Inhibitors of type I MetAPs containing pyridine-2-carboxylic acid thiazol-2-ylamide. Part 2: SAR studies on the pyridine ring 3-substituent

Systematic SAR studies on the pyridine ring 3-substituent of PCAT, an inhibitor of EcMetAP1 and ScMetAP1, revealed that 3-substituents have different selectivity for EcMetAP1 and ScMetAP1. The selective inhibitors of type I MetAP are useful tools for investigating the detailed interactions between the enzymes and their inhibitors. In addition, these findings provide useful information for the design and discovery of more potent inhibitors of type I MetAPs.     

Identification of potent type I MetAP inhibitors by simple bioisosteric replacement. Part 1: Synthesis and preliminary SAR studies of thiazole-4-carboxylic acid thiazol-2-ylamide derivatives

A series of thiazole-4-carboxylic acid thiazol-2-ylamide (TCAT, 4) derivatives were designed and synthesized according to simple bioisosteric replacement from previously reported pyridine-2-carboxylic acid thiazol-2-ylamide (PCAT) MetAP inhibitors. The preliminary SAR studies demonstrated that these TCAT series of compounds showed different activity and selectivity compared with those of the corresponding PCAT compounds. These findings provide useful information for the design and discovery of more potent inhibitors of type I MetAPs.     

LncRNA PCAT‐1 promotes tumour growth and chemoresistance of oesophageal cancer to cisplatin

Oesophageal cancer (OC) is one of the most fatal malignancies in the world, and chemoresistance restricts the therapeutic outcome of OC. Long noncoding RNA (lncRNA) was reported to play roles in multiple cancer types. Yet, the function of lncRNA in chemoresistance of OC has not been reported. A lncRNA gene, PCAT‐1, showed higher expression in OC tissues, especially higher in secondary OC compared with normal mucosa tissues. Overexpression of PCAT‐1 increased the proliferation rate and growth of OC cells. Inhibition of PCAT‐1 decreased proliferation and growth of OC cells, and increased cisplatin chemosensitivity. In a mouse OC xenograft model, PCAT‐1 inhibition repressed OC growth in vivo. Therefore, PCAT‐1 may potentially serve as a therapeutic target for treating OC. PCAT‐1 promotes development of OC and represses the chemoresistance of OC to cisplatin, and silencing of PCAT‐1 may be a therapeutic strategy for treating OC.     

lncRNA PCAT19 promotes the proliferation of laryngocarcinoma cells via modulation of the miR-182/PDK4 axis

The mechanism of tumorigenesis has not been fully identified in laryngeal cancer, which accounts for one fourth of patents with head and neck tumors. Long noncoding RNA PCAT19 has been shown to participate in the prostate cancer progression. However, little is known about the role of PCAT19 in the tumorigenesis of laryngeal cancer. In our study, it was shown that the expression levels of PCAT19 was increased in laryngeal tumor tissues and associated with decreased overall survival. Using laryngeal cancer cells lines HEp-2 and AMC-HN-8, it was demonstrated that knockdown of PCAT19 decreased the cell proliferation, increased the mitochondrial respiration, and inhibited the glycolysis. In detail, it showed that the PDK4 expression and PDHE1α phosphorylation levels were decreased upon the PCAT19 knockdown. Further studies indicated that miR-182 functioned as the bridge between PCAT19 and PDK4, which could also regulate the cellular metabolism thus affecting the cell proliferation. Furthermore, it was shown that the PCAT19/miR-182/PDK4 axis existed and regulated cell proliferation by modulating glycolysis and mitochondrial respiration. Finally, we showed that the PCAT19 knockdown decreased the tumor growth in vivo, possibly through regulating the miR-182/PDK4 axis. In conclusion, we demonstrated that lncRNA PCAT19 promoted cell proliferation and tumorigenesis by modulating the miR-182/PDK4 axis and the metabolism balance. PCAT19 might become a promising new target for laryngeal cancer therapeutics.     

Synthesis and cytotoxic activity of benzo[c][1,7] and [1,8]phenanthrolines analogues of nitidine and fagaronine

Fagaronine and nitidine are natural benzo[c] phenanthridinium alkaloids, which display antileukemic activity. Both act as topoisomerase I and topoisomerase II inhibitors. The objective of the present study was to prepare noncharged isosters of these compounds, with replacement of the aromatic A ring by a pyridine ring, present in other topoisomerase I inhibitors. Various 7,8- and 8,9-dimethoxy and metylenedioxy benzo[c][1,7] and [1,8]phenanthrolines were readily synthesized by benzyne-mediated cyclization of the corresponding substituted N-(2-halobenzyl)-5-quinolinamines or 5-isoquinolinamines. In both series, compounds bearing oxygenated substituents at positions 8 and 9 exhibited cytotoxic properties towards L1210 murine leukemia cells, which may result from their capacities to intercalate into DNA. Topoisomerase I inhibition was observed for all active compounds.     

On the irreversible destruction of reduced nicotinamide nucleotides by hypohalous acids

Degradation of the reduced pyridine nucleotides NMNH and NADH by HOCl involves two distinct stages: a fast reaction, k = 4.2 x 10(5) M(-1) s(-1), leads to generation of stable pyridine products (Py/Cl) with a strong absorption band at 275 nm (epsilon = 12.4 x 10(3) M(-1) cm(-1) in the case of NMNH); secondarily, a subsequent reaction of HOCl, k = 3.9 x 10(3) M(-1) s(-1), leads to a complete loss of the aromatic absorption band of the pyridine ring. HOBr and HOI(I(2)) react similarly. Apparent rate constants of the primary reactions of HOX species with NMNH at pH 7.2 increase in the order HOCl (3 x 10(5) M(-1) s(-1)) < HOBr( approximately 4 x 10(6) M(-1) s(-1)) < HOI(I(2))( approximately 6.5 x 10(7) M(-1) s(-1)). HOBr reacts fast also with the primary product Py/Br, k approximately 9 x 10(5) M(-1) s(-1), while the reactions of HOI and I(2) with Py/I are slower, approximately 1.4 x 10(3) M(-1) s(-1) and >6 x 10(3) M(-1) s(-1), respectively. Halogenation of the amide group of NMN(+) by HOX species is many orders of magnitude slower than oxidation of NMNH. Taurine inhibits HOCl-induced oxidation of NADH, but HOBr-induced oxidation is not inhibited because the taurine monobromamine rapidly oxidizes NADH, and oxidation by HOI(I(2)) is not inhibited because taurine is inert toward HOI(I(2)). Also sulfur compounds (GSH, GSSG, and methionine) are less efficient in protecting NADH against oxidation by HOBr and HOI(I(2)) than against oxidation by HOCl. The results suggest that reactions of HOBr and HOI(I(2)) in a cellular environment are much more selectively directed toward irreversible oxidation of NADH than reactions of HOCl. It is noteworthy that the rather inert N-chloramines react with iodide to generate HOI(I(2)), i.e., the most reactive and selective oxidant of reduced pyridine nucleotides. NMR investigations show that the primary stable products of the reaction between NMNH and HOCl are various isomeric chlorohydrins originating from a nonstereospecific electrophilic addition of HOCl to the C5&dbond;C6 double bond of the pyridine ring. The primary products (Py/X) of NMNH all exhibit similar absorption bands around 275 nm and are hence likely to result from analogous addition of HOX to the C5&dbond;C6 bond of the pyridine ring. Since the Py/X species are stable and inert toward endogeneous reductants like ascorbate and GSH, they may generally be useful markers for assessing the contribution of hypohalous acids to inflammatory injury.     

Synthesis and structural analysis of the copper(II) complexes of N2-(2-pyridylmethyl)-2-pyridinecarboxamide

Previous investigations of the potential of metal-organic compounds as inhibitors of human immunodeficiency virus type I protease (HIV-1 PR) showed that the copper(II) complex diaqua [bis(2-pyridylcarbonyl)amido] copper(II) nitrate dihydrate and the complex bis[N2-(2,3,6-trimethoxybenzyl)-4-2-pyridinecarboxamide] copper(II) behaved as inhibitors of HIV-1 PR. In a search for similar readily accessible ligands, we synthesised and studied the structural properties of N2-(2-pyridylmethyl)-2-pyridinecarboxamide (L) copper(II) complexes. Three different crystal structures were obtained. Two were found to contain ligand L simultaneously in a tridentate and bidentate conformation [Cu(L(tri)L(bi))]. The other contained two symmetry-related ligands, coordinated through the pyridine nitrogen and the amide oxygen atoms [Cu(L(bi))(2)]. A search of the Cambridge Structural Database indicated that L(tri) resulting from nitrogen bound amide hydrogen metal substitution is favoured over chelation through the amide oxygen atom. In our case, we calculated that the conformation of L(tri) is 11 kcal/mol more favourable than that of L(bi). ESI-MS experiments showed that the Cu(L(bi))(2) structure could not be observed in solution, while Cu(L(tri)L(bi))-related complexes were indeed present. The lack of protease inhibition of the pyridine carboxamide copper(II) complexes was explained by the fact that the Cu(L(bi)L(tri)) complex could not fit into the HIV-1 active site.     

Synthesis of hybrid molecules of caffeine and eudistomin D and its effects on adenosine receptors

Four hybrid molecules (1 and 12-14) of caffeine and eudistomin D, a beta-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and selectivity for adenosine receptors A(1), A(2A), and A(3) were examined. It was found that all the compounds showed better potency as adenosine receptor ligands as compared with caffeine. Among them, a compound (13) possessing a nitrogen at the delta-position of the pyridine ring (delta-N type) showed the most potent affinity for adenosine receptor A(3) subtype, while N-methylation (14) of a pyrrole ring in 13 significantly lowered the potency as adenosine receptor ligands. Compounds (1 and 12) having a nitrogen at the beta-position of the pyridine ring (beta-N type) showed lower affinity than the corresponding delta-N type compounds (13 and 14), while compounds (10, 11, and 17) lacking a pyrrole ring between the pyridine and pyrimidine rings exhibited almost no affinity to the adenosine receptor subtypes examined.     

Antimicrobial activity of some 2-amino-5-subsituted pyridine derivatives

A series of 2-amino-5-substituted pyridine derivatives were prepared and evaluated against phytopathogenic fungi and bacteria under laboratory conditions. Position 4 on the pyridine ring has notable fungicidal and bactericidal activity, greater than position 3 and/or position 6. Reaction of 1-hydroxymethyl benzotriazole with the amino group of the pyridine ring gave better fungicidal activity than substitution on the carbon of the pyridine ring (compound 4 versus 1c). Replacing the benzotriazole moiety with thiophenol exhibited the strongest fungicidal and bactericidal activity in this series (compound 3).     

Quaternary salts of 4,3' and 4,4' bis-pyridinium monooximes. Part 2: synthesis and biological activity

In continuation of our investigations of unsymmetrical bisquaternary monooximes, we synthesized four new series of compounds bridged by hexyl, heptyl, octyl and nonyl groups. All eight monooximes viz., dibromides of 1-(4-hydroxyiminomethylpyridinium)6-(3/4-carbamoylpyridinium)hexane, 1-(4-hydroxyiminomethylpyridinium)-7-(3/4-carbamoylpyridinium)heptane, 1-(4-hydroxyiminomethylpyridinium)-8-(3/4-carbamoylpyridinium)octane, 1-(4-hydroxyiminomethylpyridinium)-9-(3/4-carbamoylpyridinium)nonane as well as the corresponding bis-oximes were synthesized and characterized by spectral data. Their ability to reactivate tetraethylpyrophosphate (TEPP) inhibited mouse total brain cholinesterase was investigated and compared with the conventional oxime 2-pyridinealdoxime chloride (2-PAM). Mouse brain homogenate was used as the source of acetylcholinesterase. Among all the compounds, tested the compound with the hexylene bridge (6b) and a 3-carbamoyl group on the second pyridine ring was found to be the most active acetylcholinesterase reactivator (72%) which is greater than that of 2-PAM (56%). However, the activity was reversed; as the chain length increased from a heptylene to a nonylene bridge, they potentiated the inhibitory effect of TEPP rather than reactivation. It is interesting to note that compound 6b with a carbamoyl group at the 3rd position of the pyridine ring showed dose dependent reactivation whereas the corresponding compound 6a with the carbamoyl group present at the 4th position of the pyridine ring showed reactivation at lower concentration (30 microM) and potentiation of TEPP inhibition at higher concentrations (100 and 300 microM).     

Inhibition of copper amine oxidases by pyridine-derived aldoximes and ketoximes

The reactions of pea diamine oxidase (PSAO) and 2-phenylethylamine oxidase from Arthrobacter globiformis (AGAO) with pyridine-derived oximes were studied. Pyridine carbaldoximes and alkyl pyridyl ketoximes act as strong non-competitive inhibitors of the enzymes. The inhibition constants K(i) of these compounds vary between 10(-4) and 10(-5) M, for AGAO and some of the studied oximes were found even micromolar K(i) values. The presence of pyridine moiety in the studied compounds has remarkable influence on the inhibition potency. Elementary oximes lacking the heterocyclic ring, i.e., aliphatic (acetone oxime), alicyclic (cyclohexanone oxime) and aromatic (benzaldoxime), are considerably weaker non-competitive inhibitors (K(i) similar to 10(-3) or 10(-2) M). The position of the pyridine ring substitution by -C(R)=NOH group does not play a significant role for the inhibition potency of the studied oxime compounds. If the pyridine nitrogen is quaternised (in hydroxyiminomethyl-1-methylpyridinium iodides), the compound looses its inhibitory properties. Extended length of alkyl substituents on the ketoxime group of alkyl pyridyl ketoximes increases the K(i) value. The enzyme-bound copper represents one of possible target sites for pyridine-derived oxime inhibitors. The addition of an alkyl pyridyl ketoxime or a pyridine carbaldoxime to a native PSAO sample perturbs the absorption spectrum of the enzyme (by an absorption increase in the region 300-400 nm) that is not observed in the spectrum of reacted PSAO apoenzyme. However, an additional formation of hydrogen bonds with amino acid side-chains at the active site should be considered, namely for 3- and 4-substituted pyridine derivatives.     

Combined use of a specific probe and PCAT medium to study Burkholderia in soil

Due to its pathogenic traits and agricultural benefits, there is some challenge in detecting Burkholderia in the soil environment. In this perspective, an existing semi-selective medium, (PCAT), was combined with a Burkholderia specific molecular probe. Using the complete 16S rRNA sequences of all available Burkholderia species type strains, we selected the following sequence: 5'-ACCCTCTGTTCCGACCATTGTATGA-3'. The probe was validated against GenBank sequences, with dot blots and colony hybridization tests. A diversity study of all strains growing on a PCAT plate after plating a soil dilution (75 strains) was carried out with ARDRA analysis and colony hybridization tests. All the hybridizing strains belonged to genus Burkholderia. The major type of non-hybridizing isolates belonged to Pseudomonas (16S rRNA sequencing). Both tools were combined to compare the Burkholderia populations in a rhizosphere (maize) and a non-rhizosphere soil. Based on hybridizing PCAT isolates, we were able to show an increase in Burkholderia populations in the maize rhizosphere. This genus represented 2% and 16% of the total cultivable microflora in the non-rhizosphere and rhizosphere soils, respectively. Although PCAT was shown not to be appropriate to routinely enumerate Burkholderia populations in soil, it allowed environmental investigations at the genus level, when combined with a molecular specific probe.     

Synthesis of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives and evaluation of their topoisomerase I and II inhibitory activity,cytotoxicity, and structure–activity relationship

A series of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives were designed, synthesized, and evaluated for their topoisomerase I and II inhibition and cytotoxic activity against several human cancer cell lines. Compounds 10–19 showed moderate topoisomerase I and II inhibitory activity and 20–29 showed significant topoisomerase II inhibitory activity. Structure–activity relationship study revealed that 4-(5-chlorofuran-2-yl)-2-(thiophen-3-yl) moiety has an important role in displaying topoisomerase II inhibition.     

Type I collagen stabilization of matrix metalloproteinase-2

The activity of matrix metalloproteinase-2 (MMP-2) is regulated stringently on the posttranslational level. MMP-2 efficiently undergoes autolysis into inactive polypeptides in vitro, prompting the hypothesis that MMP-2 autolysis may function as an alternative mechanism for posttranslational control of MMP-2 in vivo. Moreover, MMP-2 binds to intact type I collagen fibrils; however, the functional consequences of this interaction have not been fully elucidated. To test the hypothesis that MMP-2 binding to type I collagen functions as a positive regulator of MMP-2 proteolytic potential, the effect of type I collagen on MMP-2 activity, inhibition by tissue inhibitor of metalloproteinase-2 (TIMP-2), and enzyme stability was examined. Here, we report that purified MMP-2 binds but does not cleave intact type I collagen. The presence of type I collagen affects neither enzymatic activity against a quenched fluorescent peptide substrate nor the kinetics of inhibition by TIMP-2. However, MMP-2 is stabilized from autolysis in the presence of type I collagen, but not by elastin, fibrinogen, or laminin. These data provide biochemical evidence that MMP-2 exosite interactions with type I collagen may function in the posttranslational control of MMP-2 activity by reducing the rate of autolytic inactivation.     

Synthesis and biological evaluation of aroylguanidines related to amiloride as inhibitors of the human platelet Na(+)/H(+) exchanger

Pyridine and benzene bioisosteres of amiloride were synthesized and evaluated for their inhibitory potency against the sodium-hydrogen exchanger (NHE) involved in intracellular pH regulation. The inhibition of NHE was determined by using the platelet swelling assay (PSA) in which the swelling of human platelets was induced by their incubation in an acid buffer (pH 6.7). Additionally, the inhibitory potency of the most active compounds was assessed by measuring the inhibition of the EIPA-sensitive (22)Na(+) uptake (UIA) by human platelets after intracellular acidosis. The results indicated that several benzene derivatives and compounds bearing an carbonylguanidine moiety in the meta position of the pyridine nitrogen were much more potent than amiloride (PSA:IC(50)=43.5 microM; UIA:IC(50)=100.1 microM), but less than EIPA, a pyrazine NHE inhibitor (PSA:IC(50)=0.08 microM; UIA:IC(50)=0.5 microM). In both biological assays (2-amino-5-bromo-pyridine-3-carbonyl)guanidine (32) was the most active molecule (PSA: IC(50)=0.8 microM, UIA : IC(50)=0.8 microM). Our investigations demonstrated that the replacement of the pyrazine ring of amiloride by a pyridine or a phenyl ring improved the NHE inhibitory potency (phenyl >pyridine >pyrazine).     

LncRNA PCAT6 promotes tumor progression in osteosarcoma via activation of TGF-β pathway by sponging miR-185-5p

Long noncoding RNAs (lncRNAs) play crucial roles in tumor development of osteosarcoma (OS). LncRNA PCAT6 was involved in the progression of multiple human cancers. However, the biological function of PCAT6 in OS remains largely unknown. We found that PCAT6 was elevated in OS tissues relative to that in their adjacent normal tissues. The upregulation of PCAT6 was positively associated with metastasis status and advanced stages and predicted poor overall and progression-free survivals in patients with OS. Functionally, silencing PCAT6 inhibited the proliferation, migration and invasion abilities of OS cells. Mechanistically, PCAT6, acting as a competitive endogenous RNA, upregulated expression of TGFBR1 and TGFBR2 to activate TGF-β pathway via sponging miR-185–5p. This study uncovers a novel underlying molecular mechanism of PCAT6-miR-185-5p-TGFBR1/2-TGF-β signaling axis in promoting tumor progression in OS, which indicates that PCAT6 may serve as a promising prognostic factor and therapeutic target again OS.     

Himbacine derived thrombin receptor (PAR-1) antagonists: structure-activity relationship of the lactone ring

The structure-activity relationship (SAR) of the lactone ring of himbacine derived thrombin receptor (PAR-1) antagonists (e.g., 2-5) is described. The effect of the lactone carbonyl group on binding to PAR-1 is dependent on the substitution pattern of the pyridine ring. A stereoselective intramolecular Michael addition reaction to the vinyl pyridine group was observed for these pyridine analogs of himbacine in basic conditions at elevated temperature.     

Inhibition studies of Brucella suis β-carbonic anhydrases with a series of 4-substituted pyridine-3-sulphonamides

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Brucella suis, BsuCA1 and BsuCA2, were investigated for their inhibition profile with a series of pyridine-3-sulphonamide derivatives incorporating 4-hetaryl moieties. BsuCA1 was effectively inhibited by these sulphonamides with inhibition constants ranging between 34 and 624?nM. BsuCA2 was less sensitive to these inhibitors, with K_Is in the range of 62?nM -?>?10?µM. The nature of the 4-substituent present on the pyridine ring was the main factor influencing the inhibitory profile against both isoforms, with 4-halogenophenylpiperazin-1-yl and 3,4,5-trisubstituted-pyrazol-1-yl derivatives showing the most effective inhibition. Some of these sulphonamides were most effective bacterial CA than human (h) CA I and II inhibitors, making them selective for the prokaryotic enzymes. Investigation of bacterial CA inhibitors may be relevant for finding antibiotics with a new mechanism of action compared to the clinically used agents for which substantial drug resistance emerged.     

KIR2DL5 can inhibit human NK cell activation via recruitment of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2)

Human NK cells use class I MHC-binding inhibitory receptors, such as the killer cell Ig-like receptor (KIR) family, to discriminate between normal and abnormal cells. Some tumors and virus-infected cells down-regulate class I MHC and thereby become targets of NK cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatases, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, to two phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). KIR2DL5 is a type II member of the KIR2D family with an atypical extracellular domain and an intracytoplasmic domain containing one typical ITIM and one atypical ITIM sequence. Although KIR2DL5 structure is expressed by approximately 50% of humans and is conserved among primate species, its function has not been determined. In the present study, we directly compared functional and biochemical properties of KIR2DL5, KIR3DL1 (a type I KIR with two ITIMs), and KIR2DL4 (the only other type II KIR, which has a single ITIM) in a human NK-like cell line. Our results show that KIR2DL5 is an inhibitory receptor that can recruit both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than KIR3DL1. Interestingly, inhibition of NK cell cytotoxicity by KIR2DL5 was blocked by dominant-negative SHP-2, but not dominant-negative SHP-1, whereas both dominant-negative phosphatases can block inhibition by KIR3DL1. Therefore, the cytoplasmic domains of type II KIRs (2DL4 and 2DL5) exhibit distinct inhibitory capacities when compared with type I KIRs (3DL1), due to alterations in the canonical ITIM sequences.