Generation of a multi-functional, target organ-specific, anti-fibrotic molecule by molecular engineering of the extracellular matrix protein, decorin

Abstract

Extracellular matrix (ECM) molecules play important roles in regulating processes such as cell proliferation, migration, differentiation and survival. Decorin is a proteoglycan that binds to ('decorates') collagen fibrils in the ECM. Decorin also interacts with many growth factors and their receptors, the most notable of these interactions being its inhibitory activity on TGF-β, the growth factor responsible for fibrosis formation. We have generated a recombinant, multi-functional, fusion-protein consisting of decorin as a therapeutic domain and a vascular homing and cell-penetrating peptide as a targeting vehicle. This recombinant decorin (CAR-DCN) accumulates at the sites of the targeted disease at higher levels and, as a result, has substantially enhanced biological activity over native decorin. CAR-DCN is an example of how molecular engineering can give a compound the ability to seek out sites of disease and enhance its therapeutic potential. CAR-DCN will hopefully be used to treat severe human diseases. LINKED ARTICLES: This article is part of a themed section on Translating the Matrix. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.1/issuetoc.

Figure 1

Structure of the engineered decorin variant. Human decorin is a proteoglycan with a monomeric protein core and a single chondroitin/dermatan sulfate GAG chain attached to it. Decorin exists as a dimer in physiological solutions. Structurally, it has a domain of 12 leucine‐rich repeats, flanked on both sides by cysteine‐rich regions. We have re‐engineered it by creating a recombinant fusion protein where a vascular homing and cell‐penetrating peptide ‘CAR’ has been cloned to the C‐terminus of the decorin (the fusion molecule is called CAR‐DCN). The outcome is a multi‐functional recombinant anti‐fibrotic molecule with enhanced biological activity and target tissue selectivity after systemic administration. CAR‐DCN structure as a dimer (from PDB 1XKU). Images were prepared with JMOL program.

Figure 2

Decorin inhibits several growth factor signalling pathways involved in fibrosis formation. The diagram illustrates all four domains of decorin as well as the GAG side chain binding to the protein core. Decorin is a proteoglycan with one GAG attachment site near the core protein N‐terminus. The major structural domain is made out of 12 leucine‐rich repeats, which are flanked by cysteine‐rich regions on both sides. Decorin interacts with a wide set of different signalling molecules; among them are different isoforms of TGF‐β, PDGF, EGFR and ErbB1–4 RTKs, myostatin (MyoS), connective tissue growth factor/CCN2 and thrombospondin (Thbs) involved in scar and fibrosis formation. The binding sites in decorin for TGF‐β, CCN2, c‐Met and EGFR neutralization reside in different parts of the decorin molecule. Thus, in theory, a single DCN molecule could simultaneously sequester several mediators of fibrosis. Owing to these multiple interactions, the anti‐fibrotic effects of decorin are likely to be the sum of a number of molecular‐binding activities.

Figure 3

Mechanism of action of multi‐functional re‐engineered decorin in inhibition of scar formation. The diagram shows the recombinant fusion protein consisting of decorin and the vascular homing and cell‐penetrating peptide CAR. The CAR‐DCN ① is a multifunctional biotherapeutic agent that inhibits numerous growth factor signalling pathways involved in fibrosis. The systemically administered molecule is targeted by CAR peptide to the inflammatory or angiogenic vasculature in any organ of the body ②. The CAR homing peptide binds to its receptor (‘zip code’) in angiogenic or inflammatory vasculature ②, and as a cell‐penetrating peptide, it delivers the fusion molecule deep in the target organ parenchyme ③. The CAR peptide then binds to heparin sulfate proteoglycans on the cell surface of the stromal cells ③. This binding facilitates the neutralization of scar‐forming isoforms TGF‐β1 and ‐β2 by the therapeutic part of the molecule, DCN ④. This mechanism results in a therapeutic response, that is, reduction of scar formation in skin wound. Diagram by Helena Schmidt; reproduced with permission from Finnish Medical Journal Duodecim (originally published in Duodecim, 2011, 127: 50–51).

Figure 4

Vascular homing peptide‐induced tissue‐selective vasodilation in PAH. (A) Vascular homing and cell‐penetrating peptide CAR homes to pulmonary arteries in disease‐specific fashion in experimental models of PAH (Urakami et al., 2011; Toba et al., 2014). A small amount of control peptide (mutant CAR peptide) binding can be seen to similar blood vessels in PAH. (B) Effect of a mixture of CAR (0.3 mg·kg⁻¹) and of the Rho‐kinase inhibitor Y27632 (1 mg·kg⁻¹) on right ventricle (RVSP, right ventricle systolic pressure) and left ventricle (SAP, systemic arterial pressure) systolic pressure in PAH. The CAR/Y27632 combination treatment induced a marked pulmonary‐specific vasodilation, i.e., a fall in RVSP with only a minimum effect on SAP, in PAH. (C) Diagram of the ‘bystander effect’, that is, target organ‐specific drug delivery, in PAH by vascular homing peptide CAR (Toba et al., 2014).