An Elastin-like Polypeptide-fused Peptide Inhibits MMP-2 Activity at nM Concentrations with High Specificity

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<b>Abstract ID 20222</b> <b>Poster Board 300</b> Chronic kidney disease (CKD) is a growing health concern. Renal fibrosis results from buildup of extracellular matrix (ECM) proteins replacing functional tissue. Matrix metalloproteinases (MMP) are endopeptidases that maintain ECM homeostasis. Studies suggest that in early stages of CKD, increases in MMP-2 activity can promote activation of inflammatory and pro-fibrotic cytokines, driving renal fibrosis. Our lab has recently created a chimeric fusion protein between an MMP-2 inhibitory peptide (MMP-2i) and the Elastin like polypeptide (ELP) drug carrier. ELP is a biopolymer created using a repetitive pentapeptide motif (VPGXG) that we9ve shown to stabilize peptide cargo in circulation and mediate delivery to the kidneys. We hypothesized the MMP-2i peptide would maintain potent and specific MMP-2 inhibition, when fused to the ELP carrier. We utilized variously sized ELP carriers, created using 63, 127, or 255 pentapeptide repeats, resulting in carrier molecular weights of 26, 50, and 99 kDa, respectively. The aims of the current study are: 1) to determine if changes in the ELP carrier size affect the inhibitory activity of MMP-2, 2) to validate that ELP-MMP2i specifically inhibited MMP-2 compared to other renally expressed MMPs, and 3) to optimize the renal delivery of ELP-MMP2i by testing various sizes of the ELP carrier. Using a fluorescent resonance energy transfer assay, the activity of MMP2 (20 ng/mL) was determined in the presence of ELP-MMP-2i or ELP control of each molecular weight. All ELP-MMP2i constructs were potent inhibitors of MMP-2 activity, and the potency of inhibition increased as the size of the ELP carrier decreased (IC₅₀= 10.5, 96.8, and 361.7 nM for the 26, 50, and 99 kDa proteins, respectively). The most potent inhibitor of MMP2, ELP₆₃-MMP2i, was also tested for inhibition of five additional renally-expressed MMPs (20 ng/mL). ELP-MMP2i had no detectable inhibition of MMP-9, and the IC₅₀ for inhibition of MMP-3, MMP-7, and MMP-10 was 85.1, 165.9, and 84.1 mM, respectively. ELP-MMP2i did inhibit MMP14 at low micromolar concentration, though an IC₅₀ could not be accurately determined. These data demonstrate that ELP-MMP2i is over 8,000x more potent for inhibition of MMP2 than most other renally-expressed MMPs. To assess plasma pharmacokinetics and biodistribution, Sprague Dawley rats (n=4/group) were dosed (600nmol/kg) either intravenously (IV) or subcutaneously (SC) with each of the ELP-MMP-2i proteins labelled with a fluorescent marker. Plasma clearance among the three proteins was size dependent in both routes of administration. Peak plasma levels were greatest with the 26 kDa protein. Bioavailability after SC administration was also greatest for the 26 kDa protein (55.3, 25.1, and 16.7% for 26, 50, and 99 kDa proteins). All proteins localized exclusive in the kidney after both IV and SC injection, and renal levels negatively correlated with the size of the ELP carrier (renal tissue concentrations of 6.2 ± 1.9, 3.6 ± 0.2, and 2.5 ± 0.4 mM for the 26, 50, and 99 kDa proteins, respectively, following IV administration (two-way ANOVA p&lt;0.0001), and 2.0 ± 0.7, 0.6 ± 0.1, and 0.5 ± 0.1 mM following SC administration (two-way ANOVA p&lt;0.0001). This study demonstrates that the ELP-fused MMP-2 inhibitory peptide maintains potent and specific MMP-2 inhibition, and the therapeutics localize to the kidney with high specificity after systemic administration. This work was supported by NIH grants R01HL095638 to GLB, R01HL138685 to RJR and F31DK130598 to AAA.