Abstract Chronic Hepatitis B Virus (HBV) affects 240-300 million people including up to 2 million Americans. Chronic HBV leads to about 800,000 deaths each year, mainly from liver failure and liver cancer. While HBV spread can be controlled by vaccines, there is no cure. Chronic HBV can be treated with reverse transcriptase inhibitors to suppress virus production and improve liver health. But, even after 5+ years of treatment, reverse transcriptase inhibitors do not lead to a cure. Now, Door Pharmaceuticals, an Indiana-based small business, proposes to take advantage of the fact that HBV tags viral nucleic acid with its capsid protein (Cp). Based on this, Door can target these nucleic acids for destruction. Door builds on its extensive experience with Cp, Cp- binding small molecules, and capsid assembly modulators (CAMs), to invent a new class of antiviral: reactive CAMs (rCAMs). rCAMs use a Cp-binding moiety to associate nucleic acid-reactive chemistry to viral DNA and RNA. This association concentrates the reactive component in the vicinity of viral nucleic acid by up to 10^5-fold greater than the bulk concentration. Furthermore, because the reactive group is targeted, it can be used in very low dosages to minimize off-target effects. Studies with purified Cp have supported our proposed rCAM mechanism. The long-term goal of this SBIR is to develop and commercialize an rCAM that is curative for chronic HBV. Door Pharmaceuticals has built a highly qualified team of scientist to develop this promising technology. In this Phase I SBIR, Door will work with its research partners at Indiana University (Zlotnick and VanNieuwenhze) to demonstrate the feasibility of Cp-specific targeting to damage HBV RNA and DNA with the following Specific Aims: Aim 1. Door will develop new rCAMs based on a single Cp-binding scaffold. In conjunction with other aims, this will support a systematic exploration of the structure-activity relationship (SAR) to optimize rCAMs. Aim 2. Determine rCAM activity with purified Cp and virus-like particles. This Aim will mitigate the risk of off-target activity with biochemical assays to test rCAMs for Cp- and capsid-binding and the ability to damage nucleic acid packaged in HBV capsids. These studies will establish the rCAM mechanism of action (MOA), a critical topic in an application for Investigational New Drug (IND) status. Aim 3. Determine rCAM efficacy in HBV-expressing cells. Door will test rCAMs in cell culture for the ability to (i) inhibit production of new virus, (ii) inhibit infection by existing virus, (iii) induce defects in packaged DNA, and (iv) damage viral covalently closed circular DNA (cccDNA), the basis of chronic infection. Proposed experiments will complement chemistry and biochemical results, leading to efficacious candidates for optimizing pharmacology. At the end of this SBIR Phase I, Door will have determined and documented a mechanism of action, selected candidate molecules for further pre-clinical testing...