Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics

The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a “fit-for-purpose” approach in instrument selection for biosensor studies.     

Bioanalysis of N-acetyl-aspartyl-glutamate as a marker of glutamate carboxypeptidase II inhibition

We report the characterization of two methods for the analysis of N-acetyl-aspartyl-glutamate (NAAG) in biological fluids. In the first method, NAAG concentrations were calculated based on differences between glutamate concentrations before and after NAAG hydrolysis with exogenous glutamate carboxypeptidase II (GCP II) using high-performance liquid chromatography (HPLC) followed by fluorescence detection. In the second method, NAAG levels were quantified directly using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analyses of NAAG levels in human cerebrospinal fluid samples using either method gave similar results within experimental error, confirming the validity of the two independent measurements. These methods will be useful in future clinical trials to assess drug-induced GCP II inhibition in biological matrices.     

Aggregation-prone Regions in HYPK Help It to Form Sequestration Complex for Toxic Protein Aggregates

Protein aggregates result from altered structural conformations and they can perturb cellular homeostasis. Prevention mechanisms, which function against protein aggregation by modulatory processes, are diverse and redundant. In this study, we have characterized Huntingtin interacting protein K (HYPK) as a global aggregation-regulatory protein. We report the mechanistic details of how HYPK's aggregation-prone regions allow it to sense and prevent other toxic protein's aggregation by forming unique annular-shaped sequestration complexes. Screenings for interacting partners of different aggregation-prone proteins identify HYPK as a global interacting partner/regulator of Huntingtin97Qexon1, α-Synuclein-A53T and Superoxide dismutase1-G93A. C-terminal hydrophobic region in HYPK makes direct contacts with aggregates to initiate the formation of sequestration complexes. HYPK acts as aggregate sensor by existing in a seeded amyloid-like state which also favors its own concentration-dependent self-oligomerization. Oligomerization of HYPK leads to annular and non-fibrillar/amorphous aggregates. Two hydrophobic segments in the C-terminus of HYPK are responsible for its own aggregations. Self-association of HYPK follows seed nucleation, in which oligomeric HYPK seeds nucleate to annular structures. Annular oligomers of HYPK fuse with each other to form amorphous aggregates. HYPK shows differential interactions with aggregation-prone and non-aggregating proteins, as it preferentially binds to aggregation-prone proteins with higher affinity than native/non-aggregating proteins. This favors the formation of HYPK's sequestration complexes both in cytosol and in ribosome. Besides having aggregation-preventive property, HYPK also reduces the cellular level of toxic proteins. In vivo, HYPK sequestration complexes prevent the formation of toxic protein aggregates to physiologically show positive impact on cell survival and restoration of normal cell physiology.     

Synthesis of kojic acid derivatives as secondary binding site probes of d-amino acid oxidase

A series of kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) derivatives were synthesized and tested for their ability to inhibit d-amino acid oxidase (DAAO). Various substituents were incorporated into kojic acid at its 2-hydroxymethyl group. These analogs serve as useful molecular probes to explore the secondary binding site, which can be exploited in designing more potent DAAO inhibitors.     

Series-type enzyme deactivations: Influence of intermediate activity on deactivation kinetics

A series-type enzyme deactivation model involving an active enzyme precursor is proposed wherein the enzyme activity is a weighted function of the active enzyme states. The active enzyme precursor may be less active, as active or more active than the initial enzyme form. The proposed model is shown to fit the soluble and immobilized enzyme deactivation data presented reasonably well. Some enzymes exhibit a ‘compensation-like’ effect. In other enzymes, if the deactivation rate coefficient for the second step, k₂, is zero, then the activity may stabilize to a value that depends upon the relative activities of the two active enzyme states.     

A modified system for thiazolinone conversion to thiohydantoin derivatives and their separation by high-pressure liquid chromatography

An improved and very simple procedure for thiazolinone conversion to thiohydantoin derivatives and their separation by reverse-phase high-pressure liquid chromatography is described. Trifluoroacetic acid (10%) in ethyl acetate has been employed as a conversion reagent to circumvent the deamidation of acid amides and methylation of acidic amino acids, with a concomitant increase in the detection limits of these residues. Additionally, a very simple procedure has been developed for the separation of phenylthiohydantoin (PTH) derivatives of amino acids. The system takes advantage of the computer-controlled precise mixing of the solvents A and B to achieve accurate pH and thus avoid the necessity of pH adjustment of a buffer. The procedure is simple and highly reproducible, and separates all the 20 known PTH amino acids. The efficiency of the method has been examined on synthetic and natural proteins/peptides, in manual and autoconversion systems, over a period of more than 18 months.