Assessment of metal-based dihydrofolate reductase inhibitors on a novel mesofluidic platform

https://doi.org/10.1016/j.snb.2022.131978Get rights and content

Highlights

  • DHFR is an attractive promising target to differentiate cancer from normal cells.

  • µSIA-LOV is developed to test DHFR inhibition of synthesized organometallic compounds.

  • Some of the metal compounds evaluated exhibit noteworthy DHFR inhibitory activity.

  • The µSIA-LOV system is a useful tool for the evaluation of DHFR enzymatic reaction.

  • Automation to assess enzymatic assays proved to be advantageous.

Abstract

A new miniaturized micro sequential injection coupled with the lab-on-valve (µSIA–LOV) technique was full-blown to perform inhibitory studies on dihydrofolate reductase (DHFR). The system was used to evaluate the DHFR inhibition activity of metal-based anticancer compounds. The metal complexes exhibited IC50 values in the range 1.3 ± 0.3–108 ± 7 μM, with half of the complexes lying in the low μM range, i.e., 1.3 ± 0.3–4.4 ± 0.2 μM. For comparison, methotrexate (MTX), a known inhibitor of DHFR, has an IC50 value of 0.38 ± 0.06 μM. The µSIA–LOV is a versatile, robust, rapid, and easy to operate, allowing automated determination of DHFR inhibition. Moreover, the automated system requires very little sample (approximately 40 µL per analysis), uses minimal reagents (5 times less than the batch procedure used), and generates very little waste (around 1.2 mL per analysis) compared with batch methods, considerably reducing costs.

Introduction

The third generation of flow analysis, also termed the lab-on-valve (LOV) concept, is an advanced mesofluidic approach that provides huge potential for instrumental miniaturization, automatization, and continuous monitoring of chemical and biochemical assays combined with automatic treatment of sample [1]. The LOV concept merges all the intrinsic benefits of flow methods with micro-flow injection analysis (μFIA) channels, using an integrated machined structure [2].

The resulting mesofluidic platform enables unattended chemical and biochemical analysis performance, independent of the matrix, chemistry, and samples involved, based on programmable pressure user-friendly software whose flow direction and rate are controlled [3].

Several detections could be associated with LOV platforms for several applications. There are some studies that reported the detection outside the LOV platform, using optic fibers that are connected to the flow cell [4] or also by chromatographic detection [5], [6]. On the other hand, there are others LOVs where the detection is inside the flow cell. This configuration is mainly used for absorbance detection but also for fluorimetry and chemiluminescence detection [7], [8], [9], [10].

Micro sequential injection coupled to lab-on-valve (µSI-LOV) platform based on the programmable flow, has been used to perform biomolecular assays, including enzymatic assays [11], [12].

The study of enzyme kinetics allows both substrates and inhibitors to be characterized [13]. Standard enzyme kinetics studies typically employ manual bench techniques. However, the irregularity of the mixing time prior to detection and the time consumed by manual operation can lead to errors. Consequently, to improve accuracy and reduce the analysis time, LOV provides some advantages. The stopped-flow injection procedure has been successfully useful to the determination of reactions rate [14], [15], [16]. Basically, the flow can be stopped for a chosen period allowing the reaction to be continuously monitored as a function of time. Hence, we decided to develop a µSIA-LOV system with a new coupled detection module using optical fibers in 90º based on stopped-flow analysis to evaluate the ability of metal-based compounds to inhibit dihydrofolate reductase (DHFR).

DHFR enzyme catalyzes the reduction of dihydrofolate to tetrahydrofolate in an NADPH-dependent manner [17]. This enzymatic reaction interferes with the various amino acids, thymidylate, and purines biosynthesis and is also an important target for anticancer drugs such as methotrexate. [18].

In recent years, significant efforts have been devoted to developing alternative metal-based drugs to platinum-based complexes currently extensively used to treat cancer [19], [20], [21]. One of the leading classes of compounds under intensive evaluation comprises ruthenium-arene complexes, which have been extensively evaluated in vivo and show highly promising properties [22]. Iron, due to its bioavailability, is another attractive element for a metal-based drug that noticeably decreases the toxic consequences of these compounds, and the chemical redox potential in physiological media, typically involving the oxidative states of FeII and FeIII. Some monoiron complexes have been studied due to their anticancer effect and functionalized ferrocenes have emerged as promising candidates [23], [24]. Recently, different categories of diiron compounds based on the {Fe2Cp2(CO)x} scaffold (x = 2 or 3) have revealed potential in this field. They display ideal characteristics for a drug candidate, such as straightforward gram-scale synthesis from cheap precursors, generally noticeable water solubility, amphiphilicity, and wide structural variability by varying the substituents on the bridging hydrocarbyl ligand (i.e., hetero-carbyne or vinyliminium). These diiron complexes exhibit cytotoxicity against cancer cells ranging from low micromolar to inactive, and a marked selectivity towards noncancerous cells [25], [26], [27]. It has been outlined that aminocarbyne complexes probably exert their action by inhibition of thioredoxin reductase enzyme [25].

Herein, we describe a simple µSIA–LOV method for the automated determination of DHFR activity and its application to a selection of metal-based compounds with a previously established anticancer activity. Results of DHFR inhibition are discussed with respect to the structure and the antiproliferative activity of the compounds.

Section snippets

Reagents

Dihydrofolate reductase (DHFR), dihydrofolic acid (DFA), Nicotinamide adenine dinucleotide phosphate (NADPH), and methotrexate were purchased from Sigma. Dimethyl sulfoxide (DMSO) and ethanol were purchased from Merck and Fisher Chemical, respectively. Milli-Q water plus system was used to get ultrapure water with specific conductivity less than 0.1 μS cm− 1. This specific water was used to prepare all the solutions and the reagents were weighed in analytical grade.

The carrier solution used in

Results and discussion

A μSIA-LOV system was developed for the automatic determination of DHFR activity and applied to a set of metal-based compounds with anticancer activity. DHFR catalyzed the reaction of dihydrofolate acid (DFA) with tetrahydrofolate (THF) using NADPH as a cofactor, and the fluorescence intensity (FI) of the reaction decreases as NADPH is concerted to NADP+.

Conclusions

A new automatic µSIA–LOV methodology was developed for the assessment of DHFR activity and used to determine the inhibitory effect of selected metal-based compounds endowed with anticancer activity. The µSIA–LOV system offers several advantages over batch methods, including a noteworthy reduction in reagents, solvents, and samples consumed and less operator involvement. Notably, the system was found to be suitable for routine analysis. The application of the µSIA–LOV method to study the

CRediT authorship contribution statement

Sarah A. P. Pereira: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization. Lorenzo Biancalana: Resources, Writing – review & editing. Fabio Marchetti: Resources, Writing – review & editing. Paul J. Dyson: Conceptualization, Resources, Writing – review & editing, Supervision M. Lúcia M. F. S. Saraiva: Conceptualization, Resources, Writing – review & editing, Supervision, Project administration, Funding

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work received financial support from the project with reference PTDC/QUI-QAN/30163/2017 - Tailored NanoGumbos: The green key to wound infections chemosensing, supported by national funds by FCT/MCTES and co-supported by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operational Competitiveness Program (COMPETE)—reference number POCI-01-0145-FEDER-030163. This work received support and help from UIDB/50006/2020 and UIDP/50006/2020 with funding from FCT/MCTES through national

Sarah A.P. Pereira is a Ph.D. student in Pharmaceutical Sciences at the Faculty of Pharmacy of the University of Porto, Portugal in collaboration with École Polytechnique Fédérale de Lausanne, Switzerland. Her current research focuses on the development of sustainable automated biochemical analytical tools for the application of the inhibitory effect of organometallic compounds in cancer-related enzymes and cancer cells toxicity assays.

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  • Cited by (0)

    Sarah A.P. Pereira is a Ph.D. student in Pharmaceutical Sciences at the Faculty of Pharmacy of the University of Porto, Portugal in collaboration with École Polytechnique Fédérale de Lausanne, Switzerland. Her current research focuses on the development of sustainable automated biochemical analytical tools for the application of the inhibitory effect of organometallic compounds in cancer-related enzymes and cancer cells toxicity assays.

    Lorenzo Biancalana received his Ph.D. in Chemistry from the University of Pisa in 2018 under the guidance of Prof. Fabio Marchetti. Since 2019, he is a Researcher at the same Institution, working on the synthesis of new transition metal complexes for catalysis and medicinal chemistry.

    Fabio Marchetti received his Ph.D. degree in Chemistry from the University of Bologna in 2003 and is currently a Full Professor of Inorganic Chemistry at the University of Pisa. He has co-authored over 200 papers in international journals, dealing with several aspects of the coordination chemistry of transition metal complexes.

    Paul J. Dyson is a Professor at the Institute of Chemical Sciences and Engineering at the EPFL and is currently the Dean of the Faculty of Basic Sciences. He received his Ph.D. degree from the University of Edinburgh in 1993 and subsequently held positions at the Imperial College of Science, Technology and Medicine and the University of York. His research interests are focused on the synthesis and properties of compounds and materials with applications in catalysis and medicine.

    M. Lúcia M. F. S. Saraiva is an Assistant Professor at the Faculty of Pharmacy of the University of Porto and a research scientist at REQUIMTE Associate Laboratory. Her main area is the development of sustainable automated (bio)analytical tools for application mainly in biomedical and environmental areas.

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