Development and Validation of a Fluorogenic Probe for Lysosomal Zinc Release.

Zinc homeostasis, which allows optimal zinc utilization in diverse life processes, is responsible for the general well-being of human beings. This paper describes developing and validating an easily accessible indole-containing zinc-specific probe in the cellular milieu. The probe was synthesized from readily available starting materials and was subjected to steady-state fluorescence studies. It showed selective sensing behavior towards Zn2+ with reversible binding. The suppression of PET (Photoinduced Electron Transfer) and ESIPT (Excited State Intramolecular Proton Transfer) elicited selectivity, and the detection limit was 0.63 µM (LOQ 6.8 µM). The zinc sensing capability of the probe was also screened in the presence of low molecular weight ligands [LMWLs] and showed interference only with GSH and ATP. It is non-toxic and can detect zinc in different cell lines under various stress conditions such as inflammation, hyperglycemia, and apoptosis. The probe could stain the early and late stages of apoptosis in PAN-2 cells by monitoring the zinc release. Most experiments were conducted without external zinc supplementation, showing its innate ability to detect zinc.

Sialic Acid Engineered Prodrug Nanoparticles for Codelivery of Bortezomib and Selenium in Tumor Bearing Mice.

Most cancer patients rarely benefit from monodrug therapy because of both cancer complexity and tumor environment. One of the main reasons for this failure is insufficient accumulation of the optimal dose at the tumorous site. Our investigation implies a promising strategy to engineer prodrug nanoparticles (NPs) of bortezomib (BTZ) and selenium (Se) using sialic acid (SAL) as a ligand to improve breast cancer therapy. BTZ was conjugated with SAL and HPMA (N-2-hydroxypropyl methacrylamide) to prepare a prodrug conjugate; BTZ-SAL-HPMA (BSAL-HP) and then fabricated into prodrug NPs with Se (Se_BSAL-HP prodrug NPs). The self-assembly of prodrug NPs functionalized with Se showed size (204.13 ± 0.02 nm) and zeta potential (-31.0 ± 0.11 mV) in dynamic light scattering (DLS) experiments and spherical shape in TEM and SEM analysis. Good stability and low pH drug release profile were characterized by Se_BSAL-HP prodrug NPs. The tumor-selective boronate-ester-based prodrug NPs of BTZ in combination with Se endowed a synergistic effect against cancer cells. Compared to prodrug conjugate, Se_BSAL-HP prodrug NPs exhibited higher cell cytotoxicity and enhanced cellular internalization with significant changes in mitochondria membrane potential (MMP). Elevated apoptosis was observed in the (G2/M) phase of the cell cycle for Se_BSAL-HP prodrug NPs (2.7-fold) higher than BTZ. In vivo studies were performed on Sprague-Dawley rats and resulted in positive trends. The increased therapeutic activity of Se_BSAL-HP prodrug NPs inhibited primary tumor growth and showed 43.05 fold decrease in tumor volume than the control in 4T1 tumor bearing mice. The surprising and remarkable outcomes for Se_BSAL-HP prodrug NPs were probably due to the ROS triggering effect of boronate ester and selenium given together.

N-2-Hydroxypropylmethacrylamide-Polycaprolactone Polymeric Micelles in Co-delivery of Proteasome Inhibitor and Polyphenol: Exploration of Synergism or Antagonism.

Breast cancer leads to the highest mortality among women resulting in a major clinical burden. Multidrug therapy is more efficient in such patients compared to monodrug therapy. Simultaneous combinatorial or co-delivery garnered significant interest in the past years. Caffeic acid (CFA) (a natural polyphenol) has received growing attention because of its anticarcinogenic and antioxidant potential. Bortezomib (BTZ) is a proteasome inhibitor and may be explored for treating breast cancer. Despite its high anticancer activity, the low water solubility and chemical instability restrict its efficacy against solid tumors. In the present study, we designed and investigated a HP-PCL (N-2-hydroxypropylmethacrylamide-polycaprolactone) polymeric micellar (PMCs) system for the simultaneous delivery of BTZ and CFA in the treatment of breast cancer. The designed BTZ+CFA-HP-PCL PMCs were fabricated, optimized, and characterized for size, zeta potential, surface morphology, and in vitro drug release. Developed nanosized (174.6 ± 0.24 nm) PMCs showed enhanced cellular internalization and cell cytotoxicity in both MCF-7 and MDA-MB-231 cells. ROS (reactive oxygen species) levels were highest in BTZ-HP-PCL PMCs, while CFA-HP-PCL PMCs significantly (p < 0.001) scavenged the ROS generated in 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay. The mitochondrial membrane potential (MMP) assay revealed intense and significant green fluorescence in both types of cancer cells when treated with BTZ-HP-PCL PMCs (p < 0.001) indicating apoptosis or cell death. The pharmacokinetic studies revealed that BTZ-HP-PCL PMCs and BTZ+CFA-HP-PCL PMCs exhibited the highest bioavailability, enhanced plasma half-life, decreased volume of distribution, and lower clearance rate than the pure combination of drugs. In the organ biodistribution studies, the combination of BTZ+CFA showed higher distribution in the spleen and the heart. Overall findings of in vitro studies surprisingly resulted in better therapeutic efficiency of BTZ-HP-PCL PMCs than BTZ+CFA-HP-PCL PMCs. However, the in vivo tumor growth inhibition study performed in tumor-induced mice concluded that the tumor growth was inhibited by both BTZ-HP-PCL PMCs and BTZ+CFA-HP-PCL PMCs (p < 0.0001) more efficiently than pure BTZ and the combination (BTZ+CFA), which may be due to the conversion of boronate ester into boronic acid. Henceforth, the combination of BTZ and CFA provides further indications to be explored in the future to support the hypothesis that BTZ may work with polyphenol (CFA) in the acidic environment of the tumor.

Benzimidazole derivatives as tubulin polymerization inhibitors: Design, synthesis and in vitro cytotoxicity studies.

A new class of benzimidazole derivatives as tubulin polymerization inhibitors has been designed and synthesized in this study. The in vitro anticancer profile of the developed molecules was reconnoitred on selected human cancer cells. The highest cytotoxicity was illustrated by compounds 7n and 7u with IC50 values ranging from 2.55 to 17.89 µM with specificity toward SK-Mel-28 cells. They displayed 5-fold less cytotoxicity towards normal rat kidney epithelial NRK52E cells, which implies that they are not harmful to normal, healthy cells. The cellular staining procedures like AO/EB, DCFDA, and DAPI were applied to comprehend the inherent mechanism of apoptosis which displayed nuclear and morphological alterations. The Annexin V binding and JC-1 studies were executed to evaluate the extent of apoptosis and the decline in mitochondrial transmembrane potential in SK-Mel-28 cell lines. Compound 7n dose-dependently arrested the G2/M phase of the cell cycle and the target-based outcomes proposed tubulin polymerization inhibition by 7n (IC50 of 5.05±0.13 µM). Computational studies were also conducted on the tubulin protein (PDB ID: 3E22) to investigate the stabilized binding interactions of compounds 7n and 7u with tubulin, respectively.

Regioselective synthesis and in vitro cytotoxicity evaluation of 3-thiooxindole derivatives: Tubulin polymerization inhibition and apoptosis inducing studies.

Herein, regiospecific nucleophilic ring-opening of spiroaziridine oxindoles has been established to afford 3-substituted-thiooxindole derivatives as anticancer agents. Among the new series, compounds 7d and 9c exhibited promising cytotoxic activity toward HCT-116 cells with IC50 values of 6.73 ± 0.36 and 6.64 ± 0.95 µM, respectively. Further, AO/EB, DCFDA, and DAPI staining studies were executed to establish the underlying apoptosis mechanism which displayed significant nuclear and morphological alterations. JC-1 staining and annexin V binding assay inferred the loss of mitochondrial membrane potential in HCT-116 cancer cells. Cell cycle analysis showed the treatment of 9c against HCT-116 cells, arrested the cell cycle in G2-M phase. In addition, tubulin binding assay revealed that compound 9c exhibited tubulin polymerase inhibition with IC50 value of 9.73 ± 0.18 µM. This inhibition of tubulin polymerase was further supported by binding interactions of 9c with tubulin through docking studies on PDB ID: 3E22.

Triazolo-linked benzimidazoles as tubulin polymerization inhibitors and DNA intercalators: Design, synthesis, cytotoxicity, and docking studies.

A simple "click" protocol was employed in the quest of synthesizing 1,2,3-triazole-linked benzimidazoles as promising anticancer agents on various human cancer cell lines such as A549, HCT116, SK-Mel-28, HT-29, and MCF-7. Compound 12j demonstrated significant cytotoxic potential towards SK-Mel-28 cancer cells (IC50 : 4.17 ± 0.09 µM) and displayed no cytotoxicity (IC50 : > 100 µM) against normal human BEAS-2B cells inferring its safety towards normal healthy cells. Further to comprehend the underlying apoptosis mechanisms, AO/EB, dichlorodihydrofluorescein diacetate (DCFDA), and 4',6-diamidino-2-phenylindole (DAPI) staining were performed, which revealed the nuclear and morphological alterations. Compound 12j displayed impairment in cellular migration and inhibited colony formation. The annexin V binding assay and JC-1 were implemented to evaluate the scope of apoptosis and the loss of the mitochondrial transmembrane potential in SK-Mel-28 cells. Cell-cycle analysis revealed that compound 12j arrested the cells at the G2/M phase in a dose-dependent manner. Target-based assays established the inhibition of tubulin polymerization by 12j at an IC50 value of 5.65 ± 0.05 µM and its effective binding with circulating tumor DNA as a DNA intercalator. The detailed binding interactions of 12j with tubulin and DNA were examined by docking studies on PDB ID: 3E22 and DNA hexamer (PDB ID: 1NAB), respectively.

Dapsone prolong delayed excitotoxic neuronal cell death by interacting with proapoptotic/survival signaling proteins.

BACKGROUND: Dapsone prevents ischemic injury, inhibits apoptosis and shows functional improvement post-ischemia. However, its effect on proapoptotic or survival proteins in delayed ischemia remains unclear. METHODS: Male adult Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by 24 h of ischemic reperfusion (I/R). Dapsone [9.375 or 12.5 mg/kg, intraperitoneally (IP)] was administered at 3, 6 and 12 h of I/R followed by behavioural assessment, brain infarction, histological alteration and cell viability study. Further, dapsone (25 and 50 µM) was added at 3, 6 and 12 h after L-glutamate (100 µM) in primary cortical culture (DIV 14) and cell viability, cytotoxicity, apoptosis was observed. Proteins expression were observed using immunocytochemistry. All experiments were performed after 24 h of I/R (In-Vivo) and 24 h of recovery post glutamate insult (In-Vitro). RESULTS: Reduced brain infarction, improved neurobehavioural functions in addition to reduction in abnormal morphological structures of ischemic brain and improvement in cell viability was observed with treatment of dapsone (12.5 mg/kg) administered upto 6 h. Similarly, dapsone (25, 50 µM) increased cell survival post glutamate insult in cortical culture (In-vitro). Further, dapsone treatment at delayed hours (6 h) reduced apoptotic nuclei and proapoptotic proteins JNK, PTEN, Calpain, Caspase 3 expression along with activation of prosurvival protein BDNF expression post-glutamate insult. CONCLUSION: Our results suggest that dapsone has the potential to limit the neuronal damage post-glutamate insult in delayed hours (6 h) through repressing proapoptotic proteins JNK, PTEN, Calpain, Caspase-3 of cerebral ischemia along with activation of pro-survival protein BDNF.

Pharmacological blockade of HDAC6 attenuates cancer progression by inhibiting IL-1ß and modulating immunosuppressive response in OSCC.

Interleukin-1-beta (IL-1ß) one of the biomarkers for oral squamous cell carcinoma (OSCC), is upregulated in tumor-microenvironment (TME) and associated with poor patient survival. Thus, a novel modulator of IL-1ß would be of great therapeutic value for OSCC treatment. Here we report regulation of IL-1ß and TME by histone deacetylase-6 (HDAC6)-inhibitor in OSCC. We observed significant upregulation of HDAC6 in 4-nitroquniline (4-NQO)-induced OSCC in mice and 4-NQO & Lipopolysaccharide (LPS) stimulated OSCC and fibroblast cells. Tubastatin A (TSA)-attenuated the OSCC progression in mice as observed improvement in the histology over tongue and esophagus, with reduced tumor burden. TSA treatment to 4-NQO mice attenuated protein expression of HDAC6, pro-and-mature-IL-1ß and pro-and-cleaved-caspase-1 and ameliorated acetylated-tubulin. In support of our experimental work, human TCGA analysis revealed HDAC6 and IL-1ß were upregulated in the primary tumor, with different tumor stages and grades. We found TSA modulate TME, indicated by downregulation of CD11b+Gr1+-Myeloid-derived suppressor cells, CD11b+F4/80+CD206+ M2-macrophages and increase in CD11b+F4/80+MHCII+ M1-macrophages. TSA significantly reduced the gene expression of HDAC6, IL-1ß, Arginase-1 and iNOS in isolated splenic-MDSCs. FaDu-HTB-43 and NIH3T3 cells stimulated with LPS and 4-NQO exhibit higher IL-1ß levels in the supernatant. Interestingly, immunoblot analysis of the cell lysate, we observed that TSA does not alter the expression as well as activation of IL-1ß and caspase-1 but the acetylated-tubulin was found to be increased. Nocodazole pre-treatment proved that TSA inhibited the lysosomal exocytosis of IL-1ß through tubulin acetylation. In conclusion, HDAC6 inhibitors attenuated TME and cancer progression through the regulation of IL-1ß in OSCC.

Development of Benzimidazole-Substituted Spirocyclopropyl Oxindole Derivatives as Cytotoxic Agents: Tubulin Polymerization Inhibition and Apoptosis Inducing Studies.

A series of spirocyclopropyl oxindoles with benzimidazole substitutions was synthesized and tested for their cytotoxicity against selected human cancer cells. Most of the molecules exhibited significant antiproliferative activity with compound 12 p being the most potent. It exhibited significant cytotoxicity against MCF-7 breast cancer cells (IC50 value 3.14±0.50 µM), evidenced by the decrease in viable cells and increased apoptotic features during phase contrast microscopy, such as AO/EB, DAPI and DCFDA staining studies. Compound 12 p also inhibited cell migration in wound healing assay. Anticancer potential of 12 p was proved by the inhibition of tubulin polymerization with IC50 of 5.64±0.15 µM. These results imply the potential of benzimidazole substituted spirocyclopropyl oxindoles, notably 12 p, as cytotoxic agent for the treatment of breast cancer.

Targeted intracellular delivery of antitubercular bioactive(s) to Mtb infected macrophages via transferrin functionalized nanoliposomes.

The packaging of antimicrobials/chemotherapeutics into nanoliposomes can enhance their activity while minimizing toxicity. However, their use is still limited owing to inefficient/inadequate loading strategies. Several bioactive(s) which are non ionizable, and poorly aqueous soluble cannot be easily encapsulated into aqueous core of liposomes by using conventional means. Such bioactive(s) however could be encapsulated in the liposomes by forming their water soluble molecular inclusion complex with cyclodextrins. In this study, we developed Rifampicin (RIF) - 2-hydroxylpropyl-ß-cyclodextrin (HP-ß-CD) molecular inclusion complex. The HP-ß-CD-RIF complex interaction was assessed by using computational analysis (molecular modeling). The HP-ß-CD-RIF complex and Isoniazid were co-loaded in the small unilamellar vesicles (SUVs). Further, the developed system was functionalized with transferrin, a targeting moiety. Transferrin functionalized SUVs (Tf-SUVs) could preferentially deliver their payload intracellularly in the endosomal compartment of macrophages. In in vitro study on infected Raw 264.7 macrophage cells revealed that the encapsulated bioactive(s) could eradicate the pathogen more efficiently than free bioactive(s). In vivo studies further revealed that the Tf-SUVs could accumulate and maintain intracellular bioactive(s) concentrations in macrophages. The study suggests Tf-SUVs as a promising module for targeted delivery of a drug combination with improved/optimal therapeutic index and effective clinical outcomes.

Pharmacological blockade of HDAC3 accelerates diabetic wound healing by regulating macrophage activation.

AIMS: Here, we report the effect of histone deacetylase 3 (HDAC3) inhibition associated with macrophage activation, IL-1ß expression, angiogenesis and wound healing in diabetic mice. MAIN METHODS: To determine the expression of HDAC3 in diabetic mice wounds, hyperglycemia was induced in C57BL/6 mice with streptozotocin followed by induction of 6 mm wounds. To understand the effect of HDAC3 selective inhibitor, BG45, wound tissues were isolated for analysing M1/M2 markers expression, immune cells infiltration, angiogenesis and healing factors expression. CD11b+F4/80+ cells were sorted from the wound tissues and analysed for the expression of M1/M2 markers using RT-qPCR and flow cytometer. In cell based assays, HDAC3 expression was measured in macrophages stimulated with high glucose (HG) plus LPS. Macrophages treated with BG45 and HG + LPS were analysed for the expression of pro-IL-1ß, mature IL-1ß, oxidative stress and pro-inflammatory (M1) and anti-inflammatory (M2) factors. KEY FINDINGS: HDAC3 was found to be upregulated in impaired diabetic mice wounds and in macrophages stimulated with HG + LPS. Topical application of BG45 loaded gel accelerated the wound healing in diabetic mice and was evident by improved expression of Collagen-1A, IL-10, TGF-ß, and angiogenesis (CD31, VEGF). BG45 treatment decreased the expression of IL-1ß, TNF-α, and IL-6 (M1 phenotype), reduced oxidative stress and promoted the expression of Arginase-1 and YM1/2 (M2 phenotype) in macrophages treated with HG + LPS. BG45 also improved the expression of CD11b+F4/80+CD206+ cells in wound tissues and reduced expression of inflammatory markers. SIGNIFICANCE: HDAC3 is upregulated in diabetic mice wounds and HDAC3 selective inhibitor promotes the wound healing by regulating macrophage activation, angiogenesis and IL-1ß.

HDAC5 RNA interference ameliorates acute renal injury by upregulating KLF2 and inhibiting NALP3 expression in a mouse model of oxalate nephropathy.

Krüppel-like factor 2 (KLF2) and NLR family pyrin domain containing 3 (NALP3) are important regulators of macrophage activation in the context of various pathological conditions. NALP3 also plays an important role in the maturation of IL-1 ß which is central to the pathogenesis of acute oxalate nephropathy. The functional role of KLF2 and regulation of both KLF2 and NALP3 in the pathogenesis of acute oxalate nephropathy is comparably less studied. Here, we explored the regulation of KLF2 and NALP3 by Histone deacetylase 5 (HDAC5) in oxalate crystals stimulated macrophages, and in the pathogenesis of acute oxalate nephropathy in mice. We observed upregulated expression of HDAC5 along with IL-1ß, Caspase1, and NALP3, while the expression of KLF2 was downregulated in stimulated macrophages and in the renal tissue of mice with acute oxalate nephropathy. We formulated chitosan HDAC5 siRNA nanoparticles to deliver the siRNA in in-vitro and in-vivo settings. siHDAC5 treated cells exhibited decreased expression of IL-1ß, and TNF-α in the supernatant, and reduced expression of NALP3, Pro-caspase1, active caspase1, Pro-IL-1ß, and IL-1ß in cell lysate. Concurrently, the expression of KLF2 was upregulated in HDAC5 depleted cells upon stimulation with crystals. Mice treated with siHDAC5 nanoparticles showed protection against renal impairment with improved renal function (plasma BUN and creatinine levels), reduced inflammation (IL-1ß expression), reduced accumulation of neutrophils, reduced tubular injury, reduced acute renal injury markers (KIM-1, NGAL-1), reduced expression of NALP3, Pro-caspase1, active caspase1, Pro-IL-1ß, and IL-1ß. Whereas, the expression of KLF2 was significantly upregulated by depletion of HDAC5 in mice.

HDAC6 inhibition attenuates renal injury by reducing IL-1ß secretion and RIP kinase mediated necroptosis in acute oxalate nephropathy.

BACKGROUND: Pathogenesis of acute kidney injury is driven by necro-inflammation, which is comprised of IL-1ß mediated inflammation and RIP-1 mediated tubular necroptosis. HDAC6 is reported to regulate both inflammation and cell death. In the present study, we explored the role of HDAC6 in the lysosomal exocytosis of IL-1ß and RIP-1 mediated necroptosis in the context of oxalate nephropathy. METHODS: Raw 264.7 macrophages and NRK52E stimulated with oxalate crystals and LPS with or without HDAC6 inhibitor for in vitro experiments. Acute oxalate nephropathy was induced in C57BL/6 mice by injecting sodium oxalate (75 mg/kg). For the drug intervention study, Tubastain A (TSA) was given an hour before injection of sodium oxalate. Mice were sacrificed 24 hrs after the oxalate injection, blood and kidney were harvested. Blood samples were analyzed for BUN and IL-1ß levels. Renal tissues were analyzed for histology, immunohistochemistry, RNA, and protein expression. RESULTS: HDAC6 and IL-1ß upregulated in crystal stimulated macrophages and acute oxalate nephropathy. Pre-treatment of macrophages with TSA reduced IL-1ß in supernatant without affecting the expression of pro-IL-1ß and mature IL-1ß in cell lysate. The effect of TSA on IL-1ß secretion was influenced by tubulin acetylation. Renal epithelial cell NRK52E stimulated with crystals showed upregulation of necroptosis pathway markers and concentration-dependent cell death. TSA inhibited RIP-1, RIP3, and MLKL expression along with p-MLKL in stimulated epithelial cells. TSA treatment of oxalate nephropathy mice showed decreased inflammation and tubular cell death by regulating IL-1ß and necroptosis and reduced renal injury. CONCLUSION: This study highlights the role of HDAC6 in regulating the tubulin-mediated secretion of IL-1ß and RIP kinase mediated necroptosis in acute oxalate nephropathy.

Cyclo(Val-Pro) and Cyclo(Leu-Hydroxy-Pro) from Pseudomonas sp. (ABS-36) alleviates acute and chronic renal injury under in vitro and in vivo models (Ischemic reperfusion and unilateral ureter obstruction).

The study aimed to identify small molecules having potentiality in alleviating renal injury. Two natural compounds cyclo(Val-Pro) (1) and cyclo(Leu-Hydroxy-Pro) (2) were first evaluated under acute renal injury model of ischemic reperfusion at different doses of 25, 50 and 75 mg/kg body weight. Further, the compounds were subjected to antimycin A-induced ischemic in vitro study (NRK-52E cell lines). Both the compounds significantly decreased plasma IL-1ß levels (P < 0.05). Also, the mRNA expression levels of inflammatory markers (TNF-α, IL-6 and IL-1ß) and renal injury markers (KIM-1, NGAL, α-GST and π-GST) in the renal tissues were significantly alleviated (P < 0.01) along with the improvement in histological damage and control over neutrophil infiltration as a result of ischemic reperfusion. The in vitro study revealed the protective effect against antimycin A-induced cytotoxicity (P < 0.05) and antiapoptotic effect acting through the regulation of Bax, caspase 3 (pro and cleaved) and BCL2 with reduction in Annexin+PI+ cells. Further, the compound cyclo(Val-Pro) (1) was evaluated (50 mg/kg body weight dose) in chronic unilateral ureter obstruction model of renal injury in mice and TGF-ß-induced in vitro fibrotic model (NRK-49F cell lines). Cyclo(Val-Pro) (1) significantly reduced the expression levels of fibrotic markers (collagen-1, α-SMA and TGF-ß) and showed marked alleviation of renal fibrosis (sirius red staining). Also, the proliferation of TGF-ß-induced NRK-49F cells was significantly reduced along with decreased levels of collagen-1 and α-SMA in immunohistochemistry studies. In conclusion, the compounds significantly abrogated ischemic injury by inhibiting renal inflammation and tubular epithelial apoptosis. Further, cyclo (Val-Pro) (1) exhibited significant anti-fibrotic activity through the inhibition of fibroblast activation and proliferation. Thus, these proline-based cyclic dipeptides are recommended as drug leads for treating renal injury.

Inhibition of Autotaxin Ameliorates LPA-Mediated Neuroinflammation and Alleviates Neurological Dysfunction in Acute Hepatic Encephalopathy.

Growing evidence suggests an essential role of neuroinflammation in behavioral abnormalities associated with hepatic encephalopathy (HE). Here, we report the involvement of autotaxin-lysophosphatidic acid (LPA) signaling in HE's pathogenesis. We demonstrate that the autotaxin (ATX) inhibitor PF-8380 attenuates neuroinflammation and improves neurological dysfunction in the mouse model of HE. In the thioacetamide (TAA)-induced model of HE, we found a twofold increase in the levels of ammonia in the brain and in plasma along with a significant change in HE-related behavioral parameters. Mice with HE show an increased brain weight, increased levels of tumor necrosis factor-α (TNF-α), IL-1ß (interleukin-1ß), interleukin-6 (IL-6), and LPA 18:0 in the cerebral cortex and hippocampus, and increased levels of LPA 18:0 in plasma. Treatment with the autotaxin inhibitor (ATXi) did not affect liver injury, as we observed no change in liver function markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) and no change in ammonia levels in the brain and plasma. However, ATXi treatment significantly ameliorated the neuroinflammation, reduced the levels of LPA 18:0 in the cerebral cortex and hippocampus in the brain and plasma, and reduced brain edema and the levels of IL1ß, IL-6, and TNF-α. The neurobehavioral symptoms for HE such as the cognitive and motor function deficit and overall clinical grading score were significantly improved in ATXi-treated mice. Mouse astrocytes and microglia stimulated with NH4CL with or without ATXi showed significant attenuation of oxidative stress and the neuroinflammatory effect of NH4CL in ATXi-treated cells.

PT3: A Novel Benzamide Class Histone Deacetylase 3 Inhibitor Improves Learning and Memory in Novel Object Recognition Mouse Model.

The importance of HDAC3 in transcriptional regulation of genes associated with long-term memory is well established. Here, we report a novel HDAC3 inhibitor, PT3, with an excellent blood-brain barrier permeability and ability to enhance long-term memory in mouse model of novel object recognition (NOR). PT3 exhibited higher selectivity for HDAC3 over HDAC1, HDAC6, and HDAC8 compared to the reference compound CI994. PT3 has significant distribution into the brain tissue with Cmax at 0.5 h and t1/2 of 2.5 h. Treatment with PT3 significantly improved the discrimination index in C57/BL6 mice in the NOR model. Brain tissue analysis of mice treated with PT3 for NOR test showed significant increase in H3K9 acetylation in hippocampus. Gene expression analysis by RT-qPCR of the hippocampus tissue revealed upregulation of CREB 1, BDNF, TRKB, Nr4a2, c-fos, PKA, GAP 43, PSD 95 and MMP9 expression in mice treated with PT3. Similar to the phenotype observed in the in vivo experiment, we found upregulation of H3K9 acetylation, CREB 1, BDNF, TRKB, Nr4a2, c-fos, PKA, GAP 43 and MMP9 expression in mouse neuronal (N2A) cells treated with PT3. Thus, our preclinical studies identify PT3 as a potential HDAC3 selective inhibitor that crosses the blood-brain barrier and improves the long-term memory formation in C57/BL6 mice. We propose PT3 as a candidate with therapeutic potential to treat age-related memory loss as well as other disorders with declined memory function like Alzheimer's disease.