DSTP-27 Prevents Entry of Human Cytomegalovirus

Abstract

cHuman cytomegalovirus (HCMV) can cause life-threatening diseases in neonates and immunocompromised patients. Due to multiple problems caused by the current available drugs, development of new antiviral compounds is urgently needed. In this study, we characterize the anti-HCMV spectrum and mechanism of action of the N-N=-(bis-5 nitropyrimidyl)dispirotripiperazine derivate 27 (DSTP-27). DSTP-27 exhibited strong antiviral activity against two laboratory HCMV strains with different cell tropism as well as ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates in plaque reduction assays and viral growth kinetics experiments. Interestingly, neither infectious nor noninfectious viral particles were observed by electron microscopy. Pretreatment of cell-free virus with DSTP-27 prevented virus infection. The results from time of addition assays, in which DTSP-27 was added to cells (i) before infection, (ii) during virus adsorption, or (iii) after adsorption, demonstrated an inhibitory effect on early steps of the HCMV replication cycle. This observation was confirmed by immunofluorescence as well as Western blot analysis, whereby reduced levels of the immediate early protein IE1, the processivity factor pUL44, and the tegument protein pp28 were detected. Results from attachment and penetration analyses of prechilled human embryonic lung fibroblasts revealed that virus attachment is not blocked. In addition, DSTP-27 inactivated HCMV by stable binding. Taken together, these results demonstrate that DSTP-27 (i) blocks viral penetration by interacting with the host cell and (ii) inactivates HCMV by interacting with the virus.

The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus

“…To further explore which step of virus entry is affected by podofilox treatment, a penetration experiment was performed using low pH to remove and inactivate non-entered virus [38] (Figure 4). To demonstrate the effectiveness of a low pH was to remove non-entered virus, MRC5 cells infected with AD169 IE2-YFP at 4 °C were washed with a pH 3.0 or pH 7.0 buffer and were analyzed (18 h p.i.)…”

“…The following day, the medium was replaced with 100 μL of DMEM containing 500 nM, 50 nM, or 5 nM of Podofilox or 0.01% DMSO for 1 h prior to infection with AD169 IE2-YFP (MOI 3). Cells were placed at 4 °C for 1 h to allow for viral attachment then washed with citrate buffer pH 3.0 or pH 7.0 or incubated at 37 °C for 1 h as previously described [37,38]. At 18 h p.i., the plates were analyzed with an Acumen ×3 cytometer for the number of IE2-YFP positive cells/well based on IE2-YFP fluorescent intensity/well.…”

Antiretroviral Activity Of a Novel Pyrimidyl-Di(Diazaspiroalkane) Derivative

“…Most often, adhesion occurs through specific binding to heparan sulfate proteoglycans (HSPG), which are located on the cell surface. It is known that this mechanism is used by various viral families, such as type 1 and 2 herpesviruses (HSV-1, HSV-2) [1], papillomaviruses (HPV) [2], human cytomegalovirus (HCMV) [3], some strains of the human immunodeficiency virus (HIV) [4], human respiratory syncytial virus (HRSV) [5], the hepatitis B and C viruses (HBV and HCV) [6], and others. …”

“…Previously we synthesized N,N-bis(1-oxido [1,2,5] oxadiazolo [3,4] pyrimidin-7-yl)-3,12-diaza-6,9-diazonium (5,2,5,2) dispirohexadecane dichloride 1 ( Figure ), the most well known and most extensively studied inhibitor of the adhesion process. It was shown that compound 1 and its analogues, including dispirotripiperazine, are characterized by effective reversible binding to cellular HSPG and, thus, prevent virus binding [7].…”

“…It was shown that compound 1 and its analogues, including dispirotripiperazine, are characterized by effective reversible binding to cellular HSPG and, thus, prevent virus binding [7]. Dispiro compound 1 inhibits replication in herpes viruses [8], as well as other viral families that use HS as a receptor or co-receptor [3]. However, the metabolic instability of compound 1 that is due to a degradation accompanied by the formation of nitric oxide prohibited an investigation of its biological properties [9].…”