Viral Nuclease Inhibitors: Small molecule disruptors of the UL12 alkaline nuclease display broad anti-herpes virus activity

Herpes simplex virus-1 (HSV-1) UL12 gene encodes a well-conserved 5 [->] 3 alkaline exonuclease. UL12 collaborates with the HSV single-strand DNA binding protein ICP8 to mediate recombination-dependent replication of viral DNA and is essential for the production of DNA that can be packaged into infectious virus. The UL12 gene has orthologs in the eight other human herpesviruses, including UL98 in HCMV and SOX in KSHV, which are also essential for virus production. We have developed viral nuclease inhibitors (VNIs) of HSV-1 UL12 that potently block its nuclease activity and display strong antiviral effects in cell culture. These inhibitors are also effective against alkaline nucleases from the {beta}-HHV HCMV (UL98) and the {gamma}-HHV KSHV (SOX), and we have demonstrated antiviral activity against HSV-1 and HCMV in cell culture. In this work, we describe the first crystal structure of an alphaherpesvirus alkaline nuclease (UL12.5), which was used to elucidate structure activity relationships and improve the selectivity of our inhibitors. These VNIs exhibit EC50 and IC50 values in the nanomolar to low micromolar range. Our findings highlight the potential of targeting HHV alkaline nucleases with novel small molecules, paving the way for the development of new therapies that can be broadly antiviral on their own or in combination with nucleoside analogs. Significance StatementHerpesviruses are widespread pathogens that establish lifelong infections and cause serious disease in immunocompromised individuals, neonates and older adults, yet treatment options remain limited. We report the first crystal structure of the HSV-1 alkaline nuclease UL12.5 and use it to design potent small-molecule inhibitors. These inhibitors exhibit antiviral activity against HSV-1 and HCMV, supporting alkaline nuclease as a conserved and druggable target across all herpesvirus subfamilies. The significance is threefold: it confirms alkaline nucleases as having an essential role in viral replication, provides a structural foundation for rational antiviral design and introduces a new class of inhibitors with potential as pan-herpesvirus therapeutics, alone or in combination, to overcome resistance and improve clinical outcomes.