Quantification of Intracellular Accumulation and Retention of Lysosomotropic Macrocyclic Compounds by High-Throughput Imaging of Lysosomal Changes

Abstract

Many marketed pharmaceuticals reach extremely high tissue concentrations due to accumulation in lysosomes (lysosomotropism). Quantitative prediction of intracellular concentrations of accumulating drugs is challenging, especially for macrocyclic compounds that mainly do not fit in current in silico models. We tested a unique library of 47 compounds (containing 39 macrocycles) specifically designed to cover the entire range of accumulation intensities observed with pharmaceuticals so far. For the first time, we show that intracellular concentration of compounds measured by liquid chromatography with tandem mass spectrometry correlates with the induction of phospholipidosis and inhibition of autophagy, but the highest correlation was observed with the increase of lysosomal volume (R ¼ 0.95), all measured by high-throughput imaging assays. Based only on imaging data, we developed a 5-class in vitro model for the prediction of compound accumulation with the accuracy of 81%. The measured change of total lysosomal volume can thus be used in high-throughput screening for determination of the actual intensity of intracellular accumulation of new macrocyclic compounds. The models are largely based on macrocycles, greatly improving the screening and prediction of intracellular accumulation of this challenging class. However, all tested nonmacrocyclic compounds fitted well in the models, indicating potential use of the models in broader chemical space.

Existing highly accumulating lysosomotropic drugs with potential for repurposing to target COVID-19

“…The mentioned drugs, chloroquine, hydroxychloroquine and azithromycin, highly accumulate in lysosomes/endosomes and their membranes, reaching about 100-fold higher intracellular than their extracellular concentration [ 7 , 8 ], and thus belong to a group of lysosomotropic compounds (compounds that accumulate in lysosomes). We propose that the antiviral activity of (hydroxy)chloroquine and azithromycin is shared among all strong lysosomotropic drugs and is a consequence of their extremely high accumulation in cells and membranes, and subsequently of all the processes affected by this pharmacokinetic property.…”

“…In our previous studies on a set of 47 compounds we have shown that the extent of lysosomotropic accumulation in cells correlates with the compound’s extent of induction of phospholipidosis and lysosomal swelling [ 7 ]. The correlation is so apparent that it is even possible to determine the level of accumulation from the intensity of either of these processes caused by a drug.…”

“…with less reliable prediction of accumulation intensity [ 30 ]. For several molecules in Table 2 the data on experimentally determined cellular accumulation is also provided (measured ACC) [ 7 ]. Third inclusion criterion was the strongest basic dissociation constant, pKa, higher than 7.5 which is an indication that the molecule can become protonated, and thus trapped, inside the acidic compartment in addition to eventually increasing lysosomal/endosomal pH [ 18 , 32 ].…”

“…When analysing the potential of repurposing such highly specific and potent antiviral drugs for fighting a different virus, it should be kept in mind that they would likely need a much higher concentration since their original target that they are specifically acting upon is not present in other viruses. Impact of strong lysosomotropic compounds on membranes and membrane trafficking often needs low micromolar concentrations [ 7 ]. It remains to be seen whether efficacious levels of any of CADs can be achieved in COVID-19 patients without trespassing into toxicity levels.…”

Lysosomotropic Features and Autophagy Modulators among Medical Drugs: Evaluation of Their Role in Pathologies

“…Gulbins et al, 2018 [20] Guan et al, 2019 [21] Lee et al, 2013 [22] Easwaranathan et al, 2019 [23] Azithromycin (antibiotic) Strong cationic amphiphilic drug.…”