# Solution-based studies on the contact between the complement receptor 3 ligand-binding domain and simvastatin

Morten Hulbæk Fog, Violaine Hubert, Corinne Sanglar, Marie Martin, Inès Hristovska, Szilvia Karpati, Frédéric Lerouge, Stéphane Parola, Olivier Pascual, Marlène Wiart, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Morten Hulbæk Fog, Violaine Hubert, Corinne Sanglar, Marie Martin, Inès Hristovska, et al. Solution-based studies on the contact between the complement receptor 3 ligand-binding domain and simvastatin. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2025, 1873 (6), pp.141096. 10.1016/j.bbvap.2025.141096. hal-05226565

HAL Id: hal-05226565

https://hal.science/hal-05226565v1

Submitted on 27 Aug 2025

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
# Solution-based studies on the contact between the complement receptor 3 ligand-binding domain and simvastatin

Morten Hulbæk Fog<sup>a</sup>, Violaine Hubert<sup>b</sup>, Corinne Sanglar<sup>c</sup>, Marie Martin<sup>c</sup>, Ines Hristovska<sup>d</sup>, Szilvia Karpati<sup>e</sup>, Frédéric Lerouge<sup>e</sup>, Stéphane Parola<sup>e</sup>, Olivier Pascual<sup>d</sup>, Marlène Wiart<sup>b,f</sup>, Jean-Marc Lancelin<sup>c</sup>, Thomas Vorup-Jensen<sup>a,*</sup>, Florence Guillière<sup>c,**</sup>

## ARTICLE INFO

### Keywords:

Simvastatin

Complement receptor 3

Integrin

Antagonism

I domain

Stroke

## ABSTRACT

Simvastatin is a primary cholesterol-lowering medication, but it has also been reported to possess anti-inflammatory properties. Notably, the CD18 integrins are targets for simvastatin antagonism of ligand binding, which may affect leukocyte adhesion and diapedesis. Lymphocyte-associated antigen (LFA)-1 is inhibited through an allosteric mechanism by binding the lactone form of simvastatin (simvastatin-lac) to a hydrophobic pocket in the major ligand binding domain, the alpha chain I domain. By contrast, crystallographic evidence showed that complement receptor 3 (CR3) is inhibited by simvastatin in its carboxylate form (simvastatin-carbox) chelated by an $Mg^{2+}$ ion in the $\alpha_M$I metal ion-dependent adhesion site (MIDAS). We now report that the affinity ($K_D$) of simvastatin-carbox for the $\alpha_M$I is ~650 µM, which is significantly weaker than the 50 %-inhibitory concentration of simvastatin-lac at 50 µM. The simvastatin-carbox was incapable of inhibiting CR3 binding to iC3b, nor did it exert any neuroprotective or anti-inflammatory effects in the middle cerebral artery occlusion animal model of stroke, unlike what has been reported for simvastatin-lac. From available structural data on the CR3 ligand binding domain in complex with C3d, we suggest that simvastatin-lac makes a critical ternary complex with the ligand binding domain and its ligand before engaging, in its carboxylate form, the MIDAS. In this way, both the LFA-1 and CR3 are antagonized by simvastatin-lac but through fundamentally different mechanisms.

## 1. Introduction

Integrins are cell membrane molecules with important roles in leukocyte adhesion, including guiding the cells to inflammation zones. In diseases with unwanted inflammation, integrins are targeted for therapeutic antibodies blocking the function and lowering the inflammation [1]. Small-molecule drugs have also been applied experimentally as integrin ligand-binding antagonists [2], with some having entered late-stage clinical testing [3,4]. Even so, the complexity of

integrin ligand binding, which includes extensive conformational regulation of the receptor ectodomain, leaves several questions concerning the mechanisms of antagonism [1].

Statins are among the most prescribed drugs worldwide. They block the 3-hydroxy-3-methylglutaryl-S-coenzyme A (HMG-SCoA) reductase (HMGR) with a 50 % inhibition concentration ($IC_{50}$%) in the nanomolar range [5]. As HMG-SCoA analogs, they are binding competitively to the HMGR active site, lowering the cholesterol biosynthesis with as much as 30–50 % *in vivo* [6]. The HMG-SCoA antagonism is only mediated by the

<footnote><sup>a</sup> Biophysical Immunology Laboratory, Dept. of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark</footnote>

<footnote><sup>b</sup> CarMeN Laboratory, Inserm U1060, INRA U1397, INSA Lyon, Université Lyon 1, F-69600, Oullins, France</footnote>

<footnote><sup>c</sup> Université Claude Bernard Lyon 1, CNRS, ISA, UMR5280, 5 rue de la Doua, F-69100 Villeurbanne, France</footnote>

<footnote><sup>d</sup> MeLiS, INSERM U1314 CNRS UMR5284, Université Lyon, Villeurbanne F-69100, France</footnote>

<footnote><sup>e</sup> Laboratoire de Chimie, Université de Lyon, Ecole Normale Supérieure de Lyon, CNRS UMR 5182, Université Lyon 1, Lyon F-69364, France</footnote>

<footnote><sup>f</sup> CNRS, Lyon, France</footnote>

<footnote>* Correspondence to: T. Vorup-Jensen, Biophysical Immunology Laboratory, Dept. of Biomedicine, Aarhus University, Høegh-Guldbergs Gade 10, DK-8000 Aarhus C, Denmark.</footnote>

<footnote>** Correspondence to: F. Guillière, MCF Université Lyon 1, Institut des Sciences Analytiques, UMR CNRS/Université Lyon 1 5280, Equipe BIOSYS, 5 rue de la Doua, F-69100 Villeurbanne, France.</footnote>

<footnote>E-mail addresses: vorup-jensen@biomed.au.dk (T. Vorup-Jensen), florence.guilliere@univ-lyon1.fr (F. Guillière).</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** A: Simvastatin lactone was hydrolyzed to its carboxylate form by saponification in NaOH and ethanol for 4 h at 50 °C with stirring, adapting a protocol from Sadeghi et al. [25]. The pH was later neutralized with HCl, and the sample was lyophilized. The numbering indicated on the molecules was used throughout the study. Simvastatin structures were drawn with Chemicalize (ChemAxon, Budapest, Hungary). B: Assignment of ¹H NMR spectrum of simvastatin-carbox in PBS pH 7.4. The numbering of the signals corresponds to the numbered protons of the simvastatin-carbox (insert).</figcaption></figure>

hydrolyzed form of the molecule, simvastatin-carboxylate (simvastatin-carbox). At the same time, the essentially water-insoluble prodrug, with an intact lactone ring, simvastatin-lactone (simvastatin-lac), is inactive. In addition to the influence on cholesterol synthesis, the pleiotropic effects of statins *in vivo* also include decreasing oxidative stress and inflammation [7]. The anti-inflammatory effects were initially demonstrated in kidney-transplanted patients [8] and, more recently, as part of high-dosage therapy in multiple sclerosis patients [9]. Statin therapy initiated at stroke onset in patients with large vessel occlusion is significantly associated with better functional outcomes and mortality at 90 days, independently of cholesterol-lowering [10]. In animal models of acute ischemic stroke, simvastatin is one of the most effective drugs to improve outcomes [11,12]. The protective effect of simvastatin was related, at least in part, to its immunomodulatory properties, in particular through the attenuation of microglial activity [13–15]. Detailed investigations at the atomic level demonstrated one possible mechanism for the anti-inflammatory part as relating to statins inhibiting lymphocyte function-associated antigen (LFA)-1 (also known as integrin αLβ2 or CD11a/CD18) [16]. This receptor carries a conformationally regulated ligand-binding domain, the alpha-chain I domain (αLI) [17]. The antagonistic effect of Type I statins such as lovastatin and simvastatin derives from the binding of the prodrug to an allosteric site formed by a hydrophobic pocket in the αLI in the closed, ligand-binding inactive conformation [17]. The IC50% for this binding at 2 µM has been a source of debate concerning the physiologic significance [9] since the concentration of simvastatin in human serum is only 3 nM [18]. Simvastatin-carbox did not influence LFA-1 binding [16].

Complement receptor 3 (CR3; also known as integrin αMβ2, Mac-1, complement receptor 3, or CD11b/CD18) is also inhibited by simvastatin. In this case, simvastatin-carbox acts as a competitive antagonist by coordinating a Mg²⁺ ion in the metal ion-dependent adhesion site (MIDAS) of the integrin αM-chain (CD11b) I domain (αMI) [19]. It is well-established that the intact CR3 receptor and αMI bind a broad range of structurally different ligands [1, 20, 21]. Further, within a single protein, several αMI binding sites may be found [22,23]. This finding was explained earlier by a significant affinity of αM1 for carboxylates in glutamate and aspartate side chains [22–24]. Detailed analysis of the inhibition of αM1 by simvastatin-carbox showed the IC50% differed among ligand binding sites with fast and slow dissociation rates, as presented in the complement fragments iC3b and C3d [19]. For a competitive antagonist, one explanation of these activities may involve a

combined interaction between the simvastatin-carbox, the αM1, and the αM1 ligand, together forming a ternary complex [19]. In such a complex, the αM1 ligand may regulate the access of the simvastatin-carbox to the αM1 MIDAS [19]. A better understanding requires insight into the direct binding between αM1 and simvastatin-carbox, but such data are lacking.

Here, in a nuclear magnetic resonance (NMR)-based analysis of the simvastatin-carbox affinity for αM1, we utilized the water solubility of the carboxylate to characterize the binding to CR3 and αM1. As a preliminary step, the ¹H and ¹³C signals from the simvastatin-carbox form were structure-specifically assigned. The binding of αM1 was studied qualitatively and quantitatively in one-dimensional (1D), saturation transfer difference (STD), and transferred nuclear Overhauser effect (NOE) experiments. The affinity for simvastatin-carbox of αM1 is much weaker than the previously determined IC₅₀% for simvastatin-lac inhibition of CR3 binding iC3b. Likewise, we observe a striking inability of the simvastatin-carbox to inhibit CR3 cell adhesion to iC3b. We further show that simvastatin-carbox does not exert protective effects in a mouse model of acute ischemic stroke. Together with earlier published structural data on the αM1:C3d complex [24], our data support the idea that efficient antagonism to CR3 binding is mediated by simvastatin-lac by forming a ternary complex with αM1 and its ligand.

## 2. Materials and methods

### 2.1. *Ethics*

All experimental procedures involving animals and their care were carried out following the European animal use regulations (EEC Council Directive 2010/63/EU, OJ L 276, October 20, 2010). Animal studies were approved by the local review board (“Comité d’éthique pour l’Expérimentation Animale Neurosciences Lyon”: CELYNE; CNREEA no. 42; APAFIS agreement no. #4688).

### 2.2. *Expression and purification of a ligand-binding active αM1 mutant*

A construct encoding the αM1 (residues 127–321) with the mutations C128S/I316G was kindly provided by Prof. Gregers R. Andersen, Aarhus University, and subcloned into pETM-11. BL21 (DE3). *E. coli* cells were transformed by thermal shock and grown at 37 °C in Luria Bertani (LB) broth with 50 µg/mL kanamycin. When the optical density at 600 nm reached 0.6–0.8, the expression of αM1 protein with an N-terminal
hexahistidine tag and a TEV cleavage site was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside overnight at 18 °C. All protein purification steps were performed at 4 °C. Bacterial cells were harvested by centrifugation at 3500 ×g and resuspended in 50 mM HEPES, 300 mM NaCl, 10 mM imidazole, pH 7.5 (lysis buffer) with 1 mM phenyl-methylsulfonyl fluoride. The lysis was achieved by ultrasound, and the cellular extract supernatant was recovered by centrifugation at 13,500 ×g.

The supernatant was loaded onto a Ni²⁺-charged HisTrap column (GE Healthcare), which had been previously equilibrated with the lysis buffer. The α<sub>M</sub>I was eluted with an increasing gradient of 50 mM HEPES, 300 mM NaCl, 500 mM imidazole, pH 7.5 (elution buffer). The histidine tag was then cleaved off by tobacco etch virus (TEV) protease digestion during an overnight dialysis against 2 L of lysis buffer supplemented with 0.5 mM ethylene diamine tetraacetic acid (EDTA). The cleaved α<sub>M</sub>I was recovered in the flow-through from a HisTrap column. Further protein purification was achieved by size-exclusion chromatography in a 120 mL Superdex™ 75 column (GE Healthcare) using 20 mM HEPES, 150 mM NaCl, pH 7.5, as running buffer (Suppl. Fig. 1). Before NMR experiments, the protein was further dialyzed against phosphate buffer saline, pH 7.4.

## 2.3. Simvastatin hydrolysis

Simvastatin-lac was hydrolyzed to its carboxylate form (Fig. 1A) using the protocol adapted from Sadeghi et al. [25]. Approximately 40 mg of simvastatin in its lactone form was dissolved in 1 mL of absolute ethanol. Then, 1.5 mL of 0.1 N NaOH and 7.5 mL of distilled water were added to the solution, which was subsequently incubated at 50 °C for 4 h with stirring. The pH was adjusted to 7.0 by adding 0.1 N HCl. The solution was then frozen and lyophilized. NMR verified the complete activation of simvastatin.

## 2.4. NMR-based characterization of binding between α<sub>M</sub>I and hydrolyzed simvastatin

NMR experiments were performed at 298 K in a Varian 600 MHz NMR spectrometer, equipped with a triple resonance cold probe (¹H, ¹³C, ¹⁵N) enhanced in ¹H and ¹³C, or a Bruker AVANCE III HD 600 MHz NMR spectrometer, equipped with a 5 mm inverse TXI probe H-C/N-D.

Standard 1D and 2D NMR ¹H and ¹³C experiments were used to verify the integrity and purity of simvastatin in its lactone form, to confirm the complete quantitative hydrolysis of simvastatin, and to assign the different NMR signals.

For studying the interaction between α<sub>M</sub>I and simvastatin in an equimolar proportion, a sample containing 50 µM of α<sub>M</sub>I and 50 µM of simvastatin in PBS, pH 7.4, was used. A standard 1D ¹H experiment was performed using a Varian DPFGSE water sequence with a spectral width of 15 ppm and 16,384 complex points. For the transferred NOE experiment [26], NMR samples (550 µL) were prepared with 1 mM activated simvastatin in PBS pH 7.4, 10 % D₂O (v/v), with or without 20 µM α<sub>M</sub>I protein. For the sample with simvastatin and α<sub>M</sub>I, 1 mM MgCl₂ was also added. The Varian dpfgse-noesy sequence was used. The experiments were performed with 1024 and 512 points in direct and indirect dimensions on a spectral width of 15 ppm, and the number of scans was set to 16.

To study the importance of Mg²⁺ in the interaction, 1D and Saturation Transfer Difference (STD) NMR experiments with and without EDTA were done using Varian dpfgse water and dpfgse satxfer sequences with excitation sculpting water suppression sequence on an NMR sample of 550 µL with PBS buffer (pH 7.4), 50 µL D₂O, 0.02 % (w/v) sodium azide, 20 µM α<sub>M</sub>I, 1 mM simvastatin-carbox. Initial experiments were without EDTA, then followed by experiments in 1 mM EDTA. The number of scans was set to 256 for 1D and 512 for STD.

For the determination by STD NMR experiments [26], NMR samples (600 µL) were prepared with PBS (pH 7.4) and 60 µL D₂O, 0.02 % (w/v)

sodium azide, 20 µM α<sub>M</sub>I, simvastatin-carbox (1000, 800, 600, 400, and 200 µM) and 2 mM MgCl₂. The Bruker NMR pulse sequence was stddiffesgp.3, a pseudo 2D version for STD with alternating acquisition of off- and on-resonance spectra. The off-resonance frequency was set to 40 ppm, and the on-resonance frequency to 7.60 ppm. Protein saturation was obtained with a pulse length of 37 ms. The number of scans was 64, with an acquisition time of 1.363 s. Saturation times to obtain STD curves were 0.5, 1, 2, and 3 s. Epitope mapping was performed using a sample with 1000 µM simvastatin, reporting integrals for each signal between the STD and 1D spectra. The percent of implication for each proton in the interaction with α<sub>M</sub>I was calculated as normalized to the signal presenting the biggest I<sub>STD</sub>/I<sub>1D</sub> (≡100 %). All the spectra were processed using the Bruker Topspin or Varian Vnmrj 4.0 software.

To determine the apparent K<sub>D</sub>, the spectral data were analyzed using the procedure given by Angulo et al. [27]. The final titration curves were fitted with OriginPro® 2025 (OriginLab Corporation, Northampton, MA) software using a Hill equation binding isotherm:

$$STD\ AFO = \frac{STD - AFO\ max \cdot (c_{Simvastatin-carbox})^{n_{Hill}}}{(K_D)^{n_{Hill}} + (c_{Simvastatin-carbox})^{n_{Hill}}} \quad (1)$$

where STD AFO is the STD amplification factor, STD-AFO<sub>max</sub> is the maximal amplification factor, c<sub>Simvastatin-carbox</sub> is the concentration of simvastatin-carbox, K<sub>D</sub> is the dissociation equilibrium constant, and n<sub>Hill</sub> is the Hill coefficient. The minimization iterative procedure used a Levenberg-Marquardt algorithm.

Experiments were also made with simvastatin-lac. The poor solubility in water limited the investigations to experiments using final concentrations of 200 µM simvastatin-lac and 20 µM α<sub>M</sub>I protein with 1 mM MgCl₂. To study the influence of the medium, 200 µM simvastatin-lac in PBS, pH 7.4, was prepared from 5 µL of simvastatin lactone at 20 mM in 100 % (v/v) DMSO-d₆ and 495 µL of PBS. A sample of simvastatin-lac at 200 µM in 100 % (v/v) DMSO-d₆ was also prepared. All settings for the NMR experiments with simvastatin-lac were made as for simvastin-carbox.

## 2.5. Cell adhesion assays for quantifying simvastatin antagonism to CR3 ligand binding

The ligand-binding antagonism of the lactone and hydrolyzed forms of simvastatin was tested as described earlier [28] with modifications [19]. Briefly, 96 V-well Costar™ plates (#3897; Corning) were coated with 100 µL of 1 µg/mL iC3b in PBS pH 7.4 in half of the wells. The plate was incubated for at least 1 h at 37 °C. All wells were then blocked in 200 µL 0.05 % (v/v) Tween 20™ (#817072.1000; Merck, lot #K38968072 903) in PBS, pH 7.4, by incubating for at least 1 h at 37 °C. Human CR3-expressing K562 cells were centrifuged at 230 ×g for 5 min and adjusted to a final concentration 0.5 × 10⁶ cells/mL in RPMI (#L0500-500; BioWest) with 0.1 % (v/v) GlutaMAX ×100 (#35050-061; Gibco, lot #2323049) and 2 % (v/v) heat-inactivated FCS (#F7524; Sigma-Aldrich). 2 µL of BCECF-AM (#14562; Sigma-Aldrich, lot #BCCC3061) was added per mL cell suspension. The cells were incubated at 37 °C and 5 % CO₂ for 15 min and washed twice in 5 mM d-glucose, 10 mM HEPES, 0.15 M NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, pH 7.4 (binding buffer). Simvastatin in the lactone form (cat#S6196; Sigma-Aldrich, lot #0000285379), dissolved in DMSO, or the hydrolyzed form (cat#567022; Merck) was prepared in binding buffer with 2 mM MgCl₂ at concentrations of 0, 2, 10, 20, 50, and 100 µM. 50 µL of simvastatin solution was added in triplicate to iC3b-coated and uncoated wells. 50 µL of BCECF-labelled cell suspension was added to all wells and incubated at 37 °C and 5 % CO₂ for 15 min. The plate was centrifuged in sequential steps of 10 ×g, 20 ×g, 50 ×g, 100 ×g, and 200 ×g. After each centrifugation step, the fluorescence at 535 nm (excited at 485 nm) was measured using the VICTOR X5 multimode plate reader (PerkinElmer). The fluorescence counts from 3 technical replicates were averaged (F). To correct for the concomitant influence of simvastatin on
both the binding in wells without ligand ($F_{uncoated}$) and wells with iC3b ($F_{coated}$), as explained earlier [19], we calculated the iC3b-specific binding relative to binding without statin as:

$$iC3b \ Binding\% = 100\% \cdot \frac{F_{uncoated} - F_{coated}}{F_{uncoated,0} - F_{coated,0}} \quad (2)$$

where F is the signal read for experiments with the addition of simvastatin, and $F_0$ is the signal for experiments without statin.

## 2.6. In vivo studies

Experiments were performed in 8-week-old C57Bl/6 male mice (Janvier, France) and 8–12 week-old CX3CR1eGFP/+ transgenic male mice [29]. For surgery and MRI, mice were anesthetized with isoflurane (induction: 3.5 % [v/v]; surgery: 2 %; ISO-VET, Piramal Healthcare, Morpeth, UK). For intravital two-photon microscopy, mice were anesthetized with ketamine (100 mg/kg) and medetomidine (1 mg/kg). Treatment allocation was concealed from experimenters, and all analyses were performed in a blinded manner.

In the neuroprotective study, 18 C57Bl/6 mice were subjected to ischemic stroke at Day 0 (D0). We used the model of middle cerebral artery occlusion (MCAo) by applying a 10 % (w/v) FeCl3-soaked filter-paper strip on the dura mater over the trunk of the distal MCA for 10 min [30]. To alleviate pain, buprenorphine was given subcutaneously at 0.05 mg/kg before surgery, and the wound was covered with lidocaine at the end of the surgery. Animals were randomized into treatment groups (simvastatin-carbox or vehicle, n = 9 per group). Treatments were given twice daily, subcutaneously, at 20 mg/kg of simvastatin [31]. Longitudinal MRI was performed at D0 (one hour after onset and before treatment), D1, and D2 on a 7 T horizontal-bore Bruker Avance II rodent imaging system (Bruker Biospin, Ettlingen, Germany). We acquired diffusion-weighted imaging (DWI) at D0 and T2-weighted imaging at D1 and D2. Acquisition protocol and data analysis are detailed in [31]. Global brain edema was assessed using the ipsilateral:contralateral ratio of hemisphere volumes. Infarct sizes at D1 and D2 were presented as percentages of lesion size measured on DWI at D0 for each animal.

For the neuroinflammation study, 6 CX3CR1eGFP/+ mice that express green fluorescence in macrophages/microglia were subjected to MCAo and treated twice daily with subcutaneous simvastatin-carbox at 20 mg/kg. The contrast agent NanoGd was injected intravenously at D1 at 2 mmol Gd/kg. NanoGd is a hybrid multimodal nanoparticle composed of a core of gadolinium fluoride ($GdF_3$) coated with PEG and functionalized with a Lemke-type fluorophore (LEM-A). This nanoparticle enabled engulfment by macrophages/microglia [32]. Longitudinal MRI was performed at D0 (before treatment and before NanoGd administration) and at D3 (48h post-NanoGd administration), using the same magnet and sequence as above. The volume of signal voids expressed as a percentage of lesion size was quantified as a surrogate marker of macrophage/microglial phagocytic activity [29]. Half of the simvastatin-carbox-treated mice were additionally imaged with intravital two-photon microscopy at D1 and D2 (i.e., immediately and 24 h post-NanoGd administration) through a thinned-skull cortical window.

The image acquisition protocol and data analysis are detailed in [29]. In brief, we used a two-photon microscope (Bruker Ultima) with an Insight 3× laser (Spectra Physics) tuned to 980 nm for simultaneous excitation of both fluorescent proteins, eGFP and LEM-A. A 20× water-immersion lens (0.95 N.A. Olympus). The number of CX3CR1-GFP/+ cells having internalized the nanoparticle and CX3CR1-GFP/+ cells morpho-phenotype (stroma surface, cell perimeter, ramification degree as markers of activation state) were quantified using ImageJ and custom-written MatLab® software (ver. R2018b; MathWorks, Natick, MA). The control groups consisted of 15 untreated CX3CR1eGFP mice, previously reported in [29], that followed the same longitudinal MRI protocol, 5 of which underwent intravital microscopy back-to-back with

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

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 2. A: The 1D $^1$H NMR spectrum of 50 µM simvastatin-carbox in PBS pH 7.4 alone (blue) and mixed with an equimolar amount of $\alpha_{MI}$ (red). All the signals are larger in the presence of the $\alpha_{MI}$, and some signals are shifted (indicated by black arrows). B,C: NOESY spectra for 1 mM simvastatin-carbox in PBS 7.4 alone (B), with 10 µM $\alpha_{MI}$ and 1 mM $MgCl_2$ (C). The diagonal for both spectra is phased in positive (blue). The cross-peaks appear negative (red) for simvastatin alone, whereas simvastatin with $\alpha_{MI}$ and $MgCl_2$ appear positive. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

MRI. One-way ANOVA and Wilcoxon-Mann-Whitney tests were used for statistical analysis.

## 2.7. Statistics

All statistical tests were made with Prism (GraphPad Software, Boston, MA).

# 3. Results

## 3.1. Assignment of $^1$H and $^{13}$C signals

Before conducting interaction studies with $\alpha_{MI}$, the complete activation of simvastatin-lac (Fig. 1A) to simvastatin-carboxylate in sodium
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** A: At the top are 1D ¹H NMR spectra of 1 mM simvastatin with 10 µM α<sub>M</sub>I (blue) and 1 mM EDTA (green). At the bottom, the NMR saturation transfer difference (STD) spectra of 1 mM simvastatin and 10 µM α<sub>M</sub>I (red) and with 1 mM EDTA (violet). The STD signals are lowered when EDTA is added, indicating a decreased affinity of simvastatin for α<sub>M</sub>I. An asterisk ("*") marks 1D ¹H NMR from EDTA. B: Epitope mapping of the simvastatin-carbox (1 mM) interaction with α<sub>M</sub>I (20 µM) and MgCl<sub>2</sub> (2 mM) in PBS, pH 7.4. The percentage of implication for each proton of the simvastatin in the interaction with α<sub>M</sub>I is indicated in red by each proton number. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

salt was verified by NMR. All the NMR signals ¹H and ¹³C were assigned (¹H assignment shown in Fig. 1B).

After this task, the first ¹H NMR spectrum was obtained on an equimolar mixture of αMI and simvastatin-carbox sodium salt. It presented signals of free simvastatin-carbox, which indicated some simvastatin was not bound to αMI, suggesting a relatively weak affinity. The signals of simvastatin were more prominent than in the same spectrum performed in the absence of αMI. Some signals underwent significant variations in their chemical shift (Fig. 2A). This suggested that the free simvastatin concentration was affected by the αMI domain-simvastatin formed complex.

## 3.2. Mg²⁺ contacts with simvastatin

In the nuclear Overhauser effect spectroscopy (NOESY) experiments performed in the absence or the presence of αMI, the NOE signals passed from weakly positive for simvastatin-carbox alone (Fig. 2B) to close to zero for simvastatin-carbox in the presence of αMI (Suppl. Fig. 2), indicating an interaction of simvastatin-carbox with the αMI. If Mg²⁺ ions were added, large negative NOE were observed, indicating a stronger interaction in the presence of Mg²⁺ (Fig. 2C).

To further estimate the importance of Mg²⁺ ions in the interaction of simvastatin with αMI, an initial NMR saturation transfer difference (STD) experiment was performed on a sample with 10 µM αMI and simvastatin in 100-fold excess (1 mM). In a second experiment, MgCl₂ was added to a final concentration of 1 mM. Instead, we added a final concentration of 1 mM EDTA as a control. The first STD spectrum presented signals of simvastatin, confirming that simvastatin interacted with αMI. The intensities of the different signals of simvastatin were higher in the presence of MgCl₂, indicating a stronger interaction in the presence of Mg²⁺ ions, as seen in the transferred NOESY experiment. By contrast, in the presence of EDTA, the signal intensity decreases, indicating a lower interaction (Fig. 3A). The analysis of the first STD spectrum in the presence of Mg²⁺ permitted epitope mapping of the simvastatin-carbox in the interaction with αMI. The three protons of the simvastatin-carbox most implicated in the interaction with the αMI domain were the ethylenic protons of the decalin cycle (H11, H13, H14) (Fig. 3B).

## 3.3. Estimation of the α<sub>M</sub>I solution affinity for simvastatin-carbox and simvastatin-lac

Before determining the direct affinity of simvastatin for αMI, it was

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** The binding between simvastatin and αMI. A: NMR saturation transfer difference (STD) experiments for different concentrations of simvastatin-carbox and the open conformation αMI in the presence of MgCl₂. Points represent the STD amplification factor (STF_AFo) as a function of the simvastatin concentration for the three ethylenic protons H11 (blue, triangle), H13 (black, square), and H14 (red, circle) situated in the decalin cycle of the simvastatin-carbox. These three protons are the most implicated in the interaction with αMI, according to the epitope mapping obtained, and they are isolated without overlapping NMR signals, facilitating the measurement of realistic integrals. Due to the sigmoidal form of the data, the points were fitted with a Hill model (Eq. 1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

necessary to evaluate the optimal concentration of MgCl₂ to add for the best signal-to-noise ratio in STD NMR experiments. MgCl₂ in concentrations of 0 mM, 1 mM, 5 mM, and 10 mM were tested. For concentrations of 5 mM and higher, the intensities of the signals were smaller (Suppl. Fig. 3). We also verified that simvastatin was not directly affected by Mg²⁺. From diffusion-ordered spectroscopy (DOSY) experiments [33,34] (Suppl. Fig. 4) and the apparent diffusion coefficient, it appeared that simvastatin-carbox sodium salt formed aggregates in phosphate buffer without Mg²⁺ ions. These formed micelle-like
<table><caption>**Table 1**

Results of Hill-plot fitting to STD-NMR build-up curves<sup>a</sup>.</caption><thead><tr><th>Proton<sup>b</sup></th><th>Variable</th><th>Fitted value ± SEM</th><th>p<sup>c</sup></th></tr></thead><tbody><tr><td rowspan="3">H13</td><td>STD AFo<sub>Max</sub></td><td>12.22 ± 1.88</td><td>0.0074</td></tr><tr><td>K<sub>D</sub> (µM)</td><td>526.33 ± 71.44</td><td>0.0052</td></tr><tr><td>n<sub>Hill</sub></td><td>3.14 ± 0.95</td><td>0.0500</td></tr><tr><td rowspan="3">H14</td><td>STD AFo<sub>Max</sub></td><td>14.98 ± 4.03</td><td>0.0339</td></tr><tr><td>K<sub>D</sub> (µM)</td><td>610.84 ± 178.76</td><td>0.0419</td></tr><tr><td>n<sub>Hill</sub></td><td>2.15 ± 0.64</td><td>0.0432</td></tr><tr><td rowspan="3">H11</td><td>STD AFo<sub>Max</sub></td><td>22.09 ± 15.02</td><td>0.2378</td></tr><tr><td>K<sub>D</sub> (µM)</td><td>852.30 ± 664.40</td><td>0.2897</td></tr><tr><td>n<sub>Hill</sub></td><td>1.77 ± 0.75</td><td>0.0992</td></tr></tbody></table>

<sup>a</sup> Parameters fitted after 13 iterations with Eq. 1 to paired concentrations of simvastatin-carbox and the STD amplification factor.

<sup>b</sup> Proton numbering according to Fig. 1B.

<sup>c</sup> The *p* value was calculated with two tails, Prob > |*t*|.

structures, consisting of approximately 8 simvastatin molecules at a concentration of 5 mM simvastatin. To know qualitatively if the addition of Mg<sup>2+</sup> could induce or alter any aggregation at lower concentrations of hydrolyzed simvastatin, we recorded the 2D NOESY ¹H-¹H spectrum with simvastatin-carbox in the presence of 2 mM or 10 mM of MgCl<sub>2</sub> in PBS buffer (Suppl. Fig. 5). We observed an inversion of the NOE signals between the NOESY at 2 mM MgCl<sub>2</sub> with positive normal NOEs typical for a small molecule in non-viscous medium, and 10 mM MgCl<sub>2</sub> with NOEs largely negative characteristic of larger molecular-weight aggregates. Since this Mg<sup>2+</sup>-induced simvastatin-carbox aggregation may affect the affinity determination for the α<sub>M</sub>I, the concentration of 2 mM MgCl<sub>2</sub> was chosen for determining K<sub>D</sub>, where simvastatin-carbox is indeed mono-solvated. It also matched the ratio of Mg<sup>2+</sup>: α<sub>M</sub>I used previously by Jensen et al. [19] in SPR experiments to determine the IC<sub>50</sub> of simvastatin for α<sub>M</sub>I complexed to different ligands (iC3b, C3d, ICAM).

To determine K<sub>D</sub> for the interaction between α<sub>M</sub>I and simvastatin-carbox, STD experiments were performed at different concentrations of simvastatin-carbox in the presence of MgCl<sub>2</sub>. Using the approach by Angulo et al. to determine the K<sub>D</sub> value [27], curves were obtained for the three most implicated protons in the interaction (H11, H13, and H14 in Fig. 3B) with good resolution in the NMR spectrum. Rather than a hyperbolic form expected for a classical 1:1 binding model, the curves had a sigmoidal form, suggesting a possible cooperative interaction (Fig. 4). From the application of the Hill equation, the apparent mean K<sub>D</sub> obtained for the three protons (Table 1) was 663 µM at 25 °C with a root-mean-square deviation of 169 µM. The Hill coefficient for the three protons ranged from 2 to 3, indicating positive cooperativity. The fitting was optimal for H13 and H14 but weaker for H11, probably reflecting the lesser engagement of this proton (Fig. 3B).

We also attempted to estimate the binding of simvastatin-lac to α<sub>M</sub>I. The low water solubility [35] of simvastatin-lac required pre-dissolving of the compound in DMSO that cannot guarantee a correct final concentration for STD analysis. In STD experiments together with α<sub>M</sub>I, the 1D spectrum had a poor signal-to-noise ratio when at 200 µM (Suppl. Fig. 6 A) unlike the spectrum for simvastatin-carbox (Suppl. Fig. 6B) confirming the poor solubility of the lactone in PBS. The presence of signals of simvastatin-lac in STD spectrum qualitatively indicates an interaction with α<sub>M</sub>I. To confirm the solubility problem, we compared at 200 µM that the simvastatin-lac alone in 100 % DMSO had a far better signal-to-noise ratio than the pre-dilution at the 1 to 100 factor in PBS (Suppl. Fig. 7 A,B). Taken together, the solubility problems precluded a precise quantification of the affinity of simvastatin-lac for α<sub>M</sub>I; however, STD detected a weak interaction at the supplied high (200 µM) simvastatin-lac concentration.

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** The influence of simvastatin on cell adhesion. A,B: Diagrams showing the normalized inhibition of binding between human CR3-expressing K562 cells and the ligand iC3b as a function of different amounts of simvastatin lactone (A) and simvastatin-carbox (B). The cell-iC3b binding was challenged with increasing centrifugation steps at 10 ×g, 20 ×g, 50 ×g, 100 ×g, and 200 ×g. Results for the application of simvastatin-lac and simvastatin-carbox were plotted as the mean (symbol) and SEM (error bar) from 3 independent experiments.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Simvastatin does not decrease infarct size or edema at the acute stage of stroke. A: Schematic representation of the ischemic brain for a pMCAo mouse, with the occlusion site (black star), the ischemic core (dotted blue lines), and the perilesional area (dotted yellow lines). B: Study design. C,D: Infarct size evaluated by longitudinal MRI (C) and Brain edema evaluated by longitudinal MRI in control (n = 9) and simvastatin (n = 8) mice (D). Bars indicate the mean values, and error bars the SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

### 3.4. Simvastatin-lac and simvastatin-carbox as antagonists of cell-expressed CR3 iC3b binding

We compared the binding to the isolated $\alpha_{MI}$ of simvastatin to the inhibition of cell-membrane-expressed CR3 binding to iC3b in an adhesion assay used previously [19]. In the present study, simvastatin-lac and simvastatin-carbox were compared.

As reported earlier, native simvastatin showed a persistent inhibition over a range of applied centrifugation forces. Inhibition was observable throughout all centrifugation steps. At 50 ×g, the $IC_{50}$ was ~50 µM (Fig. 5A), corroborating the earlier finding [19]. The $IC_{50}$ remained essentially unaltered by increased centrifugation.

The simvastatin-carbox form showed no convincing inhibition of the cell binding, neither at low, nor at high centrifugation. At low spinning force (10 ×g), the binding remained at 100 % in the applied concentration interval of simvastatin-carbox from 2 to 100 µM (Fig. 5B), i.e., the same concentration range used for simvastatin-lac. Increasing the spinning force resulted in a marked offset, down to 50 % binding, even at the lowest applied simvastatin-carbox concentration; however, the level of binding remained essentially unaffected by the increased simvastatin-carbox concentration up to 50 µM. At 100 µM, the addition of simvastatin-carbox appeared to increase binding (Fig. 5B).

### 3.5. In vivo studies

While the protective effects of simvastatin-lac in murine stroke models are well established [11], similar data are lacking on simvastatin-carbox.

To investigate neuroprotective effects, we used the model of middle cerebral artery occlusion (MCAo) [30] (Fig. 6A). Eighteen C57Bl/6 mice were subjected to ischemic stroke at Day 0 (D0), followed by MRI. Treatments with simvastatin or vehicle were administered twice on D1 and D2, followed by MRI (Fig. 6B). One mouse did not have a lesion at

baseline MRI (before treatment) and was therefore excluded from the neuroprotection study. There was no difference between groups in the edema-corrected infarct size (Fig. 5C) or brain edema (Fig. 6D) as assessed by longitudinal MRI.

We also wanted to investigate the impact of simvastatin-carbox on inflammation in the brain, especially the myeloid subset, which is known to be activated in stroke lesions [29,36]. This subset includes microglial cells, a macrophage-like cell type rich in CR3 [1]. For testing of microglial activation, we used a total of six CX3CR1eGFP mice treated with Gd nanoparticles (NanoGd) as contrast agents (Fig. 7A) [29]. However, one simvastatin-treated CX3CR1eGFP mouse died before the end of the longitudinal follow-up and was excluded from the neuro-inflammatory study.

The morphology of microglia was not altered by simvastatin-carbox treatment (data not shown). There was no difference in the percentage of CX3CR1eGFP+ cells that had engulfed NanoGd in the lesion after 24 h or 48 h (Fig. 7B,D) between treated and non-treated mice. In agreement with this finding, there was no difference in NanoGd-induced signal voids on MRI at 72 h between treated and non-treated mice (Fig. 7C,E).

## 4. Discussion

Small-molecule drugs that inhibit the function of integrins have been in focus for more than two decades [2,3]. In addition to the medical interest in integrin antagonists, their use also contributed to unraveling the allosteric control of the large ectodomains and ligand recognition of these receptors [1,2]. NMR obtained important information from studies of the $\alpha_{L1}$ in complex with lovastatin [16,37]. Similar literature on other CD18 integrins has been sparser. The $\alpha_{MI}$ has been characterized earlier by NMR, both as an isolated I domain stabilized in the open, ligand-binding conformation [38] and, more recently, in complex with protein ligands [22]. Nevertheless, insight into the contact with small molecules in solution has been lacking. From mapping the solution
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Simvastatin does not inhibit phagocytosis activity at the acute stage of stroke in dMCAo. A: Study design with time scale in days (D0-D3). Simvastatin-carbox was injected at 1, 6, 25, 31, 49, and 55 h into the experiments. The time points for injection of NanoGd⁺ and imaging are also indicated. B: Percentage of NanoGd⁺/CX3CR1eGFP⁺ cells in the ischemic lesion at Day 1 (D1) and D2 in control (n = 5) and simvastatin (n = 2) mice (Scale bar: 20 µm). C: Percentage of signal voids (dotted blue lines) in the ischemic lesion (dotted yellow lines) at D3 in control (n = 15) and simvastatin (n = 5) mice (Scale bars: 1 mm). In Panels D and E, Bars indicate the mean values and error bars the SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

structure between simvastatin-carbox and the α<sub>M</sub>I and determining the affinity, our findings now suggest a more complex scheme of competitive antagonism than previously thought.

Significant similarities are apparent when comparing the NMR epitope mapping of the interaction between α<sub>M</sub>I and simvastatin-carbox in solution with the crystallographic structure of the complex [19]. In the crystallographic structure, Arg208 and Phe248 in α<sub>M</sub>I are oriented towards the unsaturated region of the decalin bicycle of simvastatin through hydrophobic contacts (Fig. 8A). Likewise, in the STD NMR experiments, ethylenic protons of the simvastatin decalin bicycle are the most involved in the interaction with the α<sub>M</sub>I. Further, the protons of C2 and C3, close to the COO⁻ of the simvastatin-carbox, are also 30 % involved in the interaction. The NMR experiments also confirmed the interaction between simvastatin-carbox and the MIDAS Mg<sup>2+</sup> ion in solution, initially revealed by the crystallographic structure [19]. It leans on past and recent evidence identifying α<sub>M</sub>I as a binder of carboxylated ligands [22,23,38].

A logical question is how simvastatin-lac interacts with the α<sub>M</sub>I. However, simvastatin-lac is poorly soluble in aqueous environments, even in comparison to lovastatin [35,39] used for NMR inhibition studies of α<sub>L</sub>I [16]. In our experiments, simvastatin-lac in PBS produced only noisy 1D spectra when applied to experiments like those for simvastatin-carbox. In consequence, we could not derive a quantitative affinity although the interpretable spectrum for STD experiments at 200 µM showed interaction with a weak affinity in the range of the STD experimental conditions in the order of K<sub>D</sub> ~ 10<sup>-6</sup>-10<sup>-3</sup> M [40].

Likewise, simvastatin-carbox is not an inhibitor of intact CR3 binding to iC3b. Indeed, the influence of simvastatin-carbox in the CR3 cell adhesion assay was difficult to reconcile with any classic scheme for

receptor-ligand binding antagonism. The compound had some, likely non-specific, effects. However, it never quenched the cell adhesion below 50 %. In these respects, the behavior of simvastatin-carbox was different from that of simvastatin-lac. Simvastatin-lac inhibited binding to iC3b in a concentration-dependent manner, with an IC<sub>50</sub> of 50 µM, and reached complete inhibition at 100 µM, as reported earlier [19].

Our findings are important in understanding the mode of simvastatin inhibition of integrin ligand binding. Previously, it was observed that not all types of CR3 ligand binding could be inhibited by simvastatin, e. g., the binding to ICAM-1. In contrast, inhibition of iC3b and C3d binding was robustly achieved [16,19]. However, even for these ligands, the analysis of binding kinetics using the open-conformation α<sub>M</sub>I showed that interactions characterized by slow off-rates were inhibited, while interactions with faster kinetics were essentially untouched by simvastatin-lac [19]. Together, these observations suggest that the ligand for the α<sub>M</sub>I influences susceptibility to simvastatin binding antagonism.

All structural evidence obtained by crystallography [19] and NMR [22] indicates that it is the open-conformation α<sub>M</sub>I that binds ligand carboxylates. This conformation is expected for α<sub>M</sub>I in the context of the intact CR3 receptor when Mn<sup>2+</sup> is added [41] as done in our cell adhesion experiments. Consequently, one would expect the simvastatin-carbox, and not the simvastatin-lac, to act as an antagonist, contrary to what is shown here. The simvastatin-lac has no hydrophobic pocket in the open conformation α<sub>M</sub>I equivalent to the closed conformation α<sub>L</sub>I [16]. As one consequence, there seems to be no prominent site outside the MIDAS [19] for the CR3 contact with simvastatin in our experiments, nor did our NMR experiments support any such possibility. Furthermore, the involvement of the ligand in regulating inhibition suggests
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** A: Complex between the α<sub>M</sub>I domain and simvastatin-carbox shown as a ribbon diagram (top) and molecular lipophilicity potential (MLP) surface map (bottom). MLP was calculated with algorithms [46–48] in UCSF ChimeraX 1.7.1. [49]. The carboxylate group of simvastatin (dark blue) is coordinated with MIDAS (yellow) of the α<sub>M</sub>I domain (green). Simvastatin further interacts with Arg208 (cyan) and Phe246 (orange) through the decalin cycle. Bar denotes lipophilicity. Drawn with PDB ID: 4XW2 [19] B: The binding interface of the ligand C3d (grey). Asp1247 (cyan) is involved in the binding to the α<sub>M</sub>I domain. Hydrophobic patches are seen lining the binding interface. Phe1246 (orange) is highlighted. Drawn with PDB ID: 4M76. C: Complex between the α<sub>M</sub>I domain (green) and C3d (grey). Asp1247 (cyan) is coordinated with MIDAS (yellow). Hydrophobic patches, comprising Phe246 from the α<sub>M</sub>I domain and Phe1246 from C3d (orange), border the binding interface and a possible entry site for simvastatin. Drawn with PDB ID: 4M76 [24]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

that simvastatin-lac may not act as an allosteric antagonist within α<sub>M</sub>I, as it does for α<sub>I</sub>I [16].

To reconcile this evidence, we propose that the route to simvastatin-lac inhibition of α<sub>M</sub>I and intact CR3 involves a ternary complex between the α<sub>M</sub>I, iC3b/C3d, and simvastatin. This possibility was already suggested by Jensen et al. [19]. An analysis of the complex α<sub>M</sub>I:C3d complex [24] shows hydrophobic patches on the C3d ligand are present near the α<sub>M</sub>I MIDAS and in the vicinity of the position of the bound simvastatin-carbox [19] (Fig. 8B,C). We hypothesize that these patches explain the ability of the hydrophobic simvastatin-lac to penetrate CR3 complexes, which contain iC3b and C3d. It also agrees well with the observations from SPR [19]. The reported targeting of slowly releasing interactions between α<sub>M</sub>I and ligand may indicate a preference for antagonizing binding sites involving hydrophobic patches. These are slowly released because of the unfavorable thermodynamics of exposure to water. Furthermore, the patches would not readily permit the charged simvastatin-carbox in its soluble form to make initial contact with the C3d:α<sub>M</sub>I complex, explaining the inability of the soluble compound to antagonize the iC3b binding efficiently. Finally, the lower IC<sub>50</sub> (%) (higher affinity) for simvastatin-lac in cell adhesion studies at 50 µM, compared to the weaker simvastatin-carbox binding to α<sub>M</sub>I at ~650 µM may be explained by the involvement of the ternary complex in the former cases. Somewhere along the path of the formation of the ternary complex to the release of the α<sub>M</sub>I, the simvastatin-lac is hydrolyzed. We have no data to confirm this proposal. Still, examples from the chemistry of Mg compounds show an ability to catalyze simvastatin-lac conversion to simvastatin-carbox [42].

To evaluate the physiological relevance of our findings, we investigated the neuroprotective and immunomodulatory effects of simvastatin-carbox administration at the acute stage of MCAo. This model triggers a robust inflammatory reaction. No effects were noted

compared to control animals using *in vivo* longitudinal multimodal imaging, whether in terms of lesion size, brain edema, or microglial phagocytic activity. The latter measure is especially likely to reflect CR3 activity, at least in part [1]. By contrast, other animal studies have already used simvastatin-lac to obtain protection in the MCAo stroke model [11]. Even if some of the administered simvastatin-lac is spontaneously hydrolyzed into simvastatin-carbox, our study now supports the hypothesis that the immunomodulatory effects of simvastatin may be mediated by the lactone rather than the carboxylate form.

The reported difference between simvastatin-lac and its carboxylate form may help our understanding of simvastatin as an anti-inflammatory agent. For the two known examples of statin-mediated β₂ integrin antagonism, i.e., for integrins LFA-1 [16] and CR3 [19], we can now conclude that for both receptors, it is the hydrophobic simvastatin-lac, which is the antagonistic drug. Surprisingly, however, the mode of action involves both allosteric and competitive mechanisms.

A concern about the physiological relevance has been that the plasma concentration of simvastatin is far below the IC₅₀ reported for these integrins. This situation has routinely led to dismissing simvastatin antagonism to these integrins as relevant in anti-inflammatory effects [9,18]. At least one point emerges from our *in vitro* studies, which may hint at why the *in vivo* effects of simvastatin are difficult to predict based on the free plasma concentration of simvastatin. For the simvastatin-lac, it was suggested that its well-known association with albumin and other proteins [18] brings the molecule into an active mechanism for transferring the compound into cell membrane environments [43]. As recently noted by others [44], this observation questions whether the simvastatin plasma concentration is an adequate indicator of efficacy, at least for the membrane-expressed integrins. The hydrophobic trait of simvastatin-carbox may also play a role in this mechanism. In the absence of added Mg²⁺, we noted a formation of simvastatin-carbox
oligomers, which could be avoided in buffer with 2 mM Mg²⁺, close to the physiologic total concentration of Mg at 1 mM [45]. In the aqueous environment, these contacts are likely driven by the hydrophobic decalin ring in simvastatin-carbox. Because Mg²⁺ is affected by pH and chelated by many macromolecular species in blood, mainly albumin, the resulting free concentration of ionized Mg (Mg²⁺) is only at 0.48–0.65 mM [45]. For this reason, the homomolecular contacts of simvastatin-carbox could, at least on speculative grounds, translate into a Mg²⁺-regulated broader propensity of sustaining intermolecular hydrophobic contacts, i.e., with the decalin ring, even when these contacts initially require the more hydrophobic simvastatin-lac to form. In this scheme, the initial contact between simvastatin-lac and the C3d:αM1 complex is followed by a sustained contact with its hydrolyzed form, the simvastatin-carbox, critical for antagonizing MIDAS-bound ligands according to the earlier made crystallographic study [19]. Our study now opens up new perspectives on how hydrophobic drugs may exert their effects within the context of the flexible chemical environment of large protein complexes.

### CRediT authorship contribution statement

**Morten Hulbæk Fog:** Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis, Data curation. **Violaine Hubert:** Writing – review & editing, Visualization, Investigation, Formal analysis, Data curation. **Corinne Sanglar:** Writing – review & editing, Visualization, Investigation, Formal analysis, Data curation. **Marie Martin:** Writing – review & editing, Investigation, Formal analysis. **Ines Hristovska:** Writing – review & editing, Visualization, Investigation, Formal analysis, Data curation. **Szilvia Karpati:** Writing – review & editing, Visualization, Investigation, Formal analysis, Data curation. **Frédéric Lerouge:** Writing – review & editing, Formal analysis, Conceptualization. **Stéphane Parola:** Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. **Olivier Pascual:** Writing – review & editing, Resources, Investigation, Conceptualization. **Marlène Wiart:** Writing – review & editing, Supervision, Resources, Investigation, Funding acquisition, Conceptualization. **Jean-Marc Lancelin:** Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. **Thomas Vorup-Jensen:** Writing – review & editing, Writing – original draft, Supervision, Resources, Funding acquisition, Formal analysis, Conceptualization. **Florence Guillière:** Writing – review & editing, Writing – original draft, Supervision, Resources, Formal analysis, Conceptualization.

### Funding

This research was funded by the French National Research Agency (ANR) project NanoBrain (grant # ANR-15-CE18-0026-01). It was performed as part of the RHU MARVELOUS (ANR16-RHUS-0009) of University Claude Bernard Lyon 1 (UCBL), within the "Investissements d'Avenir" program. The study also received support from Independent Research Fund Denmark – Medical Science (3101-00104B).

### 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

We thank Prof. Gregers R. Andersen, Aarhus University, for access to plasmid DNA and Ms. Bettina W. Grumsen for excellent technical support.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbamap.2025.141096.

### Data availability

Data will be made available on request.

### References

[1] T. Vorup-Jensen, R.K. Jensen, Structural immunology of complement receptors 3 and 4, Front. Immunol. 9 (2018) 2716.

[2] M. Shimaoka, T.A. Springer, Therapeutic antagonists and conformational regulation of integrin function, Nat. Rev. Drug Discov. 2 (9) (2003) 703–716.

[3] R.J. Slack, S.J.F. Macdonald, J.A. Roper, R.G. Jenkins, R.J.D. Hatley, Emerging therapeutic opportunities for integrin inhibitors, Nat. Rev. Drug Discov. 21 (1) (2022) 60–78.

[4] D. Xu, C. Yang, W. Cao, X. Zhang, S. Yang, X. Shen, J. Xu, H. Yu, Platelet glycoprotein IIb/IIIa antagonists in ischemic stroke patients without endovascular therapy: a meta-analysis, Pharmacotherapy 44 (8) (2024) 675–691.

[5] M.S. Brown, J.R. Faust, J.L. Goldstein, I. Kaneko, A. Endo, Induction of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in human fibroblasts incubated with compactin (ML-236B), a competitive inhibitor of the reductase, J. Biol. Chem. 253 (4) (1978) 1121–1128.

[6] E.S. Istvan, J. Deisenhofer, Structural mechanism for statin inhibition of HMG-CoA reductase, Science 292 (5519) (2001) 1160–1164.

[7] J.K. Liao, U. Laufs, Pleiotropic effects of statins, Annu. Rev. Pharmacol. Toxicol. 45 (2005) 89–118.

[8] M.K. Jain, P.M. Ridker, Anti-inflammatory effects of statins: clinical evidence and basic mechanisms, Nat. Rev. Drug Discov. 4 (12) (2005) 977–987.

[9] D. Chan, S. Binks, J.M. Nicholas, C. Frost, M.J. Cardoso, S. Ourselin, D. Wilkie, R. Nicholas, J. Chataway, Effect of high-dose simvastatin on cognitive, neuropsychiatric, and health-related quality-of-life measures in secondary progressive multiple sclerosis: secondary analyses from the MS-STAT randomised, placebo-controlled trial, Lancet Neurol. 16 (8) (2017) 591–600.

[10] K. Uchida, S. Yoshimura, H. Imamura, N. Ohara, N. Sakai, K. Tanaka, H. Yamagami, Y. Matsumoto, M. Takeuchi, T. Morimoto, Effect of statin administration after onset of acute ischemic stroke with large vessel occlusion: insights from RESCUE-Japan Registry 2, J. Am. Heart Assoc. 9 (23) (2020) e017472.

[11] B. Christophe, M. Karatela, J. Sanchez, J. Pucci, E.S. Connolly, Statin therapy in ischemic stroke models: a meta-analysis, Transl. Stroke Res. 11 (4) (2020) 590–600.

[12] L. Garcia-Bonilla, M. Campos, D. Giralt, D. Salat, P. Chacon, M. Hernandez-Guillamon, A. Rosell, J. Montaner, Evidence for the efficacy of statins in animal stroke models: a meta-analysis, J. Neurochem. 122 (2) (2012) 233–243.

[13] M.A. Churchward, K.G. Todd, Statin treatment affects cytokine release and phagocytic activity in primary cultured microglia through two separable mechanisms, Mol. Brain 7 (2014) 85.

[14] P. Sun, M. Hernandez-Guillamon, M. Campos-Martorell, A. Simats, J. Montaner, M. Unzeta, M. Sole, Simvastatin blocks soluble SSAO/VAP-1 release in experimental models of cerebral ischemia: possible benefits for stroke-induced inflammation control, Biochim. Biophys. Acta Mol. basis Dis. 1864 (2) (2018) 542–553.

[15] X. Zheng, Y. Liao, J. Wang, S. Hu, G.R. Rudramurthy, M.K. Swamy, K.C. Rohit, Y. Wang, The Antineuroinflammatory effect of simvastatin on lipopolysaccharide activated microglial cells, Evid. Based Complement. Alternat. Med. 2018 (2018) 9691085.

[16] G. Weitz-Schmidt, K. Welzenbach, V. Brinkmann, T. Kamata, J. Kallen, C. Bruns, S. Cottens, Y. Takada, U. Hommel, Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site, Nat. Med. 7 (6) (2001) 687–692.

[17] M. Shimaoka, J. Takagi, T.A. Springer, Conformational regulation of integrin structure and function, Annu. Rev. Biophys. Biomol. Struct. 31 (2002) 485–516.

[18] L. Bjorkhem-Bergman, J.D. Lindh, P. Bergman, What is a relevant statin concentration in cell experiments claiming pleiotropic effects? Br. J. Clin. Pharmacol. 72 (1) (2011) 164–165.

[19] M.R. Jensen, G. Bajic, X. Zhang, A.K. Laustsen, H. Koldso, K.K. Skeby, B. Schiott, G. R. Andersen, T. Vorup-Jensen, Structural basis for simvastatin competitive antagonism of complement receptor 3, J. Biol. Chem. 291 (33) (2016) 16963–16976.

[20] T. Vorup-Jensen, On the roles of polyvalent binding in immune recognition: perspectives in the nanoscience of immunology and the immune response to nanomedicines, Adv. Drug Deliv. Rev. 64 (15) (2012) 1759–1781.

[21] C.J. Sommer-Pluss, C. Leiggener, E. Nikci, R.V. Mancuso, S. Rabbani, C. Lamers, D. Ricklin, Determining ligand binding and specificity within the beta(2)-integrin family with a novel assay platform, Biomolecules 15 (2) (2025).

[22] H. Nguyen, N.P. Podolnikova, T.P. Ugarova, X. Wang, Alpha(M)-domain of integrin Mac-1 binds the cytokine pleiotrophin using multiple mechanisms, Structure 32 (8) (2024) 1184–1196 e4.

[23] T. Vorup-Jensen, C.V. Carman, M. Shimaoka, P. Schuck, J. Svitel, T.A. Springer, Exposure of acidic residues as a danger signal for recognition of fibrinogen and
other macromolecules by integrin alphaXbeta2, Proc. Natl. Acad. Sci. USA 102 (5) (2005) 1614–1619.

[24] G. Bajic, L. Yatime, R.B. Sim, T. Vorup-Jensen, G.R. Andersen, Structural insight on the recognition of surface-bound opsonins by the integrin I domain of complement receptor 3, Proc. Natl. Acad. Sci. USA 110 (41) (2013) 16426–16431.

[25] M.M. Sadeghi, M. Collinge, R. Pardi, J.R. Bender, Simvastatin modulates cytokine-mediated endothelial cell adhesion molecule induction: involvement of an inhibitory G protein, J. Immunol. 165 (5) (2000) 2712–2718.

[26] O. Cala, F. Guilliere, I. Krimm, NMR-based analysis of protein-ligand interactions, Anal. Bioanal. Chem. 406 (4) (2014) 943–956.

[27] J. Angulo, P.M. Enriquez-Navas, P.M. Nieto, Ligand-receptor binding affinities from saturation transfer difference (STD) NMR spectroscopy: the binding isotherm of STD initial growth rates, Chemistry 16 (26) (2010) 7803–7812.

[28] M. Weetall, R. Hugo, C. Friedman, S. Maida, S. West, S. Wattanasin, R. Bouhel, G. Weitz-Schmidt, P. Lake, A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates, Anal. Biochem. 293 (2) (2001) 277–287.

[29] V. Hubert, I. Hristovska, S. Karpati, S. Benkeder, A. Dey, C. Dumot, C. Amaz, N. Chounlamountri, C. Watrin, J.C. Comte, F. Chauveau, E. Brun, P. Marche, F. Lerouge, S. Parola, Y. Berthezene, T. Vorup-Jensen, O. Pascual, M. Wiart, Multimodal imaging with NanoGd reveals spatiotemporal features of neuroinflammation after experimental stroke, Adv. Sci. (Weinh.) 8 (17) (2021) e2101433.

[30] H. Karatas, S.E. Erdener, Y. Gursoy-Ozdemir, G. Gurer, F. Soylemezoglu, A. K. Dunn, T. Dalkara, Thrombotic distal middle cerebral artery occlusion produced by topical FeCl(3) application: a novel model suitable for intravital microscopy and thrombolysis studies, J. Cereb. Blood Flow Metab. 31 (6) (2011) 1452–1460.

[31] C. Potey, T. Ouk, O. Petrault, M. Petrault, V. Berezowski, J. Salleron, R. Bordet, S. Gautier, Early treatment with atorvastatin exerts parenchymal and vascular protective effects in experimental cerebral ischaemia, Br. J Pharmacol. 172 (21) (2015) 5188–5198.

[32] S. Karpati, V. Hubert, I. Hristovska, F. Lerouge, F. Chaput, Y. Bretonniere, C. Andraud, A. Banyasz, G. Micouin, M. Monteil, M. Lecouvey, M. Mercey-Ressejac, A.K. Dey, P.N. Marche, M. Lindgren, O. Pascual, M. Wiart, S. Parola, Hybrid multimodal contrast agent for multiscale in vivo investigation of neuroinflammation, Nanoscale 13 (6) (2021) 3767–3781.

[33] G.A. Morris, Diffusion-ordered spectroscopy, in: R.K. Harris (Ed.), Encycl Magn Reson, John Wiley & Sons, Chichester, 2009.

[34] K.F. Morris, C.S. Johnson, Diffusion-ordered 2-dimensional nuclear-magnetic-resonance spectroscopy, J. Am. Chem. Soc. 114 (8) (1992) 3139–3141.

[35] A. Bartkowiak, D. Matyszewska, A. Krzak, M. Zaborowska, M. Broniatowski, R. Bilewicz, Incorporation of simvastatin into lipid membranes: why deliver a statin in form of inclusion complex with hydrophilic cyclodextrin, Colloids Surf. B: Biointerfaces 204 (2021) 111784.

[36] V. Desestret, J.C. Brisset, S. Moucharrafie, E. Devillard, S. Nataf, J. Honnorat, N. Nighoghossian, Y. Berthezene, M. Wiart, Early-stage investigations of ultrasmall

superparamagnetic iron oxide-induced signal change after permanent middle cerebral artery occlusion in mice, Stroke 40 (5) (2009) 1834–1841.

[37] R.N. Gursoy, D.S. Jois, T.J. Siahaan, Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1,12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH, J. Pept. Res. 53 (4) (1999) 422–431.

[38] H. Nguyen, T. Jing, X. Wang, The Q163C/Q309C mutant of alphaMI-domain is an active variant suitable for NMR characterization, PLoS One 18 (1) (2023) e0280778.

[39] A.T. Serajuddin, S.A. Ranadive, E.M. Mahoney, Relative lipophilicities, solubilities, and structure-pharmacological considerations of 3-hydroxy-3-methylglutaryl-coenzyme a (HMG-CoA) reductase inhibitors pravastatin, lovastatin, mevastatin, and simvastatin, J. Pharm. Sci. 80 (9) (1991) 830–834.

[40] S. Walpole, S. Monaco, R. Nepravishta, J. Angulo, STD NMR as a technique for ligand screening and structural studies, in: A.J. Wand (Ed.), Methods in Enzymology, Academic Press, Cambridge, MA, 2019, pp. 424–451.

[41] M. Sen, A.C. Koksal, K. Yuki, J. Wang, T.A. Springer, Ligand- and cation-induced structural alterations of the leukocyte integrin LFA-1, J. Biol. Chem. 293 (17) (2018) 6565–6577.

[42] G. Kato, H. Mitome, Y. Miyauchi, S. Takeda, Y. Toyota, N. Hidaka, M. Tanaka, K. Akira, A study on the chemical stability of cholesterol-lowering drugs in concomitant simple suspensions with magnesium oxide, J. Pharm. Health Care Sci. 9 (1) (2023) 32.

[43] K. Yamasaki, V.T. Chuang, T. Maruyama, M. Otagiri, Albumin-drug interaction and its clinical implication, Biochim. Biophys. Acta 1830 (12) (2013) 5435–5443.

[44] L. Prieto Garcia, A. Vildhede, P. Nordell, C. Ahlstrom, A.B. Montaser, T. Terasaki, H. Lennernas, E. Sjogren, Physiologically based pharmacokinetics modeling and transporter proteomics to predict systemic and local liver and muscle disposition of statins, CPT Pharmacometrics Syst. Pharmacol. 13 (6) (2024) 1029–1044.

[45] J.B. Bouillon-Minois, L. Khaled, F. Vitte, L. Miraillet, R. Eschalier, M. Jabaudon, V. Sapin, L. Derault, S. Kahouadji, M. Brailova, J. Durif, J. Schmidt, F. Moustafa, B. Pereira, E. Futier, D. Bouvier, Ionized magnesium: interpretation and interest in atrial fibrillation, Nutrients 15 (1) (2023).

[46] P. Broto, G. Moreau, C. Vandycke, Molecular structures: perception, autocorrelation descriptor and SAR studies. System of atomic contributions for the calculation of the n-octanol/water partition coefficients, Eur. J. Med. Chem. 19 (1984) 71–78.

[47] A.K. Ghose, V.N. Viswanadhan, J. Wendoloski, Prediction of hydrophobic (Lipophilic) properties of small organic molecules using fragmental methods: an analysis of ALOGP and CLOGP methods, J. Phys. Chem. A 102 (1998) 3762–3772.

[48] M. Laguerre, M. Saux, J.P. Dubost, A. Carpy, MLPP: a program for the calculation of molecular lipophilicity potential in proteins, Pharm. Pharmacol. Commun. 3 (1997) 217–222.

[49] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, UCSF Chimera-a visualization system for exploratory research and analysis, J. Comput. Chem. 25 (13) (2004) 1605–1612.