Slow Intracellular Diffusion and Lysosomal Sequestration of Weakly Basic Small Molecule Drugs

Core Concepts

Weakly basic small molecule drugs exhibit slow intracellular diffusion and accumulation in lysosomes, which can reduce their cellular activity and efficacy.

Abstract

The study investigated the intracellular diffusion and distribution of a series of small molecule fluorescent drugs and proteins in HeLa cells. Key findings:

Acidic and neutral proteins diffused freely in the HeLa cell cytoplasm, with diffusion coefficients (Dconfocal) of 18-30 μm²/s and complete FRAP recovery.

Negatively charged small molecules like fluorescein and CCF2 also diffused relatively rapidly in cells, with Dconfocal values reduced by 2-3 fold compared to buffer due to cellular crowding.

In contrast, weakly basic small molecule drugs like the GSK3 inhibitor, quinacrine, mitoxantrone, primaquine, and amidoquine exhibited 10-20 fold slower diffusion (Dconfocal of 0.2-2 μm²/s) and lower fractional recovery after FRAP (0.2-0.5) compared to the acidic/neutral molecules.

The slow diffusion and low FRAP recovery of basic drugs correlated with their sequestration in acidic organelles, particularly lysosomes, due to cationic ion trapping.

Blocking lysosomal acidification with Bafilomycin A1 or sodium azide only slightly improved the diffusion and FRAP recovery of the basic drugs.

In contrast, blocking protonation by N-acetylation greatly enhanced the diffusion and FRAP recovery of the basic drugs, suggesting that protonation is the primary cause of their slow intracellular diffusion.

The findings highlight an important limitation in the standard rules for small molecule drug design, as the "stickiness" of basic drugs within the cell cytoplasm is not considered. Modulating the protonation state of basic drugs may improve their intracellular availability and distribution.

Stats

Fluorescein in PBS: Dconfocal = 56.5 ± 2.4 μm²/s
Fluorescein in HeLa cytoplasm: Dconfocal = 38.5 ± 2.0 μm²/s
GSK3 inhibitor in PBS + 50 mg/mL BSA: Dconfocal = 24.3 ± 0.5 μm²/s
GSK3 inhibitor in HeLa cytoplasm: Dconfocal = 0.61 ± 0.03 μm²/s
Quinacrine in PBS: Dconfocal = 41.9 ± 3.0 μm²/s
Quinacrine in HeLa cytoplasm: Dconfocal = 2.5 ± 0.1 μm²/s

Quotes

"While all proteins diffused freely, we found a strong correlation between pKa 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."
"Protonation, facilitates the formation of membrane impermeable ionic form of the weak base small molecules. This results in ion trapping, further reducing diffusion rates of weakly basic small molecule drugs under macromolecular crowding conditions where other nonspecific interactions become more relevant and dominant."

Key Insights Distilled From

Reversing protonation of weakly basic drugs greatly enhances intracellular diffusion and decreases lysosomal sequestration

by DEY,D., Marc... at www.biorxiv.org 04-21-2023

https://www.biorxiv.org/content/10.1101/2023.04.19.537456v2

Deeper Inquiries

How could the findings on the impact of protonation state on small molecule drug diffusion and distribution be leveraged to improve the design and delivery of weakly basic drugs

The findings on the impact of protonation state on small molecule drug diffusion and distribution offer valuable insights for improving the design and delivery of weakly basic drugs. By understanding that protonation of weakly basic drugs at physiological pH leads to their sequestration in the cell cytoplasm, researchers can explore strategies to enhance the intracellular availability and distribution of these drugs. One approach could involve modifying the chemical structure of weakly basic drugs to reduce their propensity for protonation at physiological pH. For example, by introducing functional groups that lower the pKa of the drug, such as acetylation, the drug molecules may remain in a more neutral state, allowing for improved diffusion and distribution within the cell. This modification could potentially prevent ion trapping and reduce the sequestration of the drug in lysosomes, leading to enhanced intracellular activity.
Furthermore, the findings suggest that blocking protonation by using N-acetylated analogues significantly enhances the diffusion and fractional recovery of weakly basic small molecule drugs. This indicates that modifying the protonation state of the drug molecules can have a direct impact on their intracellular behavior. Therefore, future drug design efforts could focus on incorporating such modifications to optimize the intracellular availability and distribution of weakly basic drugs, ultimately improving their therapeutic efficacy.

What other cellular mechanisms or processes beyond lysosomal sequestration could contribute to the slow diffusion and low recovery of basic small molecule drugs in the cytoplasm

While lysosomal sequestration is a significant contributor to the slow diffusion and low recovery of basic small molecule drugs in the cytoplasm, other cellular mechanisms or processes may also play a role in this phenomenon. One potential factor could be nonspecific interactions with cellular components, such as proteins, nucleic acids, or membranes. Basic small molecule drugs may exhibit affinity for these cellular components, leading to their binding and reduced mobility within the cytoplasm. Additionally, the presence of macromolecular crowding in the cellular environment could hinder the diffusion of small molecules, especially those with basic properties, by increasing the likelihood of nonspecific interactions and steric hindrance.
Furthermore, the physicochemical properties of the small molecule drugs, such as their hydrophobicity and charge distribution, could influence their interaction with cellular structures and impact their diffusion rates. For example, the lipophilicity of a drug may affect its partitioning into cellular membranes, leading to altered diffusion behavior. Additionally, the size and shape of the drug molecule could influence its ability to navigate through the crowded cytoplasm and interact with specific cellular targets. Understanding these additional factors beyond lysosomal sequestration is crucial for comprehensively elucidating the mechanisms underlying the slow diffusion of basic small molecule drugs in the cytoplasm.

What implications do the differences in intracellular diffusion between proteins and small molecule drugs have for the design of drug-protein conjugates or other hybrid therapeutic approaches

The differences in intracellular diffusion between proteins and small molecule drugs have significant implications for the design of drug-protein conjugates or other hybrid therapeutic approaches. Proteins, being larger molecules with specific structural and functional properties, exhibit relatively faster diffusion rates and higher fractional recovery in the cellular cytoplasm compared to small molecule drugs. This difference in diffusion behavior suggests that drug-protein conjugates may face challenges in achieving optimal intracellular distribution and availability, especially if the drug component is a weakly basic small molecule.
To address this challenge, researchers developing drug-protein conjugates or hybrid therapeutic approaches need to consider the impact of the drug component's physicochemical properties on its intracellular behavior. Strategies to enhance the diffusion and distribution of small molecule drugs within the cell, such as modifying their protonation state or incorporating specific targeting moieties, may be necessary to ensure effective delivery and activity of the drug component in the conjugate. Additionally, optimizing the design of drug-protein conjugates to balance the properties of both components, considering factors like size, charge, and affinity for cellular structures, is essential for achieving the desired therapeutic outcomes. By leveraging the insights from the differences in intracellular diffusion between proteins and small molecule drugs, researchers can tailor the design of drug-protein conjugates to enhance their intracellular performance and therapeutic efficacy.