Chemical, computational and functional insights into the chemical stability of the Hedgehog pathway inhibitor GANT61

Article abstract of DOI:10.1080/14756366.2017.1419221

This work aims at elucidating the mechanism and kinetics of hydrolysis of GANT61, the first and most-widely used inhibitor of the Hedgehog (Hh) signalling pathway that targets Glioma-associated oncogene homologue (Gli) proteins, and at confirming the chemical nature of its bioactive form. GANT61 is poorly stable under physiological conditions and rapidly hydrolyses into an aldehyde species (GANT61-A), which is devoid of the biological activity against Hh signalling, and a diamine derivative (GANT61-D), which has shown inhibition of Gli-mediated transcription. Here, we combined chemical synthesis, NMR spectroscopy, analytical studies, molecular modelling and functional cell assays to characterise the GANT61 hydrolysis pathway. Our results show that GANT61-D is the bioactive form of GANT61 in NIH3T3 Shh-Light II cells and SuFu^?/? mouse embryonic fibroblasts, and clarify the structural requirements for GANT61-D binding to Gli1. This study paves the way to the design of GANT61 derivatives with improved potency and chemical stability.

Full text of DOI:10.1080/14756366.2017.1419221

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Chemical, computational and functional insights
into the chemical stability of the Hedgehog
pathway inhibitor GANT61
Andrea Calcaterra, Valentina Iovine, Bruno Botta, Deborah Quaglio, Ilaria
D’Acquarica, Alessia Ciogli, Antonia Iazzetti, Romina Alfonsi, Ludovica
Lospinoso Severini, Paola Infante, Lucia Di Marcotullio, Mattia Mori &
Francesca Ghirga
To cite this article: Andrea Calcaterra, Valentina Iovine, Bruno Botta, Deborah Quaglio, Ilaria
D’Acquarica, Alessia Ciogli, Antonia Iazzetti, Romina Alfonsi, Ludovica Lospinoso Severini, Paola
Infante, Lucia Di Marcotullio, Mattia Mori & Francesca Ghirga (2018) Chemical, computational and
functional insights into the chemical stability of the Hedgehog pathway inhibitor GANT61, Journal of
Enzyme Inhibition and Medicinal Chemistry, 33:1, 349-358, DOI: 10.1080/14756366.2017.1419221
To link to this article: https://doi.org/10.1080/14756366.2017.1419221
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
View supplementary material
Submit your article to this journal
View related articles
Published online: 17 Jan 2018.
Article views: 43
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 349–358
https://doi.org/10.1080/14756366.2017.1419221
RESEARCH PAPER
Chemical, computational and functional insights into the chemical stability of the
Hedgehog pathway inhibitor GANT61
Andrea Calcaterraa, Valentina Iovinea, Bruno Bottaa, Deborah Quaglioa, Ilaria D’Acquaricaa, Alessia Cioglia,
Antonia Iazzettia, Romina Alfonsib, Ludovica Lospinoso Severinib, Paola Infantec, Lucia Di Marcotulliob,d
Mattia Moric and Francesca Ghirgac
,
b
aDepartment of Chemistry and Technology of Drugs, Sapienza University of Rome, Rome, Italy; Department of Molecular Medicine, Sapienza
c
d
University of Rome, Rome, Italy; Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy; Pasteur Institute/Cenci
Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
ABSTRACT
ARTICLE HISTORY
Received 13 November 2017
Revised 14 December 2017
Accepted 15 December 2017
This work aims at elucidating the mechanism and kinetics of hydrolysis of GANT61, the first and most-
widely used inhibitor of the Hedgehog (Hh) signalling pathway that targets Glioma-associated oncogene
homologue (Gli) proteins, and at confirming the chemical nature of its bioactive form. GANT61 is poorly
stable under physiological conditions and rapidly hydrolyses into an aldehyde species (GANT61-A), which
is devoid of the biological activity against Hh signalling, and a diamine derivative (GANT61-D), which
has shown inhibition of Gli-mediated transcription. Here, we combined chemical synthesis, NMR spectros-
copy, analytical studies, molecular modelling and functional cell assays to characterise the GANT61
hydrolysis pathway. Our results show that GANT61-D is the bioactive form of GANT61 in NIH3T3 Shh-
Light II cells and SuFuꢀ/ꢀ mouse embryonic fibroblasts, and clarify the structural requirements for
GANT61-D binding to Gli1. This study paves the way to the design of GANT61 derivatives with improved
potency and chemical stability.
KEYWORDS
GANT61; Hedgehog
pathway; Gli inhibitor;
chemical stability; bioactive
form
CONTACT Mattia Mori
Lucia Di Marcotullio
Italy
mattia.mori@iit.it
lucia.dimarcotullio@uniroma1.it
Center For Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy;
Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome,
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

350
A. CALCATERRA ET AL.
the naturally occurring isoflavone Glabrescione B (GlaB) as the first
small molecule capable of impairing Gli1 activity by directly inter-
fering with its binding to DNA8. However, for the sake of clarity
GANT61 (Figure 1) has been the first small molecule reported to
inhibit Gli1 activity in living cells. It was discovered in 2007 by
Lauth et al. in a cell-based screen for small molecule inhibitors of
Gli-mediated transcription, together with GANT58 (Figure 1)35.
These two molecules proved to inhibit both Gli1- and Gli2-medi-
ated gene transactivation in a dose-dependent manner, with an
IC50 value of about 5 mM in cellular assays.
A subsequent study has highlighted the possible chemical
instability of GANT61 by designing a GANT61 diamine derivative
(GANT61-D, Figure 1) that has shown Hh inhibitory efficacy com-
parable to the parent prodrug GANT6136. The same authors have
also underlined the chemical instability of GANT61 in aqueous buf-
fers37, suggesting that GANT61-D and the pyridine 4-carboxyalde-
hyde (GANT61-A) are formed according to the pathway shown in
Figure 1.
GANT61-D has been the subject of another recent study aimed
at investigating its mode and specificity of binding to Gli proteins
and to further corroborate the druggability of Gli in cancer38.
Molecular docking simulations have identified the putative
GANT61-D binding site within residues E119 and E167, although
the authors have carried out molecular docking on the neutral
form of the GANT61-D that is expected to be less abundant than
the corresponding mono- and di-protonated forms at physiological
pH values.
Introduction
One of the most challenging tasks in drug discovery is the identifi-
cation of the active species responsible for the observed biological
activity of a drug or a drug candidate (thereafter referred as the
bioactive form). In fact, any molecular entity may undergo struc-
tural modifications due to the exposure to different cellular envi-
ronments, metabolic enzymes, reactions with redox species, and
so on. In this regard, understanding the metabolic or chemical sta-
bility of a bioactive compound may help to unravel its mechanism
of action and to optimise pharmacokinetics properties, which
increases the success rate of a drug discovery campaign1,2
.
In the last 10 years, many efforts have been spent by our own
and other research groups on the identification of small molecule
inhibitors of the Hedgehog (Hh) signalling pathway3–8, which plays
a pivotal role in the initiation, proliferation, invasion and metasta-
sis of a wide variety of cancers including basal cell carcinoma
(BCC), medulloblastoma9–11, rhabdomyosarcoma12,13, pancreatic14,
colorectal15, metastatic prostate16, small-cell lung17, breast18 carci-
nomas, and malignant gliomas. The Hh pathway is also implicated
in the regulation and maintenance of cancer stem cells (CSCs),
providing a link between the Hh signalling in the regulation of
normal stem cells and its role in CSCs maintenance19–22
.
Noteworthy, targeting CSCs has recently emerged as a profitable
anticancer strategy endowed with impressive therapeutic implica-
tions and challenges23,24
.
Smoothened receptor (Smo) is the most widely appreciated
drug target of the Hh signalling pathway. Two small molecule
antagonists of Smo have been approved by the FDA (namely,
Vismodegib in 2012 and Sonidegib in 2015) for the treatment of
metastatic or locally advanced BCC25,26, while a number of clinical
trials are currently running on additional chemotypes of Smo
Even though these prior works36–38 synergistically substantiated
that GANT61-D is the bioactive form of GANT61, they fail to pro-
vide clear and detailed information on the mechanism and kinetics
of GANT61 hydrolysis, thus limiting the further development of
GANT61 as anticancer lead. To fill this gap, and to pursue our sci-
entific interest in developing Hh-targeting small molecules, here
we combined multiple approaches including chemical synthesis,
computational modelling, analytical tools and functional studies to
provide additional insights into the chemical stability and Hh
inhibitory activity of GANT61. To this end, we synthesised both
GANT61 and GANT61-D in order to provide sufficient amounts of
pure compounds for analytical and functional assays. Afterwards,
GANT61 chemical stability was monitored by NMR spectroscopy
and hydrophilic interaction chromatography (HILIC), whereas
molecular modelling was used to evaluate the mode of binding of
antagonists27,28
.
However, drug-resistant mutations of Smo
sequence29–31, as well as Smo-independent Hh pathway activation
have been highlighted during the treatment with Vismodegib or
other clinical candidates32,33, thus raising the need to identify
novel leads capable to act on the drug-resistant forms of Smo
and/or to block the pathway downstream or independently by
Smo34. In this regard, the Glioma-associated oncogene homologue
1 (Gli1) is the final effector of the Hh pathway and has emerged
as an alternative and more promising target than Smo6.
By integrating multidisciplinary efforts in a concerted strategy,
recently we proved that Gli1/DNA interaction is a druggable target GANT61-D in its neutral as well as multiple ionisation states to
for the treatment of Hh-dependent tumours8. We also identified Gli1 zinc finger domain (Gli1ZF). Hh pathway inhibition by
N
N
S
N
N
GANT58
N
N
N
N
N
N
H2O
N
N
NH HN
+
H
O
GANT61
GANT61-D
GANT61-A
Figure 1. Chemical structures of two Gli antagonists GANT58 and GANT61 discovered in 2007 by Lauth et al. and GANT61 hydrolysis products GANT61-D and
GANT61-A.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
351
GANT61 and GANT61-D was finally evaluated in NIH3T3 Shh-Light sealed tube under an argon atmosphere. Anhydrous DMSO
II cells stably incorporating
a
Gli-responsive firefly luciferase (0.38 ml) and a 2 M solution of (CH3)2NH in THF (2.0 ml, 4.0 mmol)
were added and the mixture was heated to reflux temperature
reporter (Gli-RE), and in SuFuꢀ/ꢀ mouse embryonic fibroblasts
(MEFs) to monitor effects on the Hh signalling at a downstream
level.
(about 100 ꢂC) for 3 h. Then, after cooling, another aliquot (2.0 ml,
4.0 mmol) of 2 M solution of (CH3)2NH in THF was added and the
mixture was refluxed for other 3 h. The last addition of the
(CH3)2NH solution (2.0 ml, 4.0 mmol) was made before refluxing
the mixture overnight. The reaction mixture was poured into
water and extracted with CH2Cl2. Organic layers were collected,
dried over Na2SO4 and concentrated in vacuo to give a pale yellow
oil. The crude product was purified by column chromatography
using silica gel and 5% ethyl acetate/n-hexane as eluent to
obtain 2-(dimethylamino)benzaldehyde (2) in 65% yield (0.35 g,
Methods
General experimental procedures
Commercially available reagents were supplied by Sigma-Aldrich
(St. Louis, MO) and used without further purification. A sample of
GANT61 was purchased from Tocris Bioscience (Bristol, UK) for
comparison with the newly synthesised sample. Dry solvents were
purchased from Sigma-Aldrich except for ethanol, which was dried
by distillation from I2/Mg.
1
2.35 mmol). Pale yellow oil; H NMR (400.13 MHz, CDCl3) d 10.20 (s,
1H), 7.74 (dd, J1 ¼ 7.6 Hz, J2 ¼ 1.6 Hz, 1H), 7.46–7.41 (m, 1H), 7.02
(d, J ¼ 8.0 Hz, 1H), 6.97 (t, J ¼ 7.6 Hz, 1H), 2.89 (s, 6H); 13C NMR
(100.6 MHz, CDCl3) d 191.2, 155.9, 134.6, 131.1, 127.1, 120.8, 117.7,
45.5; ESI-MS (pos.) m/z ¼ 150 ([M þ H]þ).
1H and 13C NMR spectra have been acquired with a Bruker
Avance 400 spectrometer operating at 400.13 and 100.6 MHz,
respectively, at 300 K in CDCl3, MeOD or DMSO-d6, using 5-mm
diameter glass tubes.
1H NMR spectra of GANT61 for kinetic studies were recorded at
310 K in EtOH-d6/PBS-d(D2O) at 50:50 ratio (v/v). The deuterated
PBS buffer was prepared by mixing 0.066 M stock solution of
K2DPO4 (98 atom % D) and KD2PO4 (98 atom % D) prepared in
D2O to the correct pD ¼ 7.4, diluting the solution to the proper
phosphate buffer concentration reported for PBS, and then adding
NaCl and KCl to the final solution in order to reach the concentra-
tion of 0.137 M and 0.0027 M, respectively. The mixture was thor-
oughly equilibrated for ꢁ1 h before being used for NMR
measurements. The resulting pD was 7.4 (assuming that pD ¼ pH
meter reading þ0.4)39, otherwise it was adjusted to 7.4 by add-
ition of 37% w/w DCl or 40% w/w NaOD solutions in D2O.
Chemical shifts were expressed in ppm and coupling constants
(J) in Hertz (Hz), approximated to 0.1 Hz. Residual solvent peak
was used as an internal reference for 1H NMR and 13C NMR spec-
Synthesis of N1,N3-(bis-2-isopropylbenzyl)propane-1,3-diamine
(GANT61-D)
In
Dean–Stark separator fulfilled with molecular sieves (pore size 3 Å)
and anhydrous benzene, were introduced benzaldehyde
a three-necked round bottom flask, equipped with a
2
(0.166 g, 1.11 mmol), anhydrous benzene (15 ml) and 1,3-diamino-
propane (0.041 g, 46 ml, 0.556 mmol), freshly distilled from molecu-
lar sieves. The mixture was heated at reflux temperature and
stirred overnight. The reaction mixture, monitored by TLC (eluent:
n-hexane/ethyl acetate ¼ 90:10), showed the partial disappearance
of the starting material after 12 h. After that time, the solvent was
removed under reduced pressure and the residue was dissolved in
10 ml of dry ethanol. After cooling the solution to 0 ꢂC, NaBH4
(0.083 g, 2.226 mmol) was added and the mixture was stirred for
30 min. Then, the reaction was quenched with a saturated aque-
ous NH4Cl solution and most of the solvent (ethanol) was
removed under reduced pressure. The aqueous residue was
washed with ethyl acetate (3 ꢃ 15 ml) and the organic layers were
discarded. 2 M NaOH was added to the aqueous solution until pH
14 and a white precipitate rapidly appeared. The mixture was thus
extracted with ethyl acetate (4 ꢃ 15 ml) and the collected organic
layers were dried over Na2SO4 and concentrated in vacuo to give
a yellow oil. The crude product was purified by column chroma-
tography using silica gel and the mixture MeOH/Et3N/CH2Cl2
10:5:85 as eluent to obtain the pure GANT61-D in 60% yield
(0.11 g, 0.34 mmol). Oily transparent or yellowish liquid; 1H NMR
(400.13 MHz, MeOD) d 7.28–7.17 (m, 6H), 7.04 (td, J1 ¼ 7.2 Hz,
J2 ¼ 1.2 Hz, 2H), 3.86 (s, 4H), 2.65 (ovrlp s, 12H), 2.68–2.60 (ovrlp m,
4H), 1.75 (p, J ¼ 7.1 Hz, 2H). 13C NMR (100.6 MHz, MeOD) d 154.3,
134.4, 131.0, 129.4, 125.0, 121.0, 51.0, 48.2, 45.4, 29.5. ESI-MS (pos.)
m/z ¼ 341 ([M þ H]þ), 171 ([M þ 2H]2þ).
1
tra. Data for H NMR are reported as follows: chemical shift, multi-
plicity (ovrlp ¼ overlapped, s ¼ singlet, d ¼ doublet, t ¼ triplet,
p ¼ pentuplet, m ¼ multiplet, dd ¼ doublet of doublets, dt ¼ doub-
let of triplets, td ¼ triplet of doublets, ABq ¼ AB quartet), coupling
constant, integral. Spectra were processed with the program
MestReNova version 6.0.2–5475, FT and zero filling at 64 K.
Mass spectra were obtained using a Thermo Finnigan LCQ
Deca XP Plus mass spectrometer equipped with an electrospray
ionisation (ESI) source and a Fleet ion-trapanalyser; capillary tem-
perature 275 ꢂC, spray voltage 5.0 kV (positive mode), sheath gas
(N2) 25 arbitrary units, capillary voltage 40 V, tube lens 15 V.
Analytical liquid chromatography was performed using
a
Waters-1525 HPLC system equipped with an UV detector (Waters
2487) and an evaporative light scattering detector (ELSD) (SEDEX)
detector. The column used was the Acclaim HILIC-10, 3 mm
(150 ꢃ 4.6 mm I.D.), purchased from Thermo Scientific (Waltham,
MA). Eluent: CH3CN/NH4OAc 100 mM (pH ¼ 4.5) at 95:5 ratio (v/v).
Flow-rate: 1.0 ml/min, room temperature. Detection: UV at 254 nm,
and ELSD (P ¼ 3.0 bar, T ¼ 60 ꢂC). The crude product purifications
were carried out on silica column chromatography using Silica Gel
Fluka 60 Å (0.063–0.200 mm, 70–230 mesh). GANT61 was purified
by column chromatography on aluminium oxide active basic EMD
Millipore (0.063–0.200 mm, 70–230 mesh, pH ¼ 9–10.5).
Synthesis of GANT61
In a three-necked round bottom flask were introduced GANT61-D
(0.051 g, 0.15 mmol) in anhydrous THF (0.5 ml) and commercially
available pyridine 4-carboxyaldehyde (GANT61-A; 0.016 g,
0.15 mmol) in anhydrous THF (1.0 ml). The reaction mixture was
heated at reflux temperature and stirred for 18 h. The solvent was
removed under reduced pressure and the crude residue was puri-
Synthesis of 2-(dimethylamino)benzaldehyde (2)
Commercially available 2-fluorobenzaldehyde (1) (0.45 g, 0.38 ml, fied by column chromatography using basic alumina and ethyl
3.62 mmol) and K2CO3 (1.0 g, 7.24 mmol) were introduced in a acetate/n-hexane as eluent to obtain GANT61 in 90% yield

352
A. CALCATERRA ET AL.
(0.058 g, 0.135 mmol). White solid; 1H NMR (400.13 MHz, DMSO-d6) Hh-dependent luciferase reporter assay
d 8.55 (dd, J ¼ 4.5, 1.3 Hz, 2H), 7.67 (dd, J ¼ 4.5, 1.3 Hz, 2H), 7.47
The luciferase assay was performed in NIH3T3 Shh-Light II cells,
(dd, J ¼ 4.5, 1.3 Hz, 2H), 7.15 (m, 2H), 7.06–6.98 (m, 4H), 4.01 (s,
1H), 3.40, 3.51 (ABq, JAB ¼ 14.2 Hz, 4H), 2.81 (td, J ¼ 11.5, 3.7 Hz,
2H), 2.50 (ovrlp s, 12H), 2.18 (p, J ¼ 6.3 Hz, 2H), 1.62–1.54 (m, 2H).
13C NMR (100.6 MHz, DMSO-d6) d 152.5, 150.6, 149.5, 132.9, 128.8,
127.1, 124.4, 122.8, 118.8, 85.3, 52.00, 49.9, 44.7, 22.51. ESI-MS
(pos.) m/z ¼ 431 ([M þ H]þ), 215.7 ([M þ 2H]2þ).
stably incorporating a Gli-responsive luciferase reporter and the
pRL-TK Renilla (normalisation control). The Shh-Light II cells were
cultured in DMEM plus 10% FBS and then treated for 48 h with
SAG alone (200 nM, Alexis Biochemicals, Farmingdale, NY) or in
combination with GANT61 or GANT61-D.
Luciferase and Renilla activity were assayed with a dual-lucifer-
ase assay system according to the manufacturer’s instruction
(Biotium Inc., Hayward, CA). Results are expressed as luciferase/
Renilla ratios and represent the mean SD of three independent
experiments, each performed in triplicate.
Molecular modelling
The crystallographic structure of Gli1ZF in complex with DNA
(PDB ID: 2GLI)40 was used as a rigid receptor in molecular dock-
ing simulations performed with AutoDock4.241. Before doing
docking, DNA atoms and water molecules were manually
removed, whereas cobalt ions present in the X-ray structure
were substituted with Zn(II) ions as in the physiologically rele-
vant human Gli1. For grid generation, a box of 80 ꢃ 70 ꢃ 80
points with a grid spacing of 0.5 Å that covered the whole
Gli1ZF accessible surface was centred on the mass centre of the
protein. An arbitrary þ1 charge was set to Zn(II) ions within
each ZF, taking into consideration that coordination by amino
mRNA expression analysis
Total RNA was isolated with Trizol (Invitrogen/Life Technologies,
Carlsbad, CA) from SuFuꢀ/ꢀ MEFs cultured in DMEM plus 10%
FBS and then treated for 24 h with GANT61 or GANT61-D. RNA
was then reverse transcribed with SensiFAST cDNA Synthesis Kit
(Bioline Reagents Limited, London, UK). Quantitative real-time
PCR (Q-PCR) analysis of Gli1, b-2 microglobulin and HPRT mRNA
expression was performed on each cDNA sample using the VIIA7
Real-Time PCR System employing Assay-on-Demand Reagents
(Life Technologies, Carlsbad, CA). A reaction mixture containing
cDNA template, SensiFAST Probe Lo-ROX Kit (Bioline Reagents
Limited) and primer-probe mixture was amplified using FAST Q-
PCR thermal cycler parameters. Each amplification reaction was
performed in triplicate and the average of the three threshold
cycles was used to calculate the amount of transcript in the sam-
ple (using SDS version 2.3 software). mRNA quantification was
expressed, in arbitrary units, as the ratio of the sample quantity
to the quantity of the calibrator. All values were normalised with
two endogenous controls, b-2 microglobulin and HPRT, which
yielded similar results.
acids decreases the total point charge of
a metal ion as
observed in prior studies42–45. Docking calculations were per-
formed with AutoDock using the Lamarckian Genetic Algorithm
by running 100 GA_RUNS for each molecule and keeping all
other parameters at their default value. Electrostatic properties
of Gli1ZF were evaluated by solving the Poisson–Boltzmann
equation using APBS46.
Docking complexes were further relaxed by energy minimisa-
tion in explicit water solvent (TIP3P type) with Amber1247. The
ff12 and the GAFF force fields were used for simulating proteins
and ligands, respectively. As in docking simulations, an arbitrary
charge of þ1 and was assigned to Zn(II) ions, which were
treated by a non-bonded approach (harmonic restraint of 10 kcal/
(mol·Å2)). Energy minimisation was performed as follows, in
agreement with prior works42,48–51: (i) water molecules were
energy minimised for 100 steps with a steepest-descent algo-
rithm (SD) and subsequent 300 steps with a conjugate gradient
algorithm (CG) while keeping the solute as frozen; (ii) the sol-
vated solute was energy minimised for 1000 steps SD and 9000
steps CG. After energy minimisation, ligand theoretical affinity
was calculated by means of the Molecular Mechanics Generalised
Born Surface Area (MM-GBSA) method52,53 and the XSCORE func-
tion54. Prediction of pKa of GANT61-D was performed by MoKa
(Molecular Discovery Ltd.)55.
Results
Chemical synthesis of GANT61 and GANT61-D
As recently disclosed by Chenna et al., GANT61-D (Figure 1) is an
intermediate in the synthetic pathway to GANT6156. Therefore,
here we applied a similar chemical synthesis protocol with slight
modifications (see Scheme 1) to obtain both compounds in good
yields for analytical and functional studies.
1) 1,3-diaminopropane,
dry benzene, Dean-Stark,
reflux, 12 h
F
H
N
H
N
N
(CH3)2NH, K2CO3
O
O
NH HN
THF, dry DMSO,
reflux, 24 h
2) NaBH4, dry EtOH,
0 °C, 30 min
65%
60%
GANT61-D
1
2
N
GANT61-A,
dry THF, reflux, 18 h
N
N
90%
N
N
GANT61
Scheme 1. Synthetic pathway to obtain both GANT61-D and GANT61.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
353
1
1
Figure 2. GANT61 hydrolysis in EtOH-d6/PBS-d(D2O) 50:50 v/v at 37 ꢂC monitored by H NMR spectroscopy. (A) Time evolution of the H NMR spectrum in the aliphatic
signals region (2–4.2 ppm); peaks corresponding to the monitored proton of GANT61 and GANT61-D are labelled. (B) Evolution of the normalised areas of the selected
1H NMR signals for GANT61 and GANT61-D monitored up to 48 h incubation.
Briefly, nucleophilic aromatic substitution of commercially avail- possible the physiological pathway of GANT61 hydrolysis in living
able 2-fluorobenzaldehyde (1) with dimethylamine led to 2-(dime- cells, NMR experiments were performed at 310 K (37 ꢂC). The sing-
let centred at d ¼ 3.97 ppm in the 1H NMR spectrum of a freshly
thylamino)benzaldehyde (2) in 65% yield. Afterwards, 2 was
converted into the intermediate diimine by treatment with 1,3-dia-
minopropane. Reduction in situ with sodium borohydride afforded
GANT61-D in 39% overall yield from 2-fluorobenzaldehyde (1). To
obtain GANT61, GANT61-D was condensed with commercially
available GANT61-A (see Scheme 1), thus yielding the target com-
pound in 35% overall yield. The structures of GANT61, GANT61-D
and 2 were confirmed by 1H and 13C NMR spectroscopy and by
ESI mass spectrometry (ESI-MS). The NMR spectra of chemically
synthesised GANT61 were superimposable with those of a sample
of GANT61 purchased from Tocris Bioscience and used for com-
parison (data not shown).
prepared solution of GANT61 was attributed to the aminalic pro-
ton (N-CH-N) and was selected as a probe to monitor the kinetics
of hydrolysis of GANT61 (Figure 2(A)). In fact, this proton resonates
in a rather clean and empty region of the NMR spectrum and is
thought to be included in GANT61-A upon hydrolysis of GANT61
based on the pathway described in Figure 1. The intensity of this
signal (normalised with respect to the adjacent increasing signal)
proved to decrease during the time (Figure 2(A)), which indicates
GANT61 degradation. In parallel, a new singlet peak is formed at
4.04 ppm, which was attributed to the Ph-CH2-NH protons of the
newly formed hydrolysis product GANT61-D. Notably, the chemical
shift of the new peak from GANT61-D was shown to vary in time
as the result of H-bond interactions with the buffer, in agreement
with literature data58,59. Based on NMR data acquired up to 48 h
of incubation, a kinetic plot was built to show the hydrolysis of
GANT61 (Figure 2(B)).
Chemical stability of GANT61 checked by NMR spectroscopy
NMR is a powerful tool to monitor the chemical stability of bio-
active substances in solution and to clarify their possible degrad-
ation pathway57. Due to limited water solubility, NMR-based
kinetic studies of GANT61 were performed in a 1:1 mixture of
EtOH-d6 and deuterated PBS buffer prepared by mixing Na2DPO4
and KD2PO4 in D2O and adding proper amounts of NaCl and KCl
The data shown in Figure 2(B) clearly highlights the fast kinet-
ics of GANT61 hydrolysis. After 6 h, around 70% of the initial prod-
uct is hydrolysed into the corresponding GANT61-D, while the
plateau is reached at 24 h when around 90% of initial GANT61 is
(pD ¼ 7.4). In addition, and with the aim to reproduce as much as hydrolysed to GANT61-D. From this time point, the reaction is at

354
A. CALCATERRA ET AL.
Figure 3. HPLC-UV chromatograms acquired during the time showing the disappearance of GANT61 and the formation of GANT61-D. Sample: GANT61 in CH3CN/
100mM NH4OAc (pH 4.5) ¼ 95/5 (v/v) (1 mg/ml), column: Acclaim HILIC-10, 3 lm (150 ꢃ 4.6mm I.D.), mobile phase: CH3CN/100 mM NH4OAc (pH 4.5) ¼ 95/5 (v/v), flow-
rate: 1.0ml/min, detection: UV at 254nm. (A) t ¼ 0 min; (B) t ¼ 30 min; (C) t ¼ 24 h. The ESI-MS (pos.) spectrum of GANT61-D is included in the box for unequivocal
identification.
the equilibrium and no further increase in the relative abundance Acclaim HILIC-10, 3 mm (150 ꢃ 4.6 mm I.D.) column, which was
of GANT61-D is observed up to 48 h incubation.
operated under different combinations of aqueous/organic mobile
phases. The best mobile phase, consisting of acetonitrile/ammo-
nium acetate 100 mM (pH 4.5) ¼ 95:5 (v/v), yielded the GANT61
Chemical stability of GANT61 checked by HPLC
peak at about 2 min of retention time (see Figure
3 and
Supporting Information Figure S1 for HPLC-UV traces and
Supporting Information Figure S2 for the corresponding ELSD
chromatograms). When the sample was dissolved in pure aceto-
nitrile (1 mg/ml), the GANT61 peak was the only peak detected for
at least 1 h, but when we dissolved GANT61 in the mobile phase
(at the same above-mentioned concentration), a new peak attrib-
uted to GANT61-D was immediately detected (t ¼ 0 in Figure 4)
with a retention time equal to 4.20 min. Such a peak proved to
increase during the time, to become the main peak after 24 h
(Figure 3(C)).
For the HPLC monitoring of the chemical stability of GANT61, we
chose to resort to HILIC, which is nowadays accepted as a com-
mon separation mode60–62, essentially dedicated to the separation
of very polar compounds, such as carbohydrates, amino acids, oli-
gonucleotides, and highly polar natural products. One of the
major advantages of HILIC is the easy coupling with MS which
extends its applicability to impurity detection63. In fact, the use of
a
low aqueous/high acetonitrile mobile phase significantly
improves detection sensitivity for compounds analysed by liquid
chromatography (LC)/ESI-MS, thus overcoming the mismatch
between normal-phase LC and ESI-MS64. Also, ELSD has been
widely used in the detection of chromophores-lacking compounds,
and thus it proved to be particularly suitable to HILIC applications.
After an initial step of investigation for the most suitable chro-
The identity of the two peaks was confirmed by flow injection
analysis into an ESI-MS of the above-mentioned solutions (positive
ions mode). In particular, the injection of the acetonitrile solution
yielded a peak at m/z ¼ 430.33 (monoisotopic mass), correspond-
matographic column operating in the HILIC mode, chromato- ing to the protonated form of GANT61 (MW ¼ 429.61), i.e.
graphic studies were performed on the commercially available [M þ H]þ, while replicated analyses of the GANT61 solution in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
355
mobile phase gave a signal at m/z ¼ 341.33, which was attributed computational study has been performed earlier by Agyeman
to the mono-protonated form of GANT61-D (MW ¼ 340.51), i.e. et al.38 by means of molecular docking simulations on the neutral
[M þ H]þ.
form of GANT61-D. Different from this prior work, pKa predictions
clearly indicated that the secondary amino group of GANT61-D is
protonated under physiological conditions, with a prevalence of
the di-protonated form over the mono-protonated form of the
molecule in the pH range 7–7.555. Therefore, to explore the pos-
sible binding mode of GANT61-D to Gli1ZF in a physiological con-
text and to compare our results with previous findings, we
performed molecular docking of GANT61-D in its neutral, mono-
and di-protonated forms by using the same computational set-
tings described by Agyeman et al.38 The lowest energy pose of
the most populated cluster of each GANT61-D form was visually
inspected and used for theoretical affinity studies (see below).
Notably, the neutral form of GANT61-D was docked within the
groove between ZF2 and ZF3 (Figure 5(A)) in H-bond contacts to
Glu250 and Glu298, in excellent agreement with the results pub-
lished previously (see also the Supporting Information Figure
S3)38. For the sake of clarity, here we use the numbering scheme
of the full-length Gli1 (Glu250 and Glu298 used in this work cor-
respond to Glu119 and Glu167 of the X-ray structure, respect-
ively)40. In contrast, protonated forms of GANT61-D were docked
preferentially within ZF1 and ZF2 (Figure 5(A)) in a negatively
charged surface region of Gli1ZF (Figure 5(B)). Accordingly, we
hypothesised that the binding of GANT61-D to Gli1ZF in the most
abundant protonation forms is mainly driven by electrostatic
forces (detailed binding modes of GANT61-D are showed in
Supporting Information Figure S3). Based on molecular docking,
GANT61-D is not able to bind within the DNA-binding site of Gli1
that is located within ZF4 and ZF5 as highlighted by X-ray and
computational studies (Figure 5(B))40. Therefore, it is unlikely that
GANT61-D may directly interfere with Gli1 binding to DNA, as
instead observed for the direct Gli1/DNA interaction inhibitor
GlaB8, although we cannot rule out indirect mechanisms leading
to alteration in DNA interaction.
By plotting the HPLC-UV areas measured from the chromato-
grams acquired during the monitoring against the time (Figure 4)
it was pointed out that the GANT61 molecule undergoes the same
hydrolytic dissociation described above by NMR analysis, although
at lower pH values and hence more rapidly. However, similar to
the NMR study, about 90% of GANT61 is converted in GANT61-D
after 24 h.
Taken together, NMR and HILIC experiments clearly suggest
that GANT61 hydrolyses quickly into GANT61-D, this latter becom-
ing the most abundant species – and hence the bioactive form of
GANT61 – in physiological conditions and for incubation time
equal or higher than 6 h. Notably, at 24 h, around 90% of GANT61
is converted in GANT61-D.
Molecular modelling
In order to check whether the bioactive form of GANT61, namely
GANT61-D, could bind the Gli1ZF, we performed structure-based
molecular modelling studies. It is worth mentioning that a similar
Finally, the theoretical affinity of the three protonation forms of
GANT61-D towards Gli1ZF was predicted by means of the MM-
GBSA method and the XSCORE function53,54, upon energy mini-
misation of docking-based binding complexes in explicit water
solvent. Results reported in Table 1 unequivocally show that the
di-protonated form of GANT61-D holds the strongest affinity for
Gli1ZF, whereas the neutral form was predicted to be the lowest
Figure 4. Chemical stability of GANT61 monitored by HPLC as a function of time.
The acetonitrile (ACN) solution has been plotted on the left as a control.
Figure 5. Predicted binding mode of GANT61-D in its three different protonation states: neutral (cyan sticks), mono-protonated (magenta sticks) and di-protonated (yel-
low sticks). The Gli1ZF crystallographic structure is showed as (A) cartoon and grey transparent surface and (B) surface coloured according to the electrostatic potential
calculated by APBS. Red¼ negatively charged surface; blue¼ positively charged surface. The positively charged DNA docking site on Gli1ZF is highlighted.

356
A. CALCATERRA ET AL.
affinity species. These results further support the hypothesis of an Smo receptor25,26, drug-resistant Smo mutations and aberrant acti-
electrostatic-driven binding of GANT61-D to Gli1ZF that may target vation of Hh signalling downstream of Smo have been identified
the surface region within ZF1 and ZF2 of Gli1ZF, as well as that in clinical patients, which pointed to the serious need of alterna-
GANT61-D binds with the highest affinity as protonated species tive approaches29–33. Besides Smo, one of the most profitable Hh
compared to the neutral form. Finally, a good correlation between targets is Gli1, the final and most powerful effector of Hh signal-
pKd values predicted by XSCORE and experimental affinity meas- ling6,8. GANT61 has been identified as the first small molecule
ured previously was found38.
inhibitor of Gli1 and to date, it is used as a reference control in
multiple studies focusing on Hh signalling transduction and modu-
lation by small molecules35. The chemical instability of GANT61
was first highlighted in 2010 by Lauth et al.36,37, and further
addressed in 2014 by Agyeman et al.38. Besides these reports,
here we aim to provide additional insights into the mechanism
and kinetics of GANT61 hydrolysis and to complement the know-
ledge on the mechanism of actions of this reference Hh signalling
inhibitor. To these aims, GANT61 and its diamine derivative
GANT61-D were obtained by chemical synthesis, while the kinetics
of GANT61 hydrolysis was monitored by high-resolution analytical
tools such as NMR spectroscopy and HILIC. Results of our study
show that GANT61 hydrolysed to the diamine GANT61-D and the
aldehyde GANT61-A. This process is endowed with a rather fast
kinetics, as a plateau is reached in 24 h when about 90% of
GANT61 is converted in GANT61-D. These results unequivocally
indicate that the bioactive form of GANT61 in cell-based experi-
ments is the diamine derivative GANT61-D, particularly when incu-
bation time is equal or higher than 6 h.
To corroborate this hypothesis, and to further support NMR
and HILIC data, we performed molecular modelling and functional
studies. Multiple protonation states of GANT61-D were considered
in molecular docking simulations against Gli1ZF, including the
neutral form that – although being endowed with a very low
probability to exist in physiological conditions – has been the sub-
ject of a recent modelling study38. Results of the present computa-
tional analysis show that the protonated forms are most affine to
Gli1ZF compared to the neutral form, and bind in a negatively
charged region of the protein in correspondence of ZF1 and ZF2
thus suggesting that electrostatic forces may be crucial for inter-
molecular recognition and binding.
Biological activity
To investigate whether GANT61-D carried the ability to inhibit the
Hh signalling, we performed a luciferase functional assay and com-
pared the Hh inhibitory activity of GANT61 with that exerted by
GANT61-D. To this end, we treated NIH3T3 Shh-Light II cells, which
stably incorporate a Gli-RE65, with the synthetic Smo agonist
SAG66 alone or in combination with different concentrations of
the tested compounds. As shown in Figure 6(A), GANT61-D
antagonised the SAG-induced Hh pathway in a dose-dependent
manner and with comparable efficacy as the parent GANT61. Next,
we analysed the effect of GANT61-D in SuFuꢀ/ꢀ MEFs, in which
the constitutive activation of the pathway is a consequence of the
deletion of the negative regulator SuFu gene. GANT61-D at 10 mM
concentration significantly reduced the expression of Gli1, the final
and most powerful effector of the Hh signalling (Figure 6(B)),
already after 24 h, in agreement with the concept that the bio-
active form of GANT61 to mediate Hh inhibition is its diamine
derivative GANT61-D.
Discussion
The Hh signalling pathway is involved in many different types of
cancer and its inhibition by small molecules is nowadays consid-
ered an effective anticancer strategy4,6,65. Although the initial
enthusiasm for the approval by the FDA of two antagonists of the
Table 1. Theoretical affinity of GANT61-D to Gli1ZF.
Protonation form of
GANT61-D
DG binding MM-GBSA
Finally, the Hh inhibitory properties of GANT61 and GANT61-D
were evaluated in NIH3T3 Shh-Light II cells, which stably incorpor-
ate a Gli-RE, and in SuFuꢀ/ꢀ MEFs, in which the pathway is consti-
tutively active. In both assays, GANT61-D showed its ability to
antagonise Hh signalling and to inhibit Gli1 expression with
(kcal/mol)
XSCORE (pKd)
Neutral
Mono-protonated
Di-protonated
ꢀ25.982 0.008
ꢀ28.217 0.006
ꢀ31.972 0.002
4.50
4.60
4.65
Figure 6. (A) Luciferase reporter assay in NIH3T3 Shh-Light II cells, which shows the dose-dependent inhibition of Hh signalling by GANT61 and GANT61-D after 48 h
of treatment. (B) SuFuꢀ/ꢀ MEFs were treated for 24 h with GANT61 or GANT61-D (10 lM) or DMSO as a control. Gli1 mRNA levels were determined by qRT-PCR normal-
ised to b2-microglobulin and HPRT expression.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
357
comparable efficacy as GANT61. Our findings strongly support that
GANT61-D is the bioactive form of GANT61 able to counteract the
Hh signalling at downstream level, thus limiting the oncogenic
potential of the pathway occurring in a context of Smo-independ-
ent or Gli1 hyperactivation by alternative mechanisms.
7. Infante P, Alfonsi R, Ingallina C, et al. Inhibition of
Hedgehog-dependent tumors and cancer stem cells by a
newly identified naturally occurring chemotype. Cell Death
Dis 2016;7:e2376.
8. Infante P, Mori M, Alfonsi R, et al. Gli1/DNA interaction is a
druggable target for Hedgehog-dependent tumors. EMBO J
2015;34:200–17.
9. Berman DM, Karhadkar SS, Hallahan AR, et al.
Medulloblastoma growth inhibition by hedgehog pathway
blockade. Science 2002;297:1559–61.
10. De Smaele E, Fragomeli C, Ferretti E, et al. An integrated
approach identifies Nhlh1 and Insm1 as Sonic Hedgehog-
regulated genes in developing cerebellum and medulloblas-
toma. Neoplasia 2008;10:89–98.
11. Di Magno L, Manzi D, D’Amico D, et al. Druggable glycolytic
requirement for Hedgehog-dependent neuronal and medul-
loblastoma growth. Cell Cycle 2014;13:3404–13.
12. Ridzewski R, Rettberg D, Dittmann K, et al. Hedgehog inhibi-
tors in rhabdomyosarcoma: a comparison of four com-
pounds and responsiveness of four cell lines. Front Oncol
2015;5:130.
13. Oue T, Uehara S, Yamanaka H, et al. Hedgehog signal inhibi-
tors suppress the invasion of human rhabdomyosarcoma
cells. Pediatr Surg Int 2013;29:1153–8.
14. Feldmann G, Dhara S, Fendrich V, et al. Blockade of hedge-
hog signaling inhibits pancreatic cancer invasion and meta-
stases: a new paradigm for combination therapy in solid
cancers. Cancer Res 2007;67:2187–96.
Conclusion
In this work, we shed light on the mechanism and kinetics of
GANT61 hydrolysis, and further complement previous works in sub-
stantiating that GANT61-D is the bioactive form of GANT61 in Hh
inhibition. Molecular modelling studies identified the main pharma-
cophores of GANT61-D that are relevant for the binding to Gli1ZF
and suggested some strategies to generate optimised derivatives.
For example, molecular rigidification and the design of GANT61-D
analogues with constrained diamine linker may be undertaken to
limit the conformational freedom of the molecule; phenyl rings may
be decorated with functional groups that improve the interaction
with Gli1ZF, or with chemical moieties that may ameliorate the
pharmacokinetics profile of the lead; the aryl-dimethylamino group
may be also replaced by different chemotypes, as it seems not par-
ticularly relevant for the interaction with Gli1ZF that we found to be
driven mostly by electrostatic forces. Besides these suggestions, we
hope that results of our integrated and multidisciplinary effort will
inspire additional routes to design more potent and effective Hh
inhibitors based on the scaffold of GANT61-D.
15. Varnat F, Duquet A, Malerba M, et al. Human colon cancer
epithelial cells harbour active HEDGEHOG-GLI signalling that
is essential for tumour growth, recurrence, metastasis and
stem cell survival and expansion. EMBO Mol Med
2009;1:338–51.
16. Karhadkar SS, Bova GS, Abdallah N, et al. Hedgehog signal-
ling in prostate regeneration, neoplasia and metastasis.
Nature 2004;431:707–12.
Disclosure statement
The authors report no conflicts of interest.
Funding
Authors gratefully acknowledge Professor Francesco Gasparrini
from Sapienza University of Rome for his precious support with
HILIC measurements. We thank the financial support from
Associazione Italiana Ricerca Cancro (AIRC) Grant #IG14723 and
#IG20801, PRIN 2012–2013 (2012C5YJSK002), Progetti di Ricerca di
17. Watkins DN, Berman DM, Burkholder SG, et al. Hedgehog
signalling within airway epithelial progenitors and in small-
cell lung cancer. Nature 2003;422:313–17
18. Clement V, Sanchez P, de Tribolet N, et al. HEDGEHOG-GLI1
signaling regulates human glioma growth, cancer stem cell
self-renewal, and tumorigenicity. Curr Biol 2007;17:165–172
19. Zhao C, Chen A, Jamieson CH, et al. Hedgehog signalling is
essential for maintenance of cancer stem cells in myeloid
leukaemia. Nature 2009;458:776–9.
ꢀ
Universita Sapienza di Roma, Pasteur Institute/Cenci Bolognetti
Foundation. We would like to acknowledge networking contribu-
tion by the COST Action CM-1407 “Challenging organic syntheses
inspired by nature – from natural products chemistry to drug
discovery”.
20. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, can-
cer, and cancer stem cells. Nature 2001;414:105–11.
21. Pardal R, Clarke MF, Morrison SJ. Applying the principles of
stem-cell biology to cancer. Nat Rev Cancer 2003;3:895–902.
22. Coni S, Infante P, Gulino A. Control of stem cells and cancer
stem cells by Hedgehog signaling: pharmacologic clues from
pathway dissection. Biochem Pharmacol 2013;85:623–8.
23. Chen K, Huang YH, Chen JL. Understanding and targeting
cancer stem cells: therapeutic implications and challenges.
Acta Pharmacol Sin 2013;34:732–40.
24. Ismail F, Winkler DA. Getting to the source: selective drug tar-
geting of cancer stem cells. ChemMedChem 2014;9:885–98.
25. Dlugosz A, Agrawal S, Kirkpatrick P. Vismodegib. Nat Rev
Drug Discov 2012;11:437–8.
26. Mullard A. 2015 FDA drug approvals. Nat Rev Drug Discov
2016;15:73–6.
References
1. Masimirembwa CM, Bredberg U, Andersson TB. Metabolic
stability for drug discovery and development: pharmacoki-
netic and biochemical challenges. Clin Pharmacokinet
2003;42:515–28.
2. Shu YZ, Johnson BM, Yang TJ. Role of biotransformation
studies in minimizing metabolism-related liabilities in drug
discovery. AAPS J 2008;10:178–92.
3. Varjosalo M, Taipale J. Hedgehog: functions and mecha-
nisms. Genes Dev 2008;22:2454–72.
4. Ng JM, Curran T. The Hedgehog’s tale: developing strategies
for targeting cancer. Nat Rev Cancer 2011;11:493–501.
5. Iovine V, Mori M, Calcaterra A, et al. One hundred faces of
cyclopamine. Curr Pharm Des 2016;22:1658–81.
6. Infante P, Alfonsi R, Botta B, et al. Targeting GLI factors to
inhibit the Hedgehog pathway. Trends Pharmacol Sci
2015;36:547–58.
27. Ruat M, Hoch L, Faure H, Rognan D. Targeting of smooth-
ened for therapeutic gain. Trends Pharmacol Sci 2014;35:
237–46.

358
A. CALCATERRA ET AL.
28. Rimkus TK, Carpenter RL, Qasem S, et al. Targeting the sonic 48. Cau Y, Fiorillo A, Mori M, et al. Molecular dynamics simula-
hedgehog signaling pathway: review of smoothened and
GLI inhibitors. Cancers (Basel) 2016;8:22.
29. Yauch RL, Dijkgraaf GJ, Alicke B, et al. Smoothened mutation
tions and structural analysis of Giardia duodenalis 14-3-3
protein-protein interactions. J Chem Inf Model 2015;55:
2611–22.
confers resistance to a Hedgehog pathway inhibitor in 49. Cau Y, Mori M, Supuran CT, Botta M. Mycobacterial carbonic
medulloblastoma. Science 2009;326:572–4.
30. Atwood SX, Sarin KY, Whitson RJ, et al. Smoothened variants
explain the majority of drug resistance in basal cell carcin-
oma. Cancer Cell 2015;27:342–53.
31. Sharpe HJ, Pau G, Dijkgraaf GJ, et al. Genomic analysis of
smoothened inhibitor resistance in basal cell carcinoma.
Cancer Cell 2015;27:327.
anhydrase inhibition with phenolic acids and esters: kinetic
and computational investigations. Org Biomol Chem
2016;14:8322–30.
50. Mori M, Nucci A, Lang MC, et al. Functional and structural
characterization of 2-amino-4-phenylthiazole inhibitors of
the HIV-1 nucleocapsid protein with antiviral activity. ACS
Chem Biol 2014;9:1950–5.
32. Buonamici S, Williams J, Morrissey M, et al. Interfering with 51. Sholokh M, Improta R, Mori M, et al. Tautomers of a fluores-
resistance to smoothened antagonists by inhibition of the
PI3K pathway in medulloblastoma. Sci Transl Med
2010;2:51ra70.
cent G surrogate and their distinct photophysics provide
additional information channels. Angew Chem Int Ed Engl
2016;55:7974–8.
33. Metcalfe C, de Sauvage FJ. Hedgehog fights back: mecha- 52. Mori M, Manetti F, Botta M. Predicting the binding mode of
nisms of acquired resistance against smoothened antago-
known NCp7 inhibitors to facilitate the design of novel mod-
nists. Cancer Res 2011;71:5057–61.
ulators. J Chem Inf Model 2011;51:446–54.
34. Gonnissen A, Isebaert S, Haustermans K. Targeting the 53. Miller BR 3rd, McGee TD Jr, Swails JM, et al. MMPBSA.py: an
Hedgehog signaling pathway in cancer: beyond smooth-
efficient program for end-state free energy calculations.
ened. Oncotarget 2015;6:13899–913.
J Chem Theory Comput 2012;8:3314–21.
€
35. Lauth M, Bergstrom A, Shimokawa T, Toftgård R. Inhibition 54. Wang R, Lai L, Wang S. Further development and validation
of GLI-mediated transcription and tumor cell growth
by small-molecule antagonists. Proc Natl Acad Sci USA
2007;104:8455–60.
of empirical scoring functions for structure-based
binding affinity prediction. J Comput-Aided Mol Des 2002;
16:11–26.
36. Lauth M, Rohnalter V, Bergstrom A, et al. Correction to 55. Milletti F, Storchi L, Sforna G, Cruciani G. New and original
“Antipsychotic drugs regulate hedgehog signaling by modu-
lation of 7-dehydrocholesterol reductase levels”. Mol
Pharmacol 2011;79:793.
pKa prediction method using grid molecular interaction
fields. J Chem Inf Model 2007;47:2172–81.
56. Chenna V, Hu C, Khan SR. Synthesis and cytotoxicity studies
of Hedgehog enzyme inhibitors SANT-1 and GANT-61 as
anticancer agents. J Environ Sci Health A Tox Hazard Subst
Environ Eng 2014;49:641–7.
57. Valensin D, Cau Y, Calandro P, et al. Molecular insights to
the bioactive form of BV02, a reference inhibitor of 14-3-
3sigma protein-protein interactions. Bioorg Med Chem Lett
2016;26:894–8.
58. Mitra A, Seaton PJ, Ali Assarpour R, Williamson T.
Unprecedented concentration dependent chemical shift vari-
ation in 1H-NMR studies: a caveat in the investigations of
molecular recognition and structure elucidation. Tetrahedron
1998;54:15489–98.
€
37. Lauth M, Rohnalter V, Bergstrom A, et al. Antipsychotic
drugs regulate hedgehog signaling by modulation of 7-
dehydrocholesterol reductase levels. Mol Pharmacol
2010;78:486–96.
38. Agyeman A, Jha BK, Mazumdar T, Houghton JA. Mode and
specificity of binding of the small molecule GANT61 to GLI
determines inhibition of GLI-DNA binding. Oncotarget
2014;5:4492–503.
39. Glasoe PK, Long FA. Use of glass electrodes to measure acid-
ities in deuterium oxide. J Phys Chem 1960;64:188–90.
40. Pavletich NP, Pabo CO. Crystal structure of a five-finger GLI-
DNA complex: new perspectives on zinc fingers. Science
1993;261:1701–7.
41. Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and
AutoDockTools4: automated docking with selective receptor
flexibility. J Comput Chem 2009;30:2785–91.
42. Cirigliano A, Menta S, Mori M, et al. S. cerevisiae as a tool to
select inhibitors of the deneddylating activity of the COP9
signalosome. Yeast 2015;32:S175
59. D’Acquarica I, Calcaterra A, Sacco F, et al. Stereochemical
preference of 20-deoxycytidine for chiral bis(diamido)-
bridged basket resorcin[4]arenes. Chirality 2013;25:840–51.
60. Alpert AJ. Hydrophilic-interaction chromatography for the
separation of peptides, nucleic acids and other polar com-
pounds. J Chromatogr 1990;499:177–96.
61. Hemstrom P, Irgum K. Hydrophilic interaction chromatog-
raphy. J Sep Sci 2006;29:1784–821.
43. Mori M, Dietrich U, Manetti F, Botta M. Molecular dynamics
and DFT study on HIV-1 nucleocapsid protein-7 in complex 62. Nguyen HP, Schug KA. The advantages of ESI-MS detection
with viral genome. J Chem Inf Model 2010;50:638–50.
in conjunction with HILIC mode separations: fundamentals
44. Mori M, Massaro A, Calderone V, et al. Discovery of a new
and applications. J Sep Sci 2008;31:1465–80.
class of potent MMP inhibitors by structure-based optimiza- 63. Dejaegher B, Vander Heyden Y. HILIC methods in pharma-
tion of the arylsulfonamide scaffold. ACS Med Chem Lett
2013;4:565–9.
ceutical analysis. J Sep Sci 2010;33:698–715.
64. Lammerhofer M. HILIC and mixed-mode chromatography:
the rising stars in separation science. J Sep Sci 2010;33:
679–80.
65. Taipale J, Chen JK, Cooper MK, et al. Effects of oncogenic
mutations in Smoothened and Patched can be reversed by
cyclopamine. Nature 2000;406:1005–9.
66. Chen JK, Taipale J, Cooper MK, Beachy PA. Inhibition of
Hedgehog signaling by direct binding of cyclopamine to
smoothened. Genes Dev 2002;16:2743–8.
45. Mori M, Moraca F, Deodato D, et al. Discovery of the first
potent and selective Mycobacterium tuberculosis Zmp1
inhibitor. Bioorg Med Chem Lett 2014;24:2508–11.
46. Baker NA, Sept D, Joseph S, et al. Electrostatics of nanosys-
tems: application to microtubules and the ribosome. Proc
Natl Acad Sci USA 2001;98:10037–41.
47. Case DA, Darden TA, Cheatham TE, et al. AMBER 12. San
Francisco, CA: University of California; 2012.