biostudies-literatureDey DIsrael Science FoundationCzech Science FoundationRP97255https://www.ebi.ac.uk/biostudies/studies/S-EPMC11623935biostudies-literatureUnknown13For drugs to be active they have to reach their targets. Within cells this requires crossing the cell membrane, and then free diffusion, distribution, and availability. Here, we explored the in-cell diffusion rates and distribution of a series of small molecular fluorescent drugs, in comparison to proteins, by microscopy and fluorescence recovery after photobleaching (FRAP). While all proteins diffused freely, we found a strong correlation between p<i>K</i>_a and the intracellular diffusion and distribution of small molecule drugs. Weakly basic, small-molecule drugs displayed lower fractional recovery after photobleaching and 10- to-20-fold slower diffusion rates in cells than in aqueous solutions. As, more than half of pharmaceutical drugs are weakly basic, they, are protonated eLifeReversing protonation of weakly basic drugs greatly enhances intracellular diffusion and decreases lysosomal sequestration.PMC116239351268/1822-20319SDey DWohlrabova LSlanina TGroborz OMarciano SPoryval ASchreiber GfalseReversing protonation of weakly basic drugs greatly enhances intracellular diffusion and decreases lysosomal sequestration.For drugs to be active they have to reach their targets. Within cells this requires crossing the cell membrane, and then free diffusion, distribution, and availability. Here, we explored the in-cell diffusion rates and distribution of a series of small molecular fluorescent drugs, in comparison to proteins, by microscopy and fluorescence recovery after photobleaching (FRAP). While all proteins diffused freely, we found a strong correlation between p<i>K</i>_a and the intracellular diffusion and distribution of small molecule drugs. Weakly basic, small-molecule drugs displayed lower fractional recovery after photobleaching and 10- to-20-fold slower diffusion rates in cells than in aqueous solutions. As, more than half of pharmaceutical drugs are weakly basic, they, are protonated 2024-01-01T00:00:00Z2024 Dec2026-06-02T10:48:06.28Z2025-04-04T01:24:05.903ZS-EPMC116239353964197510.7554/eLife.97255