Engineered Coiled-Coil Protein for Delivery of Inverse Agonist for Osteoarthritis

Osteoarthritis (OA) results from degenerative and abnormal function of joints, with localized biochemistry playing a critical role in its onset and progression. As high levels of <i>all</i>-<i>trans</i> retinoic acid (ATRA) in synovial fluid have been identified as a contributive factor to OA, the synthesis of <i>de novo</i> antagonists for retinoic acid receptors (RARs) has been exploited to interrupt the mechanism of ATRA action. BMS493, a pan-RAR inverse agonist, has been reported as an effective inhibitor of ATRA signaling pathway; however, it is unstable and rapidly degrades under physiological conditions. We employed an engineered cartilage oligomeric matrix protein coiled-coil (C_cc^S) protein for the encapsulation, protection, and delivery of BMS493. In this study, we determine the binding affinity of C_cc^S to BMS493 and the stimulator, ATRA, via competitive binding assay, in which ATRA exhibits approximately 5-fold superior association with C_cc^S than BMS493. Interrogation of the structure of C_cc^S indicates that ATRA causes about 10% loss in helicity, while BMS493 did not impact the structure. Furthermore, C_cc^S self-assembles into nanofibers when bound to BMS493 or ATRA as expected, displaying 11–15 nm in diameter. Treatment of human articular chondrocytes <i>in vitro</i> reveals that C_cc^S·BMS493 demonstrates a marked improvement in efficacy in reducing the mRNA levels of matrix metalloproteinase-13 (MMP-13), one of the main proteases responsible for the degradation of the extracellular cartilage matrix compared to BMS493 alone in the presence of ATRA, interleukin-1 beta (IL-1β), or IL-1 β together with ATRA. These results support the feasibility of utilizing coiled-coil proteins as drug delivery vehicles for compounds of relatively limited bioavailability for the potential treatment of OA

Protein Engineered Triblock Polymers Composed of Two SADs: Enhanced Mechanical Properties and Binding Abilities

Recombinant methods have been used to engineer artificial protein triblock polymers composed of two <i>different</i> self-assembling domains (SADs) bearing one elastin (E) flanked by two cartilage oligomeric matrix protein coiled-coil (C) domains to generate CEC. To understand how the two C domains improve small molecule recognition and the mechanical integrity of CEC, we have constructed C_L44AEC_L44A, which bears an impaired C_L44A domain that is unstructured as a negative control. The CEC triblock polymer demonstrates increased small molecule binding and ideal elastic behavior for hydrogel formation. The negative control C_L44AEC_L44A does not exhibit binding to small molecule and is inelastic at lower temperatures, affirming the favorable role of C domain and its helical conformation. While both CEC and C_L44AEC_L44A assemble into micelles, CEC is more densely packed with C domains on the surface enabling the development of networks leading to hydrogel formation. Such protein engineered triblock copolymers capable of forming robust hydrogels hold tremendous promise for biomedical applications in drug delivery and tissue engineering