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The cation-independent mannose-6-phosphate receptor (CI-M6PR, aka insulin-like growth factor II receptor or IGFIIR) is a membrane protein that plays a central role in the trafficking of lysosomal acid hydrolases into lysosomes via mannose-6-phosphate (M6P) binding domains. In order to maintain cellular metabolic/catabolic homeostasis, newly synthesized lysosomal acid hydrolases are required to bind to M6PR for transit. Acid hydrolases secreted by cells can also be internalized via M6PR residing on the cell membrane and are transported to the lysosomes, a feature that enables enzyme replacement therapy for the treatment of several lysosomal storage disorders. Therefore, a thorough characterization of this receptor is critical to the development of lysosomal enzyme-based therapeutics that utilize M6PR for drug delivery to the lysosome. However, the extracellular domain (ECD) of M6PR is highly complex, containing 15-mannose receptor homology (MRH) domains. In addition, homodimerization of the receptor can occur at the membrane, making its characterization challenging. In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1-14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellular internalization., Competing Interests: Declaration of competing interest All authors are or were Takeda Pharmaceutical employees at the time of this study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Myostatin antagonists are being developed as therapies for Duchenne muscular dystrophy due to their strong hypertrophic effects on skeletal muscle. Engineered follistatin has the potential to combine the hypertrophy of myostatin antagonism with the anti-inflammatory and anti-fibrotic effects of activin A antagonism., Methods: Engineered follistatin was administered to C57BL/6 mice for 4 weeks, and muscle mass and myofiber size was measured. In the mdx model, engineered follistatin was dosed for 12 weeks in two studies comparing to an Fc fusion of the activin IIB receptor or an anti-myostatin antibody. Functional measurements of grip strength and tetanic force were combined with tissue analysis for markers of necrosis, inflammation, and fibrosis to evaluate improvement in dystrophic pathology., Results: In wild-type and mdx mice, dose-dependent increases in muscle mass and quadriceps myofiber size were observed for engineered follistatin. In mdx, increases in grip strength and tetanic force were combined with improvements in muscle markers for necrosis, inflammation, and fibrosis. Improvements in dystrophic pathology were greater for engineered follistatin than the anti-myostatin antibody., Conclusions: Engineered follistatin generated hypertrophy and anti-fibrotic effects in the mdx model.

Follistatin (FS) is an important regulatory protein, a natural antagonist for transforming growth factor-β family members activin and myostatin. The diverse biologic roles of the activin and myostatin signaling pathways make FS a promising therapeutic target for treating human diseases exhibiting inflammation, fibrosis, and muscle disorders, such as Duchenne muscular dystrophy. However, rapid heparin-mediated hepatic clearance of FS limits its therapeutic potential. We targeted the heparin-binding loop of FS for site-directed mutagenesis to improve clearance parameters. By generating a series of FS variants with one, two, or three negative amino acid substitutions, we demonstrated a direct and proportional relationship between the degree of heparin-binding affinity in vitro and the exposure in vivo. The triple mutation K(76,81,82)E abolished heparin-binding affinity, resulting in ∼20-fold improved in vivo exposure. This triple mutant retains full functional activity and an antibody-like pharmacokinetic profile, and shows a superior developability profile in physical stability and cell productivity compared with FS variants, which substitute the entire heparin-binding loop with alternative sequences. Our surgical approach to mutagenesis should also reduce the immunogenicity risk. To further lower this risk, we introduced a novel glycosylation site into the heparin-binding loop. This hyperglycosylated variant showed a 10-fold improved exposure and decreased clearance in mice compared with an IgG1 Fc fusion protein containing the native FS sequence. Collectively, our data highlight the importance of improving pharmacokinetic properties by manipulating heparin-binding affinity and glycosylation content and provide a valuable guideline to design desirable therapeutic FS molecules., (Copyright © 2018 The Author(s).)

Here we present an NMR-based approach to solving protein-ligand structures. The procedure is guided by biophysical, biochemical, or knowledge-based data. The structures are mainly derived from ligand-induced chemical-shift perturbations (CSP) induced in the resonances of the protein and ligand-detected saturated transfer difference signals between ligands and selectively labeled proteins (SOS-NMR). Accuracy, as judged by comparison with X-ray results, depends on the nature and completeness of the experimental data. An experimental protocol is proposed that starts with calculations that make use of readily available chemical-shift perturbations as experimental constraints. If necessary, more sophisticated experimental results have to be added to improve the accuracy of the protein-ligand complex structure. The criteria for evaluation and selection of meaningful complex structures are discussed. These are exemplified for three complexes, and we show that the approach bridges the gap between theoretical docking approaches and complex NMR schemes for determining protein-ligand complexes; especially for relatively weak binders that do not lead to intermolecular NOEs.

A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual (15)N,(1)H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.

Background: Characterization of the biopathologic events underlying the early steps of breast carcinogenesis may have a dramatic impact on reducing breast cancer mortality. Genes involved in breast tumorigenesis are localized on chromosomes 1 and 17, and numeric aberrations of these chromosomes have been correlated with breast cancer tumorigenesis and progression. According to the field cancerization hypothesis, specific chromosome aberrations may be present in breast cancer and in normal-appearing adjacent tissue. The latter changes reflect the genomic damage that follows longterm carcinogenic exposure and precede the morphologically detectable neoplastic transformation. We hypothesize that detection of these aberrations in benign breast epithelium may provide a tool for molecular risk assessment., Study Design: Using fluorescence in situ hybridization with centromere-specific probes, we determined the status of chromosomes 1 and 17 in fresh imprints of 28 samples of primary tumors and 54 samples of their surrounding uninvolved parenchyma taken from patients undergoing operations for breast carcinoma. Ten contralateral breast biopsy specimens collected from patients with previous breast carcinoma were also evaluated as a surrogate of a high-risk group to rule out the hypothesis that chromosomal aneusomy in tumor-adjacent tissue could be related to a paracrine effect of the primary tumor. Ten samples of benign breast tissue taken from patients at low risk were used as controls to define tolerance limits for aneusomy definition., Results: Using threshold values of 40% of signal loss and 13% of signal gain to define chromosome aneusomy (ie, mean + 3 SDs of the control group signals), we found the following: 1) almost all primary breast tumors were aneusomic for chromosomes 1 and 17; 2) primary breast tumor and adjacent uninvolved parenchyma shared the same pattern of chromosomes 1 and 17 aneusomy in 66.7% of patients; and 3) chromosomes 1 and 17 aneusomies in contralateral benign breast samples from high-risk patients were not different from those in primary breast tumor or adjacent tissue samples., Conclusions: These results suggest that chromosomes 1 and 17 aneusomy may represent an intermediate biomarker of breast tumorigenesis potentially useful to detect patients at high risk of breast carcinoma who may benefit from preventive interventions.