DESCRIPTION

Background
Objective
Project Description
References

Background

Per- and polyfluoroalkyl substances, commonly known as PFAS, are a large group of synthetic, lipid-like molecules consisting of a chain of carbon atoms with most of the hydrogens replaced with fluorine. [1] The highly stable carbon-fluorine bond affords PFAS resistance to heat, water, or staining, resulting in its widespread application in developing non-stick cookware, water-repellent clothing, stain-resistant fabrics and carpets, some cosmetics, some firefighting foams, and products that resist grease, water, and oil [2] . This very feature leads to their moniker of “forever chemicals”, because of their persistence in the environment when exposure occurs [3].

Health History and symptoms

Perhaps one of the greatest concerns with the pervasiveness of PFAS is bioaccumulation in living organisms and the resulting health consequences. Concern regarding the potential health effects associated with exposure began in the early 2000s when perfluorooctanesulfonate (PFOS) was detected in the blood of polar bears in the Arctic. This concern was further exacerbated when the U.S. Center for Disease Control and Prevention (CDC) reported that these same compounds were detectable in the blood of 98% of Americans (4). Since then, public health and life science research has been rapidly conducted and shown links between Perfluorooctanoic acid (PFOA) and high cholesterol, thyroid disease, pregnancy-induced hypertension, ulcerative colitis, premature births, kidney/testicular cancer, and, in children because they undergo sensitive periods of development, dyslipidemia, immunity, and renal function. [5] [6].

Specifically, PFAS is attributed to inhibition of the production of immune response cells, thus decreasing the effectiveness of vaccines and causing people to be at higher risk for sickness from everyday sources. [7]

History of occurence

Cases of mass PFAS pollution have been appearing due to increased testing. One of these cases took place in the same state as our team when in 2017, Chemours, a large chemical manufacturing plant in Fayetteville, North Carolina was found to have released over 2700 pounds of the GenX PFAS molecule through its smoke stacks. This toxic chemical infiltrated local air, water, and soil, contaminating the environment in communities over 18 miles away from the plant. Given the long-lasting nature of PFAS, the contamination has raised concern over the long-term effects it may have on the communities’ health. Already, testing has shown that residents in the area have blood PFAS levels higher than the national average. The number has only been growing as the plant continues to release PFAS into communities, mirroring the grim reality of PFAS pollution in the country. [8]

PFAS Regulation

As of January 2024, the EPA only limits the production of 329 PFAS, though companies are still able to both produce and import these chemicals by notifying the EPA of their reason for usage. 9 The first national and legally enforceable limitation on PFAS levels in drinking water was only passed April 10th 2024 10. This new standard mandates that all water treatment facilities work toward testing and limiting PFAS levels but unfortunately it only covers five individual PFAS and a few PFAS mixtures. While this is a step in the right direction it is known that there are more than 15,000 synthetic chemicals classified as PFAS by the US Environmental Protection Agency (EPA) 1

Research is ever-expanding but primarily focused on working to develop safer alternatives to PFAS and methods to remove it from our environment.

We realize there needs to be a solution, not only for future prevention of PFAS contamination to the environment and humans, but to combat the existing problems affecting people at this very moment.

Objective:

Our team aims to develop a therapeutic protein that removes PFOA and PFOS from the human bloodstream.This involves engineering a chimeric protein with two domains: one has a high affinity for PFOA while the other facilitates transcytosis of the whole peptide.

Project Description

Inspiration for our design comes from understanding where and how PFAS maneuvers in the body. When exposed to PFAS, distribution of PFAS is facilitated through noncovalent binding of serum proteins, such as Albumin 11. Human serum albumin (hSA) serves as an ideal control due to its well-documented interactions with PFAs, particularly PFOA and PFOS . By using albumin as a scaffold, we can understand how albumin interacts with PFOA in the bloodstream, which is crucial for designing an effective therapeutic strategy. The native hsA molecule has four distinct binding sites for PFOA obtained through calorimetry experiments with hsA and PFOA: FA4 (Sudlow Binding Site I), FA6, FA7 (Sudlow Binding Site II), and crevice.11 Competition experiments performed with other known hsA-binding drugs further showed FA4 as the high affinity binding site of PFOA to hsA. Further structural analysis proved increased stabilization for PFOA molecules in this binding site from a greater number of intermolecular interactions.

Having a method to bind these molecules is important, but the removal of this protein from the body is essential. Human serum albumin, especially an hsA-PFOA molecule complex, is too large to move across the interior of epithelial cells such as those found in lung vasculature. pIgR is a transmembrane protein that allows for the transport of other polymeric immunoglobulins, following the proteolytic cleavage of the pIgR protein12. A complex is taken into the cell via clathrin-mediated endocytosis where it travels along the endosomal transcytosis pathway.12 The hsA-PFOA molecule complex is incapable of undergoing transcytosis via the pIgR transmembrane protein alone. However, the PASR is a peptide created in 2018 capable of triggering the pIgR mediated transcytosis pathway we need.13

Through the creation of novel chimera protein that has the compatibility to both bind to PFOA present in the bloodstream and trigger poly-immunoglobulin receptor (pIgR) mediated transcytosis, we enable a “capture-and-remove” system to rid the body of PFOA present in the bloodstream.

Protein Engineering

Although the albumin pocket our domain is built off of should theoretically bind to albumin with a micromolar affinity, some problems arise. Firstly, it is unknown if the FA4 pocket of albumin which our binder is based off can fold stably by itself without being in the whole albumin protein, especially since there is a disulfide bridge which can cause protein folding problems in E.coli. Secondly, albumin is a very unselective binder relative to other protein binders, and our chimera protein would work best and minimize potential adverse effects if the primary thing it binds to would be PFOA. These goals can begin to be realized with computational protein engineering.

Albumin (PDB ID: 7AAI) colored red, with the FA4 pocket colored blue. PFOA molecules are shown as spheres. Other ligands were removed for visual clarity.

To modify the Albumin FA4 pocket, we use LigandMPNN (14) , a protein design tool from the Baker Lab at the University of Washington. Proteins designed with the LigandMPNN tend to be more stable than their natural counterparts ;most designs produced by LigandMPNN will be more stable without further wet lab optimization. Additionally, ProteinMPNN, the parent algorithm of LigandMPNN, was used to design binders with greater fitness with minimal wet lab optimization needed (15). With these facts in mind, we decided to keep all amino acids (except cysteine and methionine) within 7 angstroms of the PFOA ligand fixed, and let the algorithm optimize the rest of the protein. We also blocked cysteine and methionine from being incorporated into the designed sequences, since these amino acids can cause folding problems in E.coli.

We used Alphafold3 (16) to predict the structure of 120 of the sequences from the LigandMPNN algorithm, and created a gene based on the protein sequence of the top 4 designs, based on the probability of the sequences folding into the desired sequence and predicted overall stability of the protein.

Pharmacological Modeling

The main purpose of the PFOA binder is the removal of PFOA from the body, and utilizing pharmacological modeling provides a mathematical way of determining the amount of PFOA removed from the bloodstream. Specifically, having an understanding of how the variation of initial concentrations of substances, such as PFOA, the binding energies of proteins, biochemical interactions, and other parameters, can affect the PFOA removal from the bloodstream is crucial in determining the viability of our project. We developed a competition model between albumin and PFOA to look at steady state concentrations of PFOA in the bloodstream based on varying initial concentrations and binding efficiencies.

Using our Albumin binding to PFOA Model we can understand how albumin interacts with PFOA in the bloodstream which is crucial for designing an effective therapeutic strategy. Both with and without the introduction of our binder into the bloodstream, we can determine the steady state concentrations of PFOA in the bloodstream. Furthermore, we are able to test multiple combinations of initial concentrations of albumin, PFOA, and our binder coupled with parameters for binding interaction rates to determine the differences in end steady states. This method has allowed us to estimate how effective certain binding affinities have to be for significant PFOA removal along with the effects of the environment, e.g. high PFOA uptake through contaminated water, on the system dynamics.

Our focus is on optimizing the protein's binding efficiency and transcytosis rate to ensure maximum removal of PFOA.