Convallatoxin-Induced Reduction of Methionine Import Effectively Inhibits Human Cytomegalovirus Infection and Replication

Abstract

Cytomegalovirus (CMV) is a ubiquitous human pathogen that increases the morbidity and mortality of immunocompromised individuals. The current FDA-approved treatments for CMV infection are intended to be virus specific, yet they have significant adverse side effects, including nephrotoxicity and hematological toxicity. Thus, there is a medical need for safer and more effective CMV therapeutics. Using a high-content screen, we identified the cardiac glycoside convallatoxin as an effective compound that inhibits CMV infection. Using a panel of cardiac glycoside variants, we assessed the structural elements critical for anti-CMV activity by both experimental and in silico methods. Analysis of the antiviral effects, toxicities, and pharmacodynamics of different variants of cardiac glycosides identified the mechanism of inhibition as reduction of methionine import, leading to decreased immediate-early gene translation without significant toxicity. Also, convallatoxin was found to dramatically reduce the proliferation of clinical CMV strains, implying that its mechanism of action is an effective strategy to block CMV dissemination. Our study has uncovered the mechanism and structural elements of convallatoxin, which are important for effectively inhibiting CMV infection by targeting the expression of immediate-early genes. IMPORTANCECytomegalovirus is a highly prevalent virus capable of causing severe disease in certain populations. The current FDA-approved therapeutics all target the same stage of the viral life cycle and induce toxicity and viral resistance. We identified convallatoxin, a novel cell-targeting antiviral that inhibits CMV infection by decreasing the synthesis of viral proteins. At doses low enough for cells to tolerate, convallatoxin was able to inhibit primary isolates of CMV, including those resistant to the anti-CMV drug ganciclovir. In addition to identifying convallatoxin as a novel antiviral, limiting mRNA translation has a dramatic impact on CMV infection and proliferation. C ytomegalovirus (CMV) is an extremely large betaherpesvirus with over 230,000 bp and over 200 open reading frames. While the majority of CMV infections in immunocompetent hosts are asymptomatic (1), the phenomenon of latency allows the virus to establish a permanent and persistent infection. CMV is highly seroprevalent in the human population (Ͼ60%), with broad cell tropism, and can be transmitted through bodily fluids and organ transplantation and transplacentally from mother to fetus (2). Reactivation of latent CMV can cause disease in any population but is a particular source of morbidity and mortality for immunocompromised patients (3). It is estimated that treatment of CMV costs the United States $4.4 billion every year (4), and congenital CMV infections are the leading cause of permanent congenital neurological disabilities in the United States (5). The current FDA-approved therapies all target the viral DNA polymerase, and none are approved for use in pregnant women or newborns. These compounds can cause sever...

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…This IE2-YFP cell-based reporter assay was used to screen a 2080 bioactive compound library and identified one lead compound, the cardiac glycoside convallatoxin. This compound exhibited potent anti-HCMV activity (EC 50 values in the low nanomolar range) without significant cellular cytotoxicity [362,363]. However, it should be noted that convallatoxin has been discounted as a hit from a different screen due to toxicity [364].…”

“…Interestingly, other cardiac glycosides (ouabain, β-antiarin, digoxin, digitoxin) have also been reported to exhibit anti-HCMV activity [363,[365][366][367]. Inhibition of HCMV by cardiac glycosides is effective against clinical and GCV-resistant strains and exhibits additive activity when administered to cells in combination with GCV [362,363,365,366]. Members of this compound family have been used clinically for the treatment of heart conditions such as congestive heart failure, although toxicity and dosage issues mean that they are increasingly replaced with synthetic drugs such as ACE inhibitors and beta-blockers [368].…”

Elucidation of the mechanism of anti-herpes action of two novel semisynthetic cardenolide derivatives

“…The mechanism by which cardenolides affect several steps of HSV replication could be related to the inhibition of Na + / K + -ATPase in host cells. Many studies have demonstrated the modulation of Na + /K + -ATPase functions in host cells by DNA viruses (adenoviruses [18], cytomegalovirus [23,[70][71][72], and HSV [27,28]), and RNA viruses (chikungunya virus [19,73], coronaviruses [20,74,75], respiratory syncytial virus [76,77], Ebola virus [78,79], influenza virus [33,80,81], and HIV [30,31,82]). By activating signaling cascades or by altering the concentration of intracellular ions, the binding of cardenolides to Na + /K + -ATPase seems to create an unfavorable environment for viral replication.…”

“…Bertol et al [26] showed that the inhibition of Na + /K + -ATPase caused by glucoevatromonoside was correlated with virus release and viral protein synthesis due to a reduction in the K + concentration in the cell. Cohen et al [23] found that the inhibitory activity of cytomegalovirus replication by convallatoxin was due to a decrease in immediate-early gene expression and that the antiviral potency depends on the structure of cardiac glycosides and their specific interactions with Na + / K + -ATPase. It was also shown that influenza virus replication was impaired by ouabain through the inhibition of viral protein translation and a decrease in the intracellular K + concentration [37].…”

“…Among them, their cytotoxic and antitumor effects recently reviewed by Cerella et al [10], De et al [11], Diederich et al [12], Schneider et al [13] and El-Seedi et al [14], as well as their anti-inflammatory [15], antiprotozoal [16], anti-oxidant and anti-aging [17] activities can be cited. Another suggested possibility is their potential antiviral action, as reported by several authors against adenovirus [18], chikungunya virus [19], coronavirus [20,21], cytomegalovirus [22][23][24], dengue virus [25], herpes virus [26][27][28], HIV [29][30][31], human papillomarivus (HPV) [32], influenza virus [33][34][35], and respiratory syncytial virus [36] replication. The effects of six well-known cardiac glycosides (digoxin, digitoxin, ouabain, convallatoxin, G-strophanthin and lanatoside C) on viral biology and the mechanisms by which they impair the replication of different RNA and DNA viruses were recently compiled [37].…”