Biomedical subjects

G A Locatelli

Publications and source records attributed to G A Locatelli.

4 recordsLinked to original sources

PubMed2004

HIV-1 reverse transcriptase inhibitors: current issues and future perspectives.

One of the major advances in the recent history of the treatment of HIV infections has been the development of different classes of effective antiretroviral drugs. In particular, the reverse transcriptase (RT) inhibitors still represent the majority of the clinically used anti-HIV drugs and constitute the main backbone of currently employed combinatorial regimens. Highly active antiretroviral combination chemotherapy (HAART), combining RT and protease inhibitors, has proven the most effective approach to treat HIV disease, since it has been shown to markedly suppress viral replication and appearance of drug resistance for a relatively long period. These therapies, however, do not constitute a definitive cure, since they are not able to completely eradicate the virus from the infected individual. Beside drug toxicity problems, the emergence of drug resistance associated with the particular regimen employed further complicates the situation. This review will summarise the most recent achievements, as well as the future directions in the development of novel anti-RT compounds.

Animals↗

PubMed2001

Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1.

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.

Animals↗

PubMed2001

Hepatitis C virus NS3 NTPase/helicase: different stereoselectivity in nucleoside triphosphate utilisation suggests that NTPase and helicase activities are coupled by a nucleotide-dependent rate limiting step.

Hepatitis C virus (HCV) NS3 protein is a multifunctional enzyme, possessing protease, NTPase and helicase activities within a single polypeptide of 625 amino acid residues. These activities are essential for the virus life cycle and are considered attractive targets for anti-HCV chemotherapy. Beside ATP, the NS3 protein has the ability to utilise deoxynucleoside triphosphates (dNTPs) as the energy source for nucleic acid unwinding. We have performed an extensive analysis of the substrate specificities of both NS3 NTPase and helicase activities with respect to all four dNTPs as well as with dideoxynucleoside triphoshate (ddNTP) analogs, including both d-(beta) and l-(beta)-deoxy and dideoxy-nucleoside triphosphates. Our results show that almost all dNTPs and ddNTPs tested were able to inhibit hydrolysis of ATP by the NTPase activity, albeit with different efficiencies. Moreover, this activity showed almost no stereoselectivity, being able to recognise both d-(beta), l-(beta)-deoxy and ddNTPs. On the contrary, the helicase activity had more strict substrate selectivity, since, among d-(beta)-nucleotides, only ddTTP and its analog 2',3'-didehydro-thymidine triphosphate could be used as substrates with an efficiency comparable to ATP, whereas among l-(beta)-nucleotides, only l-(beta)-dATP was utilised. Comparison of the steady-state kinetic parameters for both reactions, suggested that dATP, l-(beta)-dCTP and l-(beta)-dTTP, specifically reduced a rate limiting step present in the helicase, but not in the NTPase, reaction pathway. These results suggest that NS3-associated NTPase and helicase activities have different sensitivities towards different classes of deoxy and dideoxy-nucleoside analogs, depending on a specific step in the reaction, as well as show different enantioselectivity for the d-(beta) and l-(beta)-conformations of the sugar ring. These observations provide an essential mechanistic background for the development of specific nucleotide analogs targeting either activity as potential anti-HCV agents.

Adenosine Triphosphate↗

PubMed2001

The stereoselective targeting of a specific enzyme-substrate complex is the molecular mechanism for the synergic inhibition of HIV-1 reverse transcriptase by (R)-(-)-PPO464: a novel generation of nonnucleoside inhibitors.

The human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor pyrrolopyridooxazepinone (PPO) derivative, (+/-)-PPO294, was shown to be active toward wild type and mutated HIV-1 RT and to act synergistically in combination with 3'-azido-3'-deoxythymidine (Campiani, G., Morelli, E., Fabbrini, M., Nacci, V., Greco, G., Novellino, E., Ramunno, A., Maga, G., Spadari, S., Caliendo, G., Bergamini, A., Faggioli, E., Uccella, I., Bolacchi, F., Marini, S., (1999) J. Med. Chem. 42, 4462-4470). The (+/-)-PPO294 racemate was resolved into its pure enantiomers, and the absolute configuration was determined by x-ray analysis. Only one enantiomer, (R)-(-)-PPO464, displayed antiviral activity against both the wild type and the K103N mutant HIV-1 RT and was found to interact exclusively with the reaction intermediate formed by RT complexed with both the DNA and the nucleotide substrates. Being the first compound of its class to display this behavior, (R)-(-)-PPO464 is the representative of a novel generation of nonnucleoside inhibitors. (R)-(-)-PPO464 showed significant synergism when tested in combination with other RT inhibitors and efficiently inhibited viral replication when tested against the laboratory strain HIV-1 IIIB or against either wild type or multidrug-resistant clinical isolates. Pharmacokinetic studies in mice and rats showed a more favorable profile for (R)-(-)-PPO464 than for the corresponding racemate. (R)-(-)-PPO464 was also found to easily cross the blood-brain barrier. The coadministration of the HIV-1 protease inhibitor ritonavir increased the bioavailability of (R)-(-)-PPO464, having little effect on its plasma and brain elimination rates.

Animals↗