Cytokine storms are potentially life-threatening overreactions of the immune system provoked by viral infection and other “threats.” Two key players are cytokines interleukin-6 (IL-6) and interleukin-1 (IL-1). Currently available inhibitors of IL-6 and IL-1 relieve the cytokine storm associated with rheumatoid arthritis, but not with COVID-19.
Now, scientists from the University of Washington have computationally designed protein inhibitors that may prevent the COVID-19-related cytokine storm. X-ray crystallography revealed a near-perfect match between the computational designs and their real-life counterparts, which blocked the cytokine storm in a human heart organoid. This suggests that computational design has the power to create entirely new proteins that function as viable therapeutics against the cytokine storm associated with COVID-19.
Researchers used the resources of the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory.
Cytokine storm became a household term during the COVID-19 pandemic. Also known as cytokine release syndrome (CRS), this process happens when the immune system grossly overreacts to a threat and produces too many inflammatory immune cells. A cytokine storm can also be triggered by certain autoimmune diseases and CAR-T cell therapy.
The major players in a cytokine storm are cytokines IL-6 and IL-1. They bind to receptors on the surface of inflammatory immune cells, among others, sending signals to the cell’s DNA. These signals may activate the cell, amplify production of more inflammatory cells, or recruit cells to various locations. During a cytokine storm associated with COVID-19, too many inflammatory cells are activated and directed to the lungs and heart, where they can destroy tissue and cause fatal organ failure.
Binding is essential to the signal being sent; if there is no binding, there is no signal, and no cytokine storm. A few drugs on the market currently inhibit IL-6 and IL-1 binding, but they are better suited for long-term conditions like rheumatoid arthritis rather than short-term, acute events like COVID-19. To fill the void, a team of scientists led by 2024 Nobel Prize winner David Baker set out to design proteins from scratch that could effectively inhibit IL-6 and IL-1 binding.
Both IL-6 and IL-1 rely on a third protein—GP130 in the case of IL-6, and an accessory protein in the case of IL-1—to send a signal when they bind with their receptors. The scientists used Rosetta, a proprietary protein design program, to create inhibitors that would occupy (a) binding sites on the IL-6 receptor, (b) the site on GP130 where IL-6 and its receptor would bind, and (c) the site on IL-1 where it would bind to both its receptor and the accessory protein.
After generating their initial designs, the scientists tweaked them to improve the structure and amino acid sequence, then chose the top 100,000 candidates to test experimentally. First, they expressed the designs as real-life proteins in yeast cells. Then they optimized binding affinity by mutating each of the amino acids in the proteins. Finally, they used E. coli to express the optimized proteins.
To test whether the new proteins could block cytokine signaling in human cells, scientists conducted experiments in human bone marrow and human umbilical vein endothelial cells. Their results indicated that both their IL-6 and IL-1 inhibitors blocked cytokine signaling extremely effectively.
For the scientists to assess the accuracy of their design methodology, they needed to show how closely the designed proteins hewed to the molecular structures they had intended to create. They used the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) at beamline 23-ID-D at the APS to collect data from crystals of the GP130 inhibitor bound to GP130, as well as the structures of the IL-1 and IL-6 inhibitors. The experimentally determined protein structures were nearly identical to their corresponding computational models, a confirmation that the scientists were on the right track.
Now came the final test: seeing whether the designed proteins inhibited a cytokine storm in a human heart organoid, a 3D structure grown from human cells that can mirror the cytokine storm caused by SARS-CoV-2 infection. When the team’s IL-1 inhibitor was introduced into the organoid, not only did the cytokine level drop but the cardiac damage and fibrosis caused by the cytokine storm were reduced.
The effectiveness of these computer-generated proteins, as well as the close match with their intended structures, underscores the potential of creating potent therapeutic inhibitors through de novo computational design. If advanced as therapeutics, scientists’ designs may come with numerous advantages. The inhibitor proteins are small, 65 amino acids or less. That makes them easier to produce in a lab, easier for the kidneys to filter out, and easier to manufacture. They are also more stable than most naturally-occurring proteins. And for people who don’t like shots, they can even be inhaled. – Judy Myers
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See: B. Huang1, B. Coventry1, M.T. Borowska2, D.C. Arhontoulis3, M. Exposit1, M. Abedi1, K.M. Jude2, S.F. Halabiya1, A. Allen1, C. Cordray1, I. Goreshnik1, M. Ahlrichs1, S. Chan1, H. Tunggal1, M. DeWitt1, N. Hyams4, L. Carter1, L. Stewart1, D.H. Fuller1, Y. Mei3,4, K.C. Garcia2, D. Baker1, “De novo design of miniprotein antagonists of cytokine storm inducers,” Nat Commun 15, 7064 (Aug. 2024)
Author affiliations: 1University of Washington; 2Stanford University School of Medicine; 3Medical University of South Carolina; 4Clemson University.
This research was supported by the National Institutes of Health’s National Institute on Aging, grant R01AG063845 (B.H., B.C., I.G., and L.C.); DARPA SD2 DARPA Synergistic Discovery and Design (SD2) HR0011835403 contract FA8750-17-C-0219 (L.C.); the National Institutes of Health’s National Cancer Institute, grant R01CA240339 (I.G. and M.E.); DARPA program Harnessing Enzymatic Activity for Lifesaving Remedies (HEALR) under award HR0011-21-2-0012 (B.H., I.G., and M.E.); the Defense Threat Reduction Agency Grant HDTRA1-21-1-0038 (I.G.); AMGEN CAGED BITE AMGEN (I.G.); CDA_NOVO NORDISK 3 (I.G. and L.C.); the Howard Hughes Medical Institute C19 HHMI INITIATIVE (I.G.); the National Institutes of Health’s National Cancer Institute grant R01CA114536 (M.E.); the Nordstrom Barrier Institute for Protein Design Directors Fund (B.H., M.A., I.G., and L.C.); the Open Philanthropy Project Improving Protein Design Fund (B.C. and I.G.); the Audacious Project at the Institute for Protein Design (M.E.); Dr. Eric and Ms. Wendy Schmidt, and Schmidt Futures funding from Eric and Wendy Schmidt by recommendation of the Schmidt Futures program (I.G.). The project or effort depicted was or is also sponsored by the F30 Fellowship F30HLI160055 (D.A.) and NIH R01 HL133308, R21 HL167211, R01 HL168255, and U01 HL169361 (Y.M.). Use of the SSRL, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). D.B. and K.C.G. are investigators with the Howard Hughes Medical Institute. This work was supported by NIH R01-AI51321 (K.C.G.) and the Mark Foundation (K.C.G.).
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