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17 pages, 3396 KB

Open AccessArticle

Identification of Inhibitors of the Disease-Associated Protein Phosphatase Scp1 Using Antibody Mimetic Molecules

by Tamaki Kobayashi, Kazuki Yamazaki, Junki Shinada, Masataka Mizunuma, Kazuhiro Furukawa and Yoshiro Chuman

Int. J. Mol. Sci. 2024, 25(7), 3737; https://doi.org/10.3390/ijms25073737 - 27 Mar 2024

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Abstract

Protein phosphorylation is a prevalent translational modification, and its dysregulation has been implicated in various diseases, including cancer. Despite its significance, there is a lack of specific inhibitors of the FCP/SCP-type Ser/Thr protein phosphatase Scp1, characterized by high specificity and affinity. In this study, we focused on adnectin, an antibody-mimetic protein, aiming to identify Scp1-specific binding molecules with a broad binding surface that target the substrate-recognition site of Scp1. Biopanning of Scp1 was performed using an adnectin-presenting phage library with a randomized FG loop. We succeeded in identifying FG-1Adn, which showed high affinity and specificity for Scp1. Ala scanning analysis of the Scp1-binding sequence in relation to the FG-1 peptide revealed that hydrophobic residues, including aromatic amino acids, play important roles in Scp1 recognition. Furthermore, FG-1Adn was found to co-localize with Scp1 in cells, especially on the plasma membrane. In addition, Western blotting analysis showed that FG-1Adn increased the phosphorylation level of the target protein of Scp1 in cells, indicating that FG-1Adn can inhibit the function of Scp1. These results suggest that FG-1Adn can be used as a specific inhibitor of Scp1. Full article

(This article belongs to the Collection State-of-the-Art Macromolecules in Japan)

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16 pages, 2503 KB

Open AccessArticle

Development of Antibody-like Proteins Targeting the Oncogenic Ser/Thr Protein Phosphatase PPM1D

by Megumi Ikeura, Hiroto Tashiro, Yuka Yamagata, Hikaru Saito, Tamaki Kobayashi, Masataka Mizunuma, Kazuki Yamazaki, Keisuke Baba, Kazuhiro Furukawa and Yoshiro Chuman

Processes 2022, 10(8), 1501; https://doi.org/10.3390/pr10081501 - 29 Jul 2022

Cited by 2 | Viewed by 2979

Abstract

PPM1D, a protein Ser/Thr phosphatase, is overexpressed in various cancers and functions as an oncogenic protein by inactivating the p53 pathway. Therefore, molecules that bind PPM1D are expected to be useful anti-cancer agents. In this study, we constructed a phage display library based on the antibody-like small molecule protein adnectin and screened for PPM1D-specific binding molecules. We identified two adnectins, PMDB-1 and PMD-24, that bind PPM1D specific B-loop and PPM1D430 as targets, respectively. Specificity analyses of these recombinant proteins using other Ser/Thr protein phosphatases showed that these molecules bind to only PPM1D. Expression of PMDB-1 in breast cancer-derived MCF-7 cells overexpressing endogenous PPM1D stabilized p53, indicating that PMDB-1 functions as an inhibitor of PPM1D. Furthermore, MTT assay exhibited that MCF-7 cells expressing PMDB-1 showed inhibition of cell proliferation. These data suggest that the adnectin PMDB-1 identified in this study can be used as a lead compound for anti-cancer drugs targeting intracellular PPM1D. Full article

(This article belongs to the Special Issue The Amazing World of Peptide Engineering)

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19 pages, 2585 KB

Open AccessReview

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

by Peter G. Chandler and Ashley M. Buckle

Cells 2020, 9(3), 610; https://doi.org/10.3390/cells9030610 - 4 Mar 2020

Cited by 42 | Viewed by 10276

Abstract

As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for the design of new therapeutics. In response, clinical-stage therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing between chains, multiple FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold. Full article

(This article belongs to the Special Issue Fibronectin in Health and Diseases)

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