Rational Drug Design Laboratories

To evaluate and describe the anti-herpesvirus effect of ASP2151, amenamevir, a novel non-nucleoside oxadiazolylphenyl-containing herpesvirus helicase-primase complex inhibitor.

The inhibitory effect of ASP2151 on enzymatic activities associated with a recombinant HSV-1 helicase-primase complex was assessed. To investigate the effect on viral DNA replication, we analysed viral DNA in cells infected with herpesviruses [herpes simplex virus (HSV), varicella-zoster virus (VZV) and human cytomegalovirus]. Sequencing analyses were conducted on an ASP2151-resistant VZV mutant. In vitro and in vivo antiviral activities were evaluated using a plaque reduction assay and an HSV-1-infected zosteriform-spread model in mice.

ASP2151 inhibited the single-stranded DNA-dependent ATPase, helicase and primase activities associated with the HSV-1 helicase-primase complex. Antiviral assays revealed that ASP2151, unlike other known HSV helicase-primase inhibitors, exerts equipotent activity against VZV, HSV-1 and HSV-2 through prevention of viral DNA replication. Further, the anti-VZV activity of ASP2151 (EC(50), 0.038-0.10 microM) was more potent against all strains tested than that of aciclovir (EC(50), 1.3-27 microM). ASP2151 was also active against aciclovir-resistant VZV. Amino acid substitutions were found in helicase and primase subunits of ASP2151-resistant VZV. In a mouse zosteriform-spread model, ASP2151 was orally active and inhibited disease progression more potently than valaciclovir.

ASP2151 is a novel herpes helicase-primase inhibitor that warrants further investigation for the potential treatment of both VZV and HSV infections.

Hepatitis C virus (HCV) NS3-NS4A protease is an attractive target for anti-HCV agents because of its important role in replication. An optimized fluorescence resonance energy transfer (FRET) substrate for NS3-NS4A protease, based on the sequence of the NS5A-5B cleavage site, was designed and synthesized. High-throughput screening of in-house compound libraries was performed using a FRET substrate FS10 (MOCAc-DKIVPC-SMSYK-Dnp) and MBP-NS3-NS4A fusion protein. Several hit compounds were found, including YZ-9577 (2-oxido-1,2,5-oxadiazole-3,4-diyl) bis (phenylmethanone) with potent inhibitory activity (IC50=1.6 μM) and good selectivity against other human serine proteases.

A structural and thermodynamic study of the entropic contribution of salt bridge formation to the interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wild-type Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the antigen-antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein antigen-antibody interaction through reduction of entropy loss due to dehydration and structural changes.