The effects of protein crowders on small molecule drug diffusion
Crowded environments are known to affect the diffusion of macromolecules but their effects on the diffusion of small molecules, such as drugs, are largely uncharacterized. Here, we investigate how three macromolecular protein crowders, bovine serum albumin (BSA), hen egg-white lysozyme and myoglobin, influence the translational diffusion rates and interactions of four low molecular-weight compounds: the diagnostic marker fluorescein, and three drugs, doxorubicin, glycogen synthase kinase-3 inhibitor SB216763 and quinacrine. Using Fluorescence Recovery After Photo-bleaching in Line mode (Line- FRAP), Brownian dynamics simulations and molecular docking, we find that the diffusive behavior of the small molecules is highly affected by self-aggregation, interactions with the proteins, and surface adhesion. The diffusion of fluorescein is decreased by protein crowders due to its interactions with the proteins and their surface adsorption. In contrast, the presence of protein crowders increases the diffusion rate of doxorubicin by reducing surface interactions. SB216763 shows a third scenario, where BSA, but not lysozyme or myoglobin , reduces self-aggregation, resulting in faster diffusion. Quinacrine was the only compound whose diffusion was not affected by the presence of protein crowders. The mechanistic insights gained here into the effects of interactions of small molecules with proteins and surfaces on the translational diffusion of small molecules can assist in optimizing the design of compounds for higher mobility and lower occlusion in complex macromolecular environments.