Acetylcholinesterase (AChE) is responsible for hydrolyzing the acetylcholine neurotransmitter, bringing an end point to cholinergic neurotransmission. Thus, AChE is the primary target of a wide spectrum of compounds used as pesticides, nerve agents or therapeutic drugs for neurodegenerative diseases such as Alzheimer’s disease (AD). This enzyme is heterogeneously distributed in the brain showing different activity depending on the nervous region. Therefore, the aim of this work is to report a novel technology that enables the simultaneous determination of tissue specific AChE activity, as well as the analysis and screening of specific inhibitors, by using cell membrane microarrays. These microarrays were composed of cell membranes, isolated from 41 tissues, organs and brain areas, that were immobilized over a slide, maintaining the functionality of membrane proteins. To validate this platform, demonstrating its usefulness in drug discovery as a high throughput screening tool, a colorimetric protocol to detect the membrane-bound AChE activity was optimized. Thus, rat cortical and striatal AChE activities were estimated in presence of increased concentrations of AChE inhibitors, and the donepezil effect was assessed simultaneously in 41 tissues and organs, demonstrating the major potential of this microarray’s technology.

Melanoma is a carcinoma known to evade the host immune defenses via the downregulation of the immune response. One of the molecules involved in this mechanism is programmed cell death ligand 1 (PD-L1), which interacts with its receptor, programmed cell death protein 1 (PD-1), expressed on T cells, leading to a reduction in cytokine release and cytotoxic activity, as well as a halt in T-cell proliferation. The approved therapeutic monoclonal antibodies, such as pembrolizumab, target the PD-1/PD-L1 interaction and are revolutionizing cancer treatments. We developed an assay that provides a quantitative readout of PD-1/PD-L1 interactive states between cell membranes of human immune cells (peripheral blood mononuclear cells, PBMCs) and PD-L1-expressing samples. For this purpose, cell membrane microarrays (CMMAs) were developed from membranes isolated from a HT144 cell line and melanoma samples, and PD-L1 expression was quantified using immunofluorescence (IF). CMMAs were incubated with cell membranes of PBMCs expressing PD-1, and the interaction with PD-L1 was quantified by time-resolved Förster resonance energy transfer, in the presence and absence of pembrolizumab as a blocking drug. The developed assay was able to quantify the PD-1/PD-L1 interaction, and this engagement was disrupted in the presence of the blocking antibody. This demonstrates the potential of the method to analyze monoclonal antibody drugs, as well as the functional states of immune checkpoint regulators. Furthermore, our findings provide evidence to support the future implementation of this methodology for both drug discovery and immune system monitoring in cancer, transplantation, and inflammatory and autoimmune diseases.

The production of reactive oxygen species (ROS) increases considerably in situations of cellular stress, inducing lipid peroxidation and multiple alterations in proteins and nucleic acids. However, sensitivity to oxidative damage varies between organs and tissues depending on the triggering process. Certain drugs used in the treatment of diverse diseases such as malaria have side effects similar to those produced by oxidative damage, although no specific study has been conducted. For this purpose, cell membrane microarrays were developed and the superoxide production evoked by the mitochondrial activity was assayed in the presence of specific inhibitors: rotenone, antimycin A and azide. Once the protocol was set up on cell membrane isolated from rat brain areas, the effect of six antimalarial drugs (atovaquone, quinidine, doxycycline, mefloquine, artemisinin, and tafenoquine) and two essential oils (Rosmarinus officinalis and Origanum majoricum) were evaluated in multiple human samples. The basal activity was different depending on the type of tissue, the liver, jejunum and adrenal gland being the ones with the highest amount of superoxide. The antimalarial drugs studied showed specific behavior according to the type of human tissue analyzed, with atovaquone and quinidine producing the highest percentage of superoxide formation, and doxycycline the lowest. In conclusion, the analysis of superoxide production evaluated in cell membranes of a collection of human tissues allowed for the characterization of the safety profile of these antimalarial drugs against toxicity mediated by oxidative stress.

Drug side effects are one of the main reasons for treatment withdrawal during clinical trials. Reactive oxygen species formation is involved in many of the drug side effects, mainly by interacting with the components of the cellular respiration. Thus, the early detection of these effects in the drug discovery process is a key aspect for the optimization of pharmacological research. To this end, the superoxide formation of a series of drugs and compounds with antidepressant, antipsychotic, anticholinergic, narcotic, and analgesic properties was evaluated in isolated bovine heart membranes and on cell membrane microarrays from a collection of human tissues, together with specific inhibitors of the mitochondrial electron transport chain. Fluphenazine and PB28 promoted similar effects to those of rotenone, but with lower potency, indicating a direct action on mitochondrial complex I. Moreover, nefazodone, a drug withdrawn from the market due to its mitochondrial hepatotoxic effects, evoked the highest superoxide formation in human liver cell membranes, suggesting the potential of this technology to anticipate adverse effects in preclinical phases.