A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

INTRODUCTION: Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. AIM: Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). METHODS: Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N = 35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. RESULTS: Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. CONCLUSION: Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

A ß-(1,2)-glycosynthase and an attempted selection method for the directed evolution of glycosynthases.

Understanding how enzymes mediate catalysis is a key to their reprogramming for biotechnological applications. The family 3 retaining glycosidase postulated to be involved in erythromycin self-resistance was cloned, recombinantly expressed in Escherichia coli, purified, and characterized. Bioinformatics analysis allowed the identification of the acid/base and nucleophile residues, and mutation of these residues resulted in hydrolytically inactive proteins. One mutant was able to synthesize a glycosidic linkage using α-glucosyl fluoride as a donor and macrolide antibiotics as acceptors. This shows an unprecedented application of glycosynthase technology in accomplishing a challenging ß-(1,2)-glycosylation of an amino sugar. This work also provides the first biochemical characterization of the EryBI protein and supports its role in the self-resistance mechanism involved in erythromycin biosynthesis. An in vivo selection approach was used in an attempt to spur evolution of the glycosynthase, and the results from the attempted selection method provide insight into the requirements for in vivo directed evolution of glycosynthases.

The UDP-Galp mutase catalyzed isomerization: synthesis and evaluation of 1,4-anhydro-beta-D-galactopyranose and its [2.2.2] methylene homologue.

The synthesis of 1,4-anhydro-beta-D-galactopyranose (1,5-anhydro-alpha-D-galactofuranose), a proposed intermediate in the ring contraction isomerisation catalyzed by UDP-galactopyranose mutase, together with its [2.2.2] bicyclic methylene homologue, synthesised as a possible competitive inhibitor or alternative substrate, are reported. Neither compound was found to be an inhibitor or substrate for UDP-galactopyranose mutase from Klebsiella pneumoniae.

Real-world assay variability between laboratories in monitoring of recombinant factor IX Fc fusion protein activity in plasma samples.

INTRODUCTION: Monitoring of factor IX (FIX) replacement therapy in haemophilia B relies on accurate coagulation assays. However, considerable interlaboratory variability has been reported for one-stage clotting (OSC) assays. This study aimed to evaluate the real-world, interlaboratory variability of routine FIX activity assays used in clinical haemostasis laboratories for the measurement of recombinant FIX Fc fusion protein (rFIXFc) activity. METHODS: Human FIX-depleted plasma was spiked with rFIXFc at 0.80, 0.20 or 0.05 IU/mL based on label potency. Participating laboratories tested samples using their own routine OSC or chromogenic substrate (CS) assay protocols, reagents and FIX plasma standards. Laboratories could perform more than one measurement and method, and were not fully blinded to nominal activity values. RESULTS: A total of 142 laboratories contributed OSC results from 175 sample kits using 11 different activated partial thromboplastin time (aPTT) reagents. The median recovered FIX activity for the 0.80, 0.20 and 0.05 IU/mL samples was 0.72 IU/mL, 0.21 IU/mL and 0.060 IU/mL, respectively. Across all OSC reagents, interlaboratory variability (% CV) per aPTT reagent ranged from 9.4% to 32.1%, 8.2% to 32.6% and 12.2% to 42.0% at the 0.80, 0.20 and 0.05 IU/mL levels, respectively. CS results showed excellent median recoveries at all nominal levels (87.5% to 115.0%; n = 11) with low interlaboratory variability (CV 3.6% to 15.4%). CONCLUSION: This large, real-world data set indicates that rFIXFc activity in plasma samples can be accurately measured with the majority of routine OSC and CS assay methods. Given the variation in FIX assay procedures between sites, it is important that individual laboratories qualify their in-house methods for monitoring of rFIXFc activity.

Glycosidase inhibition by macrolide antibiotics elucidated by STD-NMR spectroscopy.

A glycosynthase approach was attempted to glycodiversify macrolide antibiotics, using DesR, a family-3 retaining beta-glucosidase involved in the self-resistance mechanism of methymycin production. STD-NMR was used to probe enzyme-substrate interactions. Analysis of competitive STD-NMR experiments between erythromycin A and a chromogenic substrate (pNP-beta-d-glucose) with the hydrolytically inactive nucleophile mutants led us to discover a family of unprecedented glycosidase inhibitors. Analysis of kinetic data with wild-type DesR determined that erythromycin is a competitive inhibitor of the glucosidase (IC50 = 2.8 +/- 0.3 microM and Ki = 2 +/- 0.2 microM) with respect to the hydrolysis of pNP-beta-d-glucose. Comparable inhibitory data was obtained for clarithromycin; however, the inhibitory effect of azithromycin was weak and no significant inhibition was observed with methymycin or d-desosamine. This report documents significant inhibition of glycosidases by macrolide antibiotics and provides insight into the design of novel glycosidase inhibitors based on the macrolactone ring of macrolide antibiotics.

Enzyme-catalyzed synthesis of isosteric phosphono-analogues of sugar nucleotides.

Efficient enzymatic syntheses of isosteric phosphono analogues of sugar nucleotides have been accomplished using a thymidylyltransferase.

Synthesis of a carbasugar analogue of a putative intermediate in the UDP-galp-mutase catalyzed isomerization.

[reaction: see text] The synthesis of the carbasugar analogue of 1,4-anhydro-beta-d-galactopyranose, a proposed intermediate in the reaction catalyzed by uridine diphosphate-alpha-d-Galp mutase, in racemic form via Diels-Alder and Barton decarboxylation chemistry is reported. This compound was found not to inhibit the mutase from Mycobacterium tuberculosis, indicating that the enzyme does not possess a 1,4-anhydro-beta-d-galactopyranose binding pocket.

Identification of Novel Inhibitors of UDP-Galactopyranose Mutase by Structure-Based Virtual Screening.

UDP-galactopyranose mutase (UGM) is a flavo-enzyme involved in the bacterial cell wall biosynthesis. UGM catalyzes the reversible isomerization of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UDP-Galf is the activated precursor of galactofuranose (Galf) residues that are essential components of the cell wall of certain pathogenic bacteria such as Klebsiella pneumoniae and Mycobacterium tuberculosis. Neither UGM nor Galf residues are found in humans, making Galf biosynthesis a potential drug target for developing antibacterial agents. We report the identification of novel inhibitors of UGM by in silico docking of the LeadQuest compound database against UGM from Escherichia coli. The 13 most promising inhibitors were then evaluated against K. pneumonia and M. tuberculosis UGMs by enzyme inhibition studies. Two inhibitors were identified with IC50 values of ∼1 µM and subsequently these compounds were docked into the recently solved X-ray structure of Deinococcus radiodurans UGM. The structure-activity relationships of the initial 13 compounds evaluated as inhibitors are discussed.

Chemoenzymatic synthesis, inhibition studies, and X-ray crystallographic analysis of the phosphono analog of UDP-Galp as an inhibitor and mechanistic probe for UDP-galactopyranose mutase.

UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-D-galactopyranose into UDP-α-D-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP-phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-D-galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phosphonate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM-substrate complex structures or in other enzyme-sugar nucleotide phosphonate complexes. This complex structure provides a structural basis for the observed micromolar inhibition towards UGM. Steric clashes, loss of electrostatic stabilization between an active-site arginine (Arg305) and the phosphonate analog, and a 180° flip of the hexose moiety account for the differences in the binding orientations of the isosteric phosphonate analog and the physiological substrate. This provides new insight into the ability of a sugar-nucleotide-binding enzyme to orient a substrate analog in an unexpected geometry and should be taken into consideration in designing such enzyme inhibitors.