Lysosomal Trapping Assays

Assess lysosomal sequestration of a compound using Solvo’s high-content imaging (HCI) platform

While the plasma concentration of a drug is the most readily accessible measure of its bioavailability, its disposition – and often its effect, too – may ultimately depend on free intracellular drug concentrations in the respective tissues. Therefore, understanding the mechanisms that govern cellular unbound drug concentration is essential for reliable in vivo pharmacokinetic and pharmacodynamic predictions based on in vitro data from different systems. In addition to determining unbound drug plasma concentrations and addressing organ-specific uptake rates and partitioning dynamics, intracellular disposition of a compound can also affect its pharmacokinetics. Present in most eucaryotic tissues, lysosomes are not only compartments for cellular deposit, but fundamental elements of physiologic processes such as plasma membrane repair, recycling of cell-surface receptors, reactive oxygen species (ROS) generation, inflammation, cell survival and cell death [1].

Sequestration of molecules into the lysosomes – as summarized on Figure 1A –is driven by the pH difference between the nearly neutral cytosol (pH 7.2) and the acidic lysosomal lumen (pH 4-5), and therefore mostly affects lipophilic basic compounds (~ ClogP>2, pKa 6.5-11). These lipophilic basic compounds can freely diffuse across the phospholipid membrane in their unionized form to enter the cell and the lysosome. In the acidic environment of the lysosome (pH 4–5), the Henderson-Hasselbalch equilibrium shifts toward the compound’s protonated form (BH⁺), which form has markedly reduced permeability across lipid bilayers, and therefore gets trapped in the lysosome [1].

Figure 1. – A) Overview of the mechanism of lysosomal trapping. The basic compound (yellow circles) can diffuse from the plasma or medium to the cytosol and then passively enter the lysosome. In this more acidic environment, the equilibrium shifts towards the protonated form of the compound (marked with red +) that has much lower passive permeability and therefore gets trapped in the lysosome. B) Mechanism for detecting lysosomal trapping indirectly based on the extrusion of a fluorescent dye (red circles) from the lysosomal space, leading to a decrease in lysosomal fluorescent signal upon administration of a lysosomotropic compound (yellow circles).

Lysosomal trapping or lysosomal sequestration of a compound leads to its accumulation in the lysosomes from the cytoplasm. Such an intracellular distribution limits potential interaction of the compound with a target localized in the cytoplasm or nucleus as well as its availability for metabolic processes, especially in lysosome-rich organs such as the liver or the lungs [1]. Trapping of a compound can also affect how cellular accumulation data should be interpreted as in this case the cytoplasmic free fraction of the compound would be much lower than the total intracellular quantities. Lysosomal trapping can therefore have complex implications, including:

• Limited target engagement and efficacy issues [2-3], tumor resistance to treatment [4],
• Effective dose shift and clearance shift [2-3],
• Drug-drug interactions at the lysosomal level in case of co-administration with other lysosomotropic compounds [5],
• Adverse effects such as phospholipidosis [6] or loss of lysosomal function due to pH shift [5].

Lysosomal targeting of drugs can however also be an important strategy either for indications related to lysosomal dysfunction or to leverage lysosome-dependent cell death for cancer therapy [7].

Investigation of the lysosomal sequestration of a compound may therefore be relevant in multiple scenarios and phases of drug development. In case lysosomal interactions are either a goal or a considerable risk in the development strategy, early trapping screens can be warranted. As a mechanistic investigation assay, it can also be leveraged to understand potential discrepancies between in vitro and in vivo pharmacokinetic observations. Data on compound unbound fractions in specific cell types and subcellular organs is also often used to improve the predictive performance of in silico PBPK modelling [8-11], an approach applied more and more commonly in drug development projects. To evaluate lysosomal trapping of drugs, different methods are available including the quantification of changes in lysosomal dye accumulation by fluorescent detection as well as determination of intracellular concentration of a drug in the presence and absence of lysosome inhibitors [12]. Also, artificial lysosomal fluid is available to predict lysosomal trapping of the compounds without cells [13].

Compounds that undergo lysosomal trapping can affect sequestration of other lysosomal molecules – endogenous substrates or xenobiotics and therefore may act as an inhibitor of lysosomal function to some degree. However, there are several other mechanisms that have a broader impact on lysosomes. For example, NH₄Cl, upon its lysosomal uptake, neutralizes the intraorganellar pH, essentially eliminating the pH differences that is a prerequisite for lysosomal trapping of compounds [14-16]. Such a strong effect on pH is, however, rare among drug candidates. A few other mechanisms may also result in disruption of the lysosomal function, for example interfering with Cyclin G‑associated kinase (GAK) functions affects lysosomal dynamics [17], while inhibitors of the vacuolar type H⁺-ATPase (v-ATPase) responsible for transferring protons into the lysosome, such as Bafilomycin A1, also disrupt lysosomal acidification [18].

Lysosomal sequestration assessment using high-content imaging (HCI)

For investigating lysosomal trapping of drugs, the most commonly applied approach is based on the quantification of changes in lysosomal dye accumulation in the presence and absence of the compound of interest. The LysoTracker Red dye has been specifically developed for visualizing lysosomes as it is strongly lysosomotropic (preferentially accumulates in the lysosomes) and allows for fluorescent detection [14]. Compounds that are also subject to lysosomal sequestration compete with the dye and their trapping leads to a concentration-dependent decrease of LysoTracker signal in the lysosomes (Figure 1B). Based on this relative signal reduction compared to the dye applied alone, expressed at percent (%) remaining signal, an IC₅₀ value can be calculated to quantify the degree of lysosomal interaction.

Known lysosomotropic compounds, such as chloroquine or propranolol can be used as positive control for inhibition of lysosomal dye accumulation. It should be noted, however, that other mechanisms exist that interfere with lysosomal function (as described earlier), that may not be differentiated using this indirect approach. Decreased trapping of the LysoTracker dye may also be the result of various potential compound effects on lysosomal function. In most cases, however, dye extrusion due to competitive lysosomal trapping is assumed. If mechanisms other than competitive lysosomal accumulation are suspected, or the degree of sequestration needs to be quantified, direct comparison of test compound accumulation via LC/MS in the presence and absence of lysosomal inhibitors (e.g., Bafilomycin A1) is recommended.

Figure 2. – Cell images of human primary hepatocytes captured by High Content Imaging (HCI) using 20x magnification air objective. LysoTracker red (LTR) dye (applied at 50 nM) signal is shown in yellow with A) no lysosomal trapping inhibitor added B) lysosomes marked and identified by the HCI image analysis software for fluorescence quantification. C) LTR signal upon treatment with the NH₄Cl lysosomal function inhibitor (10 mM).

The fluorescent signal of the LysoTracker dye can be quantified using various detection methods. Solvo’s Lysosomal trapping assay leverages our High Content Imaging (HCI) platform for a considerably higher detection accuracy and sensitivity that the traditionally applied microplate reader-based approach. HCI-based cell detection allows for the detection of not only individual cells, but lysosomes as well so changes in the fluorescent output can be detected precisely at the lysosomal level as demonstrated on Figure 2. As a microplate reader detects signal from a whole well, relatively high background signals are obtained in control conditions as well where no specific lysosomal trapping occurs, this is, however, almost fully eliminated by a applying a HCI approach.

IC₅₀ values for the lysosomal trapping of the LysoTracker Red dye for multiple known lysosomotropic drugs, chloroquine, imipramine and propranolol, were generated using multiple cell types: MDCKII canine kidney cells, Caco-2 and hepatocytes from both human and rat. Inhibition results obtained with the different cell lines were similar for the tested compounds (Figure 3A), and also in line with literature findings where lysosomal trapping readouts were generated using HCI [14, 19]. Using this detection method, we were also able to determine IC₅₀ values for the inhibition of LysoTracker accumulation for Verapamil, a compound previously proven to affect lysosomal sequestration of other compounds in the blood-retinal barrier, likely via a competitive mechanism [20] (Figure 3B). Inhibition curves were also generated for Ketoconazole, a drug shown to induce phospholipidosis and predicted to accumulate in lysosomes [6] (Figure 2B).  

MDCKII, and especially Caco-2 cells reliably predicted lysosomal trapping (Figure 3B) and can be used in testing without the additional cost associated with the application of cryopreserved hepatocytes. Cryopreserved hepatocytes, however, express a number of characteristic uptake transporters, including OATP1Bs that are mostly absent in MDCKII, Caco-2, as well as most commercially available immortalized hepatic cells lines, which can contribute to intracellular compound accumulation allowing a more complex detection of compound handling in case a thorough mechanistic investigation is needed.

Figure 3. – Concentration dependent inhibition of LTR accumulation A) by Chloroquine in human hepatocytes, Caco-2 and MDCKII cells; and B) at 14 minutes by Chloroquine, Imipramine, Propranolol, Verapamil and Ketoconazole with Rosuvastatin as non-lysosomotropic control measured in primary human hepatocytes at 14 minutes. The figure shows one representative measurement for each inhibitor compound or cell type, respectively.

Comparison of readouts with HCI analysis versus traditional microplate reader detection confirmed superior sensitivity. Using HCI, attenuation of the fluorescent LysoTracker signal by both the competitively trapped chloroquine and by NH₄Cl, an inhibitor of lysosomal function, was several-folds more pronounced than in case of microplate readouts (Figure 4). This increased sensitivity resulted in significantly lower IC₅₀ values for lysosomotropic compounds when HCI was used (Figure 4B), confirming the improved overall detectability of lysosomal interactions, while the chance of generating false negative results is reduced.

Solvo’s Lysosomal trapping assay assesses the effect of the test compound on LysoTracker red accumulation using HCI-based analysis. Changes in dye signals are determined as percentage (%) remaining signal compared to a control where dye is applied alone. In parallel with the test article, NH₄Cl is applied as a negative control where all lysosomal function is abolished as well as chloroquine (or another reference probe) as an assay functionality control, and acceptance criteria are defined for these controls to ensure assay performance quality. Potential cytotoxic effects of a compound are also addressed using resazurin-based cytotoxicity tests after assay completion to ensure that signal reduction due to cell death is not mistaken for the absence of lysosomal trapping. Cell nuclear staining is done by Hoechst as an additional control for cell counting. Upon request, additional dyes, for example cell masks or dyes staining dead cells can be applied in a custom setup.

Figure 4. – A) Inhibition LTR accumulation by chloroquine or NH₄Cl in HEK293-OATP1B1-LV cells expressed as remaining fluorescent activity (%) with fluorescence detection performed using a Microplate Reader (MPR) or a High Content Imaging (HCI) platform. B) IC₅₀ values determined for inhibition of LTR accumulation by chloroquine using MPR or HCI readouts, using multiple cell types. Datapoints where reference is not marked were generated by SOLVO using human hepatocytes (hHEPs), rat hepatocytes (rHEPs), Caco-2 and MDCKII.

Solvo’s lysosomal trapping assay is available in multiple potential setups to be able to flexibly adapt to specific project needs. Screen-like tests looking at the effects of a series of test compounds in 1 or 2 concentrations can be applied to a high number of compounds at earlier stages while IC₅₀ determination or more complex and tailored investigative assay setups can be applied for mechanistic investigations or to generate solid data for PBPK modeling.

Direct lysosomal trapping assay using LC-MS/MS readouts

While for many potential uses, for example to confirm lysosomal targeting where needed or to rule out compound trapping, the identification of lysosomal interaction itself via an indirect approach is sufficient, information on the magnitude of lysosomal sequestration may also be necessary. For this, intracellular levels of the test drug itself needs to be directly quantified and compared in the presence and absence of a lysosomal function inhibitor such as Bafilomycin A1.

Our assay for detecting lysosomal trapping of compounds directly with LC-MS/MS (or liquid scintillation in case test compounds are [³H] or [¹⁴C] labelled), was to set up and optimized in Caco-2 cells and hepatocytes. The reference substrates used for the development of this assay were chloroquine, propranolol, and imipramine, of which, chloroquine has been retained as the recommended positive control in the assay at is showed the highest degree of lysosomal accumulation. As negative control, Rosuvastatin is used Test compounds as well as controls are tested in the presence and absence of a lysosomal function inhibitors.

After incubation with the substrate or test compound with and without inhibitor, the cells are lysed and the effect of the inhibitor on intracellular compound accumulation is quantified. In case a decrease in overall intracellular concentration is observed, the compound is assumed to undergo lysosomal trapping. Data is provided as % compound accumulation in the presence of the inhibitor versus the no-inhibitor control condition.

Lysosomal trapping	Lysosomal accumulation (%)
strong	50%<
weak	20%-50%
none	<20%

Table 1. Evaluation of lysosomal sequestration potential of test compounds. Lysosomal accumulation % is determined as the change (decrease) in intracellular compound concentrations upon co-incubation with the lysosomal function inhibitor, Bafilomycin A1. 

Both NH₄Cl and Bafilomycin A1 were considered as potential reference inhibitor in this assay system. Historically, NH₄CI has been more extensively used in lysosomal trapping experiments, therefore more literature data is available with this compound. However, upon completion of comparative testes using all three above listed probe substrates, we determined that in our setup, the use of Bafilomycin A1 leads to significantly improved fold-change detection of trapped compounds over control conditions where no inhibitor is applied. Therefore, we apply Bafilomycin A1 as reference inhibitor to improve sensitivity of our lysosomal trapping assays with LC-MS/MS readout (for HCI-based assays, the recommended inhibitor remains NH₄Cl as it does not require preincubation with the cells and is thus more compatible with this quick and sensitive, screen-type assay).

Figure 5. Effect of lysosomal function inhibitors NH₄Cl and Bafilomycin A1 on the intracellular accumulation of three lysosomally sequestered compounds, Propranolol, Imipramine and Chloroquine. In case of Rosuvastatin, a compound not subject to lysosomal trapping, neither of the tested inhibitors significantly affected its intracellular concentrations.

Similar results were obtained for Caco-2 and hepatocytes, with Bafilomycin A1 identified as a more potent inhibitor of lysosomal function. In case of hepatocytes, while uptake of imipramine and propranolol was somewhat variable across different hepatocyte lots, levels of chloroquine accumulation were similar, confirming its suitability as a positive control in the assay system.

Readout type	High Content Imaging (HCI)	LC-MS/MS
Principle	Indirect detection of lysosomal sequestration via Lysotracker Red dye extrusion	Direct measurement, % change in accumulation in presence of lysosomal inhibitor
Cell types available	MDCKII (canine), Caco-2 or human hepatocytes	Caco-2 or human hepatocytes
Positive control	Chloroquine
Negative control	Rosuvastatin
Recommended inhibitor	NH4Cl (Ammonium-chloride)	Bafilomycin A1
Recommended application	Earlier stage or preliminary screening - higher throughput, screen-like format available, high sensitivity	Mechanistic investigations - direct measurement of compound lysosomal trapping, lower throughput

Table 2. Overview of main assay parameters and advantages for lysosomal trapping assessment using an indirect approach with HCI-readout or a direct approach with LC-MS/MS readouts, respectively.

If you are interested in our Lysosomal Trapping assays, additional potential applications of our HCI platform or to consult with our expert team, reach out to us at .(JavaScript must be enabled to view this email address) or use our contact form!

References

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