Monitoring of phosphorylation using immobilized kinases by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry

Highlights

• Immobilized kinases packed bioreactors developed to monitor on-line phosphorylation.

• Proof of concept of one and two successive on-line phosphorylation monitoring.

• Application with synthesized acyclic nucleoside phosphonate conversion monitoring.

• Study of specific conversions regarding two enzyme strains.

Abstract

Nucleosides analogues are the cornerstone of the treatment of several human diseases. They are especially at the forefront of antiviral therapy. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphate form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. In this context, it is mandatory to develop a rapid, reliable and sensitive enzyme activity test to evaluate their metabolic pathways. In this study, we report a proof of concept to directly monitor on-line nucleotide multiple phosphorylation. The methodology was developed by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry detection. Human Thymidylate Kinase (hTMPK) and human Nucleoside Diphosphate Kinase (hNDPK) were covalently immobilized on functionalized silica beads, and packed into micro-bioreactors (40 μL). By continuous infusion of substrate into the bioreactors, the conversion of thymidine monophosphate (dTMP) into its di- (dTDP) and tri-phosphorylated (dTTP) forms was visualized by monitoring their Extracted Ion Chromatogram (EIC) of their [M − H]^- ions. Both bioreactors were found to be robust and durable over 60 days (storage at 4 °C in ammonium acetate buffer), after 20 uses and more than 750 min of reaction, making them suitable for routine analysis. Each on-line conversion step was shown rapid (<5 min), efficient (conversion efficiency > 55%), precise and repeatable (CV < 3% for run-to-run analysis). The feasibility of the on-line multi-step conversion from dTMP to dTTP was also proved. In the context of selective antiviral therapy, this proof of concept was then applied to the monitoring of specificity of conversion of two synthesized Acyclic Nucleosides Phosphonates (ANPs), regarding human Thymidylate Kinase (hTMPK) and vaccina virus Thymidylate Kinase (vvTMPK).

Introduction

Drug discovery is a lengthy process. To gain a better understanding of the activity and toxicity of pharmaceutical drugs, it is important to determine their metabolic pathways [1,2]. The monitoring of conversion efficiency helps to optimize the drug development process. One illustration of this latter is the activation of nucleotide analogues into their triphosphate active form [[3], [4], [5]]. These compounds are the cornerstone of the treatment of several human diseases and are especially at the forefront of antiviral therapy [[6], [7], [8], [9]]. Biological activities are exhibited by triphosphate nucleotides, which compete with natural substrates for incorporation in the newly synthesized DNA strand, thus causing the inhibition of viral polymerase activity (e.g. DNA chain termination) and therefore virus replication [10,11]. To ensure good stability and high availability, these compounds are delivered as uncharged molecules [12]. Two major antiviral nucleoside classes have proved to be biologically active: the nucleoside analogues [13] and the Acyclic Nucleoside Phosphonate (ANPs) analogues [7]. Since the rate-limiting step of the drug activation is the conversion of nucleoside analogues to their monophosphate form, nucleotide analogues as ANPs were designed to circumvent the initial phosphorylation activation step [14]. The phosphorylation process of a monophosphate to its triphosphate counterpart involves two successive enzymatic phosphorylation reactions catalyzed by cellular kinases [14,15]. For instance, the conversion of the endogenous thymidine monophosphate (dTMP) to thymidine triphosphate (dTTP) is catalyzed by the human Thymidylate Kinase (hTMPK) and the human Nucleoside Diphosphate Kinase (hNDPK) [11]. This activation is performed by a phosphotransfer reaction from a donor, usually the γ-phosphate of adenosine-5′-triphosphate (ATP), towards an acceptor such as the 5′-OH nucleoside or α-, β-phosphate nucleotide groups, in the presence of a chelating agent (usually Mg²⁺) [[3], [4], [5],16]. To the best of our knowledge, the main techniques used to determine the efficiency of a kinase catalyzed reaction are spectrophotometric [[17], [18], [19]], radioisotopic [[20], [21], [22]], High Performance Liquid Chromatography (HPLC) [23] and Capillary Electrophoresis (CE) [[24], [25], [26]]. Although all these techniques have shown their potential for the study of kinase activity, the development of an on-line mass spectrometric approach makes sense in view of its rapidity, substrate versatility and specificity of detection. These on-line methodologies are currently gaining interest for application in drug discovery [[27], [28], [29], [30], [31], [32]]. This liquid chromatography methodology uses biological agents as stationary phase to study enzyme/ligand interactions [33]. Enzyme-immobilized bioreactors offer usually various advantages such as purification of the biological environment (low matrix effect), preservation of activity, bioreactor stability (possible reuse), ease of analysis, easy coupling with the detection system, direct conversion monitoring and possible high throughput screening of compound mixtures. Recently, the use of on-line methodologies with a single bioreactor or multi-reactor have been presented in the field of substrate conversion monitoring [34,35] or DNA digestion [36].

In this context, the present study aimed to develop a rapid, efficient and versatile on-line methodology for the direct monitoring of nucleoside monophosphate and nucleotide diphosphate phosphorylation. As a proof of concept, individual conversion of dTMP into dTDP and dTDP into dTTP were monitored by on-line infusion into immobilized hTMPK and hNDPK bioreactors. The feasibility of the on-line two successive phosphorylation steps was also evaluated. This on-line system showed various advantages such as a rapid (less than 5 min) and efficient (more than 50%) conversion. Moreover, thanks to the dual loop system, this system is versatile (successive substrate infusion) and automatable. It also allows to directly visualize the drug conversion with the specific detection by mass spectrometry. This developed on-line methodology was then applied to the qualitative study of specificity of conversion of two chemically synthesized Acyclic Nucleotides Phosphonates (ANPs) named ANP-CH₃ and ANP-Br [37] (molecular formula shown in Fig. 1) regarding two TMPKs: vaccina virus TMPK (vvTMPK) and human TMPK (hTMPK).