Biotransformation of papaverine and in silico docking studies of the metabolites on human phosphodiesterase 10a

Article abstract of DOI:10.1016/j.phytochem.2020.112598

The metabolism of papaverine, the opium benzylisoquinoline alkaloid, with Aspergillus niger NRRL 322, Beauveria bassiana NRRL 22864, Cunninghamella echinulate ATCC 18968 and Cunninghamella echinulate ATCC 1382 has resulted in O-demethylation, O-methylgluc

Full text of DOI:10.1016/j.phytochem.2020.112598

Phytochemistry 183 (2021) 112598
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Biotransformation of papaverine and in silico docking studies of the
metabolites on human phosphodiesterase 10a
,
Duaa Eliwaa b, Mohamed A. Albadrya, Abdel-Rahim S. Ibrahimb, Amal Kabbashb,
,
,d,*
Kumudini Meepagalac, Ikhlas A. Khana d, Mona El-Aasrb, Samir A. Rossa
a National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, MS, 38677, USA
b Department of Pharmacognosy, Faculty of Pharmacy, Tanta University, Tanta, 31527, Egypt
c USDA-ARS, Natural Products Utilization Research Unit, University, MS, USA
d Division of Pharmacognosy, Department of Biomolecular Sciences, School of Pharmacy, University of Mississippi, MS, 38677, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
The metabolism of papaverine, the opium benzylisoquinoline alkaloid, with Aspergillus niger NRRL 322, Beauveria
bassiana NRRL 22864, Cunninghamella echinulate ATCC 18968 and Cunninghamella echinulate ATCC 1382 has
resulted in O-demethylation, O-methylglucosylation and N-oxidation products. Two new metabolites (4′′-O-
methyl-β-D-glucopyranosyl) 4′-demethyl papaverine and (4′′-O-methyl-β-D-glucopyranosyl) 6-demethyl papav-
erine, (Metabolites 5 and 6) together with 4′-O-demethylated papaverine (Metabolite 1), 3′-O-demethylated
papaverine (Metabolite 2), 6-O-demethylated papaverine (Metabolite 3) and papaverine N-oxide (Metabolite 4)
were isolated. The structure elucidation of the metabolites was based primarily on 1D, 2D-NMR analyses and
HRMS. These metabolism results were consistent with the previous plant cell transformation studies on
papaverine and isopapaverine and the microbial metabolism of papaveraldine. In silico docking studies of the
metabolites using crystals of human phosphodiesterase 10a (hPDE10a) revealed that compounds 4, 1, 6, 3, and 5
possess better docking scores and binding poses with favorable interactions than the native ligand papaverine.
Biotransformation
Benzylisoquinoline alkaloids
Papaverine
Aspergillus niger NRRL 322
Beauveria bassiana NRRL 22864
Cunninghamella echinulate ATCC 18968
Cunninghamella echinulate ATCC 1382
Human phosphodiesterase 10a
Molecular docking
1. Introduction
papaveraldine (Dorisse et al., 1986). Silene alba Miller E. H. L. Krause
cell suspension also transformed papaverine to 6- and 4′-mono-
Biotransformations (bioconversion or microbial transformation)
refer to the processes in which microorganisms convert organic com-
pounds into structurally related products. In other word, biotransfor-
mation deals with microbial (enzymatic) conversion of a substrate into a
product with limited number (one or few) enzymatic reactions (Hegazy
et al., 2015). The microbial transformation of natural and synthetic
drugs has been extensively used to decipher the fate of therapeutic
agents in the body, prepare novel drugs, and mediate preparation of
synthetic drugs. The microbial reactions are characterized by being
highly stereoselective and regioselective (Veeresham and Venisetty,
2003). The 1-benzylisoquinoline papaverine is a major alkaloid in the
latex of opium poppy chemo types and has been used as a non-specific
vasodilator owing to its direct action on smooth muscle (Facchini and
Hagel, 2013). Although its use to treat cerebral vasospasm has largely
been replaced by modern drugs, papaverine is still used topically and as
an injectable to treat erectile dysfunction (Facchini and Hagel, 2013). A
cell cultures of Silene alba Miller was able to transform papaverine to
demethylpapaverine (Verdeil et al., 1986). Also, Mucor ramannianus
1839 was able to transform papaveraldine into S-papaverinol and
S-papaverinol N-oxide (El Sayed, 2000). Four metabolites were sepa-
rated from rat bile treated with papaverine which are 4′-desmethyl-,
7-desmethyl-, and 6-desmethylpapaverine and 4′,6- desmethylpapaver-
ine (Belpaire et al., 1975). Papaverine is a selective inhibitor of Phos-
phodiesterase 10. Phosphodiesterase 10 (PDE10) is a dual-specificity
superfamily responsible for hydrolyzing both cAMP (Km = 0.05
μM) and
cGMP (Km = 3 M) (Li et al., 2018), which is highly expressed in the
μ
brain and has been considered as a potential target for the treatment of
several central nervous system (CNS) disorders such as Schizophrenia,
Huntington’s and psychiatric disorders that affect the basal ganglia
disease (Lee et al., 2019). In recent study PDE10A had a unique role in
the development of heart failure and pathological cardiac remodeling
(Chen et al., 2020). Taking into consideration that inhibition of PDE10A
might represent a novel therapeutic strategy for treating wide array of
diseases associated with CNS and the cardiovascular system, we used the
* Corresponding author. National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, MS, 38677, USA.
E-mail address: sross@olemiss.edu (S.A. Ross).
https://doi.org/10.1016/j.phytochem.2020.112598
Received 17 August 2020; Received in revised form 20 November 2020; Accepted 24 November 2020
Available online 25 December 2020
0031-9422/© 2020 Elsevier Ltd. All rights reserved.

D. Eliwa et al.
Phytochemistry 183 (2021) 112598
Table 1
1H NMR (400 MHz) spectroscopic data for Papaverine, metabolites 1 and 4 (in CDCl3) and metabolites 2, 3, 5 and 6 (in DMSO‑d6)a.
Position
papaverine
δH (multiplicities, J in Hz)
1
2
3
4
5
6
3
8.33, d (5.6)
8.29,d (5.5)
8.17, d (5.7)
8.38, d (5.6)
8.33, d (6.1)
8.24, d (5.7)
7.48, d (5.6)
7.45, s
8.31, d (6.1)
7.87, d (6.1)
7.55, s
4
7.53, d (5.7)
7.42, d (5.5)
7.42, d (5.7)
7.63, d (5.7)
7.83, d (6.1)
5
7.01, s
7.41, s
6.94, d (1.8)
6.74, brs
6.82, m
4.56, s
4.02, s
3.93, s
3.78, s
3.80, s
__
7.02, s
7.14, s
7.74, s
7.83, s
7.05, d (1.8)
6.80, brs
6.81, m
4.56, s
3.93, s
3.86, s
3.67, s
3.69, s
__
7.23, s
7.60, s
7.29, brs
6.80, brs
6.82, m
4.91, s
4.10, s
4.02, s
__
8
7.35, s
7.52, s
7.54, s
7.72, s
2′
6.77, d (2)
7.00, d (2)
7.00, d (1.8)
6.79, d (8.2)
7.19, d (1.6)
6.95, d (8.5)
5′
6.76, brs
6.803, brs
6′
6.74, m
6.800, brs
6.74, dd (8.2, 1.9)
4.48, s
6.82, dd (8.5, 1.6)
4.71, s
α
4.52, s
4.47, s
6-OMe
7-OMe
3′-OMe
4′-OMe
1′′
3.97, s
__
__
3.97, s
3.87, s
3.89, s
3.87, s
3.94, s
3.65, s
3.66, s
3.66, s
3.72, s
__
__
__
__
__
__
__
__
3.68, s
3.87, s
__
3.67, s
__
__
__
__
__
__
__
__
5.17, d (7.89)
3.41 (m)
3.46 (m)
3.04 (t, 9.0)
3.48 (m)
3.55 (brd, 5.9)
3.46, s
4.82, d (7.76)
3.41 (m)
3.46 (m)
3.02 (t, 9.0)
3.48 (m)
3.55 (brd, 5.9)
3.43, s
2′′
__
__
__
3′′
__
__
__
4′′
__
__
__
5′′
__
__
__
6′′
__
__
__
4′′-OMe
__
__
__
Table 2
13C NMR (100 MHz) spectroscopic data for Papaverine, metabolites 1 and 4 (in CDCl3) and metabolites 2, 3, 5 and 6 (in DMSO‑d6)a.
Position
Papaverine
δH (multiplicities, J)
1
2
3
4
5
6
1
156.9, C
139.0, CH
119.6, CH
134.6, C
105.6, CH
150.7, C
149.3, C
104.5, CH
122.9, C
131.6, C
111.3, CH
147.9, C
149.3, C
112.2, CH
120.7, CH
40.6, CH2
55.9, CH3
56.1, CH3
56.1, CH3
56.4, CH3
–
157.8, C
139.8, CH
119.4, CH
134.1, C
105.6, CH
153.2, C
150.4, C
104.7, CH
123.2, C
131.1, C
111.7, CH
147.4, C
144.9, C
114.9, CH
121.4, CH
41.8, CH2
56.1, CH3
56.2, CH3
56.4, CH3
–
157.7, C
139.7, CH
120.6, CH
133.3, C
108.7, CH
147.2, C
150.9, C
104.7, CH
127.8, C
132.2, C
112.7, CH
149.5, C
148.6, C
111.9, CH
117.9, CH
40.6, CH2
–
158.9, C
140.8, CH
120.6, CH
131.8, C
119.6, CH
152.8, C
150.4, C
106.1, CH
125.4, C
131.4, C
112.7, CH
147.2, C
148.6, C
111.9, CH
119.0, CH
40.9, CH2
55.40, CH3
55.43, CH3
55.9, CH3
56.3, CH3
–
154.0, C
129.5, CH
121.0, CH
136.8, C
105.7, CH
156.8, C
152.4, C
104.7, CH
122.3, C
127.0, C
114.2, CH
145.0, C
147.1, C
112.0, CH
121.3, CH
36.6, CH2
56.7, CH3
56.3, CH3
–
158.5, C
140.9, CH
121.0, CH
132.9, C
109.5, CH
150.1, C
149.0, C
105.3, CH
125.1, C
132.7, C
113.1, CH
147.6, C
147.7, C
112.3, CH
119.1, CH
41.2, CH2
–
55.8, CH3
55.9, CH3
56.1, CH3
99.6, CH
73.7, CH
76.1, CH
79.2, CH
77.0, CH
60.5, CH2
60.2, CH3
156.2, C
134.8, CH
119.9, CH
135.5, C
106.7, CH
155.20, C
151.5, C
105.3, CH
122.4, C
131.7, C
113.7, CH
149.3, C
145.6, C
115.8, CH
121.2, CH
38.3, CH2
56.7, CH3
56.6 CH3
56.1, CH3
–
3
4
4a
5
6
7
8
8a
1′
2′
3′
4′
5′
6′
α
6-OMe
7-OMe
3′-OMe
4′-OMe
1′′
55.4, CH3
55.5, CH3
55.7, CH3
–
56.2, CH3
–
–
100.1, CH
73.8, CH
75.9, CH
79.4, CH
76.9, CH
60.6, CH2
60.1, CH3
2′′
–
–
–
–
–
3′′
–
–
–
–
–
4′′
–
–
–
–
–
5′′
–
–
–
–
–
6′′
–
–
–
–
–
4′′-OMe
–
–
–
–
–
a
Carbon multiplicity were determined by DEPTq experiment.
microbial biotransformation to generate some papaverine related
products followed by molecular docking studies using the Schrodinger
software to analyze and understand the binding patterns of these com-
pounds with PDE10.
used in the initial screening of papaverine. Six metabolites of papaverine
were isolated from the biotransformation broth and freeze–dried
mycelia of Aspergillus niger NRRL 322, Beauveria bassiana NRRL 22864,
Cunninghamella echinulate ATCC 18968 and Cunninghamella echinulate
ATCC 1382. The metabolites were identified on the basis of their mass
and NMR spectroscopic data (Table 1 and Table 2).
The use of microbial synthesis can, in many cases, reduces delivery
times, minimize resources required, and circumvent speculative chem-
ical synthesis (Salter et al., 2019).
The molecular formula of 1 was established as C19H19NO4 by HR-
ESI-MS (SI: S7) analysis which exhibited a protonated molecular ion
peak at (m/z 326.1487 [M+H]+, calculated for C19H20NO4 which is less
than papaverine by 14 Da suggesting a demethylation product. When
comparing to the papaverine, the absence of 4′-methoxy group was
noticed in the 1H NMR (Table 1, SI: S1, SI: S2). Three methoxy groups
resonating at δH 3.65, 3.87 and 3.97 and assigned to 3′, 7 and 6 methoxy
2. Results and discussion
2.1. The biotransformation products identification
Seventy-two strains of filamentous fungi of different classes were
2

D. Eliwa et al.
Phytochemistry 183 (2021) 112598
Table 3
Structure of papaverine and its metabolites.
R1
R2
R3
Papaverine
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OH
OCH3
OCH3
OH
1
2
3
4
5
OCH3
OCH3
OCH3
OCH3
OCH3, N-oxide
OCH3
OCH3
6
OCH3
OCH3
groups, respectively. 13C NMR (Table 2, SI: S3, SI: S4) of 1 showed three
methoxy groups signals at 56.1, 56.2 and 56.4 ppm which is less than
papaverine by one methoxy and C-4′ signal was further deshielded by 4
ppm due to effect of the produced hydroxyl group. This assumption was
supported by detailed analysis of the HMBC and HSQC spectra of 1(SI:
S5, SI: S6).
product and identical to those reported for 6-O-demethyl-papaverine
(Brochmann-Hanssen et al., 1968), (Fig. 3 & Table 3).
The HRMS analysis (SI: S19) of 3 displayed a protonated molecular
ion peak [M+H]+ at m/z 356.1601, suggesting the same molecular
formula of papaverine with one additional oxygen atom (C20H21O5N).
The 13C and 1H-NMR spectra of 3 (Table 1, Table 2, SI: S13, SI: S14, SI:
S15, SI: S16) were closely related to those of papaverine and suggested
that 3 is the N-oxide derivative of papaverine. The two proton doublets
resonated at δH 8.38 and 7.63, J = 5.7 Hz was assigned to H-3 and H-4,
respectively (Table 1) was shifted by δH+0.05 and – 0.1 ppm, respec-
tively as compared to those of papaverine. The shifting of C-1 and C-3 in
3 (δ C 2.0 and δ C 1.8 ppm, respectively) as compared to those of
papaverine was consistent with the fact that metabolite-3 is the N- oxide
derivative of papaverine. This assumption was supported by detailed
analysis of the HMBC and HSQC spectra of 3 (SI: S17, SI: S18, Fig. 3,
Table 3).
All data were consistent with those reported for the 4′-O-demethy-
lated product and identical to those reported spectra for 4′-demethyl-
papaverine(Brochmann-Hanssen, Brochmann-Hanssen, Hirai and Hirai,
1968) (Fig. 3, Tables 1 and 3).
The molecular formula of 2 was established as C19H19NO4 by HRMS
(SI: S12) analysis which exhibited a protonated molecular ion peak at
(m/z 326.1424 [M+H]+, calculated for C19H20NO4, which is less than
papaverine by 14 Da suggesting demethylation. When comparing to the
papaverine, the absence of 6-methoxy group was observed in the 1H
NMR (Table 1, SI: S8, SI: S9). Three methoxy groups resonated at δH
3.66, 3.68 and 3.89 which were assigned to 3′, 4′ and 7 methoxy groups,
respectively. 13C NMR (Table 2, SI: S9) of 2 showed three methoxy group
signals at 55.4, 55.5 and 55.7 ppm which is less than papaverine by one
methoxy and C-6 signal deshielded by 2 ppm due to the produced hy-
droxyl group. This assumption was supported by detailed analysis of the
HMBC and HSQC spectra of 2 (SI: S10, SI: S11).
The molecular formula of 4 was established as C19H19NO4 by HRMS
analysis (SI: S25) which exhibited a protonated molecular ion peak at
(m/z 326.1498 [M+H]+, calculated for C19H20NO4 which is less than
papaverine by 14 Da suggesting a demethylation. Also, when comparing
to the papaverine, the absence of 3′-methoxy group was noticed in the
1H NMR (Table 1, SI: S20, SI: S21). Three methoxy groups were observed
at δH 3.87, 4.02 and 4.10 ppm which were assigned to 3′, 7 and 6
All data were consistent with that reported for the 6-O-demethylated
3

D. Eliwa et al.
Phytochemistry 183 (2021) 112598
occurrence of five methine carbon (CH) signals at δ 100.1, 79.4, 76.9,
75.9, and 73.8, one methylene carbon (CH2) signal at δ 60.6 and one
OCH3 signal at δ 60.1 in its 13C NMR spectrum (Table 2). The HMBC
(Fig. 1, SI: S36) correlations of H-1′′ (the anomeric protons) to C-4′
confirmed the attachment of the sugar moiety to C-4′ of metabolite 6.
The high J value of the anomeric proton H-1′′ suggested the presence of a
β-glycosidic linkage in 4′. Metabolite 6 exhibited a peak due to [M+H]+
at m/z 502.2138 in its high-resolution ESI-MS (SI: S37), suggesting a
molecular formula of C26H32NO9. Thus, this metabolite was identified as
(4′′-O-methyl-β-D-glucopyranosyl) 4′-demethyl papaverine (Figs. 1 and
3, Table 3).
2.2. In silico molecular docking study of the biotransformation products
on human phosphodiesterase 10a (HPDE10a)
Crystal structure of Human PDE-papaverine complex 2WEY(Ander-
sen et al., 2009) et al., 2009), was utilized for the docking study, in
which papaverine interacts with the receptor via hydrogen-bonding
interaction between the methoxy groups of the isoquinoline moiety
with Gln726 in addition to stacking interactions between the isoquino-
line group and phenylalanine at positions 696 and 729. Docking was
performed using papaverine and compounds (1–6) after ligand prepa-
ration step which generated 13 ligands from the input of 7 compounds.
In order to obtain a good sampling of the conformational space of these
ligands, we performed conformational search using Macromodel. We
obtained 2305 total entry as a result 105 for (1) 251 for (2) 183 for (3)
180 for (4) 552 for (5) 666 for (6) 368 for papaverine, the lowest energy
conformer of each compound are shown in (Table 4). The output of
docking study revealed that several compounds possessed good binding
poses with favorable protein-ligand interactions (Fig. 2). A study of the
binding modes and the docking scores revealed that five compounds (4,
1, 6, 3, and 5) possess better docking scores and binding poses with
favorable interactions than the native ligand papaverine (Table 5).
Fig. 1. Important 1H–13C-HMBC correlations of metabolites 5 and 6.
methoxy groups, respectively. 13C NMR of 4 (Table 2, SI: S2) showed
three methoxy groups signals at δ 56.2, 56.7 and 56.3 ppm which is less
than papaverine by one methoxy and C-3′ signal deshielded by 3 ppm
due to the produced hydroxyl group. This assumption was supported by
detailed analysis of the HMBC and HSQC spectra of 4. Hence, 4 was
approved as 3′-O-demethyl-papaverine (Brochmann-Hanssen and Hirai,
1968), (SI: S23, SI: S24, Figs. 1 and 3, Table 3).
3. Experimental
3.1. General experimental procedure
1D (1H, 13C and DEPTQ) and 2D (HMBC and HSQC) NMR spectra
were recorded on a Bruker model AMX 400 NMR spectrometer with
standard pulse sequences, operating at 400 MHz in 1H and 100 MHz in
13C. The chemical shift (δ) values were reported in parts per million
units (ppm) and tetramethylsilane or known solvent shifts, used as in-
ternal chemical shift references. Coupling constants (J values) were
recorded in Hertz (Hz). Standard pulse sequences were used for COSY,
HSQC, HMBC, and DEPT. High-resolution electrospray ionization mass
spectra (ESI-MS) were measured on a Micromass Q-T of Micro mass
spectrometer with a lock spray source. TLC was performed using pre-
coated TLC sheets silica gel G 254 F sheets (E. Merck, Germany) and
precoated C18–W silica TLC plates w/uv 254. Column chromatography
was carried out by a Biotage Isolera™ flash chromatography system,
silica gel (E. Merck, 70–230 mesh) and sephadex LH-20 (Sigma- Aldrich
chemical Co.) were used. All the reagents and solvents used for sepa-
ration and purification were of analytical grade. For preparative isola-
tion TLC silica gel 60 PR-18 F254S plates were used. Visualization of the
TLC plates was achieved with a UV lamp (l = 254 and 365 nm), sprayed
with Dragendorff’s and anisaldehyde/acid spray reagent. All chemicals
used were purchased from Sigma–Aldrich (St. Louis, Mo, USA). Three
solvent systems were used for TLC analysis; S1: ethyl acetate-methanol-
ammonia sol. (95: 5: 2.5); S2: dichloromethane-methanol (95: 5); S3:
methanol/water (70:30), The plates were dried and visualized under
UV-light at 254 and 365 nm and sprayed with Dragendorff’s and ani-
saldehyde/sulfuric acid spray reagents.
Metabolite 5 exhibited a protonated molecular ion peak at m/z
502.1980 [M+H]+ in its high-resolution ESI-MS(SI: S31), suggesting a
molecular formula of C26H32NO9. The molecular weight was the sum of
O-demethyl papaverine (325) and an additional moiety of 176 mass
units (consistent with O-methyl glucose). Spectroscopic data for this
compound were very similar to those of 2 (6-O-demethyl-papaverine)
and suggested that a 4-O-methylglucose moiety was introduced during
the biotransformation (Table 1, Table 2, SI: S26, SI: S27). This was
further confirmed by the presence of sugar signals at δ 99.6, 79.2, 77.0,
76.1, 73.7, 60.5 and 60.2 in its 13C NMR spectrum (Table 2, SI: S28). The
HMBC correlations of H-1′′ (the anomeric protons) to C-6 suggested that
the newly introduced 4-O-methylglucose moiety is linked to the iso-
quinoline moiety at C-6 through an oxygen atom. The anomeric proton
resonates at δ 5.17, shows large coupling constant, J = 7.9 Hz indicating
the aglycone binds to the sugar through a β-glycosidic linkage. Based on
the detailed analysis of HSQC and HMBC spectra (Fig. 1), this
biotransformation product was identified as (4′′-O-methyl-β-D-gluco-
pyranosyl) 6-demethyl papaverine (Figs. 1 and 3, Table 3, SI: S29, SI:
S30).
Metabolite 6 had the same molecular formula as 5. In its 1H NMR and
13C NMR spectra (Table 1, Table 2, SI: S32, SI: S33, SI: S34), the Spec-
troscopic data for this compound were very similar to those of 1 (4-O-
demethyl-papaverine) and suggested that a 4-O-methylglucose moiety
was introduced during the biotransformation. The presence of a 4-O-
methylglucose moiety in this metabolite was confirmed by the
4

Table 4
Lowest energy conformers of papaverine and compounds (1–6).
Papaverine
(1)
(2)
OPLS3 energy 164.702 kJ/mol
OPLS3 energy 117.608 kJ/mol
OPLS3 energy 106.589 kJ/mol
(continued on next page)

D. Eliwa et al.
Phytochemistry 183 (2021) 112598
3.2. Chemicals
Papaverine was purchased from Sigma–Aldrich Inc. (Milwaukee, WI,
USA).
3.3. Organism
Preliminary screening procedure were conducted as previously re-
ported(Lee et al., 1990). Sixty microbial cultures, obtained from the
University of Mississippi, NCNPR culture collection and twelve micro-
bial cultures were obtained from the American Type Culture collection
(ATCC, Rockville, Maryland), Northern Regional Research Laboratories
(NRRL, Peoria, Illinois), USA. The cultures were initiated to grow from
lyophilized state by adding 1 ml of sterile water to microorganism cells.
Few drops of suspended cells were streaked on sabouraud-dextrose agar
plates to check the purity. Pure cultures were maintained on slants of
either potato-dextrose agar and sabouraud-dextrose agar. The fresh
slants were incubated for few days at room temperature, before storage
in a refrigerator (4 ◦C) and sub cultured every three months. On the basis
of this screening process, several micro-organisms were found to
metabolize papaverine very well with variable efficiencies without
optimization. Aspergillus niger NRRL 322, Beauveria bassiana NRRL
22864, Cunninghamella echinulate ATCC 18968 and Cunninghamella
echinulate ATCC 1382 were the most efficient microorganisms capable of
conversion of Papaverine into metabolites.
The same procedure was used to produce large quantities of the
metabolites. The highest yield microbial strains found to biotransform
papaverine were chosen for the preparative-scale fermentation experi-
ments. Papaverine (600 mg) dissolved in acetone and then equally
divided among 2 L flasks containing 400 ml of stage II culture in a
concentration of 10 mg/50 ml culture media (w/v). All fermentation
experiments utilized a liquid media of the following composition: 20 g
dextrose, 5 g yeast extract, 5 g K2HPO4, 5 g peptone and 5 g NaCl in 1 L
of distilled water, which was sterilized at 121 ◦C for 15 min. After
maximum conversion, the experiments were terminated and rendered
alkaline with conc. Ammonium hydroxide (1 ml/30 ml culture, pH 8)
then exhaustively extracted with equal volume of ethyl acetate, filtered
off by a cheese cloth over anhydrous Na2SO4 and evaporated to dryness
under vacuum using rotary evaporator to give fermentation residues.
3.4. Microbial transformation using Cunninghamella echinulate ATCC
18968
Biotransformation of papaverine using Cunninghamella echinulate
ATCC 18968 produced one metabolite. The residue (800 mg) was dis-
solved in 30 ml of methanol/dichloromethane mixture (1:1) and
adsorbed onto 800 mg Celite and dried. The adsorbed sample was placed
onto a glass column (110 × 2 cm) packed with silica after making a
slurry in dichloromethane. The column was gradiently eluted with
dichloromethane 100% then dichloromethane: methanol (98.5:1.5,
98:2) and 10 ml fractions were collected. Fractions 40–60 were pooled
together to give compound 1. Further purification was achieved using
sephadex LH-20 column, as determined by TLC. The metabolite was
obtained in the form of white powder (70 mg, Rf 0.6 S2).
3.5. Microbial transformation using Aspergillus niger NRRL 322
Biotransformation of papaverine using Aspergillus niger NRRL 322
afforded to two metabolites (2 and 3). The residue (1 gm) was dissolved
in 50 ml of methanol/dichloromethane mixture (1:1) and adsorbed onto
1 gm Celite and dried. The adsorbed sample was placed onto a glass
column (50 × 2.5 cm) packed with silica after making a slurry in
dichloromethane. The column was isocratically eluted with dichloro-
methane: methanol (97.5:2.5) and 10 ml fractions were collected.
Fractions 20–44 were pooled together to give mixture of 2 and 3 as
determined by TLC. The metabolites were obtained in the form of
6

D. Eliwa et al.
Phytochemistry 183 (2021) 112598
Fig. 2. Docked poses of the five papaverine-based biotransformation products with better docking scores than the native ligand in the Human PDE-10 A crystal
structure, PDB: 2WEY; 4 (red), 1 (yellow), 6 (blue), 3 (blum), 5 (faded orange). (For interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
Fig. 3. Structure of papaverine and its metabolite from different Fungi.
yellowish residue (80 mg, Rf 0.3 S2).
3.6. Microbial transformation using Cunninghamella echinulate ATCC
1382
The mixture was loaded in silica biotage column (18 × 150 mm). The
column was gradiently eluted with dichloromethane 100% then
dichloromethane:2-propanol (98:2, 97:3,95:5) and 20 ml fractions were
collected. Fractions 27–31 were pooled together to give pure 2, in the
form of white powder (7 mg, Rf 0.70 S1). Fractions 65–85 were pooled
together to give pure 3, in the form of white powder (30 mg, Rf 0.61 S1).
Biotransformation of papaverine using Cunninghamella echinulate
ATCC 1382 afforded to one metabolite. The residue (900 mg) was dis-
solved in 20 ml of methanol/dichloromethane mixture (1:1) and
adsorbed onto 900 mg Celite and dried. The adsorbed sample was placed
7

D. Eliwa et al.
Phytochemistry 183 (2021) 112598
Table 5
Docking scores of the biotransformation products in the binding site of 2WEY “Human PDE-papaverine complex”.
Compound
Structure
Docking score
Glide score
Glide Emodel
Papaverine (native ligand)
ꢀ 5.335
ꢀ 5.378
ꢀ 6.654
ꢀ 36.528
4
ꢀ 6.610
ꢀ 57.572
1
ꢀ 6.305
ꢀ 6.305
ꢀ 47.271
6
ꢀ 6.213
ꢀ 6.256
ꢀ 59.876
3
ꢀ 6.128
ꢀ 6.128
ꢀ 37.724
5
ꢀ 5.938
ꢀ 6.109
ꢀ 63.128
(continued on next page)
8

D. Eliwa et al.
Phytochemistry 183 (2021) 112598
Table 5 (continued)
Compound
Structure
Docking score
Glide score
Glide Emodel
2
ꢀ 4.834
ꢀ 5.246
ꢀ 48.062
onto a glass column (110 × 2.5 cm) packed with silica after making a
slurry in dichloromethane. The column was gradiently eluted with
dichloromethane 100% then dichloromethane: methanol (99.5:0.5,
99:1) and 20 ml fractions were collected. Fractions 50–75 were pooled
together to give pure 4, as determined by TLC. The metabolite was ob-
tained in the form of white powder (8 mg, Rf 0.5 S2).
metals, create disulfide bonds, and delete water molecules beyond 5 Å
from the hetero groups, generate het state using Epik at pH 7 ± 2. The
H-bond assignment was applied using sample water orientations, using
PROPKA pH 7.0. Water molecules with less than three hydrogen
bonding distance were removed from the protein. Restrained minimi-
zation was performed using OPLS3, converged the heavy atoms to RMSD
0.3 Å.
3.7. Microbial transformation using Beauveria bassiana 22864
3.8.2. Ligand preparation
Papaverine and compounds (1–6) were sketched in Maestro then
Biotransformation of papaverine using Beauveria bassiana 22864
afforded to two metabolites: 5 and 6. The residue (1 g) was dissolved in
30 ml of methanol/dichloromethane mixture (1:1) and adsorbed onto 1
g Celite and dried. The adsorbed sample was placed onto a glass column
(110 × 2.5 cm) packed with silica after making a slurry in dichloro-
methane. The column was gradiently eluted with dichloromethane:
methanol (99:1, 95:5,94:6) and 10 ml fractions were collected. Fractions
50–119 were pooled together to give mixture of the two metabolites (80
mg), as determined by TLC. The two metabolites were separated by
¨
prepared using LipPrep module (Schrodinger, LLC) in OPLS3 force field,
generate possible states at target pH 7.0 ± 2 in Epik and generated
tautomers. The specified chirality was retained to generate at most 32
per ligand. The structure from the previous step were minimizes using
OPLS3 force field using Powellꢀ Reeves conjugate gradient (PRCG)
method with 2500 maximum iteration and gradient converge with 0.05
threshold. We have carried out the conformational searches using mixed
torsional/low-mode sampling with 1000 maximum number of steps and
100 steps per rotatable bond. A 21 kJ/mol energy cutoff was used A
conformer was considered redundant and subsequently eliminated if its
maximum atom deviation from an already-identified conformer was less
than 0.5.
preparative silica gel 60 PR-18 plates (10 × 20 cm, 250 μm) (methanol:
water, 70:30). 5 mg of the sample added in each plate. The bands were
located using UV lamp then scratched and extracted using 100%
methanol then evaporated, yielding to pure 5 in the form of white
powder (5 mg, Rf 0.69 S3) and pure 6 white powder (6 mg, Rf 0.61 S3).
3.8.3. Glide grid generation and docking
Receptor grid was generated for the prepared proteins 2WEY using
3.8. In silico molecular docking studies
¨
Glide (Schrodinger, LLC). No constraints were applied. The grid thus
generated was validated for the native ligands (papaverine) to check if
the RMSD of the docked output was <1 Å from that of the crystal
structure. All the generated conformers for the prepared ligands were
docked in the generated grid. Their docking results and binding poses
(table 6).
3.8.1. Protein preparation
Crystal structure of Human PDE-papaverine complex 2WEY
(Andersen et al., 2009) was downloaded from Protein Data Bank RCSB
PDB (Berman et al., 2000). Chain A from the crystal structure was
deleted since chain B contains the papaverine in the right binding mode
(Andersen et al., 2009), followed by preprocessing to assign bond or-
ders, use CCD database, add hydrogen bonds, create zero-order bonds to
9

D. Eliwa et al.
Phytochemistry 183 (2021) 112598
Table 6
Ligand-interaction diagram of compounds with key amino acid residues of Human PDE-10 A (PDB: 2WEY).
Papaverine
Compound 4
Compound 1
Compound 6
(continued on next page)
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D. Eliwa et al.
Phytochemistry 183 (2021) 112598
Table 6 (continued)
Compound 3
Compound 5
Compound 2
(continued on next page)
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D. Eliwa et al.
Phytochemistry 183 (2021) 112598
Table 6 (continued)
Dorisse, P., Gleye, J., Loiseau, P., Puig, P., Edy, A., Henry, M., 1986. Papaverine
biotransformation in plant cell suspension cultures. J. Nat. Prod. 51 (3), 532–536.
https://doi.org/10.1021/np50057a013.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
El Sayed, K., 2000. Microbial transformation of papaveraldine. Phytochemistry 53
(2000), 675–678. https://doi.org/10.1016/S0031-9422(99)00616-0.
Hagel, J., Facchini, P., 2013. Benzylisoquinoline alkaloid metabolism: a century of
discovery and a brave new world. Plant Cell Physiol. 54 (5), 647–672. https://doi.
org/10.1093/pcp/pct020.
Acknowledgments
Hegazy, M., Mohamed, T. ElShamy, Mahalel, U., Reda, E., Shaheen, A., Tawfik, W.,
Shahat, A., Shams, K., Abdel-Azim, N., Hammoud, F., 2015. Microbial
biotransformation as a tool for drug development based on natural products from
mevalonic acid pathway: a review. J. Adv. Res. 6 (1), 17–33. https://doi.org/
10.1016/j.jare.2014.11.009.
The project was supported by the Egyptian government, National
center of Natural product research, USA and in part by the USDA Agri-
cultural Research Service.
Lee, I.-S., ElSohly, H.N., Hufford, C.D., 1990. Microbial metabolism studies of the
antimalarial drug arteether. Pharmaceut. Res. 7 (2), 199–203. https://doi.org/
10.1021/np50062a020.
References
Lee, Y., Park, J., Leem, Y., Park, J., Kim, D., Choi, Y., Park, E., Kang, J., Kim, H., 2019.
The phosphodiesterase 10 inhibitor papaverine exerts anti-inflammatory and
neuroprotective effects via the PKA signaling pathway in neuroinflammation and
Parkinson’s disease mouse models. J. Neuroinflammation 16 (1), 246. https://doi.
org/10.1186/s12974-019-1649-3.
¨
Andersen, O., Schonfeld, D., Toogood-Johnson, I., Felicetti, B., Albrecht, C., Fryatt, T.,
Whittaker, M., Hallett, D., Barker, J., 2009. Cross-linking of protein crystals as an aid
in the generation of binary protein-ligand crystal complexes, exemplified by the
human PDE10a-papaverine structure. Acta Crystallogr. 65 (8), 872–874. https://doi.
org/10.1107/S0907444909017855.
Li, J., Chen, J., Deng, Y., Zhou, Q., Wu, Y., Wu, D., Luo, H., 2018. Structure-based design,
synthesis, biological evaluation, and molecular docking of novel PDE10 inhibitors
with antioxidant activities. Frontiers in Chemistry 6, 167. https://doi.org/10.3389/
fchem.2018.00167.
Belpaire, F., Bogaert, M., Rosseel, M., Anteunis, M., 1975. Metabolism of papaverine. I.
Identification of metabolites in rat bile. Xenobiotica 5 (7), 413–420. https://dio.
org:10.3109/00498257509056111.
Salter, R., Beshore, D., Colletti, S., Evans, L., Gong, Y., Helmy, R., Liu, Yong, Maciolek, C.,
Martin, G., Pajkovic, N., Phipps, R., Small, J., Steele, J., de Vries, R., Williams, H.,
Martin, I., 2019. Microbial biotransformation – an important tool for the study of
drug metabolism. Xenobiotica 49 (8), 877–886. https://doi.org/10.1080/
00498254.2018.1512018.
Berman, H., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T., Weissig, H., Shindyalov, I.,
Bourne, P., 2000. The protein Data Bank. Nucleic Acids Res. 28 (1), 235–242.
https://doi.org/10.1093/nar/28.1.235.
Brochmann-Hanssen, E., Hirai, E., 1968. Opium Alkaloids VII. Isolation of a new
benzylisoquinoline alkaloid. Synthesis and NMR Studies of papaveroline trimethyl
ethers. J. Pharmaceut. Sci. 57 (6), 940–943. https://doi.org/10.1002/
jps.2600570605.
Verdeil, L., Bister-Miel, F., Guignard, L., Viel, C., 1986. Papaverine biotransformation by
Silene alba cell suspension. Planta Med. 1, 1–3. https://doi.org/10.1016/S0031-9422
(00)84577-X.
Veeresham, C., Venisetty, R., 2003. Application of microbial biotransformation for the
new drug discovery using natural drugs as substrates. Curr. Pharmaceut. Biotechnol.
4 (3), 153–167. https://doi.org/10.3389/fmicb.2015.01433.
Chen, S., Zhang, Y., Lighthouse, J.K., Mickelsen, D.M., Wu, J., Yao, P., Small, E.M.,
Yan, C., 2020. A novel role of cyclic nucleotide phosphodiesterase 10A in
pathological cardiac remodeling and dysfunction. Circulation 141 (3), 217–233.
https://doi.org/10.1161/CIRCULATIONAHA.119.042178.
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