Herpes simplex virus helicase-primase inhibitors are active in animal models of human disease.

Herpes simplex virus infections are the cause of significant morbidity, and currently used therapeutics are largely based on modified nucleoside analogs that inhibit viral DNA polymerase function. To target this disease in a new way, we have identified and optimized selective thiazolylphenyl-containing inhibitors of the herpes simplex virus (HSV) helicase-primase enzyme. The most potent compounds inhibited the helicase, the primase and the DNA-dependent ATPase activities of the enzyme with IC50 (50% inhibitory concentration) values less than 100 nM. Inhibition of the enzymatic activities was through stabilization of the interaction between the helicase-primase and DNA substrates, preventing the progression through helicase or primase catalytic cycles. Helicase-primase inhibitors also prevented viral replication as demonstrated in viral growth assays. One compound, BILS 179 BS, displayed an EC50 (effective concentration inhibiting viral growth by 50%) of 27 nM against viral growth with a selectivity index greater than 2,000. Antiviral activity was also demonstrated for multiple strains of HSV, including strains resistant to nucleoside-based therapies. Most importantly, BILS 179 BS was orally active against HSV infections in murine models of HSV-1 and HSV-2 disease and more effective than acyclovir when the treatment frequency per day was reduced or when initiation of treatment was delayed up to 65 hours after infection. These studies validate the use of helicase-primase inhibitors for the treatment of acute herpesvirus infections and provide new lead compounds for optimization and design of superior anti-HSV agents.

HPMPC therapy of MCMV-induced retinal disease in the SCID mouse measured by electroretinography, a non-invasive technique.

The purpose of these studies was to investigate the use of non-invasive electroretinography for the evaluation of retinal disease and its treatment in an ocular murine cytomegalovirus (MCMV) disease model. While under anesthesia, 10(2.6)plaque forming units (pfu) of salivary gland passaged, Smith strain MCMV was injected in the anterior chamber of 6- to 8-week-old severe combined immunodeficiency (SCID) mice. At various times post-inoculation, bright-flash scotopic electroretinogram, viral titer, and histology were obtained from the injected eye. Antiviral therapy was tested using 0.1 and 5mg/kg/day subcutaneous injections of HPMPC (Cidofovir) once daily for 5 consecutive days. In infected animals, the a- and b-waves of the electroretinographic (ERG) signal were significantly reduced as of 10 days post-inoculation when compared to control animals. Therapy with HPMPC 0.1mg/kg/day subcutaneously (s.c.) once daily for 5 consecutive days was able to delay the decrease in ERG wave amplitude and inhibit viral replication, whereas 5mg/kg/day s.c. significantly protected the ERG, completely inhibited viral replication, and maintained ocular viral titer below the limit of detection for up to 17 days post-infection. The reduction of ERG activity during progression of retinal disease correlated well with reduction of disease pathology. ERG recording represents a valuable non-invasive technique to measure the progression of the retinal disease induced by MCMV and the efficacy of antiviral treatment in the ocular MCMV disease model.

Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase.

The aminothiazolylphenyl-containing compounds BILS 179 BS and BILS 45 BS are novel inhibitors of the herpes simplex virus helicase-primase with antiviral activity in vitro and in animal models of HSV disease. To verify the mechanism of antiviral action, resistant viruses were selected by serial passage or by single-step plaque selection of HSV-1 KOS in the presence of inhibitors. Three resistant isolates K138r3, K22r5, and K22r1 were found to be 38-, 316-, and 2500-fold resistant to BILS 22 BS, a potent analog of BILS 45 BS. All three viruses had growth properties in vitro similar to wild-type HSV-1 KOS but they were sensitive to acyclovir. Cutaneous and intra-cerebral inoculation of mice with K22r1 or K22r5 resulted in pathogenicity equivalent to that of HSV-1 KOS. Both isolates were fully competent for reactivation from latency following corneal inoculation. Helicase-primase purified from cells infected with resistant viruses showed decreased inhibition in an in vitro DNA-dependent ATPase assay that correlated well with antiviral resistance. Marker transfer experiments and DNA sequence analysis identified single base pair mutations clustered in the N-terminus of the UL5 gene that resulted in single amino acid changes in the UL5 protein. Taken together, the results indicate that helicase-primase inhibitors prevent HSV growth by inhibiting HSV helicase-primase through specific interaction with the UL5 protein.