FAST-TRACK PHASE I and II ABSTRACT Without treatment, chronic hepatitis B virus (HBV) infection can lead to cirrhosis of the liver, hepatocellular carcinoma or liver failure. Current therapies effectively control the disease, but are rarely curative. The goal of this proposal is to develop a therapeutic providing a “functional cure” for chronic HBV infection, i.e., a therapy producing sustained, undetectable HBV surface antigen (HBsAg) and rcDNA (a measure of virions) in serum. A functional cure will potentially benefit about 257 million people worldwide, including approximately 1.4 million individuals with chronic HBV infection in the USA. The applicant, Evrys Bio, has recently described a host- targeted vulnerability of viruses – the sirtuin family of deacylases, or SIRTs. SIRT modulators have broad- spectrum antiviral activity against multiple, diverse viruses including HBV. Of direct relevance to this proposal, Evrys has identified a SIRT2-inhibitor series with potent anti-HBV activity in cultured primary human hepatocytes, blocking the accumulation of extracellular HBV rcDNA, HBsAg and HBeAg as well as intracellular cccDNA – antiviral effects that suggest SIRT2 inhibitors have potential to contribute to a functional cure. Phase I of this proposal will demonstrate the feasibility of Evrys SIRT2-inhibitors to treat HBV: Specific Aim 1 will validate the reduction of cccDNA in infected hepatocytes. This aim will extend the results with HBV genotype D to include HBV genotype A, generalizing the conclusion that HBV is inhibited by SIRT2 inhibitors; it will confirm the qPCR-based conclusion that cccDNA levels are reduced by SIRT2 inhibitors by measuring cccDNA in Southern blot analysis; and it will delineate the relative contributions of the in vitro block to accumulation versus destabilization to the reduction of cccDNA levels by SIRT2 inhibition. Specific Aim 2 will determine a dosing strategy for an exemplar of Evrys SIRT2-inhibitors in FRG KO huHep mice, identifying a well-tolerated dose that can achieve the desired anti-HBV EC95. Specific Aim 3 will demonstrate feasibility using the exemplar to treat HBV-infected FRG KO huHep mice. Phase II will develop a prototype: Specific Aim 4 will identify a development candidate plus at least one backup for IND enabling studies from an existing series of nearly 600 Evrys SIRT2- inhibitors. Specific Aim 5 will probe the mechanisms by which the development candidate blocks HBV replication in human hepatocytes to facilitate clinical translation.