Ageing weakens autophagy, affecting cell functions and causing diseases. Boosting autophagy might be a key to counter age-related health issues.
Introduction Aging 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 aging and age-related diseases. Problem Lysosomal dysfunction is an important factor contributing to the reduction of autophagy during aging. Because 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’s lab uses cells with a mutation in a lysosomal protein (Npc1). This protein is 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's 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 a unique library of natural compounds, synthesize derivatives based on hits, and identify their biological target. Highlights - Assay with easy readout, decent throughput, good controls - 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 has established the events leading to the death of autophagy-deficient cells: 1 Accumulation of dysfunctional mitochondria 2 Stress with increased ROS and DNA damage 3 Activation of stress response pathways 4 Depletion of cellular NAD+ (and NADH) pools 5 Mitochondrial depolarization 6 Apoptotic cascade Targeting downstream processes can rescue cell death in cells/organisms with genetic loss of Atg genes and Npc1. Autophagy inducers can rescue autophagy block and cell survival in Npc1 cells, while true autophagy inducers are ineffective in rescuing autophagy or cell death in Atg5 KO cells. This presents an opportunity for a unique and rapid high-throughput cell death-based screening system. We propose initiating a drug discovery program to identify novel bioactive autophagy inducers. Next Steps: The KVP lab collected 1000+ compounds from rare plant and animal species from Russia. They have unique expertise in synthesizing these compounds and their derivatives. This virgin collection, untested for its effect on autophagy, combined with the natural occurrence of these molecules and their bioavailability, increases the chances of successful hit identification. Lead molecules will be identified by testing structurally similar derivatives of the hits identified in the KVP collection by JR and synthesized by KVP staff for structural verification. Our follow-up aim is to establish the specificity of these and other small molecules from the screens, identify their cellular targets, and characterize 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 stress sensitivity, cell death) using human fibroblasts and neurons. Additionally, we will test a focused collection 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)
- Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) and Drug Metabolism and Pharmacokinetics (DMPK) profile determined in mouse models - Routes of administration: oral, intravenous - Preliminary toxicology studies (dose-range finding, maximum tolerated dose) - IP sought for drug candidate(s)