Novel protein therapeutic joint retention strategy based on collagen-binding Avimers.

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen-binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen-binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra-articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL-1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL-1 receptor. In vitro, IL-1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra-articular IL-1-induced IL-6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL-1Ra_M26 and native IL-1Ra inhibited IL-6 output when co-administered with the IL-1 challenge, only IL-1Ra_M26 inhibited when administered 1 week prior to IL-1 challenge. Collagen-binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238-1247, 2018.

Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.

Voltage-gated Ca(2+) channels of the Ca(V)1 family initiate excitation-contraction coupling in cardiac, smooth, and skeletal muscle and are primary targets for regulation by the sympathetic nervous system in the 'fight-or-flight' response. In the heart, activation of beta-adrenergic receptors greatly increases the L-type Ca(2+) current through Ca(V)1.2 channels, which requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15). Surprisingly, the site of interaction of PKA and AKAP15 lies in the distal C-terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the proteolytically cleaved distal C-terminal domain forms a specific molecular complex with the truncated alpha(1) subunit and serves as a potent autoinhibitory domain. Formation of the autoinhibitory complex greatly reduces the coupling efficiency of voltage sensing to channel opening and shifts the voltage dependence of activation to more positive membrane potentials. Ab initio structural modelling and site-directed mutagenesis revealed a binding interaction between a pair of arginine residues in a predicted alpha-helix in the proximal C-terminal domain and a set of three negatively charged amino acid residues in a predicted helix-loop-helix bundle in the distal C-terminal domain. Disruption of this interaction by mutation abolished the inhibitory effects of the distal C-terminus on Ca(V)1.2 channel function. These results provide the first functional characterization of this autoinhibitory complex, which may be a major form of the Ca(V)1 family Ca(2+) channels in cardiac and skeletal muscle cells, and reveal a unique ion channel regulatory mechanism in which proteolytic processing produces a more effective autoinhibitor of Ca(V)1.2 channel function.

Beta-adrenergic regulation requires direct anchoring of PKA to cardiac CaV1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15.

Activation of beta-adrenergic receptors and consequent phosphorylation by cAMP-dependent protein kinase A (PKA) greatly increases the L-type Ca2+ current through CaV1.2 channels in isolated cardiac myocytes. A kinase-anchoring protein 15 (AKAP15) coimmunoprecipitates with CaV1.2 channels isolated from rat heart membrane extracts and transfected cells, and it colocalizes with CaV1.2 channels and PKA in the transverse tubules of isolated ventricular myocytes. Site-directed mutagenesis studies reveal that AKAP15 directly interacts with the distal C terminus of the cardiac CaV1.2 channel via a leucine zipper-like motif. Disruption of PKA anchoring to CaV1.2 channels via AKAP15 using competing peptides markedly inhibits the beta-adrenergic regulation of CaV1.2 channels via the PKA pathway in ventricular myocytes. These results identify a conserved leucine zipper motif in the C terminus of the CaV1 family of Ca2+ channels that directly anchors an AKAP15-PKA signaling complex to ensure rapid and efficient regulation of L-type Ca2+ currents in response to beta-adrenergic stimulation and local increases in cAMP.

A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function.

In skeletal muscle, voltage-dependent potentiation of L-type Ca(2+) channel (Ca(V)1.1) activity requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 targets PKA to L-type Ca(2+) channels has not been elucidated. Here we report that AKAP15 directly interacts with the C-terminal domain of the alpha(1) subunit of Ca(V)1.1 via a leucine zipper (LZ) motif. Disruption of the LZ interaction effectively inhibits voltage-dependent potentiation of L-type Ca(2+) channels in skeletal muscle cells. Our results reveal a novel mechanism whereby anchoring of PKA to Ca(2+) channels via LZ interactions ensures rapid and efficient phosphorylation of Ca(2+) channels in response to local signals such as cAMP and depolarization.