Current Pharmaceutical Design - Volume 10, Issue 30, 2004

In 2003 the first virus entry inhibitor, the anti-HIV peptide T20 (Fuzeon®, enfuvirtide®), was approved for treatment of advanced Human Immunodeficiency Virus Type 1 infection. T20 is an unconventional antiviral drug, as it does not target a viral replicase or protease but a conformational transition within the HIV1 fusion protein gp41 required for virus-cell membrane fusion. Beyond membrane fusion, numerous early drug targets have been identified that will allow for large scale screening or structure-based drug design. The first encounter of the virus with the host might be through binding to attachment receptors, such as the C-type lectins DC- and L-SIGN, which might play an important role of infection for a large number of enveloped viruses by capturing, concentrating and transmitting infectious virions. Once a virus reaches its target cell, a cascade of events generally starting with the interaction of viral envelope glycoproteins with specific entry receptors and co-receptors is necessary in order to trigger the virus-cell membrane fusion. The present review will highlight recent advances in the identification of new drugs and targets at the level of virus entry for three major human pathogens accounting for several hundred million infections worldwide: HIV, Dengue Virus and Hepatitis C virus.

The structures of the catalytic core of two HIV-1 encoded enzymes play a crucial role in the retroviral cycle: integrase and RNase H exhibit striking similarities. These enzymes also share a similar mechanism of catalysis. The homologies between RNase H and integrase led to studying the effect of the RNase H inhibitors on integrase. ODNs aptamers active on RNase H were shown to be strong IN inhibitors. On the contrary, compounds from the diketo acid family were previously known as integrase inhibitors. One compound of this family is able to inhibit the RNase H activity, but has no effect on integrase. Cellular topoisomerase 1 also shares a mechanism similar to that of HIV-1 integrase and RNase H. It has been reported to be present in retroviral particles and to enhance cDNA synthesis. Some topoisomerase inhibitors have been shown to be active on integrase. Moreover, topoisomerase, integrase and RNase H are inhibited by G-rich oligonucleotides. A G-quartet structure is necessary for integrase, but not for topoisomerase inhibition. This suggests that prototype structures can be exploited to develop inhibitors of two related enzymes, such as the RNase H and integrase activities of HIV-1 RT.

In the rush to develop anti-viral drugs against the human immunodeficiency virus type I (HIV-1), all the steps of the viral life cycle are potential targets of therapeutic intervention. In this review, we will explore the recent advances on strategies that aim at obstructing the formation, the release and the infectivity of newly formed virion particles from HIV-1 infected cells.

The concept of using RNA molecules as therapeutic agents is receiving increasing attention by basic science and pharmaceutical research. Over the past five years, a number of clinical trials have been initiated to evaluate the efficacy and safety of several RNA agents for the treatment of a range of conditions from cancer to infectious disease. From a molecular biology perspective, two main factors are implicated in RNA therapeutics against pathogenic RNAs: i / The activity, stability and delivery of the inactivating agent (ribozyme, RNase P, “decoy” RNA, aptamer, small interfering-RNA) and its co-localisation with the target; and ii / The properties of the RNA substrate, which, in the case of an RNA virus, most likely limit the effectiveness of the inactivating agent. The main reasons are the limited size of the viral genome and the restrictions imposed by the RNA structure and variations at the target. In the first section of this article we review three properties of the HCV RNA genome, from primary sequence to tertiary structure, which imply restrictions and opportunities for RNA-based treatment. In the second section, we briefly describe several of the RNA-based therapeutic strategies against HCV now under development.

Picornavirus infections have been a challenging problem in human health. Genome organisation of picornavirus is unique in having a long, heavily-structured, multifunctional 5'untranslated region, preceding a single open reading frame from which all viral proteins are produced. Within the 5'leader, an internal region termed ribosome entry site (IRES) regulates viral protein synthesis in a 5'-independent manner. The IRES element itself is a distinctive feature of the picornavirus mRNAs, allowing efficient viral protein synthesis in infected cells in spite of a severe modification of translation initiation factors induced by viral proteases that lead to a fast inhibition of cellular protein synthesis. Picornavirus IRES elements are strongly structured, bearing several motifs, phylogenetically conserved, which are essential for IRES activity. Together with RNA structure, RNA-binding proteins play an essential role in the activity of the IRES element, having a profound effect on viral pathogenesis. Recent data on the involvement of these conserved motifs in RNA structure and protein recognition is discussed in detail. Understanding the interplay between these two components of IRES function is crucial to develop viral strategies aimed to use the viral RNA as the target of antiviral approaches.

Rotaviruses are an important cause of human morbidity and mortality, representing the primary pathogens responsible for acute dehydrating diarrhea in children under the age of 3. The infectious rotavirus particle is made up of three concentric layers of protein, and contains a genome consisting of eleven segments of double-stranded (ds)RNA. Upon infection, RNA polymerases associated with double-layered virus particles are activated, resulting in genome transcription and extrusion of the eleven viral mRNAs from such particles. The mRNAs not only direct protein synthesis, but also serve as templates for minus-strand synthesis to yield dsRNAs. Synthesis of the dsRNAs is an event that occurs following the gene-specific packaging of viral mRNAs into core-like assembly intermediates. Electron-dense cytoplasmic inclusions, termed viroplasms, function as sites of genome packaging and replication in the infected cell. Our understanding of key events in the viral life cycle has been advanced considerably by the development of cell-free systems that support mRNA synthesis from virion-derived double-layered particles and dsRNA synthesis from virion-derived core particles. The recent expression and purification of rotavirus recombinant proteins have also allowed progress to be made in defining the roles of viral proteins in genome replication and viroplasm formation. However, our efforts towards a full description of the viral life cycle, most notably an understanding of the events occurring during gene-specific packaging, remain hampered by the lack of a cell-free packaging system and a reverse genetics systems. The lack of a reverse genetics systems also confounds efforts towards the generation of molecular engineered second-generation vaccines.

Serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. Within the class of serine proteases, HLE is one of the most destructive enzymes in the body. It is implicated in the promotion or exacerbation of a number of diseases including pancreatitis, acute respiratory syndrome, rheumatoid arthritis, atherosclerosis, pulmonary emphysema, and cystic fibrosis. Thrombin, a trypsin-like serine protease, plays a dual role in thrombogenesis, including fibrin formation and platelet activation. As a result, thrombin constitutes one of the most widely studied targets for antithrombotic strategy. Numerous inhibitors of serine proteases have been reported during the past three decades. Among them, coumarin-type molecules displayed a high inhibitory potency towards various serine proteases. At that time, halomethyl dihydrocoumarins have been shown to behave as the first general suicide inhibitors of serine protease. These molecules inhibit several proteases such as human leucocyte elastase, porcine pancreatic elastase, thrombin, urokinase and human plasmin. Isocoumarins are very effective as mechanism-based inhibitors of serine proteases. Pharmacomodulation on the 3-alkoxy- 4-chloroisocoumarins and the 3-alkoxy-7-amino-4-chloroisocoumarins led to strong inhibitors of numerous serine proteases such as HLE, human factor XIa and XIIa, thrombin, urokinase and kallikrein. Recently, a series of coumarins characterised by an alkyl, aryl ester, amide, thioester or ketone in the position 3 and an electrophilic chloromethyl moiety in the position 6 have been developed. These compounds were found to be high inhibitors of α-chymotrypin, HLE and human thrombin.

The Benzopyrones are a group of compounds whose members include coumarins and flavonoids. Dietary exposure to benzopyrones is quite significant, as these compounds are found in vegetables, fruit, seeds, nuts, coffee, tea and wine. It is estimated that the average western diet contains approximately 1g / day of mixed benzopyrones. It is, therefore, not difficult to see why extensive research into their pharmacological and therapeutic properties is underway over many years. Coumarin is a natural substance that has shown anti-tumour activity in vivo, with the effect believed to be due to its metabolites (e.g. 7-hydroxycoumarin). This review is based on recent studies of coumarins and coumarin related compounds. Therefore, the focus will be on these relevant compounds and their therapeutic importance. A recent study has shown that 7-hydroxycoumarin inhibits the release of Cyclin D1, which is overexpressed in many types of cancer. This knowledge may lead to its use in cancer therapy. Esculetin inhibits growth and cell cycle progression by inducing arrest of the G1 phase in HL-60 leukaemia cells, resulting from the inhibition of retinoblastoma protein phosphorylation. Recent studies investigating the potential of flavonoids as therapeutic agents have suggested they may have use in various therapeutic settings ranging from leukaemia treatment to the treatment of patients with HIV. Genistein is a well-known isoflavone and is a tyrosine kinase inhibitor. Studies have indicated that genistein elicits inhibitory effects on cell growth of various carcinoma cell-lines and may be a potential candidate for cancer therapy. In our research, we have investigated the effects of coumarins and coumarin-related compounds on a panel of cell-lines. The most recent work involves two cell-lines, MCF-7 a breast carcinoma and A549 a lung carcinoma. Microtitre assays were performed along with real-time analysis of cell viability using a biosensor called the Cytosensor microphysiometer. These studies suggest that both genistein and esculetin exerted the most potent inhibitory effect on cell growth in comparison to the other two compounds.

Several natural products with a coumarinic moiety have been reported to have multiple biological activities. It is to be expected that, in a similar way to isomeric flavonoids, coumarins might affect the formation and scavenging of reactive oxygen species (ROS) and influence processes involving free radical-mediated injury. Coumarin can reduce tissue edema and inflammation. Moreover coumarin and its 7-hydroxy-derivative inhibit prostaglandin biosynthesis, which involves fatty acid hydroperoxy intermediates. Natural products like esculetin, fraxetin, daphnetin and other related coumarin derivatives are recognised as inhibitors not only of the lipoxygenase and cycloxygenase enzymic systems, but also of the neutrophil-dependent superoxide anion generation. Due to the unquestionable importance of coumarin derivatives considerable efforts have been made by several investigators, to prepare new compounds bearing single substituents, or more complicated systems, including heterocyclic rings mainly at 3-, 4- and / or 7-positions. In this review we shall deal with naturally occurring or synthetically derived coumarin derivatives, which possess antiinflammatory as well as antioxidant activities.

Fluorescent probes have evolved into an extremely useful tool for the detection of ions in biological systems. The design of ion indicators is based in the proper choice of the ion chelating group as well as the chromophore moiety. The chromophores of choice should fulfill a number of requirements concerning the photostability of the group, the range of the excitation and emission wavelengths of the indicators, the Stokes shift, the fluorescence quantum yield, the excitation and / or emission wavelength shift upon coordination of the probe with its target ion, the lipophilicity of the indicators, and their possible cell toxicity. Coumarin and its analogues have been extensively used in ion detection by incorporation of the coumarin chromophore in the larger indicator framework. Coumarins fulfill all the aforementioned requirements since they are relatively photostable and their excitation and emission maxima, in many cases, are long enough to minimise “background” fluorescence of cellular components, tissues and biological fluids. They exhibit Stokes shifts large enough to avoid significant overlap of the excitation and emission spectra, their fluorescence quantum yields allow for ion detection at low indicator concentrations, and they can be introduced to cells either by microinjection or as membrane permeable derivatives without causing cell death. Synthetic approaches, aiming at the optimisation of indicator properties, have extended the conjugated coumarin system either by introduction of substituents or by expansion of the heterocyclic system. In this review, the basic rationale for the selection of the particular coumarin analogues is analysed, synthetic pathways leading to the desired structures are presented, and properties and relative advantages in the use of these probes are described.