Targeting mitochondria for drug discovery against copper toxicity

Project leaders

Elia Di Schiavi, Liudmila Puchkova

Agreement

RUSSIA - RFBR-suspended - Russian Foundation for Basic Research

Call

CNR/RFBR triennio 2018-2020 2018-2020

Department

Biology, agriculture and food sciences

Thematic area

Biology, agriculture and food sciences

Status of the project

New

Research proposal

Background
Right balance of microelements represent a serious challenge for nutrition and health care. Copper (Cu) is a vitally important nutrient required for processes as respiration, connective tissue biogenesis and neurotransmitter synthesis. However, in excess Cu represents a serious danger due to its ability to induce oxidative damage of cellular components. Anthropogenic activities resulted in the Cu contamination of the environment at levels exceeding the toxicity threshold. Thus, revealing mechanisms that either promote or counteract Cu toxicity is of great importance for human and animal health and for agrifood production. Recently, mitochondria emerged as a first target of Cu toxicity due to alteration of mitochondrial Red-Ox balance upon Cu overload.
Wilson disease (WD) represents an excellent system for Cu toxicity studies considering that easy-to-use C.elegans and cell models for WD are available. WD is caused by mutations in ATP7B pump effluxing excess Cu from hepatocytes into the bile. Loss of ATP7B leads to toxic Cu overload in liver and then in brain, causing fatal hepatic and neurologic abnormalities. Accumulating Cu hampers integrity of mitochondria, which release numerous pro-apoptotic factors, killing hepatocyte and causing liver failure in WD patients. Thus counteracting mitochondria-mediated death pathways is of particular importance in WD, but will also be a powerful biotechnological tool to counteract Cu toxicity induced by pollution in agrifood productions and constitutes the main objective of this joint proposal.
Overall Strategy
Cu-mediated mitochondrial damage could be handled using two approaches: 1) protection of mitochondria from oxidative damage/stress and 2) rapid removal of damaged mitochondria, which threaten cell viability via release of pro-apoptotic compounds. Correspondingly, RFBR and CNR teams will develop two tightly integrated lines of research outlined below.
TASK 1. PROTECTION OF MITOCHONDRIA FROM COPPER TOXICITY (RFBR)
A) Screening for sub-population of mitochondria with particular sensitivity to Cu toxicity.
We intent to employ mitochondria-specific sensors of Cu, membrane potential and oxidative stress to reveal subpopulations of mitochondria, which undergo rapid damage in response to Cu overload in WD models in C.elegans and cell (ATP7B-KO cells). Identification of such mitochondrial subpopulation is needed to have a reliable readout of efficiency of gene silencing and drugs that prevent mitochondrial dysfunction in ATP7B-deficient cells (tasks 1B, C). After identification in C.elegans and ATP7B-KO cells the search for the similar Cu-sensitive mitochondrial populations will be done in mice model of WD.
B) Identification and validation of molecular targets that increase resistance of mitochondria to Cu.
CNR will run shRNA screening to detect druggable genes whose silencing protects ATP7B-KO cells from Cu toxicity (task 2B). RFBR will validate these gene targets for ability to reduce population of Cu-sensitive mitochondria and for capacity to protect mitochondria from Cu-mediated damage in vitro (ATP7B-KO). Validated targets will be advanced for further in vivo studies first in C.elegans and then in mice.
C) Validation of drugs, reducing sensitivity of mitochondria to toxic Cu in WD.
Drugs corresponding to validated shRNA hits and obtained through FDA-approved library screening (task 2B) will be tested for their potential to limit mitochondria damage by Cu in ATP7B-KO cells and C.elegans. Best compounds will be further advanced for evaluation of their efficiency in mice models of WD.

TASK 2. REMOVAL OF DAMAGED MITOCHONDRIA TO REDUCE COPPER TOXICITY (CNR)
A) Evaluation of mitophagy as protective mechanism in WD.
Our preliminary studies indicate that mitophagy is activated in cell and animal models of WD. During the course of the project we will analyze mitochondria subpopulations for their commitment to mitophagic turnover. Moreover the impact of mitophagy activators/inhibitors on tolerance to Cu will be evaluated in C.elegans and cell model of WD.
B) High throughput screenings (HTSs) for gene targets and drugs, facilitating survival of ATP7B deficient cells through mitochondrial and/or mitophagic pathways
We will perform viability-based HTSs of druggable genome shRNA library (7494 target genes) and FDA-approved drug library (over 1300 compounds) for hits that allow ATP7B-KO cells to survive in Cu. HTS hits will be evaluated for their impact on mitochondrial function (Task 1B,C) and mitophagy in ATP7B-deficient cells.
3) Validation of mitochondria-specific gene targets and drugs in animal models of WD
Hits from HTSs with impact on mitophagy will be further tested in C.elegans for ability to counteract loss of ATP7B ortholog cua-1. The most effective FDA-approved compounds or target genes will be advanced into study in mice model of WD for ability to reduce Cu toxicity.
In sum, the proposed plan will allow us to reveal molecular targets involved in Cu toxicity and to identify FDA-approved compounds that can be rapidly repurposed for WD therapy.
Justification for collaboration
The proposed project requires joint effort of both teams and cannot be executed alone by either partner. RFBR will require expertise of CNR in C.elegans disease modeling, in mitophagy and in WD and CNR-accessible technologies (HTS and advanced microscopy). Correspondingly CNR needs the expertise of Russian partner in mitochondrial biology, Cu homeostasis and disease modeling in mice. The joint effort of both teams significantly increases the chances for successful execution of the project.
Feasibility
We believe that the proposed research plan is feasible, considering joint expertise of collaborating teams. Planned research will be executed at high-end instrumentation, from confocal microscopes to next generation sequencing and supported by institutional core services. All key resources, like shRNA, drug libraries, WD cells, C.elegans and mice model are available.

Research goals

AIM1. DISCOVERY OF MECHANISMS AND TREATMENTS THAT PROTECT MITOCHONDRIA FROM COPPER TOXICITY
Sub-aim 1A. Screening for sub-population of mitochondria with particular sensitivity to Cu toxicity.
Sub-aim 1B. Identification and validation of molecular targets that increase resistance of mitochondria to Cu in WD.
Sub-aim 1C. Validation of mitochondria-specific drugs that induce tolerance to Cu overload in WD.

AIM2. IDENTIFICATION OF MECHANISMS THAT REDUCE COPPER TOXICITY THROUGH TURNOVER OF DAMAGED MITOCHONDRIA
Sub-aim 2A. Evaluation of mitophagy as potential protective mechanism in WD.
Sub-aim 2B. High throughput screenings for gene targets and drugs, facilitating survival of ATP7B deficient cells through mitochondrial and/or mitophagic pathways
Sub-aim 2C. Validation of mitochondria/mitophagy-associated gene targets and drugs in animal models of WD

Last update: 25/04/2024