Scripps Research Engineers Protein to Block TLR4 Inflammation

Scientists at Scripps Research have engineered a small synthetic protein designed to bind directly to a specific cell-membrane receptor and reduce inflammatory signaling. The research focuses on Toll-like receptor 4, a critical protein that helps cells detect bacteria and trigger immune responses. While this mechanism is vital for fighting infections, excessive activity from the receptor has been linked to inflammatory disorders such as sepsis, arthritis, and inflammatory bowel disease. Currently, no drugs approved by the U.S. Food and Drug Administration specifically target and block this receptor.

Targeting the Membrane-Spanning Region of TLR4

Past scientific assumptions held that the regions of Toll-like receptor 4 exposed outside and inside the cell acted as the primary signaling drivers. Researchers at Scripps Research challenged that view by examining the section of the protein that sits directly within the cell membrane. “People assumed that the regions of TLR4 exposed outside and inside the cell were the main signalling drivers, but we showed that the membrane-spanning region is also critical for this function,” Colleen Maillie, a research project analyst at Scripps Research, said. Cell membranes consist of two layers of oily molecules, creating an environment starkly different from the water-based surroundings where most proteins typically operate. This unique lipid bilayer environment has historically made it difficult to understand how proteins behave within membranes or to design molecules capable of targeting them.

Designing Synthetic Proteins Through Computational Modeling

Working in the laboratories of Assistant Professor Marco Mravic and Professor Andrew Ward, the research team tested whether the membrane-embedded section of Toll-like receptor 4 could be directly targeted. “TLR4 is a key sensor of bacteria that activates and mobilises immune cells to fight infection,” Ward explained, adding that the receptor may be activated by adjuvants in vaccines to improve immune responses or inhibited to suppress inflammation, making it both a sensor and a dial to tune innate immunity. The team introduced fragments of the receptor containing its membrane-spanning region into human cells grown in the laboratory. Using a screening technique developed by the Mravic laboratory, they discovered these fragments could associate with the receptor and reduce NF-κB signaling, one of the primary inflammatory pathways activated by Toll-like receptor 4.

Researchers subsequently used computer-generated protein structures to design synthetic proteins capable of interacting more strongly with the receptor. “Scientists have been using computers to help design proteins for decades,” Mravic said. “Models for protein interactions and structures living in water have become increasingly accurate. However, for membrane proteins, they are not. There are unique atomic details underlying molecular biophysics in lipid bilayers that current equations and AI models don’t accurately capture.” Out of nine initial designs tested in living cells, eight demonstrated signs of interacting with the receptor, and three emerged as the strongest candidates. Design-6 showed the strongest evidence of interaction and substantially reduced NF-κB inflammatory signaling. “We had this theory, which we encoded into software, that maximising apolar packing would make more stable protein interactions within the greasy membrane,” Mravic said. “The software now lets us design new practical molecules with potential clinical relevance that can insert and act within the membrane.”

Advancing Beyond Human Embryonic Kidney Cells

The initial experiments relied on human embryonic kidney cells, which provide a useful medium for laboratory research but are not especially relevant to inflammation-related diseases. Future testing must expand into disease-relevant cell types, including liver and immune cells. Scientists also need to determine how synthetic proteins engineered to operate within cell membranes can be delivered in a clinical setting. “This work could be a launchpad for a new class of biologics delivered within the membrane,” Maillie said, noting that the approach represents an innovative space carrying a lot of risk and a long roadmap to the clinic.