Novel Class of Thiourea Compounds That Inhibit Herpes Simplex Virus Type 1 DNA Cleavage and Encapsidation: Resistance Maps to the UL6 Gene

Abstract

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-{4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl}-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.The herpesvirus family has many members that are human pathogens and make a significant contribution to morbidity and mortality associated with viral diseases. Based on criteria such as host cell specificity, oncogenicity, length of replication cycle, and genome arrangement, the herpesviruses have been divided into alpha-, beta-, and gammaherpesviruses (31). The alphaherpesviruses herpes simplex virus (HSV) types 1 and 2 latently infect nerve cells. HSV-1 is primarily associated with herpes labialis, and HSV-2 is associated with herpes genitalis, but both types have been associated with both diseases (28,39,47). In immunocompetent adults, these diseases often recur due to reactivation of the virus from the latent state. HSV infections of immunocompromised patients such as transplant and AIDS patients are often chronic and fatal. Current therapy for HSV disease consists of nucleoside analogs such as acyclovir (ACV) and valacyclovir, a prodrug of ACV, and pencyclovir (PCV) and its prodrug, famcyclovir. ACV and PCV are selectively phosphorylated by the viral thymidine kinase in HSVinfected cells, followed by further phosphorylation to the triphosphate by cellular kinases. Triphosphorylated ACV and PCV are both inhibitors of the viral DNA polymerase, and ACV also acts as a chain terminator when incorporated into the nascent viral DNA chain (4, 13). Drug resistance can occur in chronic infections, where replication is ineffectively curtailed by the immune system. Recently increasing numbers of drugresistant HSV strains have been isolated from immunocompromised people. The mechanism of resistance of most ACV-resistant isolates is...

Herpes Simplex Virus 1 Immediate-Early and Early Gene Expression during Reactivation from Latency under Conditions That Prevent Infectious Virus Production

“…2) and to the effect of WAY-150138 on plaque formation by wild-type strain Patton. In addition, two groups have reported finding no effect of WAY-150138 on synthesis of certain viral proteins (25,45).…”

“…The value of WAY-150138 in our proposed experiments lay in its reported ability to block production of progeny virus while allowing viral DNA replication to proceed (45). To confirm that this compound had little or no effect on viral DNA replication, the viral DNA content of cells infected in the presence of WAY-150138 was measured by dot blotting and Southern hybridization (Fig.…”

“…As the explant model is well suited for pharmacological studies, the de- Specificity and efficacy of WAY-150138 in vitro. WAY-150138 inhibits the replication of some strains of HSV-1, including Patton, with a 50% inhibitory concentration of 0.2 to 0.7 g/ml (45). Plaque reduction assays demonstrated that wild-type strain Patton was prevented from forming plaques in 10 g/ml WAY-150138, while the drug-resistant derivative and wild-type strain KOS were subject to no more than a 40% reduction in plaque number in up to 50 g/ml (Fig.…”

“…To enable examination of gene expression during reactivation in more detail, we have employed acyclovir (ACV), a well-established inhibitor of viral DNA replication, and WAY-150138, a newer inhibitor of viral DNA cleavage and packaging (45). The latter compound appears to preclude the incorporation of the UL6 portal protein and UL15 into capsids (25).…”

Synthesis and biological evaluation of some new 1,3,4-thiadiazole and 1,2,4-triazole derivatives from L-methionine as antituberculosis and antiviral agents

“…In this regard, potent and selective inhibitors of cytomegalovirus (CMV) (17), Varicella-zoster virus (VZV) (18), and herpes simplex virus type-1 (HSV-1) (19)(20)(21) were developed (Figure 3). In our previous work on thiourea derivatives (1), we reported the synthesis and antiviral evaluation of N-phenyl-N´-{4-[ (4-allyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy]phenyl}thiourea which showed moderate protection against Coxsackie virus B4 with an MIC value of 16 µg/ml (40.25 µM, SI=5) and thymidine kinase wild-type varicella-zoster virus (TK + VZV, OKA strain) with an EC 50 value of 9.9 µg/ml (Figure 3).…”