Three-Dimensional Proteome-Wide Scale Screening for the 5-Alpha Reductase Inhibitor Finasteride: Identification of a Novel Off-Target

Article abstract of DOI:10.1021/acs.jmedchem.0c02039

Finasteride, a 5-alpha reductase (5α-R) inhibitor, is a widely used drug for treating androgen-dependent conditions. However, its use is associated with sexual, psychological, and physical complaints, suggesting that other mechanisms, in addition to 5α-R inhibition, may be involved. Here, a multidisciplinary approach has been used to identify potential finasteride off-target proteins. SPILLO-PBSS software suggests an additional inhibitory activity of finasteride on phenylethanolamine N-methyltransferase (PNMT), the limiting enzyme in formation of the stress hormone epinephrine. The interaction of finasteride with PNMT was supported by docking and molecular dynamics analysis and by in vitro assay, confirming the inhibitory nature of the binding. Finally, this inhibition was also confirmed in an in vivo rat model. Literature data indicate that PNMT activity perturbation may be correlated with sexual and psychological side effects. Therefore, results here obtained suggest that the binding of finasteride to PNMT might have a role in producing the side effects exerted by finasteride treatment.

Full text of DOI:10.1021/acs.jmedchem.0c02039

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Article
Three-Dimensional Proteome-Wide Scale Screening for the 5‑Alpha
Reductase Inhibitor Finasteride: Identification of a Novel Off-Target
Silvia Giatti, Alessandro Di Domizio, Silvia Diviccaro, Eva Falvo, Donatella Caruso, Alessandro Contini,
and Roberto Cosimo Melcangi*
Cite This: J. Med. Chem. 2021, 64, 4553−4566
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ABSTRACT: Finasteride, a 5-alpha reductase (5α-R) inhibitor, is a widely used drug for treating androgen-dependent conditions.
However, its use is associated with sexual, psychological, and physical complaints, suggesting that other mechanisms, in addition to
5α-R inhibition, may be involved. Here, a multidisciplinary approach has been used to identify potential finasteride off-target
proteins. SPILLO-PBSS software suggests an additional inhibitory activity of finasteride on phenylethanolamine N-methyltransferase
(PNMT), the limiting enzyme in formation of the stress hormone epinephrine. The interaction of finasteride with PNMT was
supported by docking and molecular dynamics analysis and by in vitro assay, confirming the inhibitory nature of the binding. Finally,
this inhibition was also confirmed in an in vivo rat model. Literature data indicate that PNMT activity perturbation may be correlated
with sexual and psychological side effects. Therefore, results here obtained suggest that the binding of finasteride to PNMT might
have a role in producing the side effects exerted by finasteride treatment.
more than 90 and 70%, respectively.7 Accordingly, most
INTRODUCTION
■
common complaints reported during FIN use are linked to
Finasteride (FIN), the prototypical 5-alpha reductase (5α-R)
inhibitor, is a widely used drug with clinical relevance to treat
androgen-dependent conditions, including benign prostate
sexual functions in both observational and clinical studies.8−11
In addition, depression, self-harm/suicide ideation,12,13 and
sexual dysfunction14 have been observed during FIN treat-
hyperplasia (BPH) and androgenetic alopecia (AGA). In
2017, US prescriptions for FIN were more than 9 million,1
ment. In support, the American Food and Drug Administration
Adverse Event Reporting System (FAERS) included the
with the global market projected to increase approximately
2.5% from 2020 to 2026. This compound will be increasingly
presence of such side effects.15 FIN can be consumed at a
dose of 5 mg/day to treat BPH, while a dose of 1 mg/day is
requested due to the growing prevalence of BPH, linked to the
used for AGA. When the FAERS database is analyzed for FIN
rising of the geriatric population and to the surge in the
considering the dose, the retrieved side effects fall into three
demand due to increasing hair loss issues in males globally.
categories: sexual, psychological, and physical complaints.16
Even though FIN is considered a safe medication, it causes
negative side effects.2,3 However, the molecular mechanisms
Interestingly, the lowest dose (1 mg/day) presented the
behind such adverse events are unknown. FIN blocks the
Received: November 24, 2020
Published: April 12, 2021
conversion of testosterone into dihydrotestosterone (DHT)
through competitive inhibition of type I and II 5α-R, with high
selectivity for type II in humans. 5α-R type II (5α-R2) is
present in the outer root sheaths of hair follicles, the
epididymis, vas deferens, seminal vesicles, and prostate.4−6
Thus, in men, FIN reduces prostatic and serum DHT levels by
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.jmedchem.0c02039
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Figure 1. SPILLO-PBSS screening and ranking of the available structural proteome of Homo sapiens. Plot resulting from the SPILLO-PBSS
screening and ranking of the available human structural proteome (17,925 holo- and apo-protein 3D structures from the RCSB Protein Data Bank
in January 2019, without 100% sequence identity redundancies). Proteins are ranked in a decreasing order according to their score.
highest number of reports. Among sexual function complaints,
decreased or loss of libido, disorders of ejaculation, erectile
dysfunction, testicular atrophy, orgasmic disorders, and
hypogonadism were reported with the 1 mg/day dose. The
same dose induced psychological alterations, such as an
increase in self-harm, slow cognition and psychological
pathology, changes in emotional effect, and sleep disturbances.
In addition, when analyzing the physical domain, AGA subjects
reported rash and metabolic abnormalities. However, patients
with BPH that use a higher dose of FIN (i.e., 5 mg/day)
described gynecomastia as the most frequent adverse event due
to pharmacological treatment.16 Overall, considering the side
effects described, the blockade of the conversion of peripheral
testosterone may not be the only factor responsible for the
symptomatology reported after the 1 mg/day dose.
In the present study, a 3D proteome wide-scale in silico
screening of a human protein database retrieved from the
RCSB Protein Data Bank was performed using SPILLO
potential binding sites searcher (SPILLO-PBSS, https://www.
spilloproject.com),17 aimed at identifying alternative off-target
interactions of FIN. In contrast to traditional structure-based
approaches (e.g., molecular docking simulations), this software
takes into account protein flexibility and recognizes potential
targets and off-targets of any small molecule, even when the
protein binding sites are strongly distorted (e.g., too open,
occupied, or even fully closed) compared to a suitable
conformation for the binding. SPILLO-PBSS has been recently
used in similar tasks where it was able to identify off-target
proteins (OTPs) of a new compound that was found to reduce
migration and invasiveness in U87 glioblastoma cell lines.18
Then, the reliability of SPILLO-PBSS predictions was further
assessed in silico by means of docking and molecular dynamics
(MD) simulations aimed at testing the stability of FIN
interactions with the predicted OTP, while providing insights
on its binding mode. In silico experiments were followed by in
vitro biochemical analysis that confirmed the interaction
between FIN and the identified OTP. Finally, an innovative
multilevel cross-organism transferability analysis (MCOTA)
was performed to rationally choose a suitable model organism
for subsequent molecular in vivo analysis that, in turn, further
confirmed the proposed interaction.
Overall, our study identifies a novel FIN-interacting protein
with the supposed ability to participate in the side effects
observed in FIN users.
RESULTS
■
Protein Database Screening and Ranking by SPILLO-
PBSS. With the aim of identifying FIN OTPs that could
correlate with the side effects reported in the FAERS
database,16 the SPILLO-PBSS software was used to screen
and rank the whole structural protein database of Homo sapiens
available (January 2019). Results are summarized by the plot
in Figure 1, in which points correspond to proteins ranked in a
decreasing order according to their scores. The nonlinearity of
the curve highlights the presence of a minority of proteins with
scores clearly higher than all others, corresponding to the
potential OTPs of FIN.
It should be noted that SPILLO-PBSS is designed to identify
PBSs, where the interaction between the small molecule and
the protein is mediated by proteogenic residues only, whereas
other elements such as cofactors, nucleic acids, or cell
membranes are currently not included in the model. Thus, it
is not surprising that the enzyme 5β-R, an expected target for
FIN,19 was not present in the top-ranked positions. Indeed, the
binding site of 5β-R contains the NADP cofactor that plays an
important role in mediating its interaction with FIN. The same
considerations would also apply to 5α-R2, the therapeutic
target of FIN, whose structure was not yet available in the PDB
when the calculation was performed. However, the limitations
of the model discussed above have no influence on the
reliability of the OTPs found, in which the interaction with the
drug is entirely mediated by amino acid residues.
Of the top-ranked potential OTPs identified by the software,
we focused our attention on the phenylethanolamine N-
methyltransferase (PNMT, PDB code: 4MIK). Indeed, a
possible link was identified, as explained below, between a
perturbation of the activity of this enzyme and some of the
most common adverse effects of FIN.
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Figure 2. PNMT enzymatic reaction and competitive inhibition hypothesis. The enzymatic reaction (panel A) catalyzed by PNMT is reported
along with the 2D formula of FIN (panel B). Norepinephrine and S-adenosyl-L-methionine (panel CPDB ID: 3HCD) and FIN (panel DPDB
ID: 4MIK) within their corresponding binding sites in the same PNMT region are also shown, as obtained by X-ray diffraction and SPILLO-PBSS
calculation, respectively (drawings rendered using VMD31).
PNMT as a Potential OTP for FIN. Among the top-ranked
potential OTPs of FIN, namely, in the fifth position out of
17,925 (SPILLO-PBSS score = 83.498%), PNMT (PDB code:
4MIK, UniProtKB AC: P11086) warranted further inves-
tigation. In fact, this off-target immediately caught our
attention due to its close connection with the onset of the
most common previously cited adverse effects of FIN, in
particular, those related to sexual functions. For this reason, we
decided to start our experimental validations from PNMT,
while an in-depth analysis and validations for other top-ranked
OTPs will be the matter of future studies.
PNMT principally catalyzes the conversion of norepinephr-
ine to epinephrine (Figure 2A) and is mainly involved in the
stress response.20,21 Epinephrine is strongly correlated with
mood alteration and depression.22−25 In addition, several
studies indicate a role for this hormone in the control of
erection.26−30 Overall, these findings prompted us to consider
PNMT as a potentially relevant factor in FIN side effects.
Competitive Inhibition Hypothesis. A PBS for FIN
similar to a reference binding site (RBS) (see Table S1 for a
direct comparison between the FIN RBS and PBS) was
identified within the PNMT 3D structure (PDB code: 4MIK).
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In particular, this PBS turned out to largely overlap with the
catalytic site of the enzyme that, in this specific structure, is
occupied by the bisubstrate inhibitor JIL. Within the PNMT
binding site, norepinephrine and S-adenosyl-L-methionine are
converted to epinephrine and S-adenosyl-L-homocysteine
(Figure 2A), respectively. We thus hypothesized that FIN
(Figure 2B) inhibits the catalytic activity of the enzyme by
competing with norepinephrine and S-adenosyl-L-methionine
for the same binding region (Figure 2C,D). PNMT structures
shown in Figure 2C,D correspond to PDB files 3HCD and
4MIK, respectively. In the former, PNMT is cocrystallized with
norepinephrine and S-adenosyl-L-homocysteine, which we
virtually transformed into S-adenosyl-L-methionine for the
sake of clarity.
Docking and MD Simulations. It should be noted that
SPILLO-PBSS is not a molecular docking program and is able
to identify PBSs even when they are occupied by a ligand or in
a closed conformation. Indeed, SPILLO-PBSS compares the
3D structure of the RBS with that of any PBS present in the
target protein on a similarity basis. A tolerance (5.5 Å) is
applied to take into account any conformational rearrange-
ments that may occur to the binding site upon binding, thus
implicitly considering some degree of side chain and backbone
flexibility.17,18 As an output, SPILLO-PBSS provides a
structure between the query ligand and the identified off-
target protein, as is found in the PDB, so steric clashes might
be present (see Figure S1), and the binding mode might not be
ideal (see Table S2). For these reasons, the hypothetical
complex between human PNMT (hPNMT) and FIN
generated by SPILLO-PBSS was initially subjected to a
geometry minimization. Then, we redocked FIN using MOE,
and the top docking poses were refined by MD simulations.
The top three poses of FIN (hereafter referred to as P1, P2,
and P3) obtained by docking and the binding geometry
obtained from the SPILLO-PBSS search (here referred as P0)
were subjected to 200 ns of MD simulation in an explicit
solvent, followed by binding energy calculations using the
Nwat-MMGBSA method32−34 including 30 explicit waters
around the ligand. The crystallographic complex described in
4MIK,35 where the bisubstrate ligand (referred as JIL in the
PDB file) is bound to hPNMT, was also subjected to MD
simulations using the same protocol adopted for FIN
complexes. The binding energies reported in Table 1 show
Additionally, pose P0 resulted in the most favored, even if
the energy difference between the considered poses is not large
enough to make an unequivocal choice among the predicted
binding modes. However, by inspecting the representative
geometry of the most populated cluster obtained from the
analysis of P0−P3 MD trajectories (Figure S2), we observed
that FIN is similarly oriented in P0−P2 with respect to the
hPNMT binding pocket. Thus, poses P1 and P2 can be
considered metastable states along the binding path eventually
leading to P0, where FIN is deeply buried in the binding
pocket. Conversely, a flipped orientation is observed in pose
P3, with the δ-lactam ring occupying the same position of the
N-tert-butylcarbamoyl moiety, as found for P0−P2, and vice
versa. The most relevant hydrogen bonds (H-bonds) found by
analysis of the MD trajectories of P0 and P1−P3 are reported
in Tables S3 and S4, respectively. Considering P0, the most
relevant H-bond, with an occupancy (occ %) higher than 94%,
is observed between the amino group of the Lys57 side chain
and the carbonyl group of the N-tert-butoxycarbonyl moiety of
FIN (Table S3). A second interaction involving the same
moiety is observed between the amido NH and the side-chain
oxygen of Tyr35. Interestingly, a rather relevant H-bond
bridged by a water molecule is found between the lactam
carbonyl of the 4-aza-5α-androsten-3-one group and both the
backbone carbonyl of Tyr27 and the side chain NH2 of Asn106
(occ % = 74.0). Hydrophobic interactions are also observed
between the lipophilic core of FIN and Phe30, Trp123,
Phe182, and Leu229 (Figure S2). P1 is similar to P0 in
orientation with respect to the binding site but shifted outward
(Figure S2). Indeed, the main H-bond is observed between the
lactam NH and the side chain of Asp101 (occ % = 98.3, Table
S4).
The same residue is also involved in a water-bridged
interaction with the lactam CO of FIN (occ % = 87.2),
which also interacts with Phe102 NH through a water
molecule (occ % = 40.2). Another relevant water bridge is
observed between the N-tert-butoxycarbonyl and OH of Tyr85
(occ % = 76.8), while a water-mediated interaction with the
side chain of Lys57 is only occasionally sampled (occ % =
11.8). P2 is the pose with the highest predicted binding energy
and thus, is considered the least reliable. The lactam moiety of
FIN overlays with the 6-aminopurine group of JIL that resides
in the outermost zone of the binding site (Figure S2C). Only
one direct H-bond is observed between the lactam CO of
FIN and the backbone NH of Val159 (occ % = 96.6, Table
S4). Instead, three water-bridges are found. The first involves
the lactam CO and the side chain of Asp158 (occ % = 90.0),
the others are found between the N-tert-butoxycarbonyl group
and Pro32 and Phe182 (occ % = 80.9 and 72.5, respectively).
Among the predicted poses, P3 is the one that differs the most
since it is flipped along the longitudinal axes (Figure S2D) but
still has a rather low binding energy (Table 1). However, this
low binding energy can be explained by considering the
chemical properties of FIN. Indeed, the two amide groups,
separated by the hydrophobic polycyclic core, confer a rather
symmetrical electrostatic distribution to FIN itself (Figure S4).
Thus, an interaction profile with hPNMT that is comparable to
that described for poses P0−P2 can also be observed for P3
(Table S4). H-bonds with Tyr27 and Tyr35 can indeed be
observed but involving the carbonyl group of the N-tert-
butoxycarbonyl moiety (occ % = 99.7) and the lactam NH
(occ % = 87.5), respectively. Moreover, an additional H-bond
is observed between the exocyclic carbonyl and the OH of
Table 1. Binding Energies Computed by Nwat-MMGBSA
Analysis (Nwat = 30) of the Last 20 ns of the MD
Trajectories of P0−P3 and JIL
ΔEbind (kcal/mol)
P0
P1
P2
P3
JIL
−58.4 3.7
−55.3 4.6
−53.6 3.9
−55.8 3.9
−75.3 5.7
that the crystallographic ligand JIL is significantly more potent
than FIN, which is expected considering that JIL fills the rather
long binding pocket of hPNMT better than FIN (Figure S2A).
However, all four binding poses obtained for FIN were stable
enough during the 200 ns of MD simulation (Figure S3) to
support the capability of FIN to bind hPNMT, as predicted by
SPILLO-PBSS.
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Tyr40 (occ % = 42.8), while two water-mediated bridges are
found between the lactam NH and Asp101 side chain (occ % =
35) and Phe102 backbone (occ % = 35).
To better understand how FIN can bind to hPNMT, a
comparison was made between our predictions and the
experimental binding modes determined for FIN bound to
5α-R2, whose crystal structure was recently released,36 and 5β-
R (Figure 3). In all three structures, the binding of FIN with
the enzyme is mediated by several hydrophobic contacts and
by H-bonds involving the cyclic and exocyclic amido groups. In
the 5α-R2 complex (Figure 3A), direct H-bonds are found
between Arg114 (donor) and the FIN N-tert-butylcarbamoyl
carbonyl (acceptor) and between Glu57 (acceptor) and the
FIN δ-lactam NH (donor). A distance of 12.5 Å is measured
between Arg114 and Glu57.
These direct H-bonds between FIN and the receptor are not
observed with 5β-R (Figure 3B). Although H-bonds are not
essential for binding, when hydrophobic interactions are
possible, they are important for ligand efficiency and
specificity.37,38 However, crystallographic waters are found
making H-bonds with the same donor and acceptor groups of
FIN, and the distance measured between the two water
oxygens is 12.7 Å. When studying the hPNMT structure, a
cationic and an anionic residue, Lys57 and Asp101,
respectively, were found able to interact with FIN similarly
to 5α-R2. In particular, Lys57 directly interacts with the N-tert-
butylcarbamoyl carbonyl of FIN, whereas Asp101 interaction
with the drug is mediated by a bridging water connecting the
FIN NH group to the Asp101 side chain. Here, the distance
between Lys57 and Asp101 is 15.5 Å (Figure 3C), 3.0 Å larger
than the distance measured between 5α-R2 Arg114 and Glu57,
but the interaction is not lost. Indeed, the distance measured
between the Lys57 and water HOH277, directly connected to
FIN NH, is of 13.0 Å. Within the second most favored pose
(P1) predicted for FIN bound to hPNMT (Figure S2B), Lys57
and Asp101 are at a distance (15.8 Å) similar to that measured
in P0, but in P1, FIN is directly bound to Asp101 and a water
bridge mediates its interaction with Lys57.
In Vitro hPNMT Activity Assay. To verify whether FIN
might make contact with hPNMT enzyme through an
inhibitory interaction, an in vitro inhibition assay was set up.
First, the basal hPNMT activity (between 5.43 and 9.28 pmol/
min/μg) was obtained and was in line with the expected
activity indicated by the supplier. Then, FIN was tested at the
concentration of 50 μM to evaluate the inhibitory activity in
vitro (Figure 4). In parallel, the known PNMT inhibitor
LY7833539 was tested at the same concentration (50 μM) as a
positive control. The concentrations tested were selected based
on previous protocols evaluating the unknown compounds’
inhibitory activity on PNMT activity.39,40 In these reports,
inhibitors were tested at the same concentration of the methyl
donor (S-adenosyl-L-methionine). Thus, based on the protocol
indicated by the supplier, the selected compound’s concen-
tration was 50 μM, as S-adenosyl-L-methionine.
As reported in Figure 4A, LY78335 inhibited hPNMT
activity by nearly 60%. The same concentration of FIN (i.e., 50
μM) was able to reduce hPNMT activity by more than 30%.
Based on the published Ki,39 in the present assay, LY78335
was able to inhibit less than expected PNMT activity. On the
other hand, it is important to recall that the published
assays39,40 were conducted under very different experimental
conditions, and this affected the final inhibitory activity.
Moreover, this situation is very far from the physiological
Figure 3. Complexes of FIN and 5α-R2, 5βR and hPNMT. FIN is
depicted by a tube representation with carbon atoms colored in
orange. Panel A represents the complex of the drug with 5α-R2 (PDB
ID: 7BW1); Panel B represents the complex of the drug with 5βR
(PDB ID: 3G1R); panel C represents the complex of the most
favored pose of the drug bound to hPNMT, as obtained by
simulations. H-bonds involving FIN and receptor or solvent atoms are
represented by dashed light-blue lines including a cylinder, whose
length is proportional to the H-bond estimated strength. Donor−
acceptor distances are reported for H-bonds between FIN and
receptors. Selected geometrical distances between anchor points
(enzyme atoms or bridging waters) of FIN to each system are
represented by plain black lines along with corresponding values.
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Figure 4. hPNMT activity. Human recombinant enzyme activity was detected by fluorescence in the presence of a vehicle (DMSObasal PNMT
activity) of LY78335 or FIN. Panel A: PNMT activity in the presence of a high-affinity inhibitor LY78335 (50 μM) or FIN (50 μM). The columns
represent the mean SEM of duplicate reactions. Panel B: dose−response curve in the presence of a high-affinity inhibitor LY78335 (black
rhombus; from 6.4 nM to 50 μM) or FIN (gray triangle; from 6.4 nM to 50 μM). The symbols represent the mean SEM of duplicate reactions.
Figure 5. Adrenal catecholamine and PNMT protein levels. Catecholamine levels were detected by liquid chromatography tandem mass
spectrometry analyses in control (Ctrl; n = 6) and FIN-treated animals (FIN, n = 6). Adrenal PNMT protein levels were detected by western
blotting in control (Ctrl; n = 6) and FIN-treated animals (FIN, n = 6). Panel (A) norepinephrine levels; panel (B) epinephrine levels; panel (C)
representative blot of PNMT (25 kDa) and GAPDH (37 kDa); panel (D) quantification of PNMT protein levels. The columns represent the mean
SEM after normalization for the starting tissue (panel A and B) or for GAPDH (panel D). Data were analyzed by Student’s t-test. *p ≤ 0.05 vs
Ctrl.
condition in adrenal glands. Thus, the concentrations here
reported are intended only for a comparison of the inhibitory
ability of the compounds in this particular assay and do not
want to describe any physiological situation. To test if the assay
we designed is reliable, a dose−response curve was prepared
(Figure 4B). As reported, several concentrations (from 6.4 nM
to 50 μM) of LY78335 and FIN were able to inhibit PNMT
activity to different extents. The results indicated that LY78335
inhibits with higher potency PNMT activity than FIN, as
expected.
Rational Design of Animal Testing. To evaluate if the
described in vitro inhibitory effect was also maintained in vivo,
further animal tests have been rationally designed in
compliance with the guidelines provided by the 3R
principles.41 In fact, the precise knowledge of the human
SPILLO-PBSS-predicted OTP for FIN, along with the
structural details concerning its PBS provided by the software,
made it possible to perform an innovative MCOTA aimed at
identifying the most suitable model organism for the in vivo
tests from a pool of possible model organisms (i.e.,
Caenorhabditis elegans, Danio rerio, Drosophila melanogaster,
Mus musculus, and Rattus norvegicus). In particular, a basic
check for the presence of the PNMT gene allowed to primarily
discard C. elegans, D. rerio, and D. melanogaster as the PNMT
gene is excluded from their genome. Then, an overall
comparison between the hPNMT sequence and PNMT
sequences of M. musculus and R. norvegicus, followed by a
local 3D structural comparison between their PBSs for FIN,
provided a positive assessment for the use of these two model
organisms in our tests, with a slight advantage for R. norvegicus.
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A final SPILLO-PBSS calculation was then performed to
confirm that R. norvegicus and M. musculus PNMTs belong to
the target zone of the global FIN OTPs ranking. Thus, only
these two structures were included in the calculations
performed previously, within the human protein database.
Namely, R. norvegicus and M. musculus PNMTs ranked fifth
(causing the hPNMT to scale down to sixth) and seventh (out
of 17,927), respectively. Thus, analysis by MCOTA
determined that C. elegans, D. rerio, and D. melanogaster were
not suitable for our tests, while indicating that R. norvegicus and
M. musculus were rather reliable and nearly equivalent (see
Table S5). R. norvegicus was chosen because of the team’s
experience with this model organism during previous studies
concerning FIN.42,43
Catecholamine and PNMT Adrenal Content. Based on
the results obtained by MCOTA, adult male Sprague-Dawley
rats were treated with FIN, as reported.42,43 As described
previously,43 the dose and timing applied in this experiment
have been selected based on the ability to affect DHT plasma
levels and prostate gland weights. Indeed, plasma DHT levels
and prostate gland weights were both reduced in FIN-treated
rats, thus indicating an effective FIN treatment (43 and data
not shown). As reported above, PNMT enzyme is responsible
for epinephrine production and the adrenal gland represents
the main source of this hormone.44 Therefore, epinephrine and
norepinephrine levels were evaluated by liquid chromatog-
raphy tandem mass spectrometry analyses in the adrenal glands
of FIN-treated rats and compared to vehicle-treated animals.
As reported in Figure 5, norepinephrine levels significantly
increased after drug treatment (Figure 5A), while those of
epinephrine decreased in a statistically significant way (Figure
5B). The data obtained are in line with the hypothesis of a
reduced enzymatic activity due to FIN inhibition.
gland by liquid chromatography−tandem mass spectrometry
(LC−MS/MS). In support of our hypothesis, results revealed a
reduction in the levels of epinephrine, with a concomitant
increase of norepinephrine levels, coupled to no effect on
PNMT protein levels. These findings suggest that the altered
hormonal levels were due to a reduction of the enzymatic
activity rather than to decreased levels of PNMT.
When this study was conducted, the crystal structure of FIN
complexed with its principal target 5α-R2 was not available in
the PDB, where only the complex with 5β-R was present.19
The former enzyme is irreversibly inhibited by FIN that forms
a covalent bond with the NADPH cofactor. A subnanomolar
affinity was determined for the NADP−dehydrofinasteride
(NADP−DHF) adduct, thus explaining the irreversible
inhibition. Conversely, FIN inhibits 5β-R with a low μM
affinity,19 and no covalent adduct is formed. A crystal structure
of 5α-R2 complexed to NADP-DHF has been recently
released,36 allowing a comparison between the experimental
binding modes determined for FIN bound to either 5α-R2 or
5β-R and the predicted binding mode for hPNMT (Figure 3).
In the three structures, it can be observed that FIN binding
with the enzyme is mediated by several hydrophobic contacts
and by H-bonds involving the cyclic and exocyclic amido
groups. Thus, it is possible that a distance of 12.5 Å between a
H-bond donor and a H-bond acceptor is needed for optimal
ligand−receptor interactions. This distance is met for 5α-R2
only, the enzyme for which FIN is highly selective. The same is
not observed in 5β-R, but FIN can still bind, even if with
micromolar affinity, due to hydrophobic interactions and
water-mediated H-bonds. Concerning hPNMT, the H-bond
donor and acceptor are at the right position, but their distance
is too long for optimal interactions. However, water can
accommodate this distance by forming bridges between FIN
and Lys57 or Asp101.
To determine if the FIN effect on PNMT was due to a
deficit in its protein expression levels, western blotting was
performed. No differences in protein levels of PNMT enzyme
were detected in the adrenal glands of FIN-treated rats in
comparison to vehicle-treated animals (Figure 5C,D). Overall,
the data obtained after in vivo FIN treatment indicate a
decreased conversion of norepinephrine into epinephrine in
the adrenal glands of drug-treated rats, reasonably due to a
reduced enzymatic activity.
PNMT represents the final step in the pathway generating
epinephrine, thus identifying adrenergic cells. The PNMT
expression has been found in different regions of the nervous
system, including the medulla oblongata, brainstem, locus
coeruleus, diencephalon, and sympathetic ganglia,45 as well as
in other tissues,70−75 such as in the heart, spleen, thymus,
retina,46 and mainly, in chromaffin cells of the adrenal
medulla.44
While epinephrine is responsible in controlling the
peripheral stress response, norepinephrine seems to have
major relevance in the brain.
DISCUSSION AND CONCLUSIONS
■
To our knowledge, this is the first report demonstrating that
other proteins besides 5α-R may be OTPs of FIN. The 3D
proteome-wide scale SPILLO-PBSS in silico screening
identified hPNMT as a top-ranked protein possibly able to
interact with the drug. Furthermore, SPILLO-PBSS showed
that the binding site of FIN within hPNMT overlaps with that
of norepinephrine and S-adenosyl-L-methionine. Therefore, it
is possible that FIN, competing with physiological substrates,
may reduce the catalytic activity of PNMT in converting
norepinephrine to epinephrine. Docking and MD simulations
elucidated possible binding modes of FIN within the hPNMT
binding site and provided further elements supporting the
stability of the predicted interaction. The in silico predictions
were confirmed by a biochemical assay, indicating an inhibition
of hPNMT by FIN in vitro. To evaluate whether this effect is
maintained in vivo, animal tests were designed by MCOTA in
compliance with the 3R principles. Thus, the ability of FIN in
inhibiting PNMT was tested in vivo by treating male rats and
analyzing norepinephrine and epinephrine levels in the adrenal
Focusing on the peripheral actions of these two catechol-
amines, it is important to recall the involvement of epinephrine
in the “fight or flight” response.47 To response a stressful
situation (emotional, environmental, or physical), the adrenal
medulla rapidly releases the stored epinephrine into the
general circulation.21 This, in turn, leads to the subsequent
release of cortisol in humans. The cooperation among these
hormones produces different physiological responses involving
central and sympathetic nervous system functions. Indeed,
norepinephrine and epinephrine are involved in the increase in
the heart rate and blood pressure. Metabolically, they increase
fat metabolism and glucose blood levels to sustain the energy
demand of the body. Exogenous epinephrine administration
increases blood glucose by inhibition of insulin release,
stimulation of glucagon release, hepatic glycogenolysis, and
hepatic and renal gluconeogenesis.48 Both norepinephrine and
epinephrine stimulate β3 receptors to promote fat metabolism,
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while epinephrine stimulates the thermogenic response in
humans.49
known gut microbiota−brain axis, the involvement of altered
microbiota and depression−anxious conditions reported by
FIN users should be taken into account.
The noradrenergic system is also involved in the regulation
of male sexual functions.50,51 The central control of erection is
linked to ascending signals to the brain and descending ones to
the spinal cord.51 In addition, adrenergic innervation is
strongly present in penile arteries and veins and in cavernosal
smooth muscle.52 In support, vascular control by the
autonomic nervous system regulates erection.53 The first
phase of the erection cycle in humans is the flaccid state; then,
upon stimuli, the penis reaches tumescence until complete
rigidity, to conclude with the detumescence phase, back to the
initial flaccid condition. Adrenergic control is involved in all
erection phases.54 Indeed, norepinephrine release in the penis
induces the flaccid state by contracting the trabecular smooth
muscle.55 Accordingly, in healthy men, a reduction in
norepinephrine levels is associated with penile tumescence
and erection, while an increased level of this hormone is
associated with the transition from rigidity to detumescence.26
Thus, norepinephrine is involved in transforming the penis
from the erect to flaccid state.56 In contrast to norepinephrine,
epinephrine levels are increased in the tumescence phase in
relation to the flaccid condition and then decreased in the rigid
and detumescence phases.27 Thus, these two hormones have
opposite functions in penile erection. In human psychogenic
and neurogenic erectile dysfunctions, the rigidity phase cannot
be achieved. Interestingly, in these conditions, plasma
norepinephrine levels are high in flaccidity, tumescence and
detumescence phases, suggesting an impairment in adrenergic
signals.27 In summary, the balance of epinephrine and
norepinephrine is crucial for achieving an erection.76,77 Thus,
based on the results we obtained, it is possible that FIN
administration, by affecting PNMT enzymatic activity, is
involved in the sexual problems reported by FIN users.
Interestingly, norepinephrine may also influence the gut
microbiota, the community of bacteria that reside in the
mammalian gut and have profound effects on physiology and
disease.57 In particular, gut microbiota is involved in essential
functions, such as digestion, vitamin production, and defense
against pathological bacteria.25 On the other hand, alterations
of microbial composition in the gastrointestinal tract are
associated with many pathological situations, including
depression,58 anxiety,59 and other neurological patholo-
gies.60,61 A direct interaction among the host and gastro-
intestinal bacteria has been proposed, with catecholamines as
major modulators of this communication.62 Indeed, given that
the communication is bidirectional, many studies have focused
on gut bacteria production of catecholamines and their
influence on brain functions.25,62 However, how host-produced
catecholamines can influence the gut microbiome has been less
explored. In vitro evidence suggests that norepinephrine
promotes the growth of Gram-negative bacteria and, in
general, increases virulence and facilitates bacterial invasion.63
In addition, Houlden and colleagues described altered
microbiota composition in an experimental model of stroke,
where increased norepinephrine levels have been observed.64
Interestingly, FIN administration altered gut microbiota
composition in male rats.42 Whether the impaired microbial
composition was a consequence of presumed altered levels of
norepinephrine or rather a direct action of FIN on the gut
community remains to be assessed. However, the possible
influence of catecholamine in the rat experimental model could
not be ruled out.78−83 In this context, in relation to the well-
In conclusion, data presented here indicate that the 5α-R
inhibitor FIN is also able to interact with PNMT. This concept
is supported by 3D proteome wide-scale screening, by docking
and MD simulations, by an in vitro biochemical assay, and in
vivo analysis. We believe that the present findings may help in
explaining the various side effects reported by FIN users, in
particular those related to sexual function and gut-microbiota
alterations. In future studies, it will be critical to further explore
the consequences of FIN−PNMT interaction. For instance,
recent observations identified PNMT genetic variants and
polymorphisms in human subjects. Interestingly, some
haplotypes resulted in decreased activity or accelerated
degradation and different abilities to produce epinephrine in
the basal condition or during exercise.65,66,84,85 Probably,
carriers of PNMT variants might react differently to FIN
administration, inducing different responses. Finally, consider-
ing that FIN may cross the blood−brain barrier, it will be
crucial to explore the possible influence of this drug on brain
PNMT67−69 and the subsequent pathology.
EXPERIMENTAL SECTION
■
Drugs and Reagents. FIN (Merck Life Science S.r.l., Milano,
Italy, Catalog #F1293), dopamine-1,1,2,2-d4 hydrochloride (Merck
Life Science S.r.l., Milano, Italy, Catalog # 655651), MES buffer
(Merck Life Science S.r.l., Milano, Italy), DL-Norepinephrine hydro-
chloride (Merck Life Science S.r.l., Milano, Italy, Catalog # A7256), S-
adenosyl-L-methionine (AdoMet) (Merck Life Science S.r.l., Milano,
Italy, Catalog # A7007), reduced glutathione, and ThioGlo 3
fluorescent thiol reagent (Covalent Associates, Inc., Catalog #
T003) were obtained. All recombinant human enzymes and the
PNMT inhibitor LY78335 (Catalog # 4060) were purchased from
Bio-Techne, Milano, Italy: PNMT (rhPNMT, Catalog # 7854MT),
adenosyl homocysteinase/AHCY (rhAHCY, Catalog # 6466AH),
adenosine deaminase/ADA (rhADA, Catalog # 7048AD).
The purity of LY78335 and FIN compounds was declared to be 99
and ≥98%, respectively, by the manufacturers.
Antibodies: PNMT (Novus Biologicals, Centennial, CO, USA,
NBP2-00688); GAPDH (Santa Cruz, Dallas, Texas, US, sc-25778).
Protein Database Preparation. The protein database used for
SPILLO-PBSS screening was generated by collecting all human
protein 3D structures available in the RCSB Protein Data Bank
(update January 2019) experimentally solved by either X-ray
diffraction or solution NMR, excluding 100% sequence identity
redundancies. It included 17,925 holo- and apoprotein 3D structures.
Biological assemblies for proteins showing multimeric structures were
then generated by the MakeMultimer program (http://watcut.
uwaterloo.ca/tools/makemultimer/index) according to the BIOMT
transformation matrices included in the PDB files. For multimodel
PDB files from solution NMR experiments, only the first model was
included in the database. No further refinements of the protein
structure were conducted to improve the quality of protein 3D
structures in the database.
RBS Generation. The RBS used by SPILLO-PBSS to search the
protein database for potential OTPs of FIN was obtained using
molecular modeling techniques and the standard RBS generation
protocol described in the SPILLO-PBSS paper.17 It included 18
amino acid residues directly interacting with the drug without any
cofactor or water-mediated contact. The detailed amino acid
composition of the RBS is reported in Table S1.
In Silico Screening and Ranking of the Protein Database. An
unbiased and systematic search for FIN PBSs within all protein 3D
structures included in the database was performed by SPILLO-PBSS.
Calculations were performed using a rotation step of 30° and a grid
spacing of 2.0 Å, with the geometric tolerance set to 5.5 Å. SPILLO-
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PBSS analyzed all proteins of the database, and a ranking of the PBSs
for the drug was obtained for each protein and stored by the program.
Then, the protein database was ranked according to the highest PBS
score, representing the highest similarity to the corresponding RBS,
obtained from each analyzed protein 3D structure.
vehicle dimethyl sulfoxide (DMSO) were preincubated separately for
30 min at 25 °C, with the hPNMT enzyme. For each experiment, a
duplicate reaction of experimental points and a blank (without
hPNMT), hereafter referred to as “reactions”, was performed. Then, a
substrate mixture containing 50 μM (final concentration) AdoMet
and 125 μM DL-norepinephrine hydrochloride was incubated to all
experimental points for 30 min at 25 °C. The reactants were boiled at
95−100 °C for 5 min and then cooled in ice for 3 min. An equal
volume of the hydrolysis mixture (6.25 μg/mL rhAHCY and 0.625
μg/mL rhADA) was added to reactions. After 1 h incubation at 37 °C,
the samples were plated into a black multiwell in duplicate and 50 μM
of ThioGlo 3 fluorescent thiol reagent was added to reactions and to
the standard curve points prepared with glutathione. The sealed plate
was incubated at RT for 5 min in the dark, and excitation and
emission wavelengths of 380 and 445 nm (top read), respectively,
were read with an EnSpire workstation (PerkinElmer). For the dose−
response curve, the following (final) concentrations have been tested:
6.4 nM, 32 nM, 160 nM, 800 nM, 4 μM, 20 μM, and 50 μM.
The specific activity (pmol/min/μg) was calculated as follows:
thiol produced (pmol)/[incubation time (min) x amount of enzyme
(μg)]. All absorbance values were adjusted for the blank measure-
ment, and then, the values obtained were interpolated with the
standard curve. The result obtained for each sample represents the
thiol produced. Since each experiment was performed as a duplicate,
the final result was the mean of the two values obtained. Finally, the
specific activity of PNMT + vehicle was put at 100%, and the activity
of PNMT with LY78335 or FIN was calculated in relation to PNMT
+ vehicle.
Docking. The receptor model used for docking was derived from
the PDB entry 4MIK35 that describes the crystal structure of hPNMT
in complex with an inhibitor (hereafter referred to as JIL, as named in
the PDB file) at a resolution of 1.95 Å. Chain B, where the N-
terminus is better resolved, was used to prepare the model.
Crystallographic water molecules and ligands other than the inhibitor
were initially removed. Then, the structure was processed by the
QuickPrep tool of MOE,86 using the default settings. QuickPrep
automatically corrects any inconsistency of the PDB file and
protonates the complex at pH = 7. The last step consists of a
tethered geometry minimization, where tethers (10 kcal/mol/Å) are
applied to the receptor atoms up to 8 Å from the ligand, while keeping
the farthest ones fixed. The receptor coordinates were then saved for
further studies. Docking was initially tested on the JIL ligand using the
MOE software. The optimized protocol used the Triangle Matcher
algorithm86 with a timeout of 30,000 s and many returned poses
increased to 100,000. A first scoring was performed using the London
dG function, and rescoring was done on the top 100 poses using the
induced fit method coupled to the GBVI/WSA dG function.87 The
top ranked pose showed a root-mean-squared deviation (RMSD) to
the crystallographic ligand of 1.9 Å and was then considered reliable
enough to be applied to the docking of FIN.
Molecular Dynamics. After visual inspection, the top three
docking poses of hPNMT complexed to FIN (hereafter referred to as
P1, P2, and P3, respectively, according to the ranking obtained from
docking), with the structure of the complex directly obtained from the
SPILLO-PBSS run (hereafter referred to as P0), were subjected to
MD simulations. The model of JIL bound to hPNMT, obtained as
described above, was also subjected to the same protocol as a
reference. All MD simulations and analyses were performed using the
Amber18 and AmberTools18 software packages.88 Charge parameter-
ization of JIL and FIN was performed by the antechamber using the
restrained electrostatic potential (RESP) method.89 After the
solvation of the complexes by an octahedral TIP3P box90 up to 10
Å from the solute, the charge of each system was neutralized by
adding four Na+ ions. MD simulations were then conducted using
pmemd.cuda.91 The ff14SB92 and gaff93 force fields were adopted for
protein and ligands, respectively. A protocol consisting in multiple
equilibration steps up to a final temperature of 300 K at a constant
volume and temperature (NVT) and constant pressure and
temperature (NPT) was adopted as detailed in previous works,94,95
followed by 200 ns of NPT production run. After analysis of the
variation of the RMSD versus time during the whole trajectory
(Figure S1), H-bond (donor−acceptor distance cutoff = 3.5 Å; donor-
H-acceptor angle cutoff = 135 deg) and clustering (five clusters based
on mass-weighted RMSD were generated using the average linkage
algorithm) analyses were performed using cpptraj88 on the last 20 ns
of each MD trajectory, where the RMSD was better converged. MD
simulations were performed twice, and comparable results were
obtained. Binding energies were computed using the Nwat-MMGBSA
method.32−34 As suggested by the authors, 30 explicit water
molecules, that are the closest to ligand atoms in each selected
frame, were included in the calculation as part of the receptor (Nwat
= 30), while the entropic contribution to the binding energy was
neglected.
Multilevel Cross-Organism Transferability Analysis. The
MCOTA consisted of the following checks and analyses performed
on a pool of possible model organisms (i.e., C. elegans, D. rerio, D.
melanogaster, Mus musculus, and R. norvegicus):
(i) Basic check (Table S5, column I). A basic check for the
presence/absence of the PNMT gene in the considered model
organisms was performed by the UniProtKB webserver.
(ii) Overall protein sequence comparison (Table S5, column II).
An assessment of the degree of similarity and identity
(calculated by the EMBOSS Needle webserver https://www.
ebi.ac.uk/Tools/psa/emboss_needle/) between the hPNMT
(UniProtKB: P11086) and the same protein in M. musculus
(UniProtKB: P40935) and R. norvegicus (UniProtKB: P10937)
provided a preliminary evaluation of these two model
organisms. However, although this analysis is informative
regarding an overall comparison between sequences, it cannot
detect local differences in the PBS amino acid composition,
which may affect the way the PNMT from different organisms
interact with FIN.
(iii) Local 3D structural comparison (Table S5, column III). A
search (calculations carried out by the SPILLO-PBSS
software) for the PBSs on the PNMT of M. musculus and R.
norvegicus allowed to assess their similarity with the PBS found
on the hPNMT and to point out any local differences (e.g., a
different amino acid composition of the PBSs) that may affect
their interaction with FIN. After a first visual check aimed at
confirming the same position of the PBSs on the various
protein structures and the spatial orientation of FIN within the
PBSs, the SPILLO-PBSS scores were compared, which
simultaneously consider several features of the PBSs, including
their amino acid composition and the stabilizing contribution
of each amino acid residue to the ligand binding.17 Since no
PNMT 3D structure of the two model organisms was available
in the RCSB Protein Data Bank, it was first necessary to
generate the homology models, calculated using the SWISS-
MODEL webserver96 using the hPNMT 3D structure (PDB
code: 4MIK) as a template; then, the SPILLO-PBSS software
was applied (with the same settings previously used for the
protein database screening) to analyze the PNMT 3D
structures of M. musculus and R. norvegicus.
In Vitro hPNMT Activity Assay. A first set of experiments was
performed to evaluate the in vitro enzymatic hPNMT activity. The
assay was performed by following the protocol developed by R&D,
Bio-Techne, available on-line, to measure PNMT S-adenosyl-L-
methionine-dependent ability to transfer a methyl group to
norepinephrine. The assay is based on the measurement of the thiols
present after PNMT methylation of norepinephrine; thus, it
represents an indirect quantification of PNMT activity. Then, an in
vitro hPNMT inhibition assay was set, and the protocol was modified
as follows. In brief, the known PNMT inhibitor LY78335, FIN or
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incubated with a rabbit polyclonal antibody against GAPDH, as the
housekeeping protein; both antibodies were incubated overnight at 4
°C. After extensive washing, the filters were incubated with an
antimouse and antirabbit horseradish peroxidase-conjugated secon-
dary antibody, respectively. After washing, bound antibodies were
detected with the ECL method. A Chemi-Doc TM XRS+ system
(Bio-Rad, Segrate, Italy) was used to acquire chemiluminescent
signals, while Image Lab TM software, version 3.0 (Bio-Rad, Segrate,
Italy), was used for their quantification. The mean control value
within a single experiment was set to 100, and all other values were
expressed as percentage. The values of controls from different
experiments were all within 10%.
Statistical Analyses. The quantitative data obtained by the
catecholamine experiments were analyzed by inferential statistical
analysis in accordance with the experimental protocols and the data
(i.e., Student’s t-test). p ≤ 0.05 was considered significant. Analyses
were performed using GRAPHPAD PRISM, version 4.00 (GraphPad
Inc., La Jolla, Calif., USA).
(iv) Proteome-scale ranking-position evaluation (Table S5, column
IV). The ranking positions of PNMT of M. musculus and R.
norvegicus have been evaluated with respect to the interaction
with FIN. This made it possible to assess whether they belong
to the target zone and how they eventually rank with respect to
hPNMT. The new ranking including the two model organism
PNMTs was obtained by integrating the SPILLO-PBSS scores
calculated in the previous step into the original ranking of the
protein database.
Animals and Treatments. Male Sprague-Dawley rats (150−175
g at arrival, Charles River Laboratories, Lecco, Italy) were housed in
the animal care facility of the Dipartimento di Scienze Farmacologiche
̀
e Biomolecolari (DiSFeB) at the Universita degli Studi di Milano,
Italy. All animals were kept in standard rat cages with food and tap
water available ad libitum and under controlled temperature (21
4
°C), humidity (40−60%), room ventilation (12.5 air changes per
hour), and light cycles (12 h light/dark cycle; at 7 a.m./off 7 p.m.).
The rats were acclimated to the new environment for 7 days, and
then, after they were 2 months old, they were randomly divided into
two experimental groups: (i) control, vehicle-treated rats (Ctrl) and
(ii) FIN-treated rats. All experimental procedures were performed in
strict accordance with the Italian and EU regulation on animal welfare
and were previously approved by our institutional animal use and care
committee (OPBA office) by the Italian Ministry of Health
(authorization 1083/2015-PR) and followed national (D.L. no. 26,
March 4, 2014, G.U. no. 61 March 14, 2014) and international laws
and policies (EEC Council Directive 2010/63, September 22, 2010:
Guide for the Care and Use of Laboratory Animals, United States
National Research Council, 2011).
PDB ID Codes. PNMT: 4MIK.
PNMT (cocrystallized with norepinephrine and S-adenosyl-L-
homocysteine): 3HCD 5α-R2: 7BW1.
5βR: 3G1R.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02039.
■
sı
Amino acid residues included in the RBS of finasteride
and the corresponding PBS amino acid residues, steric
clashes between amino acids of hPNMT and FIN, most
relevant H-bonds and water-mediated bridges between
FIN and hPNMT, MCOTA on five possible model
organisms, RMSD vs the time profile for the 200 ns MD
trajectory of P0−P3 and JIL, and representation of the
electrostatic map generated for finasteride using the
default options within the MOE software (PDF)
FIN and vehicle treatments have already been described.43 In brief,
FIN (1 mg/rat/day) dissolved in a vehicle (5% ethanol in sesame oil)
or vehicle only was subcutaneously (sc) administered to the animals
daily for 20 days, and all rats were sacrificed 24 h after the last
treatment. The adrenal glands were dissected, rapidly frozen, and
stored at −80 °C for subsequent analyses.
Catecholamine Quantification by Liquid Chromatography−
Tandem Mass Spectrometry Analysis. For catecholamine (i.e.,
norepinephrine and epinephrine) analysis, one adrenal gland was
analyzed as reported by Su and colleagues97 with modifications. In
brief, the tissue was homogenized using a Tissue Lyser II (Qiagen,
Hilden, Germany) in ice-cold methanol after weighing precisely (1 g
of tissue in 4 mL of methanol) supplemented with an internal
standard, dopamine-1,1,2,2-d4 hydrochloride. Then, to remove
particulate matter, the adrenal homogenates were centrifuged at
20,817g for 20 min at 4 °C. The supernatant was evaporated to
dryness under a stream of nitrogen. The dry pellet was reconstituted
with 300 μL of water, vortex-mixed for 10 s, and 300 μL of
chloroform−isopropanol (100:30, v/v) was added. After vortex-
mixing for 2 min, the mixture was centrifuged at 1187g for another 5
min. The upper aqueous layer was filtered with a 0.2 μm filter (SRC
grade, regenerated cellulose membrane filter, CHMLAB Group), and
then, for norepinephrine and epinephrine analysis, 5 μL of the sample
volume was injected. To obtain the chromatographic separation of the
analytes, a column for HPLC Luna Omega 5 μm PS C18 100 Å was
used (Phenomenex, California, USA). The model of the mass
spectrometer used is API 3500 (AB Sciex, USA), equipped with an
electrospray source and triple quadrupole analyzer, interfaced with a
pump for the HPLC model EXION SL (Sciex, USA).
Western Blotting. Snap-frozen adrenal glands were homogenized
in a Tissue Lyser II (Qiagen, Hilden, Germany) in a cold lysis buffer
(phosphate-buffered saline, pH 7.4, added with 1% Nonidet P-40 and
with a protease cocktail inhibitor), then sonicated, and centrifuged to
remove particulate matter. After quantification, equal amounts of
proteins for each sample were loaded into polyacrylamide electro-
phoresis gel and then electroblotted to a nitrocellulose membrane.
For immunoblot detection, the filter was cut, and membranes were
blocked on an orbital shaker at room temperature in PBS with added
0.1% Tween 20 and 10% nonfat dried milk. Successively, the 25 kDa
part of the filter was incubated with mouse monoclonal antibody
against PNMT, while in parallel, the 37 kDa part of the filter was
Residual PNMT activity (%) with 50 μM FIN (CSV)
Coordinates of the binding site residues (4.5 Å from the
ligand) of the representative geometry obtained by
clustering the MD trajectory of JIL (PDB)
P0 (PDB)
P1 (PDB)
P2 (PDB)
P3 (PDB)
Coordinates of the SPILLO-PBSS output for PNMT-
FIT (PDB)
AUTHOR INFORMATION
Corresponding Author
■
Roberto Cosimo Melcangi − Department of Pharmacological
and Biomolecular Sciences, Universita degli Studi di Milano,
̀
20133 Milano, Italy; orcid.org/0000-0003-0861-8967;
Phone: +39-02-50318238; Email: roberto.melcangi@
unimi.it; Fax: +39-02-50318202
Authors
Silvia Giatti − Department of Pharmacological and
̀
Biomolecular Sciences, Universita degli Studi di Milano,
20133 Milano, Italy
Alessandro Di Domizio − Department of Pharmacological
̀
and Biomolecular Sciences, Universita degli Studi di Milano,
20133 Milano, Italy; SPILLOproject, 20037 Milano, Italy;
orcid.org/0000-0003-1867-2449
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Silvia Diviccaro − Department of Pharmacological and
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Biomolecular Sciences, Universita degli Studi di Milano,
20133 Milano, Italy
Eva Falvo − Department of Pharmacological and Biomolecular
̀
Sciences, Universita degli Studi di Milano, 20133 Milano,
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578−589.
Alessandro Contini − Dipartimento Di Scienze
̀
Farmaceutiche, Universita degli Studi di Milano, 20133
Milano, Italy; orcid.org/0000-0002-4394-8956
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02039
Author Contributions
S.G. and A.D.D. contributed equally to this work. SPILLO-
PBSS analysis/MCOTA: A.D.D.; docking/MD: A.C.; in vitro
assay: S.G; LC/MS−MS: S.D. and D.C; WB: E.F; Writing
S.G., A.D.D, A.C., and R.C.M.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(11) Tsunemi, Y.; Irisawa, R.; Yoshiie, H.; Brotherton, B.; Ito, H.;
Tsuboi, R.; Kawashima, M.; Manyak, M. Long-term safety and
efficacy of dutasteride in the treatment of male patients with
androgenetic alopecia. J. Dermatol. 2016, 43, 1051−1058.
(12) Altomare, G.; Capella, G. L. Depression circumstantially related
to the administration of finasteride for androgenetic alopecia. J.
Dermatol. 2002, 29, 665−669.
This work was supported by the MIUR “Progetto Eccellenza”,
PON “Ricerca e Innovazione” PerMedNet-project
ARS01_01226, Post-Finasteride Foundation, and SoE “Linea
3PSR 2020Bando SEED”. The authors also acknowledge
Professor M. J. Tetel for paper revision and F. Giavarini for
technical support.
(13) Irwig, M. S. Depressive symptoms and suicidal thoughts among
former users of finasteride with persistent sexual side effects. J. Clin.
Psychiatr. 2012, 73, 1220−1223.
ABBREVIATIONS
■
5α-R, 5-alpha reductase; AGA, androgenetic alopecia; BPH,
benign prostate hyperplasia; DHT, dihydrotestosterone;
DMSO, dimethyl sulfoxide; FAERS, Food and Drug
Administration Adverse Event Reporting System; MCOTA,
multilevel cross-organism transferability analysis; MD, molec-
ular dynamics; NVT, constant volume and temperature; NPT,
constant pressure and temperature; OTPs, off-target proteins;
PNMT, phenylethanolamine N-methyltransferase; PBSs, po-
tential binding sites; RBS, reference binding site; RESP,
restrained electrostatic potential; rhADA, recombinant human
adenosine deaminase; rhAHCY, recombinant human adenosyl
homocysteinase; RMSD, root-mean-squared deviation;
SPILLO-PBSS, SPILLO potential binding sites searcher
̇
̆
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