The Subcellular Distribution of Small Molecules: From Pharmacokinetics to Synthetic Biology

Abstract

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.

Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs

“…Chemotypes have been identified that improve cellular uptake,(70) including conjugation of polyarginine,(71) as well as those that influence localization. (72) Such data could be incorporated into a traditional medicinal chemistry approach or chemical similarity searching. Interestingly, the permeability(73) and localization of multivalent compounds can be improved by changing the scaffold, in particular the module used to separate RNA-binding modules, and by the RNA-binding module itself.…”

Investigation of Polyethylenimine/DNA Polyplex Transfection to Cultured Cells Using Radiolabeling and Subcellular Fractionation Methods

“…Some studies have also tracked radiolabeled polyplexes in additional organelles using subcellular fractionation methods. While fluorescence microscopy can be affected by environmental factors, such as pH and temperature 25 , radioactivity can provide substantially increased sensitivity and robustness. Furthermore, the use of radiolabeled compounds allows for mass balance calculations, so that 100% of the material applied to the sample can be tracked.…”

Molecular Imaging of Intracellular Drug–Membrane Aggregate Formation

“…Nevertheless, the sequestration of drugs within intracellular organelles can also affect pharmacokinetics and pharmacodynamics after systemic delivery 1–3 . Among several known organelle sequestration mechanisms, ion-trapping of drugs within acidic compartments, i.e.…”

“…Protonation of weakly basic molecules leads to increased solubility and decreased membrane permeability, and promotes retention within lysosomes and endosomes. In the case of lipophilic cations, the electrical potential present across the inner membrane of mitochondria drives accumulation of membrane permeant lipophilic cations into this organelle 1, 3, 6, 7 . Beyond transmembrane pH gradients (i.e.…”

“…MDCK cells were selected because they are considered a robust in vitro assay system for drug transport studies. While these cells originated from the epithelia of the distal renal tubules, they are also used as a model to study the passive diffusive component of intestinal drug absorption 2 and to study drug bioaccumulation associated with ion-trapping 13 and phospholipidosis 3, 14 . Taking advantage of clofazimine’s red color, the drug’s absorbance, fluorescence, and biodistribution properties could be readily measured.…”