Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00871

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop se

Full text of DOI:10.1021/acs.molpharmaceut.7b00871

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
Molecular docking-based design and development of a highly
selective probe substrate for UDP-glucuronosyltransferase 1A10
Risto Olavi Juvonen, Sanna Rauhamäki, Sami Kortet, Sanna P. Niinivehmas, Johanna Troberg,
Aleksanteri Petsalo, Juhani Huuskonen, Hannu Raunio, Moshe Finel, and Olli T. Pentikainen
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00871 • Publication Date (Web): 08 Feb 2018
Downloaded from http://pubs.acs.org on February 14, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Molecular Pharmaceutics is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
Molecular docking-based design and development of a highly selective probe
substrate for UDP-glucuronosyltransferase 1A10
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
4
1Risto O. Juvonen, Sanna Rauhamäki, 2,3Sami Kortet, Sanna Niinivehmas, Johanna
Troberg, Aleksanteri Petsalo, Juhani Huuskonen, Hannu Raunio, 4Moshe Finel, 2,5Olli T.
1
3
1
Pentikäinen
1 School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Box 1627,
FIꢀ70211 Kuopio, Finland
2 University of Jyvaskyla, Department of Biological and Environmental Science, P.O. Box
35, FIꢀ40014 University of Jyvaskyla, Finland
3 University of Jyvaskyla, Department of Chemistry, P.O. Box 35, FIꢀ40014 University of
Jyvaskyla, Finland
4 Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of
Helsinki, P.O. Box 56, FIꢀ00014 University of Helsinki, Finland
5 Institute of Biomedicine, Faculty of Medicine, University of Turku, FIꢀ20014 University of
Turku, Finland
* Corresponding authors: risto.juvonen@uef.fi (in vitro), olli.pentikainen@utu.fi (modeling
and synthesis)
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 2 of 41
1
2
3
4
5
Keywords: 7ꢀhydroxycoumarin, UDPꢀglucuronosyltransferase, glucuronidation, in silico,
fluorescence, drug metabolism
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 3 of 41
Molecular Pharmaceutics
1
2
3
Abstract
4
5
6
7
8
Intestinal and hepatic glucuronidation by the UDPꢀglucuronosyltransferases (UGTs) greatly
affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation
reactions in the intestine, but not in the liver. Here, our aim was to develop selective,
fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models
were constructed, used to design new substrates and subsequently six novel C3 substituted (4ꢀ
fluorophenyl, 4ꢀhydroxyphenyl, 4ꢀmethoxyphenyl, 4ꢀdimethylaminophenyl, 4ꢀmethylphenyl
or triazole) 7ꢀhydroxycoumarin derivatives were synthetized from inexpensive starting
materials. All tested compounds could be glucuronidated to nonꢀfluorescent glucuronides by
UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10ꢀ
H210M, was prepared based on the newly constructed model. Glucuronidation kinetics of the
new compounds, in both wildꢀtype and mutant UGT1A10 enzymes, revealed variable effects
of the mutation. All six new C3 substituted 7ꢀhydroxycoumarins were glucuronidated faster
by human intestine than by liver microsomes supporting the results obtained with
recombinant UGTs. The most selective 4ꢀdimethylaminophenyl and triazole C3 substituted 7ꢀ
hydroxycoumarins could be very useful substrates in studying the function and expression of
the human UGT1A10.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Keywords: 7ꢀhydroxycoumarin derivative, UDPꢀglucuronosyltransferase, in silico,
fluorescence, drug metabolism
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 4 of 41
1
2
3
Introduction
4
5
6
7
8
The extents of absorption and firstꢀpass metabolism in the intestine and liver strongly affect
the bioavailability of drugs and other orally ingested xenobiotic compounds 1ꢀ2. Although
hepatic metabolism is the major determinant of firstꢀpass metabolism for most drugs,
intestinal metabolism is critical for the bioavailability of certain compounds, particularly
those that could be directly conjugated, like phenols and flavonoids 3ꢀ5. Such xenobiotics
typically contain a nucleophilic functional group, usually a hydroxyl group, that can accept
an endogenous conjugating moiety, particularly glucuronic acid or sulfone. More rarely the
conjugating group is methyl, acetyl or amino acid 6.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The UDPꢀglucuronosyltransferase enzymes (UGTs, EC 2.4.1.17) catalyze about 35% of the
drug conjugation reactions and are abundantly expressed among the intestinal conjugating
enzymes 4ꢀ5, 7. They catalyze transfer of the glucuronic acid moiety from UDPꢀglucuronic
acid cofactor onto hydroxyl, amine, carboxylic acid, thiol or thioacid groups of the aglycone
8
substrates, reactions that are commonly called glucuronidation . There is a significant
9ꢀ11
difference in the expression profile of individual UGTs between the intestine and liver
.
While about ten different UGTs are abundantly expressed in the liver, only UGT1A1,
UGT1A10, UGT2B7 and UGT2B17 are expressed in the small intestine to significant
amounts at the protein level 10, 12ꢀ14. Unfortunately, the high activity and importance of the
intestinal UGT1A10 was (and still is) often underestimated due to common use of poorlyꢀ
active commercial UGT1A10 13.
UGT1A10 glucuronidates many drugs and xenobiotics 13, 15ꢀ17. Glucuronidation of estriol at
the 3ꢀOH and of estrone could be used as selective reactions for UGT1A10, but their
measurements require chromatographic separation of the resulting glucuronide from the
substrate 16, 18. Likewise, dopamine is a UGT1A10 selective substrate, but its low affinity19
ACS Paragon Plus Environment

Page 5 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
has limited its use to qualitative measurements only14. Accordingly, availability of more
convenient marker substrates for UGT1A10 would foster evaluation of glucuronidation
reactions, particularly if the assays were easy and fast to perform.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The interactions between substrates and xenobiotic metabolizing enzymes are increasingly
being studied by in silico modeling 20ꢀ23. Currently, the lack of crystal structures of the Nꢀ
terminal domain of any mammalian UGT impedes structureꢀactivity relationship and
mechanistic studies. UDPꢀglucuronic acid binds at the Cꢀterminal domain, which is highly
homologous among different UGTs and evolutionary conserved. The structure of the latter
domain has been solved by Xꢀray crystallography 24. However, that available crystal structure
does not provide sufficient data to predict binding of acceptor substrates, e.g. a drug
molecule, as substrates bind to the Nꢀterminal domain, which is more variable than the Cꢀ
terminal domain among UGT enzymes. In silico models for this domain were constructed
previously 20ꢀ21, but we need better ones in order to analyze substrateꢀenzyme interactions in
UGTs at the atomic level.
The hydroxyl group at C7 on the coumarin scaffold renders the compound fluorescent and
glucuronidation of this hydroxyl abolishes the fluorescence (Figure 1) 25. Substituents at
positions such as C3 or C4 of the coumarin do not quench the 7ꢀhydroxycoumarin
derivatives’ fluorescence, but modify its intensity, depending on the substituent’s chemical
nature. The 7ꢀhydroxyl group on coumarin is also a good functional group for
glucuronidation by many UGTs. Therefore, fluorescent 7ꢀhydroxycoumarin derivatives
provide an opportunity to design novel fluorescent substrates for UGTs, using molecular
modelling as the starting point.
[Figure 1]
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 6 of 41
1
2
3
4
5
6
7
8
9
In this study, we first constructed homology models for all the human UGT enzymes of
subfamily 1A and used them to design fluorescent 7ꢀhydroxycoumarin derivatives in silico.
We then synthetized six compounds and developed a convenient multiwell plate assay
protocol, based on fluorescence decrease, to test their glucuronidation rate. The results led to
the identification of several 3ꢀsubstituted 7ꢀhydroxycoumarins as selective substrates for the
human UGT1A10. A UGT1A10 mutant was prepared based on the model and as a test for it.
Subsequently, glucuronidation kinetic analyses of the 3ꢀsubstituted 7ꢀhydroxycoumarins, by
wildꢀtype and mutant UGT1A10, were carried out using the same multiwell plate assay
protocol.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 7 of 41
Molecular Pharmaceutics
1
2
3
Experimental section
4
5
6
7
8
Materials: Alamethicin, trichloroacetic acid, UDPGA sodium salt, 7ꢀhydroxycoumarin
(99%), 7ꢀhydroxyꢀ(4ꢀtrifluoromethyl)coumarin (99%) and bovine serum albumin were from
SigmaꢀAldrich (Mannheim, Germany). Formic acid (99 %) and MgCl2 were from Riedelꢀde
Haen (Vantaa, Finland). Acetonitrile (Ultra gradient HPLC grade), methanol (HPLC gradient
grade) and glycine were from J.T. Baker (Deventer, the Netherlands). Ethanol (≥99.5 %, Etax
Aa) was from Altia (Helsinki, Finland). Water was deionized by MilliQ gradient A10.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methods
Modeling. To enable structureꢀbased design of UGT1A10 selective substrates, all nine
UGT1Aꢀenzymes were modeled. Sequences of human UGT1A enzymes were gathered from
the UniProt Knowledgebase at www.uniprot.org (UniProt Consortium, 2015). The accession
codes for the retrieved UGT1As were: Q9HAW8 (1A10), O60656 (1A9), Q9HAW9 (1A8),
Q9HAW7 (1A7), P19224 (1A6), P35504 (1A5), P22310 (1A4), P35503 (1A3) and P22039
(1A1). To identify template protein structures for homology modeling purposes, the retrieved
UGT sequences were used in blast searches against the protein data bank (PDB) structures.
24
26
27
28
Based on the results of these searches, structures 2O6L , 3HBF , 3WC4 and 2C1Z
were selected as templates for homology modeling. The sequence alignment for the modeling
was produced in two steps. First a protein structureꢀbased sequence alignment was derived
for the selected four protein structures by using Vertaa in Bodil 29 and the 2C1Zꢀstructure as a
template, since it gave the best match for UGT1A10 and contained both the Nꢀ and Cꢀ
termini. In the second step the above listed nine UGT1A sequences were aligned, using
30
Bodil, against the structural alignment, using STRMAT110 matrix
with 40 as the gap
penalty. The alignment was adjusted for occasional variations in sequence length and used to
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 8 of 41
1
2
3
4
5
create models for each UGT1A, as well as in model construction that was performed using
Modeller version 9.15 31.
6
7
8
9
Molecular docking. The ligands that were selected for docking studies were prepared by
using LigPrep (version 3.3, Schrödinger, LLC, New York, NY, 2015). The shapes and
electrostatic properties of the substrate binding sites of modelingꢀproduced UGT1A enzymes
were analysed with Panther 32 and molecular docking was performed with PLANTS 33.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis. All the synthesis reactions were carried out using commercial materials and
reagents without further purification, unless otherwise noted. Reaction mixtures were heated
using the CEM Discovery microwave apparatus. All reactions were monitored by thin layer
1
13
chromatography (TLC) on silica gel plates. H NMR and C NMR data were recorded on a
Bruker Avance 400 MHz spectrometer or Bruker Avance III 300 MHz spectrometer.
Chemical shifts are expressed in parts per million values (ppm) and are designated as s
(singlet), br s (broad singlet), d (doublet), dd (double douplet), t (triplet). Coupling constants
(J) are expressed as values in hertz (Hz). The mass spectra were recorded using Micromass
LCT ESIꢀTOF equipment. Elemental analyses were done with Elementar Vario EL III
elemental analyzer. All compounds tested present more than 95 % purity.
General procedure for the synthesis of coumarin derivatives
The coumarin derivatives 1ꢀ6 were synthesized using PerkinꢀOglialor condensation reaction
(Scheme 1). The method was developed from the earlier published procedures and transferred
to a microwave reactor. 34
ACS Paragon Plus Environment

Page 9 of 41
Molecular Pharmaceutics
1
2
3
Scheme 1. The general procedure for the synthesis of coumarin derivatives
4
5
6
7
8
A typical procedure: a mixture of salicylaldehyde derivative (2 mmol) and phenyl acetic acid
derivative (2.1 mmol), acetic acid anhydride (0.6 ml) and triethylamine (0.36 ml) were placed
in a microwave reactor tube and heated at 100ꢀ170 °C in the microwave apparatus for 10ꢀ20
min. After cooling, 2 ml of 10% NaHCO3 solution was added and the precipitate was filtered,
dried and recrystallized from EtOH/H2O or acetone/H2O mixture. The acetyl group(s) were
removed by treating the compound with MeOH/NaOH(aq) solution for 30ꢀ60 min at r.t. The
solution was acidified with HCl (aq.) and the precipitate was filtered and recrystallized if
needed. Experimental data for 7ꢀacetoxyꢀ3ꢀ(4ꢀfluorophenyl)ꢀ2Hꢀchromenꢀ2one, 7ꢀhydroxyꢀ3ꢀ
(4ꢀfluorophenyl)ꢀ2Hꢀchromenꢀ2ꢀone (1), 7ꢀacetoxyꢀ3ꢀ(4ꢀacetoxyphenyl)ꢀ2Hꢀchromenꢀ2one,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7ꢀhydroxyꢀ3ꢀ(4ꢀhydroxyphenyl)ꢀ2Hꢀchromenꢀ2one
(2),
and
7ꢀacetoxyꢀ3ꢀ(4ꢀ
methoxyphenyl)coumarin, 7ꢀhydroxyꢀ3ꢀ(4ꢀmethoxyphenyl)coumarin (3) were already
published elsewhere. 34
7ꢀhydroxyꢀ3ꢀ(4ꢀ(dimethylamino)phenyl)ꢀ2Hꢀchromenꢀ2ꢀone (4) 35
In the first step 7ꢀacetoxyꢀ3ꢀ(4ꢀ(dimethylamino)phenyl)ꢀ2Hꢀchromenꢀ2one was obtained.
Yield: 70%; 1HꢀNMR (400 MHz, d6ꢀDMSO) δ: 2.31 (s, 3H, CH3CO), 2.95 (s, 6H, (CH3)2Nꢀ),
6.77 (d, J3 = 9.0 Hz, 2H, Hꢀ2’, Hꢀ6’), 7.14 (dd, J3 = 8.4 Hz, J4 = 2.2 Hz, 1H, Hꢀ5), 7.26 (d,
J4 = 2.2 Hz, 1H, Hꢀ8), 7.63 (d, J3 = 9.0 Hz, 2H, Hꢀ3’, Hꢀ5’) 7.76 (d, J3 = 8.5 Hz , 1H, Hꢀ5),
8.11 (s, 1H, Hꢀ4); 13CꢀNMR (100.6 MHz, d6ꢀDMSO) δ: 20.85, 39.84, 109.44, 111.58, 117.76,
118.57, 121.57, 126.00, 128.82, 129.11, 136.46, 150.45, 151.90, 152.77, 159.74, 168.85.
In the second step 7ꢀhydroxyꢀ3ꢀ(4ꢀ(dimethylamino)phenyl)ꢀ2Hꢀchromenꢀ2one (4) was
obtained.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 10 of 41
1
2
3
4
5
6
7
8
9
Yield: 85% yellow solid; 1HꢀNMR (400 MHz, d6ꢀDMSO) δ: 2.94 (s, 6H, (CH3)2Nꢀ), 6.72 (d,
J4 = 2.3 Hz, 1H, Hꢀ8), 6.75 (d, J3 = 9.0 Hz, 2H, Hꢀ2’, Hꢀ6’), 6.79 (dd, J3 = 8.4 Hz, J4 = 2.3
Hz, 1H, Hꢀ5),), 7.55 (d, J3 = 8.5 Hz , 1H, Hꢀ5), 7.58 (d, J3 = 9.0 Hz, 2H, Hꢀ3’, Hꢀ5’), 7.99 (s,
1H, Hꢀ4); 13CꢀNMR (100.6 MHz, d6ꢀDMSO) δ: 39.92, 101.59, 112.33, 113.16, 122.30,
122.32, 129.34, 137.83, 150.07, 154.27, 160.30, 160.41. ESIꢀMS: m/z (rel. abund. %) 304 (M
+ Na+); elemental anal. for C17H15N1O3, calc. C% 72.58, H% 5.37, N% 4.98, found C%
72.45, H% 5.40, N% 5.15.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7ꢀhydroxyꢀ3ꢀ(pꢀtolyl)ꢀ2ꢀHꢀchromenꢀ2ꢀone (5) 36
In the first step 7ꢀacetoxyꢀ3(pꢀtolyl))ꢀ2Hꢀchromenꢀ2one was obtained.
1
Yield: 70%; HꢀNMR (300 MHz, d6ꢀDMSO) δ: 2.32 (s, 3H, CH3CO), 2.35 (s, 3H, CH3ꢀPh),
7.17 (dd, J3 = 8.4 Hz, J4 = 2.2 Hz 1H, Hꢀ6), 7.27 (d, J3 = 8.0 Hz, 2H, Hꢀ2’, Hꢀ6’), 7.29 (d, J4
= 2.2 Hz, 1H, Hꢀ8), 7.63 (d, J3 = 8.2 Hz, 2H, Hꢀ3’, Hꢀ5’) 7.80 (d, J3 = 8.5 Hz , 1H, Hꢀ5), 8.21
(s, 1H, Hꢀ4); 13CꢀNMR (100.6 MHz, d6ꢀDMSO) δ: 20.77, 20.82, 109.56, 117.37, 118.66,
125.99, 128.25, 128.76, 129.30, 131.60, 138.09, 139.37, 152.51, 153.28, 159.47, 168.74.
In the second step 7ꢀhydroxyꢀ3(pꢀtolyl))ꢀ2Hꢀchromenꢀ2one (5) was obtained
1
Yield: 85% brownish solid; HꢀNMR (300 MHz, d6ꢀDMSO) δ: 13CꢀNMR (75 MHz, d6ꢀ
DMSO) δ:20.74, 101.65, 112.01, 113.30, 122.07, 128.04, 128.69, 129.80, 132.14, 137.37,
140.38, 154.77, 160.05, 161.05. ESIꢀMS: m/z (rel. abund. %) 275 (M + Na+); elemental anal.
for C16H12O3, calc. C% 76.18, H% 4.79, found C% 75.95, H% 4.83.
7ꢀhydroxyꢀ3ꢀ(1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (6)
In the first step 2ꢀoxoꢀ3ꢀ(1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)ꢀ2Hꢀchromenꢀ7ꢀyl acetate was obtained.
ACS Paragon Plus Environment

Page 11 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
Yield: 65% slightly brown solid; 1HꢀNMR (400 MHz, d6ꢀDMSO) δ: 2.33 (s, 3H, CH3ꢀCO),
7.27 (dd, J3 = 8.5 Hz, J4 = 2.2 Hz, 1H, Hꢀ6), 7.44 (d, J4 = 2.2 Hz 1H, Hꢀ8), 7,98 (d, J3 =8.5
Hz, 1H, Hꢀ5), 8.33 (s, 1H, Hꢀ3’), 8.61 (s, 1H, Hꢀ4), 9.20 (s, 1H, Hꢀ5’); 13CꢀNMR (100.6
MHz, d6ꢀDMSO) δ: 20.87, 110.02, 116.19, 119.50, 123.04, 130.10, 131.81, 144.40, 152.06,
152.21, 153.12, 155.73, 168.76. FTꢀIR (KBr, cmꢀ1): 3416, 1723, 1617, 1208, 1129. mp. 190–
192 °C. ESIꢀMS: m/z (rel. abund. %): calculated for (M + Na+)=294.0485, measured
294.0499, ꢁ=ꢀ1.4 mDa
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In the second step the acetyl group was removed by treating the above mentioned compound
with MeOH/K2CO3 solution.
Yield: 92 % slightly brown solid; 1HꢀNMR (400 MHz, d6ꢀDMSO) δ: 6.85 (d, J4 = 2.3 Hz,
1H, Hꢀ8), 6.90 (dd, J3 = 8.5 Hz, J4 = 2.3 Hz, 1H, Hꢀ6), 7.74 (d, J3 = 8.6 Hz, 1H, Hꢀ5), 8.27
(s, 1H, Hꢀ3’), 8.49 (s, 1H, Hꢀ4), 9.10 (s, 1H, Hꢀ5’), 10.83 (br s, 1H, OH). 13CꢀNMR (100.6
MHz, d6ꢀDMSO) δ: 102.07, 110.42, 114.23, 119.72, 130.70, 134.11, 144.24, 151.78, 153.96,
156.37, 162.01. FTꢀIR (ATR, cmꢀ1): 1724, 1623, 1511, 1328, 1129 811. mp. decomp. > 312
°C. ESIꢀMS: m/z (rel. abund. %): calculated for (M ꢀ H+)=228.0415, measured 228.0408,
ꢁ=0.7 mDa
UGTs and microsomes. Recombinant human UGTs 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10,
2A1, 2A2, 2A3, 2B4, 2B10, 2B7, and 2B17 were produced, as Hisꢀtagged proteins, in
baculovirusꢀinfected insect cells as previously described 37ꢀ39. The relative expression level of
each of these recombinant UGTs was evaluated by immunodetection, using monoclonal
antibody against the Hisꢀtag, as described elsewhere40. A numeric value of 1.0 was given to
the expression level of UGT1A8 and the relative expression level of each of the other UGTs
was related to this value. Normalized activities were obtained by dividing the glucuronidation
rate values by the relative expression level of the tested UGT. In addition, UGTs 1A4, 2B10
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 12 of 41
1
2
3
4
5
6
7
8
9
and 2B15 were purchased from Corning Life Sciences (New York, USA) and are marked, in
Figure 5, with a “C” to indicate that they are commercial enzymes. The expression levels of
the UGTs in the commercial samples could not be determined, so their protein concentration
was used to calculate the reaction rate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A commercial HLM pool (cat no:452210, BD Gentest, Bedford, MA, USA) and human
intestine microsomes (HIM) pool (lot. no 1110189, XenoTech, Kansas City, KS, USA) were
purchased. Pig liver samples were prepared from untreated female pigs that were used for
practicing surgical procedures at the University of Kuopio (currently: University of Eastern
Finland, Kuopio campus). Other animal liver microsomes were prepared as described
previously41. The Ethics Committee for Animal Experiments, University of Kuopio approved
these experiments.
Mutagenesis of UGT1A10
The UGT1A10 mutant 1A10ꢀH210M was prepared according to the QuikChange
methodology, using the cloned UGT1A10 in pFastBac42 as a template and the following two
oligonucleotides:
1. 5’ꢀACTTTCAAGGAGAGAGTATGGAACATGATCGTGCACTTGGAGGACCATTTꢀ3’
2. 5’ꢀ AAATGGTCCTCCAAGTGCACGATCATGTTCCATACTCTCTCCTTGAAAGTꢀ3’
The entire coding sequence of UGT1A10 in the mutant clone was sequenced and,
subsequently, recombinant baculovirus was prepared and used to express the mutant enzyme
in baculovirusꢀinfected SF9 insect cells.
Absorbance and fluorescence spectra of C3 substituted 7-coumarins
ACS Paragon Plus Environment

Page 13 of 41
Molecular Pharmaceutics
1
2
3
The absorbance spectra of 10 µM coumarin derivatives at 100 mM TrisꢀHCl pH 7.4
4
5
6
containing 10
%
dimethylsulfoxide were measured using
a
Hitachi Uꢀ2000
7
8
9
Spectrophotometer (Tokyo, Japan). Excitation and emission fluorescence spectra of 0.1 µM
coumarin derivatives in 100 mM TrisꢀHCl pH 7.4 were measured using a Shimadzu RFꢀ5000
spectrophotofluorometer (Tokyo, Japan). The excitation spectra was from 200 to 420 nm at
460 nm emission and the emission spectra was from 400 to 600 nm at 390 nm excitation
(Data in supplementary Table S1 and Figures S1 and S2). The effect of pH on fluorescence
intensity was determined at 405 nm excitation and 460 nm emission, in the presence of either
1.5 % trichloroacetic acid, 100 mM phosphate buffers at pH 5, 6, 7, 8 and 9, or 1.6 M
glycineꢀNaOH pH 10.4.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Glucuronidation reactions
The incubation mixtures for glucuronidation assays contained 100 mM TrisꢀHCl buffer pH
7.4, 2.5 mM MgCl2, 0.5 mM UDPGA, recombinant UGT or microsomes as the enzyme
source and 0 – 15 µM of the test aglycone substrate. When the incubation mixtures contained
microsomes, alamethicin was used at a final concentration of 12.5 µg/ml, but it was not
included in the recombinant enzyme assays 43. In the first experiments three negative control
samples were tested, namely i) without the substrate 7ꢀhydroxycoumarin derivative, ii)
without the cofactor UDPGA, or iii) without the enzyme source. In subsequent experiments,
the control samples lacked the enzyme source since it gave the highest fluorescence
background. Preliminary experiments were done under different conditions than most of the
later assays, namely in 2.5 ml of 100 mM phosphate buffer pH 7.4, containing 500 µg pig
liver microsomes, 1.0 µM compound 6 and 0.5 mM UDPGA, at room temperature.
Fluorescence spectra (excitation 200 ꢀ 420 nm; emission 400 – 600 nm) or fluorescence
decrease at 390 nm excitation and 460 nm emission, were measured in these preliminary
experiments, using a Shimadzu RFꢀ5000 spectrophotofluorometer.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 14 of 41
1
2
3
4
5
6
7
8
9
Most of the glucuronidation assays were carried out in 96 multiwell plate format, and
incubations were carried out in 100 µl and at 37°C, in the presence of TrisꢀHCl buffer pH 7.4,
UDPGA and the tested 7ꢀhydroxycoumarin derivative, at the indicated concentrations.
Fluorescence decline in the multiwell plate experiments was monitored every other minute,
for 40 min, using an excitation filter at 405 nm and detection at 460 nm, in a Victor2 1420
Multilabel counter (PerkinElmer, Life Sciences, Turku, Finland). The fluorescence values
were transformed into molarity using the aglycone substrates for making the respective
standard curves. Slopes of the substrate concentration decrease per minute were calculated
using linear regression analysis, in which the linear part of the kinetic assay indicated the
glucuronidation rate. Enzyme catalyzed glucuronidation rate was calculated by subtracting
the blank value from the full reaction value. The intraꢀassay variability of the kinetic assays
was 6 % when compound 6 was used as the aglycone substrate. Kinetic analyses were also
performed in the same 96 multiwell plates format, with excitation filter at 405 nm and
detection at 460 nm, using 6ꢀ8 different substrate concentrations per substrate and two
different protein concentrations for both the wildꢀtype and mutant UGT1A10. The higher
concentrations, 13.5 mg/L for UGT1A10 and 12.0 mg/L for the UGT1A10 mutant, were used
when the substrates were HFC, compounds 4 and 6, whereas the lower protein
concentrations, 6.75 and 6.0 mg/L for UGT1A10 and the mutant, respectively, were used
when the substrates were compounds 1, 2, 3, and 5.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In endꢀpoint determinations, the glucuronidation reactions were stopped by the addition of
150 µl 1.6 M glycineꢀNaOH buffer, pH 10.4, followed by fluorescence measurements at 405
nm excitation and 460 nm emission. There was a good correlation between kinetic and endꢀ
point assays (data not shown).
HPLC-MS analysis of 7-hydroxy-3-triazolecoumarin glucuronide. For further analysis of
7ꢀhydroxyꢀ3ꢀtriazolecoumarin glucuronide, 10 µM of compound 6 were incubated in 100 µl
ACS Paragon Plus Environment

Page 15 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
of 100 mM TrisꢀHCl pH 7.4 buffer containing 5 mM MgCl2, 1 mM UDPGA and either 30 ꢀ
40 µg recombinant UGT, 30 µg HLM or 20 µg HIM, for 1 h at 37 C. The reactions were
stopped by the addition of 300 µl methanol, centrifugation and then the supernatant was
divided into two. One part, 100 µl supernatant, was mixed with 150 µl 1.6 M glycineꢀNaOH
pH 10.4 and subjected to fluorescence measurements as described above for the 96ꢀwell
plate. The other part, 250 µl supernatant, was stored at – 20 C until analysis by HPLCꢀMS.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
An Agilent 1200 Series Rapid Resolution LC System (Agilent Technologies, Waldbronn,
Germany) was used for the chromatographic separation, equipped with a reversedꢀphase C8
column (Brownlee Supra, 3 µm, 50 × 2.1 mm, Perkin Elmer). The mobile phases were (A)
0.1% aqueous formic acid and (B) acetonitrile. A linear gradient from 10% to 90% B in five
min was applied, followed by one min isocratic elution with 90% B and column reꢀ
equilibration, yielding a total analysis time of nine min. The flow rate was 0.3 mL/min,
injection volume 3 µL and column oven temperature 30 °C. For the MS detection, a Finnigan
LTQ ion trap mass spectrometer (Thermo, San Jose, CA) was used in the positive
electrospray (ESI) mode. A divert valve was used to direct eluent flow to the waste for 1 min
at the beginning and at the end of the gradient run. The MS analysis was carried out using the
following parameters: spray voltage 4 kV, sheath gas 30 (instrument units), aux gas 15,
sweep gas 3, capillary temperature 250 °C, capillary voltage 43 V, tube lens 100 V. The
collision energy used for MS/MS was 30 V. The acquired full scan MS range was m/z 60 –
600. Ions used for the detection of the aglycone and glucuronide conjugate were m/z 230
[M+H]+ and m/z 406 [M+H+176]+, respectively. Data was acquired and processed using
Xcalibur software package.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 16 of 41
1
2
3
Results
4
5
6
Modeling UGT1A enzymes
7
8
9
Template structures for homology modeling of the UGT enzymes of subfamily 1A were
selected based on sequence similarity, using the blast option in Uniprot. Structures that
contained both the sugarꢀnucleotide cofactor and a small aglycone molecule were beneficial
for the model and, therefore, structures (pdbꢀcodes) 2C1Z/X, 2O6L, 3HBF, and 3WC4 were
selected. The conserved lipophilic region that spans the endoplasmic reticulum membrane
and ends in the “cytoplasmic tail” were excluded from the model because the focus of the
model construction was on the catalytic site and its surrounding area. In addition, other
structures outside the catalytic site of the model, including loops and amino acid side chain
conformations, were not optimized. Optimizing effords were considered detrimental for the
quality of the models as the sequence similarity between the templates and the UGT
sequences was very low. In other words, the models were not considered to be good enough
for molecular dynamics simulations in order to evaluate binding energy or other kinetic
parameters. On the other hand, an essential part of the model building was to predict binding
of the 7ꢀhydroxycoumarin derivatives so that glucuronidation will occur at the 7ꢀhydroxyl
group and for this purpose the models were adequate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The homology models were used to design novel and UGTꢀselective fluorescent 7ꢀ
hydroxycoumarin substrate molecules, whose fluorescence will decrease upon
glucuronidation (Figure 1). Docking of the 7ꢀhydroxycoumarin scaffolds (Figure 2AꢀC) into
different UGT1A models yielded several possible conformations. One of them oriented the 7ꢀ
hydroxy group toward the bound UDPꢀglucuronic acid (UDPGA) cofactor at the catalytic site
and, simultaneously, the coumarin core was optimally placed into a nearby cavity in the
catalytic site (Figure 2D). In our models, the coumarin scaffold could be stabilized through its
ACS Paragon Plus Environment

Page 17 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
C2ꢀcarbonyl oxygen, accepting a hydrogen bond from Q101 in UGT1A8 (Figure 2C),
UGT1A9 and UGT1A10 (Figure 2A). Even stronger stabilizing interactions could be formed
between the coumarin carbonyl at C2 and R102 of UGT1A6 (Figure 2C). On the other hand,
D103 in UGT1A1 (Figure 2B), E104 in UGT1A3 and UGT1A4, or P101 in UGT1A7, are not
capable of forming similar stabilizing hydrogen bond interactions at this site, suggesting that
the coumarin core would not be an optimal substrate for these UGTs. Furthermore, R103 of
UGT1A7, UGT1A8 and UGT1A9, counteracted the stabilizing interactions and partially
blocked the binding of the coumarin scaffold to these UGTs due to its size (Figure 2C).
Unlike significantly bulkier R103 of UGT1A8 (Figure 2C), the Q103 of UGT1A10 at this
site could donate a hydrogen bond to the oxygen at position 1 of the coumarin scaffold
(Figure 2A).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[Figure 2]
Based on these homology models, there was an additional, but variable in size, free space
available in the active site of each UGT1A enzyme, next to position 3 of the coumarin core.
When combined with the 7ꢀhydroxycoumarin docking site analyses (see above), this
additional space was large enough in UGT1A1 and UGT1A10 to accommodate fiveꢀ or sixꢀ
membered ring substituents (Figure 2D). In contrast, a bulkier phenylalanine (F212) is
present in the UGT1A6 model at this position, clearly hindering binding of any 3ꢀsubstituted
7ꢀhydroxycoumarins. The properties of this binding site were quite different also between
UGT1A1 and UGT1A10. While UGT1A1 has a hydrophobic methionine (M213) facing this
site (Figure 2B), UGT1A10 has a histidine at the corresponding site (H210; Figure 2A). On
the opposite side of this additional space, UGT1A10 has an alanine (A100), while UGT1A1
has an asparagine (N102) aligned to the same position. This may mean that in UGT1A10
there is enough space for rather large 3ꢀsubstituted 7ꢀhydroxycoumarins, whereas UGT1A1
would have problems accommodating larger subsitutions at this position of the 7ꢀ
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 18 of 41
1
2
3
4
5
hydroxycoumarin scaffold. At the other end of the UGT1As “spectrum”, UGT1A6 has a
glutamic acid (E101) aligned at the same site, which would limit the cavity even further.
6
7
8
9
Based on docking of a virtual library into the UGT1A10 model, the most promising
molecules were 7ꢀhydroxycoumarin derivatives with the following substituents at the C3
position: 4ꢀfluorophenyl (1), 4ꢀhydroxyphenyl (2), 4ꢀmethoxyphenyl (3), 4ꢀ
dimethylaminophenyl (4), 4ꢀmethylphenyl (5) or triazole (6). Among these, the triazole (6)
derivative could be stabilized by H210 in UGT1A10, while the corresponding methionine in
UGT1A1 (M213) would not be able to form hydrogen bonds with it. In contrast, the addition
of a phenyl moiety to the C3ꢀposition would allow binding by both UGT1A1 and UGT1A10,
as the hydrophobic methionine in UGT1A1 is quite an ideal companion, while the histidine in
UGT1A10 could change its conformation according to the donor functionality, as with
triazole. Based on these observations and considerations, the six new coumarin derivatives
were designed and synthesized (Figure 3: compounds 1ꢀ6). The selected compounds were
made from inexpensive starting materials, producing 3ꢀsubstituted coumarins with 5ꢀ or 6ꢀ
membered rings.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[Figure 3]
Glucuronidation of the 3-substituted 7-hydroxycoumarins
The absorbance and fluorescence spectra of compounds 1 – 6 were very similar showing,
however, some differences in intensity as well as excitation and emission peaks among them
(Figure 4A, Supplementary Figures 1 and 2, Supplementary Table 1.). The fluoresecence was
pH dependent, emitting strongly in neutral and alkaline solutions.
In the first glucuronidation experiments, the fluorescence intensity of all the C3 substituted 7ꢀ
hydroxycoumarin derivatives, compounds 1 – 6, decreased when they were incubated in the
ACS Paragon Plus Environment

Page 19 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
presence of pig liver microsomes and UDPGA, in a buffer containing 100 mM TrisꢀHCl pH
7.4 and 5 mM MgCl2 (Figure 4B). No significant fluorescence decrease was observed in the
negative controls, namely in the absence of either microsomes, a coumarin derivative such as
6, or UDPGA. The fluorescence changes were linearly dependent on the amount of
microsomes (Figure 4C), indicating that the biosynthesis of C3 substituted 7ꢀ
hydroxycoumarin derivatives to nonfluorescent glucuronide conjugates was catalyzed by one
or more UDPꢀglucuronosyltransferase enzymes in the microsomes. The presence of
alamethicin increased glucuronidation rates in this microsomal sample, whereas the addition
of more than 5 % (v/v) dimethyl sulfoxide, acetonitrile or ethanol decreased it (Figure 4D).
An example for glucuronide formation under such incubation condition, using 6 as the
substrate, is presented in Figure S3.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[Figure 4]
The above results with 6 and pig liver microsomes suggested that the glucuronidation rates of
the new 7ꢀhydroxycoumarin derivatives could be accurately determined under our
standardized assay conditions, using different enzyme sources. Subsequently, the
glucuronidation rates of all the new C3 substituted 7ꢀhydroxycoumarin derivatives by nearly
all the human UGTs, including enzymes that are not commercially available, were
determined (Figure 5). For comparison and additional controls, the glucuronidation of 7ꢀ
hydroxycoumarin and 4ꢀtrifluoromethylꢀ7ꢀhydroxycoumarin (HFC), that carry no C3
substitution, by the recombinant enzymes were also measured (Figure 5).
[Figure 5]
The human UGTs screen revealed that all six newly synthesized C3 substituted 7ꢀ
hydroxycoumarins were glucuronidated by UGT1A10 more rapidly than by other UGT
forms. Furthermore, 2, 4, 5 and 6 were selective for UGT1A10, as other UGTs catalyzed their
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 20 of 41
1
2
3
4
5
6
7
8
9
glucuronidation at very low rates. The remaining two new substrates, 1 and 3, were
glucuronidated, in addition to UGT1A10, also by UGT1A1, at rates of about 40 % and 20 %,
respectively, of the corresponding UGT1A10 rate (Figure 5). The glucuronidation profiles of
the new derivatives differed significantly from the control substrates, 7ꢀhydroxycoumarin and
HFC, which were glucuronidated primarily by UGT1A6 (7ꢀhydroxycoumarin), or UGT1A6
and UGT1A10 (HFC), as well as by few other UGTs at lower rates (Figure 5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In addition to recombinant UGTs, the glucuronidation rates of all the new C3 7ꢀ
hydroxycoumarin derivatives, along with 7ꢀhydroxycoumarin and HFC, were measured in
human liver microsomes (HLM) and human intestinal microsomes (HIM). The results
showed that all the new compounds were glucuronidated by HIM at higher rates than by
HLM (Figure 6). Furthermore, 2, 4 and 6 were not glucuronidated by HLM at all, or only at
very low rates, whereas 3 and 5 were conjugated by HLM to about 20 – 25% of the rate
exhibited by HIM. 1 was glucuronidated by HLM at about 40% of the corresponding rate in
HIM (Figure 6). Kinetics of compound 6 glucuronidation confirmed that glucuronidation is
more specific in intestinal than hepatic microsomes, as its Kmꢀvalue was 82 (49ꢀ115) µM and
Vmax 4.0 (2.6ꢀ5.4) µmol/(min* g prot) in human hepatic microsomes and its Kmꢀvalue was 12
(8.8ꢀ15) µM and Vmax 3.5 (2.9ꢀ4.0) µmol/(min * g prot) in human intestinal microsomes.
Adding albumin to incubation mixture increased both Kmꢀ and Vmaxꢀvalues in instestinal
microsomes. In sharp contrast to glucuronidation of the new 7ꢀhydroxycoumarin derivatives,
the “parent compound” 7ꢀhydroxycoumarin, as well as HFC, were glucuronidated to much
higher rates by HLM than by HIM (Figure 6).
[Figure 6]
Effect of H210 to M210 mutation on UGT1A10 activity
ACS Paragon Plus Environment

Page 21 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
The developed model, by being explicit about the role of certain key UGT1A10 residues in
the binding of the designed 7ꢀhydroxycoumarin derivatives, also allowed testing it by
mutagenesis. Hence, we prepared mutant 1A10ꢀH210M, a mutant of UGT1A10 in which
H210 was changed to methionine, the corresponding residue in UGT1A1. The mutant was
expressed in insect cells, similarly to all the other recombinant UGTs that were used in this
study, and its activity toward the new 7ꢀhydroxycoumarin derivatives was tested. This was
combined with kinetic analyses of the glucuronidation of these compounds by both
UGT1A10 and UGT1A10ꢀH201M, sideꢀbyꢀside (Fig. 7 and Table 1). The results
demonstrated clear effects of the mutation on the glucuronidation kinetics of most
compounds. A decrease of Vmax occurred in all but 2 and 6, in which the Km values were
considerably increased (Table 1). Changes in the Km values of the other compounds were
variable, however, an increase in the case of HFC, no change for 5 and a decrease in 1, 3 and
4 (Table 1).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[Table 1.]
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 22 of 41
1
2
3
Discussion
4
5
6
7
8
In this study we constructed predictive homology models for the human UGT1A enzymes in
order to design selective substrates for them. The models indicated several key characteristics
that differ among the active sites of individual UGT1A enzymes. These differences were
exploited for the design and synthesis of six new 3ꢀsubstituted 7ꢀhydroxcoumarin derivatives.
All the new compounds were good substrates for UGT1A10 and four of them, namely 2, 4, 5
and 6, were selective substrates for this UGT (Fig. 5). Among the clear advantages of these
new 7ꢀhydroxcoumarin derivatives as UGT1A10 substrates are their extensive fluorescence
and simple synthesis from lowꢀcost starting materials that make them suitable for quick and
convenient activity measurements in a high throughput format.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Currently, no highꢀresolution, or even lowꢀresolution, structure of a fullꢀlength UGT is
available from Xꢀray crystallography or cryoꢀelectron microscopy. Although their 3Dꢀmodel
construction is challenging, homology models of UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8,
1A9 and 1A10 were constructed in this study. These models suggested that the active site of
UGT1A10 is sufficiently different from the other UGTs to enable design and synthesis of
selective substrates for it.
We had two goals in designing these substrates: they should be selective for UGT1A10 and
determination of their glucuronidation should be based on easily measurable fluorescence
change during the assay. As a starting point, the 7ꢀhydroxycoumarin scaffold was selected,
since it is a common UGT substrate and its derivatives have intense fluorescence 44. In
addition, 7ꢀhydroxycoumarin and 7ꢀhydroxyꢀ4ꢀtrifluoromethylcoumarin have both been
shown to be glucuronidated by several different UGTs 25. The hydroxyl group of 7ꢀ
hydroxycoumarin was oriented toward the UDPGA in the active site of the UGT1A10 model,
indicating that there is space in the active site for an additional six or five ring substituent at
ACS Paragon Plus Environment

Page 23 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
position C3 of the 7ꢀhydroxycoumarin scaffold (Figure 2). Thus, we synthetized six new
UGT1A10 substrates, all of which were 7ꢀhydroxycoumarin derivatives with various
substituents at position C3 of the coumarin scafold.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
All the new 7ꢀhydroxycoumarin derivatives are highly fluorescent and their fluorescence
decreases upon enzymatic glucuronidation (see Figure 4 for an example with compound 6).
A concern in these assays was nonꢀlinearity of the fluorescence at substrate concentrations
above 20 µM. However, fluorescence changes of these substrates were selective, sensitive
and quantitative enough for measurements below this concentration limit, as the amount of
UGT1A10 enzyme could be adjusted to yield linear glucuronidation rates.
The selectivities of the 7ꢀhydroxycoumarin derivatives for UGT1A10 differed substantially.
While some of them, such as 2, 4 and 6, were highly selective, others, like 1 and 3, were also
glucuronidated by UGT1A1 at considerable rates (Figure 5). Previous studies have shown
that fluorescent derivatives of Nꢀbutylꢀ4ꢀphenylꢀ1,8ꢀnaphthalimide are UGT1A1 selective
substrates 45.
11
Since UGT1A10 is an extrahepatic enzyme expressed at high level in the intestine,
the
results obtained with recombinant UGTs could be tested with HLM and HIM. Although these
microsomal preparations contain several different UGTs each, HLM lacks a functional
UGT1A10 while HIM contains it 11. Indeed, the glucuronidation rates in HLM of 4 and 6, as
well as 2, were very low or below the detection limit. Glucuronidation of 1, 3 and 5 took
place in HLM at rates up to 40% of the rates in HIM (Figure 6). These results also suggest
which of the new compounds would be most useful for studies on UGT1A10 activity in
samples from human tissues that express or may express this enzyme. The current results
point at 2, 4 and 6 as good candidates, 2 due to the lack of detectable activity in HLM, 4
based on the combination of high rate with high selectivity, and 6 due to its high selective
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 24 of 41
1
2
3
fluorescence intensity (Figure 6 and supplementary Figures S1 and S2). It may be added here
4
5
6
13
that we recently reported that the commonly used commercial UGT1A10 has low activity .
7
8
9
In this study we have used the UGT1A10ꢀH preparation, not the commercial UGT1A10.
Researches should not expect to get similar results to those reported here for UGT1A10 when
working with the commonly used commercial UGT1A10. On the other hand, experiments
with commercial HIM and HLM are expected to reproduce the current results.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
It is interesting to understand why 6 has a remarkably lower (less than 30 %) intrinsic
glucuronidation clearance (Vmax/Km) by UGT1A10 than the other new 7ꢀhydroxycoumarin
derivatives, 1 – 5 (Table 1). Examination of the substituents at C3 suggests that 6 with a
triazole derivative at C3 was glucuronidated less efficiently than derivatives containing
hydrophobic or other types of hydrophilic substituents. The reason for this could be that the
triazole causes stronger interactions than the other substituents in the active site of
UGT1A10, resulting in slower release of the formed glucuronide.
The present modeling work indicates specific residues that are expected to lead to substrate
selectivity of UGT1A10. We have tested one of the predicted residues, H210, by changing it
to M210, the corresponding residue in UGT1A1, expressed the mutant UGT1A10ꢀM210 and
studied the glucuronidation kinetics of all the new compounds by both the wildtype (i.e.
UGT1A10ꢀH210) and mutant enzymes. The results revealed changes in the Km and/or Vmax
values of the glucuronidation kinetics of all the compounds, but with clear differences among
them (Figure 7 and Table 1). Another unexpected observation was the effect of the mutation
on HFC glucuronidation rate, which may suggest that the effect of the mutation is larger than
expected by the model, or that substrate(s) binding by UGT1A10 (also) involves an induced
fit mechanism.
ACS Paragon Plus Environment

Page 25 of 41
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
In conclusion, in this study 3D molecular models of the UGT1As were constructed and used
for the design and synthesis of six new fluorescent UGT substrates. A new multiwellꢀbased
method that takes advantage of the fluorescence of the compounds and their fluorescence
decrease upon glucuronidation, was established to measure glucuronidation rates. Of the new
compounds, 4ꢀdimethylaminophenyl (4) and triazole (6) C3 substituted 7ꢀhydroxycoumarins
appeared to be the most selective substrates for UGT1A10, an important and often
underestimated extrahepatic human UGT. It is concluded that the selectivity of the new
coumarin derivatives for UGT1A10 depends on the chemical character of their substituent at
C3. These new compounds should enable better, faster, and easier determination of
UGT1A10 activity in tissues than was earlier possible. In addition, their further chemical
modification could stimulate the development of new tools to explore the active site of
different UGT enzymes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

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

Page 27 of 41
Molecular Pharmaceutics
1
2
3
Acknowledgements
4
5
6
7
8
We thank Ms Hannele Jaatinen for excellent expertise in laboratory work. Finnish IT Centre
for Science (CSC) is acknowledged for generous computational grant (O.T.P.: jyy2516 and
jyy2585). We thank Johanna Mosorin for her valuable contribution in the preparation of
recombinant UGTs. The financial support of the academy of Finland (grant no. 137589) and
the Sigrid Juselius Foundation (grant no. 4704583) are highly acknowledged.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Author Contributions
R.O.J, H.R., and O.T.P designed the study. S.R., S.N. and O.T.P. performed the modelling.
S.K. and J.H. performed the synthesis of molecules. A.P. performed HPLCꢀMS analysis.
R.O.J. performed the experimental measurements. M.F. provided the UGT enzymes and with
J.T. prepared the mutant. All the authors contributed to writing of the manuscript.
Declaration of Interest
The authors declare no conflict of interests.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 28 of 41
1
2
3
4
5
6
7
Table 1. MichaelisꢀMenten (MM) kinetic constants of UGT1A10 and UGT1A10mutant catalyzed 7ꢀhydroxyl glucuronidation for the 3ꢀ
substituted 7ꢀhydroxycoumarins.
8
9
UGT1A10
Compound Km (95 %
confidence
UGT1A10mutant
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
V
max (95 %
V
max / Km
Nonꢀlinear Km (95 % confidence
V
max (95 %
V
max / Km
Nonꢀ
linear
MM
confidence
[L/min/g
prot]
MM
model R2
interval) [µM]
confidence
interval)
[L/min/g
prot]
interval) [µM] interval)
[µmol/min/g
[µmol/min/g
prot)
model
R2
prot)]
10.7 (1.7ꢀ19.6) 31 (17ꢀ46)
2.9
2.7
2.7
0.9813
0.9788
0.8028
3.5 (0ꢀ9.3)
8.7 (3.4ꢀ14)
2.5
0.8726
0.9965
0.9695
1
2
3
2.2 (1ꢀ3.4)
5.9 (4.9ꢀ6.9)
7.8 (5.5ꢀ10.2)
3.8 (0.97ꢀ6.7)
6.3 (5.4ꢀ7.3) 0.81
8.3 (0ꢀ31.4)
22.3 (0 – 53.4)
9.5 (6.8ꢀ
12.3)
2.5
2.8 (0ꢀ6.5)
2.8 (0.7ꢀ4.9)
7.0 (3.1ꢀ11)
15.3 (7.3ꢀ24)
16.2 (12ꢀ20)
5.3 (3.7ꢀ8)
5.5
0.9215
0.9571
0.9879
0.9991
4.7 (1.5ꢀ7.9)
4.6 (1.5ꢀ7.8)
26.8 (0ꢀ80)
32.9 (0ꢀ102)
6.5 (4.4ꢀ8.6) 1.4
5.9 (4.1ꢀ7.6) 1.3
0.9846
0.9750
0.9733
0.9781
4
5.8
5
0.76
6.3 (0ꢀ15.9)
0.24
6
14 (10.7ꢀ17.3) 15.1 (12.7ꢀ17.5) 1.1
11.7 (0ꢀ31.7) 0.36
HFC
ACS Paragon Plus Environment

Page 29 of 41
Molecular Pharmaceutics
1
2
3
Figure legends
4
5
6
7
8
Figure 1. Glucuronidation of 7ꢀhydroxycoumarin derivatives. Fluorescent 3ꢀsubstituted 7ꢀ
hydroxycoumarins are glucuronidated to nonꢀfluorescent glucuronide conjugates by UGT
enzymes. The decrease in fluorescence can be measured conveniently in different kinds of
experimental setups.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Docking of 7ꢀhydroxycoumarin and its derivatives into the UGT1A10 model. (A)
Molecular docking placed the 7ꢀhydroxycoumarin with the 7ꢀhydroxy facing the catalytic site
formed by H37 and UDPGA, enabling the glucuronidation reaction and the subsequent
decrease in fluorescence. (B) In UGT1A1, D103 and L106 are not capable of forming similar
beneficial interactions with the coumarin core as the UGT1A10 model. Although N102 might
be able to form beneficial interactions with certain compounds, the cavity might not be large
enough for more sizable substitutions due to N102 and M213. (C) UGT1A8 has otherwise the
same amino acid residues aligning the cavity as UGT1A10, except for R103. Due to its size,
R103 might impair the binding of the coumarin core. (D) 6 fills the binding cavity (orange
solvent accessible surface) of the UGT1A10 model (green solvent accessible surface).
Docking suggests that the 7ꢀhydroxycoumarin and 6 have a similar binding mode at their
identical core. In addition, the C3 substituent of 6 fits tightly to the additional space at the
outward facing end of the binding cavity.
Figure 3. Novel (1ꢀ6) and control 7ꢀhydroxycoumarin and 7ꢀhydroxyꢀ4ꢀ
trifluoromethylcoumarin (HCF) substrates of UGT1A enzymes in this study.
Figure 4. Decrease of 7ꢀhydroxycoumarin fluorescence during glucuronidation. Excitation
and emission fluorescence spectra of 0.1 µM 7ꢀhydroxyꢀ3ꢀtriazolecoumarin at 100 mM
phosphate pH 7.4 buffer (panel A); decrease in 10 µM 7ꢀhydroxyꢀ3ꢀtriazolecoumarin
fluorescence in the presence of 0.7 mg/mL pig liver microsomal protein, 0.5 mM UDPGA, 5
ACS Paragon Plus Environment