Single-molecule-binding studies of antivirals targeting the hepatitis C virus core protein.

IMPORTANCE: The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals.

Treatment of Chronic Hepatitis B Virus Infection Using Small Molecule Modulators of Nucleocapsid Assembly: Recent Advances and Perspectives.

On the basis of the recent advance of basic research on molecular biology of hepatitis B virus (HBV) infection, novel antiviral drugs targeting various steps of the HBV life cycle have been developed in recent years. HBV nucleocapsid assembly is now recognized as a hot target for anti-HBV drug development. Structural and functional analysis of HBV nucleocapsid allowed rational design and improvement of small molecules with the ability to interact with the components of HBV nucleocapsid and modulate the viral nucleocapsid assembly process. Prototypes of small molecule modulators targeting HBV nucleocapsid assembly are being preclinically tested or have moved forward in clinical trials, with promising results. This Review summarizes the recent advances in the approach to develop antiviral drugs based on the modulation of HBV nucleocapsid assembly. The antiviral mechanisms of small molecule modulators beyond the capsid formation and the potential implications will be discussed.

Discovery of Novel Hepatitis B Virus Nucleocapsid Assembly Inhibitors.

Hepatitis B virus (HBV) core protein is a small protein with 183 amino acid residues and assembles the pregenomic (pg) RNA and viral DNA polymerase to form nucleocapsids. During the last decades, several groups have reported HBV core protein allosteric modulators (CpAMs) with distinct chemical structures. CpAMs bind to the hydrophobic HAP pocket located at the dimer-dimer interface and induce allosteric conformational changes in the core protein subunits. While Type I CpAMs, heteroaryldihydropyrimidine (HAP) derivatives, misdirect core protein dimers to assemble noncapsid polymers, Type II CpAMs, represented by sulfamoylbenzamides, phenylpropenamides, and several other chemotypes, induce the assembly of empty capsids with global structural alterations and faster mobility in native agarose gel electrophoresis. Through high throughput screening of an Asinex small molecule library containing 19 920 compounds, we identified 8 structurally distinct CpAMs. While 7 of those compounds are typical Type II CpAMs, a novel benzamide derivative, designated as BA-53038B, induced the formation of morphologically "normal" empty capsids with slow electrophoresis mobility. Drug resistant profile analyses indicated that BA-53038B most likely bound to the HAP pocket but obviously modulated HBV capsid assembly in a distinct manner. BA-53038B and other CpAMs reported herein provide novel structure scaffolds for the development of core protein-targeted antiviral agents for the treatment of chronic hepatitis B.

Past, Current, and Future Developments of Therapeutic Agents for Treatment of Chronic Hepatitis B Virus Infection.

For decades, treatment of hepatitis B virus (HBV) infection has been relying on interferon (IFN)-based therapies and nucleoside/nucleotide analogues (NAs) that selectively target the viral polymerase reverse transcriptase (RT) domain and thereby disrupt HBV viral DNA synthesis. We have summarized here the key steps in the HBV viral life cycle, which could potentially be targeted by novel anti-HBV therapeutics. A wide range of next-generation direct antiviral agents (DAAs) with distinct mechanisms of actions are discussed, including entry inhibitors, transcription inhibitors, nucleoside/nucleotide analogues, inhibitors of viral ribonuclease H (RNase H), modulators of viral capsid assembly, inhibitors of HBV surface antigen (HBsAg) secretion, RNA interference (RNAi) gene silencers, antisense oligonucleotides (ASOs), and natural products. Compounds that exert their antiviral activities mainly through host factors and immunomodulation, such as host targeting agents (HTAs), programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors, and Toll-like receptor (TLR) agonists, are also discussed. In this Perspective, we hope to provide an overview, albeit by no means being comprehensive, for the recent development of novel therapeutic agents for the treatment of chronic HBV infection, which not only are able to sustainably suppress viral DNA but also aim to achieve functional cure warranted by HBsAg loss and ultimately lead to virus eradication and cure of hepatitis B.

Synthesis and in Vitro Screening of New Series of 2,6-Dipeptidyl-anthraquinones: Influence of Side Chain Length on HIV-1 Nucleocapsid Inhibitors.

2,6-Dipeptidyl-anthraquinones are a promising class of nucleic acid-binding compounds that act as NC inhibitors in vitro. We designed, synthesized, and tested new series of 2,6-disubstituted-anthraquinones, which are able to bind viral nucleic acid substrates of NC. We demonstrate here that these novel derivatives interact preferentially with noncanonical structures of TAR and cTAR, stabilize their dynamics, and interfere with NC chaperone activity.

HIV-1 gag: an emerging target for antiretroviral therapy.

The advances made in the treatment of HIV-1 infection represent a major success of modern biomedical research, prolonging healthy life and reducing virus transmission. There remain, however, many challenges relating primarily to side effects of long-term therapy and the ever-present danger of the emergence of drug-resistant strains. To counter these threats, there is a continuing need for new and better drugs, ideally targeting multiple independent steps in the HIV-1 replication cycle. The most successful current drugs target the viral enzymes: protease (PR), reverse transcriptase (RT), and integrase (IN). In this review, we outline the advances made in targeting the Gag protein and its mature products, particularly capsid and nucleocapsid, and highlight possible targets for future pharmacological intervention.

The inhibitory effect of the hepatitis B virus singly-spliced RNA-encoded p21.5 protein on HBV nucleocapsid formation.

Hepatitis B virus (HBV) is the smallest DNA virus and the major cause of acute and chronic hepatitis. The 3.2 kb HBV viral genome generates four major species of unspliced viral transcript as well as several alternatively spliced RNAs. A 2.2 kb singly-spliced RNA is the most abundant spliced RNA and is widely expressed among all HBV genotypes. The expression of the singly-spliced RNA, as well as that of its encoded protein HBSP, is strongly associated with hepatopathology during HBV infection. Here, we report a novel inhibitory role of a p21.5 protein, which is encoded by a 2.2 kb singly-spliced RNA, in the modulation of HBV replication. We show that overexpression of the singly-spliced RNA is able to efficiently inhibit HBV replication. Furthermore, a mutation in the ATG start codon of the precore region completely abolishes the inhibitory effect of the singly-spliced RNA, indicating that a viral protein (p21.5) derived from the singly-spliced RNA is the mediator of the inhibition. Furthermore, p21.5 is able to form a homodimer that interacts with core dimers forming hybrid viral assembly components. Sucrose gradient fractionation revealed that co-expression of p21.5 resulted in a spread distribution pattern of core proteins ranging from low to high sucrose densities. When compared with p22, p21.5 is almost ten times more efficient at destabilizing HBV nucleocapsid assembly in Huh7 cells overexpressing either p21.5 or p22 protein. Moreover, in vivo expression of p21.5 protein by tail vein injection was found to decrease the amount of nucleocapsid in the livers of HBV-expressing BALB/c mice. In conclusion, our study reveals that the HBV 2.2 kb singly-spliced RNA encodes a 21.5 kDa viral protein that significantly interferes with the assembly of nucleocapsids during HBV nucleocapsid formation. These findings provide a possible strategy for elimination of HBV particles inside cells.

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay allows the rapid identification of HIV-1 nucleocapsid inhibitors.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Retrospective on the all-in-one retroviral nucleocapsid protein.

This review aims at briefly presenting a retrospect on the retroviral nucleocapsid protein (NC), from an unspecific nucleic acid binding protein (NABP) to an all-in-one viral protein with multiple key functions in the early and late phases of the retrovirus replication cycle, notably reverse transcription of the genomic RNA and viral DNA integration into the host genome, and selection of the genomic RNA together with the initial steps of virus morphogenesis. In this context we will discuss the notion that NC protein has a flexible conformation and is thus a member of the growing family of intrinsically disordered proteins (IDPs) where disorder may account, at least in part, for its function as a nucleic acid (NA) chaperone and possibly as a protein chaperone vis-à-vis the viral DNA polymerase during reverse transcription. Lastly, we will briefly review the development of new anti-retroviral/AIDS compounds targeting HIV-1 NC because it represents an ideal target due to its multiple roles in the early and late phases of virus replication and its high degree of conservation.

Unexploited viral and host targets for the treatment of human immunodeficiency virus type 1 infection.

To date, all approved drugs for the treatment of infection by human immunodeficiency virus type 1 (HIV-1) target either of two viral enzymes, reverse transcriptase or protease. Drugs targeting different macromolecules could improve upon current shortcomings (ex, drug resistance, metabolism, toxicity, formulation) and provide foundations for novel combination therapies. This review will focus on the two key challenges for any new target--target validation (demonstrating the role in the disease), and target tractability (the likelihood of identifying modulators of that target that have drug-like properties). For this discussion, drug-like molecules are orally active, relatively small organic molecules. All of the virally-encoded proteins (other than reverse transcriptase and protease) and the host targets that have been postulated to be critical for HIV-1 proliferation will be reviewed.

Fractions of chemically oversulphated galactosaminoglycan sulphates inhibit three enveloped viruses: human immunodeficiency virus type 1, herpes simplex virus type 1 and human cytomegalovirus.

A series of chemically oversulphated galactosaminoglycans (SO3H:COOH ratio > or = 2) were tested in vitro as antiviral agents against human immunodeficiency virus type 1 (HIV-1), the aetiological agent of AIDS, and against herpes simplex virus type 1 and human cytomegalovirus, two agents responsible for opportunistic infections in HIV-infected people. The oversulphated derivatives displayed an increase in activity ranging from one to four orders of magnitude against the three viruses, as compared to the natural parent compounds (SO3H:COOH, ratio approx. 1). The antiviral activity of these polyanions appears to be favoured by a high degree of sulphation and a high molecular mass. An oversulphated dermatan, with a SO3H:COOH ratio of 2.86 and molecular mass of 23.2 kDa, was the most potent anti-HIV-1 compound (EC50 0.04 microgram/ml). A second oversulphated dermatan, with a SO3H:COOH ratio of 2.40 and molecular mass of 25 kDa, displayed the highest activity against HSV-1 (EC50 0.01 microgram/ml). An oversulphated chondroitin, with a SO3H:COOH ratio of 2.80 and molecular mass of 17.3 kDa, was the strongest anti-HCMV agent (EC50 0.4 microgram/ml). In view of the absence of the side-effects typical of heparin-like compounds, a combination of these derivatives could have therapeutic potential.