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

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 homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvβ6 and αvβ8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvβ6 and the αvβ8 integrins. In a lung fibrosis mouse model, the αvβ6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

SUBMITTER: Roy A

PROVIDER: S-EPMC10500007 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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

Roy Anindya A Shi Lei L Chang Ashley A Dong Xianchi X Fernandez Andres A Kraft John C JC Li Jing J Le Viet Q VQ Winegar Rebecca Viazzo RV Cherf Gerald Maxwell GM Slocum Dean D Poulson P Daniel PD Casper Garrett E GE Vallecillo-Zúniga Mary L ML Valdoz Jonard Corpuz JC Miranda Marcos C MC Bai Hua H Kipnis Yakov Y Olshefsky Audrey A Priya Tanu T Carter Lauren L Ravichandran Rashmi R Chow Cameron M CM Johnson Max R MR Cheng Suna S Smith McKaela M Overed-Sayer Catherine C Finch Donna K DK Lowe David D Bera Asim K AK Matute-Bello Gustavo G Birkland Timothy P TP DiMaio Frank F Raghu Ganesh G Cochran Jennifer R JR Stewart Lance J LJ Campbell Melody G MG Van Ry Pam M PM Springer Timothy T Baker David D

Nature communications 20230913 1

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 homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a genera ...[more]

PMID: 37704610

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Project description:Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.

Project description:BackgroundThe αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many solid tumors, can promote the activation of transforming growth factor-β (TGFβ), a potent immunosuppressive cytokine, by interacting with the RGD sequence of the latency-associated peptide (LAP)/TGFβ complex. We have previously described a chromogranin A-derived peptide, called "peptide 5a", which recognizes the RGD-binding site of both αvβ6 and αvβ8 with high affinity and selectivity, and efficiently accumulates in αvβ6- or αvβ8-positive tumors. This study aims to demonstrate that peptide 5a can inhibit TGFβ activation in tumors and suppress tumor growth.MethodsPeptide 5a was chemically coupled to human serum albumin (HSA) to prolong its plasma half-life. The integrin recognition properties of this conjugate (called 5a-HSA) and its capability to block TGFβ activation by αvβ6+ and/or αvβ8+ cancer cells or by regulatory T cells (Tregs) were tested in vitro. The in vivo anti-tumor effects of 5a-HSA, alone and in combination with S-NGR-TNF (a vessel-targeted derivative of tumor necrosis factor-a), were investigated in various murine tumor models, including pancreatic ductal adenocarcinoma, fibrosarcoma, prostate cancer, and mammary adenocarcinoma.ResultsIn vitro assays showed that peptide 5a coupled to HSA maintains its capability of recognizing αvβ6 and αvβ8 with high affinity and selectivity and inhibits TGFβ activation mediated by αvβ6+ and/or αvβ8+ cancer cells, as well as by αvβ8+ Tregs. In vivo studies showed that systemic administration of 5a-HSA to tumor-bearing mice can reduce TGFβ signaling in neoplastic tissues and promote CD8-dependent anti-tumor responses. Combination therapy studies showed that 5a-HSA can enhance the anti-tumor activity of S-NGR-TNF, leading to tumor eradication.ConclusionPeptide 5a is an efficient tumor-homing inhibitor of αvβ6- and αvβ8-integrin that after coupling to HSA, can be used as a drug to block integrin-dependent TGFβ activation in tumors and promote immunotherapeutic responses.

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Project description:Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2) maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2. These inhibitors should prove useful for further chromatin biology investigations.

Dataset Information

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

Publications

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

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