Antiviral Research: Strategies in Antiviral Drug Discovery

Highly active antiretroviral therapy (HAART) based on the combination of different classes of inhibitors has dramatically improved the prognosis of human immunodeficiency virus type 1 (HIV-1) infection after its establishment. This chapter focuses on the viral entry processes that play crucial roles in HIV-1 replication and describes inhibitors thereof. It talks about three kinds of inhibitor named as fusion inhibitors, coreceptor inhibitors and viral attachment inhibitors. Maraviroc was approved for use in combination with other antiretroviral medications for the treatment of R5 HIV-1 in adults whose viral loads remain detectable despite existing antiretroviral treatment or who have multidrug-resistant HIV-1. HIV-1 variants resistant to Cyanovirin-N (CV-N) or cross-resistant to additional carbohydrate-binding agents were generated and examined for their biological properties. A study on resistance to CV-N demonstrated that the resistant variants had increased susceptibility to immunoglobulins and sera obtained from HIV-1-infected patients and particularly to V3-loop-directed monoclonal antibodies. The maraviroc-resistant R5 HIV-1 retained full susceptibility to SCH-C and aplaviroc, suggesting that although the CCR5 binding sites for these agents are similar, their impacts on the surface conformation of the receptor are different. The results of the study also suggest that the envelope proteins of maraviroc-resistant viruses are able to recognize and utilize inhibitor-bound CCR5, which involves the ordered accumulation of mutations in the viral envelope, both in the V3 loop and elsewhere within gp120. In addition to the inhibitors described in the chapter, several antibodies have been shown to interact with the molecules essential for HIV-1 entry to the host cells.

More than 20 different antiretroviral agents have been approved for human immunodeficiency virus (HIV) treatment. These compounds target distinct stages in the life cycle of this retrovirus that include (i) its entry into the cytoplasm, which marks the beginning of the infection; (ii) the process of reverse transcription, i.e., the conversion of the single-stranded RNA genome into double-stranded DNA; (iii) the integration of proviral, double-stranded DNA into the host chromosome; and (iv) the processing of viral precursor proteins at later stages. These steps are vital for viral replication, and with the exception of the entry process, each of the aforementioned reactions involves viral enzymes, i.e., the reverse transcriptase (RT), the integrase, and the protease, respectively, that can be targeted by antiretroviral drugs. This chapter focuses on nucleoside analogue RT inhibitors (NRTIs) in the context of mechanisms of action and resistance and on the implications for the development of future strategies designed to counteract resistance. All approved NRTIs show a broad spectrum of antiviral activity against HIV-1, HIV- 2, and sometimes even hepatitis B virus (HBV), which points to structurally highly related active sites. A given mutation or mutational cluster can affect susceptibility to different NRTIs to various degrees, which makes it difficult to group the mutations. It will be interesting to investigate how established and novel NRTIs can be most effectively combined with new classes of compounds with the ultimate goal of further reducing the risk of resistance development, while maintaining high standards regarding problems associated with toxicities and dosing.

This chapter discusses the potential of HIV Tat, Rev, and their cellular cofactors as drug targets. Tat interacts with numerous transcriptional regulatory factors and presumably by virtue of its interaction with transcription activation response (TAR) RNA, recruits these factors to the human immunodeficiency virus type 1 (HIV-1) promoter. Thus, HIV-1 transcription directed from the viral long terminal repeat (LTR) is balanced by the actions of kinases and phosphatases, and both can be potential targets for drug development. Tat promotes the elongation of viral transcripts by increasing the occupancy time of CDK9/cyclin T1 on the HIV-1 LTR. Small-molecule inhibitors of Tat-TAR RNA interaction, small-molecule inhibitors of CDKs such as CDK9 or CDK2, and inhibitors that disrupt the interaction of Tat with additional host cell factors such as p300/CREB binding protein-associated factor (PCAF) and/or inhibit cellular activities of the host cell factors could all be viable anti-HIV drug candidates. Both Tat and TAR RNA are essential for activated HIV-1 transcription, and were thus the first candidates to be considered for drug design that targeted HIV-1 transcription. Antiviral development against HIV-1 regulatory proteins Tat and Rev represents a conceptual work in progress. In the arena of inhibiting Tat and Rev cellular cofactors, the concerns over host toxicity have not been fully resolved. Future efforts are needed to address the two major challenges of mechanistic specificity and functional toxicity.

This chapter focuses on Hepatitis C virus (HCV) polymerase as a drug target for designing non-nucleoside inhibitors, and discusses details related to HCV sequence variation and quasispecies, HCV polymerase structure and mechanism, classes of polymerase inhibitors, inhibitors currently under development, the nature of drug resistance, and the need for combination therapy. Among the non-nucleoside inhibitors, compounds with thiophene carboxylic acid, pyranoindole, dihydropyranone, or phenylalanine scaffolds have been shown to bind to HCV polymerase in the thumb allosteric pocket by X-ray cocrystallization structures. C316Y and G554D appear to be the most important resistance mutations, suggesting overlapping resistance with HCV-796 and likely binding to the HCV polymerase active-site priming nucleotide pocket. Both sequence variations between genotypes and the quasispecies nature of HCV may contribute to drug resistance. Patients may have low levels of preexisting variants in their quasispecies that are resistant to antiviral drugs. These preexisting variants may be selected upon drug treatment and quickly become the dominant species. Many groups have reported on resistant mutants selected with various HCV polymerase inhibitors in vitro using the HCV replicon system. GSK reported several mutants against a benzothiadiazine (Compound 4), including M414T and N411S. Due to the high replication rate of HCV and errorprone nature of HCV polymerase, monotherapy will lead to the selection of preexisting resistance mutations and ultimately to the inability to eradicate HCV.

This chapter summarizes many targets for orthopoxviruses that might be exploited in the discovery of additional agents for potential diseases and indicates the mechanisms of action of a number of compounds. The viral DNA polymerase performs a critical role in viral replication, and its susceptibility to nucleoside analogs has made it the dominant target for the development of antiviral drugs. The synthesis of deoxynucleotides is also catalyzed by the heterodimeric ribonucleotide reductase encoded by F4L and I4L. Ribonucleotide reductase converts ribonucleotides to the corresponding deoxyribonucleotides at the level of the diphosphate and supplies deoxyribonucleoside triphosphates to support DNA replication in the cytoplasm. A number of small molecules are known to broadly target protein kinases, and it is possible that medicinal chemistry efforts could identify selective inhibitors. Immature virions containing genomic DNA undergo complex maturation that leads to the condensation of virus cores and involves proteolytic events, the loss of D13, and the formation of new disulfide linkage catalyzed by the viral redox system. The complex replication cycle of the orthopoxviruses requires the concerted activities of numerous viral proteins. The chapter also summarizes each of the stages of viral replication, notes the viral proteins that perform critical functions, and identifies specific inhibitors that affect these processes.

Severe acute respiratory syndrome (SARS) is the first new infectious disease of this century, caused by a novel human coronavirus (SARS-CoV), and the disease is associated with severe pulmonary pathological features leading to high mortality. This chapter talks about different kinds of inhibitors such as small-molecule inhibitors, peptide, and papain-like proteinase inhibitors. SARS-CoV is a positive-sense single-stranded (ss) RNA virus. The 30-kb genome is predicted to encode at least 10 open reading frames (ORF), some of which encode proteins involved in virus entry into cells. Receptor-virus interaction can be inhibited using two approaches. First, develop inhibitors that block the cellular receptor with which the virus attachment protein interacts. Second, block the domain in the virus attachment protein that binds to the cellular receptor. Researchers have determined the crystal structures of human coronavirus 3CLpro and suggested that the rhinovirus 3CLpro inhibitor AG7088 could serve as a starting point for an anti-SARS drug based on the theoretical homology model of SARS-CoV 3CLpro. Researchers have showed that the RNAi targeting of the coronavirus RdRp using synthesized short hairpin expression plasmids significantly reduced the expression of target protein in 293 cells and HeLa cells and blocked plaque formation of SARS-CoV in Vero E6 cells. Development of inhibitors of the innate immune response might also be worthwhile, with the caveat that humans, like animals, are reservoirs of mixed infections, latent, chronic, and acute, and that down-regulating an immune response to ameliorate a SARS infection may exacerbate a coexisting infection from another infectious agent.

Recently, the most significant progress in knowledge of hepatitis C virus (HCV) biology and interaction with host cells has concerned the role of lipids for genome replication, assembly and egress, and entry into cells. The major hepatic consequence of HCV infection is the progression to cirrhosis and its complications, such as ascites, hepatic insufficiency, and hepatocellular carcinoma (HCC). With the identification in 1989 of HCV as the infectious agent responsible for non-A/non-B hepatitis and the development of specific detection tests, it has become possible to monitor the efficacy of interferon (IFN)-α treatment. With the increased survival associated with the use of highly active antiretroviral therapy (HAART), morbidity and mortality from HCV-induced liver disease have started to increase significantly. Microarray results from liver biopsy tissue taken before therapy in a cohort of patients given pegIFN and ribavirin were recently reported. In this study, patients who were subsequently identified as non-responders had high baseline expression of IFN-stimulated genes (ISGs), whereas responders to therapy more closely resembled healthy controls. Kempf et al. illustrated the need for good knowledge of drug metabolism to improve the efficacy of a drug in vivo. In this study it was shown that a pharmacokinetic enhancement of telaprevir and boceprevir could be achieved by codosing with ritonavir, a potent inhibitor of the cytochrome P450 3A, which is involved in the metabolism of both drugs.

Phenotypic susceptibility assays are used in the laboratory to measure the susceptibility of a virus to an antiviral agent. They are used for some viruses, such as human immunodeficiency virus type 1 (HIV-1), in clinical settings to help construct the drug regimen most active against an individual patient’s virus. This chapter summarizes the technology behind commonly used antiviral drug susceptibility assays for HIV-1 and the interpretation of results generated by them. Currently there are two approved anti-HIV drugs that target virus entry: the fusion inhibitor enfuvirtide (ENF) and the CCR5 coreceptor antagonist maraviroc. The chapter describes phenotypic assays for determining HIV-1 coreceptor tropism. Assays that determine HIV-1 viral tropism in a patient are needed to allow the clinician to make an informed decision as to whether a CCR5 co-receptor antagonist is an appropriate drug choice for a given patient. Two standardized MT-2 assay approaches have been described. In the first, viral stocks from stimulated patient lymphocytes co-cultured with lymphocytes from an HIV-negative donor are generated. The second method utilizes direct cocultivation of patient lymphocytes with MT-2 cells, followed by microscopic examination. Currently approved, interferon/ribavirin-based treatment of hepatitis C virus (HCV) infection is incompletely effective and is associated with significant toxicity and tolerability issues. Approaches to measure HCV drug susceptibility can be broadly classified into four categories: (i) intact virus assays, (ii) replicon-based recombinant virus assays (RVAs), (iii) cell-based (nonreplicon) assays, and (iv) enzymatic assays. Phenotypic assays are useful during preclinical development, clinical development, and potentially in clinical practice.

This chapter provides a general introduction and overview of the interferon (IFN) pathway. Separate sections of this chapter address the different types of IFN, activation of IFN-α/β expression, IFN-mediated signal transduction, antiviral mechanisms, and viral resistance to the IFN-α/β response. The IFNs are classified into three types (I, II, and III) based on receptor usage, sequence homology, and chromosome location of their genes. The chapter primarily focuses on the type I IFNs, as these cytokines are most closely associated with antiviral activity and therapeutic use. Like RNA, cytosolic DNA also induces the production of IFN-β. Therefore, in addition to RNA viruses, a mechanism also exists to detect the genomes from DNA viruses in the cytoplasm of infected cells. The Toll-like receptors (TLRs) are critical for recognizing different molecular patterns that are unique to viral or bacterial pathogens. As with the cytoplasmic sensors, specificity for pathogen-derived nucleic acids by the TLRs is at least in part provided at the level of nucleic acid modifications. IFN-α/β is important for certain aspects of the adaptive immune response and serves as one example of a bridge between innate and adaptive immunity. In addition to antiviral and immunomodulatory functions, IFN-α/β can also negatively influence cellular proliferation. Future research into improved IFN-based antiviral therapies might focus on improving the antiviral properties of these cytokines while decreasing the unwanted side effects. This goal might be accomplished in a number of distinct ways.

The human alphaherpesviruses include human herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV). HSV-1 and HSV-2 are responsible for primary and recurrent herpetic lesions of the mouth and genital tract, as well as more serious and potentially life-threatening infections of the eye and central nervous system. This chapter discusses alternative targets for antiherpesviral therapy. The roles of various viral glycoproteins in the attachment and penetration steps at the outset of the infection cycle are only now being identified; however, as with human immunodeficiency virus (HIV), the processes of attachment, receptor recognition, and penetration are likely be excellent targets for the development of antiviral therapy. Several reports have described a class of agents that inhibit the adsorption of the virus to host cells. Viral genes are classified as immediate early, early, or late and are transcribed from both strands of the viral genome by cellular RNA Pol II. The authors have proposed that recombination-dependent DNA replication plays an important role in viral DNA replication. Procapsids formed in the nucleus are competent to undergo encapsidation of the viral genome accompanied by a major conformational change in its structure from a sphere to a more angular shape.