Amphipathic DNA Polymers Exhibit Antiherpetic Activity In Vitro and In Vivo

Abstract

Phosphorothioated oligonucleotides have a sequence-independent antiviral activity as amphipathic polymers (APs). The activity of these agents against herpesvirus infections in vitro and in vivo was investigated. The previously established sequence-independent, phosphorothioation-dependent antiviral activity of APs was confirmed in vitro by showing that a variety of equivalently sized homo-and heteropolymeric AP sequences were similarly active against herpes simplex virus type 1 (HSV-1) infection in vitro compared to the 40mer degenerate parent compound (REP 9), while the absence of phosphorothioation resulted in the loss of antiviral activity. In addition, REP 9 demonstrated in vitro activity against a broad spectrum of other herpesviruses: HSV-2 (50% effective concentration [EC 50 ], 0.02 to 0.06 M), human cytomegalovirus (EC 50 , 0.02 to 0.13 M), varicella zoster virus (EC 50 , <0.02 M), Epstein-Barr virus (EC 50 , 14.7 M) and human herpesvirus types 6A/B (EC 50 , 2.9 to 10.2 M). The murine microbicide model of genital HSV-2 was then used to evaluate in vivo activity. REP 9 (275 mg/ml) protected 75% of animals from disease and infection when provided 5 or 30 min prior to vaginal challenge. When an acid-stable analog (REP 9C) was used, 75% of mice were protected when treated with 240 mg/ml 5 min prior to infection (P < 0.001), while a lower dose (100 mg/ml) protected 100% of the mice (P < 0.001). The acid stable REP 9C formulation also provided protection at 30 min (83%, P < 0.001) and 60 min (50%, P ‫؍‬ 0.07) against disease. These observations suggest that APs may have microbicidal activity and potential as broad-spectrum antiherpetic agents and represent a novel class of agents that should be studied further.Microbicides are intended to prevent the transmission of sexually transmitted infections (STIs) from one sexual partner to another. The ideal microbicide would be completely safe and broadly effective against a variety of STIs (reviewed in reference 9). Despite recent setbacks, including failed trials of nonoxynol 9, Savvy (C31G), and cellulose sulfate (6), there are more than 30 other microbicides being investigated (9). Because genital herpes infections are not only one of the most common STIs but also have recently been shown to have profound effects on the spread of human immunodeficiency virus (HIV), they have been another major target for microbicides (reviewed in references 11 and 8). Thus, several preclinical microbicide evaluations have used animal models of genital herpes for the initial testing (20).Recently, the use of a novel class of compounds, phosphorothioate oligonucleotides (PS-ONs), has been suggested as a promising microbicide approach (17). The presence of sequence-independent antiviral activity with PS-ONs in HSV has been previously described (10,12,13). Recently, this sequenceindependent antiviral activity of PS-ONs was more clearly elucidated in HSV and other viruses (17,22,27) and was shown to be derived from the chemical nature of PS-ONs amphipathic polymers, independent of the ...

Nucleic Acid Polymers Inhibit Duck Hepatitis B Virus InfectionIn Vitro

“…Thus, while subtle differences between the entry-inhibitory activity of REP 2006 and REP 2031 cannot be clarified based on the results presented here, it is clear that both these NAPs possess significant entry-inhibitory activity. The SAR of NAPs in DHBV infection is highly congruent to that observed in other hepatotropic viruses (CMV and HCV) (7,8,11) and type 1 viruses (HSV-1 and -2, LCMV, and HIV-1) (6,10,12). In each of these previously reported cases, this unique SAR defines the antiviral target in each virus as a large amphipathic protein domain of similar size that functions during viral entry.…”

“…These NAPs are constructed from oligonucleotides in which phosphorothioation of a nonbridging oxygen atom in the phosphodiester linkage, traditionally used as a modification to stabilize oligonucleotides against nuclease attack, is used to enhance the amphipathic properties of oligonucleotides (5). Phosphorothioation has been shown to be strictly essential for the antiviral activity of NAPs in several in vitro systems (6)(7)(8)(9)(10)(11)(12). The water-soluble yet amphipathic properties of NAPs are derived from inherent chemical properties of phosphorothioate oligonucleotides (PS-ONs) that are independent of the sequence of nucleotides present.…”

“…In HIV-1 and LCMV, their interaction with viral fusion proteins is consistent with their ability to block viral entry. The involvement of analogous amphipathic protein structures in the surface glycoproteins of many viruses may underlie the broad-spectrum antiviral activity of NAPs against other viruses, such as herpes simplex virus 2 (HSV-2), cytomegalovirus (CMV), and hepatitis C virus (HCV) (6)(7)(8)11), where NAPs have also been shown to act as viral entry inhibitors.…”

“…In all cases where the antiviral activity of NAPs has been examined, striking similarities in the structure-activity relationship (SAR) have been demonstrated (6)(7)(8)(10)(11)(12). The broad-spectrum antiviral activity of NAPs exhibits similar size-dependent properties in different model systems, with NAPs of Ͻ20 nucleotides (nt) in length having minimal activity, NAPs between 30 and 40 nt having increased activity with increasing length, and NAP activity becoming maximal with NAPs of Ͼ40 nt.…”

Nucleic Acid Polymers Prevent the Establishment of Duck Hepatitis B Virus Infection In Vivo

“…Analogous amphipathic interactions may underlie the ability of NAPs to block entry of herpes simplex virus 2 (HSV-2), cytomegalovirus (CMV), and hepatitis C virus (HCV) (16)(17)(18).…”