Developing Novel NLRX1-Based Immuno-Oncology Therapeutics Biotherapeutics Inc (BTI) is a clinical-stage biotech company that synergistically combines the power of advanced computational modeling with translational experimentation to accelerate the development of novel products for precision medicine and health. This SBIR application results from promising data on nucleotide- binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) as a new cancer therapeutic target. Our Product: BTI has identified the first family of small-molecule compounds that bind and inhibit the novel regulatory molecule, NLRX1. The goal of this project is to validate NLRX1 as an immuno-oncology target and develop novel NLRX1-based, oral first-in-class immuno-oncology therapeutics. Background: The incidence of cancer continues to grow while cancer is the second leading cause of death in the United States. In particular, immunogenic cancers, those with the highest potential to be treated with immune- based therapeutics, result in over 500,000 new cases per year and over 230,000 deaths. Thus, there is an unmet clinical need for safer, more efficacious cancer therapeutics. While activation of NLRX1 has shown efficacy in autoimmune disease, inhibition of NLRX1 can increase effector responses to promote oncolytic reactions, de- sensitize immune cells to checkpoint receptors and reduce autophagy. NX-43, our top NLRX1 inhibitor, exerts strong therapeutic efficacy in two models of colorectal cancer (CRC). This Phase I application will characterize the NX-43-mediated effects on survival and validate its safety and specificity to NLRX1. The Aims are to: AIM 1. Assess the direct and indirect cytotoxicity of NX-43 on CT-26 cells by measurement of cell viability and apoptosis in vitro in the presence and absence of lymph node lymphocytes. AIM 2. Determine the therapeutic efficacy of NX-43 in vivo in the CT-26 and AOM/DSS models of CRC to promote survival and induce intratumoral immune activation. AIM 3. Evaluate the ADME-Tox and pharmacokinetics of NX-43 in off-target binding assays, pharmacokinetic analysis, 7-d dose ranging toxicity studies in rats and ADME-Tox assays. Expected Outcomes: Validation of NX-43 as a lead antagonistic molecule for targeting and inhibiting NLRX1 through: i) 50% reduction in cancer cell viability at ≤ 100 µM; ii) significant improvement of survival by oral NX- 43 treatment in mouse models of CRC; and iii) a benign safety profile with NOAEL ≥ 500 mg/kg oral in rats. SBIR Phase II will assess the efficacy of combination therapies with NX-43, evaluate therapeutic effects in additional models of cancer and advance NX-43 to IND-enabling GLP toxicology studies in rats. Commercial Application: Success in this project will launch a new drug development pipeline centered on NLRX1-inhibiting immuno-oncology therapeutics. BTI’s new NLRX1-targeting oral immune-oncology therapeutics could disrupt a market of over $8.5B in colorectal cancer and an addition...