Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anticancer therapeutics. Current therapeutics target EGFR's kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFR's ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

The Candida albicans virulence factor candidalysin polymerizes in solution to form membrane pores and damage epithelial cells.

Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence factor candidalysin (CL) which damages target cell membranes. However, the mechanism that CL uses to permeabilize membranes is unclear. We reveal that CL forms membrane pores using a unique mechanism. Unexpectedly, CL readily assembled into polymers in solution. We propose that the basic structural unit in polymer formation is a CL oligomer, which is sequentially added into a string configuration that can close into a loop. CL loops appear to spontaneously insert into the membrane to become pores. A CL mutation (G4W) inhibited the formation of polymers in solution and prevented pore formation in synthetic lipid systems. Epithelial cell studies showed that G4W CL failed to activate the danger response pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of CL pores by atomic force microscopy revealed co-existence of simple depressions and more complex pores, which are likely formed by CL assembled in an alternate oligomer orientation. We propose that this structural rearrangement represents a maturation mechanism that stabilizes pore formation to achieve more robust cellular damage. To summarize, CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.

The fungus Candida albicans is the most common cause of yeast infections in humans. Like many other disease-causing microbes, it releases several virulent proteins that invade and damage human cells. This includes the peptide candidalysin which has been shown to be crucial for infection. Human cells are surrounded by a protective membrane that separates their interior from their external environment. Previous work showed that candidalysin damages the cell membrane to promote infection. However, how candidalysin does this remained unclear. Similar peptides and proteins cause harm by inserting themselves into the membrane and then grouping together to form a ring. This creates a hole, or 'pore', that weakens the membrane and allows other molecules into the cell's interior. Here, Russell, Schaefer et al. show that candidalysin uses a unique pore forming mechanism to impair the membrane of human cells. A combination of biophysical and cell biology techniques revealed that the peptide groups together to form a chain. This chain of candidalysin proteins then closes in on itself to create a loop structure that can insert into the membrane to form a pore. Once embedded within the membrane, the proteins within the loops rearrange again to make the pores more stable so they can cause greater damage. This type of pore formation has not been observed before, and may open up new avenues of research. For instance, researchers could use this information to develop inhibitors that stop candidalysin from forming chains and harming the membranes of cells. This could help treat the infections caused by C. albicans.