De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8

bioRxiv. 2023 Jun 12:2023.06.12.544624. doi: 10.1101/2023.06.12.544624.

Anindya Roy ¹, Lei Shi ¹ ², Ashley Chang ³, Xianchi Dong ⁴ ⁵, Andres Fernandez ⁶, John C Kraft ¹, Jing Li ⁴, Viet Q Le ⁴, Rebecca Viazzo Winegar ³, Gerald Maxwell Cherf ⁷ ⁸, Dean Slocum ³, P Daniel Poulson ³, Garrett E Casper ³, Mary L Vallecillo-Zúniga ³, Jonard Corpuz Valdoz ³, Marcos C Miranda ¹ ⁹, Hua Bai ¹, Yakov Kipnis ¹ ¹⁰, Audrey Olshefsky ¹ ¹¹, Tanu Priya ¹² ¹³, Lauren Carter ¹, Rashmi Ravichandran ¹, Cameron M Chow ¹, Max R Johnson ¹, Suna Cheng ¹, McKaela Smith ¹, Catherine Overed-Sayer ¹⁴ ¹⁵, Donna K Finch ¹⁴ ¹⁶, David Lowe ¹⁴ ¹⁷, Asim K Bera ¹, Gustavo Matute-Bello ¹⁸, Timothy P Birkland ¹⁰, Frank DiMaio ¹, Ganesh Raghu ¹⁰, Jennifer R Cochran ⁷, Lance J Stewart ¹, Melody G Campbell ⁶, Pam M Van Ry ³, Timothy Springer ⁴, David Baker ¹ ¹⁹

Affiliations

1 Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, WA 98195, USA.
2 Current Address: Encodia Inc, 5785 Oberlin Drive, San Diego, CA 92121.
3 Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.
4 Program in Cellular and Molecular Medicine, Children's Hospital Boston, and Departments of Biological Chemistry and Molecular Pharmacology and of Medicine, Harvard Medical School, Boston, United States.
5 Current address: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China; Engineering Research Center of Protein and Peptide Medicine，Ministry of Education.
6 Division of Basic Sciences, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA.
7 Department of Bioengineering, Stanford University, Stanford CA 94305.
8 Current Address: Denali Therapeutics, South San Francisco, CA, USA.
9 Current Address: Department of Medicine Solna, Division of Immunology and Allergy, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.
10 Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Washington, Seattle, Washington.
11 Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
12 Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA.
13 Current Address: Department of Pharmacology, Northwestern University Feinberg School of Medicine; Chicago, IL 60611, USA.
14 Research and Early Development, Respiratory and Immunology, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom.
15 Current Address: Bioscience COPD/IPF, Research and Early Development, Respiratory and Immunology, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK.
16 Current Address: Alchemab Therapeutics Ltd, Cambridge, United Kingdom.
17 Current Address: Evox Therapeutics Limited, Oxford Science Park, Medawar Centre, East Building, Robert Robinson Avenue, Oxford, OX4 4HG.
18 Center for Lung Biology, Division of Pulmonary, Critical Care and Sleep Medicine, University of Washington.
19 Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.

PMID: 37398153 DOI: 10.1101/2023.06.12.544624

Abstract

The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated Cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related Integrin proteins and Other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD Integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvβ6 and αvβ8 with high selectivity. The αvβ6 and αvβ8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over Other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvβ6 Inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvβ6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvβ8 inhibitor maintains the constitutively fixed extended-closed αvβ8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvβ6 Inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed Integrin binding proteins with high selectivity.

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