Ageing is associated with the decline in the capacity of the autophagy pathway to degrade dysfunctional and damaging cellular components, such as protein aggregates and mitochondria. Dysfunctional autophagy, in turn, undermines other cellular functions including DNA repair, metabolism and survival. Therefore, activation of autophagy is considered a promising therapeutic approach to combat ageing and age-related diseases.
Problem Lysosomal dysfunction is an important factor contributing to the reduction of autophagy during aging. As dysfunctional lysosomes interfere with autophagy at the terminal stage, stimulation of autophagy initiation can be ineffective to rescue autophagy. Additionally, current methods to measure autophagy are rather unreliable, slow, and with complicated readouts, making the screening of compounds that promote autophagy less efficient. Opportunity To model lysosomal dysfunction, Prof. Korolchuk lab uses cells with a mutation in a lysosomal protein (Npc1) associated with neurodegenerative diseases. When these cells are subjected to metabolic stress, they suffer cell death due to dysfunctional autophagy, providing an easy readout for an autophagy assay (cells dead/cells alive). To identify true autophagy activators, Prof. Korolchuk lab uses cells that lack initiation of autophagy and are therefore not rescuable by autophagy inducers in parallel with Npc1 KO cells. The Korolchuk lab will use this innovative method to screen an unique library of natural compounds, synthesize derivatives based on hits and identify their biological target. Highlights - Assay with easy readout, decent throughput, good controls and targeting one of the most important processes in cellular aging (autophagy) - Solid evidence supporting their approach - The platform would allow collaboration with other projects targeting autophagy/mitophagy - Strong and productive scientific team interested in the IP/NFT model and in company formation
Extensive work leading to this proposal identified a unique phenotype of cells with dysfunctional autophagy in tissue culture (Autophagy promotes cell survival by maintaining NAD(H) levels | Research Square, https://www.biorxiv.org/content/10.1101/2020.01.31.928424v1, under revision for Nat Cell Biol). In normal glucose-containing medium autophagy KOs upregulate glycolysis at the expense of mitochondrial respiration. When glucose is replaced with galactose, which results in zero net ATP production through glycolysis and cells are forced to respire, autophagy deficient cells become apoptotic. This phenotype is common for Npc1 KO MEFs where autophagosome maturation is impaired as well as for cells with the loss of core autophagy genes (Atg5, Atg7 and FIP200/Rb1cc1). We established the sequence of events leading to cell death of autophagy deficient cells: 1) accumulation of dysfunctional mitochondria; 2) stress in the form of increased ROS and DNA damage; 3) activation of stress response pathways (NADases of Sirtuin and PARP family); 4) depletion of cellular NAD+ (and NADH) pools; 5) mitochondrial depolarisation; 6) apoptotic cascade. Our data with yeast, fly, and human stem cell-derived neuronal models of autophagy deficiency indicate that the mechanisms of cell death described in MEFs is evolutionarily conserved from yeast to humans (Autophagy promotes cell survival by maintaining NAD(H) levels | Research Square, https://www.biorxiv.org/content/10.1101/2020.01.31.928424v1). Targeting the processes downstream of autophagy dysfunction (mitochondrial dysfunction, hyperactivity of NADases, NAD boosting, mitochondrial re-polarisation) can rescue cell death in cells/organisms with both the genetic loss of Atg genes and Npc1. On the other hand, autophagy inducers can rescue autophagy block and cell survival in Npc1 cells, whilst in Atg5 KO cells true autophagy inducers are not able to rescue autophagy or cell death. This provides us with an opportunity for a unique and rapid high throughput cell death-based screening system. Compounds that are capable of activating autophagy and overriding the autophagy block in Npc1 KO cells rescue cell death, whilst their dependence on functional autophagy is indicated by the lack of effect in Atg5 KO cells. Additionally, we generated and optimised for HTS wild type or Npc1 KO MEFs with an inducible tet-on-tet-off expression of a luciferase-tagged autophagy flux reporter p62. As a proof of principle, we performed a screen using a library of FDA-approved drugs, provided by the Newcastle High Throughput Facility. The screen produced 12 small molecules previously not known as autophagy inducers that effectively activated autophagy flux. Finally, we generated and optimised wild type or Npc1 KO cells stably expressing “traffic-light” EGFP-RFP-LC3 for high throughput high content screening. Both platforms will be used for secondary/tertiary screening purposes. We propose to initiate a drug discovery programme with the aim of identifying novel bioactive autophagy inducers. Next Steps KVP lab collected 1000+ compounds initially isolated from rare plant and animal species from northern Russia and have a unique expertise in the synthesis of these compounds and their structural derivatives. This is a virgin collection that has never been tested for its effect on autophagy and, combined with the natural occurrence of these molecules and therefore bioavailability, it increases the chances of successful hit identification. In addition to chemical synthesis, KVP also brings in vivo testing capacity, subject to initial identification of drug leads as outlined below. We will initially test a small (~200) collection of compounds which are readily available and represent a diverse chemical variety present in the library. The hits from the initial screen will be processed through secondary/tertiary readouts. Lead molecules will be identified by testing structurally similar derivatives of the hits identified in the KVP collection by our long-term collaborator JR and synthesised by KVP staff for structural verification, followed by validation of their effect on autophagy in several standard assays building a preliminary structure activity relationship (SAR). Our follow up aim will be to establish the specificity of these and other small molecules from the screens, identify their cellular targets (though Samsara platform), and characterise their mechanism of action in autophagy. We will also investigate the potential of these molecules to alleviate cellular defects caused by lysosomal dysfunction (e.g. mitochondrial deficit, DNA damage, increased sensitivity to stress and cell death) using human fibroblasts and neurons. In parallel, we will test a focused collections of small molecules based on the structure of our lead compounds to establish a robust SAR for future translation into preclinical models.
- Screen a diverse library of naturally occurring bioactive compounds (~200) in cell survival assays (Atg5 vs Npc1 KO) 🡪 dose response effect - Hits selection based on their chemical diversity, chemical tractability, and physicochemical parameters (JR) - Hit verification screening 🡪 Orthogonal assays (Luc-p62 clearance, traffic light LC3)
- Second round of synthesis based on lead series ~12 derivatives - Testing the ability of lead compounds to alleviate cellular defects caused by lysosomal dysfunction (e.g. mitochondrial deficit, DNA damage, increased sensitivity to stress and cell death) - Determination of biological target (Samsara) - Screening of derivatives, SAR determination, identification of drug leads (KPV, JR, VK)
- ADMET, DMPK profile determined in mouse models - Pharmacokinetics, tissue distribution, excretion - Routes of administration: PO, IV - Preliminary tox studies (dose-range finding, MTD) - IP sought for drug candidate(s)