Cell and Material‐Specific Phage Display Peptides Increase iPS‐MSC Mediated Bone and Vasculature Formation In Vivo

Biomimetically designed materials matching the chemical and mechanical properties of tissue support higher mesenchymal stem cell (MSC) adhesion. However, directing cell‐specific attachment and ensuring uniform cell distribution within the interior of 3D biomaterials remain key challenges in healing critical sized defects. Previously, a phage display derived MSC‐specific peptide (DPIYALSWSGMA, DPI) was combined with a mineral binding sequence (VTKHLNQISQSY, VTK) to increase the magnitude and specificity of MSC attachment to calcium‐phosphate biomaterials in 2D. This study investigates how DPI‐VTK influences quantity and uniformity of iPS‐MSC mediated bone and vasculature formation in vivo. There is greater bone formation in vivo when iPS‐MSCs are transplanted on bone‐like mineral (BLM) constructs coated with DPI‐VTK compared to VTK (p < 0.002), uncoated BLM (p < 0.037), acellular BLM/DPI‐VTK (p < 0.003), and acellular BLM controls (p < 0.01). This study demonstrates, for the first time, the ability of non‐native phage‐display designed peptides to spatially control uniform cell distribution on 3D scaffolds and increase the magnitude and uniformity of bone and vasculature formation in vivo. Taken together, the study validates phage display as a novel technology platform to engineer non‐native peptides with the ability to drive cell specific attachment on biomaterials, direct bone regeneration, and engineer uniform vasculature in vivo.Non‐native peptides derived from a combinatorial phage display are engineered to increase iPS‐MSC attachment on biomaterials and increase the quantity and uniformity of bone and vasculature formation in vivo. Findings validate phage display as a new technology platform to engineer the interface between selective cell populations and specific biomaterial chemistries.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149285/1/adhm201801356_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149285/2/adhm201801356.pd

Muscle glycogen remodeling and glycogen phosphate metabolism following exhaustive exercise of wild type and laforin knockout mice

Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease

Quantifying the efficiency of hydroxyapatite mineralising peptides

We present a non-destructive analytical calibration tool to allow quantitative assessment of individual calcium phosphates such as hydroxyapatite (HAP) from mixtures including brushite. Many experimental approaches are used to evaluate the mineralising capabilities of biomolecules including peptides. However, it is difficult to quantitatively compare the efficacy of peptides in the promotion of mineralisation when inseparable mixtures of different minerals are produced. To address this challenge, a series of hydroxyapatite and brushite mixtures were produced as a percent/weight (0–100%) from pure components and multiple (N=10) XRD patterns were collected for each mixture. A linear relationship between the ratio of selected peak heights and the molar ratio was found. Using this method, the mineralising capabilities of three known hydroxyapatite binding peptides, CaP(S) STLPIPHEFSRE, CaP(V) VTKHLNQISQSY and CaP(H) SVSVGMKPSPRP, was compared. All three directed mineralisation towards hydroxyapatite in a peptide concentration dependent manner. CaP(V) was most effective at inducing hydroxyapatite formation at higher reagent levels (Ca2+ = 200mM), as also seen with peptide-silk chimeric materials, whereas CaP(S) was most effective when lower concentrations of calcium (20mM) and phosphate were used. The approach can be extended to investigate HAP mineralisation in the presence of any number of mineralisation promoters or inhibitors

Discovery and Development of Small-Molecule Inhibitors of Glycogen Synthase

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure–activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation

Targeting Pathogenic Lafora Bodies in Lafora Disease Using an Antibody-Enzyme Fusion

Lafora disease (LD) is a fatal childhood epilepsy caused by recessive mutations in either the EPM2A or EPM2B gene. A hallmark of LD is the intracellular accumulation of insoluble polysaccharide deposits known as Lafora bodies (LBs) in the brain and other tissues. In LD mouse models, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Therefore, LBs have become a therapeutic target for ameliorating LD. Herein, we demonstrate that human pancreatic α-amylase degrades LBs. We fused this amylase to a cell-penetrating antibody fragment, and this antibody-enzyme fusion (VAL-0417) degrades LBs in vitro and dramatically reduces LB loads in vivo in Epm2a−/− mice. Using metabolomics and multivariate analysis, we demonstrate that VAL-0417 treatment of Epm2a−/− mice reverses the metabolic phenotype to a wild-type profile. VAL-0417 is a promising drug for the treatment of LD and a putative precision therapy platform for intractable epilepsy