Crystal structure and receptor-interacting residues of MYDGF - a protein mediating ischemic tissue repair.

Myeloid-derived growth factor (MYDGF) is a paracrine-acting protein that is produced by bone marrow-derived monocytes and macrophages to protect and repair the heart after myocardial infarction (MI). This effect can be used for the development of protein-based therapies for ischemic tissue repair, also beyond the sole application in heart tissue. Here, we report the X-ray structure of MYDGF and identify its functionally relevant receptor binding epitope. MYDGF consists of a 10-stranded ß-sandwich with a folding topology showing no similarities to other cytokines or growth factors. By characterizing the epitope of a neutralizing antibody and utilizing functional assays to study the activity of surface patch-mutations, we were able to localize the receptor interaction interface to a region around two surface tyrosine residues 71 and 73 and an adjacent prominent loop structure of residues 97-101. These findings enable structure-guided protein engineering to develop modified MYDGF variants with potentially improved properties for clinical use.

Optimizing NBE PK/PD assays using the Gyrolab Affinity Software; conveniently within the bioanalyst's existing workflow.

AIM: The fully automated microfluidics-based Gyrolab is a popular instrument for the bioanalysis of protein therapeutics; requiring minimal sample and reagent volumes. Gyros offers affinity software for determining binding affinity in solution using a high-throughput method and miniaturized reactions. RESULTS: Using this affinity software, multiple CTGF-targeting reagents were characterized on the Gyrolab after <100% target coverage was seen in a cynomolgus pharmacokinetic/PD study dosed with anti-CTGF antibodies. The results uncovered magnitude differences in binding affinities between the dosed antibody, target and assay reagents. CONCLUSION: The binding affinity values were used to investigate reduced target coverage and results highlight potential of the affinity software for incorporation into the bioanalyst's existing Gyrolab workflow for characterizing reagents and optimizing pharmacokinetic/PD bioanalytical assays.

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

A high-throughput scintillation proximity assay for sphingosine-1-phosphate lyase.

The emergence of sphingosine-1-phosphate lyase (SPL) as a promising therapeutic target for inflammatory diseases has heightened interest in the identification of small molecules that modulate its activity. The enzymatic activity of SPL is typically measured using radiometric or fluorescence-based assays that require a lipid extraction step, or by direct quantitation of reaction products using mass spectrometry (MS). To facilitate testing large numbers of compounds to identify SPL modulators, we developed a robust scintillation proximity assay (SPA) that is compatible with high-throughput screening (HTS). This assay employs recombinant human full-length SPL in insect cell membrane preparations to catalyze the conversion of biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) to trans-2-hexadecenal-biotin and ethanolamine [(33)P]phosphate. To validate the SPA and confirm the fidelity of its measurement of SPL enzyme activity, we developed a Rapid-Fire MS method that quantitates nonradiolabeled S1P-biotin. In addition, we developed a simple, scalable method to produce S1(33)P-biotin in quantities sufficient for HTS. The optimized SPA screen in 384-well microplates produced a mean plate-wise Z'-statistic of 0.58 across approximately 3,000 plates and identified several distinct structural classes of SPL inhibitor. Among the inhibitors that the screen identified was one compound with an IC50 of 1.6 µM in the SPA that induced dose-dependent lymphopenia in mice.

The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function.

Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1.

Bioluminescent method for assaying multiple semicarbazide-sensitive amine oxidase (SSAO) family members in both 96- and 384-well formats.

Vascular adhesion protein-1 (VAP-1), also known as semicarbazide-sensitive amine oxidase (SSAO) or copper-containing amine oxidase (AOC3, EC 1.4.3.6), catalyzes oxidative deamination of primary amines. One endogenous substrate has recently been described (Siglec 10), and although its mechanism of action in vivo is not completely understood, it is suggested to play a role in immune cell trafficking, making it a target of interest for autoimmune and inflammatory diseases. Much of the enzymology performed around this target has been conducted with absorbance, fluorescent, or radiometric formats that can have some limitations for high-throughput screening and subsequent compound profiling. The authors present the use of a bioluminescent assay, originally developed for monoamine oxidase enzymes, in a high-throughput format. It can be used for related SSAOs such as AOC1 given their substrate similarity with VAP-1. The authors also demonstrate that it is compatible with different sources of VAP-1, both purified recombinant and VAP-1 overexpressed on live cells.

Kinetic characterization and in vitro toxicity evaluation of a luciferase less susceptible to HPV chemical inhibition.

Enzyme-based in vitro toxicity assays are often susceptible to inhibition by test compounds. A mutant luciferase selected to be less susceptible to inhibition by chloroform (CNBLuc03-06) and other high production volume (HPV) chemicals, consisting of three point mutations was created and characterized. The mutant luciferase was less inhibited by chloroform, other HPV chemicals and common surfactant release reagents (Triton-X and SDS) compared to the wild-type. Inhibition was shown to be competitive. CNBLuc03-06 was a factor of 1.5-3.2 more active than wild type and exhibited a much higher affinity for ATP. CNBLuc03-06 was more thermostable than wild-type and also more active at pH values higher than 10. Both luciferases exhibited a significant tradeoff between activation and susceptibility to chemical inhibition in the presences of the reducing agent DTT. Inhibition to HPV chemicals was eliminated using an "optimum" formulation of DTT and co-solvent ethanol. The performance of CNBLuc03-06 in cell-based in vitro toxicity assays was shown to be superior to the current commercial formulation.

Creating a mutant luciferase resistant to HPV chemical inhibition by random mutagenesis and colony-level screening.

Firefly luciferase covers a wide range of applications. One common usage of the bioluminescence assay is the measurement of intracellular concentration of adenosine triphosphate (ATP) for cell viability. However, inhibition of the enzyme reaction by chemicals in the assay has so far limited the application of luciferase for high production volume (HPV) chemical testing. The objective of this research was to obtain a mutant luciferase with increased stability to inhibition by HPV chemicals, yet retaining specific activity comparable to, or better than, wild-type luciferase. The enzymatic properties of the wild-type luciferase were improved by random mutagenesis and colony-level screening. In this paper, the detailed process of creating mutant luciferases for testing the toxicity of HPV chemicals is described. As a result, two mutant luciferases were created, with different degrees of improved tolerance to inhibition by chloroform and other HPV chemicals.