Despite decades of scientific advancements, serious diseases, such as cancer and infectious diseases, continue to pose global challenges to human health. The discovery of small-molecule inhibitors that target disease-relevant proteins remains a central objective in modern drug discovery. This dissertation focuses on the application of structure-based drug design to develop small-molecule inhibitors targeting viral enzymes for antiviral therapy and oncoproteins for anticancer treatment. The first two studies investigate the cap-dependent endonuclease (EN) of two arenaviruses (Lassa and Machupo viruses). These viruses are associated with severe viral hemorrhagic fevers, high mortality rates (15-35%), and potential pandemic risk. A fluorescence resonance energy transfer (FRET)-based assay was employed to monitor enzymatic activity using a dual-labeled RNA analog labeled with the 6-Carboxyfluorescein (6-FAM) fluorophore and a Black Hole Quencher (BHQ1) as a substrate. Upon incubation with our enzymes and their cofactors (Mn2+/Mg2+), cleavage of the substrate separated the fluorophore from the quencher, resulting in increased fluorescence proportional to enzymatic activity. Substrate cleavage was further validated by gel electrophoresis. This assay was adapted for high-throughput screening of >40 compounds to identify inhibitors. Inhibition levels were quantified by calculating the percentage of reduction in fluorescence relative to control conditions. Two compounds, BW-148 and BW-149, were identified as lead inhibitors with IC50 values of 48.44 µM (R2= 0.98) and 63.8 µM (R2= 0.99), respectively. Binding studies of BW-148 and Machupo endonuclease yielded a dissociation constant (KD) of 11.6 µM. A combination of computational and X-ray crystallography techniques was used to study structures of protein-inhibitor complexes. BW-148 interacts with the endonucleases by coordinating one or both of its metals, forming key polar interactions with residues D89/90, K115/116, and nonpolar/hydrophobic interactions with surrounding residues. These structural insights provide a foundation for iterative optimization of inhibitor design. This strategy was also applied to studying the interaction between the anticancer compounds MA242 and SP141 with the RING domain of MDM2. Hence, this study infers, from structure-activity relationships, a guide to iterative compound optimization of BW-148 for inhibition of arenavirus cap-dependent endonuclease, and of MA242 and SP141 for cancer therapies.