Benextramine and derivatives as novel human monoamine oxidases inhibitors: an integrated approach

Received Date : 28-Feb-2019

Revised Date : 02-Jun-2019

Accepted Date : 08-Jul-2019

Benextramine and derivatives as novel human monoamine oxidases

inhibitors: an integrated approach

Maria Luisa Di Paolo^1,2, Giorgio Cozza¹, Andrea Milelli³, Francesca Zonta⁴, Stefania Sarno⁴,

Elirosa Minniti⁵, Fulvio Ursini¹, Michela Rosini⁵, and Anna Minarini⁵

¹Department of Molecular Medicine, University of Padova, Padova, Italy,

marialuisa.dipaolo@unipd.it

²Consorzio Interuniversitario “Istituto Nazionale Biostrutture e Biosistemi” Roma, Italy

³Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Rimini, Italy

⁴Department of Biomedical Sciences, University of Padova, Padova, Italy

⁵Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna,

Bologna, Italy

Corresponding authors

Maria Luisa Di Paolo, Department of Molecular Medicine, Biological Chemistry

section, Via G. Colombo 3, University of Padova, 35131 Padova, Italy; tel: ++39-049-

8276119; e-mail: marialuisa.dipaolo@unipd.it;

https://www.medicinamolecolare.unipd.it/

Anna Minarini, Department of Pharmacy and Biotechnology, Via Belmeloro 6, Alma Mater

Studiorum-University of Bologna, 40126 Bologna, Italy; tel: ++39-051-2099709;

anna.minarini@unibo.it; http://www.fabit.unibo.it/

Giorgio Cozza, Department of Molecular Medicine, Biological Chemistry section, Via G.

Colombo 3, University of Padova, 35131 Padova, Italy; tel: ++39-049-8276154; e-mail:

giorgio.cozza@unipd.it; https://www.medicinamolecolare.unipd.it/

Running title: Benextramine derivatives are MAOs inhibitors

Article type : Original Articles

Keywords: benextramine; monoamine oxidases; inhibitors; polyamines analogues, docking

studies;

This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which may

lead to differences between this version and the Version of Record. Please cite this article as

doi: 10.1111/febs.14994

Abbreviations: AD, Alzheimer’s disease; BZA, benzylamine; DC₅₀: death concentration

50%; DMSO, dimethyl sulphoxide; DTT, 1,4-dithiothreitol; hr-MAO, human recombinant

monoamine oxidase; MTDL, multi-target-directed ligand; NEM, N-ethylmaleimide; Par,

pargyline; PD, Parkinson’s disease; p-Tyr, p-tyramine; TMS, tetramethylsilane.

Abstract

The two human MAO isoforms (namely MAO A and MAO B) are enzymes involved in the

catabolism of monoamines, including neurotransmitters, and for this reason are well-known

and attractive pharmacological targets in neuropsychiatric and neurodegenerative diseases,

for which novel pharmacological approaches are necessary. Benextramine is a tetraamine

disulfide mainly known as irreversibleα-adrenergic antagonist, but able to hit additional

targets involved in neurodegeneration. As the molecular structures of monoamine oxidases

contain nine cysteine residues, the aim of this study was to evaluate benextramine and eleven

structurally related polyamine disulfides as potential MAO inhibitors. Most of the compounds

were found to induce irreversible inactivation of MAOs with inactivation potency depending

on both the polyamine structure and the enzyme isoform. The more effective compounds

generally showed preference for MAO B. Structure-activity relationships studies revealed the

key role played by the disulfide core of these molecules in the inactivation mechanism.

Docking experiments pointed to Cys323, in MAO A, and Cys172, in MAO B, as target of

this type of inhibitors thus suggesting that their covalent binding inside the MAO active site

sterically impedes the entrance of substrate towards the FAD cofactor. The effectiveness of

benextramine in inactivating MAOs was demonstrated in SH-SY5Y neuroblastoma cell line.

These results demonstrated for the first time that benextramine and its derivatives can

inactivate human MAOs exploiting a mechanism different from that of the classical MAO

inhibitors and could be a starting point for the development of pharmacological tools in

neurodegenerative diseases.

INTRODUCTION

Monoamine oxidases (MAOs) are mitochondrial FAD-containing enzymes that catalyse the

oxidative deamination of biogenic amine neurotransmitters (dopamine, noradrenaline,

adrenaline and serotonin), and a wide variety of xenobiotic amines, including therapeutic

drugs.

The two MAO isoenzymes, MAO A and MAO B (70% of sequence identity), differ for their

substrate specificities, inhibitor sensitivities and tissue localization [1, 2].

Being involved in the catabolism of neurotransmitters, MAOs are well-known

pharmacological targets in various neurological, psychiatric and neurodegenerative diseases,

such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) [3, 4]. Indeed, MAO

inhibitors decrease the catabolism of neurotransmitters and the subsequent generation of

corresponding aldehydes, ammonia and hydrogen peroxide (reaction products), which have

been linked to conditions of oxidative stress that can contribute to the onset of various

diseases [5].

Different MAO inhibitors are or have been used in clinical practice as antidepressant and in

the treatment of PD [6, 7]. Plenty of evidences suggest that the therapeutic potential of MAO

inhibitors (such as deprenyl and rasagiline) are also related to their ability to hit other

biochemical targets [8]. Indeed, their therapeutic actions in the treatment of

neurodegenerative disorders have been linked to the reduction of the oxidative stress,

regulation of gene expression and induction of antiapoptotic and prosurvival factors [3, 4, 8-

11]. The development of multi-target-directed ligands (MTDLs), such as ladostigil, able to hit

simultaneously MAO B and acetylcholinesterase (AChE), strengthens their therapeutic

potential in AD and other neurodegenerative disease [12, 13]. Additionally, in recent years

MAOs (mainly MAO A) emerged as potential therapeutic targets in other pathologies, such

as prostate cancer and cardiovascular diseases [14, 15], opening up new horizons for MAO

inhibitors.

In search of novel types of MAO inhibitors, our previous studies highlighted that polyamine

based analogues are capable to inhibit human MAOs [16, 17]. In particular, ITC-BzDD (N-

benzyl-12-isothiocyanato-N-methyldodecan-1-amine) was found to act as MAO B

competitive inhibitor, and as MAO A irreversible inhibitor (Fig.1) [17].

By exploring the molecular structure of MAOs, it emerged that both the isoforms contain

nine Cys residues, one bound to the FAD-cofactor and eight in the reduced form: kinetic,

structural and site-directed mutagenesis studies supported that some of these Cys may be

important in maintaining the fully functional enzyme [18-20]. To probe the role of Cys

residues on MAOs activity, the most used compounds were the “classical” N-ethylmaleimide

(NEM) and its derivatives [19], and 2,2’dipiridyl disulfide [21]. Importantly, MAO A was

generally found more easily inactivated by thiol oxidation compared to MAO B [19].

Given these premises, benextramine (1, Fig.1), characterized by a cystamine core in its

structure, was herein selected to be studied on MAOs. Compound 1 is a tetraamine disulfide

mostly known as an irreversible α-adrenergic antagonist [22-26], also able to hit additional

targets involved in neurodegenerative processes, such as muscarinic receptors and AChE

enzyme [27, 28]. It was demonstrated that compound 1 exerts its action towards the α-

adrenergic receptor thanks to a disulfide-thiol interchange reaction between the disulfide

moiety of the cystamine core and a Cys residue of the receptor, leading to a disulfide covalent

bond formation. In this context, emerging studies emphasise the role of Cys residues as redox

molecular switches in various signalling pathways and pathologies [29, 30], such as

neurodegeneration [31]. Although compound 1 has been used as a template to design

polyamine analogues as MTDLs against neurodegenerative diseases [27, 28], no data is

available on its effects on MAOs.

Thus, the aim of this study was to evaluate compound 1 and a series of structurally related

polyamine disulfides (2-12) (Table 1) as human MAO inhibitors. The results obtained by

exploiting a multidisciplinary approach including kinetic studies integrated with docking

experiments and in cell validation, showed that most of the tested compounds induced

irreversible inactivation of both MAO isoforms. Compound 1 showed the highest k_inactvalue

for MAO A, and the “short” analogue 12, was found to be the most potent MAO B

inactivator. By the comparison of the inactivation potency (k_inact/K_IE), compounds 1 and 9

were found to be MAO B selective. The importance of the disulfide bridge in the mechanism

of inactivation of these molecules was demonstrated by testing carbon analogues of

compound 1. Docking experiments highlighted the role of Cys323 (in MAO A) and Cys172

(in MAO B) as targets of compound 1 and its derivatives. This investigation led to discovery

of a new class of MAO inhibitors with an unprecedented mechanism of action. This

discovery may have important implication in the design of new MAO inhibitors as

therapeutic agents or bio-pharmacological investigation tools.

RESULTS

Rational of compound selection

In order to investigate the structural features required for an optimal interaction of a

polyamine disulfide scaffold with the MAO A and MAO B active sites, compound 1, together

with polyamine disulfide analogues 2-12, (Table 1), were tested as potential MAO A and B

substrates and inhibitors. The structural modifications applied to compound 1 concerned the

variation of the length of the methylene linkers between the nitrogen atoms (compounds 2-5),

the replacement of the o-methoxybenzyl groups on the terminal amine functions with

catechol (compound 6 and 9), pyrrole (compound 8) or pyridine (compound 7) rings, and a

decrease of the number of amine functions in the polyamine backbone, leading to the

asymmetrically substituted triamine (compound 10) and to diamines (compounds 11 and 12).

To boost the crucial role of the disulfide moiety in MAO interaction, Met-666 and

methoctramine, two carbon analogue of compound 1 (Fig. 1) and the new diamine 13 (Table

1), featuring the hydroxylated analogue of compound 1 truncated in two halves, were also

included in the study.

Screening of the effects of compound 1 and its analogues on monoamine oxidase activity

To evaluate the potential effects of the polyamine analogues on human recombinant -MAOs

(hr-MAOs), compounds 1-13 (Table 1) were firstly evaluated as potential substrates. No

significant MAO activity was detected testing all the analogues in the 0.1-1 mM range of

concentration, with the exception of the compound 10, characterized by a free primary amino

group. However, compound 10 behaved as a very poor substrate for MAO A (enzyme

activity less than 1% compared to 1 mM, (p-Tyr).

Consequently, compounds 1-13, Met-666 (carbon analogue of 1), cystamine (core moiety of

1), and NEM (reference-standard reactive compound for thiols) were tested as potential

inhibitors.

The kinetic parameters (K_mand V_max) of MAO A and MAO B were determined in the

presence of 100 M compounds added in the assay solution immediately before the substrate.

The results are shown in Table 2 as the ratio between the V_maxor K_mvalues with respect to

control samples (in absence of any compounds). Most of these molecules affect both V_max

and K_m, depending on the MAO isoform. The double-reciprocal plots reported as examples in

Fig. 2 indicate that the most effective compounds decreased mainly MAO B substrate affinity

(Fig. 2B) (K_m/K_m0 1.6-2 with compounds 1 and 9 and K_m/K_m0 6 with compound 4, Table

2) whereas, in the case of MAO A, both the V_maxand/or K_mvalues were affected (Fig. 2A).

To understand the mechanism of action of these polyamine disulfides, their time-dependent

inhibitory activity was investigated. MAOs were pre-incubated with 100 M of each

compound, in absence of substrate, and the residual enzyme activity was determined at

various times, after dilution and addition of the substrate. Residual enzyme activity was

mostly found to decrease with increasing incubation time, suggesting that most of the

polyamine disulfides behaved as time-dependent MAO inactivators (see example of

compound 1 in Fig 3 and the calculated apparent inactivation constant values, k_obs’ in Table

2). Moreover, in our experimental conditions, these compounds were more potent than NEM

and cystamine, being compounds 1, 4 and 12 the most effective molecules. In agreement with

previous studies [19], NEM was found to inactivate MAO A faster than MAO B, whereas

cystamine behaved as a very weak MAO B inactivator. Moreover, the substitution of the

cystamine core of compound 10 with the ethanolamine moiety, leading to compound 13,

determined the loss of the inactivation capability toward both the MAO isoforms.

Concerning the length of the spacer between the inner and outer amine functions of the

tetraamine backbone, compound 2 and 3, respectively, the shorter and longer homologous of

compound 1, showed almost the same MAO inactivation profile. Increasing the length of the

spacer up to 12 methylene units, as in compound 4, resulted in a significant rise of the

inactivation potency, suggesting that more hydrophobic chains boost the reactivity against

MAOs. Elongation of the cystamine core of compound 2 with two methylene units led to

compound 5, resulting in a significant decrease of MAO inhibitory ability, particularly for

MAO A, with an apparent inversion of selectivity (5 is more effective on MAO B and

compound 2 is more effective on MAO A). The replacement of the terminal o-

methoxybenzyl groups of compound 1 with other aromatic rings resulted in a reduction of the

activity: compound 7, bearing the pyridine ring at the two terminal ends, is the less potent

compound of the series. The substitution with a pyrrole ring has similar effect to that with the

catechol ring (compound 6) on MAO B; however, the introduction of the catechol moiety

increased the selectivity for MAO B compared to the lead compound 1. This apparent MAO

B selectivity and decreased MAO A inhibitory activity was further enhanced in compound 9,

characterized both by a “lengthened” cystamine core and the catechol groups at the terminal

ends.

The reduction in the number of amine functions in the polyamine backbone, as in the

asymmetric triamine 10 and in the diamine 11, decreased their inactivation capability mainly

towards MAO A in comparison with compound 1. Compound 12, where the o-

methoxybenzyl groups of compound 11 are replaced with o-methoxyphenoxyethyl moieties,

turned out to be the second more potent compound in inactivating MAO B.

On this basis, compounds 1, 4 and 12 (the most potent ones), compound 9 (the most selective

for MAO B) and the triamine 10 were selected for a more detailed kinetic characterization as

time-dependent inhibitors.

Kinetic characterization of the most potent and selective compounds

The inactivation rate constant values were determined at various concentrations of

compounds 1, 4, 9, 10 and 12 with both MAOs isoforms. From the plots k_obs’ vs inhibitor

concentrations, saturation kinetics were found with compounds 1, 9, 10 and 12. For these

molecules, the two steps mechanism of inactivation, which consider a first step of formation

of the reversible complex (EI) followed by the second step of the chemical reaction with the

production of the irreversibly inactivated enzyme (E-X*) (Eqn.1-2, in Material and Methods),

was found to fit the experimental data. In Fig. 4 the analysis of the kinetic data obtained with

1 vs MAOs (panels A-D) and with 12 vs MAO B (panels E,F) are shown as examples. The

calculated k_inact(inactivation constant rate) and K_IE(inhibition constant that represents the

enzyme-inhibitor dissociation constant) are reported in Table 3.

In the case of compound 4, saturation was not reached (example in Fig. 4, panels G, H), thus,

from the slope of the plots k_obs’ vs inhibitor concentration, only the k_inact/K_IEparameter was

calculated. Notably, also inactivation of MAO A by NEM showed a similar non-saturable

behaviour (Fig. 4, panels I, J), in agreement with other studies [21]. Under our experimental

conditions, inactivation of MAO A by NEM (k_inact/K_IE= 400 min^-1M^-1) is about one order of

magnitude slower respect to compounds 1, 4 and 12.

From Table 3 it appears that all the tested compounds show higher affinity (lower K_IEvalues)

for MAO B than for MAO A, resulting compound 1 the most potent MAO B inhibitor (K_IE=

6 M for MAO B vs K_IE= 175 M for MAO A). On the other side, the highest inactivation

rate constants (at saturating concentration of compounds) were found with compound 12

(k_inact= 1.42 min^-1vs MAO B) and 1 (k_inact= 0.98 min^-1vs MAO A). Consequently, the best

inhibitory potency (k_inact/K_IE), which represents the rate of inactivation under not-saturating

concentration of inhibitor, was found with compound 12 (k_inact/K_IE=109 x10³M^-1min^-1vs

MAO B), followed by compound 4 (k_inact/K_IE=51x10³M^-1min^-1vs MAO A). By considering

k_inact/K_IE, the most selective MAO B inhibitors were the catechol derivative 9 (MAO A:MAO

B 1:27) and compound 1 (MAO A:MAO B 1:7), but only 1 maintained a high inactivation

potency for both MAO isoforms.

The unsymmetrical substituted cystamine derivative 10, lacking one of the N-(2-

methoxybenzyl) hexanamine moieties of 1 was found to inactivate both MAOs with lower

selectivity and potency than its precursor 1. In particular, the k_inactvalue for MAO A is halved

and its k_inact/K_IEfor MAO B is decreased by a factor of 5, in comparison to 1.

Inhibition and reversibility mechanisms of compound 1

To understand the role of the disulfide bond in inactivating MAOs, we focused our

investigation on compound 1, showing the best balance between selectivity and inhibitory

potency vs MAO isoforms. After incubation and reduction with 1,4-dithiothreitol (DTT), the

disulfide bond of compound 1 was broken in half producing two molecules of thiols. The

inactivation capability was tested by comparing the effect of 100 M compound 1 and of 100

M of DTT pre-reduced compound 1 on MAOs activity. Intriguingly, the products derived

by the reduction of compound 1 inactivated MAO A and MAO B about 5 and 8 times faster,

respectively than the entire molecule (Fig. 5).

To note that cystamine showed no increase in the inactivation rate constant vs MAOs after

DTT treatment that led to cysteamine formation. These results suggested that the formation of

a disulfide bond between the sulfhydryl group of the reduced form of compound 1 and a Cys

residue of MAOs could be a possible mechanism of MAOs inactivation and that this

interaction takes place faster with respect to the one with the entire disulfide molecule.

To evaluate the reversibility of the chemical bond between compound 1 and MAOs, the

inactivated enzymes were extensively dialyzed. After dialysis, no recovery of MAO A

activity was detected, whereas, in the case of MAO B, about 41% of activity was found

(Table 4), suggesting that the irreversible inhibition induced by compound 1 versus MAO B

is less efficient, as demonstrated also by the lower k_inact(see Table 3). The irreversible and

partial reversible inactivation of MAO A and MAO B, respectively, were confirmed also by

using compound 4, bearing the longest chain within this series (data not shown).

To note that, after inactivation by compound 1, MAOs enzymatic activity was not recovered

by DTT treatment (Table 4). This behaviour suggested that the disulfide bridge formed

between compound 1 and a Cys of MAOs, could be buried, and, consequently, DTT-

inaccessible.

To further confirm the role of the disulfide moiety of 1 in inactivating MAOs, Met-666, the

carbon analogue of compound 1, was assayed. Unlike compound 1, if Met-666 is incubated

with MAOs, a decrease in the enzyme activity over time was not observed (Table 2).

Moreover, if tested without pre-incubation, Met-666 and compound 1 (100 M) showed

similar effects on K_m(Table 2, first columns). These results demonstrated that the substitution

of the disulfide moiety of the irreversible inhibitor 1 with two methylene groups, as in Met-

666, led to a weak reversible inhibitor of MAOs. In particular, Met-666 decreased V_max(K_I=

790 ± 190 M) and increased K_mvalues (K_EI>1mM) of MAO A (Fig. 6A) and increased K_m

values of MAO B (K_EI= 540 ± 130 M (Fig. 6B). Similar results were obtained also with

methoctramine (Met-686, molecular structure in Fig. 1), another carbon analogue of 1,

characterized by an eight methylene inner chain.

All these data confirmed the key role played by the disulfide moiety in the irreversible

mechanism of human MAO inactivation by compound 1.

Protection experiments to prove that polyamine disulfides are “active-site-directed

inhibitors”

The reported experimental evidence clearly demonstrated that compound 1 and its analogues

inactivate MAOs through the disulfide moiety; however, it remains unclear if the mechanism

of inactivation involves Cys located in the MAO active site and if the polyamine disulfides

are to be considered active-site-directed inhibitors. To answer to this question, the ability of a

competitive inhibitor or of a substrate to protect against compound 1 inactivation was

evaluated [32].

First of all, inactivation by 1 was performed in the presence of the competitive inhibitor isatin

[2, 33]. The apparent inactivation rate constant of MAOs (k’_obs) obtained with the lead

compound 1 was slowed down in the presence of isatin (Table 5), by a factor of 6 and 4 in the

case of MAO A and B, respectively. Similar results were obtained also with compound 4, the

longest analogue of the series.

Furthermore, the effect of the substrate on the inactivation rate was evaluated by using the

compounds with the highest inactivation potency (k_inact/K_IE), compound 4 for MAO A, and

compound 12 for MAO B. In the presence of saturating concentrations of substrate (1 mM p-

Tyr for MAO A and 10 mM benzylamine for MAO B) both compounds 4 and 12 lost most of

their capability to inactivate MAOs (Table 5).

These results strongly support the fact that the active site of MAOs is the main target of

compound 1 and its analogues, thus involving Cys residues located in the catalytic cavity.

Docking studies

The above reported biochemical investigations suggested that the binding of compound 1, 4

and 12 might occur in the cavity of MAOs active site, through the formation of a covalent

disulfide bond with a Cys of MAOs. With the aim to evaluate this hypothesis and to

determine the binding mode of these compounds, docking studies were carried out using

MAO A and MAO B crystal structures.

Before the docking procedure, a Site Finder strategy (see Material and Methods) was applied

to select all the possible binding sites of MAO A and MAO B. Interestingly, in both cases the

most suitable interaction zone was superimposed with the catalytic binding site of the two

enzymes, accordingly to the mechanism of action of the molecules above discussed. The

catalytic sites of both MAO A and MAO B are characterized by a hydrophobic tunnel and a

small hydrophilic region located close to the FAD cofactor. Several cysteines are located

inside the catalytic tunnel but only a couple of them are exposed towards the catalytic site, in

particular, MAO A Cys323 and MAO B Cys172, located in the opposite side of the two

cavities, are not conserved. Intriguingly, MAO A Cys323 (Thr314 in MAO B) is located in a

motif almost identical except for the Cys to Thr substitution (MAO A: DYCGCMIIED;

MAO B: DYCGTMIIDG); similarly, MAO B Cys172 (Asn171 in MAO A) belongs to a

very conserved sequence (MAO B: FVNLCVTAE; MAO A: FVNINVTSE). To note that

even if one single cysteine is conserved in both MAO A and MAO B (Cys321 and Cys312,

respectively), these amino acids are not accessible, accordingly to the Site finder approach.

Indeed, MAO A Cys321 is shielded by Cys323 and Leu97, while MAO B Cys312 by Thr314

and Leu88.

To obtain structural information about the residues involved in the interaction inside the

active site of MAOs and, since, under reductive conditions, compound 1 resulted in a more

efficient MAO inactivator, compounds 1, 9 and 12 were docked against MAO A and MAO B

crystal structures as half-molecules, representing the thiol derivatives.

Docking results suggested that thiol molecules derived from the disulfide compounds 1 and

12 bind MAO A and MAO B active sites by exploiting two different hydrophobic cavities,

located inside the tunnel, close each other but not superimposable. In particular, in MAO A,

the two compounds insert their o-methoxybenzyl head into a hydrophobic region assembled

by Pro113, Leu97, Ile 325 and the alkyl chain of Gln99 (Fig. 7A and C). On the other side,

the aromatic heads of compounds 1 and 12 fit into a hydrophobic cavity built by Trp119,

Leu164, Ile199, Leu167 in MAO B (Fig. 7B and D). While the heads of the two compounds

are structurally conserved, sharing the same binding pocket in MAO A or in MAO B, the

thiol tails present huge differences in length and chemical composition, thus reflecting an

unrelated binding motif towards MAO A Cys323 and MAO B Cys172. Indeed, the shorter

thiol chain of compound 12 is perfectly directed versus MAO A Cys323 by the hydrophobic

interaction with Phe208 and Ile335, further stabilized by the hydrogen bond between the

amino group of compound 12 and the carbonyl group of Phe208 (2.7 Å, Fig. 7A). In MAO B

Ile316, Tyr326 and Leu171 address the sulfhydryl group of the thiol compound deriving from

12 towards Cys172 (Fig. 7B). Interestingly, the docking pose versus MAO B is partially

superimposable with the crystallographic pose of pioglitazone (PDB code: 4A79, Fig S1 A).

To note that the in silico pK values of the two complexes MAO A/compound 12 and MAO

B/compound 12 are very similar (7.5 and 7.8, respectively).

On the contrary, the bulkier chain of compound 1 is not able to efficiently interact with MAO

A Cys323 due to the short distance between Cys323 and the hydrophobic cavity bearing the

methoxyphenyl ring of 1 (Fig. 7C). However, in the case of MAO B, compound 1 can easily

accommodate his chain directing it versus Cys172, by exploiting the same hydrophobic

interactions discussed for compound 12 (Fig. 7D). The difference binding poses of compound

1 against MAO A and B are reflected also on the in silico pK values. Indeed, while MAO A/1

complex present a pK of 6.2, MAO B/1 seems more efficient with a value (pK = 8.0) close to

the one observed for MAO B/12 complex.

In the case of compound 9, the docking pose presents substantial differences. The substitution

of the o-methoxyl group of the benzyl moiety with two hydroxyl groups drives the compound

to interact in a more hydrophilic and deep region of MAO catalytic tunnel by performing

direct contact with the FAD molecule (Fig. 7E and F). This interaction is further stabilized by

hydrophobic contacts with MAO A Phe352 and Tyr407 as well as with MAO B Phe343 and

Tyr398 (Fig. 7E and F). The thiol chain of compound 9 is directed versus MAO A Cys323

and MAO B Cys172, being the interaction with the first one (Fig. 7E), apparently less

efficacious compared to the second one (Fig. 7F). This event is also reflected in the in silico

pK values (MAO A/9 pK = 6.5; MAO B/9 pK = 7.1), even if they are close to the limit of

significance cut off.

Interestingly, the in silico pK values above reported showed the same trends and selectivity

as the K_IEdetermined by the kinetic approach: compound 12 binds with similar “affinity” to

both MAOs, while compounds 1 and 9 show higher affinity for MAO B. To note that also the

whole polyamine disulfides considered in the in silico analysis are able to bind to MAO A

and MAO B. However, the pK values calculated suggested that the binding of the whole

molecules to MAOs are less efficient compared to those of the corresponding thiols,

supporting the results obtained in the biochemical experiments.

In summary, the docking results suggests that compounds 1, 9 and 12 can efficiently interact

with the catalytic sites of MAO A and MAO B, directing their thiol chains versus specific

cysteines. To confirm the compatibility of the docking poses with the possible formation of a

disulfide bridge, covalent docking has been performed for compound 1 versus MAO A and B

(Fig. S1 B and C). The results obtained are superimposable with the previously reported for

non-covalent docking; slight differences are explainable by the application of an optimization

process to the final covalent complexes.

Toxicity evaluation and in-cell MAO inhibition of compound 1

After the kinetic and computational studies on the interaction of compound 1 and derivatives

with human-recombinant MAOs, the effect of this novel type of MAO inhibitors on a suitable

cellular model has been prioritized. Since the previously identified targets of compound 1 are

involved in neurodegenerative diseases, the SH-SY5Y neuroblastoma cells, one of the most

used model of neuronal function [34], were chosen as cellular model. To note that this cell

line, as most of the neuronal cell models, expresses mainly MAO A [35] (see also

https://www.proteinatlas.org). MAOs activity was preliminary tested in cellular lysates, using

pre-incubation with clorgyline (irreversible and specific inhibitor of MAO-A) and pargyline

(irreversible inhibitor of both MAO A and MAO B). Clorgyline was able to completely

abolish MAOs activity, confirming that MAO A is the main type of MAO isoform expressed

in SH-SY5Y cells, as previously reported [35].

Before evaluating the effect on MAOs activity, the cytotoxicity of the compounds was tested.

The most efficient compounds 1 and 12 were used for these studies. Their effects were

compared to that of methoctramine, a polyamine analogue lacking the disulfide moiety,

already studied on the SH-SY5Y cells [36].

The cells were incubated for 12 and 24 h in the presence of increasing concentration of the

above reported compounds and then the cell viability was determined. From the DC₅₀(Death

Concentration 50) values calculated from plots shown in Fig. 8, it clearly appears that

compound 1 has a much lower cytotoxic effect than compound 12 (DC₅₀= 252  28 M vs

DC₅₀= 25  7 M and DC₅₀= 208  10 M vs DC₅₀= 22  4 M with compound 12, at 12

and 24 h, respectively); the DC₅₀= 72  10 M obtained with methoctramine (at 24 h) was in

good agreement with the previously reported value [36]. On this basis, only the less toxic

compound 1, was tested as potential MAO inhibitor on the cellular system; pargyline was

also assayed as standard MAO inhibitor. After incubation of cells with compound 1 for 3 and

24 h, a significant decrease in MAOs activity was found (Fig. 9A). After 24 h, an IC₅₀of

about 13 M was found, a concentration too low to induce significant citotoxicity. No

decrease in MAO A protein content was found by western blotting and immunostaining

analysis (Fig. 9B). From Fig. 9B, an increase of about 30% of MAO A protein was observed

at the highest compound 1 concentration (250 M). An increase in MAO A expression was

previously reported in the same cell line with other MAO inhibitors (selegiline and rasagiline,

MAO B inhibitors) [37].

The above reported results confirm the effectiveness of compound 1 in the inactivation of

MAO A in a cellular system, at not cytotoxic concentrations.

DISCUSSION

This study demonstrated for the first time that benextramine (1) and its derivatives can

inactivate human MAO A and MAO B isoforms, exploiting a mechanism of inactivation

different from that of the classical MAO inhibitors. While the traditional MAO inhibitors act

by targeting the FAD cofactor, the mechanism herein proposed for benextramine involves a

time-dependent inactivation by the formation of disulfide bridge with the thiol group of a Cys

of the enzyme.

The inactivation rate constant values with compounds 1-13 were found dependent on: a) the

polyamine disulfide skeleton, b) the aromatic substituent on the two terminal ends of the

molecule and c) the MAO isoform. In particular, the o-methoxybenzyl moieties at the

terminal ends of the tetraamine disulfide skeleton are responsible of a stronger potency than

other substitutions; on the other side, the elongation of two methylene groups in the inner

cystamine core, decreases the inactivation potency versus MAO A to favour the selectivity

towards MAO B.

By considering k_inact/K_IE(index of inactivation efficiency), the diamine 12, the shortest

analogue of the series, was found to be the most potent MAO B inactivator (k_inact/K_IE= 1x10⁵

M^-1min^-1), and the tetraamine 4, the longest one, appears to be the more potent MAO A

inactivator (k_inact/K_IE= 57x10³M^-1min^-1); however, both the compounds are poorly selective.

A good selectivity for MAO B was found for compounds 1 and 9 (MAO A: MAO B, 1:7 and

1:27, respectively).

By comparing these kinetic results with that previously found with ITC-BzDD (Fig. 1) [17],

compounds 1, 4 and 12 resulted more potent than ITC-BzDD as MAO A inactivator in terms

of k_inact/K_IEor k_inact. Furthermore, although ITC-BzDD (Fig. 1) behaves with a different

inhibition mechanism, that is as competitive inhibitor of MAO B, its K_IEvalue towards MAO

B is comparable with those found with the inactivator compounds 1 and 12.

Moreover, by comparing the kinetic behaviour of the standard and selective inhibitors

targeting the FAD cofactor, clorgyline for MAO A and deprenyl for MAO B, 1 analogues

show a lower inactivation efficiency (k_inact/K_IE, in the range 10⁶-10⁸M^-1min^-1for clorgyline

and deprenyl vs k_inact/K_IxE, in the range 10⁴-10⁵M^-1min^-1), but good inactivation rate constant

values (k_inactabout 0.9 min^-1with clorgyline and 0.08 min^-1with deprenyl vs k_inact= 1-1.4 min

^-1with 1 and 12). Additionally, the inactivation efficiency of 1 analogues is higher than that

of other thiol reactive reagents (2,2'-dipyridyl disulfide, k_inact/K_i1.2-0.3 x 10³M^-1min^-1for

MAO A [21] and NEM, k_inact/Ki 0.4 x 10³M^-1min^-1). These data suggest that the mechanism

of chemical inactivation by compound 1 derivatives could be considered as a potential new

mode of inactivating MAOs.

The inactivation by compound 1 was found irreversible for MAO A and partially reversible

for MAO B. This partial reversibility is probably explained by the stronger but reversible

physical interaction of compound 1 with MAO B and by a concomitant slower chemical

reactivity or reversibility of the chemical reaction (k_inact= 0.25 min^-1for MAO B vs 0.98 min^-

¹in MAO A).

By the comparison of the inhibitory behaviour of Met-666 vs compound 1 and compound 13

vs compound 10, respectively, it was demonstrated the crucial role played by the disulfide

bridge in the inactivation process. Indeed, in absence of this feature, inactivation did not

occur and the compounds act as poor reversible inhibitor (Met-666), suggesting the formation

of a disulfide bond between compound 1 and MAOs.

Previous structural studies demonstrated the presence of eight reduced Cys residues in both

MAO proteins [19] and other authors reported that the mutation/chemical modification of

some of them may affect enzyme activity [20, 21]; so the Cys residues of MAOs are the best

candidates to react with the disulfide moiety of compound 1 and its derivatives.

The protection by inactivation in the presence of a competitive inhibitor or of a substrate

strongly supported that these compounds target a Cys residue of MAO active site cavity. The

docking experiments pointed to the not conserved Cys323 (in MAO A) and Cys172 (in MAO

B) as targets (Fig. 7): the covalent disulfide bond formed between the molecules and these

Cys residues of MAO active sites should impede/occlude the entrance of substrate towards

the FAD-cofactor, resulting in enzyme inactivation.

However, alternative binding or additional binding to Cys at remote sites, such as on the

enzyme surface, may not be excluded, even if the not recovery of enzyme activity, after DDT

treatment suggests that the Cys involved in the binding of compound 1 and its analogues are

solvent inaccessible.

Therefore, concerning the mechanism of MAO inactivation by compound 1 derivatives, two

ways are here proposed: a) the pre-reduced disulfide (half-molecule of the compound) could

react with the Cys of MAO active site via a sulfenic acid intermediate (–SOH), which might

form inside the active site in air-exposed aqueous solutions [38]; b) the disulfide core of the

compound could react with the Cys-SH of MAO active site through a classical reaction of

thiol-disulfide interchange [38].

The first way is supported by the extremely efficacious enzyme inactivation by the DTT-

reduced compounds 1 and 10, which demonstrated that the free -SH groups formed are much

more efficient than the whole disulfide-containing molecule in inactivating both MAOs.

Under physiological conditions, in a cellular system, the presence of reducing agents, such as

glutathione, could speed up the reduction of compound 1 analogues before reacting with

MAO (bound to the mitochondrial membrane).

On the other side, the known flexibility of the molecular structure of the enzyme [40] and of

the polyamine backbone might allow the disulfide compound/Cys interaction in the active

site, through a thiol-disulfide interchange reaction, even if docking experiments suggested

better binding affinities for the thiol compound (half molecule), compared to the disulfide.

Indeed, these results could be impaired by the semiflexible docking protocol, in which the

compounds enjoy full degree of freedom, while the protein is completely rigid and not

subjected to induced fitting.

The efficacy of compound 1 in inactivating MAO A was confirmed also in a model of

neuronal cells, where inactivation occurs at concentration one order of magnitude lower than

its cytotoxic concentration (IC₅₀= 13 M vs DC₅₀= 208 M).

In conclusion, the main finding of this study is that human MAOs are novel targets for the

tetraamine disulfide benextramine and its derivatives, exploiting a novel inactivation

mechanism of MAOs.

Starting from the above reported results, further studies are in progress to design novel

analogues with a better selectivity and reactivity than compound 1, for a future potential

development as pharmacological tools in neurodegenerative diseases.

MATERIALS AND METHODS

Chemicals and reagents

All chemicals were of analytical grade and purchased from Fluka-Sigma-Aldrich S.r.l. (Milan,

Italy) with the exception of Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine),

purchased from Molecular Probes/Invitrogen (Invitrogen s.r.l, San Giuliano Milanese (MI,

Italy).

Human recombinant MAO A and MAO B expressed in baculovirus infected BT1 cells (5

mg/mL) and horseradish peroxidases were purchased from Fluka-Sigma-Aldrich s.r.l. (Italy).

Rabbit polyclonal to MAO A was from Abcam and anti-actin was from Sigma-Aldrich S.r.L.

(Milan, Italy)

Synthesis of compounds 1-13

Compounds 1-12 were synthesized following improved procedures respect to those previously

reported [22-24, 26, 39]. We describe here the modified synthesis of compound 10 and the

synthesis of the new compound 13 (Scheme 1). The detailed procedure is reported in the SI

(NMR spectra in Fig.S2).

Briefly, 2-methoxybenzaldeyde was subjected to a classical reductive amination with 6-

aminohexanol to furnish compound 14. The secondary nitrogen was protected with Boc (15)

and the hydroxy functionality was activated with tosyl chloride (16). Reaction between 16

and cystamine led to intermediate 17, which was subjected to acidic deprotection to remove

Boc group giving compound 10 as hydrochloride salt. Reaction between 16 and ethanolamine

led to compound 18, which was subjected to acidic deprotection to give compound 13 as

hydrochloride salt. Benextramine can be obtained adopting the same procedure described for

17, by using 16 and cystamine in 2:1 equivalent ratio, followed by deprotection of Boc

groups.

Monoamine oxidase activity experiments

Amine oxidase assay methods

Amine oxidase activity was determined by measuring H₂O₂generation rate by a peroxidase-

coupled continuous assay. The Amplex Red reagent was used as fluorogenic substrate for

horseradish peroxidase [40]. No significant interference of the various polyamine analogues

on fluorescence intensity was observed.

All experiments were carried out in 50 mM Hepes-NaOH, 50 mM KCl, 120 mM NaCl, pH

7.4 and at 37°C.

Assays were carried out in a final volume of 800 l, in the presence of Amplex Red (100

M) and horseradish peroxidase type II (5 UmL^-1). Benzylamine (BZA) and p-tyramine (p-

Tyr) were used as substrates for MAO B and MAO A, respectively. Initial velocities were

determined by measuring the increase in fluorescence intensity (_exc= 563 nm and _em= 586

nm); H₂O₂generation rate was calculated from the change in fluorescence intensity, by

means of calibration curves obtained by serial dilution of stock solution of H₂O₂. In particular,

since the fluorescence intensity generated by the system peroxidase-Amplex Red-hydrogen

peroxide, decreases in the presence of high tyramine concentrations, the reaction rates of

MAO A, at various tyramine concentrations, were calculated using specific calibration curves

built for each concentration of the substrate used for the enzyme activity assay. If not

differently reported, hr-MAO A and hr-MAO B concentrations in the assay solutions were 3

and 6 g/ml, respectively.

In experiments carried out in the presence of DTT, which was found to interfere with the

Amplex Red- peroxidase assay method, MAO activity was determined by the method using

kynuramine as substrate [41]. Briefly: enzyme stock solutions, after incubation in the presence

of DTT (with or without inhibitor), were diluted with assay buffer to get a final concentration

0.003 mg/mL for MAO-A and 0.006 mg/mL for MAO-B; 300 M kynuramine (final

concentration) was added to the 200 l of assay solution (0.1 M potassium phosphate buffer,

pH 7.4, 0.1 mM EDTA). After 30 min incubation at 37°C, the reaction was stopped by

addition of 2 M NaOH (80 l) and 480 L of distilled water. Kynuramine deaminated by

MAO spontaneously cyclizes to give 4-hydroxyquinoline, the amount of which was

determined by the fluorescence intensity of the peak of its emission spectra (_exc= 330 nm and

_em330-530 nm), using a specific calibration curve built with the standard 4-

hydroxyquinoline.

This kynuramine assay was also used for the assay of MAO activity in SH-SY5Y cell lysates

(25 L of cell lysates in 200 L of the assay solution). MAO activity was expressed as

pmol_4HQmin^-1mg _protein^-1. To verify the presence of MAO A activity in this cells, SH-SY5Y

lysates, were pre-incubated for 15 min at 37 °C with clorgyline (1 M, to inhibit MAO A) and

pargyline (250 M, to inhibit both MAO A and MAO B); then the substrate (kynuramine) was

added and the residual MAO activity was determined.

Inactivation of MAOs by compound 1 pre-treated with DTT

Compound 1 (1mM) was pre-incubated with DTT (20 mM) in Hepes buffer (pH 7.4) for 15

min at 37°C. In these conditions, the disulfide bond is reduced breaking compound 1 in two

half-molecules characterized by the presence of thiol groups. After reduction, experiments of

inactivation of MAOs were carried out using 100 M of compound 1, or 100 M of DTT-

reduced compound 1. MAOs without any compounds and MAOs with only DTT (at 2 mM

final concentration, as in the sample with the reduced compound 1), were run under the same

experimental conditions and were taken as control samples. MAOs activity studies were

carried out using the assay method with kynuramine as substrate.

The effect of DTT-oxidation product on MAOs, was evaluated using DTT-pretreated

cystamine under the same experimental conditions.

Reversibility experiments

MAO A and MAO B (0.025 mg/mL) were incubated in the presence of compound 1 or 4 200

M. When the residual activity was decreased and constant at its minimum value (about zero

for MAO A and about 30%, in the case of MAO B), the samples were extensively dialyzed

against several changes of buffer (100 mM potassium phosphate, pH 7.20, at 4 °C). Control

samples of MAOs were run simultaneously, under the same experimental conditions. After

dialysis, the residual MAO activity and protein concentration were determined, to calculate

the residual specific activity (nmol_H2O2min^-1mg^-1_protein).

The reversibility was also tested by adding DTT to the MAOs previously inactivated by 1.

DTT was added at a final concentration of 10 mM, for about 20 min, at 36°C. The residual

enzyme activity was tested after dilution (1:40) of the enzyme- 1 solution, before adding

substrate. Control samples of enzyme, in the presence and absence of DTT were also run and

treated under the same experimental conditions to verify the effect of DTT on the free

enzyme.

Protein assay

The protein content of samples was measured by the Bradford method, with bovine serum

albumin as standard [42].

In silico analysis

The crystal structure of human MAO A and MAO B were retrieved from the Protein Data

Bank (PDB codes: 2Z5X and 4A79, respectively) and processed in order to remove ligands

and water molecules. Hydrogen atoms were added to the protein structures using standard

geometries with the MOE program [43]. To minimize contacts between hydrogens, the

structures were subjected to Amber99 force-field minimization until the rms (root mean

square) of conjugate gradient was <0.1 kcal•mol⁻¹•Å⁻¹(1 Å = 0.1 nm) keeping the heavy

atoms fixed at their crystallographic positions. Compound 1, and the selected derivatives 12

and 9 were built using MOE, considering the molecules in both oxidized and reduced states;

Gasteiger partial charges (MOE Suite) were added.

Before the molecular docking procedure a Site Finder Approach [43] was performed; this

tool, by identifying regions of tight atomic packing (using Alpha Shapes, a generalization of

convex hulls developed in [44], allows the identification of pockets and surface sites and was

successfully applied in different target analysis [45, 46]. In the case of MAO A and MAO B,

the procedure suggests a cavity, superimposable with the catalytic tunnel of both the

enzymes, as the best interaction zone for the molecules considered in this work.

Using the Site Finder indications, a preparation step for the docking procedure was performed

with Glide package (Schrodinger suite) [47]. A set of co-crystallized inhibitors of MAO A

and MAO B were used to optimize the docking protocol, determining a final Glide grid (outer

grid dimensions: 14Å x 28Å x 16Å for MAO A; 18Å x 25Å x 15Å for MAO B). Compounds

1, 12 and 9 were docked in the defined sites and grids by using the XP (extra precision)

procedure. The in silico association constant (pK), estimated using MOE suite, was optimized

exploiting the crystal structures available during the docking preparation phase. It is

expressed as the summation of a directional hydrogen-bonding term, a directional

hydrophobic interaction term, and an entropic term; higher values indicate more affinity with

a significance cut off of 0.5.

Covalent docking of compound 1 versus MAO A and MAO B was performed by using

CovDock tool [47]. The optimization of the final covalent complexes was performed using

Prime [47].

Cell cultures

Human SH-SY5Y neuroblastoma cells (A.T.C.C., Manassas, VA) were cultured in DMEM

medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 2 mM L-glutamine,

100 U/mL penicillin and 100 mg/mL streptomycin (Sigma-Aldrich) and maintained in a 5%

CO₂humidified atmosphere at 37 °C and grown until they reached 70% confluence for a

maximum of 20 passages.

After cell treatment with the various compounds, cells were gathered, washed with phosphate

saline buffer (PBS) and after centrifugation pellets were lysated in 20 mM Hepes-NaOH, pH

7.5, 1mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitor cocktail (about 10

million cells/1mL of lysis buffer) and frozen in liquid nitrogen. Samples were stored at -

80 °C until use.

Cell viability assay

Log-phase cells were seeded into the 96-wells plate (15.000 cells/well) and were allowed for

the attachment overnight followed by the treatment with increasing concentrations of

compound 1 and methoctramine for 12 h and 24 h. Cellular proliferation was quantified with

colorimetric methods based on the metabolic reduction of the soluble yellow 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye to its insoluble formazan

[48]. The absorbance was read on Titertek Multiskan Plus plate reader (Flow Laboratories,

Sutton, U.K.) using 540 nm wavelength.

Results were normalized to time point zero (t = 0 h), and the concentration inducing 50% of

cell death (DC₅₀) was calculated with respect to DMSO treated controls, with Prism 7.0a

software.

Western blot analysis

Cells treated at the reported concentrations of 1 and pargyline for 3h and 24h were

trypsinized and lysed by addition of 20 mM Hepes (pH 7.5), 1 mM EDTA and protease and

phosphatase inhibitor cocktails and frozen in liquid nitrogen. Protein concentration was

determined by the Bradford method. 30 g of protein lysates were subjected to 9% SDS-

PAGE, blotted on Immobilon-P membranes (Merck-Millipore), processed in western blot

with the indicated primary antibodies and detected by enhanced chemiluminescent detection

system (ECL). Re-probing of a blot with new primary antibodies was done after 3 washing

with phosphate buffered saline with 0.1% Tween-20 (Sigma-Aldrich).

Immunostained bands were quantified by means of a Kodak-Image-Station 4000MM-PRO

and analysis with Carestream Molecular Imaging software (New-Haven, CT).

Differences among treatments were evaluated by analysis of variance (ANOVA) followed by

Bonferroni post-test, using Prism 7.0a; p<0.05 was considered significant.

Kinetic and statistical analysis

Steady-state kinetic parameters (V_maxand K_m) were calculated by fitting the Michaelis-

Menten equation to the experimental data (initial rate of reactions vs substrate concentrations)

by non-linear regression analysis, with Sigma Plot software, version 9.0 (Jandel Scientific,

San Rafael, CA, USA) and the value of the kinetic parameter obtained from the best fit and

its S.E. are reported.

Time-dependent inhibition studies. Recombinant MAO A and MAO B (0.02-0.05 mg/mL) in

the Hepes buffer, were pre-incubated for periods from 0 to about 50 minutes, at 37 °C in the

presence and in the absence of inhibitor (control sample). After various time intervals of pre-

incubation (at least 5 times after mixing of enzyme and inhibitor), the residual MAOs enzyme

activity of samples was determined: an aliquot of sample was withdrawn and diluted in the

assay buffer (50-fold dilution) and, after 1 minute, substrate was added under “saturating”

condition, (p-Tyr 1 mM and BZA 10 mM for MAO A and B, respectively).

All time-dependent inhibition experiments were monitored relative to a control sample,

which was run exactly as the experiment except without inhibitor added (only an equivalent

volume of solvent used for the tested inhibitor, was added).

The enzyme activity of this control sample (v₀) was set to 100% and for each time point the

ratio between residual enzyme activities (v_+I) relative to the control sample (v₀), was

calculated. The “apparent inactivation rate constant” values (k_obs’), for each inhibitor

concentration, were calculated by linear regression analysis of the data of logarithm of

residual activity (ln (v_+I/v₀)) versus time. The k_obs’ values were plotted versus [I] and, in the

case of a saturation behaviour, the Kitz and Wilson model, shown in the following equation,

was found to be appropriate to fit the time-dependent inhibition data (k_obs’ vs inhibitor

concentration):

K_EI

k_inact

EI

(1)

EI*

E + I

In this scheme, a rapid reversible interaction between free enzyme (E) and inhibitor (I) is

followed by a slower, irreversible reaction, which transforms the reversible enzyme-inhibitor

complex (EI) into an irreversible enzyme inhibitor complex (EI*). The inactivation rate

constant (k_inact) and the enzyme-inhibitor dissociation constant (K_IE), were calculated by

fitting eqn. 2 to the data of “apparent inactivation rate constants” (k_obs’) versus [I]₀(non-

linear regression analysis, with Sigma Plot software, version 9.0)

k_inact[I]₀

k '

=

obs

(K_EI[I]₀)

(2)

If saturation kinetics is not observed, only the apparent bimolecular rate constant k_inact/K_IE

was calculated from the slope of the plot of k _obs’ against [I]₀.

Unless stated otherwise, the correlation coefficient for linear regression was 0.98 or greater.

All experiments were repeated independently at least three times, and the experimental data

were expressed as the mean ± standard deviation (SD).

Statistical analyses of data from cellular experiments were conducted using Student's t-test.

Values of *p < 0.05 or **p < 0.01 were defined as statistically significant. All analyses were

carried out with the GraphPad Prism program (GraphPad Software, Inc., San Diego, CA).

Error bars on all graphs consider 95% confidence intervals.

For calculating the IC₅₀value of compound 1 in the cellular system, curve fitting was

performed by non-linear regression analysis using the standard “dose-response” curve

equation and the Sigma Plot software.

Acknowledgments The Authors thank the “International Polyamine Foundation - ONLUS”

for the availability to look up in the Polyamines documentation and the Modelling Section

(MMS, University of Padova) for the kind support.

This research was supported by institutional grants from the University of Padova, Italy

(60A06-1707/15 to M.L.D.P. and COZZ_SID18_01 to G.C.) and by the institutional grant

from the University of Bologna, Italy "University of Bologna - RFO" to A.M.

Conflict of interest: the authors declare they have no conflicts of interests with the contents

of this article.

Author contributions

MLDP, AM (Anna Minarini) and GC, conceived the project; MLDP conducted all kinetic

investigations, analysed all the results and wrote the manuscript; GC conducted the

computational studies, analysed and discussed all the results and participated in writing the

manuscript; AM (Andrea Milelli) contributed to the synthesis of compounds and to writing

the paper; FZ carried out cell cultures and analysis of cellular data; SS contributed to design

and analyse all cellular experiments; EM carried out the synthesis of compounds; FU

contributed to the analysis and discussion of the results; MR contributed to the analysis of

results; AM (Anna Minarini) designed the compounds, carried out their synthesis, analysed

and discussed all the results and participated in writing the manuscript. All authors have

given approval to the final version of the manuscript.

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Supporting information

Additional supporting information may be found online in the Supporting Information section

at the end of the article.

Synthesis of compounds 10 and 13.

Fig. S1. Superimposition between compound 12 and pioglitazone in MAO B; covalent

docking of benextramine (1) against MAOs.

Fig. S2. NMR spectra of compounds 10 and 13.

1

A) H and ¹³C NMR spectra of compound 10

1

B) H and ¹³C NMR spectra of compound 13

Table 1. Molecular structures of benextramine (1) and its analogues.

Compound

Structure

Benextramine (1)

Modification of the length of methylene linkers between the amino groups

2

3

4

Elongation of the cystamine core

5

Replacemtent of the o-methoxybenzyl groups

6

7

8

9

Decrease in the number of amino groups

10

11

12

Replacement of the disulfide core

13

MET666

Cystamine

NEM

For each group of molecules, a brief description of the rational modifications is reported. The

reference compounds cystamine (core moiety) and NEM (-SH reactive compound) are also

shown.

Table 2. Preliminary screening of compounds 1-13 as potential inhibitors vs hr-MAOs.

Compound hr-MAO A

s

hr-MAO B

V_max/V_max0

0.90

hr-MAO A hr-MAO B

V_{max Vmax0}

/

K_m/K_m0

k_obs’

(min^-1)

k_obs’

(min^-1)

(100 M)

0.77

1.5

1.89

1

0.3500.039

0.2170.031

Effect of the length of methylene linkers between the nitrogen atoms

0.85

0.9

0.95

0.72

0.47

2.21

2.02

6.3

2

3

4

0.3870.012

0.3780.043

5.81.0

0.1970.070

0.1050.027

2.430.50

0.78

1.23

n.d.^(a)

n.d. ^(a)

Effect of the elongation of the cystamine core

1.28 0.88 1.55

0.93

5

0.0540.005

0.1180.003

Effect of the replacement of the o- methoxybenzyl groups

1.0

1.04

1.0

1.20

1.10

1.7

1

1.2

2.02

1.20

1.6

0.060±0.06

0.0030.01

0.0780.013

0.2840.100

6

7

8

0.0390.003

0.0170.002

0.0770.010

0.0240.005

1.0

1

1.0

9

Effect of the decrease in the number of amine functions

0.76

0.87

0.64

1.08

0.8

0.92

1

1.25

1.14

1.87

10

11

12

0.2070.027

0.1660.017

0.4500.105

0.1760.020

0.33 0.10

0.94

0.77

0.9400.280

Effect of the replacement of the disulfide core

0.0030.002^(b0.0010.001

0.90

1.10

1.5

1

1.10

1.74

1

13

)

0.0030.002^(b0.0010.001

1

Met-666

CYSTAMINE

NEM

1

)

1

0.0020.001^(b0.0100.002

)

1

0.8

1.2

0.0270.003

0.0050.002

Steady-state kinetic parameters in the absence of compounds were: Vmax₀,_p-Tyr= 110 ± 9

nM/min and Km₀,_p-Tyr= 165± 30 for MAO A; Vmax₀,_BZA= 109 ± 11 nM/ and Km₀,_BZA= 280

± 45 for MAO B. The “apparent inactivation rate constant” values (k_obs’) of the various

compounds for MAOs were determined by the linear regression analysis of data of residual

activity (ln(v_+I/v_o) vs time)(r≥0.98). Parameter ± S.D are reported (n=3)

^(a)n.d.: not determinable, MAO A was immediately inactivated.

(b)

k ’ equivalent to that of control samples in absence of compound

obs

Table 3. Inactivation rate constant and inhibition constant values of the most effective

polyamine disulfides.

Selectivity

MAO A/MAO B

Compoun

d

MAO A

MAO B

k_inact

K_IE

(M)

k_inact/K_IE

(M^-1min^-

k_inact

K_IE

(M)

k_inact/K_IE

k_inactk_inact/K_IE

(min^-1)

(M^-1min^-1)

1

)

0.98±0.0

1

5.6±0.1

x10³

0.25±0.0

1

40.0± 0.4

x10³

175±14

n.d.

6.3±0.6

n.d.

3.9:1

n.d.

1:7.1

2.8:1

1:27

1

4

9

51 ±7

18.3±6

x10^3(a)

n.d.

x10^3(a)

0.04±0.0

1

0.5±0.1

x10³

0.19±0.0

1

13.8±1.0

x10³

77±25

14±2

1:4.8

0.40±0.0

1

4.0±0.5

x10³

0.22±0.0

1

7.8± 1.7

x10³

97±9

20±4

30±5

13±2

1:1.9

1:3.9

10

12

1.8:1

1:2.5

0.57±0.0

4

28.0±7.0 1.42±0.0

x10³

109.0±17.0

x10³

6

The kinetic parameters of inactivation were calculated fitting equation 2 to experimental data

(k’_obsof inactivation vs inhibitor concentrations), by non-linear regression analysis (parameter

± standard error values are shown).

n.d.: not determinable, saturation was not reached in the explored compound 4 concentration

range (2-100 M)

^(a)This parameter is the slope value obtained by the linear regression of the data of the plot

k’_obsvs compound 4 concentrations.

Table 4. Irreversibility of the inactivation of MAOs by compound 1

RECOVERED MAO ACTIVITY

( %)

A) Effect of dialysis on MAOs inactivated by compound 1

Sample

hr-MAO A

hr-MAO B

100^a

100^b

Control

0

41±5

Compound 1-inactivated MAO

B) Effect of DTT on MAOs inactivated by compound 1

100

11011

364

Control

Control + DTT

876

0

Compound 1-inactivated MAO

Compound 1- inactivated MAO after DTT

244

A) Effect of dialysis: MAOs were inactivated by compounds 1 (200 M) and then extensively

dialyzed; after dialysis, the recovered MAO activity was measured. A control MAO samples

was run under the same experimental conditions.

B) Effect of DTT: MAO A and MAO B were inactivated by pre-incubation with compound 1

(200 M for 15 min at 36°C); then DTT 10 mM was added. After additional 20 min of

incubation, samples were diluted and the residual MAO activity was tested, using the

kynuramine assay method (final concentration in the assay: compound 1 5 M and DTT 0.25

mM)

Means ±SD are shown (n=3).

-1

^aMAO A specific activity: 30 nmol min^-1mg_protein

^bMAO B specific activity: 16 nmol min^-1mg_protein

-1

Table 5. Protective effect of a competitive inhibitor and of the substrate on inactivation

of MAOs by compounds 1 and its derivatives.

A) Effect of the competitive inhibitor Isatin

k_obs’

k_{obs’+ISATIN}

[Isatin]^a

M)

Enzyme

[Compound]

k_obs’ (min^-1)

- isatin + isatin

[1]

100

12

5.8

3.8

MAO A

MAO B

100 M

[4]

0.3500.039 0.0600.005

0.2170.031 0.0570.007

100

12

5.8±1.4

3.0±0.3

2

MAO A

MAO B

100 M

2.35±0.52

0.52±0.08

4.5

B) Effect of the substrate

Substrate

k_obs’ (min^-1)

Enzyme

[Compound]

[4]

+ substrate

- substrate

p-Tyr

MAO A

MAO B

5 M

[12]

0.920.14

0.530.07

No inactivation

BZA

0.310.03

No inactivation

p-Tyr

MAO A

MAO B

5 M

0.3490.08

0.1050.01

BZA

A) Effect of the competitive inhibitor isatin on inactivation by compound 1 and 4.

Inactivation experiments were performed under standard conditions, in the presence and

absence of isatin.

B) Effect of substrate on inactivation of MAOs by compounds 4 and 12. Inactivation

experiments were performed under the standard conditions, in the presence and absence of

substrate ([S]>>Km: 1 mM p-Tyr for MAO A and 10 mM BZA)

The “apparent inactivation rate constant” values (k_obs’) for MAOs were determined by linear

regression analysis of data of residual activity (ln(v_+I/v_o)) vs time. Error bars represent the SD

of the parameters (n = 3 replicates).

^a)Isatin concentrations were 6-7 times higher than its corresponding K_Ifor the free enzyme

([E-Isatin]/[E]>6). In our experimental conditions: Ki = 16±3 M and Ki = 2.3±0.5 M for

MAO A and MAO B, respectively, in agreement with those previously reported [33].

Scheme 1. i) a. o-OMe-benzaldehyde, EtOH, reflux, 5 h, b. NaBH₄, EtOH, rt, , 16h,79%

yield; ii) Boc₂O, CH₂Cl₂, rt, 16 h, quantitative yield; iii) tosyl chloride, Et₃N, DMAP,

CH₂Cl₂, 0 °C, 1.5 h, 81% yield; iv) cystamine (for 17) or ethanolamine (for 18), DMF, Et₃N,

rt, 3 days, 36-60% yield; v) 4M HCl/dioxane, 0 °C, 40 min, 81% yield. Boc = -COOC(CH₃)₃;

Ts = -SO₂C₆H₄CH

Fig. 1. Structures of the prototype of the tetraamine disulfides, benextramine (1), its carbon

analogues (Met-666 and methoctramine) and of the previously studied MAO inhibitor ITC-

BzDD [17].

Fig. 2. Effect of compounds 1, 4, 9 and 12 on kinetic parameters (K_mand V_max) of hr-MAOs.

Lineweaver-Burk plots of oxidative deamination of substrate catalysed by hr-MAO A (A)

and hr-MAO B (B), in the absence (●) and presence of 100 M compounds 1 (●),4 (■), 9

(▲) or 12 (▲). Steady-state kinetic experiments were carried out without pre-incubating the

enzyme with the compound. Solid lines were calculated by linear-regression analysis of data

(r > 0.99).

Fig. 3. Time dependent inactivation of MAOs by compound 1

Decay of MAO activity in the presence of 100 M compound 1 vs pre-incubation time; solid

lines are the results of the best-fitting of the equation for an exponential decay at two

parameters (V_(t=0)and k_obs’) to the experimental data (non-linear regression analysis, with

Sigma Plot software, version 9.0; r ≥ 0.99). No decay of MAO activity was found in control

samples (dashed lines).

p-Tyr 1 mM and BZA 10 mM were used as substrate to assay the residual MAO A and MAO

B activity, respectively. Error bars represent the S.D. of the data (n = 3 replicates).

Article Doi

Benextramine and derivatives as novel human monoamine oxidases inhibitors: an integrated approach

DOI: 10.1111/febs.14994

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Article abstract of DOI:10.1111/febs.14994

Full text of DOI:10.1111/febs.14994

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