Highly Selective MIF Ketonase Inhibitor KRP-6 Diminishes M1 Macrophage Polarization and Metabolic Reprogramming.

Macrophage polarization is highly involved in autoimmunity. M1 polarized macrophages drive inflammation and undergo metabolic reprogramming, involving downregulation of mitochondrial energy production and acceleration of glycolysis. Macrophage migration inhibitory factor (MIF), an enigmatic tautomerase (ketonase and enolase), was discovered to regulate M1 polarization. Here, we reveal that KRP-6, a potent and highly selective MIF ketonase inhibitor, reduces MIF-induced human blood eosinophil and neutrophil migration similarly to ISO-1, the most investigated tautomerase inhibitor. We equally discovered that KRP-6 prevents M1 macrophage polarization and reduces ROS production in IFN-γ-treated cells. During metabolic reprogramming, KRP-6 improved mitochondrial bioenergetics by ameliorating basal respiration, ATP production, coupling efficiency and maximal respiration in LPS+IFN-γ-treated cells. KRP-6 also reduced glycolytic flux in M1 macrophages. Moreover, the selective MIF ketonase inhibitor attenuated LPS+IFN-γ-induced downregulation of PARP-1 and PARP-2 mRNA expression. We conclude that KRP-6 represents a promising novel therapeutic compound for autoimmune diseases, which strongly involves M1 macrophage polarization.

Synthesis and evaluation of a new class of MIF-inhibitors in activated macrophage cells and in experimental septic shock in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with enzymatic activities. Anti-inflammatory effects of MIF enzyme inhibitors indicate a link between its cytokine- and catalytic activities. Herein the synthesis, docking, and bioactivity of substituted benzylidene-1-indanone and -1-tetralone derivatives as MIF-tautomerase inhibitors is reported. Many of these substituted benzylidene-1-tetralones and -indan-1-ones were potent MIF-tautomerase inhibitors (IC50 < 10 µmol/L), and the most potent inhibitors were the 1-indanone derivatives 16 and 20. Some of these compounds acted as selective enolase or ketonase inhibitors. In addition, compounds 16, 20, 26, 37 and 61 efficiently inhibited NO, TNFα and IL-6 production in lipopolysaccharide-induced macrophages. Compound 20, 37 and 61 also inhibited ROS generation, and compound 26 and 37 abolished activation of NF-κB. Compound 37 significantly augmented hypothermia induced by high dose of lipopolysaccharide in mice. The possible mechanisms of action were explored using molecular modelling and docking, as well as molecular dynamics simulations.

Tetralone derivatives are MIF tautomerase inhibitors and attenuate macrophage activation and amplify the hypothermic response in endotoxemic mice.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine playing crucial role in immunity. MIF exerts a unique tautomerase enzymatic activity that has relevance concerning its multiple functions and its small molecule inhibitors have been proven to block its pro-inflammatory effects. Here we demonstrate that some of the E-2-arylmethylene-1-tetralones and their heteroanalogues efficiently bind to MIF's active site and inhibit MIF tautomeric (enolase, ketolase activity) functions. A small set of the synthesised derivatives, namely compounds (4), (23), (24), (26) and (32), reduced inflammatory macrophage activation. Two of the selected compounds (24) and (26), however, markedly inhibited ROS and nitrite production, NF-κB activation, TNF-α, IL-6 and CCL-2 cytokine expression. Pre-treatment of mice with compound (24) exaggerated the hypothermic response to high dose of bacterial endotoxin. Our experiments suggest that tetralones and their derivatives inhibit MIF's tautomeric functions and regulate macrophage activation and thermal changes in severe forms of systemic inflammation.

Study on the Synthesis, Antioxidant Properties, and Self-Assembly of Carotenoid-Flavonoid Conjugates.

Flavonoids and carotenoids possess beneficial physiological effects, such as high antioxidant capacity, anticarcinogenic, immunomodulatory, and anti-inflammatory properties, as well as protective effects against UV light. The covalent coupling of hydrophobic carotenoids with hydrophilic flavonoids, such as daidzein and chrysin, was achieved, resulting in new amphipathic structures. 7-Azidohexyl ethers of daidzein and chrysin were prepared in five steps, and their azide-alkyne [4 + 2] cycloaddition with pentynoates of 8'-apo-ß-carotenol, zeaxanthin, and capsanthin afforded carotenoid-flavonoid conjugates. The trolox-equivalent antioxidant capacity against ABTS•+ radical cation and self-assembly of the final products were examined. The 1:1 flavonoid-carotenoid hybrids generally showed higher antioxidant activity than their parent flavonoids but lower than that of the corresponding carotenoids. The diflavonoid hybrids of zeaxanthin and capsanthin, however, were found to exhibit a synergistic enhancement in antioxidant capacities. ECD (electronic circular dichroism) and UV-vis analysis of zeaxanthin-flavonoid conjugates revealed that they form different optically active J-aggregates in acetone/water and tetrahydrofuran/water mixtures depending on the solvent ratio and type of the applied aprotic polar solvent, while the capsanthin derivatives showed no self-assembly. The zeaxanthin bis-triazole conjugates with daidzein and with chrysin, differing only in the position of a phenolic hydroxyl group, showed significantly different aggregation profile upon the addition of water.

Novel Cell Wall Antifungals Reveal a Special Synergistic Activity in pbr1 Mutants Resistant to the Glucan Synthesis Antifungals Papulacandins and Echinocandins.

A series of 4-(arylmethylene)-3-isochromanones have been prepared with base-catalyzed Knoevenagel condensation starting from 3-isochromanone and aromatic aldehydes. The outcome of the reaction- the isomeric composition of the products depends on the aromatic aldehyde applied. These reactions afforded mostly the more stable E-diastereoisomer, but some condensations resulted in the Z-diastereoisomer or mixture of the stereoisomers (1-16). The products showed antifungal effect against some pathogenic fungi. We wanted to extend this study and to synthesize a new generation of 4-(arylmethylene)-3-isochromanones. These condensations led mostly to E-diastereoisomers (17-30). The structure verifications were performed by FT IR, 1H and13C NMR methods. Both the 1-16 and the novel 17-30 compounds have been screened against the three yeast models, fission yeast Schizosaccharomyces pombe (wild-type, and pbr1-6 and pbr1-8 mutants resistant to specific cell wall synthesis inhibitors), budding yeast Saccharomyces cerevisiae (wild-type and pbr1-1) and pathogenic yeast Candida albicans (wild-type, ATCC 26555, 90028 and SC5314). Osmotic protection with sorbitol attenuated the in vivo inhibition in living cells suggesting a cell wall-specific antifungal effect. Moreover, the S. pombe wild-type and mutant strains were tested for their resistant or sensitive in vitro ß(1,3)-glucan synthase (GS) activity. We found both in vivo in living cells and in vitro in the enzymatic GS assay a synergistic effect of higher sensitivity of the pbr1 mutants resistant to the specific GS inhibitors papulacandins and echinocandins. These results may provide new insights into new strategies of combined antifungal therapy of GS inhibitors directed against spontaneous mutants resistant to echinocandins.

Mannich Ketones as Possible Antimycobacterial Agents.

Twenty-three known unsaturated and fused Mannich ketones and their reduced derivatives (amino alcohols) were selected for an antituberculotic study. They were screened against several mycobacterial strains including Mycobacterium tuberculosis, M. xenopi, and M. gordonae, and minimum inhibitory concentration values were also determined using the standard antituberculotic drug isoniazid (INH) as a reference. Structure-activity relationships were also studied. The mode of action of the test compounds was investigated using transmission electron microscopy, high-performance liquid chromatography, and matrix-assisted desorption/ionization mass spectrometry. Several test substances proved to be as potent as INH, but their antimycobacterial spectra were broader than that of INH. Our findings suggest that their mode of action is probably through the inhibition of mycobacterial cell wall biosynthesis.

3-benzylidene-4-chromanones: a novel cluster of anti-tubercular agents.

In a quest for developing novel anti-tubercular agents, a series of 3-benzylidene-4-chromanones 1a-l were evaluated for growth inhibition of Mycobacterium tuberculosis H37Rv. Three promising compounds 1d, g, j emerged as the lead compounds with the IC50 and IC90 values of less than 1 µg/mL. Evaluation of the potent compounds 1d, g, j and k against Vero monkey kidney cells revealed that these compounds are far more toxic to M. tuberculosis than to Vero cells. Structure-activity relationships demonstrated that 3-benzylidene-4-chromanones are more potent against M. tuberculosis than the related 2-benzylidene cycloalkanones and the meta substituted chromanone derivatives are more active than their ortho- and para-counterparts. Some guidelines for amplifying the project are presented.

Antifungal activity of fused Mannich ketones triggers an oxidative stress response and is Cap1-dependent in Candida albicans.

We investigated the antifungal activity of fused Mannich ketone (FMK) congeners and two of their aminoalcohol derivatives. In particular, FMKs with five-membered saturated rings were shown to have minimum inhibitory concentration (MIC90s) ranging from 0.8 to 6 µg/mL toward C. albicans and the closely related C. parapsilosis and C. krusei while having reduced efficacy toward C. glabrata and almost no efficacy against Aspergillus sp. Transcript profiling of C. albicans cells exposed for 30 or 60 min to 2-(morpholinomethyl)-1-indanone, a representative FMK with a five-membered saturated ring, revealed a transcriptional response typical of oxidative stress and similar to that of a C. albicans Cap1 transcriptional activator. Consistently, C. albicans lacking the CAP1 gene was hypersensitive to this FMK, while C. albicans strains overexpressing CAP1 had decreased sensitivity to 2-(morpholinomethyl)-1-indanone. Quantitative structure-activity relationship studies revealed a correlation of antifungal potency and the energy of the lowest unoccupied molecular orbital of FMKs and unsaturated Mannich ketones thereby implicating redox cycling-mediated oxidative stress as a mechanism of action. This conclusion was further supported by the loss of antifungal activity upon conversion of representative FMKs to aminoalcohols that were unable to participate in redox cycles.

Novel dating method to distinguish between forensic and archeological human skeletal remains by bone mineralization indexes.

The fast, high-throughput distinction between paleoanthropological remains and recent forensic/clinical bone samples is of vital importance in the field of medicolegal science. In this paper, a novel screening method has been described, using the crystallinity index (C.I.) and carbonate-phosphate index (C/P) as a means to distinguish between archeological and forensic anthropological skeletal findings. According to the Fourier transform infrared spectroscopy analyses, the archeological bone samples are characterized by a range of C.I. between 2.84 and 3.78 and by low C/P values of 0.10-0.33, while the C.I. and C/P ranges of forensic skeletal remains are 2.55-3.18 and 0.38-0.88, respectively. Significant (p < 0.05) changes were observed in C/P as well as C.I. values between the groups of forensic and archeological skeletal samples. The suggested dating method needs only a few milligramms of bone tissue; thus, it can be extremely useful for distiguishing ancient and recent bone fragments.

Protective effect of the poly(ADP-ribose) polymerase inhibitor PJ34 on mitochondrial depolarization-mediated cell death in hepatocellular carcinoma cells involves attenuation of c-Jun N-terminal kinase-2 and protein kinase B/Akt activation.

BACKGROUND: 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11) was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose)-polymerase (PARP), protein kinase B/Akt and mitogen activated protein kinase (MAPK) activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. RESULTS: We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2), and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. CONCLUSIONS: These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

Amphotericin B and fluconazole susceptibility of Candida species determined by cell-chip technology.

The aim of this study was to apply the microfluidic cell-chip technology for susceptibility testing. The cell-chip technology was tested with ATCC Candida strains to determine their viability and susceptibility against amphotericin B and fluconazole. Fungal cells were labelled by Sytox Green, and measurements were carried out in the cell chips of the Agilent Bioanalyzer 2100 system. Results obtained by the chip technology were compared with the standard macrodilution method and conventional flow cytometry. Determination of minimum inhibitory concentration values was based on the differentiation between living and dead cells. The cell-chip method was found to be suitable for the detection of Candida cells, for the differentiation between dead and living cells and for the determination of amphotericin B and fluconazole susceptibility of fungal cells. The minimum inhibitory concentration values obtained by the standard macrodilution, the flow cytometry and the cell-chip method showed good correlation.

Macrophage migration inhibitory factor (MIF) tautomerase inhibitors as potential novel anti-inflammatory agents: current developments.

Macrophage migration inhibitory factor (MIF), the pro-inflammatory cytokine, first described in 1966, plays an essential role in both, innate and adaptive immune response. It has been implicated in tumour growth and angiogenesis and it exerts an antagonistic action against glucocorticoid immunosuppressive effect. Its perplexing enzymatic tautomerase activity has attracted considerable interest in the last decade. It has been suggested, that a multitude of autoimmune/inflammatory/neoplastic disease states might benefit from therapeutic measures, targeting MIF. Hence, small molecule inhibitors of MIF are relentlessly sought as potential anti-inflammatory (antitumour) agents, while a true in vivo substrate for MIF still remains unidentified. One of the first studied MIF inhibitor group was the D-dopachrome family, and its carboxyderivatives have shown good inhibitory effect, as well as the fluorosubstituted phenylpyruvic acid class. The substance ISO-1 of isoxazoline skeleton was the first small molecular inhibitor of MIF, not related to its known substrates. N-acetyl-p-benzoquinone, an acetaminophen metabolite and its synthetic derivatives exerted submicromolar IC(50) values. An acetylenic compound, the 2-oxo-4-phenyl-3-butynoate is a potent active-site-directed irreversible inhibitor of the phenyl pyruvate tautomerase activity of MIF. Some oxygen heterocycles, coumarines and chromenes, have also drawn attention as MIF inhibitors. The alpha,beta-unsaturated carbonyl compounds constitute a large novel class of MIF inhibitors. Several potent inhibitors were found among the cinnamic acid derivatives, thealpha,beta-unsaturated cyclic ketones, and the natural curcuminoids. Some other plant derived compounds were also studied. One of the latest developments in the field is the synthesis of AVP-13546, an exceptionally potent inhibitor. The structural pattern of MIF enzyme inhibitors exhibits wide variety; compounds having quite different molecular backbones belong to the MIF inhibitor family. In this paper, the separate classes of MIF inhibitors are discussed.

Antifungal unsaturated cyclic Mannich ketones and amino alcohols: study of mechanism of action.

The antifungal activity of some known unsaturated Mannich ketones and their amino alcohols has been reported and the mechanism of antifungal action has been studied. The inhibition of the fungal ergosterol, chitin, protein synthesis and pseudohypha formation was investigated. According to our results, Mannich ketones can influence the development of pseudohyphae of Candida albicans strains. In addition, they are able to induce significant changes in the protein composition of this fungal strain. Some of our Mannich ketones have shown inhibitory effect on the fungal chitin synthase enzyme. QSAR studies were also carried out.

Analysis of pathological and non-pathological human skeletal remains by FT-IR spectroscopy.

In this study, we report the chemical analyses of various non-pathological, tuberculosis and syphilis infected bone samples from different burial environments by Fourier transform infrared spectroscopy (FT-IR), in the framework of a general study of diagenesis. Dating human skeletal remains is one of the most important and yet unreliable aspects of forensic anthropology. In this paper, a new method has been suggested, using the crystallinity index and carbonate-phosphate index as a means of distinction between recent and archaeological, anthropological bone samples. Pathological bone samples were analyzed with the same method to see if changes in crystallinity interfere with the process of dating.

Design, synthesis and antiproliferative activity of some 3-benzylidene-2,3-dihydro-1-benzopyran-4-ones which display selective toxicity for malignant cells.

A series of 3-benzylidene-4-chromanones 1a-l were prepared and their cytotoxicity towards human Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 lymphoid leukemia cells were compared to the previously generated biodata in these three assays for the isosteric 2-benzylidene-1-tetralones 2a-l. Over 40% of the compounds in series 1 were more potent than their counterparts in series 2, while equipotency was noted in one-third of the comparisons made. In general the IC(50) values of 1a-l towards the human T-lymphocytes were in the low micromolar range. Molecular modelling revealed differences in shapes of representative molecules in series 1 and 2 which may contribute to the variation in cytotoxic potencies. Most of the compounds in series 1 displayed greater potencies towards HSC-2, HSC-3, HSC-4 and HL-60 neoplasms than HGF, HPC, and HPLF normal cells and were well tolerated in mice.

MIF tautomerase inhibitor potency of alpha,beta-unsaturated cyclic ketones.

The pro-inflammatory cytokine, macrophage migration inhibitory factor (MIF), is currently enjoying a renewed interest owing to its recently revealed functions. Among these its enzymatic tautomerase activity remains the most perplexing. There is a notion that some aspects of MIF signaling might involve its catalytic action. Though a true in vivo substrate for MIF has not been identified yet small molecule inhibitors of MIF are sought currently as potential anti-inflammatory agents. We have reported earlier that ketone bodies and some plant phenols feature acidic CH groups that appear to be good markers of their inhibitor potency toward MIF phenylpyruvate tautomerase. These molecules, like phenylpyruvate itself, belong to the keto-carboxylic acids or to the alpha,beta-unsaturated ketones. Some ketones of similar structure have earlier been reported to have anti-inflammatory effect. In this paper we report tautomerase inhibition by certain synthetic alpha,beta-unsaturated cyclic ketones, a novel class of small molecule MIF inhibitors.

Recent advances in antifungal agents.

New antifungals are needed in the medicine because of more aggressive and invasive diagnostic and therapeutic methods used, rapid emergence of resistant and new opportunistic fungi, increasing number of patients suffering from immunosuppressive situations e.g., AIDS, transplantation, cancer, etc. Several classes of new antifungal agents are discussed here including some new members of known families. Voriconazole, posaconazole and ravuconazole, are novel triazoles that inhibit the ergosterol synthesis. These drugs overcome problems associated with the ineffectivity of fluconazole against some Aspergillus spp. or the variable bioavailability of itraconazole. Echinocandins (caspofungin, anidulafungin and micafungin) represent a new family of antifungal agents that inhibit 1,3-beta-glucan synthase. Nikkomycins targeting the chitin synthase, show activity against Histoplasma capsulatum and Blastomyces dermatitidis. Sordarin derivatives that block the fungal protein synthesis can be considered as a promising new class of antifungal agents for the treatment of Candida and Pneumocystis infections.

Lipophilicity and antiproliferative activity profiling of 2-benzylidencycloalkanones.

High performance liquid chromatographic (HPLC) method has been developed to separate the members of a library including 24 benzylidenecycloalkanone-type structures and to characterize their lipophilicity. The experimental lipophilicity data (k) of the compounds have been compared with their calculated lipophilicity parameters (CLOGP). In general, good correlations between the measured and calculated lipophilicities have been found and these results were in good accordance with our previously data obtained in case of structurally related molecular libraries. In addition, cytotoxicity screening has been performed to determine the antiproliferative activity of these compounds. Some of the investigated compounds possessed noticeable inhibitory potential. Based on the correlation between the antiproliferative activity and experimentally determined lipophilicity of the molecules investigated, limited structural demands to obtain more potent compounds can be exhibited to support the synthetic design.

Reactions of 3-isochromanone with aromatic aldehydes--microwave assisted condensations performed on solid basic inorganic supports.

An improved Knoevenagel condensation of 3-isochromanone and aromatic aldehydes can be achieved by microwave irradiation on solid supports in the presence of various catalysts. This synthetic method offers some major advantages, especially the possibility to change the ratio of E/Z isomers.

Analysis of the vibrational spectra of new OH-containing E-4-arylmethylene-3-isochromanones and 3-arylcoumarins.

A combined experimental and theoretical approach is presented to structural characterization of fairly large, newly synthesized organic molecules in order to enhance the effectiveness of their instrumental analysis by vibrational spectroscopy. The method consists of measurement of FT-IR and Raman spectra of the reaction products and subsequent ab initio or DFT quantum mechanical calculations (prediction) of the vibrational spectra for any anticipated structural varieties of the synthesized molecules. Comparison of the measured and computed frequencies as well as the observed and simulated spectra is performed to resolve any uncertainties in identifying the reaction products. Vibrational frequency and normal mode calculations based on scaled quantum mechanical (SQM) force fields performed at the DFT/B3LYP/6-31G* level of theory are demonstrated to provide a wealth of information that have been used in this work to ascertain the molecular structure, probable conformation and H-bond properties of three new isochromanone or coumarin derivatives, namely: 3-([2'-hydroxymethyl]-phenyl)-coumarin (1), E-4-(3'-hydroxyphenylmethylene)-3-isochromanone (2), and 2-[(2'-hydroxymethyl)phenyl]-3H-naphto[2,1-b]pyran-3-one (3).