SUMMARY/ABSTRACT Despite advances in high-throughput screening technologies, initial hits require extensive optimization by medicinal chemistry to yield molecules with the potency and selectivity needed for biological applications, creating a significant bottleneck in small molecule drug discovery and chemical probe development efforts. In this R35 MIRA proposal, I outline my research program consisting of two complementary research directions aimed at expanding the scope of methodologies for chemical probe development toward underexplored biological targets and, ultimately, accelerating drug discovery. Research A is focused on improving the throughput of medicinal chemistry by expanding our first-of-its-kind high-throughput medicinal chemistry (HTMC) platform, which is based on Sulfur(VI) Fluoride Exchange (SuFEx) click-chemistry reactions. Although I previously demonstrated successful application of my HTMC platform to several well-characterized model proteins, additional work is needed to enable targeting of underexplored proteins, including those that lack distinct binding sites. In this research, we will address the challenge of developing chemical probes against underexplored proteins by coupling our platform with a covalent ligand approach, both at the target protein level (A1) and in a proteome-wide manner (A2). Successful completion of this work will expand the biological targets accessible to our HTMC workflow, thereby accelerating probe development against underexplored proteins and enabling elucidation of their biological functions and roles in human health and disease. In Research B, we will develop strategies for synthesis and evaluation of subcellular location selective–probes. Modulation of small molecule subcellular distribution, introduced by Christian de Duve in the 1970s, remains a promising yet underexplored strategy in chemical biology and medicinal chemistry, largely due to the technical challenge of developing subcellular-selective chemical probes. Our long-term goal is to establish a comprehensive medicinal chemistry workflow to develop subcellular-selective probes and drugs. In this grant period, we will develop a SuFEx-enabled method for extracellular-selective chemical probe synthesis (B1), demonstrate approaches to assess organelle-selective target engagement (B2), and explore the potential of modulating organelle selectivity as a medicinal chemistry strategy to improve cellular potency (B3). Development of chemical probes with subcellular resolution will overcome a longstanding hurdle in medicinal chemistry and provide crucial insights into spatio-specific protein functions in disease mechanisms. In total, the workflows and approaches that result from the proposed research program will accelerate and expand the scope of chemical probe development in a proteome-wide and spatio-defined manner, fundamentally advancing the fields of chemical biology and medicinal chemistry. Application of the developed technologies and c...