Activity and mechanism of action of HDVD, a novel pyrimidine nucleoside derivative with high levels of selectivity and potency against gammaherpesviruses

Abstract

A novel nucleoside analogue, 1-[(2S,4S-2-(hydroxymethyl)-1,3-dioxolan-4-yl]5-vinylpyrimidine-2,4(1H,3H)-dione, or HDVD, was evaluated against a wide variety of herpesviruses and was found to be a highly selective inhibitor of replication of the gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). HDVD had also a pronounced inhibitory activity against murine herpesvirus 68 (MHV-68) and herpes simplex virus 1 (HSV-1). In contrast, replication of herpesvirus saimiri (HVS), HSV-2, and varicella-zoster virus (VZV) was weakly inhibited by the compound, and no antiviral activity was determined against human cytomegalovirus (HCMV) and rhesus rhadinovirus (RRV). The HDVD-resistant virus phenotype contained point mutations in the viral thymidine kinase (TK) of HSV-1, MHV-68, and HVS isolates. These mutations conferred cross-resistance to other TK-dependent drugs, with the exception of an MHV-68 mutant (E358D) that exhibited resistance only to HDVD. HSV-1 and HVS TK-mutants isolated under selective pressure with bromovinyldeoxyuridine (BVDU) also showed reduced sensitivity to HDVD. Oral treatment with HDVD and BVDU was assessed in an intranasal model of MHV-68 infection in BALB/c mice. In contrast to BVDU treatment, HDVD-treated animals showed a reduction in viral DNA loads and diminished viral gene expression during acute viral replication in the lungs in comparison to levels in untreated controls. The valyl ester prodrug of HDVD (USS-02-71-44) suppressed the latent infection in the spleen to a greater extent than HDVD. In the present study, HDVD emerged as a highly potent antiviral with a unique spectrum of activity against herpesviruses, in particular, gammaherpesviruses, and may be of interest in the treatment of virus-associated diseases.

Fig 1

Chemical structure of thymidine analogs HDVD, its valine ester prodrug (USS-02-71-44), and BVDU.

Fig 2

Mutations associated with drug resistance in the TK of herpesviruses. Mutations found in the TK of HDVD- and BVDU-resistant HSV-1, HVS, and/or MHV-68. Black boxes indicate highly conserved regions. Nts, nucleotides; Del C, deletion of cytosine.

Fig 3

Kinetic analysis of MHV-68 gene expression in lungs and spleen of BALB/c mice. The graph demonstrates the relative quantification (RQ) of gB (lytic) and ORF73 (latent) expression in the lungs, as well as in the spleen of MHV-68-infected mice, over a time period of 18 days. The grey bars represent the days p.i. relevant to study the effect of antiviral agents. Gene expression levels were normalized to the endogenous control GAPDH. The error bars indicate the calculated maximum and minimum expression levels (RQ_Max and RQ_Min, respectively) from the mean expression level (RQ). The RQ_Max was calculated as ΔΔC_T + s, and the RQ_Min was calculated as ΔΔC_T − s, where C_T is threshold cycle and s is the standard deviation of the ΔΔC_T value.

Fig 4

Analysis of MHV-68 viral DNA load in BALB/c mice treated with HDVD or BVDU. Mice were infected by intranasal route with 10⁴ PFU of MHV-68 and treated with PBS (untreated), HDVD, or BVDU for five consecutive days, starting the day of infection. Viral DNA loads were determined by qPCR in lungs (A and D), MLNs (B and E), and spleen (C and F) at day 6 and day 12 p.i. In each group 5 mice were used, and the limit of detection was 100 copies per mg of tissue. Values are given as the mean viral copy number per mg of tissue ± standard deviation. Statistical significance was calculated using a Mann-Whitney U test: *, P < 0.05; **, P < 0.01.

Fig 5

MHV-68 DNA load in the spleen of USS-20-71-55-treated mice at day 12 p.i. MHV-68-infected BALB/c mice (10⁴ PFU) were treated twice a day with PBS (untreated), or 100 mg/kg of USS-02-71-44, or BVDU for five consecutive days, starting the day of infection. Values are given as the mean viral copies per mg of tissue ± standard deviations. Statistical significance was calculated using a Mann-Whitney U test. **, P < 0.01.

Fig 6

Effects of HDVD and BVDU on lytic (gB) and latent (ORF73) gene expression in lungs and spleen of infected mice. Total RNA was extracted from the tissues at day 6 and/or day 12 p.i. and RT-qPCR was performed (A to C). The upper panels represent the level of gB and ORF73 expression relative to the untreated control and normalized to the endogenous control GAPDH. The error bars indicate the calculated maximum and minimum expression levels (RQ_Max and RQ_Min, respectively) that represent standard error of the mean expression level (RQ value). The RQ_Max was calculated as ΔΔC_T + s, and the RQ_Min was calculated as ΔΔC_T − s, where C_T is threshold cycle and s is the standard deviation of the ΔΔC_T value. The lower panels represent the ΔC_T values obtained from each mouse in the untreated and treated groups and on which we performed statistical analysis using a Mann-Whitney U test. *, P < 0.05.

Fig 7

Histopathological analysis of lungs and spleen of uninfected, infected, and treated infected mice. Hematoxylin- and eosin-stained lung tissues are shown on day 6 and day 12 of control mice, MHV-68-infected mice, and HDVD- and BVDU-treated mice. Histopathology of the spleen at day 12 is shown in the right panels, and arrows indicate the presence of prominent macrophages. Magnification is indicated on the figure.