Project Summary We propose to develop human immunodeficiency virus (HIV) theranostics for the central nervous system (CNS) based on the intrinsic chemical exchange saturation transfer (CEST) contrasts of antiretroviral drugs. Theranostics is a combination of the terms therapeutics and diagnostics that enables the biodistribution measurements of antiretrovirals (ARVs) in target organs using in vivo imaging techniques such as magnetic resonance imaging (MRI). HIV theranostics benefits the research of antiretroviral therapy in three ways. First, HIV theranostics will be a powerful tool for the development of ARVs targeting CNS viral reservoirs. Second, HIV theranostics will help develop strategies to minimize the off-target effects of ARVs. Third, In vivo imaging of long term PK and BD is critical for the development of long-acting drugs. Moreover, HIV theranostics enables real- time monitoring of CNS drug levels making it possible to design personalized treatments tailored for individual patients. Compared to traditional theranostic technologies that tag drugs with imaging agents, CEST is an intrinsic property of an ARV, therefore no extrinsic chemical agents are required for its imaging. This eliminates the limitations associated with imaging agents including limited therapy effectiveness and imaging sensitivity, and possible toxicity. We posit that the combined CEST effects of the protons in a drug molecule generate a unique CEST characteristic termed as CEST fingerprint that enables in vivo drug detection with sensitivity and specificity. We will first characterize the CEST fingerprints of first-line HIV ARVs, and develop MRI methods based on the drug CEST fingerprints for in vivo detection. Aim 1: To characterize the CEST fingerprints of ARVs (3TC, ABC, TDF and DTG). We hypothesize that exchangeable protons of the drugs generate unique CEST fingerprints. The CEST effects of the protons in a drug molecule will be measured and combined to build the drug's CEST fingerprint. The CEST fingerprint of each drug will be further validated and determined in cells using human monocyte-derived macrophages (MDMs). Aim 2: To measure in vivo CNS biodistribution of ARVs in humanized mice using CEST MRI. We posit that MRI utilizing CEST fingerprints offer sensitivity and specificity for in vivo measurements of ARVs. To test the hypothesis, MRI data acquisition and analysis methods will be developed based on the ARV CEST fingerprints measured in Aim 1, and optimized for in vivo detection in infected humanized mice that treated with combined antiretroviral therapy (DTG/ABC/3TC or DTG/3TC/TDF). Brain tissue drug levels measured using liquid chromatography mass spectrometry (LC-MS/MS) will be used to validate CEST MRI results. A successful outcome of the study is a noninvasive MRI technique that measures brain ARV levels. The technique will provide a powerful tool for the development of treatments that mitigate CNS complications of HIV infection and ameliorate the ...