A semi-quantitative cell-based phenotypic assay for determining the lysosomal accumulation of small molecule antimalarials

Published by Gus Rosania on 18 December 2007 - 3:22pm

One of the reasons why scientists in the past have not been able to develop small molecule drugs targeted to subcellular organelles is the lack of suitable assays to monitor the absolute concentration of small molecules within those organelles. Although measuring the absolute concentration of small molecules within organelles is still very difficult experimentally, the availability of large chemical libraries of small molecules have permitted the use of semi-quantitative (or qualitative) experimental readouts, to test the validity of mathematical models predicting subcellular distribution of small molecules. In this context, Nan Zheng, a second year PhD graduate student that recently joined the lab, is interested in developing a bioassay to report the accumulation of small molecules in lysosomes. Such an assay would allow validating the results of cell-based molecular transport simulations as they pertain to the lysosomal accumulation of small drug-like molecules, such as falcipain-2 inhibitors being developed as antimalarial agents (falcipain-2 is a resident lysosomal enzyme).

In the past, it had been observed that small lysosomotropic molecules (such as the antimalarial drug chloroquine) induce a vacuolation phenotype in cells that are incubated with drug. This vacuolation phenotype is the result of the osmotic swelling and expansion of the lysosomal compartment, as a result of the accumulation of the drug molecules in the organelle. Back in 1974, Charles DeDuve described this phenomenom, and attributed it to an ion trapping mechanism. According to this mechanism, the lumen of the lysosome is acidic (pH 5) in relation to the cytosol (pH 7.4). Weakly basic molecules (such as molecules containing amine functionality) exist mostly in neutral form in the cytosol, while in the lysosomes they exist mostly in protonated, charged form. Because the protonated charged form of the molecule is largely membrane impermeant while the neutral form is membrane permeant, the pH gradient across the lysosomal membrane results in a chemical potential that drives the accumulation of the weakly basic molecule in the lumen of the lysosome.

While one may expect that most weakly basic molecules would tend to accumulate in lysosomes, one would also expect them to do so to different extents based on differences in the ionization constants and membrane permeability of the different ionic species. But, perhaps most importantly, pH gradient across the lysosomal membrane is a generated by the lysosomal H+ATPase, a protein that uses ATP hydrolysis to pump protons from the cytosol into the lysosomal lumen. Thus, the lysosomal accumulation of small molecules should also be highly sensitive to the metabolic status of the cell. If a small molecule accumulates in mitochondria or other organelles in a non-specific fashion (in addition to accumulating in lysosomes), it will compromise the metabolic status of the cell in a manner that decreases the production of ATP, ultimately leading to the dissipation of the lysosomal pH gradient. In this manner, only those molecules that selectively accumulate in lysosomes “while not accumulating in other organelles- should be able to induce lysosomal vacuolation phenotype. Accordingly, the goal of Nans project will be to fine-tune our molecular transport simulators so as to be able to rank molecules in terms of their ability to induce lysosomal swelling, considering not only the ion trapping mechanism, but also the selectivity of the molecules for accumulating in lysosomes vs. other organelles.