The systemic pharmacokinetics and pharmacodynamics of small molecules are determined by subcellular transport phenomena. Although approaches used to study the subcellular distribution of small molecules have gradually evolved over the past several decades, experimental analysis and prediction of cellular pharmacokinetics remains a challenge. In this article, we surveyed the progress of subcellular distribution research since the 1960s, with a focus on the advantages, disadvantages and limitations of the various experimental techniques. Critical review of the existing body of knowledge pointed to many opportunities to advance the rational design of organelle-targeted chemical agents. These opportunities include: 1) development of quantitative, nonfluorescence-based, whole cell methods and techniques to measure the subcellular distribution of chemical agents in multiple compartments; 2) exploratory experimentation with nonspecific transport probes that have not been enriched with putative, organelle-targeting features; 3) elaboration of hypothesis-driven, mechanistic and modeling-based approaches to guide experiments aimed at elucidating subcellular distribution and transport; and 4) introduction of revolutionary conceptual approaches borrowed from the field of synthetic biology combined with cutting edge experimental strategies. In our laboratory, state-of-the-art subcellular transport studies are now being aimed at understanding the formation of new intracellular membrane structures in response to drug therapy, exploring the function of drug-membrane complexes as intracellular drug depots, and synthesizing new organelles with extraordinary physical and chemical properties.

To explore the extent to which current knowledge about the organelle-targeting features of small molecules may be applicable towards controlling the accumulation and distribution of exogenous chemical agents inside cells, molecules with known subcellular localization properties (as reported in the scientific literature) were compiled into a single data set. This data set was compared to a reference data set of approved drug molecules derived from the DrugBank database, and to a reference data set of random organic molecules derived from the PubChem database. Cheminformatic analysis revealed that molecules with reported subcellular localizations were comparably diverse. However, the calculated physicochemical properties of molecules reported to accumulate in different organelles were markedly overlapping. In relation to the reference sets of Drug Bank and Pubchem molecules, molecules with reported subcellular localizations were biased towards larger, more complex chemical structures possessing multiple ionizable functional groups and higher lipophilicity. Stratifying molecules based on molecular weight revealed that many physicochemical properties trends associated with specific organelles were reversed in smaller vs. larger molecules. Most likely, these reversed trends are due to the different transport mechanisms determining the subcellular localization of molecules of different sizes. Molecular weight can be dramatically altered by tagging molecules with fluorophores or by incorporating organelle targeting motifs. Generally, in order to better exploit structure-localization relationships, subcellular targeting strategies would benefit from analysis of the biodistribution effects resulting from variations in the size of the molecules.