Current Pharmaceutical Design - Volume 12, Issue 11, 2006

Over 400 pathogenic viruses that infect humans have been identified. Moreover, newly emerging viruses, such as SARS-CoV, and avian flu, post a constant threat. Unfortunately, only limited approved treatments exist for viral infections. Therefore, it is urgent and crucial to develop more effective and nontoxic antiviral agents. In preparation this issue, we have sought to provide useful information for scientists with an interest in antiviral research. These studies review the current status of drug discovery based on specific molecular targets in virus infection, or focus on specific virus families. In the first article, Hsu and Coworkers [1] focused on viral protease as the molecular targets for the development of antiviral agents. Viral protease usually plays an essential role in viral replication. Viral polypeptide must be further cleaved by viral protease to create mature, functional protein. The fact that several FDA-approved protease inhibitors can effectively treat HIV-1 patients and a class of hepatitis C virus protease inhibitors can reduce HCV RNA levels in patient's plasma highlighted the considerable potential of viral protease as the molecular target for antiviral drug discovery. This paper not only summarizes the classification, biochemical property, and functional role of viral protease, but also reviews the specific protease inhibitors for each virus. Notably, some protease inhibitors can inhibit more than one virus, for example, an anti-HIV agent also acts as a SARS-CoV 3CLpro inhibitor. Consequently, understanding the molecular features of each viral protease can help in developing effective antiviral therapies. Frick and Lam [2] discussed recent progress in antiviral research based on viral helicase as the molecular target. Helicases are essential for viral genome replication, transcription and translation. Numerous potent helicase inhibitors have been demonstrated to reduce viral replication in cell cultures and animal models, such as SF1 helicase inhibitors against HSV and SARS-CoV; SF2 helicase inhibitors against HCV, West Nile virus and JEV; and SF3 helicase inhibitors against HPV. This article also discusses in detail the classification, structure and function of viral helicases. All viral helicases share a common motor function fueled by ATP hydrolysis, but differ in precisely how the motor moves the protein and its cargo on a nucleic acid chain. The mechanism of these helicases inhibitors is based on influencing rates of helicase-catalyzed viral DNA or RNA unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or disrupting the interaction of a helicase with a required cofactor. Tsai and coworkers [3] focused on the current antiviral research based on viral polymerase as the molecular target. Polymerase has been classified into four classes, RNA-dependent RNA polymerase (RDRPs), DNA-dependent RNA polymerase (DDRPs), RNA-dependent DNA polymerase (reverse transcriptase) and DNA-dependent DNA polymerase (DNA pol). All of these polymerases share common structures and functions, but have different specific features for different viral polymerases. For example, cap-snatching activity is a specific and unique feature for influenza viral RDRP. This article discusses in details not only about the inhibition modes of antiviral compounds targeting to viral polymerase, but also addresses the resistance mechanism of these antiviral agents. The success of anti-HSV therapy by acyclovir and anti-HIV therapy by AZT strongly indicated that intensive structural and functional studies of viral polymerases are potentially important in developing more effective antiviral therapy. Schang [4] reviewed antiviral drug discovery for herpes simplex viruses (HSV). The first efficacious and safe antivirals were developed against HSV. However, the emergence of drug-resistant viral strains creates some limitations in viral polymerase inhibitors. Schang pointed out that cellular proteins are currently considered to be alternative potential targets for novel anti-HSV agents. Schang described the discovery of pharmacological CDK inhibitors (PCIs). CDKs are a family of cellular protein kinases that are employed for HSV replication. CDK2 has been shown to be nonessential for mammals, and has overcome concerns regarding the restriction of PCIs. Furthermore, PCIs hinder the selection of drug-resistant mutants, and are effective against strains that have developed resistance to conventional antiviral drugs. PCIs are also active against various unrelated viruses, which makes them promising as drugs against new emerging viral diseases. Tanikawa [5] discussed the current development of antivirals against hepatitis viruses, with an emphasis on HBV and HCV. Estimates indicate that 350 million people are infected with HBV and 170 million people with HCV.........

Proteases fulfill multiple roles in health and disease, and considerable interest has been expressed in the design and development of synthetic inhibitors of disease-related proteases. Virus-encoded proteases have been shown to be involved in the replication of many viruses. The success of anti-HIV-1 therapy using specific and potent protease inhibitors suggests that viral proteases can be the valid molecular targets for the development of antiviral drugs against other viruses. Intensive genetic and biochemical studies have been conducted on viral proteases and new insights and results are rapidly emerging. This work reviews features of viral proteases with respective to the development of effective antiviral therapy.

Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a required cofactor.

Viral DNA and RNA polymerases are enzymes, which are responsible for copying the genetic materials of viruses and are therefore central components in the life cycles of viruses. The polymerases are essentially required for the replication of viruses. The reverse transcriptase (RT) of the retroviruses and the hepadnaviruses is the sole viral enzyme required for the synthesis of DNA from viral RNA. Viral polymerases are therefore an extremely favorable target for the development of antiviral therapy. The success of anti-HIV-1 therapy using inhibitors specifically targeting HIV RT suggests that other viral polymerases can be the valid molecular targets for the design of antiviral drugs. Intensive structural and functional studies of viral polymerases have been conducted and have opened new avenues for the development of more effective antiviral therapy. This review summarizes the insights gained from recent structural and functional studies of antiviral agents, which target viral polymearses. The primary focus will be on hepatitis C virus (HCV), herpesviruses, HIV, hepatitis B virus (HBV) and influenza virus.

Since the 1950's, herpes simplex viruses (HSV) have played prominent roles in the development of antivirals. The first efficacious antivirals, as well as the first safe for systemic use, were developed against HSV. It was only in 1995 when the first antiviral against a target not validated first with HSV was approved for clinical use (sequinavir). The early successes of targeting HSV DNA replication were most influential in developing the concept that antiviral drugs must target viral proteins. Such concept has ensured the safety of current antiviral drugs, which all target viral proteins. Current antivirals have proven to have no major negative effects on non-infected cells or treated patients. Because of their success widespread and use, however, these drugs have been found to have some limitations. Perhaps the clinically most important one is their ability to promptly select for drug-resistant viral strains. Owing to such limitations, cellular proteins are now considered as valid targets for novel antiviral drugs. The discovery that pharmacological CDK inhibitors (PCIs) inhibit HSV replication through novel mechanisms played a major role in this new appreciation of cellular proteins as potential targets for antivirals. This appreciation is reflected in the scheduling of PCIs to enter clinical trials as antivirals (against HIV). Herein, we will review the roles of HSV as model viruses in the discovery and development of antiviral drugs. The major focus will be on the recent discovery on the activities of PCIs against HSV and other viruses.

Hepatitis B virus (HBV)- or hepatitis C virus (HCV)- associated liver diseases are now one of the important health problems in the world because of the high numbers of patients and the serious consequences. Recently, however, relatively effective treatments with antiviral agents have become available. Interferon (IFN), lamivudine and adefovir are now approved for treatment of HBV-associated liver diseases and they have been shown to be fairly effective. The goal of treatments for HBV-associated liver disease is to achieve a clinical cure in as short a period as possible without producing resistance mutation of the virus. Several nucleotide analogues with more potent antiviral activities are now in clinical trials. In the case of HCV-associated liver diseases, Pegylated IFN (Peg IFN) + ribavirin combination therapy is the standard and most effective treatment with a sustained response of 60-70%. The goal of the treatments for these liver diseases is to induce the complete eradication of the infected virus and at present new anti HCV drugs targeting the molecular segments of the virus are under development. It is expected that the complete eradication of infected virus will be possible in most cases in the near future.

Picornaviruses are important human pathogens causing severe morbidity and some mortality with the potential to cause worldwide crippling disease. Currently, there are few treatments for many of the viruses in the Picornaviridae, For rhinoviruses, there are no approved treatments, although ruprintrivir looks promising in clinical trials and pyridazinyl oxime ethers may prove useful. Poliovirus treatments are needed to supplement the World Health Organization's polio eradication plan in order to treat infections caused by reversion of the attenuated vaccine virus and to supplement vaccine coverage control in polio endemic areas. However, no promising compounds for treatment of poliovirus have been developed due to the efficacy of the vaccines in use. Broad-spectrum inhibitors developed for other picornavirus may be useful for poliovirus infections. Coxsackievirus infections in children and in infants are being treated with pleconaril with some efficacy in reducing mortality and improving recovery, albeit the treatment is often on a compassionate use basis. There are no therapies for echovirus infections. Very little drug discovery research is being done to develop inhibitors for echovirus infections, probably due to the broad-spectrum inhibition exhibited by capsid binding agents and protease inhibitors discovered for treatment of other picornaviruses. For example, pyridazinyl oxime ethers are inhibitory to most echoviruses. Treatments for enterovirus infections are also limited, although in a small clinical trial, milrinone seemed to reduce mortality and improve recovery from EV71-induced pulmonary edema. Thus, these results strongly emphasize the need for the development of potent and nontoxic compounds for the treatment of picornavirus infections.

Substantial progress in the development of new anti-viral drugs has taken place in recent years. Most of these new drugs belong to three groups of compounds, nucleoside analogs, thymidine kinase-dependent nucleotide analogs and specific viral enzyme inhibitors. Although these drugs revolutionized the treatment of several viral diseases, the involvement of the immune system is crucial for complete recovery and prevention of re-infection. New advances in the understanding of immune regulation mechanisms, mainly the role of cytokines, led to the development of several new immunologically- based anti-viral drugs and treatments. The most studied group of immunomodulators is the cytokines, some of which were shown to act as potent stimulators of immune responses. Other, non-cytokine immune modulators have also been successfully employed in both humans and experimental animals as anti-viral drugs of which several are currently in clinical trials. Advances in genetic engineering and transgenic mouse technologies facilitated the production of humanized as well as authentic human anti-viral monoclonal antibodies. Some of these antibodies proved to be clinically efficacious and are commercially produced as anti-viral drugs. As is often the case in anti-viral treatments, a combination of conventional and an immune-mediated anti-viral drugs or a combination therapy involving immunomodulators, therapeutic vaccines, immune intervention and even gene therapy might prove most efficacious as a treatment for a particular virus. Most of the advances made in anti-viral treatments have also been applied to the development of new vaccines. Some of the classical attenuated viruses are being replaced by recombinant attenuated viruses. Recombinant viral vaccines containing genes encoding other viral antigens and/or cytokines are being tested as new vaccines. Several chimeric viral vaccines have proven efficacious in experimental animals and are now in different phases of clinical trials. This review will encompass the major new developments in this field, including some that have not yet been subjected to human trials.

In recent years pharmaceutical design has been facing the needs expressed by new therapeutic methodologies such as gene therapy, targeted delivery and closely related diagnostic fields as contrast enhancing agents for ultrasonic investigations. In this context pharmaceutical research has diversified the efforts toward a more integrated approach where the efficacy of an active molecule is enhanced and assisted by the surrounding carrier. Usually this drug platform is a hydrogel matrix, a multicomponent system constituted by an aqueous solution and a polymeric moiety imparting different functions to the matrix, as responsiveness to external stimuli, affinity to receptors, controlled drug release. Such devices represent one of the leading topics of the soft condensed matter recent research, a domain where physics, chemistry and bioengineering cross each other with the aim to achieve an integrated description of these materials. In this respect modern drug design will make use more and more of concepts proper of soft condensed polymer and colloidal sciences. In this review we will describe the state-of-art in the field of the matrices used in innovative drug formulations with a particular emphasis on the implications to pharmaceutical design along with the experimental and theoretical investigation tools worked out in the last decade.

The pig and especially the minipig are becoming increasingly used as a test animal both in pharmacological and toxicological testing of new compounds. The minipig is used because of its size, it is easy to handle and less test substrate is required. When using an animal species for testing it is of importance to know if the test animal's posses the same abilities to metabolize drugs as humans. Some of the P450 enzymes have been characterized in the pig regarding substrate specificity, inhibition and regulation. The porcine enzymes CYP1A, CYP2A and CYP3A all metabolize the same test substrates as the human enzymes, whereas the enzymes CYP2B, CYP2D, and CYP2E in pig on the other hand seem to be different from the human enzymes concerning metabolism of the well know test substrates. Some of the porcine enzymes have been sequenced i.e. CYP1A, CYP2A, CYP2B, CYP2D, CYP2E and CYP3A and not surprisingly the porcine CYPs that metabolize the human test substrates are about 75% identical in cDNA sequences. What is needed is inhibitory antibodies against each of the porcine enzymes, in order to test whether a test compound is metabolized by one or the other enzyme. Until now chemical inhibitors have been used, but they are rarely 100% specific. Anti-human inhibitory antibodies have also been used, but they may not recognize the porcine enzyme and therefore will not inhibit the reaction. Antibodies for immunoblotting would also make it possible to estimate how much of the total P450 the individual enzymes comprise. From what is known about the porcine P450, it can be concluded that the pig seems to be a good test species if CYP1A, CYP2A or CYP3A are involved in the metabolism of the test compound, depending on the contribution of other enzymes in competing pathways.