Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation

Abstract

Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson’s disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs’ design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at −80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

“…Previous stabilization studies carried out for hSCOMT lysates obtained from Escherichia coli showed that DTT and glycerol were effective to stabilize the protein, maintaining its activity [ 28 ]. Moreover, a low concentration of [Ch][DHP] 7.5 mM incorporated in the defined multicompetent buffer (150 mM NaCl, 1 mM MgCl 2 , and 50 mM Tris-Cl, pH 8.0) for a period of 32.4 h at −80 °C has been shown as the ideal conditions to maximize the activity recovery of hMBCOMT [ 25 ]. In fact, all the results obtained for recombinant human COMT lysates were helpful to define the proper stabilizers and respective concentrations that should be used in the development of a further formulation to maintain and/or improve hSCOMT stability after the main purification step, as described by our research group [ 29 , 30 ].…”

“…In particular, from previous results with the membrane isoform of recombinant human COMT in 10 mM Tris-HCl pH 7.8, 30% glycerol, 100 mM cysteine, and 300 mM trehalose, it was shown that protein remains active for 72 h when stored at −80 °C, presenting also an increment in the protein activity recovery of 125% (data not shown). More recently, the same isoform in the presence of 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM MgCl 2 , 7.5 mM of [Ch][DHP], 15 mM cysteine, 5 mM trehalose, and 5% glycerol maintained its activity up to 32.4 h when stored at −80 °C, presenting also an increase in its activity recovery of approximately 140%, with a drastic reduction in the main additive (cysteine and trehalose) concentrations [ 25 ]. It was also reported that for a long storage period of 72 h at −80 °C, hMBCOMT still presents an increment of 27% on its original activity recovery.…”

“…Once, during hSCOMTVal108-6His crystallization trials, the formation of cysteine crystals was observed when a concentration of 100 mM was used, lower concentrations of additives (15 mM cysteine, 5 mM trehalose, and 5% glycerol) were applied in order to stabilize the enzyme as successfully tried with the membrane isoform hMBCOMTVal158 [ 25 ]. Moreover, two distinct Ionic Liquids (ILs) classes, choline, with [Ch][DHP], and imidazole, with [C 4 mim]Cl, were evaluated in hSCOMTVal108-6His lysates in terms of percentage of activity recovery at the two storage temperatures (4 °C and −80 °C), as shown in Figure 6 .…”

“…Herein, taking advantage of the TSA methodology, we aim to screen the soluble isoform of COMT against generic buffer formulations to identify stabilizing conditions, comprising salts, the ideal pH, and simple additives (namely, cysteine, trehalose, glycerol, and ionic liquids), which have already successfully been explored with the membrane COMT isoform [ 25 ]. Considering the similarities shared by both isoforms, Mg 2+ and S-Adenosyl-L-methionine (SAM) dependency, as well as an identical catalytic mechanism, SCOMT (24.7 kDa, 221 residues) has higher K m and V max values, being responsible for the O -methylation of catechols at a higher concentration in the peripheric tissues [ 26 ].…”

Influence of Metal Salts Addition on Physical and Electrochemical Properties of Ethyl and Propylammonium Nitrate

“…The possibility of availing materials with physical properties tailored to a specific application has been key for using them in other applications, such as stabilization and activation of biocatalysts, biomass treatment processes, synthesis of biopolymer-based hydrogels, bioseparation processes, environmental problem solving or the development of new ILs from natural products [7]. More specifically, in biomedicine and the pharmaceutical industry, important advances have also been made in the formulation of advanced materials for the generation of artificial tissues [8], drug transport or the formulation of active pharmaceutical ingredients [9,10], enzyme stabilization [11], biocides [12], etc. These applications are only a small sample of the potential of these materials and the importance of research in future applications.…”