The multifaceted role of macrophages during acute liver injury.

The liver is situated at the interface of the gut and circulation where it acts as a filter for blood-borne and gut-derived microbes and biological molecules, promoting tolerance of non-invasive antigens while driving immune responses against pathogenic ones. Liver resident immune cells such as Kupffer cells (KCs), a subset of macrophages, maintain homeostasis under physiological conditions. However, upon liver injury, these cells and others recruited from circulation participate in the response to injury and the repair of tissue damage. Such response is thus spatially and temporally regulated and implicates interconnected cells of immune and non-immune nature. This review will describe the hepatic immune environment during acute liver injury and the subsequent wound healing process. In its early stages, the wound healing immune response involves a necroinflammatory process characterized by partial depletion of resident KCs and lymphocytes and a significant infiltration of myeloid cells including monocyte-derived macrophages (MoMFs) complemented by a wave of pro-inflammatory mediators. The subsequent repair stage includes restoring KCs, initiating angiogenesis, renewing extracellular matrix and enhancing proliferation/activation of resident parenchymal and mesenchymal cells. This review will focus on the multifaceted role of hepatic macrophages, including KCs and MoMFs, and their spatial distribution and roles during acute liver injury.

Distinct spatial distribution and roles of Kupffer cells and monocyte-derived macrophages in mouse acute liver injury.

Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple roles in the liver are not fully understood. We aimed herein to map the intrahepatic macrophage populations and their function(s) during acute liver injury. We used flow cytometry, gene expression analysis, multiplex-immunofluorescence, 3D-reconstruction, and spatial image analysis to characterize the intrahepatic immune landscape in mice post-CCl4-induced acute liver injury during three distinct phases: necroinflammation, and early and late repair. We observed hepatocellular necrosis and a reduction in liver resident lymphocytes during necroinflammation accompanied by the infiltration of circulating myeloid cells and upregulation of inflammatory cytokines. These parameters returned to baseline levels during the repair phase while pro-repair chemokines were upregulated. We identified resident CLEC4F+ Kupffer cells (KCs) and infiltrating IBA1+CLEC4F- monocyte-derived macrophages (MoMFs) as the main hepatic macrophage populations during this response to injury. While occupying most of the necrotic area, KCs and MoMFs exhibited distinctive kinetics, distribution and morphology at the site of injury. The necroinflammation phase was characterized by low levels of KCs and a remarkable invasion of MoMFs suggesting their potential role in phagoctosing necrotic hepatocytes, while opposite kinetics/distribution were observed during repair. During the early repair phase, yolksac - derived KCs were restored, whereas MoMFs diminished gradually then dissipated during late repair. MoMFs interacted with hepatic stellate cells during the necroinflammatory and early repair phases, potentially modulating their activation state and influencing their fibrogenic and pro-repair functions that are critical for wound healing. Altogether, our study reveals novel and distinct spatial and temporal distribution of KCs and MoMFs and provides insights into their complementary roles during acute liver injury.

Novel Functions of Integrins as Receptors of CD154: Their Role in Inflammation and Apoptosis.

CD154, an inflammatory mediator also known as CD40 ligand, has been identified as a novel binding partner for some members of the integrin family. The αIIbß3, specifically expressed on platelets, was the first integrin to be described as a receptor for CD154 after CD40. Its interaction with soluble CD154 (sCD154) highly contributes to thrombus formation and stability. Identifying αIIbß3 opened the door for investigating other integrins as partners of CD154. The αMß2 expressed on myeloid cells was shown capable of binding CD154 and contributing as such to cell activation, adhesion, and release of proinflammatory mediators. In parallel, α5ß1 communicates with sCD154, inducing pro-inflammatory responses. Additional pathogenic effects involving apoptosis-preventing functions were exhibited by the CD154-α5ß1 dyad in T cells, conferring a role for such interaction in the survival of malignant cells, as well as the persistence of autoreactive T cells. More recently, CD154 receptors integrated two new integrin members, αvß3 and α4ß1, with little known as to their biological significance in this context. This article provides an overview of the novel role of integrins as receptors of CD154 and as critical players in pro-inflammatory and apoptotic responses.

New tilomisole-based benzimidazothiazole derivatives as anti-inflammatory agents: Synthesis, in vivo, in vitro evaluation, and in silico studies.

New tilomisole-based benzimidazothiazole derivatives were designed and synthesized in this work. Their anti-inflammatory activity was assessed through the in vivo carrageenan rat paw edema model, and the in vitro COX inhibition assay. Compounds 13, 20, 30, 40, 43, and 46 demonstrated values of inhibition of induced edema in the in vivo assay comparable to celecoxib. All the synthesized compounds expressed their activity on COX-2 enzyme more than COX-1, proving their advantageous selectivity. In addition, compounds 13, 16, 20, 25, and 46 displayed lower IC50 values than celecoxib as a reference drug against COX-2 enzyme; having values of 0.09, 13.87, 32.28, 33.01, and 5.18 nM respectively vs 40.00 nM for celecoxib. Particularly, the most active compound (13) with its extreme potency (400 folds more potent than celecoxib) exhibited a notable high selectivity index (SI = 159.5). In silico studies, including ADMET prediction, compliance to Lipinski's rule of five, and molecular docking into the active site of both COX isozymes were conducted for the synthesized compounds. The results suggested that these compounds are good candidates for orally active drugs, and docking revealed higher number of interactions with COX-2 for 13 as the most active compound compared with COX-1 reflecting its advantageous selectivity and explaining its extreme potency.

Synthesis and Biological Evaluation of Thiazole-Based Derivatives as Potential Acetylcholinesterase Inhibitors.

Nineteen new thiazole-based derivatives were synthesized and their structures characterized with analytical and spectral data. The in vitro assessment of their acetylcholinesterase (AChE) inhibitory activity revealed that compounds 10 and 16 produced potent AChE inhibitory activities with IC50 values of 103.24 and 108.94 nM, respectively. Compounds 13, 17, 18, 21, 23, 31, and 33 displayed moderate activity with 25-50% relative potency compared to the known potent AChE inhibitor donepezil. Molecular docking studies of the active compounds docked within the active site cavity of AChE showed a binding orientation similar to that of donepezil, with good predicted binding affinities. These compounds could therefore be considered as potential lead compounds for the development of new and potentially improved AChE inhibitors.

Monoclonal Antibody Targeting the CD154 Cleavage Site Inhibits CD40-Dependent and -Independent Cleavage of CD154 from the Cell Surface.

In addition to the membrane-bound molecule, soluble CD154 (sCD154) is also detected at high levels in the medium of activated T cells and platelets and in the serum of patients suffering from different inflammatory diseases. This sCD154 is the result of cleavage of the full-length molecule between the glutamic acid residue at position 112 (E112) and methionine at position 113 (M113) and can be derived from the intracellular milieu and from cleavage of cell surface molecules. We have recently reported that substitution of both E112 and M113 by alanine inhibits intracellular and CD40-induced membrane cleavage of CD154 and procures to CD154 an increased biological function as compared with cleavable CD154. Thus, in this study, and in the aim of developing tools inhibiting cleavage of CD154 from the cell surface, we generated a panel of anti-human CD154 mAbs. One of the derived mAbs that did not alter the binding of sCD154 to CD40, named in this study Clone 8 mAb, totally lost its binding activity against cells expressing CD154 mutated at its E112 and M113 residues. Treatment with Clone 8 mAb was shown to completely abolish CD40-dependent and -independent cleavage of CD154 from the cell surface. Our study is highlighting the development and characterization of an innovative therapeutic tool capable of inhibiting the release/cleavage of CD154 from cells and thus maintaining its availability on the cell surface and the high probably of increasing its potency as an activator of CD40-induced responses.

Design, synthesis, and antitumor activity of PLGA nanoparticles incorporating a discovered benzimidazole derivative as EZH2 inhibitor.

PURPOSE: Targeting enhancer of zeste homolog 2 (EZH2) can represent a hopeful strategy for oncotherapy. Also, the use of PLGA-based nanoparticles as a novel and rate-controlling carrier system was of our concern. METHODS: Benzimidazole derivatives were synthesized, and their structures were clarified. In vitro antitumor activity was evaluated. Then, a modeling study was performed to investigate the ability of the most active compounds to recognize EZH2 active sites. Compound 30 (Drug) was selected to conduct pre-formulation studies and then it was incorporated into polymeric PLGA nanoparticles (NPs). NPs were then fully characterized to select an optimized formula (NP4) that subjected to further evaluation regarding antitumor activity and protein expression levels of EZH2 and EpCAM. RESULTS: The results showed the antitumor activity of some synthesized derivatives. Docking outcomes demonstrated that Compound 30 was able to identify EZH2 active sites. NP4 exhibited promising findings and proved to keep the antitumor activity of Compound 30. HEPG-2 was the most sensitive for both Drug and NP4. Protein analysis indicated that Drug and NP4 had targeted EZH2 and the downstream signaling pathway leading to the decline of EpCAM expression. CONCLUSIONS: Targeting EZH2 by Compound 30 has potential use in the treatment of cancer especially hepatocellular carcinoma.

CD154 Resistant to Cleavage from Intracellular Milieu and Cell Surface Induces More Potent CD40-Mediated Responses.

In addition to the membrane-bound form, CD154 also exists as a soluble molecule originating from an intracellular and membrane cleavage. We have previously shown that CD154 cleavage from T cell surface is mediated by CD40 and involves the action of ADAM10/ADAM17 enzymes. In the aim of defining the importance of CD154 maintained on cell surface, we generated a CD154 mutated at the cleavage site. Our data show that the double mutation of E112 and M113 residues of CD154 abolishes its spontaneous release and the CD40-mediated cleavage from cell surface but does not affect its binding to CD40. We also demonstrated that both the release of CD154 from the intracellular milieu and its CD40-mediated cleavage from cell surface are highly dependent on ADAM10/ADAM17 enzymes. The CD154-EM mutant was shown capable of inducing a more prominent apoptotic response in susceptible B cell lines than the wild-type (WT) form of the molecule. In addition, human B cells cultured in the presence of the CD154-EM mutant exhibited upregulated proliferative responses compared with the CD154-WT. The CD154-EM mutant was also shown to trigger differentiation of human B cells, reflected by an increased Ig production, more significantly than CD154-WT. Thus, our data strongly suggest that cleavage-resistant CD154 is a more prominent stimulant than the cleavable form of the molecule. Therefore, a maintained expression of CD154 on cell membrane and a disturbed cleavage of the molecule could be a mechanism by which CD154 is involved in some pathological conditions and should be revisited.

DNA binding studies of novel diazatruxenones analogs as potential anticancer agents: Synthesis, antitumor investigation, DNA binding, SAR and molecular modeling calculation.

A series of polycyclic skeleton of truxene and triazatruxene analogs has been synthesized and evaluated for antitumor and DNA binding activities. The synthesized structures were confirmed by different spectroscopic techniques such as IR, 1HNMR, 13CNMR, and mass spectroscopy. The antitumor screening was performed adopting the NCI protocol against 60 different cell lines. Compounds 2 and 8 proved to be the most active ones among the other target compounds. In a trial to investigate the mechanism of action of the target compounds, DNA binding activity was also investigated. Compounds 3f, 4-8 exhibited good binding activity explaining their mechanism. In addition, molecular modeling studies were also performed for more clearance of the data obtained from the biological screening.

Cyclohepta[b]thiophenes as Potential Antiproliferative Agents: Design, Synthesis, In Vitro, and In Vivo Anticancer Evaluation.

Several thiophene featuring compounds are known for their promising antiproliferative activity. Prompted by the urgent need to identify new potent anticancer agents, 16 compounds of benzamides, benzylamines, and urea analogues incorporating a cyclohepta[b]thiophene scaffold were synthesized and biologically evaluated with a cell proliferation assay using the A549 nonsmall cell lung cancer cell line. Compound 17 demonstrated both potent and broad-spectrum anticancer activity with submicromolar 50% growth inhibition (GI50) values. It also showed superior antiproliferative activity (vs nocodazole) in OVACAR-4, OVACAR-5, CAKI-1, and T47D cell lines with GI50 values of 2.01 (vs 22.28), 2.27 (vs 20.75), 0.69 (vs 1.11), and 0.362 (vs 81.283) µM, respectively. Additionally, compound 17 displayed minimal cytotoxicity based on 50% lethal concentration (LC50) values toward all tested cell lines. Further cell-based mechanistic studies of compound 17 revealed its ability to induce cell cycle arrest of A549 cells as evidenced by dose dependent G2/M accumulation. Furthermore, induction of early apoptosis along with activation of caspase 3, 8, and 9 were confirmed in A549 cells treated with compound 17. Targeting tubulin polymerization may explain the mechanism of the antiproliferative activity of compound 17 based on cell cycle analysis, detected apoptosis, and in vitro inhibition of tubulin polymerization. In vitro data were further supported by in vivo antitumor efficacy studies of compound 17 in a CT26 murine model for which the results showed a reduction in the tumor growth compared to untreated mice. Overall, compound 17 has the potential to function as a promising candidate for further development of potent anticancer chemotherapeutics.

New thiazolopyrimidine as anticancer agents: Synthesis, biological evaluation, DNA binding, molecular modeling and ADMET study.

In the present study, new series of thiazolopyrimidine derivatives was synthesized as purine analogs. The structures of the products were confirmed through spectroscopic techniques such as NMR and mass spectrometry. In addition, the synthesized compounds were evaluated as antitumor active agent through NCI screening protocol against 60 different cell lines under 9 different panels. Furthermore, DNA binding activity of the compounds was also evaluated. The results revealed that compound 35 proved to be the most active member of the tested series and it is promoted to the 5-dose testing where it gives GI50, TGI and LC50 values of 1.07, 6.61, 34.7 µM respectively. Furthermore, it also proved to have a good DNA binding activity with value that is comparable with that produced by doxorubicin which was used as positive standard. In addition, compound 27 was proved to be the most active DNA binding agent with binding affinity 28.38 ± 1.1. The pharmacokinetic properties were also calculated. Molecular docking studies suggested binding mode of compounds 27 and 35 to DNA minor groove via hydrogen bonding interaction. The anticancer activity of compounds 27 and 35 may be attributed to DNA binding.

Targeting EGFR tyrosine kinase: Synthesis, in vitro antitumor evaluation, and molecular modeling studies of benzothiazole-based derivatives.

New benzothiazole-based derivatives were synthesized in the present work with the aim of evaluating their antitumor activity. They were in vitro tested against hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT-116), mammary gland cancer (MCF-7), prostate cancer (PC-3), and epithelioid carcinoma (HeLa). The results of the in vitro antitumor evaluation revealed that the most active compounds were 39, 40, 51, 56, and 61 exhibiting IC50 values comparable to the reference drug lapatinib. The most active compounds were further subjected to EGFR inhibitory activity assay to rationalize their potency mode. Notably, the most active antitumor compounds 39 and 40 represented the most potent inhibitors to EGFR with IC50 values of 24.58 and 30.42 nM respectively in comparison with 17.38 nM for lapatinib as a standard drug. Molecular modeling studies were also conducted for the synthesized compounds, including docking into EGFR active site and surface mapping. Results proved the superior binding of the hydrazone derivatives 39 and 40 with EGFR suggesting them as good candidates for targeted antitumor therapy through EGFR kinase inhibition.

CD154 inhibits death of T cells via a Cis interaction with the α5ß1 integrin.

CD154 plays a major role in the pathogenesis of several autoimmune and inflammatory diseases. In addition to CD40, soluble CD154 (sCD154) binds to other receptors namely αIIbß3, αMß2, α5ß1 and αvß3 integrins. We have previously reported that binding of sCD154 to α5ß1 integrin expressed on several human T cell lines is capable of inhibiting Fas-induced cell death. In the current study, we show that such effect of the sCD154/α5ß1 interaction is not restricted to the cell death response induced by Fas but could also be exhibited toward other death signals such as TRAIL and TNF- α. We also demonstrate that sCD154 is capable of inhibiting Fas-mediated death of human activated T cells, more importantly of CD4+ than CD8+ T ones. Our data also show that membrane-bound CD154 and α5ß1 integrin expressed on the surface of distinct cells failed to influence cell death responses. However, when membrane-bound CD154 and α5ß1 are expressed on the surface of same cell, their interaction was capable of down regulating cell death. CD154 was shown to co-localize with the α5ß1 integrin on the surface of these cells. These data strongly suggest a cis-type of interaction between CD154 and α5ß1 when both are expressed on the same cell surface, rather than a trans-interaction which usually implicates the ligand and its receptor each expressed on the surface of a distinct cell. Taken together, these findings add to the list of roles through which CD154 is contributing to the pathogenesis of autoimmune-inflammatory diseases, i.e. by protecting T cells from death and enhancing their survival.

Marine-Inspired Bis-indoles Possessing Antiproliferative Activity against Breast Cancer; Design, Synthesis, and Biological Evaluation.

Diverse indoles and bis-indoles extracted from marine sources have been identified as promising anticancer leads. Herein, we designed and synthesized novel bis-indole series 7a-f and 9a-h as Topsentin and Nortopsentin analogs. Our design is based on replacing the heterocyclic spacer in the natural leads by a more flexible hydrazide linker while sparing the two peripheral indole rings. All the synthesized bis-indoles were examined for their antiproliferative action against human breast cancer (MCF-7 and MDA-MB-231) cell lines. The most potent congeners 7e and 9a against MCF-7 cells (IC50 = 0.44 ± 0.01 and 1.28 ± 0.04 µM, respectively) induced apoptosis in MCF-7 cells (23.7-, and 16.8-fold increase in the total apoptosis percentage) as evident by the externalization of plasma membrane phosphatidylserine detected by Annexin V-FITC/PI assay. This evidence was supported by the Bax/Bcl-2 ratio augmentation (18.65- and 11.1-fold compared to control) with a concomitant increase in the level of caspase-3 (11.7- and 9.5-fold) and p53 (15.4- and 11.75-fold). Both compounds arrested the cell cycle mainly in the G2/M phase. Furthermore, 7e and 9a displayed good selectivity toward tumor cells (S.I. = 38.7 and 18.3), upon testing of their cytotoxicity toward non-tumorigenic breast MCF-10A cells. Finally, compounds 7a, 7b, 7d, 7e, and 9a were examined for their plausible CDK2 inhibitory action. The obtained results (% inhibition range: 16%-58%) unveiled incompetence of the target bis-indoles to inhibit CDK2 significantly. Collectively, these results suggested that herein reported bis-indoles are good lead compounds for further optimization and development as potential efficient anti-breast cancer drugs.

Antitumor properties of certain spirooxindoles towards hepatocellular carcinoma endowed with antioxidant activity.

In the current medical era, spirooxindole motif stands out as a privileged heterospirocyclic scaffold that represents the core for a wide range of bioactive naturally isolated products (such as Strychnofoline and spirotryprostatins A and B) and synthetic compounds. Interestingly, no much attention has been paid to develop spirooxindole derivatives with dual antioxidant and anticancer activities. In this context, a series of spirooxindoles 6a-p was examined for their anticancer effect towards HepG2 hepatocellular carcinoma and PC-3 prostate cancer cell lines. Spirooxindole 6a was found to be an efficient anti-proliferative agent towards both HepG2 and PC-3 cells (IC50 = 6.9 and 11.8 µM, respectively). Afterwards, spirooxindole 6a was assessed for its apoptosis induction potential in HepG2 cells, where its pro-apoptotic impact was approved via the significant elevation in the Bax/Bcl-2 ratio and the expression levels of caspase-3.

Towards breast cancer targeting: Synthesis of tetrahydroindolocarbazoles, antibreast cancer evaluation, uPA inhibition, molecular genetic and molecular modelling studies.

A series of some new tetrahydroindolocarbazole derivatives has been synthesized. The structure of the synthesized compounds has been confirmed by different spectroscopic techniques such as IR, NMR, elemental analysis and mass spectrometry. The target compounds were evaluated for their antitumor activity against breast cancer cell line MCF-7, their GI% and their LC50 have been determined. Six of the synthesized compounds exhibited GI% values against MCF-7 cell lines exceeding 70% ranging from 71.9 to 85.0% in addition that compound 11 expressed GI% values of 99.9% and considered the most active derivatives among the synthesized ones. Compound 11 showed a remarkable decrease of u PA level to 3.5 ng/ml compared to DOX. Compound 5, 11 and 15 showed significant decrease in expression of MTAP and CDKN2A, in addition to a remarkable decrease in DNA damage comet assay method. Molecular modeling studies were performed to interpretate the behavior of active ligands as uPA inhibitors.

Assessment of lipophilicity of newly synthesized celecoxib analogues using reversed-phase HPLC.

BACKGROUND: Lipophilicity is a physicochemical property of an essential importance in medicinal chemistry; therefore, fast and reliable measurement of lipophilicity will affect greatly the drug discovery process. RESULTS: A series of N-benzenesulfonamide-1H-pyrazoles, oximes and hydrazones as celecoxib analogues was investigated with regard to their retention behavior using reversed-phase high performance liquid chromatography (RP-HPLC). The mobile phases employed for this study consist of a mixture of water and methanol in different proportions. In addition, the stationary phase utilized for this separation was C18 silanized silica gel and using 200 nm as a detection wavelength. The retention behavior of the investigated compounds was determined based on practical determination of log k at different concentrations of methanol (as an organic modifier) in the mobile phase ranging from 60 to 80%. It was observed that the retention of these compounds (expressed as log k) decreased in a linear manner with increasing the concentration of methanol. High correlation coefficients (more than 0.90 in most cases) were obtained for the relationship between the volume fraction of the organic solvent and the retention values represented as log k w. A comparative evaluation was carried out between chromatographically-obtained lipophilicity parameters (represented as lipophilicity chromatographic index log k w or isocratic chromatographic hydrophobicity index, φ 0) and the computationally calculated log P values (miLogP, ALOGP, ACD/Labs and ALOGPs). CONCLUSION: It was found that a good correlation exists between the experimental and computed log P values. In the future, these results can give a deep insight about the anti-inflammatory and analgesic activity of the newly synthesized compounds.

Synthesis, biological evaluation and in silico studies with 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides as novel selective carbonic anhydrase IX inhibitors endowed with anticancer activity.

In the presented work, we report the synthesis of a series of 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides 7a-fvia the Erlenmeyer-Plöchl reaction. All the prepared imidazolones 7a-f were evaluated as inhibitors of human (h) carbonic anhydrases (CA, EC 4.2.1.1) cytosolic isoforms hCA I and II, as well as transmembrane tumor-associated isoforms hCA IX and XII. All the tested hCA isoforms were inhibited by the prepared imidazolones 7a-f in variable degrees with the following KIs ranges: 673.2-8169 nM for hCA I, 61.2-592.1 nM for hCA II, 23-155.4 nM for hCA XI, and 21.8-179.6 nM for hCA XII. In particular, imidazolones 7a, 7e, and 7f exhibited good selectivity towards the tumor-associated isoforms (CAs IX and XII) over the off-target cytosolic (CAs I and II) with selectivity index (SI) in the range of 6.2-19.4 and 3.3-8, respectively. Moreover, imidazolones 7a-f were screened for their anticancer activity in one dose (10-5 M) assay against a panel of 60 cancer cell lines according to US-NCI protocol. Furthermore, 7a, 7e and 7f were evaluated for their anti-proliferative activity against colorectal cancer HCT-116 and breast cancer MCF-7 cell lines. Furthermore, 7e and 7f were screened for cell cycle disturbance and apoptosis induction in HCT-116 cells. Finally, a molecular docking study was carried out to rationalize the obtained results.