Design, synthesis and biological evaluation of novel bioactive nucleosides and nucleotides

At the present there are 36 approved antiviral drugs in the UK of which half are nucleoside analogues. However, the emergence of drug resistance and of new virus strains necessitates new drugs. In particular in this thesis, different nucleoside analogues were studied as potential antivirals. One of the major issues related to nucleoside analogues is the emergence of resistance due to a lack of bioactivation to the monophosphate form. To overcome this issue, the phosphoramidate ProTide technology can be applied. This strategy allows the delivery of the monophosphate form directly inside the cell. Bicyclic nucleoside analogues are a new class of anti-varicella zoster agents of which Cfl743 is the most potent anti-varicella zoster compounds reported to date. Its 5'-valyl derivative, FV100, is currently in phase II clinical trials. A series of derivatives to increase the activity and to investigate the mechanism of action of this new class of compound are reported. Moreover, attempts to improve the scale up synthesis of FV100 are described. Ribavirin is a broad spectrum antiviral drug. The application of the ProTide approach to this compound was not successful. Enzymatic and molecular modelling studies have been performed in order to understand the lack of activity. Acyclovir and its esters are currently the treatment of choice for herpes simplex and varicella-zoster infections. The application of the ProTide technology gave surprising results. In fact, these compounds have been found to be active against HIV, whilst ACV itself did not show any activity. Moreover, these compounds retained activity versus thymidine kinase deficient strains against which acyclovir lost activity. These striking results prompted us to investigate other different nucleoside analogues, through a virtual screening using reverse transcriptase, guanylate or adenylate kinase and human polymerase y. The selected nucleoside analogues from this study include: ganciclovir, penciclovir and their derivatives. ProTides of these are thus pursued

From ligand to complexes: inhibition of HIV-1 Integrase by beta-diketo acid metal complexes

Recently, a class of compounds bearing a β-diketo acid moiety have emerged as the most promising lead in anti-HIV-1 IN drug discovery. It is believed that the β-diketo acid pharmacophoric motif could be involved in a functional sequestration of one or both divalent metal ions, which are critical cofactors at the enzyme catalytic site. This would subsequently block the transition state of the IN-DNA complex. In this scenario, it is of paramount importance to acquire information about the mode of action of diketo acids, which could then be useful in the design of new compounds as IN inhibitors

Investigation of furo[2,3-h]- and pyridazino[3,4-f]cinnolin-3-ol scaffolds as substrates for the development of novel HIV-1 integrase inhibitors

With the aim to develop novel HIV-1 integrase inhibitors, we obtained a set of condensed ring systems based on the furo[2,3-h]cinnolin-3(2H)-one and pyridazino[3,4-f]cinnolin-3-ol scaffolds bearing a potential chelating pharmacophore, which can be involved in the inhibition mechanism of the enzyme. Herein, we report the design, synthesis, structural investigation and preliminary biological results of these heteroaromatic systems

Synthesis of bis-amides and hydrazide-containing derivatives of malonic acid as HIV-1 integrase inhibitors

With the aim to identify novel and/or unified putative pharmacophore required for activity we selected and formally combinated the main structural motifs of I and II together to the hydrazide fragment of compounds III and IV, previously reported as new class of selective IN inhibitors having antiviral activity. Also, the possibility to generate a potential metal chelating pharmacophore has been considered. With this in mind, we designed two sets of symmetrical and unsymmetrical bis-amides and hydrazide derivatives of malonic acid

d-Tubocurarine and Berbamine: Alkaloids That Are Permeant Blockers of the Hair Cell's Mechano-Electrical Transducer Channel and Protect from Aminoglycoside Toxicity

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell’s stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 µM) or berbamine (≥1.55 µM) protect zebrafish hair cells in vivo from neomycin (6.25 µM, 1 h). Protection of zebrafish hair cells against gentamicin (10 µM, 6 h) was provided by ≥25 µM dTC or ≥12.5 µM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 µM, 48 h) by 20 µM berbamine or 25 µM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 µM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 µM (dTC) and 2.8 µM (berbamine) in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell’s basolateral K + current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants

Virtual Screening of acyclovir derivatives as potential antiviral agents: design, synthesis, and biological evaluation of new acyclic nucleoside ProTides

Following our findings on the anti-human immunodeficiency virus (HIV) activity of acyclovir (ACV) phosphate prodrugs, we herein report the ProTide approach applied to a series of acyclic nucleosides aimed at the identification of novel and selective antiviral, in particular anti-HIV agents. Acyclic nucleoside analogues used in this study were identified through a virtual screening using HIV-reverse transcriptase (RT), adenylate/guanylate kinase, and human DNA polymerase γ. A total of 39 new phosphate prodrugs were synthesized and evaluated against HIV-1 (in vitro and ex vivo human tonsillar tissue system) and human herpes viruses. Several ProTide compounds showed substantial potency against HIV-1 at low micromolar range while the parent nucleosides were not effective. Also, pronounced inhibition of herpesvirus replication was observed. A carboxypeptidase-mediated hydrolysis study was performed for a selection of compounds to assess the formation of putative metabolites and support the biological activity observed

Design and synthesis of novel DNA binders

In this context, molecular recognition of DNA by polycyclic heterocycles having a planar structure bearing appropriate side chains have been widely investigated. In the course of our work aimed at developing novel heterocycles of pharmaceutical interest, we designed and synthetized several templates as potential substrate in drug design

Docking ligando-DNA e validazione della capacità intercalante di nuovi derivati "two-armed"

In questo contesto è risultato di grande interesse cercare di studiare la capacità intercalante e le modalità di binding di noti ligandi (Actinomicina D, Etidio bromuro, Distamicina) attraverso uno studio computazionale con tecniche di docking che potesse risultare di utile supporto nella ricerca di nuovi agenti intercalanti. E’ stato quindi messo a punto un versatile metodo di docking tramite l’utilizzo del programma AutoDock operando in diverse condizioni sperimentali

Design, synthesis and biological evaluation of a new series of carvedilol derivatives that protect sensory hair cells from aminoglycoside-induced damage by blocking the mechanoelectrical transducer channel

Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious bacterial infections but have use-limiting side effects including irreversible hearing loss. Here, we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo zebrafish assays and investigated its mechanism of protection which, we found, may be mediated by a block of the hair cell’s mechanoelectrical transducer (MET) channel, the major entry route for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues were prepared and evaluated for their effect against AG-induced damage as well as their affinity for the MET channel. One derivative was found to confer greater protection than carvedilol itself in cochlear cultures and also to bind more tightly to the MET channel. At higher concentrations, both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting a good therapeutic window under in vivo conditions

Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues

For most viruses, there is a need for antimicrobials that target unique viral molecular properties. Acyclovir (ACV) is one such drug. It is activated into a human herpesvirus (HHV) DNA polymerase inhibitor exclusively by HHV kinases and, thus, does not suppress other viruses. Here, we show that ACV suppresses HIV-1 in HHV-coinfected human tissues, but not in HHV-free tissue or cell cultures. However, addition of HHV-6-infected cells renders these cultures sensitive to anti-HIV ACV activity. We hypothesized that such HIV suppression requires ACV phosphorylation by HHV kinases. Indeed, an ACV monophosphorylated prodrug bypasses the HHV requirement for HIV suppression. Furthermore, phosphorylated ACV directly inhibits HIV-1 reverse transcriptase (RT), terminating DNA chain elongation, and can trap RT at the termination site. These data suggest that ACV anti-HIV-1 activity may contribute to the response of HIV/HHV-coinfected patients to ACV treatment and could guide strategies for the development of new HIV-1 RT inhibitors