George R. Painter

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

β-d-N4-Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is a representative member of the New World alphaviruses. It is transmitted by mosquito vectors and causes highly debilitating disease in humans, equids, and other vertebrate hosts. Despite a continuous public health threat, very few compounds with anti-VEEV activity in cell culture and in mouse models have been identified to date, and rapid development of virus resistance to some of them has been recorded. In this study, we investigated the possibility of using a modified nucleoside analog, β-d-N4-hydroxycytidine (NHC), as an anti-VEEV agent and defined the mechanism of its anti-VEEV activity. The results demonstrate that NHC is a very potent antiviral agent. It affects both the release of genome RNA-containing VEE virions and their infectivity. Both of these antiviral activities are determined by the NHC-induced accumulation of mutations in virus-specific RNAs. The antiviral effect is most prominent when NHC is applied early in the infectious process, during the amplification of negative- and positive-strand RNAs in infected cells. Most importantly, only a low-level resistance of VEEV to NHC can be developed, and it requires acquisition and cooperative function of more than one mutation in nsP4. These adaptive mutations are closely located in the same segment of nsP4. Our data suggest that NHC is more potent than ribavirin as an anti-VEEV agent and likely can be used to treat other alphavirus infections. IMPORTANCE Venezuelan equine encephalitis virus (VEEV) can cause widespread epidemics among humans and domestic animals. VEEV infections result in severe meningoencephalitis and long-term sequelae. No approved therapeutics exist for treatment of VEEV infections. Our study demonstrates that β-d-N4-hydroxycytidine (NHC) is a very potent anti-VEEV compound, with the 50% effective concentration being below 1 μM. The mechanism of NHC antiviral activity is based on induction of high mutation rates in the viral genome. Accordingly, NHC treatment affects both the rates of particle release and the particle infectivity. Most importantly, in contrast to most of the anti-alphavirus drugs that are under development, resistance of VEEV to NHC develops very inefficiently. Even low levels of resistance require acquisition of multiple mutations in the gene of the VEEV-specific RNA-dependent RNA polymerase nsP4.

A simple in vitro assay to evaluate the incorporation efficiency of ribonucleotide analog 5′ -triphosphates into RNA by human mitochondrial DNA-dependent RNA polymerase

ABSTRACT There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5′-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5′-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5′-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5′-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of >105; a potentially toxic compound has a D value of >104 but <105; and a toxic compound has a D value of <104. This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

Evolving therapies for the treatment of HCV viral hepatitis.

Hepatitis is a general term referring to inflammation of the liver, a condition that can result from infection with a virus, bacterium, or a parasitic organism, or alternatively from noninfectious causes such as alcohol, drugs, or autoimmune disease. Viral infections are responsible for over half of all diagnosed cases of hepatitis. While a number of viruses replicate in multiple organs including the liver, hepatitis viruses are defined as those that show a primary tropism for the liver. These viruses include types A, B, C, D, E, and F (not confirmed). Of these, types A, B, and C are the most common. Infection with these viruses can lead to acute disease characterized by symptoms of nausea, abdominal pain, fatigue, malaise, and jaundice. Severe cases of acute viral hepatitis can rapidly progress to acute liver failure. Additionally, hepatitis B virus (HBV) and hepatitis C virus (HCV) infection can lead to chronic infection. Patients who are chronically infected with HBV or HCV have a high probability of developing cirrhosis and/or hepatocellular carcinoma. Chronic hepatitis carriers remain infectious and can transmit the disease for many years postinfection. While the population impact of HBV infection has long been appreciated (it is still the leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma worldwide), the epidemiology of HCV infection and its significance to world health has only become clear in the last two decades.1 HCV infection is extremely widespread and has put an enormous burden on the world healthcare system. In July of 2013, the World Health Organization (WHO) reported that HCV is found worldwide with some countries having chronic infection rates in excess of 5% of the population (Egypt, for example, has the largest known burden of HCV infection with a 10% incidence in persons aged 15 to 59).2 Seroprevalence data suggests that as many as 185 million people are infected worldwide and consequently are at risk of developing cirrhosis and/or hepatocellular carcinoma. It is estimated that an additional 3 to 4 million people will be infected with HCV every year with 350,000 people dying from HCV related liver disease annually. The virus is most commonly transmitted through (1) receipt of contaminated blood products or organ transplants, (2) contaminated syringes and needle-stick injuries in health care settings, (3) injection drug abuse, and (4) transmission to a neonate by a hepatitis C infected mother. Complete eradication of a pathogenic virus is the goal of antiviral therapy, but this has often proven to be difficult if not impossible to achieve. However, a sustained virologic response (SVR) to treatment for HCV infection, as measured by viremia, has been shown to significantly reduce liver-related morbidity, as well as all-cause mortality in chronically infected patients. Until 2011, the standard of care was treatment with a combination of interferon alpha given once weekly as a subcutaneous injection and ribavirin given orally twice daily for 24 to 48 weeks. Fewer than half of those infected with genotype 1 virus (there are six genotypes of virus, 1 through 6, with multiple subtypes in each genotype) were cured by this treatment regimen, and many patients were ineligible or unable to tolerate it. Clearly, additional treatment options were needed. Two direct acting antiviral agents, boceprevir and telaprevir, that selectively target the virally encoded NS3/4A protease received United States FDA regulatory approval in 2011. Both of these drugs are indicated in combination with pegylated interferon and ribavirin, and their inclusion in the treatment regimen led to higher rates of SVR. However, because of sequence variability in the NS3 protease across genotypes, these drugs are only indicated for the treatment of genotype 1. By mid-2012, additional direct acting antivirals targeting the NS3/4A protease, the NS5B polymerase, and the NS5A protein (involved in formation of the viral replicase complex) were in various stages of clinical development. In late 2013, the United States Food and FDA-granted approvals for two additional HCV drugs. Sofosbuvir, a first-in-class NS5B inhibitor, was approved in combination with pegylated interferon alpha and ribavirin for patients infected with genoytpes 1 and 4, and in combination with ribavirin alone for patients infected with genotypes 2 and 3 (use of the drug in combination with ribavirin alone is also allowed in patients infected with genotype 1 who are interferon ineligible). The NS3/4A protease inhibitor, simeprivir, also received approval for treatment of patients infected with genotype 1 virus, including patients with cirrhotic livers, in combination with pegylated alpha interferon and ribavirin. On the basis of clinical results with the approved drugs and on data emerging from additional drugs in late stage clinical trials, there is considerable optimism that an interferon alpha and ribavirin sparing combination can be identified that has pangenotypic activity and that has a high SVR, even in patients with advanced liver disease. Despite these considerable advances in the treatment of chronic HCV infection, more research and development effort is needed. Clinical experience with the treatment of HIV, HBV, and various herpesvirus infections has shown that over time multiple drugs and drug combinations are necessary for continued control of viral replication and continued interruption of the pathogenic course of disease. These additional drugs and combination regimens were needed due to the pharmacokinetic and metabolic variability found in larger populations, as well as the impact of various coincident medical conditions on drug efficacy. Also, although the development of drug resistance and treatment breakthrough has not yet proven to be as big of an issue for HCV as it has been for other viruses, there is no guarantee that it will not become an issue as drugs are more widely used. In addition, the global burden of HCV infection is mostly found in Africa, the Middle East, and Asia where HCV genotypes 4, 5, and 6 are common. These genotypes are relatively uncommon in the West where pivotal treatment trials for the new drugs were conducted. Consequently, it is not clear if the currently approved drugs or the drugs currently under development will be effective in these populations. It is also true that the current standard of care treatments that are available in developed nations are not affordable in these regions, and more affordable medicines will be required. As a consequence of this, the editors of ACS Medicinal Chemistry Letters thought it would be timely to put together a special issue of the journal dedicated exclusively to the current research and patents in this very important area. I was pleased to serve as the Guest Editor for this special issue.