# Molecular Mechanisms of Programmed Necrosis Execution

> **NIH NIH R35** · UT SOUTHWESTERN MEDICAL CENTER · 2024 · $215,117

## Abstract

PROJECT SUMMARY
Necroptosis is a genetically encoded necrotic cell death that is overactivated in many pro-inflammatory human
diseases, including cancer, inflammatory bowel disease, liver injury, pancreatitis, neurodegenerative disorders,
and a diverse range of viral, bacterial, and fungal infections. The necroptosis signaling cascade is mediated by
the sequential activation of RIPK1 and RIPK3 kinases downstream of pro-inflammatory ligands such as TNF or
microbe-associated molecules. MLKL (Mixed Lineage Kinase Like protein) is a pseudokinase that tetramerizes
upon phosphorylation by RIPK3 to form water-permeable pores that drive cell membrane rupture. The parent
award has been given for investigating the molecular mechanisms of programmed necrosis execution, focusing
on the regulation of MLKL by upstream kinases and E3 ubiquitin ligases. Current methods for accurate real-time
quantification of cell death execution kinetics are labor-intensive, expensive, and low-throughput. The
differentiation between late apoptotic and necrotic cell death is challenging, complicating cell death data analysis
and interpretation. To this end, within the scope of the R35, we request the Incucyte® S3 live-cell imaging system
for real-time and reliable quantification of cell death kinetics. This system will enable us to perform label-free
quantification of cell death, using machine-learning algorithms of the system, thereby bypassing any need for
fluorescent probes, allowing us to perform cell death quantitation in a non-perturbing manner in experiments
where only one subtype of cell death is observed. Incucyte® S3 will also allow us to perform cell death kinetics
experiments over prolonged periods in real-time and differentiate between apoptosis and necroptosis. Moreover,
this system will allow us to develop a method for monitoring the activation kinetics of kinases that mediate
necroptosis using a fluorescent-protein-based reporter system to support our mechanistic studies within the
scope of the R35 project. In summary, this sophisticated platform for continuous real-time monitoring of cell
health and cell signaling will enable us with advanced, unique, and rigorous methodological capabilities for
studying the molecular mechanisms of programmed necrosis execution, as it will allow us to perform highly
efficient, quantitative, multiplexed, and cost-effective cell death quantification and kinase activity monitoring
experiments. Thus, this equipment will significantly boost our efforts in deciphering the mechanisms that govern
the execution of programmed necrosis and identifying novel druggable targets for therapeutic interventions
aimed at necroptosis blockade in human inflammatory diseases where it is overactivated.

## Key facts

- **NIH application ID:** 11036184
- **Project number:** 3R35GM146861-02S1
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Ayaz Najafov
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $215,117
- **Award type:** 3
- **Project period:** 2022-09-20 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11036184

## Citation

> US National Institutes of Health, RePORTER application 11036184, Molecular Mechanisms of Programmed Necrosis Execution (3R35GM146861-02S1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/11036184. Licensed CC0.

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