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PubMed2002

Influence of compound structure on affinity, sequence selectivity, and mode of binding to DNA for unfused aromatic dications related to furamidine.

In the course of a program aimed at developing sequence-specific gene-regulatory small organic molecules, we have investigated the DNA interactions of a new series of nine diphenylfuran dications related to the antiparasitic drug furamidine (DB75). Two types of structural modifications were tested: the terminal amidine groups of DB75 were shifted from the para to the meta position, and the amidines were replaced with imidazoline or dimethyl-imidazoline groups, to test the importance of both the position and nature of positively charged groups on DNA interactions. The interactions of these compounds with DNA and oligonucleotides were studied by a combination of biochemical and biophysical techniques. Absorption and CD measurements suggested that the drugs bind differently to AT and GC sequences in DNA. The para-para dications, like DB75, bind into the minor groove of poly(dAT)(2) and intercalate between the base pairs of poly(dGC)(2), as revealed by electric linear dichroism experiments. In contrast, the meta-meta compounds exhibit a high tendency to intercalate into DNA whatever the target sequence. The lack of sequence selectivity of the meta-meta compounds containing amidines or dimethyl-imidazoline groups was also evident from DNase I footprinting and surface plasmon resonance (SPR) experiments. Accurate binding measurements using the BIAcore SPR method revealed that all nine compounds bind with similar affinity to an immobilized GC sequence DNA hairpin but exhibit very distinct affinities for the corresponding AT hairpin oligonucleotide. The minor groove-binding para-para compounds have a high specificity for AT sequences. The biophysical data clearly indicate that shifting the cationic substituents from the para to the meta position results in a loss of specificity and change in binding mode. The strong AT selectivity of the para-para compounds was independently confirmed by DNase I footprinting experiments performed with a range of DNA restrictions fragments. In terms of AT selectivity, the compounds rank in the order para-para > para-meta > meta-meta. The para dications bind preferentially to sequences containing four contiguous AT base pairs. Additional footprinting experiments with substrates containing the 16 possible [A.T](4) blocks indicated that the presence of a TpA step within an [A.T] (4) block generally reduces the extent of binding. The diverse methods, from footprinting to SPR to dichroism, provide a consistent model for the interactions of the diphenylfuran dications with DNA of different sequences. Altogether, the results attest unequivocally that the binding mode for unfused aromatic cations can change completely depending on substituent position and DNA sequence. These data provide a rationale to explain the relationships between sequence selectivity and mode of binding to DNA for unfused aromatic dications related to furamidine.

Amidines↗
PubMed2007

Solution behaviour and biological activity of bisamidine complexes of platinum(II).

A series of platinum(II) amidine complexes were previously prepared with the aim of obtaining a new class of platinum-based antitumour drugs. This series includes compounds of the type cis--[PtCl2{Z-HN=C(NHMe)Me}2] and trans-[PtCl2{Z-HN=C(NHMe)Me}2] (1, 2), cis-[PtCl2{E-HN=C(NMe2)Me}2] and trans-[PtCl2{E-HN=C(NMe2)Me}2] (3, 4), cis-[PtCl2{Z-HN=C(NHMe)Ph}2] and trans-[PtCl2{Z-HN=C(NHMe)Ph}2] (5, 6), and cis-[PtCl2{HN=C(NMe2)Ph}2] and trans-[PtCl2{HN=C(NMe2)Ph}2] (7, 8). The reactions with dimethyl sulfoxide were studied for complexes 5-8; the formation of cationic species containing coordinated dimethyl sulfoxide was demonstrated by NMR experiments and electrospray ionization mass spectrometry. In this work, the amidine platinum(II) complexes were tested for their in vitro cytotoxicity on a panel of various human cancer cell lines. The results indicate that the benzamidine complex 8 was the most effective derivative also circumventing acquired cisplatin resistance as demonstrated by chemosensitivity tests performed on cisplatin-sensitive and cisplatin-resistant cell lines. The studies concerning the cellular DNA damage on both parental chemosensitive and resistant sublines suggest for the new trans-amidine complex a different mechanism of action compared with that exhibited by cisplatin.

Amidines↗
PubMed2004

Theoretical prediction of gas-phase infrared spectra of imidazo[1,2-a]pyrazinediones and imidazo[1,2-a]imidazo[1,2-d]pyrazinediones derived from glycine.

Imidazo[1,2-a]pyrazine-3,6-diones and imidazo[1,2-a]imidazo[1,2-d]pyrazine-3,8-diones can be produced by pyrolysis of simple amino acids. While such bicyclic and tricyclic amidines were detected and characterized by IR spectroscopy for some alpha-substituted amino acids, the parent systems composed of glycine fragments are unknown up to now. IR spectra for five amidines derived from glycine were calculated by using different semi-empirical (PM3, AM1, MNDO and MINDO/3), HF, and hybrid DFT (B3LYP, B3P86 and B3PW91) methods in conjunction with 6-31G(d) basis set (for HF and DFT). Vibration frequencies in the experimental IR spectra were predicted based upon the B3LYP data, by correcting the calculated wavenumbers by a scaling factor of 0.959. The behavior of most characteristic bands (nu(CX), nu(NH), etc.) and their shifts with respect to such bands in the spectra of alanine and alpha-aminoisobutyric acid derivatives studied before, are discussed. Performance of the semi-empirical methods was tested, bearing in mind possible future needs for IR spectra predictions for larger molecular systems of similar chemical nature; the use of MINDO/3 and MNDO is recommended. A basis set effect on the B3LYP fundamental vibration frequencies for hexahydroimidazo[1,2-a]pyrazine-3,6-dione was studied by varying Pople basis sets from minimal STO-3G to 6-311++G(d, p). No significant improvements were found beyond the 6-31G(d) basis set, which thus can be recommended to predict IR spectra for the amidines and similar molecules.

Amidines↗
PubMed1990

Structural analysis of d(GCAATTGC)2 and its complex with berenil by nuclear magnetic resonance spectroscopy.

The structures of d(GCAATTGC)2 and its complex with berenil in solution were analyzed by two-dimensional 1H NMR spectroscopy. Intra- and internucleotide nuclear Overhauser effect (NOE) connectivities demonstrate that the octanucleotide duplex is primarily in the B conformation. Binding with berenil stabilizes the duplex with respect to thermal denaturation by about 10 degrees C, based on the appearance of the imino proton signals. The berenil-d(GCAATTGC)2 system is in fast exchange on the NMR time scale. The two-dimensional NMR data reveal that berenil binds in the minor groove of d(GCAATTGC)2. The aromatic drug protons are placed within 5 A of the H2 proton of both adenines, the H1', H5', and H5" of both thymidines, and the H4', H5', and H5" of the internal guanosine. The amidine protons on berenil are also close to the H2 proton of both adenines. The duplex retains an overall B conformation in the complex with berenil. At 18 degrees C, NOE contacts at longer mixing times indicate the presence of end-to-end association both in the duplex alone and also in its complex with berenil. These intermolecular contacts either vanished or diminished substantially at 45 degrees C. Two molecular models are proposed for the berenil-(GCAATTGC)2 complex; one has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen, O2, of the external thymines, and the other has hydrogen bonds between the drug amidine protons and the purine nitrogen, N3, of the internal adenines. Quantitative analysis of the NOE data favors the second model.

Amidines↗
PubMed2002

Crystal structures of two potent nonamidine inhibitors bound to factor Xa.

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.

Amidines↗
PubMed2002

Design, synthesis, and activity of a novel series of factor Xa inhibitors: optimization of arylamidine groups.

A novel series of diaryloxypyridines have been designed as selective nanomolar factor Xa (fXa) inhibitors for use as anticoagulants. In this paper, we describe our efforts to identify an additional interaction and a replacement for the distal amidine group that binds in the S3/S4 pocket of fXa. Introduction of a hydroxyl group para to the proximal amidine group increases the potency vs fXa by 1-2 orders of magnitude, which is the result of a hydrogen bond to Ser195 of the catalytic triad. A methyl imidazoline and a dimethylamide are good alternatives for the second amidine. These substitutions have increased the selectivity vs the related serine proteases trypsin and thrombin. The synthesis, in vitro activity, and hypothetical modes of binding to fXa based on trypsin crystallographic data are outlined.

Amidines↗
PubMedDate not supplied

Quantitation using enhanced signal tags: a technique for comparative proteomics.

Differential amidination of N-termini and lysine residues provides the basis for a novel approach to protein quantitation using MALDI mass spectrometry. Because the amidination of lysines increases their basicity and therefore MALDI ionization yields, the method is called quantitation using enhanced signal tags (QUEST). Amidine labels differ by methylene groups, leading to 14 Da mass differentials. The utility of QUEST is demonstrated while analyzing the digests of two model proteins using MALDI-TOF mass spectrometry.

Amidines↗
PubMed1998

Behavior of amino acids when volatilized in the presence of silica gel and pulverized basaltic lava.

To evaluate the types of amino acid thermal transformations caused by silicate materials, we studied the volatilization products of Aib, L-Ala, L-Val and L-Leu under temperatures of up to 270 degrees C in the presence of silica gel as a model catalyst and pulverized basaltic lava samples. It was found that silica gel catalyzes nearly quantitative condensation of amino acids, where piperazinediones are the major products, whereas lava samples have much lower catalytic efficiency. In addition bicyclic and tricyclic amidines and several products of their subsequent thermal decomposition have been identified using the coupled technique of GC-FTIR-MS and HPLC-PB-MS, with auxiliary computer simulation of IR spectra and NMR spectroscopy. The decomposition is due to dehydrogenation, elimination of the alkyl substituents and dehydration as well as cleavage of the bicyclic ring system. The imidazole ring appears to be more resistant to thermal decomposition as compared to the pyperazine moiety, giving rise to the formation of different substituted imidazolones. The amidines were found to hydrolyze under treatment with concentrated HCl, releasing the starting amino acids and thus behaving as amino acid anhydrides. The thermal transformations cause significant racemization of amino acid residues. Based on our observations, the formation of amidine-type products is suggested to be rather common in the high-temperature experiments on amino acid condensation.

Amidines↗
PubMed1998

Stability of alkoxycarbonylamidine prodrugs.

PURPOSE: Alkoxycarbonylamidine prodrug modification was used to mask the positively-charged amidine moiety of an Arg-Gly-Asp peptidomimetic and enhance oral bioavailability. The aqueous stability of ethoxycarbonylamidine (ECA), ethanethiocarbonylamidine (ETCA) and phenoxycarbonylamidine (PCA) prodrugs was examined. METHODS: Degradation was followed by RP-HPLC and rate constants were determined from a degradation scheme defined by product analysis. RESULTS: ECA gave a pH of maximum stability at pH approximately 7 and was independent of pH below pH approximately 4. A novel degradation pathway of ECA, conversion to ethoxycarbonyl- aminocarbonyl, was observed below pH 7. The relative rates below pH 7 were ECA approximately ETCA < PCA, in the same order of decreasing pKa of the conjugate acid of the substituted amidino group. Base-catalyzed cleavage of ECA to yield the amidine derivative gave the relative rates ECA < ETCA < PCA, in agreement with the decreasing pKa of the leaving groups. CONCLUSIONS: The observed rate constants at all pHs were small enough that only 5-30% (depending on the substituent) undesirable degradation is predicted during transit time of the gut. The spontaneous post-absorptive conversion to the amidine drugs at neutral pH is predicted to be 6x greater for the PCA than the ECA prodrugs.

Amidines↗
PubMed2004

Protective effects of benzisothiazolylamidines on IL-1 beta induced alterations in human articular chondrocyte metabolism.

The in vitro effects on human articular chondrocytes were evaluated for a series of N-benzo[d]isothiazol-3-yl-amidines, bearing as pharmacophoric moiety the nonacidic isosteric nitrogen analogue of the carboxylic group. The aim was to verify their effectiveness in articular diseases, such as arthritis. Human chondrocytes were treated with IL-1beta in the presence of a series of N-benzo[d]isothiazol-3-yl-amidines at a concentration of 100 microg/mL. After 120 h, the amount of glycosaminoglycans (GAGs), the production of nitric oxide (NO) and the inhibition of metalloproteinases (MMP-3) and prostaglandin (PGE2) were measured. Nitrite production induced by inflammatory IL-1beta on cultured chondrocytes was inhibited by the N-benzo[d]isothiazol-3-yl-amidines tested, in particular by N-benzo[d]isothiazol-3-yl-benzamidine, which was the most active. Concerning the effects on GAGs, all the tested benzisothiazolylamidines, and in particular N-benzo[d]isothiazol-3-yl-acetamidine, prevented the depletion of proteoglycan induced by IL-1beta. Inhibitory effects of the tested compounds on MMP-3 activity and on PGE2 production were also observed.

Amidines↗
PubMed2005

Characterization and partial purification of the rat and human enzyme systems active in the reduction of N-hydroxymelagatran and benzamidoxime.

The enzymic basis for intracellular reduction of N-hydroxylated amidines to their corresponding amidines, and hydroxylamines to their corresponding amines, is unknown. The hydroxylated amidines can be used as prodrug moieties, and an understanding of the enzyme system active in the reduction can contribute to more efficient drug development. In this study, we examined the properties of this enzyme system using benzamidoxime and N-hydroxymelagatran as substrates. In rats and humans, the hepatic enzyme system was localized in mitochondria as well as in microsomes, using preferably NADH as cofactor. Potassium cyanide, N-methylhydroxylamine, p-hydroxymercuribenzoate, and desferrioxamine were efficient inhibitors, whereas typical cytochrome P450 (P450) inhibitors were ineffective. In rats, the highest specific activity was found in liver, adipose tissue, and kidneys, whereas in humans, the specific activity in the preparations of adipose tissue examined was lower. A sex difference was observed in rat liver, where 4-fold higher activity was seen in microsomes from female rats. No gender differences were present in any other tissue investigated. Partial purification of the hepatic system was achieved using polyethylene glycol fractionation followed by Octyl Sepharose chromatography at low detergent concentrations, whereas the enzyme was denatured after complete solubilization. The unique appearance of the enzyme activity in adipose tissue, together with the cyanide sensitivity and the failure of typical P450 inhibitors to impede the reaction, indicates that the enzyme system active in reduction of benzamidoxime and N-hydroxymelagatran formation is not of cytochrome P450 origin, but likely consists of an NADH-dependent electron transfer chain with a cyanide-sensitive protein as the terminal component.

Adipose Tissue↗
PubMed2006

Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells.

Cationic liposomes can efficiently carry nucleic acids into mammalian cells. This property is tightly connected with their ability to fuse with negatively charged natural membranes (i.e. the plasma membrane and endosomal membrane). We used FRET to monitor and compare the efficiency of lipid mixing of two liposomal preparations--one of short-chained diC14-amidine and one of long-chained unsaturated DOTAP--with the plasma membrane of HeLa cells. The diC14-amidine liposomes displayed a much higher susceptibility to lipid mixing with the target membranes. They disrupted the membrane integrity of the HeLa cells, as detected using the propidium iodide permeabilization test. Morphological changes were transient and essentially did not affect the viability of the HeLa cells. The diC14-amidine liposomes were much more effective at either inducing lipid mixing or facilitating transfection.

Amidines↗
PubMed1976

Exposure of free amino groups in the coupling factor of energized spinach chloroplasts.

Spinach chloroplasts were incubated in the dark with methyl acetimidate in order to amidinate and thereby protect free amino groups. An energy-dependent attack on the coupling factor (CF1) of these amidinated chloroplasts by trinitrobenzenesulfonate was apparent in a second reaction, as long as the reagent was applied in the light or after an acid-base transition. Trinitrophenyl residues approached one each on the alpha and beta subunits, and two to three on the gamma subunit polypeptide of CF1. Accompanying trinitrophenylation was an inhibition of the ATPase activity of CF1 due to a major decrease in the affinity for ATP; however, neither the maximal ATPase rate nor the ability of the protein to serve as a coupling factor for EDTA-extracted chloroplasts was affected. Trinitrophenylation and consequent inhibition of ATPase were 50% prevented by the presence of phosphate, or ADP, or ATP during exposure to trinitrobenzenesulfonate. The protective effects of adenylates were additive with those of phosphate. The ratio of trinitrophenyl groups on the three subunits concerned was the same whether phosphate or ATP was providing 50% protection, or whether neither was present. It is inferred that a conformational change occurs in the amidinated coupling factor when a proton activity gradient is placed across the membranes, and effective ligands tend to prevent the resulting exposure of free amino groups from a previously hidden location.

Adenosine Triphosphatases↗
PubMed2004

Odd-even chain length-dependent order in pH-switchable self-assembled layers.

The reversible self-assembly of a series of bipolar amphiphiles, alpha,omega-bis(3- or 4-amidinophenoxy)alkanes (chain length n = 5-12), on mercaptoalkanoic acid-functionalized gold surfaces (chain length n = 10, 11, 14, 15) has been studied by in-situ ellipsometry, IR reflection absorption spectroscopy (IRAS), and atomic force microscopy (AFM). The layer order, amphiphile orientation, and tendency to form bilayers depends on the position of the amidine substituent, the alkyl chain length of both the amidine amphiphile and the underlying acid self-assembled monolayer (SAM), and whether the amidine alkyl chain contained an even or odd number of methylene groups. Thus, para-substituted bisbenzamidines containing more than six methylene groups (n>6) and with an odd number (n = 7, 9, 11) tended to form bilayered structures, whereas those containing an even number formed monolayers when adsorbed on SAMs of the long-chain acids (n = 14, 15). This behavior also correlated with the average tilt angle of the benzene moieties of the amphiphiles, as estimated by IRAS. The odd-numbered chains gave lower tilt angles than the even-numbered ones, and a possible model that accounts for these results is proposed. IRAS also revealed a higher order of the odd-numbered chains and an increasing hydrogen-bonding contribution with increasing chain length. Additional evidence for the proposed bilayered assemblies and their reversibility was obtained by AFM. Images obtained from the assembly of decamidine on a SAM of mercaptohexadecanoic acid in a pH 9 borate buffer revealed domains of similar size to that of the underlying acid SAM (20-30 nm), but less densely packed. By acidifying the solution, the second layer was destabilized and a very smooth layer with few defects appeared. Further acidification to pH 3 also destabilized the first layer.

Journal Article↗
PubMed1997

A cationic lipid for rapid and efficient delivery of plasmid DNA into mammalian cells.

Cationic lipids are widely used for gene transfer into cultured eukaryotic cells. However, lipids with potent transfection activity are often associated with high levels of cytotoxicity, and also require serum-free conditions for optimal performance. These characteristics in many cases result in unsatisfactory transfection efficiency. In this report, we describe a new cationic amphiphile, N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (Amidine). Amidine requires only 1-2 hour incubation intervals to produce maximal transfection efficiency, and can transfect cells in the presence of serum. Such characteristics significantly minimize cytotoxicity, and also provide time flexibility for researchers. We routinely obtain over 80% transfection efficiency as evidenced by use of an enhanced green fluorescence protein (EGFP) as the reporter. These studies demonstrate the utility of Amidine for rapid and efficient transfection of mammalian cells.

Animals↗
PubMed2000

A thermodynamic and structural analysis of DNA minor-groove complex formation.

As part of an effort to develop a better understanding of the structural and thermodynamic principles of DNA minor groove recognition, we have investigated complexes of three diphenylfuran dications with the d(CGCGAATTCGCG)(2) duplex. The parent compound, furamidine (DB75), has two amidine substituents while DB244 has cyclopentyl amidine substituents and DB226 has 3-pentyl amidines. The structure for the DB244-DNA complex is reported here and is compared to the structure of the DB75 complex. Crystals were not obtained with DB226 but information from the DB75 and DB244 structures as well as previous NMR results on DB226 indicate that all three compounds bind in the minor groove at the AATT site of the duplex. DB244 and DB75 penetrate to the floor of the groove and form hydrogen bonds with T8 on one strand and T20 on the opposite strand while DB226 forms a complex with fewer interactions. Binding studies by surface plasmon resonance (SPR) yield -delta G degrees values in the order DB244>DB75>DB226 that are relatively constant with temperature. The equilibrium binding constants for DB244 are 10-20 times greater than that for DB226. Isothermal titration calorimetric (ITC) experiments indicate that, in contrast to delta G degrees, delta H degrees varies considerably with temperature to yield large negative delta Cp degrees values. The thermodynamic results, analyzed in terms of structures of the DNA complexes, provide an explanation of why DB244 binds more strongly to DNA than DB75, while DB266 binds more weakly. All three compounds have a major contribution to binding from hydrophobic interactions but the hydrophobic term is most favorable for DB244. DB244 also has strong contributions from molecular interactions in its DNA complex and all of these factors combine to give it the largest-delta G degrees for binding. Although the factors that influence the energetics of minor groove interactions are varied and complex, results from the literature coupled with those on the furan derivatives indicate that there are some common characteristics for minor groove recognition by unfused heterocyclic cations that can be used in molecular design.

Base Sequence↗
PubMed2006

Design, synthesis, and biological testing of potential heme-coordinating nitric oxide synthase inhibitors.

Based on computer modeling of the active site of nitric oxide synthases (NOS), a series of 10 amidine compounds (9-18) was designed including potential inhibitors that involve the coordination of side-chain functional groups with the iron of the heme cofactor. The most potent and selective compound was the methylthio amidine analogue 9, which was more potent than L-nitroarginine with 185-fold selectivity for inhibition of neuronal NOS over endothelial NOS. It also exhibited time-dependent inhibition, but did not involve the mechanism previously proposed for other amidine inhibitors of NOS. None of the compounds, however, exhibited heme-binding characteristics according to absorption spectroscopy.

Animals↗
PubMed2026

Mechanistic characterization and inhibition of PAD4-dependent NETosis following experimental neonatal hypoxic-ischemic brain injury.

Neonatal hypoxia-ischemia (HI) remains a major global cause of neonatal morbidity and mortality. To develop effective therapeutic strategies, a deeper understanding of the acute inflammatory processes following HI is required. As the role of peripheral immune cell infiltration is poorly understood we used single nuclei RNA sequencing to investigate acute sequalae post-HI. This revealed a distinct neutrophil population characterized by increased expression of markers associated with neutrophil extracellular trap (NET) formation. The study evaluated the therapeutic potential of inhibiting NETosis formation using the peptidylarginine-deiminase-4 (PAD4) inhibitor Cl-amidine. We demonstrated that while oxygen-glucose deprivation (OGD) induces NET-formation and matrix-metalloproteinase-9 release in neutrophils, this is significantly reduced by the PAD4 inhibitor Cl-amidine. Second, in a co-culture model, inhibition of NETosis during OGD conditions improved neuronal survival. Third, Cl-amidine treatment reduced brain tissue loss and decreased expression levels of NETosis-related proteins 48&#xa0;h post-HI in vivo. Moreover, behavioral testing performed six weeks after HI revealed improved motor function in the treatment group. This study highlights the detrimental role of neutrophil activation and NETosis in exacerbating brain injury following neonatal HI. Targeting NET formation in the acute phase after HI may represent a promising therapeutic approach to improve both immediate and long-term neurological outcomes.

Animals↗