their structural optimization process and structure-activity relationship, providing clues for their future development direction.

Utility of Chimeric Mice with Humanized Livers for Predicting Hepatic Organic Anion-Transporting Polypeptide 1B-Mediated Clinical Drug-Drug Interactions.

The influence of transporters on the pharmacokinetics of drugs is being increasingly recognized, and drug-drug interactions (DDIs) via modulation of transporters could lead to clinical adverse events. Organic anion-transporting polypeptide 1B (OATP1B) is a liver-specific uptake transporter in humans that can transport a broad range of substrates, including statins. It is a challenge to predict OATP1B-mediated DDIs using preclinical animal models because of species differences in substrate specificity and abundance levels of transporters. PXB-mice are chimeric mice with humanized livers that are highly repopulated with human hepatocytes and have been widely used for drug metabolism and pharmacokinetics studies in drug discovery. In the present study, we measured the exposure increases [blood AUC (area under the blood/plasma concentration-time curve) and Cmax] of 10 OATP1B substrates in PXB-mice upon coadministration with rifampin, a potent OATP1B specific inhibitor. These data in PXB-mice were then compared with the observed DDIs between OATP1B substrates and single-dose rifampin in humans. Our findings suggest that the DDIs between OATP1B substrates and rifampin in PXB-mouse are comparable with the observed DDIs in the clinic. Since most OATP1B substrates are metabolized by cytochromes P450 (CYPs) and/or are substrates of P-glycoprotein (P-gp), we further validated the utility of PXB-mice to predict complex DDIs involving inhibition of OATP1B, CYPs, and P-gp using cyclosporin A (CsA) and gemfibrozil as perpetrators. Overall, the data support that the chimeric mice with humanized livers could be a useful tool for the prediction of hepatic OATP1B-mediated DDIs in humans. SIGNIFICANCE STATEMENT: The ability of PXB-mouse with humanized liver to predict organic anion-transporting polypeptide 1B (OATP1B)-mediated drug-drug interactions (DDIs) in humans was evaluated. The blood exposure increases of 10 OATP1B substrates with rifampin, an OATP1B inhibitor, in PXB-mice have a good correlation with those observed in humans. More importantly, PXB-mice can predict complex DDIs, including inhibition of OATP1B, cytochromes P450 (CYPs), and P-glycoprotein (P-gp) in humans. PXB-mice are a promising useful tool to assess OATP1B-mediated clinical DDIs.

Metformin enhances endogenous neural stem cells proliferation, neuronal differentiation, and inhibits ferroptosis through activating AMPK pathway after spinal cord injury

BackgroundInadequate nerve regeneration and an inhibitory local microenvironment are major obstacles to the repair of spinal cord injury (SCI). The activation and differentiation fate regulation of endogenous neural stem cells (NSCs) represent one of the most promising repair approaches. Metformin has been extensively studied for its antioxidative, anti-inflammatory, anti-aging, and autophagy-regulating properties in central nervous system diseases. However, the effects of metformin on endogenous NSCs remains to be elucidated.MethodsThe proliferation and differentiation abilities of NSCs were evaluated using CCK-8 assay, EdU/Ki67 staining and immunofluorescence staining. Changes in the expression of key proteins related to ferroptosis in NSCs were detected using Western Blot and immunofluorescence staining. The levels of reactive oxygen species, glutathione and tissue iron were measured using corresponding assay kits. Changes in mitochondrial morphology and membrane potential were observed using transmission electron microscopy and JC-1 fluorescence probe. Locomotor function recovery after SCI in rats was assessed through BBB score, LSS score, CatWalk gait analysis, and electrophysiological testing. The expression of the AMPK pathway was examined using Western Blot.ResultsMetformin promoted the proliferation and neuronal differentiation of NSCs both in vitro and in vivo. Furthermore, a ferroptosis model of NSCs using erastin treatment was established in vitro, and metformin treatment could reverse the changes in the expression of key ferroptosis-related proteins, increase glutathione synthesis, reduce reactive oxygen species production and improve mitochondrial membrane potential and morphology. Moreover, metformin administration improved locomotor function recovery and histological outcomes following SCI in rats. Notably, all the above beneficial effects of metformin were completely abolished upon addition of compound C, a specific inhibitor of AMP-activated protein kinase (AMPK).ConclusionMetformin, driven by canonical AMPK-dependent regulation, promotes proliferation and neuronal differentiation of endogenous NSCs while inhibiting ferroptosis, thereby facilitating recovery of locomotor function following SCI. Our study further elucidates the protective mechanism of metformin in SCI, providing new mechanistic insights for its candidacy as a therapeutic agent for SCI.

An RNA interference approach for functional studies in the sea urchin and its use in analysis of nodal signaling gradients

Dicer substrate interfering RNAs (DsiRNAs) destroy targeted transcripts using the RNA-Induced Silencing Complex (RISC) through a process called RNA interference (RNAi). This process is ubiquitous among eukaryotes. Here we report the utility of DsiRNA in embryos of the sea urchin Lytechinus variegatus (Lv). Specific knockdowns phenocopy known morpholino and inhibitor knockdowns, and DsiRNA offers a useful alternative to morpholinos. Methods are described for the design of specific DsiRNAs that lead to destruction of targeted mRNA. DsiRNAs directed against pks1, an enzyme necessary for pigment production, show how successful DsiRNA perturbations are monitored by RNA in situ analysis and by qPCR to determine relative destruction of targeted mRNA. DsiRNA-based knockdowns phenocopy morpholino- and drug-based inhibition of nodal and lefty. Other knockdowns demonstrate that the RISC operates early in development as well as on genes that are first transcribed hours after gastrulation is completed. Thus, DsiRNAs effectively mediate destruction of targeted mRNA in the sea urchin embryo. The approach offers significant advantages over other widely used methods in the urchin in terms of cost, and ease of procurement, and offers sizeable experimental advantages in terms of ease of handling, injection, and knockdown validation.

Antibody conjugated targeted nanotherapy epigenetically inhibits calpain-mediated mitochondrial dysfunction to attenuate Parkinson’s disease

Many neurodegenerative and psychiatric malignancies like Parkinson’ disease (PD) originate from an imbalance of 17β-Estradiol (E2) in the human brain. However, the peripheral side effects of the usage of E2 for PD therapy and less understanding of the molecular mechanism hinder establishing its neurotherapeutic potential. In the present work, systemic side effects were overcome by targeted delivery using Dopamine receptor D3 (DRD3) conjugated E2-loaded chitosan nanoparticles (Ab-ECSnps) that showed a promising delivery to the brain. E2 is a specific calpain inhibitor that fosters neurodegeneration by disrupting mitochondrial function, while B-cell-specific Moloney murine leukemia virus integration region 1 (BMI1), an epigenetic regulator, is crucial in preserving mitochondrial homeostasis. We showed the administration of Ab-ECSnps inhibits calpain's translocation into mitochondria while promoting the translocation of BMI1 to mitochondria, thereby conferring neurotherapeutic benefits by enhancing cell viability, increasing mitochondrial DNA copy number, and preserving mitochondrial membrane potential. Further, we showed a novel molecular mechanism of BMI1 regulation by calpain that might contribute to maintaining mitochondrial homeostasis for attenuating PD. Concomitantly, Ab-ECSnps showed neurotherapeutic potential in the in vivo PD model. We showed for the first time that our brain-specific targeted delivery might regulate calpain-mediated BMI1 expression, thereby preserving mitochondrial homeostasis to alleviate PD.

Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5′-untranslated region (UTR), and a 233 bp 3′-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37–335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.

Chemoreactomic study of fonturacetam effects: molecular mechanisms of influence on adipose tissue metabolism

Objective: to conduct chemoreactomic, pharmacoinformatic and chemoneurocytological analyzes of the properties of racetams (piracetam, aniracetam, pramiracetam, levetiracetam, fonturacetam).Material and methods. Chemoreactomic, pharmacoinformatic and chemoneurocytological methods of molecule properties analyzis are based on chemoreactomic methodology – the latest direction in the application of machine learning systems in the field of postgenomic pharmacology. Analysis of pharmacological capabilities of molecules within the framework of chemoreactomic methodology is carried out by comparing the chemical structure of racetam molecules with the structures of molecules for which pharmacological properties were studied using training artificial intelligence algorithms based on big data information presented in PubChem, HMDB, STRING, PharmGKB databases. Based on the entire complex of differences between molecules in interactions with receptor proteins, an “anti-obesity” score was calculated for each one as a serial number of this molecule in descending order by corresponding IC50, EC50 chemoreactomic constants values.Results. The lipolytic effect is predicted specifically for fonturacetam as a result of activation by this molecule of β3-adrenoceptors, adenosine receptors, glucagon-like peptide, sphingosine phosphate and peroxisome proliferators, as well as specific inhibition of cannabinoid, opioid, histamine, glutamate, nociceptin, orexin and neuropeptide Y receptors. Due to these mechanisms fonturacetam will contribute to normalizing appetite and improving adipose tissue metabolism. The total lipolytic effect score was calculated for all established interactions with receptors and amounted to 4.3±0.9 for fonturacetam, 3.0±1.4 for pramiracetam, and 2.5±1.5 for all other molecules.Conclusion. The results of the analysis suggest that the lipolytic effects of fonturacetam (Actitropil – Pharmstandard, Russia) will be much stronger than for other racetams (piracetam, aniracetam, pramiracetam, levetiracetam). Chemoreactomic analysis of fonturacetam indicated new mechanisms of pharmacological action of the molecule, providing a decrease in excess appetite and body weight normalization. Fonturacetam is the only nootropic drug indicated for the treatment of obesity.

Targeting the immunoproteasome in hypothalamic neurons as a novel therapeutic strategy for high-fat diet-induced obesity and metabolic dysregulation

ObjectiveObesity represents a significant global health challenge characterized by chronic low-grade inflammation and metabolic dysregulation. The hypothalamus, a key regulator of energy homeostasis, is particularly susceptible to obesity’s deleterious effects. This study investigated the role of the immunoproteasome, a specialized proteasomal complex implicated in inflammation and cellular homeostasis, during metabolic diseases.MethodsThe levels of the immunoproteasome β5i subunit were analyzed by immunostaining, western blotting, and proteasome activity assay in mice fed with either a high-fat diet (HFD) or a regular diet (CHOW). We also characterized the impact of autophagy inhibition on the levels of the immunoproteasome β5i subunit and the activation of the AKT pathway. Finally, through confocal microscopy, we analyzed the contribution of β5i subunit inhibition on mitochondrial function by flow cytometry and mitophagy assay.ResultsUsing an HFD-fed obese mouse model, we found increased immunoproteasome levels in hypothalamic POMC neurons. Furthermore, we observed that palmitic acid (PA), a major component of saturated fats found in HFD, increased the levels of the β5i subunit of the immunoproteasome in hypothalamic neuronal cells. Notably, the increase in immunoproteasome expression was associated with decreased autophagy, a critical cellular process in maintaining homeostasis and suppressing inflammation. Functionally, PA disrupted the insulin-glucose axis, leading to reduced AKT phosphorylation and increased intracellular glucose levels in response to insulin due to the upregulation of the immunoproteasome. Mechanistically, we identified that the protein PTEN, a key regulator of insulin signaling, was reduced in an immunoproteasome-dependent manner. To further investigate the potential therapeutic implications of these findings, we used ONX-0914, a specific immunoproteasome inhibitor. We demonstrated that this inhibitor prevents PA-induced insulin-glucose axis imbalance. Given the interplay between mitochondrial dysfunction and metabolic disturbances, we explored the impact of ONX-0914 on mitochondrial function. Notably, ONX-0914 preserved mitochondrial membrane potential and attenuated mitochondrial ROS production in the presence of PA. Moreover, we found that ONX-0914 reduced mitophagy in the presence of PA.ConclusionsOur findings strongly support the pathogenic involvement of the immunoproteasome in hypothalamic neurons in the context of HFD-induced obesity and metabolic disturbances. Targeting the immunoproteasome highlights a promising therapeutic strategy to mitigate the detrimental effects of obesity on the insulin-glucose axis and cellular homeostasis. This study provides valuable insights into the mechanisms driving obesity-related metabolic diseases and offers potential avenues for developing novel therapeutic interventions.

Abstract Tu054: Deficiency or targeting inhibition of histone lysine demethylase KDM5B in vivo for potential therapeutic effects on both HFrEF and HFpEF

Heart failures with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF) have been recognized as the leading cause of morbidity and mortality worldwide. Histone lysine demethylase KDM5B, an important anti-tumor drug target, has been involved in several cardiovascular diseases. However, its role in heart failure remains largely unknown. In the current study, we found that the expression of KDM5B were significantly enhanced and KDM5B’s downstream substrate: the methylation level of H3K4me3 was decreased in the mouse hearts of both trans-aortic constriction (TAC)-induced HFrEF and high fat diet (HFD) with Nω-nitro-L-arginine methyl ester (L-NAME) (two-hit)-induced HFpEF. By generating a tamoxifen-induced cardiomyocyte- (CM-KO) and a myofibroblast- (MF-KO) specific KDM5B Cre-loxP conditional knockout mice, specifically, we further found that either KDM5B CM-KO or MF-KO mouse significantly ameliorated pathological ventricular remodeling, fibrosis and rescued the cardiac dysfunction phenotypes induced by TAC or the two-hit models, specifically. Furthermore, pretreatment of a KDM5B specific inhibitor TK-129, which was previously identified by our group, also significantly attenuated myocardial hypertrophy, fibrosis and improved cardiac dysfunction in the two mouse heart failure models. Our findings demonstrated, for the first time, that genetic deficiency of KDM5B in either cardiomyocytes or myofibroblasts in vivo or pharmacological inhibition of KDM5B display a similar therapeutic effect in the pathological cardiac remodeling and heart failure in both pressure overload-induced HFrEF and HFpEF mouse models, suggesting that KDM5B is a potential therapeutic target for these two different HFs.

The Secreted Aminopeptidase of Pseudomonas aeruginosa (PaAP).

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in compromised hosts. P. aeruginosa infections are difficult to treat because of the inherent ability of the bacteria to develop antibiotic resistance, secrete a variety of virulence factors, and form biofilms. The secreted aminopeptidase (PaAP) is an emerging virulence factor, key in providing essential low molecular weight nutrients and a cardinal modulator of biofilm development. PaAP is therefore a new potential target for therapy of P. aeruginosa infections. The present review summarizes the current knowledge of PaAP, with special emphasis on its biochemical and enzymatic properties, activation mechanism, biological roles, regulation, and structure. Recently developed specific inhibitors and their potential as adjuncts in the treatment of P. aeruginosa infections are also described.