Internalization of methotrexate conjugates by folate receptor-Î±

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Internalization of methotrexate conjugates by folate receptor-# Eugénia Nogueira, Marisa P. Sárria, Nuno Gonçalo Azoia, Egipto Ferreira Antunes, Ana Loureiro, Diana Guimarães, Jennifer Noro, Alexandra Rollett, Georg M. Guebitz, and Artur Cavaco-Paulo Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00607 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on November 20, 2018

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Biochemistry

1

Internalization of methotrexate conjugates by folate

2

receptor-α

3

Eugénia Nogueira, § ‡ Marisa P. Sárria, §,¥ †‡ Nuno G. Azoia, § ψ‡ Egipto Antunes, §Ana Loureiro,

4

§

5

Paulo§ *

6

§

7

Portugal

8

¥

9

of Minho, Campus of Gualtar, 4710-057 Braga, Portugal

Diana Guimarães, § Jennifer Noro, § Alexandra Rollett,Փ Georg Guebitz,Փ and Artur Cavaco-

Centre of Biological Engineering, University of Minho, Campus of Gualtar, 4710-057 Braga,

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University

10

Փ

11

Vienna, A-3430 Tulln, Austria

Institute for Environmental Biotechnology, University of Natural Resources and Life Sciences,

12

13

ABSTRACT

14

The folate antagonist methotrexate is a cytotoxic drug used in the treatment of several cancer types.

15

Methotrexate entry into the cell is mediated by two main transport systems: the reduced folate

16

carrier and membrane-associated folate receptors. These transporters differ considerably in their

17

mechanism of (anti)folate uptake, substrate specificity and tissue specificity. Although the

ACS Paragon Plus Environment

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mechanism of action of the reduced folate carrier is fairly well-established, that of the folate

2

receptor has remained doubtful. The development of specific folate receptor-targeted antifolates

3

would be accelerated if additional mechanistic data becomes available. In this work, we used two

4

fluorescent-labeled conjugates of methotrexate, differently linked at the terminal groups, to clarify

5

the uptake mechanism by the folate receptor-α. The results demonstrate the importance of

6

methotrexate amino groups in the interaction with the folate receptor-α.

7 8

Table of Contents

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Biochemistry

1

INTRODUCTION

2

The folate antagonist methotrexate (MTX) was introduced in oncology as a cytotoxic

3

antiproliferative agent that has been used in the treatment of various cancer types for decades. 1 It

4

is an antimetabolite that inhibits dihydrofolate reductase (DHFR), which is an enzyme involved in

5

the biosynthetic pathway of nucleotides, and MTX is therefore highly toxic to rapidly dividing

6

cancer cells.2

7

The first pivotal step in the cellular pharmacology of MTX is its entry into the cell, which can be

8

mediated by two general transport systems: the reduced folate carrier (RFC) and membrane-

9

associated

folate

receptors

(FRs).

These

transport

routes

are

physiologically

and

10

pharmacologically relevant for immunocompetent cells because they facilitate the uptake of

11

natural reduced folate cofactors and folate antagonists, such as MTX.3 The RFC and FRs differ

12

considerably in their mechanism of (anti)folate uptake (transmembrane carrier versus

13

endocytosis), substrate specificity (low-affinity folic acid/high-affinity MTX versus high-affinity

14

folic acid/low-affinity MTX), and tissue specificity (constitutive versus restricted expression)

15

(Figure 1).

16


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Figure 1. Schematic representation of folate receptor-mediated endocytosis and methotrexate

3

influx. The folic acid has a 100-200 times greater affinity to the folate receptor than to the reduced

4

folate carrier. Inversely, the reduced folate carrier-binding affinity to methotrexate is higher.

5 6

The membrane-spanning anion transporter RFC, has a constitutive expression in essentially all

7

human tissues, providing a high-affinity bi-directional exchange mechanism for reduced-folate

8

cofactors and MTX (Km of 1-10 μM). Inversely, the RFC-binding affinity to folic acid is low (Ki

9

of 200-400 μM).4-5

10

A second folate transport mechanism is mediated by FR, a particulate form of folate binding

11

proteins, which are anchored to the cell membrane by glycosylphosphatidylinositol (GPI) residues.


4

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Biochemistry

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FRs have a mass of 38–40 kDa and are coded by two genes (FR α and β) with differential tissue

2

expression. The α- isoform of FR is overexpressed in specific types of cancer (e.g. ovarian

3

cancer),6 while the β isoform on activated macrophages.7 The third member of this gene family, γ-

4

type, is a secretory protein, from hematopoietic cells, due to the lack of signal for GPI attachment.8

5

Unlike RFC, FR exhibits a much greater affinity for folic acid and 5-methyltetrahydrofolate (Kd

6

1–10 nM), but lower for other reduced folates and MTX (Kd 10–300 nM). FR also have different

7

properties in terms of energy, ion, and pH dependence.4 Folate binding to FR is decreased after

8

energy depletion and in absence of chloride anion, but does not diminish until the pH falls below

9

5.0.9

10

The functional activity of the RFC-mediated cellular traffic of antifolates with antitumor activity

11

in vivo is fairly well-established.5 On the contrary, the exact role and mechanism of action of the

12

FRα has remained unclear.10 In 2013, the structure-activity relationship for molecular (high-

13

affinity) recognition of folate and folate-conjugates by FRα was disclosed.11 It was demonstrated

14

that FRα has a globular structure stabilized by eight disulphide bonds and contains a folate-binding

15

pocket formed of conserved residues (across all receptor subtypes).11 The molecular model unveils

16

that glutamate moiety is solvent-exposed and projects from the receptor’s pocket entrance,

17

enabling cargo attachment without obstructing FRα binding.11 This structure helps explain the low-

18

MTX binding affinity to FRα, since it shows that there is a steric impediment of a specific amino

19

group that interferes with the binding to the receptor.

20

Knowing the molecular recognition details, and considering that MTX is transported into cells via

21

the FR rather than through facilitative transporters (expressed in normal tissues), damage to normal

22

cells is not expectable.12 Therefore, FRα-targeted therapy should focus on overcoming affinity and

23

specificity to MTX, over RFC. This could be achieved through engineering of molecules that take


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advantage of the many solvent-filled cavities that surround ligands in the folate receptor binding

2

cleft.12

3

In the present study, we elucidate the uptake mechanism of MTX by the FRα. Two fluorescently

4

labelled conjugates of MTX, differently linked at the terminal groups, were used for cellular

5

uptake, and molecular docking and molecular dynamics simulations were performed to validate

6

their findings.

7 8

METHODS

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Fluorescent labeling of MTX

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MTX was labeled with the amino-reactive fluorescent dye Red Mega 520 NHS-ester (fluorescent

11

red), as previously described for folic acid.13 Briefly, 140 μl of MTX-solution (3.7 mg/ml in a 3:1

12

mixture of 50 mM bicarbonate buffer, pH 9 and DMF) were mixed with 28 μl of fluorescent red

13

solution (20 mg/ml in DMF), and 200 μl of DMF were added. The mixture was shaken for 4 h at

14

25°C under light protection. The product was purified by size exclusion chromatography using a

15

HiTrap Desalting column (GE Healthcare Europe GmbH, Vienna, Austria) installed on an Äkta

16

Purifier system (Amersham Pharmacia Biotech, Uppsala, Sweden), with 50 mM potassium

17

phosphate plus 100 mM NaCl, pH7 as eluent, at the flow rate of 1 ml/min, to remove excess

18

fluorescent red. Fractions, which showed absorbance at 520 nm for fluorescent red, and 368 nm

19

for MTX, were collected. Electro spray ionization (ESI) was performed in a mass detector Thermo

20

Finnigan LxQ (Linear Ion Trap). Mass detector susceptible of analysis in full scan mode, SIM and

21

MS/MS with negative ionization. Mass spectra range was between 50 and 2000. Capillary voltage

22

was used as 29 V. MTX concentrations were determined photometrically by comparison with a


6

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Biochemistry

1

calibration curve. According to manufacturer instructions (Sigma-Aldrich Co.; Saint Louis, MO,

2

USA), fluorescent Mega dyes have a large stoke’s shift among excitation and emission maxima,

3

compatible to the same excitations conditions (light source or filter arrangement) than the

4

fluorescent green fluorescein isothiocyanate (FITC). As so, the commercial fluorescent green

5

conjugate of MTX (Molecular Probes® ‫ ׀‬Life Technologies), linked through the carboxyl groups,

6

was further considered for the cellular uptake experiments.

7 8

Cell culture conditions

9

The epithelial cancer cell line KB (ATCC®CCL-17™), and the human melanoma cells MDA-

10

MB-435S (ATCC®HTB-129™) were obtained from the American Type Culture Collection. KB

11

cell line was maintained in Roswell Park Memorial Institute (RPMI)-1640 Medium (without folic

12

acid) containing 2 mM of L-glutamine, 2 mg/mL sodium bicarbonate supplemented with 10%

13

(v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin solution. MDA-MB-435S

14

cell line was maintained in Dulbecco's modified Eagle medium (DMEM) containing 4 mM L-

15

glutamine, 4.5 g/L glucose and 1.5 g/L sodium bicarbonate supplemented with 10% (v/v) of FBS

16

and 1% (v/v) penicillin/streptomycin solution. Cells were maintained at 37 ºC in a humidiﬁed

17

atmosphere of 5% CO2. Cells were routinely subcultured over 2/3 days.

18 19

FR confirmation by real-time qPCR

20

Cell lysates of different-origin cancer lines were collected and total RNA was extracted using the

21

“SV Total RNA Isolation System”, according to the manufacture’s protocol (Promega


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Corporation, USA). Following column elution with 30 μl of RNase-free water, RNA concentration

2

was determined by measuring the absorbance at 260 nm in a NanoDrop ND-1000

3

spectrophotometer (A260=1↔40μg/ml) (Nanodrop Technologies, USA). The ratio A260/ A280

4

was calculated to estimate RNA purity. Reverse transcription was performed using the “iScriptTM

5

cDNA synthesis kit” (Bio-Rad Laboratories, USA). Unique blended oligo(dT) and random

6

hexamer primers were incubated according to the manufacturer’s protocol as follows: 5 min at

7

25ºC, 60 min at 42ºC, 5 min at 85ºC and hold at 4ºC. For confirmation of FRα and RFC expression

8

levels, TaqMan® probe-based Gene Expression assays (Applied Biosystems®, Spain) were

9

considered: ID Hs01124177_m1 and Hs00953345_m1, respectively. Real-time qPCR reactions

10

were performed in CFX96™ Real-Time PCR Detection System, C1000TM thermal cycler (Bio-

11

Rad Laboratories, USA). The amplification process used the following conditions: UNG

12

incubation 50 ºC 2 min, polymerase activation 95 °C 10 min, denaturation 95 °C 15 s,

13

annealing/extension 60 °C 60 s, for 50 cycles (data collection). A stable endogenous control gene,

14

the Cyclin-dependent kinase Inhibitor 1A, CDKN1A (TaqMan® assays ID Hs00355782_m1;

15

Applied Biosystems®, Spain), was used for normalization of the expression values.

16 17

Uptake experiments

18

The FRα positive cells (KB cancer cell line), and MDA-MB-435S cells (negative control) were

19

harvest from the culture vessel during their S-phase (i.e. until a monolayer, with confluence greater

20

than 60%, was obtained), and seeded at a density of 100 000 cells/mL (24-wells microplates) for

21

the uptake experiments. After an overnight period to ensure optimal adhesion, the culture medium

22

was removed, and adherent cells were washed twice with acidic phosphate-buffered saline (pH


8

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Biochemistry

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3.5) to remove bound folic acid. The MTX conjugates were diluted in Hanks' balanced salt solution

2

(folate free), at a non-toxic (data not shown) concentration of 6 μM (adjusted for antifolate

3

concentration), and incubated in cancer cells during 4 h. The same experiment was repeated using

4

media containing 113 μM free folic acid (Sigma) and calcium folinate (TCI) to determine the effect

5

of FR and RFC blockade, respectively. The cultures were maintained under a humidified

6

atmosphere containing 5% CO2, in a 37ºC incubator. The internalized concentration of MTX

7

conjugates was determined, considering the fluorescence intensity measured (Perkin Elmer LS-

8

50B Luminescence Spectrometer) upon lysis of the exposed-cells. All values were corrected by

9

the ratio intensity/molarity, according calibration curves of fluorescent-labeled conjugates.

10 11

Molecular Docking Simulations

12

The computational models of the FRα, RFC and MTX have different origins. The FRα model

13

corresponds to the 4KM6 entry on RCSB protein databank,14 that is an X-ray diffraction of human

14

FRα (FOLR1) at acidic pH. The software Modeller (https://salilab.org/modeller)15 was used to

15

obtain a 3D model of RFC protein, through homology with a structure of a transmembranar glucose

16

transporter obtained by X-ray diffraction (4ZW9 entry on RSCB protein databank),16 due to the

17

non-existence of a full crystallographic model of RFC. Finally, the MTX structure was obtained

18

using

19

(https://atb.uq.edu.au/)17 using AM1 optimized geometry and MOPAC charges.

20

The models of FRα, RFC and MTX were preprocessed with Autodock tools18 to correct the

21

charges, the hydrogens and the bonds flexibility when needed. Later, Autodock Vina19 was used

22

to perform the docking simulations between the FRα and MTX, as well as between RFC and MTX

the

“Automated

Topology

Builder

(ATB)

and

Repository”

online

server


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to evaluate the binding affinity between them. All docking calculations were performed with one

2

box of 5x5x5 nm centered on the FR active site and the exhaustiveness was varied between 80 and

3

800.

4 5

Molecular Dynamics Simulations

6

The simulations were performed with the GROMACS 4.6.520 package using the GROMOS 54A7

7

force field.21-22 The initial configurations of the protein-MTX conjugates were the result of docking

8

simulations with higher affinity binding energies. These structures were solvated and simulated

9

during a few nanoseconds (~10ns). The bonds lengths of the proteins were constrained with

10

LINCS23 and those for water with SETTLE.24 Nonbonded interactions were calculated using a

11

twin-range method, with short and long-range cutoffs of 0.8 and 1.4 nm, respectively. Neighbor

12

searching was carried out up to 1.4 nm and updated every two steps. A time step of integration of

13

2 fentoseconds was used. A reaction field correction for the electrostatic interactions was applied

14

using a dielectric constant of 54.25 The single point charge model26 was used for water molecules.

15

The initial systems were energy minimized for 2000 steps using the steepest descent method, with

16

all heavy atoms harmonically restrained using a force constant of 10³ kJ/mol nm².

17

The systems were initialized in the canonical ensemble (NVT) for 50 picoseconds, with all heavy

18

atoms harmonically restrained using a force constant of 10³ kJ/mol nm². The simulation was then

19

continued for picoseconds in the isothermal–isobaric ensemble (NPT), with the heavy atoms

20

harmonically restrained with the same force constant. Finally, for allowing the equilibration of the

21

system properties, the simulations were further extended in the NPT ensemble with position

22

restrains applied only to the α-carbons. Pressure control was implemented using the Berendsen


10

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Biochemistry

1

barostat,27 with a reference pressure of 1 bar, 0.5 picoseconds of relaxation time, and isothermal

2

compressibility of 4.5x10⁻⁵ bar⁻¹. Temperature control was set using the V-rescale27-28 thermostat

3

at 300 K with temperatures coupling constants of 0.025 picoseconds in the first two initialization

4

steps and with 0.1 picoseconds for the rest of the simulations. At least three replica systems were

5

simulated for each condition, from 50 nanoseconds using different initial velocities taken from a

6

Maxwell–Boltzman distribution at 300k. GROMACS tools were used to evaluate several

7

properties of the systems, such as the pressure, temperature, surface accessible solvent area,

8

providing insights on the correct modeling of the proteins-MTX interaction.

9

In addition, pulling simulations were performed with FR-MTX and RFC-MTX conjugates, with a

10

constant force of 1000 KJ/mol.nm² and with 0.1 nm/ns rate applied to the MTX molecule. The

11

force applied in the MTX molecule oblige the molecule to exit from the tested proteins active-

12

sites. The MTX had its carboxyl groups termination facing the solvent in both cases. Later

13

umbrella sampling methodology29 was applied to calculate the potential of mean force for the

14

process of MTX molecules exiting the FRα and RFC proteins.

15 16

RESULTS AND DISCUSSION

17

In this work, two fluorescently labeled conjugates of MTX, differently linked at the terminal

18

groups (Figure 2), were considered for cellular uptake. Since high levels of FRα were reported in

19

a wider spectrum of epithelial origin tissues,30 human epithelial adenocarcinoma cells of the

20

mammary gland were selected for exposure to the antifolate conjugates, in comparison to a

21

negative control. The FRα expression levels of the different-origin cancer cells were quantified

22

using a standardized real-time qPCR detection system. The concentration of the conjugates


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internalized (adjusted for MTX concentration) was determined, considering the fluorescence

2

measured in the lysates of the exposed cells. To ascertain FRα “targetability”, molecular docking

3

simulations of the complex FRα/MTX were performed.

4

5

6 7

Figure 2. The (most probable) molecular structures of the tested conjugates (A) RedMega-HN-

8

MTX-COOH and (B) H2N-MTX-CO-FITC. Fluorescent red dye can also be linked to the amino

9

group marked with a circle, but since the conjugate was purified by size-exclusion

10

chromatography, the occurrence of a 2:1 (Mega dye:MTX) conjugate is excluded, as it would have

11

a larger size and therefore a shorter retention time. The MTX-Red Mega 520 conjugate was


12

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Biochemistry

1

analysed by ESI and the mass peak observed (m/z= 951.02) correspond to the theoretical molecular

2

weight (data not shown). The fluorescent green dye is linked to the carboxyl groups of MTX as

3

presented, according to the manufacturer’s information (Molecular Probes® ‫ ׀‬Life Technologies).

4 5

This study adds information to the current understanding regarding the structural underpinnings of

6

the transport of antifolate-based derivatives. Moreover, the interference of the chemical nature of

7

their free terminal-groups on the affinity/specificity of the molecular interactions involved in their

8

cellular uptake was evaluated. The functional chemical groups of the MTX conjugates implicated

9

on the binding to the surface-associated receptor, and their potential as “tool” to surmount the

10

membrane-spanning anion transporter, avoiding significant side-effects and dose-limiting

11

toxicities, is discussed.

12

Prior to cellular uptake experiments, FRα expression levels of the different-origin human cancer

13

cells were confirmed. Real-time qPCR data analysis revealed higher expression levels of FRα in

14

the KB cancer cell line, and negligible levels in the cell line MDA-MB-435S (Figure 3A) 11.

15

Although posttranscriptional regulation can occur, previous studies show that protein expression

16

of FR31-32 and RFC33-34 in several cancer cell lines is directly proportional to mRNA levels.

17


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Figure 3. (A) Analysis of FRα and RFC expression levels in human cancer cell lines. (B) Uptake

3

concentrations of the fluorescent-labeled MTX-conjugates by cancer cell lines: FRα-positive KB

4

and FRα-negative MDA-MB-435S. RFC expression levels and uptake of H2N-MTX-CO-FITC

5

conjugate were also analysed after incubation of cells with folic acid and calcium folinate. Values

6

are presented as mean ± standard error of two independent experiments. Differences were tested

7

for statistical significance by a one-way ANOVA (* P < 0.005, *** P < 0.001).

8 9

The uptake concentration of the MTX conjugates, differently linked at the terminal functional

10

groups, showed significant interaction among the selected factors. For FRα-positive cells, KB,

11

significant statistical differences were observed among the internalized concentration of the two

12

MTX conjugates, whereas for the FRα-negative, MDA-MB-435S, negligible differences were

13

observed (Figure 3B). Comparable amounts of both MTX conjugates, with either terminals

14

blocked, were uptaken similarly by FRα-negative cells. Noteworthy, although H2N-MTX-CO-

15

FITC internalization remains unaltered after incubation with high affinity FR substrate (folic

16

acid)35, as expected, it is possible to observe an internalization increase after incubation with high


14

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Biochemistry

1

affinity RFC substrate (calcium folinate)36. Once RFC expression remains similar after incubation

2

with high affinity RFC substrate (Figure 3A), one can speculate that other uptake mechanism is

3

present or is activated in the presence of huge quantity of the calcium folinate.

4

Higher concentrations of H2N-MTX-CO-FITC conjugate were uptaken by FRα-positive cells than

5

that of RedMega-HN-MTX-COOH conjugate (Figure 3B). This result provide a proof of concept

6

of higher passage of MTX via FRα when the amino groups are free. Furthermore, as expected,

7

H2N-MTX-CO-FITC conjugate internalization decreases after incubation with high affinity FR

8

substrate, folic acid, remaining unaltered after incubation with high affinity RFC substrate, calcium

9

folinate.

10

The receptors FRα and FRβ have high sequence identity (82%) and similarity (92%), being their

11

binding sites highly conserved, with only two divergent residues (V129α/F123β and

12

K158α/R152β)12. Although we did not perform studies with cell lines expressing FRβ, a similar

13

behaviour to FRα would be expected.

14

The ability of the conjugate with free amino groups to bind to FRα was further confirmed by

15

molecular docking and molecular dynamics simulations, as showed in Figure 4. Molecular docking

16

experiments initially suggested that MTX is capable to bind to FRα using either the terminal amino

17

group, Figure 4A, or the terminal carboxylic group, Figure 4B. However, stabilization of these

18

docking structures in water, using molecular dynamics simulations tools, showed that the complex

19

is stable only if the amino groups are facing the FRα pocket and not the opposite.


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Page 16 of 32

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Figure 4. Molecular docking and molecular dynamics simulation. (A) Docking result showing

3

MTX at the active site of the FRα with the amino groups facing the protein. The docking result

4

was the start point of the molecular dynamics. (B) Docking result showing MTX at the active site

5

of the FRα with the carbonyl groups facing the protein. The docking result was the start point of

6

the molecular dynamics. (C) System A after a simulation run of 1ns to unsure stabilization. This

7

was the starting point of the umbrella pulling procedure. (D) System B after a simulation run of

8

1ns to unsure stabilization. The configuration derived from the docking was not stable, and after a

9

short simulation MTX in no longer at the active site of the receptor.

10


16

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Biochemistry

1

Taking into account the molecular dynamics simulations, the binding energy between the two

2

transport systems and MTX was calculated, using umbrella sampling technic to calculate the

3

potential of mean force. The final result demonstrate that the complex among MTX and RFC is

4

stronger, with a binding energy of approximately 65 Kcal mol-1, comparatively with FRα, which

5

binding energy is approximately 15 Kcal mol-1 (Figure 5).

6

7 8

Figure 5. Potential of mean force of MTX with RFC and FRα.

9 10

In summary, the obtained outcomes provide a rationale stand for designing more specific drugs

11

for selective approaches. Modulation of the MTX terminal groups might be a potentially useful

12

strategy for improved mediated transport targeting through FRα system on cancer cells (Figure 6).


17

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 32

1

Major outcomes anticipated from this study are the development of the next generation of FR-

2

targeted therapeutics.

3

4 5

Figure 6.

6

mechanisms of MTX conjugates, in FRα-negative and -positive cells.

Schematic representation of the proposed mechanism showing internalization

7 8

AUTHOR INFORMATION

9

Corresponding Author

10

*E-mail: [email protected].

11

Present Addresses

12

† International Iberian Nanotechnology Laboratory, 4715-330 Braga, Portugal

13

ψ Centre for Nanotechnology and Smart Materials (CeNTI), 4760-034, Vila Nova de Famalicão,

14

Portugal


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Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1

Author Contributions

2

‡These authors contributed equally.

3

Funding

4

Egipto Antunes (SFRH/BD/122952/2016) and Jennifer Noro (SFRH/BD/121673/2016) holds

5

scholarships from Portuguese Foundation for Science and Technology (FCT). This study was

6

supported by the FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and

7

COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-

8

0145-FEDER-000004) funded by the European Regional Development Fund under the scope of

9

Norte2020 - Programa Operacional Regional do Norte. This work has also received funding from

10

the European Union 7th Framework Programme (FP7/2007-2013), under grant agreement NMP4-

11

LA-2009-228827 NANOFOL and Horizon 2020 research and innovation programme under grant

12

agreement NMP-06-2015- 683356 FOLSMART.

13

Notes

14

The authors declare no competing financial interest.

15 16

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Biochemistry

Figure 1. Schematic representation of folate receptor-mediated endocytosis and methotrexate influx. The folic acid has a 100-200 times greater affinity to the folate receptor than to the reduced folate carrier. Inversely, the reduced folate carrier-binding affinity to methotrexate is higher. 296x209mm (100 x 100 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. The (most probable) molecular structures of the tested conjugates (A) RedMega-HN-MTX-COOH and (B) H2N-MTX-CO-FITC. Fluorescent red dye can also be linked to the amino group marked with a circle, but since the conjugate was purified by size-exclusion chromatography, the occurrence of a 2:1 (Mega dye:MTX) conjugate is excluded, as it would have a larger size and therefore a shorter retention time. The MTX-Red Mega 520 conjugate was analysed by ESI and the mass peak observed (m/z= 951.02) correspond to the theoretical molecular weight (data not shown). The fluorescent green dye is linked to the carboxyl groups of MTX as presented, according to the manufacturer’s information (Molecular Probes® ‫ ׀‬Life Technologies). 260x100mm (96 x 96 DPI)


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Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2. The (most probable) molecular structures of the tested conjugates (A) RedMega-HN-MTX-COOH and (B) H2N-MTX-CO-FITC. Fluorescent red dye can also be linked to the amino group marked with a circle, but since the conjugate was purified by size-exclusion chromatography, the occurrence of a 2:1 (Mega dye:MTX) conjugate is excluded, as it would have a larger size and therefore a shorter retention time. The MTX-Red Mega 520 conjugate was analysed by ESI and the mass peak observed (m/z= 951.02) correspond to the theoretical molecular weight (data not shown). The fluorescent green dye is linked to the carboxyl groups of MTX as presented, according to the manufacturer’s information (Molecular Probes® ‫ ׀‬Life Technologies). 260x100mm (96 x 96 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. (A) Analysis of FRα and RFC expression levels in human cancer cell lines. (B) Uptake concentrations of the fluorescent-labeled MTX-conjugates by cancer cell lines: FRα-positive KB and FRαnegative MDA-MB-435S. RFC expression levels and uptake of H2N-MTX-CO-FITC conjugate were also analysed after incubation of cells with folic acid and calcium folinate. Values are presented as mean ± standard error of two independent experiments. Differences were tested for statistical significance by a oneway ANOVA (* P < 0.005, *** P < 0.001). 113x93mm (300 x 300 DPI)


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Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3. (A) Analysis of FRα and RFC expression levels in human cancer cell lines. (B) Uptake concentrations of the fluorescent-labeled MTX-conjugates by cancer cell lines: FRα-positive KB and FRαnegative MDA-MB-435S. RFC expression levels and uptake of H2N-MTX-CO-FITC conjugate were also analysed after incubation of cells with folic acid and calcium folinate. Values are presented as mean ± standard error of two independent experiments. Differences were tested for statistical significance by a oneway ANOVA (* P < 0.005, *** P < 0.001). 108x94mm (300 x 300 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Molecular docking and molecular dynamics simulation. (A) Docking result showing MTX at the active site of the FRα with the amino groups facing the protein. The docking result was the start point of the molecular dynamics. (B) Docking result showing MTX at the active site of the FRα with the carbonyl groups facing the protein. The docking result was the start point of the molecular dynamics. (C) System A after a simulation run of 1ns to unsure stabilization. This was the starting point of the umbrella pulling procedure. (D) System B after a simulation run of 1ns to unsure stabilization. The configuration derived from the docking was not stable, and after a short simulation MTX in no longer at the active site of the receptor. 199x173mm (300 x 300 DPI)


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Biochemistry

Figure 5. Potential of mean force of MTX with RFC and FRα. 359x198mm (100 x 100 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Schematic representation of the proposed mechanism showing internalization mechanisms of MTX conjugates, in FRα-negative and -positive cells. 199x80mm (100 x 100 DPI)


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Internalization of methotrexate conjugates by folate receptor-Î±

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