Pyrazinoporphyrazines with externally appended pyridine rings. 13. Structure, UV-visible spectral features, and noncovalent interaction with DNA of a positively charged binuclear (Zn(II)/Pt(II)) macrocycle with multimodal anticancer potentialities.

We investigated with spectroscopic techniques the noncovalent interaction of a bimetallic water-soluble (Zn(II)/Pt(II)) porphyrazine hexacation, [(PtCl(2))(CH(3))(6)LZn](6+), and its octacationic analogue [(CH(3))(8)LZn](8+), lacking the cis-platin-like functionality, with a 21-mer double strand (ds) 5'-d[GGG(TTAGGG)(3)]-3'/3'-d[CCC(AATCCC)(3)]-5', as model for B-DNA. Both hexacation and octacation tend to aggregate in water. The structure as well as the ground and excited-state electronic properties of the Zn(II)/Pt(II) hexacation [(PtCl(2))(CH(3))(6)LZn](6+) in water solution were investigated using density functional theory (DFT) and time-dependent DFT (TDDFT) methods. TDDFT calculations of the lowest excited states of [(PtCl(2))(CH(3))(6)LZn](6+) in water provided an accurate description of the Q-band spectral region. In particular, the calculated optical spectra were in agreement with the experimental ones, obtained in the presence of micelles favoring complete disruption of the aggregates. The model for dsDNA binding that emerges from the analysis of UV-vis absorption and time-resolved fluorescence data shows the presence of complexes of 1 dsDNA molecule with 1, 2, and 4 macrocycles. Comparing the results for the hexacation [(PtCl(2))(CH(3))(6)LZn](6+) with those for the [(CH(3))(8)LZn](8+)octacation, we observed a higher degree of monomerization for the [(PtCl(2))(CH(3))(6)LZn](6+) derivative.

Complexes of the antitumoral drugs Doxorubicin and Sabarubicin with telomeric G-quadruplex in basket conformation: ground and excited state properties.

We studied the binding of two anthracycline drugs, Doxorubicin and Sabarubicin, to a model telomeric sequence 5'-d[GGG(TTAGGG)(3)]-3' (21-mer), assuming the basket G-quadruplex (G4) conformation in Na(+)-rich aqueous solution. We used an approach that combines spectroscopic and microcalorimetric techniques to obtain information about ground and excited state properties of the most stable complexes. Both drugs bind to the 21-mer in basket conformation and complexes of 1:1 and 2:1 drug : 21-mer stoichiometry coexist in solution. Binding constants were determined from fluorescence and isothermal titration calorimetry experiments. For both drugs association is driven by enthalpy and disfavoured by entropy in the case of two sequential binding events to different sites. The drug fluorescence is completely quenched in the 1:1 complex, most likely by electron transfer from the guanine system to the anthraquinone moiety, while part of the emission survives in the 2:1 complex. Circular dichroism (CD) of the individual complexes is dominated by the G-quadruplex signal in the UV and by the anthracycline signal in the near-UV and Vis region. The experimental CD spectra combined with conformational calculations at MM level and quantum mechanical calculation of the rotational strength of the electronic transitions afforded information on the binding geometries.

Tetra-2,3-pyrazinoporphyrazines with externally appended pyridine rings. 10. A water-soluble bimetallic (Zn(II)/Pt(II)) porphyrazine hexacation as potential plurimodal agent for cancer therapy: exploring the behavior as ligand of telomeric DNA G-quadruplex structures.

The behavior of a bimetallic water-soluble (Zn(II)/Pt(II)) porphyrazine hexacation as ligand of G-quadruplex (G4) structures adopted by a human telomeric DNA sequence has been examined with different spectroscopic techniques. In K(+) rich solution the hexacationic Zn(II) porphyrazine ligand bearing a peripheral cis-platin-like functionality changes the G-quadruplex conformational equilibrium of the human telomeric sequence 5'-d[AGGG(TTAGGG)(3)]-3' and drives it exclusively toward a very stable parallel G4 form in the complex with 2:1 stoichiometry. An increase of the melting temperature of more than 20 °C is observed in this complex compared to the G4 alone. On the contrary ligand binding to G-quadruplex of the same telomeric sequence in Na(+) rich solution neither markedly influences the predominant basket conformation nor confers increased thermal stability to the G4 structure.

Tetra-2,3-pyrazinoporphyrazines with externally appended pyridine rings. 9. Novel heterobimetallic macrocycles and related hydrosoluble hexacations as potentially active photo/chemotherapeutic anticancer agents.

New homo- and heterobimetallic porphyrazine complexes of general formula [(M'Cl(2))LM] (L = tetrakis-2,3-[5,6-di-(2-pyridyl)pyrazino]porphyrazinato dianion), with M = Zn(II), Mg(II)(H(2)O), or Pd(II) in the central cavity and one M'Cl(2) unit (M' = Pd(II), Pt(II)) peripherally coordinated at the pyridine N atoms of one of the dipyridinopyrazine fragments, were prepared and characterized by elemental analyses and IR/UV-visible spectroscopy. Related water-soluble salt-like species, carrying the hexacations [(PtCl(2))(CH(3))(6)LM](6+) (neutralized by I(-) ions), were also prepared and similarly characterized. Retention of clathrated water molecules is a common feature of all the compounds. A detailed (1)H and (13)C NMR investigation in dimethylformamide (DMF-d(7)) and dimethyl sulfoxide (DMSO-d(6)) provided useful information on the type of arrangement in the neutral and hexacationic species of the metalated dipyridinopyrazine fragments, in which the metal centers (Pd(II)/Pt(II)) are bound to the pyridine N atoms ("py-py" coordination) with formation of N(2(pyr))PdCl(2) or N(2(pyr))PtCl(2) coordination sites, the latter one featuring a cis-platin-like functionality. Data obtained in DMF solution of the quantum yield (Φ(Δ)) for the generation of singlet oxygen, (1)O(2), the cytotoxic agent in photodynamic therapy (PDT), indicate that all the neutral and charged complexes, among them particularly those carrying centrally Zn(II) or Pd(II), exhibit excellent photosensitizing properties, this qualifying the externally platinated complexes as potential bimodal PDT/chemotherapeutic anticancer agents. Fluorescence data (Φ(F)) provided additional information on the photoactivity of all the species studied. The following companion paper describes the observed interaction of the Zn(II) hexacation [(PtCl(2))(CH(3))(6)LZn](6+) with a G-quadruplex (G4) structure of the telomeric DNA sequence 5'-d[AGGG(TTAGGG)(3)]-3' in water.

A cationic Zn(II) porphyrazine induces a stable parallel G-quadruplex conformation in human telomeric DNA.

A water soluble Zn(II) porphyrazine drives the conformational equilibrium of the G-quadruplex of a human telomeric sequence exclusively towards a parallel conformation upon complexation.

Affinity of the anthracycline antitumor drugs Doxorubicin and Sabarubicin for human telomeric G-quadruplex structures.

Combining various techniques in solution we proved that Doxorubicin, also called Adriamycin, and Sabarubicin, also known as MEN 10755, bind to the human telomeric sequence, 5'-d[GGG(TTAGGG)(3)]-3' (21-mer), assuming a G-quadruplex structure in the presence of K(+). Complexes of drugs with the 21-mer in 1 : 1 and 2 : 1 stoichiometry coexist in solution. Association constants were obtained from titration experiments and confirmed by isothermal titration calorimetry. The fluorescence of the drugs was quenched upon complexation. UV circular dichroism (CD) spectra of the complexes were characterized by the G-quadruplex signal and indicated that drug binding influences the equilibrium between quadruplex conformations. The visible CD spectra were exclusively due to the drug and show differences in the complexation modes of the two drugs. Spectroscopic and thermodynamic parameters of the 1 : 1 complexes point to drug stacking with the G-quadruplex top or bottom tetrad. Thermodynamic data suggests that the binding of the second drug molecule in the 2 : 1 complex may occur in a groove. Complexation caused a small increase in the thermal stability of the G-quadruplex main conformation, shifting T(m) from 62 to 67 °C.

Effect of Cu(II) on the complex between kanamycin A and the bacterial ribosomal A site.

The solution structure of kanamycin A interacting with a ribosomal A-site fragment was solved by transferred-NOE techniques and found to agree with the structure of the complex observed in the crystal. Despite the fast exchange conditions found for the interaction, the bound form was identified by NOESY spectroscopy. At 600 MHz, NOE effects are only observed for the RNA-associated antibiotic. Dissociation constants were measured by NMR spectroscopy for two sites of interaction (K(d1)=150+/-40 microM; K(d2)=360+/-50 microM). Furthermore, the effects of the Cu(II) ion on the antibiotic, on the RNA fragment that mimics the bacterial ribosomal A site, and on the complex formed between these two entities were analyzed. The study led to the proposal of a model that localizes the copper ion within the kanamycin-RNA complex.

New anti-viral drugs for the treatment of the common cold.

Human Rhinovirus (HRV) is the most important aetiologic agent of common cold in adults and children. HRV is a single-stranded, positive sense RNA virus and, despite the high level of conservation among different serotypes, sequence alignment of viral protease 3C with mammalian protease reveals no homology. Thus, protease 3C is an optimal target for the development of anti-HRV agents. In the present work we investigated the design, the synthesis and the development of new potential reversible inhibitors against HRV protease 3C. Docking studies on the crystallized structure of HRV2 protease 3C led us to the design and the synthesis of a series of 3,5 disubstituted benzamides able to act as analogues of the substrate. We also developed 1,3,5 trisubstituted benzamides where aromatic substitutions on the aryl ring led us to investigate the importance of pi-pi interaction on the stabilization of protease 3C-inhibitor complex. All structures were tested for enzymatic inhibition on HRV14 protease 3C. Results highlighted the inhibitory activity of compounds 13, 14, and 20 (91%, 81%, and 85% at 10 microM, respectively), with the latter exhibiting an ID(50) (dose that inhibits 50% of the viral cytopathic effect) on HRV-14=25 microg/ml.

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy.

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).

Excision of incorporated nucleotide analogue chain-terminators can diminish their inhibitory effects on viral RNA-dependent RNA polymerases.

Bovine viral diarrhea virus (BVDV) is amongst the best-characterized members of the Flaviviridae, that includes the hepatitis C virus (HCV). The virally encoded RNA-dependent RNA polymerase (RdRp) plays a crucial role during replication and therefore represents an important target for the development of antiviral drugs. Here we studied biochemical mechanisms associated with the inhibition of BVDV RNA synthesis by 2'-hydroxyl, 3'-deoxynucleoside triphosphates (3'-dNTPs). All four nucleotide analogues are effectively incorporated and act as chain-terminators. However, relatively low, physiologically relevant concentrations of pyrophosphate (PPi) are sufficient to drive the reaction backwards, which results in primer unblocking and rescue of RNA synthesis. Metal ion requirements for nucleotide incorporation and pyrophosphorolysis are similar; the efficiency of both reactions is higher with Mn2+ as compared to Mg2+. Complexes containing chain-terminated primer strands are stable in the presence of heparin, which increases the probability that pyrophosphorolysis occurs before the enzyme can dissociate from its nucleic acid substrate. In contrast to the reverse transcriptase of the human immunodeficiency virus type-1 (HIV-1 RT), the BVDV RdRp may not recruit NTP pools as PPi donors. Conversely, we found that the efficiency of primer unblocking is severely compromised in the presence of increasing concentrations of the NTP that is complementary to the next template position. These data suggest that the incoming NTP can access its designated binding site, which, in turn, prevents the catalytically competent complexation of PPi. The results of this study provide novel insights into mechanisms involved in pyrophosphorolysis associated with viral RdRps, and suggest that the excision reaction is likely to be an important parameter that can affect susceptibility to nucleotide analogue inhibitors directed against viral RdRps.

Sepharose-bound, highly sulfated glycosaminoglycans can capture HIV-1 from culture medium.

In the search for new strategies against HIV-1 and on the basis of a number of previous studies reporting on the capacity of certain polyanionic compounds to influence the replication of HIV-1, we prepared a few chemically oversulfated dermatan and chondroitin sulfates. Four of these compounds and two samples of heparin were bound to activated Sepharose through either their carboxylic groups, or their aldehydic groups, or their deacetylated primary amino groups. Some of these so-derivatised resins, packed into columns, proved able to remove HIV-1 IIIB, a laboratory adapted strain, and one clinical primary isolate from an AIDS patient, from infected cell culture medium. The resins bind the virus very tightly and could be useful for capturing the virus from infected fluids.

Susceptibility to highly sulphated glycosaminoglycans of human immunodeficiency virus type 1 replication in peripheral blood lymphocytes and monocyte-derived macrophages cell cultures.

In the search for new drugs against human immunodeficiency virus type 1 (HIV-1), the replication of III(B) and BaL strains, and of seven primary isolates from AIDS patients, cultured both in peripheral blood lymphocytes (PBLs) and in monocyte-derived macrophages (MACs), was investigated in the presence of two dermatan sulphate and heparin at 10 microg/ml. The three polysaccharides effectively inhibited the replication of III(B) in PBLs and of BaL in MACs, while producing either a slight inhibition or an unexpected large increase in the replication of the seven primary isolates, especially in MAC cultures. In one case, stimulation was found in PBLs and, at lower doses, also with BaL in MACs. Co-receptor use, adaptation to C8166 T cell line, partial sequence of the gp120 V3 loop, variation in positive charge distribution and number of potential glycosylation sites along the V3 loop were assessed for each strain. No explanation could be found for the different susceptibility of the viruses to the polysaccharides. Their presence probably brings about both inhibitory and stimulatory effects, the final outcome depending on the virus, cells and polysaccharide.

Fluorescence anisotropy in the frequency domain by an optical microscope.

Fluorescence anisotropy decay spectroscopy is a suitable tool for investigating the size and the shape of biological molecules. We coupled this technique to an optical microscope in order to reduce the excitation volume and to allow its application to spatially inhomogeneous samples. Phase modulated measurements of the fluorescence anisotropy decay were performed by feeding an intensity modulated linearly polarized laser beam to the epifluorescence port of a microscope. Here we report the test of the dynamic response of the microscope by comparing the lifetime and fluorescence polarization anisotropy decays obtained in cuvettes in a standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor for the objective depolarization is introduced in the best-fit functions for the data analysis of the decays, the results obtained on the two setups are comparable. Some applications are reported here on long DNA tracts as well on short DNA fragments containing structural anomalies.

Aptamers as innovative diagnostic and therapeutic agents in the central nervous system.

Aptamers are short non-naturally occurring single stranded DNA or RNA able to bind tightly, due to their specific three-dimensional shapes, to a multitude of targets ranging from small chemical compounds to cells and tissues. Since their first discovery, aptamers became a valuable research tool and show great application to fundamental research, drug selection and clinical diagnosis and therapy. Thanks to their unique characteristics (low size, good affinity for the target, no immunogenicity, chemical structures that can be easily modified to improve their in vivo applications), aptamers may represent a valid alternative to antibodies particularly for the treatment of neurological disorders that urgently needs modalities for drug delivery through the blood brain barrier. Aptamers have excellent potential as reagents for the targeted delivery of active drug substances, either through direct conjugation to the aptamer, or through their encapsulation in aptamer-coated vesicles. We will review here the recent and innovative methods that have been developed and the possible applications of aptamers as inhibitors or tracers in neurological disorders and brain cancer.

Pyrophosphorolytic excision of nonobligate chain terminators by hepatitis C virus NS5B polymerase.

Nonobligate chain terminators, such as 2'-C-methylated nucleotides, block RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3'-hydroxyl group can be excised in the presence of pyrophosphate (PP(i)), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2'-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2'-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2'-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2'-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.

Control of template positioning during de novo initiation of RNA synthesis by the bovine viral diarrhea virus NS5B polymerase.

The RNA-dependent RNA polymerase of the hepatitis C virus and the bovine viral diarrhea virus(BVDV)is able to initiate RNA synthesis denovo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5'-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3'-end of the template can remain opposite to the 5'-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3'-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotide-primed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme.

Diminished RNA primer usage associated with the L74V and M184V mutations in the reverse transcriptase of human immunodeficiency virus type 1 provides a possible mechanism for diminished viral replication capacity.

The emergence of drug resistance-conferring mutations can severely compromise the success of chemotherapy directed against human immunodeficiency virus type 1 (HIV-1). The M184V and/or L74V mutation in the reverse transcriptase (RT) gene are frequently found in viral isolates from patients treated with the nucleoside RT inhibitors lamivudine (3TC), abacavir (ABC), and didanosine (ddI). However, the effectiveness of combination therapy with regimens containing these compounds is often not abolished in the presence of these mutations; it has been conjectured that diminished fitness of HIV-1 variants containing L74V and M184V may contribute to sustained antiviral effects in such cases. We have determined that viruses containing both L74V and M184V are more impaired in replication capacity than viruses containing either mutation alone. To understand the biochemical mechanisms responsible for this diminished fitness, we generated a series of recombinant mutated enzymes containing either or both of the L74V and M184V substitutions. These enzymes were tested for their abilities to bypass important rate-limiting steps during the complex process of reverse transcription. We studied both the initiation of minus-strand DNA synthesis with the cognate replication primer human tRNA(3)(Lys) and the initiation of plus-strand DNA synthesis, using a short RNA primer derived from the viral polypurine tract. We observed that the efficiencies of both reactions were diminished with enzymes containing either L74V or M184V and that these effects were significantly amplified with the double mutant. We also show that release from intrinsic pausing sites during reverse transcription appears to be a major obstacle that cannot be efficiently bypassed. Our data suggest that the efficiency of RNA-primed DNA synthesis represents an important consideration that can affect viral replication kinetics.