# Using CE to confirm the activity of fluorescent miRFP670-LIMK1 protein produced for MST assays directly in cell lysate

Solweig Chartier, Bérengère Claude, Rouba Nasreddine, Alexandra Launay, Marion Martins, Villalonga-Rosso Elodie, Béatrice Vallée, Muriel Sebban, Gaël Coadou, Reine Nehmé

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## To cite this version:

Solweig Chartier, Bérengère Claude, Rouba Nasreddine, Alexandra Launay, Marion Martins, et al. Using CE to confirm the activity of fluorescent miRFP670-LIMK1 protein produced for MST assays directly in cell lysate. Analytica Chimica Acta, 2025, 1360, pp.344145. 10.1016/j.aca.2025.344145. hal-05304644

HAL Id: hal-05304644

https://hal.science/hal-05304644v1

Submitted on 8 Oct 2025

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# Using CE to confirm the activity of fluorescent miRFP670-LIMK1 protein produced for MST assays directly in cell lysate

<figure><img src="image_4.png" /></figure>

Solweig Chartier<sup>a</sup>, Bérengère Claude<sup>a, b</sup>, Rouba Nasreddine<sup>a, b</sup>, Alexandra Launay<sup>a</sup>, Marion Martins<sup>a, b</sup>, Villalonga-Rosso Elodie<sup>b, d</sup>, Béatrice Vallée<sup>b, d</sup>, Muriel Sebban<sup>c, d</sup>, Gaël Coadou<sup>c</sup>, Reine Nehmé<sup>a, *, d</sup>

## HIGHLIGHTS

* Successful electrophoretic separation of ADP from cell lysate components.

* Evaluation of miRFP670-LIM Kinase activity *via* capillary electrophoresis (CE).

* CE assays confirm activity of miRFP670-LIMK1 despite the fluorescent tag.

* Fluorescent miRFP670-LIMK1 is still inhibited by TH-257 as confirmed by CE.

* Kinetic and affinity assays, directly in cell lysates, using CE and MST.

## GRAPHICAL ABSTRACT

### Using CE to confirm the activity of fluorescent miRFP670-LIMK1 protein produced for MST assays directly in cell lysate

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CE: catalytic activity confirmation

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MST: binding confirmation

## ARTICLE INFO

### Keywords:

Capillary electrophoresis

Cell lysate

Fusion protein

LIM kinases

Microscale thermophoresis

## ABSTRACT

LIMK1 is a promising therapeutic target for multiple pathologies. Its holistic study seems crucial, as none of its inhibitors has passed clinical trials. The need for new fluorescent probes for the study of enzyme/ligand interactions is always increasing with the development of new analytical tools. A method for the assessment of LIMK activity directly in cell lysate is needed to ensure that interaction studies can be performed close to native conditions and by using active enzymes.

Here, we introduce a study of LIM Kinase activity and inhibition monitored directly in cell lysate by capillary electrophoresis (CE). The miRFP670-LIMK1 was prepared because it is a fluorescent fusion protein emitting in the red spectrum and is thus suitable for microscale thermophoresis (MST) affinity assays. A PDADMAC-coated capillary was mandatory to avoid adsorption issues. The results directly confirm, for the first time, that miRFP670-LIMK1 is still active in the cell lysate even when the miRFP670 fluorescent tag is attached to its C-terminal. The inhibition of miRFP670-LIMK1 activity in the presence of TH-257 was also confirmed by CE. The dissociation constant, $K_d$, of the interaction miRFP670-LIMK1/TH-257 was then evaluated by MST, again directly

<footnote><sup>a</sup> Institut de Chimie Organique et Analytique (ICOA), CNRS FR 2708 – UMR 7311, Université d'Orléans, 45067, Orléans, France</footnote>

<footnote><sup>b</sup> Centre de Biophysique Moléculaire, CNRS UPR4301, Affiliated with Université d'Orléans, Rue Charles Sadron Cedex 2, 45071, Orléans, France</footnote>

<footnote><sup>c</sup> Normandie Université, COBRA, UMR6014 & FR3038 CNRS, Université de Rouen, INSA de Rouen, Mont-Saint-Aignan Cedex, 76821, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: reine.nehme@univ-orleans.fr (R. Nehmé).</footnote>
in the cell lysate without any purification step. Submicromolar cellular on-target activity was revealed ($K_d$ = 9 nM), confirming that TH-257 is suitable as a chemical probe for LIMK1.

This study presents an innovative application of CE as a direct approach to verify, in a straightforward and simple manner, without purification or radioactivity, if an enzymatic target maintains its functionality after labeling. This work also highlights the complementarity of CE and MST in advancing pharmaceutical development under conditions that closely mimic those *in vivo*.

# 1. Introduction

LIMK1 and LIMK2, the two members of the LIM Kinases family, play central roles in regulating the actin cytoskeleton by phosphorylating cofilin on serine 3 and inactivating its actin depolymerization activity [1]. Dysregulation of LIMK behavior can lead to several pathologies, including cancer [2-4], neurofibromatosis [5] and neuronal diseases [6] (such as Alzheimer's disease and Parkinson's disease). LIMK1 is highly expressed in neural tissues [7]. Dendritic spines are critical for brain function, including memory and learning, and LIMK1 plays a crucial role in their deterioration. LIMK1 inhibition enhances the size and density of dendritic spines and has anti-Alzheimer's disease effects [6]. Studying LIMK1 inhibition is therefore pivotal in the search for drugs to treat neuronal diseases. Furthermore, cofilin dysregulation is broadly known to cause several neurodegenerative pathophysiologies [8], including Alzheimer's disease [9,10]. Studying LIMKs' activity and inhibition constitutes a major node in the search for new drug candidates [11]. Adenosine triphosphate (ATP) is involved in a myriad of cellular processes [12]. As a substrate of several enzymes, including kinases, it has already been studied by capillary electrophoresis (CE) in the case of tau phosphorylation by glycogen synthase kinase 3-β [13] and the PI3K/Akt/mTOR signaling pathway [14]. The major degradation product of ATP is adenosine diphosphate (ADP), which is obtained after the transfer, by a kinase, of a phosphate group from ATP to its substrate. Kinase substrates include proteins, lipids, sugars, nucleosides/nucleotides, and small molecules or secondary metabolites.

To evaluate an enzyme's catalytic activity, it is essential to monitor the chemical changes that occur during the conversion of a substrate to its product, which, in some enzyme assays, can be quantified directly by taking advantage of the product's absorbance characteristics. Currently, the most common method for managing the vast array of enzymatic activities is to classify enzymes based on the kinds of chemical reactions they catalyse [15]. In the case of kinases, the reaction occurring is the catalysis of the transfer of a phosphate group from one molecule to another: radioactive phosphate incorporation ($^{31}$P) is widely used to monitor this reaction [16]. The use of radioactivity-based assays is limited by safety and environmental concerns. Another technique is immunoprecipitation: mass fingerprinting analysis can be performed after immunoprecipitation using anti-phospho antibody, which identifies the phosphorylation motif site by AMP-activated protein kinase [17]. The most straightforward method used to monitor enzymatic activity is monitoring the increasing concentration of the reaction product in the reaction medium. For example, quantitative indirect spectrophotometric analysis in the reaction catalyzed by glucose 6-phosphate dehydrogenase at 340 nm by Warburg optical test: one product, reduced nicotinamide adenine dinucleotide (NADH), absorbs light at this wavelength, while the reactant, $NAD^+$ (oxidized form), does not. Therefore, the reaction can be monitored by observing the increase of the absorbance at 340 nm or *vice versa* [18]. Other techniques rely on spectroscopy. For example, because the signals from ATP and phosphocreatine occur at distinct chemical shifts, creatine kinase activity may be measured using magnetic resonance spectroscopy (MRS) and imaging (MRI), which are techniques based on $^{31}$P [19]. These spectrometric methods are generally fast but may lack the sensitivity and separation capabilities needed for complex samples.

Using separative techniques such as liquid chromatography (LC) or CE for enzymatic assays, mainly those conducted in complex matrix such

as cell lysates, provides enhanced specificity and resolution, allowing for the separation and quantification of individual products or substrates. These techniques reduce matrix effects, improving accuracy by separating the analytes from interfering substances. LC is based on the retention of analytes by a stationary phase in a column, leading to their separation. This technique is suitable for enzymatic activities [20] but requires the use of organic solvents, which can negatively impact online assays [21]. CE is a separative method that enables quick, highly efficient, and economical analyses [22,23]. This analytical tool was chosen for the present study, but working with an uncoated capillary can be tricky when working with biomolecules in complex matrices [24]. The coating of the capillary with a cationic polymer (e.g. poly (diallyldimethylammonium), PDADMAC [13,25] or hexadimethrine bromide, Polybrene [26]), an anionic polymer (e.g. polystyrene sulfonate, PSS [27]) or even a neutral polymer (e.g. polyethylene oxide, PEO) limits the adsorption of macromolecules on the capillary wall.

For the first time, we present a direct evaluation of miRFP670-LIMK1 activity, in cell lysate without any purification step, *via* CE to confirm whether this kinase retains its catalytic activity despite the presence of the fluorescent miRFP670 tag on its C-terminal end. Ensuring that an enzyme retains its activity after labelling is crucial for the development of pharmaceutical drugs based on the enzyme inhibition under conditions closest to those *in vivo*. This fusion protein, which emits in the red spectrum, is expected to be valuable for any assay requiring a fluorescent LIM Kinase. Indeed, some affinity assays involve the use of fluorescence, as is the case when microscale thermophoresis (MST) is employed [28,29]. The fluorescent partner can be obtained by producing a fusion fluorescent protein of the enzyme, for example with Green Fluorescent Protein (GFP) [30] or with red fusion miRFP670 protein (recently proposed by our group). However, because fusion proteins are large macromolecules, they can hinder the binding process of the enzyme to its target or to inhibitors. Moreover, cofilin (or any actin-depolymerizing factor), especially not directly present in the HEK293 cell lysate, has never been studied by CE. These samples could cause significant undesirable adsorption phenomena on bare-silica capillaries. In this work, a systematic optimization of the conditions used to study the enzymatic reaction of miRFP670-LIMK1 with ATP and cofilin by CE in the absence and presence of a reference inhibitor, TH-257, was performed (Fig. 1). The dissociation constant, $K_d$, of the interaction of miRFP670-LIMK1/TH-257 was subsequently evaluated by MST to obtain complementary results confirming the affinity between these two partners.

# 2. Materials and methods

## 2.1. Chemicals

All reagents were of analytical grade and were used as received without any further purification. Ammonia ($NH_4OH$, 28 %), dimethylsulfoxide (DMSO, purity 99 %), glacial acetic acid ($CH_3COOH$, purity >99.7 %) and sodium hydroxide ($NaOH$, purity 98.5–100 %) were purchased from VWR International (Fontenay-sous-Bois, France). Syringes and polytetrafluoroethylene (PTFE) syringe filters (pore size 0.45 µm) were purchased from Agilent Technologies (Les Ulis, France). Adenosine 5'-diphosphate sodium salt (ADP, purity >95 %), adenosine 5'-triphosphate disodium salt hydrate (ATP, purity >99 %), magnesium chloride ($MgCl_2$), anhydrous, purity >8 %), ethane-1,2-diyldinitrilo
tetra-acetic acid (EDTA, 0.5 M, pH 8.0), glycerol (purity 99 %) β-glycero-phosphate disodium salt hydrate ((HOCH₂)₂CHOP(O) (ONa)₂ xH₂O), 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N'-[(2-ethanesulfonic acid) or HEPES (C₈H₁₈N₂O₄S, purity >99.5 %), hydrochloric acid (HCl, 36.5–38.0 %, pH < 1.0), phenylmethanesulfonyl fluoride (PMSF, C₇H₇FO₂S, purity >99.0 %), orthophosphoric acid (H₃PO₄, 85 %), sodium fluoride (NaF, purity >99 %), poly (diallyldimethylammonium chloride (PDADMAC 20 % w/v), and sodium pyrophosphate dibasic (Na₂H₂P₂O₇, purity >99.0 %), TH-257 or N-Benzyl-N-butyl-4-(N-phenylsulfamoyl)benzamide, N-Butyl-4-[(phenylamino)sulfonyl]-N-(phenylmethyl)benzamide (C₂₄H₂₆N₂O₃S, purity >98 %) Triton X-100, TH-257 (C₂₄H₂₆N₂O₃S) and cell culture media were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France). Aprotinin (Apro), leupeptin, sodium chloride (NaCl, ultrapure grade), p-Nitrophenylphosphate (pNPP), tris(hydroxymethyl)amino-methane (Tris) (C₄H₁₁NO₃, ultrapure grade), and okadaic acid (AcOK, purity >98 % HPLC) were purchased from Euromedex (Souffelweyersheim, France). Sodium orthovanadate (OV) from LC laboratories (Woburn, USA) was used. Ultrapure water (18 MΩ-cm) was purified with a Milli-Q apparatus from Merck (Semoy, France).

## 2.2. Buffer solutions

All solutions were prepared with ultrapure water and filtered through a 0.45 µm PVDF filter. The pH of the various solutions was measured using a MeterLab PHM201 portable pH meter (Radiometer Analytical, Villeurbanne, France).

**HEK293 cell lysis buffer:** HEK293 cells that overexpressed fluorescent LIMK1 fusion proteins, miRFP670-LIMK1, were lysed with a lysis buffer composed of 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.1 % Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na₃VO₄, 20 mM p-nitrophenyl phosphate, 20 mM β-glycerophosphate, 10 µg/mL aprotinin, 0.05 µg/mL okadaic acid, 1 µg/mL leupeptin, and 1 mM PMSF.

**Background electrolyte buffers (BGE):** BGEs containing ammonia/acetic acid at ionic strengths of 50 mM or 80 mM (pH 10.0) were obtained by diluting the appropriate volume of acetic acid (1 M) and ammonia (1 M) in ultrapure water. The Tris/phosphate containing 5 mM MgCl₂ (pH 7.2, 180 mM ionic strength) was prepared by diluting the appropriate amount of phosphoric acid (85 %) in ultrapure water and weighing the appropriate quantity of Tris and MgCl₂.

**Incubation buffers (IB):** Different IBs were prepared by weighing the appropriate amount of HEPES (25 mM) and MgCl₂ (0, 2.5 and 10 mM) in ultrapure water. The pH of the IB was adjusted to 7.2 with NaOH 1 M.

**MST buffer:** The lysis buffer previously described was also used as MST buffer for the affinity studies ($K_d$ measurement).

**Tris-buffered saline or TBS buffer:** This buffer, used for the preparation of cofilin solutions, was composed of 25 mM Tris, 2.7 mM KCl and 137 mM NaCl at pH 7.4.

## 2.3. Cloning of miRFP670-LIMK1

To construct miRFP670-LIMK1, LIMK1 was amplified by PCR from the plasmid pcDNA3-LIMK1 [31] with the following primers CGACGTGCTAGCGCCACCATGAGGTTGACGCTACTTTGT and CTGAGCACCGGTAAGTCGGGGACCTCAGGGTGGGC. The PCR product was purified, digested with the restriction enzymes NheI and AgeI, and cloned into the miRFP670 plasmid (#79987 from Addgene, Watertown, USA) opened with the same restriction enzymes NheI and AgeI using T4 DNA ligase. The ligation mix was transformed into competent cells. Minipreparation of 6 colonies were performed and their profile was analyzed upon digestion by NheI and AgeI. Two of the correct clones were checked by sequencing by Eurofins Genomics (Ebersberg, Germany).

## 2.4. Cell culture

HEK293 (ATCC, CRL1573) were cultured under 5 % CO₂ at 37 °C in low-glucose Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum. Cells were transiently transfected with 10 µg of expression plasmid/100-mm dish using calcium phosphate method. They were cultured for 48 h before performing any experiment. Cells were regularly checked for mycoplasma contamination using a luminescence-based kit (Lonza, MycoAlertTM Mycoplasma Detection Kit).

## 2.5. Cell lysis

Culture medium of the plates containing transfected HEK293 cells adherent to the dish was removed. Cells were washed twice with Phosphate Buffer Saline. At the second wash, buffer was carefully removed and cells were lysed directly in the dish by adding 0.5 mL of lysis buffer (composition described previously) on top of them. The lysis buffer was spread all over the cells covering the plate, and the plate was incubated on ice for 10 min. Lysed cells were then scraped delicately to collect all of them, and transferred into a 1.5 mL microtube. This tube was centrifuged for 10 min at 10 000×g. The supernatant was then collected in a new microtube. This corresponds to the cell lysate stock solution that has been then used for further CE experiments.

## 2.6. Stock and working solutions

The cell lysate stock solutions, containing miRFP670-LIMK1, were prepared and stored in the lysis buffer containing glycerol at 30 % (v/v). Working solutions at 64 nM for CE-assays and 32 nM for MST-assays were obtained by dilution in either the IB or the lysis buffer.

The TH-257 stock solution was prepared at a concentration of 4 mM in 100 % DMSO. Working solutions at 100 nM for CE-assays and 1 µM for MST-assays were obtained by dilution in either the IB or the lysis buffer.

<figure><img src="image_7.png" /><figcaption>**Fig. 1.** Representation of the reaction catalyzed by miRFP670-LIMK1 for the phosphorylation of the cofilin substrate in the presence of the phosphate-donor ATP and the co-factor Mg²⁺, leading to a phosphorylated cofilin and ADP. The influence of TH-257, a type III inhibitor, on the reaction is also represented.</figcaption></figure>
Stock solutions of the substrate ATP at 25 mM and the product ADP at 2 mM were prepared in the IB. They were diluted in the incubation buffer (IB) to the desired concentrations: 0.1 mM and 4 mM for ATP. The response function of the UV detector was evaluated by plotting the time-corrected peak areas (CPA) against ADP concentrations. For this, working standard solutions were prepared by diluting the ADP stock solution in the IB. Linearity was assessed using six concentrations: 0, 25, 50, 100, 150, and 200 µM. Each solution was analyzed 3 times (n = 3). The linearity was assessed by least squares regression and determination coefficient (r²) was evaluated. Recombinant cofilin, purchased from Upstate cell signaling Inc., was transferred in a TBS buffer at 135 µM. A cofilin working solution of 81 µM was obtained by diluting the stock solution in the appropriate IB.

All stock solutions were stored at -20 °C.

## 2.7. Instrumentation and operating conditions

### 2.7.1. Measurement of miRFP670-LIMK1 concentration in HEK293 cells

The concentration of miRFP670-LIMK1 in the cell lysate was determined to be 453 nM using a double-beam UV-visible spectrometer (UV-1800, Shimadzu, Kyoto, Japan) and a 0.5 mL quartz cuvette with a 1 cm optical path length. Blank assays were conducted with the lysis buffer. The concentration of miRFP670-LIMK1 was estimated using Beer-Lambert law (equation (1)) at 642 nm, the specific excitation wavelength of miRFP670, to avoid interference with other species in the cell lysate:

$$A_{642} = \varepsilon_{642} \times l \times C \quad (1)$$

where A$_{642}$ is the absorbance of the sample at λ$_{max}$ of the miRFP670 tag; ε is the molar absorption coefficient of miRFP670 at 642 nm (87 400 L/mol/cm) [32]; l is the optical path of the cuvette (1 cm); and C is the molar concentration (mol/L) of analyzed miRFP670-LIMK1 in HEK293 lysate.

### 2.7.2. Capillary electrophoresis (CE)

The CE system used was a Beckman P/ACE MDQ (AB Sciex, USA) CE apparatus equipped with a photodiode array detector. The UV spectrum was acquired between 200 and 400 nm, with a step of 1 nm. Beckman 32 karat software (Beckman Coulter, USA) was used to pilot the CE system and for signal acquisition. CE analyses were performed in a fused-silica capillary (60 cm total length, 50 cm effective length, 50 µm i.d.) purchased from Polymicro Technologies (Phoenix, AZ, USA). New capillaries were conditioned with 1 M NaOH for 20 min and water for 5 min. All the rinse cycles were carried out at 50 psi. Hydrodynamic injection was performed by applying a pressure of 0.5 psi for 5 s (~4 nL). The volume of the injected plug was estimated using CE expert Lite from Sciex (https://sciex.com/ce-features-and-benefits/ce-expert-lite). Separation was conducted at 25 °C by applying a voltage of +15 kV for bare silica capillaries and at -30 kV for cationic PDADMAC-coated capillaries. ATP and ADP detection was obtained by UV absorption at 254 nm. CE analysis repeatability of ATP and ADP was investigated by monitoring the relative standard deviations (RSD) of the migration times (% RSD$_{tm}$) and of the CPA (% RSD$_{CPA}$).

Pressure-mediated analysis was conducted to reveal adsorption issues on the inner capillary surface by applying a pressure of 5 psi for 5 min, followed by detection at both 214 and 254 nm.

### 2.7.3. PDADMAC coating

New capillaries were conditioned with 1 M NaOH for 20 min and water for 5 min. They were then coated by rinsing with a cationic PDADMAC solution (0.2 % w/v) for 10 min from the anode side followed by 10 min from the cathodic side. After a waiting period of 5 min, the capillary was rinsed with water for 5 min and then with BGE for 10 min. The coating was stabilized by applying an electric field of +30 kV for 10 min [14,24]. The electroosmotic flow was anodic; it was

measured by using DMSO as a neutral marker [33]. The separation was conducted at -30 kV (reverse polarity). Between runs, the capillary was flushed for 1 min with water, 2 min with PDADMAC 0.2 % w/v, and 3 min with BGE.

### 2.7.4. Enzymatic reaction monitored by CE-UV

A blank assay was conducted to establish the baseline of the experiment. For this purpose, a reaction mixture containing 4 mM ATP and 64 nM miRFP670-LIMK1 (a dilution of the stock cell lysate containing 30 % v/v glycerol by ~5) was prepared in the IB. To study the enzymatic reaction, in the absence of the inhibitor, 81 µM of cofilin substrate was added to the previous solution. Finally, a solution of 100 nM TH-257 was added to the previous enzymatic mixture to study the inhibition of the enzymatic reaction. 5 µL of each preparation was collected immediately after mixing to control the reaction media at t = 0 min (t0) for each experiment. Each of the three previously prepared Eppendorf tubes was subsequently placed in a thermomixer at 37 °C with stirring at 450 rpm. After 210 min of incubation (t210), the reaction media were removed from the thermomixer and centrifuged for 2 min at 2000×g, after which the supernatant was collected with a pipette. The resulting mixture was centrifuged for 2 min at 2000×g, and the supernatant was again collected and centrifuged one last time, for a total of three centrifugations before supernatant collection. The reaction was stopped by these three successive centrifugation steps. The solutions were then either immediately analyzed by CE or stored at -20 °C for later analysis.

The CPA of ADP obtained from the electropherograms were converted to concentrations using the linear regression equation. The spontaneous non-enzymatic degradation of ATP was calculated for the blank assay, i.e. in the absence of the substrate cofilin, by taking into account the concentration of ADP present in the cell lysate at t0 (Fig. 3a) and at 210 min (equation (2)):

$$\% \text{ Degradation} = \left( \frac{[\text{ADP}]_{t210} - [\text{ADP}]_{t0}}{[\text{ADP}]_{t0}} \right) \times 100 \quad (2)$$

With: [ADP]$_{t210}$ and [ADP]$_{t0}$, the concentration of ADP obtained, in the absence of cofilin, after 210 min and 0 min of incubation, respectively. % Degradation is positive in the case of ATP degradation and null in the case of no ATP degradation.

The enzymatic reaction to control the so-called 100 % activity of miRFP670-LIMK1 was calculated via equation (3):

$$\% \text{ Enzymatic reaction} = \left( \left( \frac{[\text{ADP}]_{C \ t210} - [\text{ADP}]_{C \ t0}}{[\text{ADP}]_{C \ t0}} \right) - \left( \frac{[\text{ADP}]_{t210} - [\text{ADP}]_{t0}}{[\text{ADP}]_{t0}} \right) \right) \times 100 \quad (3)$$

where [ADP]$_{C}$$_{t210}$ and [ADP]$_{C}$$_{t0}$ are the concentrations of ADP obtained in the presence of cofilin after 210 min and 0 min of incubation, respectively.

The inhibition of miRFP670-LIMK1 activity was calculated as follows (equation (4)):

$$\% \text{ Inhibition} = \left( 1 - \left( \frac{[\text{ADP}]_{t210} - [\text{ADP}]_{t0}}{[\text{ADP}]_{C \ t210} - [\text{ADP}]_{C \ t0}} \right) \right) \times 100 \quad (4)$$

Where [ADP]$_{C}$$_{t210}$ and [ADP]$_{C}$$_{t0}$ are the concentrations of ADP obtained after 210 min of incubation in the presence and absence of the inhibitor, respectively. [ADP]$_{C}$$_{t0}$ and [ADP]$_{C}$$_{t0}$ are the concentrations of ADP detected due to spontaneous hydrolysis at 0 min of incubation in the presence and absence of the inhibitor, respectively.

### 2.7.5. Affinity measurement by MST

The binding affinity between miRFP670-LIMK1 and TH-257 was determined using microscale thermophoresis (MST). MST experiments were performed on a Monolith® NT.115pico (NanoTemper Technologies GmbH, Munich, Germany). HEK293 cell lysate containing 30 % (v/v)
glycerol was diluted five times in the lysis buffer specified in section 2.2. A solution of 2 µM TH-257 was prepared in the lysis buffer by diluting the corresponding stock solution. A 16-point serial dilution was made in the lysis buffer containing the same amount of DMSO as the highest concentration of the inhibitor, that is, 0.025 % v/v DMSO, to ensure that the only variable was the inhibitor concentration. The concentration range of the inhibitor TH-257 was 30 pM to 1 µM. Each capillary contained a constant final concentration of miRFP670-LIMK1 in the cell lysate of 32 nM.

# 3. Results and discussion

## 3.1. Optimization of ATP and ADP separation

The incubation buffer (IB) used for the enzymatic reaction, the BGE used for CE-UV separation and the coating of the capillary inner surface were optimized to achieve satisfactory separation in terms of resolution, analysis time and repeatability. The effect of Mg²⁺ complexation on the separation of ATP and ADP was principally considered. As the molecules that were to be monitored were ATP and ADP, their separation was first investigated without neither miRFP670-LIMK1 nor cofilin in the cell lysate. The optimal wavelength at which the UV-detector monitored the samples was 254 nm, in accordance with their UV spectrum.

### 3.1.1. ATP and ADP analysis using a standard bear fused silica capillary

The first set of experiments was conducted using bare fused silica capillaries and 50 mM BGE ammonia/acetic acid (pH 10.0). Three IBs were prepared with 25 mM HEPES at pH 7.2, one without Mg²⁺, one containing 2.5 mM Mg²⁺ and one containing 10 mM Mg²⁺. Solutions of 0.1 mM ATP and 0.1 mM ADP were prepared in each of the aforementioned IBs. ATP and ADP separation was not possible in the absence of Mg²⁺ since they had similar electrophoretic mobility of -30.3 TU (As shown (Table 1). Complexation between cationic metals and nucleotides has been previously described and investigated by CE [34,35]. Furthermore, it is well established that the ATP-Mg²⁺ complex facilitates their binding towards kinase [36,37]. Upon the addition of 2.5 mM Mg²⁺, the peaks of ADP and ATP were well resolved (Rs = 4.0) since Mg²⁺ forms more stable complexes with ATP than with ADP, with different stoichiometries [34,38]. When 10 mM Mg²⁺ was added to the IB, the separation was highly improved. However, the EOF intensity was decreased and, consequently, a longer analysis time, up to 21 min, was noticed (Table 1). This may be explained by the capillary history since several analyses with different buffers were conducted successively [39]. Increasing the BGE ionic strength from 50 mM to 80 mM further extended the analysis time to 26 min. When ammonia/acetic acid was used at 50 mM (pH 10.0), the repeatability of the analyses was acceptable, with RSD<sub>tm</sub> < 1.2 % and RSD<sub>CPA</sub> < 3 % (n = 6).

The analysis of both the cellular lysate and the cofilin protein was then performed. Pressure-mediated analysis was conducted to reveal any adsorption of the cell lysate components and/or the cofilin protein. For this purpose, 4 nL of the cell lysate were hydrodynamically injected in a bare silica capillary filled with the optimal BGE (ammonia/acetic acid at 50 mM, pH 10.0) and pushed under a pressure of 5 psi for 5 min. A small peak was observed as expected at 1.8 min, in accordance with the Poiseuille law. This experiment was repeated 6 times, and the results remained the same, confirming the absence of adsorption for the cell extracts in a bare silica capillary. Then, cofilin was injected and detected at 1.8 min under the same conditions (Fig. 2). However, the intensity of the cofilin peak obtained during the second injection was almost three times lower than that of the first injection. This peak was almost completely lost after the third injection which confirms that cofilin strongly tends to adsorb on the capillary. Monitoring the phosphorylation of cofilin by miRFP670-LIMK1 in cell lysates via CE was unfeasible unless adsorption issues were remedied. Therefore, the implementation of a cationic coating of the silica capillaries was proposed for the remainder of the study.

### 3.1.2. ATP and ADP analysis using a capillary coated with a cationic layer of PDADMAC

Poly (diallyldimethylammonium chloride) or PDADMAC, which is a

<figure><img src="image_8.png" /><figcaption>Fig. 2. Cofilin adsorption on uncoated silica capillary walls revealed by pressure-mediated analysis because of poor repeatability between the first injection (a) and the second injection (b). Experimental conditions: 5 psi for 5 min; detection at 214 nm; cofilin at 135 µM in TBS buffer (pH 7.4); uncoated silica capillary (60 cm × 50 cm × 50 µm i.d.).</figcaption></figure>

**Table 1**

Optimization of ATP and ADP separation with different BGEs and IB. The results are presented in terms of the migration time of DMSO and EOF mobility, the migration time and electrophoretic mobility of ATP and ADP, and the resolution between ATP and ADP. Capillary: 60 cm × 50 cm × 50 µm i.d. Tiselius unit: 1 TU = 10⁻⁹ m²V⁻¹s⁻¹. Separation: +15 kV (26 µA).

<table><thead><tr><th></th><th></th><th colspan="4">Bare silica capillary (+15 kV)</th><th>PDADMAC-coated capillary<br>(-30 kV)</th></tr></thead><tbody><tr><td rowspan="2">BGE</td><td>Ammonia/acetic acid (pH 10.0) at an ionic strength (mM)</td><td>50</td><td>50</td><td>50</td><td>80</td><td>-</td></tr><tr><td>equal to:<br>Tris/phosphate (pH 7.2) + 5 mM MgCl<sub>2</sub> at an ionic strength<br>(mM) equal to:</td><td></td><td>-</td><td></td><td>-</td><td>180</td></tr><tr><td>IB</td><td>HEPES 25 mM at pH 7.2 + MgCl<sub>2</sub> at a concentration (mM)</td><td>0</td><td>2.5</td><td>10</td><td>10</td><td>10</td></tr><tr><td></td><td>equal to:<br>DMSO</td><td>6.99 (47.7)</td><td>7.56 (44.1)</td><td>8.75 (38.1)</td><td>10.07 (33.1)</td><td>7.31 (-22.8)</td></tr><tr><td rowspan="2">t<sub>m</sub> min (µ<sub>eof</sub><br>TU)</td><td>ATP</td><td>19.15</td><td>16.96</td><td>18.40</td><td>23.58</td><td>4.40 (-15.1)</td></tr><tr><td>ADP</td><td>(-30.3)</td><td>(-24.4)</td><td>(-20.0)</td><td>(-19.0)</td><td>4.87 (-11.5)</td></tr><tr><td rowspan="2">Rs</td><td>ATP/ADP</td><td>0</td><td>4.0</td><td>26.5</td><td>26.4</td><td>6.9</td></tr></tbody></table>
<figure><img src="image_9.png" /><figcaption>**Fig. 3.** Electropherogram of ATP degradation to ADP by miRFP670-LIMK1 in HEK293 cell lysates after 210 min of incubation. (a) Blank assay (in blue): ATP + miRFP670-LIMK1 (absence of substrate); (b) Enzymatic reaction control (100 % activity, in red): ATP + miRFP670-LIMK1 + cofilin (substrate); (c) Inhibition assay (in black): ATP + miRFP670-LIMK1 + cofilin + TH-257 (a type III inhibitor).</figcaption></figure>

Enzymatic reaction conditions: IB: 25 mM HEPES + 10 mM MgCl₂ (pH 7.2); ATP at 4 mM; miRFP670-LIMK1, in HEK293 cell lysate, at 64 nM; cofilin at 81 µM in TBS; TH-257 at 100 nM. The mixture was incubated for 210 min at 37 °C.

CE conditions: BGE: Tris/phosphate +5 mM MgCl₂ (pH 7.2, 180 mM). PDADMAC-coated capillary: 60 cm total length, 50 cm detection length, 50 µm i.d. Cathodic injection: 0.5 psi for 5 s (~4 nL). Separation: 30 kV at 25 °C. Detection at 254 nm. Peak identification was performed according to the migration order in the capillary: AMP, ATP, and ADP. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

cationic polymer, was used to coat the silica capillary surface dynamically and thus to prevent cofilin adsorption by shielding the silanol groups [40]. The stability of PDADMAC monolayer coatings is known to be limited at high pH values [25,41–43]. Therefore, the pH of the BGE needs to be decreased. Tris/phosphate at pH 7.2, the same as the pH of the IB, was used. Nonetheless, between runs, it was imperative to flush the capillary with PDADMAC 0.2 % w/v to ensure regeneration of the coating [42]. At a working pH of 7.2, cofilin is positively charged because its pI is approximately 8.6 [44,45], whereas the inner capillary surface is negatively charged. Thus, tenacious adsorption onto silanol groups is still expected unless the inner surface is masked by positively charged polyelectrolytes such as PDADMAC [25].

When a PDADMAC cationic coating is employed, the EOF intensity is known to be relatively high and anodic [40], thereby aligning with the direction of the anionic ATP and ADP analytes, which significantly reduces their analysis time. This means that ATP and ADP peak resolutions are likely compromised. Indeed, even when a BGE Tris/phosphate (pH 7.2) with an ionic strength of 180 mM was used, the resolution of ATP/ADP was not sufficient (R ~1.1). For this purpose, building upon the results of the previous section which confirmed the significant role of Mg²⁺ in the separation of ATP and ADP, a new BGE consisting of Tris/phosphate containing 5 mM MgCl₂ was tested. The ionic strength was constant at 180 mM to avoid excessive Joule heating (resulting current of ~67 µA) and therefore peak dispersion and reduced separation efficiency [46].

The results are summarized in Table 1. Briefly, the EOF was detected at 7.31 min, which corresponds to ~22.8 TU (anionic EOF). It was very repeatable (RSD<sub>tm DMSO</sub> < 0.3%; n = 6), confirming the stability of the PDADMAC coating. The t<sub>m</sub> values of ATP and ADP were highly reduced to 4.40 min (~15.1 TU) and 4.87 min (~11.5 TU), respectively, with a satisfactory resolution of 6.9 and an efficiency of 10⁴ theoretical plates. It is worth noting that despite the reversal of the direction of the EOF, there was no inversion of the ATP/ADP migration order compared with that of the assays with uncoated capillaries. This can be explained, as indicated by the calculated electrophoretic mobilities, by the complexation with Mg²⁺ added to the BGE (and not only in the IB) as well as the change in the working pH [34,35,47].

These conditions were chosen for the remainder of the study. To

summarize, the IB (pH 7.2) was 25 mM HEPES + 10 mM MgCl₂, the BGE (pH 7.2) was Tris/phosphate + 5 mM MgCl₂ at an ionic strength of 180 mM, and a PDADMAC-coated capillary was used.

The linearity of the ADP response was evaluated over the 0–200 µM range using least squares regression, yielding the following equation: CPA<sub>ADP</sub> = 8779.3 × [ADP] + 2.5668. A strong correlation was observed, with a determination coefficient of r² = 0.9991. This equation was subsequently used to calculate ADP concentrations from the CPAs obtained from the electropherograms.

## 3.2. Enzymatic activity of miRFP670-LIMK1 in cell lysate monitored by CE-UV

The specifics of the enzymatic reaction conditions are detailed in section 2.7.4. First, a blank enzymatic assay was conducted at 37 °C by mixing miRFP670-LIMK1 in cell lysates with ATP in the absence of cofilin substrate. This assay was repeatable (RSD<sub>CPA</sub> of ADP ~ 2%; n = 3) and demonstrated that even after 210 min of incubation, there was only a 6.7 % (equation (2)) increase in the quantity of ADP compared with the initial quantity present in the cell lysate at t0 (Table 2). This slight increase can be attributed to spontaneous, non-enzymatic degradation of ATP occurring at a reaction temperature of 37 °C. An example of electropherogram for the blank assay is presented in Fig. 3a, which shows that the peaks of highly concentrated ATP and a small quantity of ADP due to spontaneous hydrolysis were detected under these conditions with excellent resolution.

To verify whether LIMK1 is still active despite the fluorescent protein

<caption>**Table 2**</caption>

Results obtained from the miRFP670-LIMK1 enzymatic and inhibition assays. The enzymatic and CE conditions are detailed in section 2.7.4 and in Fig. 3.

<table><thead><tr><th></th><th>Blank assay</th><th>Enzymatic reaction</th><th>Inhibition assay</th></tr></thead><tbody><tr><td>[ADP] @ t0 (µM)</td><td>44.0</td><td>52.6</td><td>45.4</td></tr><tr><td>[ADP] @ t210 (µM)</td><td>47.0</td><td>108.9</td><td>58.8</td></tr><tr><td>Δ[ADP] (t0 - t210)</td><td>3.0</td><td>56.4</td><td>13.4</td></tr><tr><td>Degradation (%)</td><td>6.7</td><td>100.5</td><td>23.8</td></tr><tr><td>Inhibition (%)</td><td>n.r.</td><td>n.r.</td><td>76.2</td></tr></tbody></table>
miRFP670 fused to its C-terminal end, miRFP670-LIMK1 in the cell lysate was mixed with ATP and its substrate cofilin in the incubation buffer and incubated at 37 °C for 210 min. Fig. 3b shows that the migration time, resolution and efficiency were in the same order of magnitude as those of the blank assay reaction, with good peak symmetry. Between t0 and t210, the CPA of ADP increased corresponding to an increase of the concentration of ADP from 3.0 to 56.4 µM (Table 2), which demonstrates that miRFP670-LIMK1 has indeed transferred a phosphate group from ATP, leading to a drastic increase in ADP in the reaction media: miRFP670-LIMK1 is still active despite its fluorescent tag. This means that the attachment of miRFP670 close to the LIMK1 active site does not prevent ATP from accessing the hinge region of LIMK1, where competitive inhibitors interact [48]. Using equation (3), the activity of miRFP670-LIMK1 was found to be 100.5 % (Table 2), taking into account the spontaneous degradation estimated in the previous experiment (blank assay).

Notably, when the Myelin Basic Protein (MBP), a nonspecific kinase substrate that is used as a universal substrate for many *in vitro* enzymatic activity assays was used, phosphorylation by LIM kinase was not detected since the ADP concentration in the media did not increase over time. The use of cofilin, an accurate substrate of LIMKs, is mandatory for activity assays.

Since miRFP670-LIMK1 activity was demonstrated by CE, in the complex media that is cell lysate, the ability of miRFP670-LIMK1 to be inhibited by a reference LIMK1 inhibitor, TH-257, was investigated by adding 100 nM TH-257 to the reaction mixture. This concentration of TH-257 is in the same order of magnitude as its half-maximal inhibitory concentration ($IC_{50}$) on LIMK1 [49,50]. The migration times and efficiency were comparable to those obtained with the blank and control/enzymatic reactions, confirming the reproducibility of the developed CE-based method (Fig. 3c). The presence of TH-257 decreased the production of ADP because, between t0 and t210, the concentration of ADP increased by only 13.4 µM vs. 56.4 µM for the enzymatic reaction in the absence of TH-257. This result directly demonstrates that miRFP670-LIMK1 was inhibited by this compound (Table 2). The percentage of residual activity of the enzyme was 23.8 %, which corresponds to an inhibition of 76.2 % (equation (4)). This value is acceptable since the tested concentration in this study is in the same range as the $IC_{50}$ reported in previous interaction studies. Importantly, the $IC_{50}$ values can vary depending on the experimental conditions used, mainly the cell line and the concentration of the target in the reaction media. Consequently, direct comparisons between our findings and those in the literature can be challenging. Moreover, rather than being active in the cell lysate, miRFP670-LIMK1 can interact with TH-257, an allosteric inhibitor: the binding pocket of LIMK1 is probably not affected by the presence of miRFP670, even if it is attached close to the active site (near the kinase domain).

A key advantage of this study is the ability to analyze targets directly in cell lysates without purification. However, this approach poses challenges for accurately quantifying the miRFP670-LIMK1 fusion protein. Estimations based on the absorbance of the miRFP670 tag may be influenced by structural changes upon fusion, and in the absence of purified protein, its exact extinction coefficient remains unknown. Conventional methods such as SDS-PAGE and Western blotting did not allow reliable quantification under these conditions, and no alternative methods reported in the literature proved more effective. Nonetheless, the potential error introduced is considered negligible and does not affect the conclusions drawn from the CE-based LIMK1 assay developed.

In the forthcoming section, the equilibrium dissociation constant ($K_d$) of miRFP670-LIMK1/TH-257 will be evaluated using MST to confirm and quantify the interaction affinity between these partners in the cell lysate without any purification. $K_d$ is a crucial parameter in assessing the binding propensity of a drug to its target, offering a measure that is less influenced by variations in the target concentration. Hence, $K_d$ will complement the activity assays conducted in this study, which relies on CE-UV analysis.

### 3.3. Affinity of miRFP670-LIMK1/TH-257 in cell lysate monitored by MST

First, TH-257 autofluorescence at 1 µM (the highest concentration studied) was checked and found to be negligible, with fewer than 500 counts (as per the manufacturer's instructions). The specific steps are detailed in section 2.7.5. The MST binding assay was then conducted, and a dose-response curve was obtained from two merged sets of data (Fig. 4). The signal-to-noise (S/N) ratio, i.e. the amplitude of the signal divided by the average of the replicate, was equal to 6. This value is greater than 5, which is suitable for the manufacturer's recommendations [51]. Fig. 4 shows that the dose-response curve of TH-257 towards miRFP670-LIMK1 has a sigmoid shape with a well-defined lower and higher plateau. The negative control, where the miRFP670 tag is mixed with TH-257 instead of miRFP670-LIMK1, showed a straight curve (red curve), corroborating the fact that the interactions between miRFP670-LIMK1 and TH-257 in the cell lysate occur specifically with LIMK1 and not with miRFP670. A one-site binding model was used to fit the data shown in Fig. 4, and the $K_d$ value of miRFP670-LIMK1/TH-257 was 9 nM. Since the dissociation constant is lacking from the literature for this target/ligand couple, this result was contextualized by referring to the closest $K_d$ that we can compare to that of TH-257 and purified Kin 1 (the catalytic site of LIMK1), determined by isothermal titration calorimetry (ITC) at 20 °C, which is equal to 64 nM [49]. The $K_d$ determined in this study for full-length miRFP670-LIMK1 in cell lysate by MST was on the same order of magnitude as the one obtained by ITC, despite the dissimilarity of assay conditions.

## 4. Conclusion

In this study, a straightforward approach was developed to evaluate the catalytic activity of LIM Kinases, particularly the fluorescent protein miRFP670-LIMK1, directly in cell lysates *via* CE-UV without any purification step. The developed CE-based enzymatic assay was used to confirm, for the first time, that the miRFP670-LIMK1 in the cell lysate is still active even when the miRFP670 fluorescent tag is attached to its C-terminal end. The presence of the TH-257, a LIM kinase reference inhibitor, also resulted in significant inhibition of the miRFP670-LIMK1.

The optimal conditions for the electrophoretic separation of ATP and ADP in the presence of HEK293 cell lysate were a BGE Tris/phosphate containing 5 mM $MgCl_2$ (pH 7.2, 180 mM ionic strength) and an IB of HEPES containing 10 mM $MgCl_2$ (pH 7.2, 60 mM ionic strength), a PDADMAC-coated capillary and a voltage of -30 kV. These conditions allowed fast separation in only 5 min, with good repeatability, resolution and efficiency. MST was used to quantify the interaction affinity between miRFP670-LIMK1 and TH-257 by successfully measuring the $K_d$ value directly in the cell lysate ($K_d$ = 9 nM). The complementarity of the CE and MST approaches confirms that LIMK1 linked to the miRFP670 tag retains its enzymatic ability and capacity to be inhibited by an allosteric inhibitor. The miRFP670 fluorescent fusion protein was thus revealed as a convenient fluorescent tag that can be used serenely for LIM kinase affinity assays in cell lysates. The submicromolar cellular on-target activity confirmed that TH-257 is suitable as a chemical probe for LIMK1.

## Funding information

This work has been financially supported by Labex SynOrg "ANR-11-LABX-0029", the Région Centre Val de Loire (PhD fellowship of S. Chartier).

## CRediT authorship contribution statement

**Solweig Chartier:** Writing – original draft, Methodology, Investigation, Formal analysis. **Bérengère Claude:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Rouba**
<figure><img src="image_10.png" /><figcaption>Fig. 4. Dose-response curve for the binding interaction between miRFP670-LIMK1 and TH-257 (green) compared with the control, where only miRFP670 was mixed with TH-257 (brown). The concentration of miRFP670-LIMK1 was kept constant at 32 nM, whereas the concentration of TH-257 varied from 30 pM to 1 µM. Each point represents the mean of two capillary readings and the corresponding error bars representing standard deviation values. The dose-response curve was fitted to a one-site binding model provided with MO affinity software to obtain the K<sub>d</sub> value. MST conditions: 37 °C; LED power 20 %; medium MST power. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

**Nasreddine:** Writing – review & editing, Supervision, Resources, Investigation. **Alexandra Launay:** Resources, Investigation. **Marion Martins:** Resources, Investigation. **Villalonga-Rosso Elodie:** Resources, Investigation. **Béatrice Vallée:** Writing – review & editing, Supervision, Resources, Methodology, Investigation. **Muriel Sebban:** Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition, Conceptualization. **Gaël Coadou:** Writing – review & editing, Supervision, Methodology, Investigation. **Reine Nehmé:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors have no interest to declare.

Acknowledgments

The authors would also like to thank the projects CChemBio (FEDER-FSE 2014-2020-EX003677), Techsab (FEDER-FSE 2014-2020-EX011313), QUALICHIM (APR-IA-PF 2021-00149467), RTR Motiv-health (2019-00131403) and the Labex programs SYNORG (ANR-11-LABX-0029) and IRON (ANR-11-LABX-0018-01) for their financial support of ICOA, UMR 7311, University of Orléans, CNRS. The authors would like to thank the University of Rouen Normandy, the Centre National de la Recherche Scientifique (CNRS), INSA Rouen Normandy, the European Regional Development Fund (ERDF), LabexSynOrg (ANR-11-LABX-0029), Carnot Institute I2C, the graduate school for research XL-Chem (ANR-18-EURE-0020XL CHEM), and Region Normandie. The authors would like to thank C. Maffre for the technical support.

Data availability

Data will be made available on request.

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