An AFM investigation of oligonucleotides anchored on unoxidized crystalline silicon surfaces.

Carboxylic terminated monolayers have been covalently attached on phosphorous doped crystalline (100) silicon surfaces using a cathodic electro grafting technique. The functionalization concentration and efficiency have been evaluated with different techniques. In particular, topographic images, performed with an atomic force microscope, were used to optimize the protocol in order to obtain a surface whose characteristics of uniformity and reproducibility are ideal for a bio-electronic device. Phase lag images of the functionalized surfaces were also performed, and show non-topographic structures that have been interpreted as areas of different molecule self-orientation. Poly-thymine oligonucleotides have been anchored on such a surface to form a nano-biosensing device capable to react selectively with a specific target molecule, a poly-adenine oligonucleotide. AFM images of high density (approximately 3x10(12) mol/cm2) single strand and double strand covered samples show toroidal shaped structures formed by the self-assembly of the oligonucleotides on the silicon surface.

Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2'-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein.

The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found.

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins.

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.

Long chain analogs of physostigmine as potential drugs for Alzheimer's disease: new insights into the mechanism of action in the inhibition of acetylcholinesterase.

Heptylphysostigmine is in advanced clinical trial as a drug for Alzheimer's disease. 8-Morpholinooctylphysostigmine and 8-(cis-2,6-dimethylmorpholino)octylphysostigmine are currently undergoing pre-clinical evaluation. The mechanism of action of these compounds in the inhibition of acetylcholinesterase has been investigated. All the examined compounds display non competitive-like kinetics of inhibition. There are no reversible components in the observed inhibition: the whole inhibitory effect is due to the time-dependent pseudo-irreversible carbamylation of the active site. Yet the observed time course of the inhibition does not match a simple second order kinetics. An influence of the quaternary structure of the enzyme on the more complex kinetics of carbamylation is hypothesized. Reactivation experiments on the inhibited enzyme show long lasting inhibitory effects for these compounds. The higher duration of the anticholinesterase effect of the morpholino derivatives compared to heptylphysostigmine should provide the basis for their higher therapeutic potential.

4-Deoxypyrido[1',2':1,2]imidazo[5,4-c]rifamycin SV derivatives. A new series of semisynthetic rifamycins with high antibacterial activity and low gastroenteric absorption.

A series of 4-deoxypyrido[1',2':1,2]imidazo[5,4-c]rifamycin SV derivatives (6-11) were prepared that demonstrated high antibacterial activity suitable for an intestinal disinfectant. These compounds are zwitterionic in nature and are poorly absorbed through the gastroenteric tract but maintain the ability to cross the bacterial cell wall. X-ray crystallographic data are presented to demonstrate the zwitterionic nature of these compounds. The structure-activity relationship of this novel series of antibiotics is discussed and the derivative with the highest ratio between subcutaneous and oral activity (6) was selected for clinical development. At the outset of this work several 3-(quaternary ammonium bromides) (1-5) were prepared and tested for antibacterial activity. These compounds were demonstrated to be too polar to even cross the bacterial cell wall but led to the synthesis of 6-11.

Acetazolamide-like carbonic anhydrase inhibitors with topical ocular hypotensive activity.

New carbonic anhydrase (EC 4.2.1.1) inhibitors were synthesized as potential drugs for the topical treatment of glaucoma. They were obtained by substituting the acetyl group of acetazolamide and methazolamide with bicarboxylic acids of different chain length (C4-C6). The terminal carboxyl was either kept free or esterified with alcohols of different size (C1-C12). A gamma-aminovaleric derivative was also prepared. All compounds proved active as carbonic anhydrase inhibitors in vitro, with an average IC50 of about 0.5 microM. Some proved also to be topically active in vivo in lowering the artificially elevated intraocular pressure in rabbits. The most active compound, carrying a succinic acid side chain, is the most soluble in aqueous buffers. Its duration of action is about 8 h and it is under evaluation as a topical antiglaucoma drug. It is hypothesized that the duration of action could be longer in compounds having both the same high water solubility and partition coefficient.

Fractions of chemically oversulphated galactosaminoglycan sulphates inhibit three enveloped viruses: human immunodeficiency virus type 1, herpes simplex virus type 1 and human cytomegalovirus.

A series of chemically oversulphated galactosaminoglycans (SO3H:COOH ratio > or = 2) were tested in vitro as antiviral agents against human immunodeficiency virus type 1 (HIV-1), the aetiological agent of AIDS, and against herpes simplex virus type 1 and human cytomegalovirus, two agents responsible for opportunistic infections in HIV-infected people. The oversulphated derivatives displayed an increase in activity ranging from one to four orders of magnitude against the three viruses, as compared to the natural parent compounds (SO3H:COOH, ratio approx. 1). The antiviral activity of these polyanions appears to be favoured by a high degree of sulphation and a high molecular mass. An oversulphated dermatan, with a SO3H:COOH ratio of 2.86 and molecular mass of 23.2 kDa, was the most potent anti-HIV-1 compound (EC50 0.04 microgram/ml). A second oversulphated dermatan, with a SO3H:COOH ratio of 2.40 and molecular mass of 25 kDa, displayed the highest activity against HSV-1 (EC50 0.01 microgram/ml). An oversulphated chondroitin, with a SO3H:COOH ratio of 2.80 and molecular mass of 17.3 kDa, was the strongest anti-HCMV agent (EC50 0.4 microgram/ml). In view of the absence of the side-effects typical of heparin-like compounds, a combination of these derivatives could have therapeutic potential.

DNA, RNA and hybrid RNA-DNA oligomers of identical sequence: structural and dynamic differences.

A 27-mer sequence was synthesised as DNA duplex (DD), RNA duplex (RR), and RNA-DNA (RD) hybrid in order to characterise their structural and dynamic features. The hydrodynamic radius (Rh) and the rise (b) values of the three samples were consistent with the conformations predicted by CD analysis. The value of the torsional constant (alpha) of the samples containing RNA was approximately twice that of the DD sample and followed the order: DD < RD < RR. The same order was observed in the thermodynamic stability and in the reduction of the electrophoretic mobility. gamma-Ray footprinting analysis was carried out to resolve the individual strand conformation in the hybrid. The RNA strand preserved its conformation, while the DNA strand showed local deformations mainly at TA and TG steps.

Influence of an 8-oxoadenine lesion on the structural and dynamic features of a 30-mer DNA fragment with and without a mismatch.

PURPOSE: To elucidate the influence of the oxidative lesion 7,8-dihydro-8-oxoadenine (8-oxoA) on the structural and dynamic features of a 30-mer DNA fragment, and to understand if differences occur when C is positioned opposite 8-oxoA instead of T. MATERIALS AND METHODS: Two 30-mer DNA oligomers with or without the 8-oxoA and two complementary oligomers with C or T base opposite the lesion site were synthesized and annealed. Duplexes named AT, A*T, AC and A*C were characterized by means of circular dichroism and UV denaturation measurements. gamma-Ray footprinting experiments were performed to give insight into their fine three-dimensional structure. Elastic torsional constants were derived by following the decay of the fluorescence polarization anisotropy (FPA) of the ethidium-DNA complexes measured by multifrequency-phase fluorometry. RESULTS: The introduction of one oxidative lesion in a 30-mer DNA oligomer with and without a mismatch did not cause relevant changes in their overall conformation and slightly modified their elastic properties. Small energetic differences were revealed by thermodynamic analysis in the sample bearing both the oxidative lesion and the mismatch. Minor variations in the cleavage pattern due to the hydroxyl radicals in the A*T sample were observed and present along the entire DNA fragment length. In the A*C sample, by contrast, there was a major modification in the cleavage pattern extending for about 11 bases around the lesion, especially towards the 5'-end. CONCLUSIONS: Differences in the fine structure and in the elastic properties between the A*T and A*C samples were observed, while their overall conformation was unchanged. The results are consistent with the hypothesis that the observed local changes of the double helix structure in A*C are due to the pairing of the oxidized adenine in a syn conformation with the cytosine.

Rifamycins inhibit human neutrophil functions: new derivatives with potential antiinflammatory activity.

In our study we investigated the effect of rifamycin SV, rifamycin B, rifampicin and five semisynthetic derivatives on human neutrophil functions such as locomotion, superoxide production and degranulation stimulated by specific agonists. All compounds were tested at concentrations ranging from 10(-9) to 10(-5) M. Among the newly synthesized compounds the most active we found to be the derivatives carrying an acidic substituent at C3: these significantly lowered the superoxide generation induced by PMA throughout the entire concentration range, whereas rifamycin SV, rifamycin B and rifampicin were effective only at the highest concentrations. Moreover, chemotactic movement was significantly attenuated by derivative R4, rifamycin B and rifamycin SV at high doses; granule enzyme release was unaffected by all compounds.

3-(Carboxyalkylthio) rifamycin S and SV derivatives inhibit human neutrophil functions.

In order to further investigate the potential of rifamycins as antiinflammatory drugs, twenty-five semisynthetic rifamycins were tested at concentrations ranging from 10(-9) to 10(-5) M on in vitro human neutrophil functions such as locomotion, superoxide anion production, and degranulation, under different stimulatory conditions. They were also tested as antiproliferative agents on peripheral blood lymphocytes. The present semisynthetic derivatives are in general characterized by their carrying a hydrophilic substituent; they are rifamycin S or rifamycin SV derivatives carrying at C(3) either a carboxyalkyl side-chain or a glycosyl side-chain. Derivatives of the former group displayed inhibitory activities covering the whole range of activities tested, suggesting that the sum of these different effects could support their antiinflammatory activity in vivo. These derivatives, carrying a free carboxyl, are more water soluble than rifamycin SV at physiological pH, and may serve as antiinflammatory drugs for local administration, alternative to rifamycin SV, possibly giving higher efficacy and reduced side effects of pain and tissue swelling.

Inhibition of human leukocyte elastase by chemically and naturally oversulfated galactosaminoglycans.

Several samples of oversulfated chondroitin and dermatan were obtained by chemical sulfation and by SAX-HPLC enrichment. The starting products and oversulfated products were tested as potential inhibitors of human leukocyte elastase, an enzyme hypothesized to be involved in the etiology of diseases such as emphysema, atherosclerosis, and rheumatoid arthritis. Chemical oversulfation (SO3H/COOH 1.6-3.2), preferentially occurring at C-6 of galactosamine residues, was found generally to increase the inhibitory power on elastase. Chemically oversulfated galactosaminoglycans thus have potential as therapeutic agents, considering that they produce non-significant effects on the hemocoagulative system. Two naturally oversulfated dermatans sulfate (SO3H/COOH ca. 1.2), mainly oversulfated at C-2 of iduronic acid residues, showed comparatively higher anticoagulant activity (in the HC-II mediated thrombin inhibition test).

The synthesis of 4-deoxypyrido[1',2'-1,2]imidazo[5,4-c]rifamycin SV derivatives.

Two series of new semisynthetic rifamycin SV derivatives have been prepared. One of them bears a quaternary ammonium salt at C3 (1-5), and the other a pyridoimidazo system condensed at C3 and C4 (6-11). While compounds 1-5 had poor antibacterial activity in vitro, compounds 6-11 were found to be highly active in vitro but poorly absorbed in vivo. They could thus have potential as agents in the therapy of intestinal infections. The synthesis and the 1H NMR structure determination of these new compounds are reported.

Chemical modifications of the aliphatic bridge of ansamycins. Synthesis and activity of 25-deacetoxy-25-epi-hydroxyrifamycin S.

Rifamycins are supposed to bind to, and inhibit the bacterial DNA-dependent RNA polymerase (DDRP) by the formation of hydrogen bonds through O (1), O (2), O (9), O (10). Therefore, with the aim of increasing the intrinsic activity of rifamycin S (1), the 25-deacetoxy-25-epi-hydroxyrifamycin S (8), was synthesized, which displays an additional hydroxyl available for the inhibiting interaction with the bacterial enzyme. The configuration and conformation of the new compound were as expected, but the biological evaluation did not confirm the hypothesis.

Chemical modifications of the aliphatic bridge of ansamycins. 3. Synthesis and activity of 21-epi-rifamycin S.

Rifamycins inhibit bacterial DNA-dependent RNA polymerase through the formation of non-covalent bonds by the oxygenated groups at C(1), C(8), C(21), and C(23). These must be unhindered and underivatized, with the antibiotic in a proper overall molecular conformation. The present study shows that contrary to previous conclusions the availability of the hydroxyl group at C(21) is not as important as that of the other three groups. In support of this is the observation that 21-epi-rifamycin S is partially active, both on the isolated DNA-dependent RNA polymerase and on some Gram-positive bacterial strains.

X-ray crystal structure of 4-deoxy-3'-bromopyrido[1',2'-1,2]imidazo[5,4-c]rifamycin S.

This paper reports the determination of the X-ray molecular structure of 4-deoxy-3'-bromopyrido[1',2'-1,2]imidazo[5,4-c]rifamycin S, carried out in order to unequivocally define the general structure of a new series of rifamycin SV derivatives, which are potent antibacterial agents, and are not absorbed at the gastroenteric level. They have been prepared by Alfa Farmaceutici, Bologna, by condensing 2-aminopyridine derivatives to 3-bromorifamycin S. The solid state X-ray study has confirmed the structure proposed on the basis of 1H NMR studies in solution. It has also shown that the newly formed pyridoimidazo system is in a mesomeric betaine form, the pyrido nitrogen being positively charged and the imidazo nitrogen being negatively charged. This feature is believed responsible for the pharmacokinetic behavior of these new drugs, one of which, denoted either as rifamycin L 105 or rifaximin, is actually under clinical trial as a topical intestinal disinfectant.

Metabolites of microorganisms. 229. Absolute configuration of naphthomycin A determined by X-ray analysis and chemical degradation.

The relative and absolute configurations of naphthomycin A were elucidated by an X-ray structural analysis of a methylation product, 25-O-methylnaphthomycin A iminomethyl ether. The absolute configuration was confirmed by degradation (O3, NaBH4) to (S)-butane-1,2,4-triol.

Oligonucleotide labeling: synthesis of a new spin-labeled 2'-deoxyguanosine analogue.

In order to achieve an EPR sensitive probe for DNA, 3-carboxy-Proxyl free radical was linked to O-6 of dG through a five-atoms-tether. The modified base was incorporated into a 30-mer ODN, then annealed to its complementary DNA strand. Hydrodynamic parameters show only a slight destabilization with respect to the equivalent unlabeled hybrid. EPR could monitor the hybrid formation showing a progressive enlargement of the upfield signal in passing from the labeled ss- to the ds-30-mer.

Chondroprotective action of chondroitin sulfate. Competitive action of chondroitin sulfate on the digestion of hyaluronan by bovine testicular hyaluronidase.

Lysosomal hyaluronidase is responsible for the degradation of hyaluronan, a component of the extracellular matrix, in degenerative disorders of the joints. It has been hypothesized that the administration of chondroitin sulfate (both a component of the extracellular matrix and a substrate for hyaluronidase) could compete for this enzyme and reduce the degradation process. The present study shows that a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate is a good competitor of hyaluronan for hyaluronidase. The digestion of hyaluronan is reduced in proportion to the amount of competing chondroitin. The competitive ability is dependent on the 4-sulfate, 6-sulfate composition of the chondroitin mixture. Mixtures richer in the 4-sulfate isomer are more effective. The enzymatic reactions have been monitored by HPLC and PAGE.

Synthesis of 2-hydroxyacetyl-7-acetyl-xanthone, a new xanthone derivative endowed with antianaphylactic, analgesic, and antiinflammatory activities.

2-Hydroxyacetyl-7-acetylxanthone (V), a new xanthone derivative, was synthesized in four steps starting from xanthene, by two synthetic approaches. The new compound displayed antianaphylactic activity in the PCA test and in the anaphylaxis shock test in rats. It also displayed analgesic activity in the writhing test in mice, and antiinflammatory activity in the carrageenin edema test in rats. The activity of the new compound has been tentatively interpreted on the basis of its chemical and structural analogy with known drugs.