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A class of DNA binding antibiotics endowed with antiviral and antitumor properties is reviewed. Starting from the original natural products, namely distamycin and netropsin, new compounds have been recently synthesized with the aim to obtain agents with specific affinity for defined DNA sequences and with different interaction mechanism (reversible or irreversible).
The Human Genome Project as well as sequencing of the genomes of other organisms offers a wealth of DNA targets for both therapeutic and diagnostic applications, and it is important to develop additional DNA binding motifs to fully exploit the potential of this new information. We have recently found that an aromatic dication, DB293, with an amidine-phenyl-furan-benzimidazole-amidine structure can recognize specific sequences of DNA by binding in the minor groove as a dimer [Wang, L., Bailly, C., Kumar, A., Ding, D., Bajic, M., Boykin, D. W., and Wilson, W. D. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 12-16]. The dimer binding is strong, highly cooperative and, in contrast to many closely related heterocyclic dications, has both GC and AT base pairs in the minor groove binding site. The aromatic heterocycle stacked dimer is quite different in structure from the polyamide-lexitropsin type compounds, and it is a dication while all lexitropsin dimers are monocations. The heterocyclic dimer represents only the second small molecule class that can recognize mixed sequences of DNA. To test the structural limits on the new type of complex, it is important to probe the influence of compound charge, chemical groups, and structural features. The effects of these compound molecular variations on DNA complex formation with several DNA sequences were evaluated by DNase I footprinting, CD and UV spectroscopy, thermal melting, and quantitative analysis with surface plasmon resonance biosensor methods. Conversion of the amidines to guanidinium groups does permit the cooperative dimer to form but removal of one amidine or addition of an alkyl group to the amidine strongly inhibited dimer formation. Changing the phenyl of DB293 to a benzimidazole or the benzimidazole to a phenyl or benzofuran also inhibited dimer formation. The results show that formation of the minor groove stacked-dimer complex is very sensitive to compound structure. The discovery of the aromatic dimer mode offers new opportunities to enhance the specificity and expand the range of applications of the compounds that target DNA.
The reactivity of two sterically bulky amidines, ArNC(R)N(H)Ar (Ar=2,6-diisopropylphenyl; R=H (HFiso); tBu, (HPiso)) towards LiMH4, M=Al or Ga, [AlH3(NMe3)], and [GaH3(quin)] (quin=quinuclidine) has been examined. This has given rise to a variety of very thermally stable aluminum and gallium hydride complexes. The structural motif adopted by the prepared complexes has been found to be dependent upon both the amidinate ligand and the metal involved. The 1:1 reaction of HFiso with LiAlH4 yielded dimeric [{AlH3(mu-Fiso)Li(OEt2)}2]. Amidine HFiso reacts in a 1:1 ratio with [AlH3(NMe3)] to give the unusual hydride-bridging dimeric complex, [{AlH2(Fiso)}2], in which the Fiso- ligand is nonchelating. The equivalent reaction with the bulkier amidine, HPiso, yielded a related hydride-bridging complex, [{AlH2(Piso)}2], in which the Piso- ligand is chelating. In contrast, the treatment of [GaH3(quin)] with one equivalent of HFiso afforded the four-coordinate complex [GaH2(quin)(Fiso)], in which the Fiso- ligand acts as a localized monodentate amido-imine ligand. The 2:1 reactions of HFiso with [AlH3(NMe3)] or [GaH3(quin)] gave the monomeric complexes [MH(Fiso)2], which are thermally robust and which exhibit chelating amidinate ligands. In contrast, HPiso did not give 2:1 complexes in its reactions with either of the Group 13 trihydride precursors. For sake of comparison, the reactions of [AlH3(NMe3)] and [GaH3(quin)] with the bulky carbodiimide ArN=C=NAr and the thiourea Ar(H)NC(=S)N(H)Ar were examined. These last reactions afforded the five-coordinate thioureido complexes, [MH{N(Ar)C[N(H)(Ar)]S}2], M=Al or Ga.
New derivatives of doxorubicin and daunorubicin with amidine group bonded to daunosamine at C-3' atom and bearing the morpholine ring attached to the amidine group have been recently synthesized. Their cytotoxic activities and effects on RNA synthesis in vitro were assayed. The drug concentrations inhibiting mouse leukaemia L1210 cell growth to 50% were about two- and three fold higher for the derivatives compared to doxorubicin and daunorubicin respectively. Inhibition of phage T7 RNA polymerase by the non-covalently interacting derivatives was also slightly lower than that by the parent compounds. As doxorubicin and daunorubicin, their amidine derivatives in the presence of dithiothreitol and Fe(III) ions are activated and covalently bind to DNA. The adducts formed affect RNA polymerase activity. Several bands corresponding to prematurely terminated RNA chains are observed by means of polyacrylamide gel electrophoresis. The patterns of bands are virtually identical for all the anthracyclines studied here and are similar to the terminations induced by actinomycin D. This observation is consistent with a notion that the adducts are formed at guanine in GpC sequences which are also binding sites of actinomycin D. A substantial difference between daunorubicin and its amidine derivative is shown by means of high performance liquid chromatography. The derivative undergoes rapid rearrangements in the presence of dithiothreitol and Fe(III) ions, while daunorubicin is stable for several hours under these conditions. The results presented here indicate that the amidine derivatives despite bulky morpholine substitution exhibit biological activity in the systems used here.
The activation of bovine prothrombin is known to be accompanied in purified systems by proteolytic reactions catalyzed by the product, thrombin. These reactions, which are directed principally towards the prothrombin substrate and the Factor V cofactor, are eliminated if the lysine residues of prothrombin are chemically modified beforehand with methyl acetimidate. Amidinated prothrombin in which the usual lysine content has been reduced by 75% is cleaved completely by Factor Xa to give thrombin which has little or no activity towards fibrinogen, the thrombin-sensitive bond in prothrombin, or Factor V, but with normal activity towards the synthetic chromogenic substrate D-Phe-Pipecolyl-L-Arg-p-nitroanilide. The formation of thrombin could therefore be studied spectrophotometrically by discontinuous assays of thrombin without complication by the proteolytic feedback activity of this enzyme. Such assays showed that the rate of appearance and yield of thrombin is the same whether from native or amidinated prothrombin, in spite of the lack of proteolytic activity in the product from the latter. Native and amidinated prothrombin have identical fluorescence emission spectra (lambda ex = 280 nm) which, upon activation, show a broadening and a red shift of the peak of emission from 330 to 336 nm. This change is different from the quenching known to occur when prothrombin binds Ca2+ and is contingent upon cleavage by Factor Xa. When monitored at the 370 nm band, the shift is seen as an increase in fluroescence intensity which, when the concentrations of Factor Xa and Factor V are adjusted appropriately, has the same general appearance as a progress curve obtained by discontinuous assay of thrombin activity. However, the curves obtained with the native zymogen appear to contain a component due to proteolysis by the accumulating product. in the case of amidinated zymogen, this is not longer so: the curves reflect only proteolysis by Factor Xa. In addition, a comparison of the fluorescence shift with the time course of thrombin appearance shows that the shift results mainly from the cleavage of intact prothrombin by Factor Xa, with little or no contribution from later events in the activation pathway. Modified Stern-Volmer plots for the quenching of fluorescence (lambda ex = 295 nm) of the intact amidinated zymogen and its activation products by sodium iodide allow the conclusion that activation results in the exposure to solvent of tryptophan residues that were previously sheltered. However, there is a variation in the pattern of quenching with the protein concentration, suggesting that these residues may be sheltered in the zymogen by intermolecular rather than intramolecular interactions.
We describe here a new cationic amphiphile, N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), which interacts with plasmid DNA and generates hydrophobic stable complexes resistant against DNase I. In partition experiments between two non-miscible phases, DNA was transferred into an organic phase upon complex formation with diC14-amidine-containing vesicles. Finally, vesicles made of a diC14-amidine and phosphatidylethanolamine (PE) (1:1, mol:mol) mixture or pure diC14-amidine were efficient in mediating transfection of adherent (CHO) and suspension (K562) cell lines, using the chloramphenicol acetyltransferase (CAT) gene as reporter.
We have developed a series of potent and selective factor VIIa inhibitors based on the 2-[5-(5-carbamimidoyl-1H-benzoimidazol-2-yl)-6-hydroxy-biphenyl-3-yl]-succinic acid scaffold. These amidine-containing compounds have low oral bioavailability. Herein, we describe our efforts to improve the oral bioavailability of the parent amidine via a prodrug strategy where the amidine basicity and polarity were reduced with either an alkoxy-amidine or a carbamate prodrug.
Protein arginine deiminase 4 (PAD4) is a transcriptional coregulator that catalyzes the calcium-dependent conversion of specific arginine residues in proteins to citrulline. Recently, we reported the synthesis and characterization of F-amidine, a potent and bioavailable irreversible inactivator of PAD4. Herein, we report our efforts to identify the steric and leaving group requirements for F-amidine-induced PAD4 inactivation, the structure of the PAD4-F-amidine x calcium complex, and in vivo studies with N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a PAD4 inactivator with enhanced potency. The PAD4 inactivators described herein will be useful pharmacological probes in characterizing the incompletely defined physiological role(s) of this enzyme. In addition, they represent potential lead compounds for the treatment of rheumatoid arthritis because a growing body of evidence supports a role for PAD4 in the onset and progression of this chronic autoimmune disorder.
Protein arginine deiminase 4 (PAD4) is a calcium-dependent transcriptional corepressor that has been implicated in the onset and progression of rheumatoid arthritis. Herein we describe the synthesis and in vitro evaluation of a fluoroacetamidine-containing compound, N-alpha-benzoyl-N5-(2-fluoro-1-iminoethyl)-l-ornithine amide, 1, hereafter referred to as F-amidine, that is the most potent PAD4 inhibitor ever described. Additional studies described herein indicate that F-amidine can also inhibit PAD4 activity in vivo. The bioavailability of this compound suggests that F-amidine will be a powerful chemical probe of PAD4 function that can be used to dissect the roles of this enzyme in both rheumatoid arthritis and transcriptional control. The fact that inhibition is of an irreversible nature suggests that, with appropriate functionalization, F-amidine analogues will be robust activity-based protein-profiling and proteomic capture reagents.
1. The structure of human plasma low-density lipoprotein(s) [LD lipoprotein(s)] was investigated by several immunological and optical techniques. The effects of delipidation and of chemical modification by 3-carboxypropionylation, acetylation, diazotization and amidination were examined. A sensitive double-antibody radioimmunoassay for human LD lipoproteins is presented and is used to assess the extent of immunochemical modification. A computer best-fit analysis is used to analyse circular-dichorism (c.d.) spectra. These methods permit comparisons of the relative effects of chemical modification and delipidation of LD lipoprotein under similar experimental conditions. 2. 3-Carboxypropionylation, acetylation or diazotization produces qualitative and quantitative changes in the immunochemical properties of LD lipoprotein. Amidination causes minor changes detected by radioimmunoassay but not by double-diffusion experiments. In general, the order of effectiveness in displacing (125)I-labelled LD lipoprotein is amidinated>diazo or acetyl>3-carboxypropionyl derivatives. 3. The order of the extent of conformational alteration induced in apoLD lipoprotein and LD lipoprotein, as judged by c.d. analyses, was 3-carboxypropionylation>diazotization>acetylation or amidination. 4. Delipidation of LD lipoprotein results in immunological alterations that are qualitatively detected by antisera to LD lipoprotein. Four of five antisera to apoLD lipoprotein form precipitin lines of identity between native LD lipoprotein and apoLD lipoprotein in double-diffusion experiments. An anti-(apoLD lipoprotein) serum that forms precipitin lines of complete identity between LD lipoprotein and apoLD lipoprotein reacts differently with these two antigens in radioimmunoassay. ApoLD lipoprotein is only one-fourth to one-half as effective as LD lipoprotein, on a protein basis, in the displacement of (125)I-labelled LD lipoprotein from this anti-(apoLD lipoprotein). 5. Conformational analysis indicates that apoLD lipoprotein retains a high proportion of the structural integrity of the native lipoprotein. Delipidation induces a small decrease in the content of beta-structure and a small increase in disordered structure, without greatly affecting the alpha-helical content. Chemical modification produces more severe conformational changes of apoLD lipoprotein than of LD lipoprotein. Computer analysis of the c.d. spectra of apoLD lipoprotein indicates that addition of high concentrations of sodium decyl sulphate abolishes most of the beta-conformation concomitant with increases in alpha-helical and disordered structure. 6. There is parallelism between the alteration of the charge of LD lipoprotein and apoLD lipoprotein and the extent of immunochemical and conformational changes.
N-Hydroxylated amidines (amidoximes) can be used as prodrugs of amidines. The prodrug principle was developed in our laboratory for pentamidine and had been applied to several other drug candidates. One of these compounds is melagatran, a novel, synthetic, low molecular weight, direct thrombin inhibitor. To increase the poor oral bioavailability due to its strong basic amidine functionality selected to fit the arginine side pocket of thrombin, the less basic N-hydroxylated amidine was used in addition to an ethyl ester-protecting residue. The objective of this investigation was to study the reduction and the hydrolytic metabolism of ximelagatran via two mono-prodrugs (N-hydroxy-melagatran and ethyl-melagatran) to melagatran by in vitro experiments. New high-performance liquid chromatography methods were developed to analyze all four compounds. The biotransformation of ximelagatran to melagatran involving the reduction of the amidoxime function and the ester cleavage could be demonstrated in vitro by microsomes and mitochondria from liver and kidney of pig and human, and the kinetic parameters were determined. So far, one enzyme system capable of reducing N-hydroxylated structures has been identified in pig liver microsomes, consisting of cytochrome b(5), NADH-cytochrome b(5) reductase, and a P450 isoenzyme of the subfamily 2D. This enzyme system also reduces ximelagatran and N-hydroxy-melagatran. The participation of recombinant human CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 with cytochrome b(5) and b(5) reductase in the reduction can be excluded. In summary, ximelagatran and N-hydroxy-melagatran are easily reduced by several enzyme systems located in microsomes and mitochondria of different organs.
Daunorubicin (DRB) and doxorubicin (DOX) in the presence of formaldehyde (CH2O) form covalent adducts with DNA. A G-specific adduct is formed by producing an aminal bridge between the C-3' of daunosamine and the C-2 of guanine. New derivatives of DRB, DOX and epidoxorubicin (EDOX) with an amidine group bonded to the C-3' of the daunosamine moiety, with either a morpholine or hexamethyleneimine ring attached to the amidine group, were studied in this paper. DNase I footprinting and analyses with restriction endonucleases were applied to compare the specificity of adduct formed by the amidine derivatives and their parent compounds. These approaches provide consistent results, proving that a GC pair is required for covalent binding of anthracycline derivatives to DNA and that different flanking sequences are able to modify the sequence preference of the drugs. The 5'-GC-3', 5'-CG-3' and 5'-TC-3' sequences were protected most efficiently by the parent compounds and their morpholine derivatives and some increased protection of 5'-TC-3' sequence was observed for morpholine analogues. Hexamethyleneimine derivatives bind to DNA with much lower efficiency. Finally, the sequence specificity of anthracycline derivatives was correlated with their ability to inhibit binding of transcription factors Sp1 and AP-1 to their DNA recognition sequences. The anthracycline derivatives were more potent in inhibiting Sp1 binding to its cognate GC box than in preventing AP-1 from binding to its mixed A.T and G.C site. Overall, the results indicate that the amidine derivatives of anthracyclines show similar, but not identical sequence specificity as parent compounds, though they exert their effect at a higher concentration.
In this report, we show that intravenous (i.v.) injection into mice of a complex made of the cationic lipid diC14-amidine and the luciferase reporter plasmid (pCMV-luc) results in efficient gene expression in several organs but elicits an inflammatory response characterized by a release of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) into the serum of treated animals. A single preinjection of free diC14-amidine liposomes improves the i.v. transfection efficiency of the diC14-amidine/protamine/pCMV-luc complex as much as 40 times. This improvement is correlated with the ability of free liposomes to inhibit TNF-alpha but not IFN-gamma production resulting from complex injection. TNF-alpha-rich serum obtained from mice injected with diC14-amidine/protamine/pCMV-luc complex inhibits luciferase expression in transfected mouse lung endothelial (MLE) cells cultured in vitro, whereas IFN-gamma has no effect. This inhibitory effect can be partly abolished by treating the mouse serum with a specific anti-TNF-alpha antibody. These data point out that cationic lipids are potent inhibitors of the inflammatory response to the CpG motifs in plasmid DNA. This property is shown to enhance the in vivo transfection efficiency.
The presence of the Zn2+ ion dramatically enhances the inhibition of trypsin and tryptase by amidine-modified benzimidazole inhibitors via coordination to both the catalytically active Ser195 hydroxyl and His57 imidazole residues of the enzyme and the nitrogens of the amidine-modified benzimidazole inhibitor (Janc, J. W.; Clark, J. M.; Warne, R. L.; Elrod, K. C.; Katz, B. A.; Moore, W. R. Biochemistry 2000, 39, 4792-4800). Some new 5-amidino-2-substituted benzimidazoles were synthesized and compared to known related molecules to explore systematically the metal-mediated inhibition of bovine trypsin as a function of coordinating groups and metal ions. These compounds take advantage of the favorable interaction between the amidine group on one side of the inhibitor and the Asp189 carboxylate in the binding pocket of the enzyme. The 5-amidino-2-substituted benzimidazoles all demonstrated similar inhibition constants (Ki) of 20-50 microM in the absence of metal ions. In the presence of Zn2+, inhibition increased to varying extents, depending upon the group substituted at the 2 position of the benzimidazole. The largest increase in inhibition in the presence of Zn2+ was seen with (5-amidino-2-benzimidazolyl)-2-benzimidazolylmethane with an apparent inhibition constant (Ki') of 0.37 +/- 0.06 nM, giving a 59,000-fold increase in inhibition when Zn2+ is present. Other metal ions, including Mn2+, Sc3+, and Hg2+, also increased the inhibition by several of the benzimidazole derivatives synthesized. The compound bis(2-benzimidazolyl)methane (BBIM) was also examined because it lacks the amidine group that provides a favorable hydrogen-bonding interaction with Asp189 in the binding pocket of trypsin. In the absence of metal ions, BBIM did not have a detectable affinity for trypsin; however, in the presence of Zn2+, a Ki' of 127 +/- 3 nM was observed. This result demonstrates that an affinity for the enzyme in the absence of metal ions is not required for potent metal-mediated inhibition, greatly expanding the possibilities for metal mediation of nonmetalloenzymes.
Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.
In cats under chloralose anaesthesia the reflex fall of blood pressure and heart rate caused by injection of veratrine, amidines, diphenhydramine, or ethyl acetoacetate was accompanied by a fall in cardiac output. After veratrine and amidines there was a fall in mean pulmonary arterial pressure and after veratrine no significant change in pulmonary vascular resistance. After diphenhydramine and ethyl acetoacetate there was a rise in mean pulmonary arterial pressure and after diphenhydramine an increase in pulmonary vascular resistance. The effects of veratrine and amidines, but not those of diphenhydramine and ethyl acetoacetate, were abolished by section of the vagi. The main change leading to the fall of cardiac output after amidines was bradycardia.
Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.
Amidination of the available lysine residues of the complex between RNase A and human placental RNase inhibitor has been performed with methyl acetimidate; the conditions of the derivatization preserve the complex functionally intact. Resistance of epsilon-acetimidyllysine residues to hydrolysis by trypsin allowed, after peptide mapping, the identification of lysine residues 7, 31, 41, 61, and 91 as those which were fully protected by the inhibitor from amidination. Lysine residue 37 was partially protected from amidination. In the presence of poly(A), lysine residues 41 and 61 of RNase A were fully protected from amidination, while lysine residues 7, 31, 37, 91, and 104 were only partially protected; the enzyme retained full activity. The results permit identification of lysine residues located in the binding domain of RNase A for the inhibitor. This region is not identical with, but does overlap, the binding domain for poly(A).