A High-Throughput Screening of a Natural Products Library for Mitochondria Modulators

Abstract

Mitochondria, the energy hubs of the cell, are progressively becoming attractive targets in the search for potent therapeutics against neurodegenerative diseases. The pivotal role of mitochondrial dysfunction in the pathogenesis of various diseases, including Parkinson's disease (PD), underscores the urgency of discovering novel therapeutic strategies. Given the limitations associated with available treatments for mitochondrial dysfunction-associated diseases, the search for new potent alternatives has become imperative. In this report, we embarked on an extensive screening of 4224 fractions from 384 Australian marine organisms and plant samples to identify natural products with protective effects on mitochondria. Our initial screening using PD patient-sourced olfactory neurosphere-derived (hONS) cells with rotenone as a mitochondria stressor resulted in 108 promising fractions from 11 different biota. To further assess the potency and efficacy of these hits, the 11 biotas were subjected to a subsequent round of screening on human neuroblastoma (SH-SY5Y) cells, using 6-hydroxydopamine to induce mitochondrial stress, complemented by a mitochondrial membrane potential assay. This rigorous process yielded 35 active fractions from eight biotas. Advanced analysis using an orbit trap mass spectrophotometer facilitated the identification of the molecular constituents of the most active fraction from each of the eight biotas. This meticulous approach led to the discovery of 57 unique compounds, among which 12 were previously recognized for their mitoprotective effects. Our findings highlight the vast potential of natural products derived from Australian marine organisms and plants in the quest for innovative treatments targeting mitochondrial dysfunction in neurodegenerative diseases.

Keywords: high-throughput screening; mitochondria modulators; natural products.

Figure 1

Flow chart of the study.

Figure 2

A schematic chromatogram illustrating the HPLC fractionation of a representative positive biota. Dotted lines indicate the time periods for each fraction.

Figure 3

High-throughput screening of 4224 fractions obtained from NatureBank generating 108 hits. The two red lines indicate a relative Z-score of −2.5–2.5. Grey dots: fractions, blue dots: cells treated with vehicle only.

Figure 4

High-throughput screening of 108 fractions, leading to 20 fractions from 11 biotas. Grey dots: fractions, blue dots: cells treated with vehicle only. The two red lines indicate a relative Z-score of −2.5–2.5.

Figure 5

Dose–response curves for rotenone, MPP⁺, 6-OHDA, and DMSO in SH-SY5Y cells. Assays were performed in three biological repeats, with two technical repeats for each biological repeat.

Figure 6

MTT assay of fractions at (A) 100 µg/mL, (B) 50 µg/mL, and (C) 25 µg/mL. * p < 0.05 when fractions are compared to cells + DMSO + 6-OHDA.

Figure 7

(A) Dose–response curve for SH-SY5Y cells exposed to 6-OHDA using the mitochondrial membrane potential (MMP) assay. (B) Analysis of selected active fractions identified in the MTT assay through the MMP assay. All the analyzed fractions, except for fraction 7 from Dendrilla sp., showed significant mitochondrial protective effects compared to the control group of cells treated with DMSO and 6-OHDA (** p < 0.01).

Figure 8

Structure of compounds with known mitoprotective activity.