Transforming growth factor beta orchestrates PD-L1 enrichment in tumor-derived exosomes and mediates CD8 T-cell dysfunction regulating early phosphorylation of TCR signalome in breast cancer.

Tumor cells promote immune evasion through upregulation of programmed death-ligand 1 (PD-L1) that binds with programmed cell death protein 1 (PD1) on cytotoxic T cells and promote dysfunction. Though therapeutic efficacy of anti-PD1 antibody has remarkable effects on different type of cancers it is less effective in breast cancer (BC). Hence, more details understanding of PD-L1-mediated immune evasion is necessary. Here, we report BC cells secrete extracellular vesicles in form of exosomes carry PD-L1 and are highly immunosuppressive. Transforming growth factor beta (TGF-ß) present in tumor microenvironment orchestrates BC cell secreted exosomal PD-L1 load. Circulating exosomal PD-L1 content is highly correlated with tumor TGF-ß level. The later also found to be significantly associated with CD8+CD39+, CD8+PD1+ T-cell phenotype. Recombinant TGF-ß1 dose dependently induces PD-L1 expression in Texos in vitro and blocking of TGF-ß dimmed exosomal PD-L1 level. PD-L1 knocked down exosomes failed to suppress effector activity of activated CD8 T cells like tumor exosomes. While understanding its effect on T-cell receptor signaling, we found siPD-L1 exosomes failed to block phosphorylation of src family proteins, linker for activation of T cells and phosphoinositide phospholipase Cγ of CD8 T cells more than PD-L1 exosomes. In vivo inhibition of exosome release and TGF-ß synergistically attenuates tumor burden by promoting Granzyme and interferon gamma release in tumor tissue depicting rejuvenation of exhausted T cells. Thus, we establish TGF-ß as a promoter of exosomal PD-L1 and unveil a mechanism that tumor cells follow to promote CD8 T-cell dysfunction.

Circulating Exosomes Control CD4+ T Cell Immunometabolic Functions via the Transfer of miR-142 as a Novel Mediator in Myocarditis.

CD4+ T cells undergo immunometabolic activation to mount an immunogenic response during experimental autoimmune myocarditis (EAM). Exosomes are considered key messengers mediating multiple T cell functions in autoimmune responses. However, the role of circulating exosomes in EAM immunopathogenesis and CD4+ T cell dysfunction remains elusive. Our objective was to elucidate the mechanism of action for circulating exosomes in EAM pathogenesis. We found that serum exosomes harvested from EAM mice induced CD4+ T cell immunometabolic dysfunction. Treatment with the exosome inhibitor GW4869 protected mice from developing EAM, underlying that exosomes are indispensable for the pathogenesis of EAM. Furthermore, by transfer of EAM exosomes, we confirmed that circulating exosomes initiate the T cell pathological immune response, driving the EAM pathological process. Mechanistically, EAM-circulating exosomes selectively loaded abundant microRNA (miR)-142. We confirmed methyl-CpG binding domain protein 2 (MBD2) and suppressor of cytokine signaling 1 (SOCS1) as functional target genes of miR-142. The miR-142/MBD2/MYC and miR-142/SOCS1 communication axes are critical to exosome-mediated immunometabolic turbulence. Moreover, the in vivo injection of the miR-142 inhibitor alleviated cardiac injury in EAM mice. This effect was abrogated by pretreatment with EAM exosomes. Collectively, our results indicate a newly endogenous mechanism whereby circulating exosomes regulate CD4+ T cell immunometabolic dysfunction and EAM pathogenesis via cargo miR-142.

Phase 1 study of the protein deubiquitinase inhibitor VLX1570 in patients with relapsed and/or refractory multiple myeloma.

This phase 1 study sought to characterize the safety, tolerability, and pharmacokinetic behavior of VLX1570, a small molecule inhibitor of the deubiquitinases (DUBs) that remove sterically bulky ubiquitin chains from proteins during processing in the19S regulatory subunit of the proteasome, in patients with relapsed and refractory multiple myeloma (MM). Fourteen patients were treated with escalating doses of VLX1570 ranging from 0.05 to 1.2 mg/kg as a brief intravenous (IV) infusion on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle. Due to its poor aqueous solubility, VLX1570 was formulated in polyethylene glycol, polyoxyethylated castor oil, and polysorbate 80 and administered as a brief intravenous (IV) infusion via a central venous catheter. Anti-myeloma effects were noted at doses at or above 0.6 mg/kg, however, two patients treated at the 1.2 mg/kg dose level experienced severe, abrupt, and progressive respiratory insufficiency, which was associated with diffuse pulmonary infiltrates on imaging studies, similar to those rarely noted with bortezomib and other inhibitors of the 20S proteasome, culminating in death. Although the contribution of VLX1570's formulation to the pulmonary toxicity could not be ruled out, the severity and precipitous nature of the toxicity and the steep relationship between dose and toxicity, the study was discontinued. Despite the severe pulmonary toxicity noted with VLX1570, efforts directed at identifying DUB inhibitors with greater therapeutic indices appear warranted based on the unique mechanism of action, robustness of preclinical antitumor activity, and activity of the DUB inhibitors in MM resistant to PIs targeting the 20S proteasome subunit.

The cationic small molecule GW4869 is cytotoxic to high phosphatidylserine-expressing myeloma cells.

We have discovered that a small cationic molecule, GW4869, is cytotoxic to a subset of myeloma cell lines and primary myeloma plasma cells. Biochemical analysis revealed that GW4869 binds to anionic phospholipids such as phosphatidylserine - a lipid normally confined to the intracellular side of the cell membrane. However, interestingly, phosphatidylserine was expressed on the surface of all myeloma cell lines tested (n = 12) and 9/15 primary myeloma samples. Notably, the level of phosphatidylserine expression correlated well with sensitivity to GW4869. Inhibition of cell surface phosphatidylserine exposure with brefeldin A resulted in resistance to GW4869. Finally, GW4869 was shown to delay the growth of phosphatidylserine-high myeloma cells in vivo. To the best of our knowledge, this is the first example of using a small molecule to target phosphatidylserine on malignant cells. This study may provide the rationale for the development of phosphatidylserine-targeting small molecules for the treatment of surface phosphatidylserine-expressing cancers.

6-Benzylidene-2-[4-(pyridin-3-ylcarboxy)benzylidene]cyclohexanones: A novel cluster of tumour-selective cytotoxins.

Novel cytotoxins 3-5 containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore are disclosed. The compounds in series 3 and 5 have the potential to liberate niacin which may reduce some of the side effects of antineoplastic compounds. 3a-c emerged as the most potent cytotoxic compounds with IC50 values in the low micromolar range against human Molt4/C8 and CEM CD4+ T-lymphocytes as well as murine L1210 leukemia cells. QSAR studies revealed that cytotoxic potencies were negatively correlated with the magnitude of the Hammett sigma values of the aryl substituents. The compounds 3a-e displayed tumour-selective toxicity against human HL-60, HSC-2, HSC-3 and HSC-4 neoplasms as compared to human HGF, HPC and HPLF nonmalignant cells. A representative potent compound 3a caused PARP1 cleavage and G0/G1 cell cycle arrest in HSC-2 cells. These compounds are well tolerated in mice at doses up to and including 300mg/kg of the compounds and no mortalities were noted after 4h. The stability studies undertaken did not reveal that a representative compound 3a underwent hydrolysis to the related phenol 2a. Some guidelines for further analog development of the novel esters 3 were made.

PAC down-regulates estrogen receptor alpha and suppresses epithelial-to-mesenchymal transition in breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options and very poor prognosis following progression after standard chemotherapeutic regimens. Therefore, novel molecules and therapeutic options are urgently needed for this category of patients. Recently, we have identified PAC as a curcumin analogue with potent anti-cancer features. METHODS: HPLC was used to evaluate the stability of PAC and curcumin in PBS and also in circulating blood. Cytotoxicity/apoptosis was assessed in different breast cancer cell lines using propidium iodide/annexinV associated with flow cytometry. Furthermore, immunoblotting analysis determined the effects of PAC on different oncogenic proteins and pathways. Additionally, the real time xCELLigence RTCA technology was applied to investigate the effect of PAC on the cellular proliferation, migration and invasion capacities. RESULTS: PAC is more stable than curcumin in PBS and in circulating blood. Furthermore, we have shown differential sensitivity of estrogen receptor-alfa positive (ERα(+)) and estrogen receptor alfa negative (ERα(-)) breast cancer cells to PAC, which down-regulated ERα in both cell types. This led to complete disappearance of ERα in ERα(-) cells, which express very low level of this receptor. Interestingly, specific down-regulation of ERα in receptor positive cells increased the apoptotic response of these cells to PAC, confirming that ERα inhibits PAC-dependent induction of apoptosis, which could be mediated through ERα down-regulation. Additionally, PAC inhibited the proliferation and suppressed the epithelial-to-mesenchymal transition process in breast cancer cells, with higher efficiency on the TNBC subtype. This effect was also observed in vivo on tumor xenografts. Additionally, PAC suppressed the expression/secretion of 2 important cytokines IL-6 and MCP-1, and consequently inhibited the paracrine procarcinogenic effects of breast cancer cells on breast stromal fibroblasts. CONCLUSION: These results indicate that PAC could be considered as important candidate for future therapeutic options against the devastating TNBC subtype.

Pharmacologic suppression of inflammation by a diphenyldifluoroketone, EF24, in a rat model of fixed-volume hemorrhage improves survival.

An exaggerated release of proinflammatory cytokines and accompanying inflammation contributes to the development of multiple organ failure after hemorrhagic shock. Here, we tested the nuclear factor (NF) κ-light-chain-enhancer of activated B cell (NF-κB)-mediated transcriptional control of inflammatory pathways as a target in the management of hemorrhage-induced inflammation. We performed a study in a rat model of fixed-volume hemorrhage to investigate the anti-inflammatory effects of the diphenyldifluoroketone EF24 [3,5-bis(2-fluorobenzylidene)piperidin-4-one], an NF-κB inhibitor, in lung tissue. EF24 treatment (0.4 mg/kg) significantly prevented the upregulation of inflammatory biomarkers in rats subjected to 50% hemorrhage and preserved the pulmonary histology in hemorrhaged rats. The lung tissue from treated rats showed marked suppression of the hemorrhage-mediated induction of Toll-like receptor 4, phospho-p65 NF-κB, inducible nitric-oxide synthase, heme oxygenase-1, and cyclooxygenase-2 (COX-2). The hemorrhage-induced COX-2 activity was also significantly inhibited by the EF24 treatment. At the same time, EF24 induced nuclear factor (erythroid-derived 2)-like 2-mediated protective mechanisms against oxidative stress. EF24 also reduced hemorrhage-induced lung myeloperoxidase activity. The plasma levels of proinflammatory tumor necrosis factor-α, interleukin (IL)-6, IL-1α, and IL-1ß were lower in EF24-treated rats than in untreated rats. Moreover, there was a significant reduction in the pulmonary expression of high-mobility group B1 protein. These biochemical effects were accompanied by a significant improvement in the survival of rats administered with EF24 as compared with the rats receiving vehicle control (P < 0.05). Overall, the results suggest that EF24 attenuates hemorrhage-induced inflammation and could serve as a salutary anti-inflammatory agent in resuscitation strategies.

Autophagy and cathepsin L are involved in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model.

AIM: 2-(3',5'-Dimethoxybenzylidene) cyclopentanone (DMBC) is a novel synthetic compound with antinociceptive activities. The aim of this study was to investigate the roles of the autophagic-lysosomal pathway in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model. METHODS: Mouse acetic acid-writhing test and hotplate test were used to assess the antinociceptive effects of DMBC, 3-MA (autophagy inhibitor) and Clik148 (cathepsin L inhibitor). The drugs were administered peripherally (ip) or centrally (icv). RESULTS: Peripheral administration of 3-MA (7.5-30 mg/kg) or Clik148 (10-80 mg/kg) produced potent antinociceptive effect in acetic acid-writhing test. Central administration of 3-MA or Clik148 (12.5-50 nmol/L) produced comparable antinociceptive effect in acetic acid-writhing test. Peripheral administration of DMBC (25-50 mg/kg) produced potent antinociceptive effects in both acetic acid-writhing and hotplate tests. Furthermore, the antinociceptive effect produced by peripheral administration of DMBC (50 mg/kg) in acetic acid-writhing test was antagonized by low doses of 3-MA (3.75 mg/kg) or Clik148 (20 mg/kg) peripherally administered, but was not affected by 3-MA or Clik148 (25 nmol/L) centrally administered. CONCLUSION: Activation of central autophagy and cathepsin L is involved in nociception in mice, whereas peripheral autophagy and cathepsin L contributes, at least in part, to the antinociceptive effect of DMBC in mice.

Cardiopulmonary arrest and resuscitation disrupts cholinergic anti-inflammatory processes: a role for cholinergic α7 nicotinic receptors.

Cardiac arrest is a leading cause of death worldwide. While survival rates following sudden cardiac arrest remain relatively low, recent advancements in patient care have begun to increase the proportion of individuals who survive cardiac arrest. However, many of these individuals subsequently develop physiological and psychiatric conditions that likely result from ongoing neuroinflammation and neuronal death. The present study was conducted to better understand the pathophysiological effects of cardiac arrest on neuronal cell death and inflammation, and their modulation by the cholinergic system. Using a well validated model of cardiac arrest, here we show that global cerebral ischemia increases microglial activation, proinflammatory cytokine mRNA expression (interleukin-1ß, interleukin-6, tumor necrosis factor-α), and neuronal damage. Cardiac arrest also induces alterations in numerous cellular components of central cholinergic signaling, including a reduction in choline acetyltransferase enzymatic activity and the number of choline acetyltransferase-positive neurons, as well as, reduced acetylcholinesterase and vesicular acetylcholine transporter mRNA. However, treatment with a selective agonist of the α7 nicotinic acetylcholine receptor, the primary receptor mediating the cholinergic anti-inflammatory pathway, significantly decreases the neuroinflammation and neuronal damage resulting from cardiac arrest. These data suggest that global cerebral ischemia results in significant declines in central cholinergic signaling, which may in turn diminish the capacity of the cholinergic anti-inflammatory pathway to control inflammation. Furthermore, we provide evidence that pharmacological activation of α7 nicotinic acetylcholine receptors provide significant protection against ischemia-related cell death and inflammation within a clinically relevant time frame.

Antihyperglycaemic activity of 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal in diabetic mice.

We have recently generated lipophilic D-xylose derivatives that increase the rate of glucose uptake in cultured skeletal muscle cells in an AMP-activated protein kinase (AMPK)-dependent manner. The derivative 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal (EH-36) stimulated the rate of glucose transport by increasing the abundance of glucose transporter-4 in the plasma membrane of cultured myotubes. The present study aimed at investigating potential antihyperglycaemic effects of EH-36 in animal models of diabetes. Two animal models were treated subcutaneously with EH-36: streptozotocin-induced diabetes in C57BL/6 mice (a model of insulin-deficient type 1 diabetes), and spontaneously diabetic KKAy mice (Kuo Kondo rats carrying the A(y) yellow obese gene; insulin-resistant type 2 diabetes). The in vivo biodistribution of glucose in control and treated mice was followed with the glucose analogue 2-deoxy-2-[(18) F]-D-glucose; the rate of glucose uptake in excised soleus muscles was measured with [(3) H]-2-deoxy-D-glucose. Pharmacokinetic parameters were determined by non-compartmental analysis of the in vivo data. The effective blood EH-36 concentration in treated animals was 2 µM. It reduced significantly the blood glucose levels in both types of diabetic mice and also corrected the typical compensatory hyperinsulinaemia of KKAy mice. EH-36 markedly increased glucose transport in vivo into skeletal muscle and heart, but not to adipose tissue. This stimulatory effect was mediated by Thr(172) -phosphorylation in AMPK. Biochemical tests in treated animals and acute toxicological examinations showed that EH-36 was well tolerated and not toxic to the mice. These findings indicate that EH-36 is a promising prototype molecule for the development of novel antidiabetic drugs.

New 2-amino-pyridinyl-N-acylhydrazones: Synthesis and identification of their mechanism of anti-inflammatory action.

AIMS: The main aim of this paper was the synthesis and the evaluation of the anti-inflammatory activity of LASSBio-1828 (an amino-pyridinyl-N-acylhydrazone) and its respective hydrochloride, based on a p38α MAPK inhibitor (LASSBio-1824) previously synthesized by our group. MAIN METHODS: The compounds were tested regarding their cell viability effect and on acute models of inflammation such as formalin-induced licking test, cell migration and inflammatory mediators quantification. KEY FINDINGS: Treatment with the compounds inhibited p38α, reduced inflammatory pain, cell migration and inflammatory mediators that participate on the MAPK pathway such as TNF-α and IL-1ß. SIGNIFICANCE: Taken together, these results suggest that the synthesis of the corresponding hydrochloride of LASSBio-1828 enhanced its potency as a p38 inhibitor, and also that this compound could be considered a good anti-inflammatory drug candidate after further studies.

Distinct Roles of α7 nAChRs in Antigen-Presenting Cells and CD4+ T Cells in the Regulation of T Cell Differentiation.

It is now apparent that immune cells express a functional cholinergic system and that α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating T cell differentiation and the synthesis of antigen-specific antibodies and proinflammatory cytokines. Here, we investigated the specific function α7 nAChRs on T cells and antigen presenting cells (APCs) by testing the effect of GTS-21, a selective α7 nAChR agonist, on differentiation of CD4+ T cells from ovalbumin (OVA)-specific TCR transgenic DO11.10 mice activated with OVA or OVA peptide323-339 (OVAp). GTS-21 suppressed OVA-induced antigen processing-dependent development of CD4+ regulatory T cells (Tregs) and effector T cells (Th1, Th2, and Th17). By contrast, GTS-21 up-regulated OVAp-induced antigen processing-independent development of CD4+ Tregs and effector T cells. GTS-21 also suppressed production of IL-2, IFN-γ, IL-4, IL-17, and IL-6 during OVA-induced activation but, with the exception IL-2, enhanced their production during OVAp-induced activation. In addition, during antigen-nonspecific, APC-independent anti-CD3/CD28 antibody-induced CD4+ polyclonal T cell activation in the presence of respective polarizing cytokines, GTS-21 promoted development of all lineages, which indicates that GTS-21 also acts via α7 nAChRs on T cells. These results suggest 1) that α7 nAChRs on APCs suppress CD4+ T cell activation by interfering with antigen presentation through inhibition of antigen processing; 2) that α7 nAChRs on CD4+ T cells up-regulate development of Tregs and effector T cells; and that α7 nAChR agonists and antagonists could be potentially useful agents for immune response modulation and enhancement.

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.

Pharmacokinetic Limitations on Effects of an Alpha7-Nicotinic Receptor Agonist in Schizophrenia: Randomized Trial with an Extended-Release Formulation.

The aim of the trial was to assess whether extending plasma levels of the alpha7-nicotinic acetylcholine receptor (nAChR) agonist 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXB-A) over time enhances its cognitive effects in schizophrenia. Both smoking and non-smoking patients were studied, to determine whether effects differ between these two groups. Forty-three smokers and thirty-seven non-smokers who met DSM-IV criteria for schizophrenia were enrolled in a double-blind, randomized, placebo-controlled 1 month trial. DMXB-A 150 mg was formulated with hypromellose to produce extended release over 4 h and administered four times daily. The primary outcome (the Neurocognitive Composite of the MATRICS Consensus Cognitive Battery) and secondary outcomes (the MATRICS Attention-Vigilance Domain and P50 gating), showed no significant effect. Plasma levels were obtained 2.5 h post administration. In non-smokers, levels were similar to those reached transiently with 75-150 mg DMXB-A immediate-release formulations twice daily, which were earlier shown to be effective doses. However, the extended-release formulation produced no cognitive or clinical effect either in non-smokers or smokers. The 10-fold lower DMXB-A plasma levels in smokers suggest that chronic smoking enhances DMXB-A metabolism. Pro-cognitive effects of DMXB-A may result from transient increases in cell signaling that are limited by receptor tachyphylaxis. Future efforts to improve cognition in schizophrenia by enhancing alpha7 nAChR function may require consideration of these pharmacokinetic limitations.

Tyrosine kinase inhibition ameliorates the hyperdynamic state and decreases nitric oxide production in cirrhotic rats with portal hypertension and ascites.

Tumor necrosis factor-alpha (TNF) causes vasodilatation and a hyperdynamic state by activating nitric oxide (NO) synthesis. Tyrphostins, specific inhibitors of protein tyrosine kinase (PTK), block the signaling events induced by TNF and NO production. A hyperdynamic circulatory syndrome (HCS) is often observed in portal hypertension (PHT). TNF and NO seem to mediate these hemodynamic changes. The aim of this work was to study the effect of PTK inhibition on the systemic and portal hemodynamics, TNF and NO production, in cirrhotic rats with portal hypertension. Rats with liver cirrhosis induced by chronic inhalation of carbon tetrachloride were used. Animals were treated daily with tyrphostin AG 126 (alpha-cyano-(3-hydroxy-4-nitro) cinnamonitrile) or placebo for 5 d. Mean arterial pressure (MAP), heart rate (HR), and portal pressure (PP) were measured by indwelling catheters. Cardiac output (CI) and stroke volume (SV) were estimated by thermodilution, systemic vascular resistance (SVR) was calculated (MAP/CI), and portal systemic shunting (PSS) was quantitated using radioactive microspheres. Serum and mesenteric lymph node (MLN) TNF levels were measured using an immunoassay kit, and serum NOx was determined photometrically by its oxidation products. The AG 126-treated group showed a statistically significant increase in MAP and SVR, and decreases in CI, SV, MLN TNF, and serum NO oxidation products nitrite and nitrate (NOx) in comparison with the placebo-treated rats. No significant differences were noticed in HR, PP, PSS, or serum TNF. Significant correlations were observed between MAP and NOx, MAP and MLN TNF, PSS and NOx, and serum TNF and serum NOx. The HCS observed in PHT seems to be mediated, at least in part, by TNF and NO by the activation of PTKs and their signaling pathways. PTK activity inhibition ameliorates the hyperdynamic abnormalities that characterize animals with cirrhosis and PHT.

3-Furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic receptor, reverses schizophrenia-like cognitive and social deficits in rats.

The cognitive impairments and negative symptoms experienced by schizophrenia patients still await effective treatment. Alpha7 nicotinic acetylcholine receptors (α7 nAChRs) have gain considerable attention in this regard. It has been recently proposed that positive allosteric modulators (PAMs) of α7 nAChRs may represent an alternative strategy to that based on orthosteric agonists. The aim of the present study is to evaluate the efficacy of PAM-2 (3-furan-2-yl-N-p-tolyl-acrylamide) against cognitive deficits and negative-like symptoms in a rat model of schizophrenia based on administration of ketamine, a NMDAR antagonist. The activity of PAM-2 was compared to that elicited by DMXBA, an α7 nAChR partial agonist. For this purpose, the attentional set-shifting task (ASST) and the novel object recognition task (NORT) were used. The efficacies of PAM-2 and DMXBA against ketamine-induced social withdrawal were assessed using the social interaction test (SIT). The results demonstrated that PAM-2 and DMXBA ameliorated ketamine-induced cognitive impairments on the ASST and NORT as well as produced pro-social activities in the SIT. Moreover, the co-administration of inactive doses of PAM-2 and antipsychotic drugs, clozapine or risperidone, reversed ketamine-induced deficits. The present findings provide further support for the concept that α7-PAMs could be used either alone or in combination with antipsychotics for schizophrenia therapy.

LASSBio-1829 Hydrochloride: Development of a New Orally Active N-Acylhydrazone IKK2 Inhibitor with Anti-inflammatory Properties.

Inhibitor of nuclear factor κB kinase 2 (IKK2) is suggested to be a potential target for the development of novel anti-inflammatory and anticancer drugs. In this work, we applied structure-based drug design to improve the potency of the inhibitor (E)-N'-(4-nitrobenzylidene)-2-naphthohydrazide (LASSBio-1524, 1 a: IC50 =20 µm). The molecular model built for IKK2 together with the docking methodology employed were able to provide important and consistent information with respect to the structural and chemical inhibitor characteristics that may confer potency to IKK2 inhibitors, providing important guidelines for the development of a new N-acylhydrazone (NAH) derivative. (E)-N'-(4-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzylidene)-2-naphthohydrazide hydrochloride (LASSBio-1829 hydrochloride, 10) is a 7-azaindole NAH able to inhibit IKK2 with an IC50 value of 3.8 µm. LASSBio-1829 hydrochloride was found to be active in several pharmacological inflammation tests in vivo, showing its potential as an anti-inflammatory prototype.

Synergistic antitumor activity of rapamycin and EF24 via increasing ROS for the treatment of gastric cancer.

Mechanistic/mammalian target of rapamycin (mTOR) has emerged as a new potential therapeutic target for gastric cancer. Rapamycin and rapamycin analogs are undergoing clinical trials and have produced clinical responses in a subgroup of cancer patients. However, monotherapy with rapamycin at safe dosage fails to induce cell apoptosis and tumor regression which has hampered its clinical application. This has led to the exploration of more effective combinatorial regimens to enhance the effectiveness of rapamycin. In our present study, we have investigated the combination of rapamycin and a reactive oxygen species (ROS) inducer EF24 in gastric cancer. We show that rapamycin increases intracellular ROS levels and displays selective synergistic antitumor activity with EF24 in gastric cancer cells. This activity was mediated through the activation of c-Jun N terminal kinase and endoplasmic reticulum stress (ER) pathways in cancer cells. We also show that inhibiting ROS accumulation reverses ER stress and prevents apoptosis induced by the combination of rapamycin and EF24. These mechanisms were confirmed using human gastric cancer xenografts in immunodeficient mice. Taken together, our work provides a novel therapeutic strategy for the treatment of gastric cancer. The work reveals that ROS generation could be an important target for the development of new combination therapies for cancer treatment.

Coinhibition of the deubiquitinating enzymes, USP14 and UCHL5, with VLX1570 is lethal to ibrutinib- or bortezomib-resistant Waldenstrom macroglobulinemia tumor cells.

The survival of Waldenstrom macroglobulinemia (WM) tumor cells hinges on aberrant B-cell receptor (BCR) and MYD88 signaling. WM cells upregulate the proteasome function to sustain the BCR-driven growth while maintaining homeostasis. Clinically, two treatment strategies are used to disrupt these complementary yet mutually exclusive WM survival pathways via ibrutinib (targets BTK/MYD88 node) and bortezomib (targets 20 S proteasome). Despite the success of both agents, WM patients eventually become refractory to treatment, highlighting the adaptive plasticity of WM cells and underscoring the need for development of new therapeutics. Here we provide a comprehensive preclinical report on the anti-WM activity of VLX1570, a novel small-molecule inhibitor of the deubiquitinating enzymes (DUBs), ubiquitin-specific protease 14 (USP14) and ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5). Both DUBs reside in the 19 S proteasome cap and their inhibition by VLX1570 results in rapid and tumor-specific apoptosis in bortezomib- or ibrutinib-resistant WM cells. Notably, treatment of WM cells with VLX1570 downregulated BCR-associated elements BTK, MYD88, NFATC, NF-κB and CXCR4, the latter whose dysregulated function is linked to ibrutinib resistance. VLX1570 administered to WM-xenografted mice resulted in decreased tumor burden and prolonged survival (P=0.0008) compared with vehicle-treated mice. Overall, our report demonstrates significant value in targeting USP14/UCHL5 with VLX1570 in drug-resistant WM and carries a high potential for clinical translation.

The neutral sphingomyelinase-2 is involved in angiogenic signaling triggered by oxidized LDL.

Capillaries of the external part of the normal arterial wall constitute the vasa vasorum network. In atherosclerotic lesions, neovascularization occurs in areas of intimal hyperplasia where it may promote plaque expansion, and intraplaque hemorrhage. Oxidized LDL that are present in atherosclerotic areas activate various angiogenic signaling pathways, including reactive oxygen species and the sphingosine kinase/sphingosine-1-phosphate pathway. We aimed to investigate whether oxidized LDL-induced angiogenesis requires neutral sphingomyelinase-2 activation and the neutral sphingomyelinase-2/sphingosine kinase-1 pathway. The role of neutral sphingomyelinase-2 in angiogenic signaling was investigated in Human Microvascular Endothelial Cells (HMEC-1) forming capillary tube on Matrigel and in vivo in the Matrigel plug assay in C57BL/6 mice and in the chicken chorioallantoic membrane model. Low concentration of human oxidized LDL elicits HMEC-1 capillary tube formation and neutral sphingomyelinase-2 activation, which were blocked by neutral sphingomyelinase-2 inhibitors, GW4869 and specific siRNA. This angiogenic effect was mimicked by low concentration of C6-Ceramide and was inhibited by sphingosine kinase-1 inhibitors. Upstream of neutral sphingomyelinase-2, oxidized LDL-induced activation required LOX-1, reactive oxygen species generation by NADPH oxidase and p38-MAPK activation. Inhibition of sphingosine kinase-1 blocked the angiogenic response and triggered HMEC-1 apoptosis. Low concentration of oxidized LDL was angiogenic in vivo, both in the Matrigel plug assay in mice and in the chorioallantoic membrane model, and was blocked by GW4869. In conclusion, low oxLDL concentration triggers sprouting angiogenesis that involves ROS-induced activation of the neutral sphingomyelinase-2/sphingosine kinase-1 pathway, and is effectively inhibited by GW4869.