Carboranes increase the potency of small molecule inhibitors of nicotinamide phosphoribosyltranferase

Article abstract of DOI:10.1021/jm300740t

Herein we report the use of carboranes to significantly increase the potency of small molecule inhibitors of nicotinamide phosphoribosyltranferase (Nampt), an enzyme that is central to metabolism and cell survival. We compare the inclusion of carborane wi

Full text of DOI:10.1021/jm300740t

Brief Article
pubs.acs.org/jmc
Carboranes Increase the Potency of Small Molecule Inhibitors of
Nicotinamide Phosphoribosyltranferase
Mark W. Lee, Jr.,*,† Yulia V. Sevryugina,† Aslam Khan,† and Shui Q. Ye‡
†Departments of Chemistry and Radiology, University of Missouri, Columbia, Missouri 65211, United States
‡Department of Pediatrics and Department of Biomedical and Health Informatics, Children’s Mercy Hospitals and Clinics, University
of Missouri School of Medicine, Kansas City, Missouri 64108, United States
S
* Supporting Information
ABSTRACT: Herein we report the use of carboranes to significantly increase the potency of small molecule inhibitors of
nicotinamide phosphoribosyltranferase (Nampt), an enzyme that is central to metabolism and cell survival. We compare the
inclusion of carborane with other similarly sized substituents and demonstrate that, compared with their purely organic
counterparts, these molecules exhibit up to 10-fold greater antiproliferative activity against cancer cells in vitro and a 100-fold
increase in Nampt inhibition.
rate limiting enzyme in the mammalian NAD+ recycling
pathway, converting nicotinamide to nicotinamide mononu-
INTRODUCTION
■
Over the past decade, there has been an increasing interest in
cleotide (NMN). Owing to the many disparate physiological
the use of carboranes as pharmacophoric units in drug design.1
roles and cellular compartmentalization of NAD+, this vital
Carboranes, which are icosahedral clusters composed of boron,
carbon, and hydrogen, demonstrate high chemical stability and
enzyme has been given different names (visfatin, pre-B cell
colony enhancing factor (PBEF), NAmPRTase, and Nampt). It
may be readily incorporated into small molecules as highly
has recently been elucidated that Nampt activity plays a central
symmetrical analogues of aromatic hydrocarbons.2 Because
role in metabolism, cellular proliferation, cell survival, and
each of the 12 B−H or C−H vertices may be derivatized
through extensively developed substitution chemistry, these
inflammatory response, making this enzyme a new and
intriguing target for the treatment of many diseases, including
clusters may serve as rigid scaffolds upon which to build
cancer, Alzheimer’s, diabetes, and arthritis.11−15
molecules with well-defined, three-dimensional conformations.3
FK866 1, a molecule identified through library screening
While early biomedical applications of carborane chemistry
focused on the development of boron-delivery agents for use in
methods, is the first known specific and highly potent small
molecule inhibitor of Nampt.16 Although this drug has no
boron neutron capture therapy (BNCT),4 more recent work
primary effect on cellular energy metabolism, its activity causes
has focused on the incorporation carboranes into the structures
of new pharmacological agents. In such examples, the
a gradual depletion of cellular NAD+ levels, inducing
apoptosis.16 The molecular basis for the inhibition of Nampt
hydrophobic nature of carboranes was exploited for the
development of novel bioactive molecules.5−8 In addition to
hydrophobic interactions, it has been elucidated that carboranes
bind strongly with biomolecules through a unique, strong form
of hydrogen bonding.9 This dihydrogen, or proton−hydride,
bond may occur between a hydrogen on a typical proton donor
and a hydrogen bound to an electropositive boron atom.10 The
strength of these individual dihydrogen bonds has been
calculated to average 20 kJ mol−1 and may total more than
67 kJ mol−1 for a single carborane cluster bound to multiple
donors and over 200 kJ mol−1 for metallacarboranes.9
Consequently, these noncovalent interactions may be much
stronger than typical biomolecular hydrogen bonds. Therefore,
the use of the carborane moiety in drug design might prove
superior to similarly sized organic groups in some circum-
stances.
by 1 is understood, as the crystal structure for the drug−
enzyme complex has been determined.17 Located near the
protein dimer interface, each of the two active sites is accessible
only through a narrow tunnel, the dimensions of which
appeared to be ideally suited for a drug molecule bearing a
carborane moiety. When 1 is bound to Nampt, its
benzoylpiperidine moiety partially blocks this tunnel.17 While
Nampt possesses residues at the benzoylpiperidine−enzyme
interface that could potentially form hydrogen bonds with a
ligand, no such drug−enzyme interactions are apparent. We
postulated that a drug presenting a carborane moiety in this
position might prove superior to benzoylpiperidine or other
similarly sized organic groups, owing to the size and
hydrophobicity of carboranes, as well as their potential to
form strong dihydrogen bonds with the available amino acid
residues. Among the adjacent residues is an arginine, which has
been calculated to form strong interactions with carboranes.9
Figure 1 depicts a molecular model of the binding of 1 in the
Before now, no group has systematically compared the three
isomers of carborane with other similarly sized organic groups
in the structure of a drug molecule with the purpose of
improving activity. Here, we report such a comparison. For this
purpose, we chose to construct inhibitors of nicotinamide
phosphoribosyltranferase (Nampt). This protein is the first and
Received: May 27, 2012
Published: August 13, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
included a structure bearing an unsubstituted butylene moiety
2 and other structures in which benzoylpiperidine was replaced
by a phenyl ring 3, adamantane 4, ortho-carborane 5, meta-
carborane 6, or para-carborane 7. We evaluated the activities of
1−7 by performing cell viability assays against three human
cancer cell lines: A549 (lung), T47D (breast), and DLD1
(colon). We employed the MTT assay19 because this test
provides a direct measure of mitochondrial activity and enables
comparison of our results with those previously reported for
1.16 Figure 3 depicts the concentration−response curves
Figure 1. Molecular model of the binding of 1 (green) in the active
site of Nampt. Here, the benzoylpiperidine moiety of 1 has been
replaced with a carborane to illustrate the residues immediately
surrounding this volume.
active site of Nampt where the benzoylpiperidine moiety has
been replaced by a carborane. In this model, the carborane is
surrounded by several hydrophobic residues, including tyrosine,
valine, and isoleucine. As is apparent, arginine 349 is also in
proximity to the carborane. The performance of docking
studies using carborane derivatives is not facile, as none of the
available software packages (including AutoDock, Surflex,
FlexX, and Glide) have the parameters necessary to describe
the hexavalent boron atoms found in polyhedral boranes or
carboranes. However, one group has recently reported efforts in
this area.18
Figure 3. Concentration dependent cell viability exhibited by 1−4, 6,
7 against the A549 human lung cancer cell line.
measured against the A549 cell line for five of the test
compounds and 1. For clarity, the curve for 5 is omitted, as this
agent exhibited activities between those of 1 and 7.
As evidence for our hypothesis, the absence of benzoylpiper-
idine in 2 resulted in a near-complete loss of activity,
exemplifying the necessity for a bulky, hydrophobic moiety at
this position. The addition of a phenyl ring in place of
benzoylpiperidine in 3 resulted in an ∼100-fold increase in
activity over 2. Previous studies that utilized carborane as a
pharmacophoric unit often describe the substitution of a phenyl
ring with a carborane in a drug structure.5−8 Owing to extensive
electron delocalization, carboranes may be described as three-
dimensional analogues of benzene.20 Remarkably, analogues 5−
7, which bear a carborane moiety at the same position as the
phenyl ring in 3, exhibited activities 3−4 orders of magnitude
higher.
RESULTS AND DISCUSSION
■
To ascertain the contribution of the benzoylpiperidine moiety
of 1 to drug activity, we have designed and synthesized a
number of analogues in which this moiety was absent or
replaced by other groups (Figure 2). These compounds
As a hydrophobic, polycyclic compound, adamantane is
similar in size and bulk to the carboranes (differing in mass by
only 10 amu) and it is often employed in drug design studies.
An analogue bearing this substituent might be expected to
exhibit activities comparable to those of the carborane-based
analogues 5−7. The inclusion of an adamantyl group in 4
produced an agent with significant activity, exhibiting cell-line-
dependent IC50 between 89 and 295 nM, further demonstrating
the utility of a bulky, hydrophobic group in this location.
However, the carborane-based agents demonstrated activities
100- to 500-fold higher than 4.
Remarkably, all three carborane-based analogues demon-
strated potencies higher than 1 in each of the three cell lines
tested. Table 1 lists the IC50 values for each of the significantly
potent agents in our study.
Figure 2. Molecular structures of Nampt inhibitors investigated in this
study.
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Journal of Medicinal Chemistry
Brief Article
Table 1. Measured Half-Maximal Inhibitory Concentrations
(IC50) for Agents 1, 4−7 against the Human Tumor Cell
Lines A549 (Lung), DLD1 (Colon), and T47D (Breast)
IC50 (nM)
compd
A549
DLD1
T47D
1 (FK866)
1.62 0.04
88.9 1.1
0.92 0.02
0.41 0.01
0.99 0.03
3.14 0.11
20.3 0.44
1.69 0.06
0.31 0.01
2.20 0.12
3.20 0.16
186.4 4.1
1.29 0.13
0.32 0.02
0.58 0.03
4
5
6
7
The molecule demonstrating the highest potency, 6, bears a
meta-carborane moiety and exhibited subnanomolar IC50
concentrations in each of the three cell lines, corresponding
to a 10-fold increase in potency over 1 in DLD1 and T47D and
a 4-fold increase in A549. While the three isomers of carborane
are essentially identical in size, their dipole moments differ
because of the relative positions of the C−H vertices. The
magnitudes of these dipole moments range between a high of
4.53 D for the ortho isomer and a low of 0 D for the para
isomer.21 If the carborane moieties interacted with the enzyme
simply through hydrophobic interactions, then one might
expect that the para-isomer 7, the most hydrophobic of the
three, would demonstrate the highest potency. However, the
meta-carborane-based analogue 6 exhibited significantly higher
activity in each of the three cell lines, indicating that other
carborane−enzyme interactions are involved. These data,
coupled with the significantly higher potency of the
carborane-based agents over those incorporating a phenyl,
adamantyl, or benzoylpiperidine moiety, indicate that strong
carborane−enzyme interactions are formed, possibly through
the formation of dihydrogen bonds.
Figure 4. Concentration-dependent cell rescue. A549 cells were
treated with 10 nM 1, 5, 6, or 7. Error bars represent the mean SD.
Cell viability was measured using the MTT assay; each measurement
was repeated four times. A nearly complete rescue of the cells was
afforded with increasing concentrations of the Nampt product NMN.
Nampt, an enzyme that has only recently come into focus as a
central link connecting metabolism, cancer, and inflammation.
Given the tremendous unmet need for more efficacious and
affordable chemotherapeutic agents, our findings further
demonstrate that these clusters may provide a manner in
which to improve the efficacy and specificity of new drugs, one
not provided through the use of organic chemistry alone.
Each of our new molecules are structurally similar to 1, with
each sharing the pyridinylacrylamide moiety. We presumed
that, as in the case for 1, the basis for the observed
antiproliferative activity was the inhibition of Nampt activity.
To confirm this, we performed cell rescue assays using the
Nampt enzymatic product NMN against the A549 and DLD1
cell lines. Cells were treated with 10 nM solutions of each test
compound, a concentration high enough to kill most of the
cells. Increasing concentrations of NMN were added, and cell
viability was assayed. Figure 4 depicts the concentration-
dependent rescue effects of NMN. At high NMN concen-
trations, a nearly complete rescue was afforded in each of the
three cell lines. The assay results were virtually identical among
the three carborane-based agents 5−7 and 1, indicating that
Nampt is the common target for each of these agents.
EXPERIMENTAL SECTION
■
General Methods. See Supporting Information for details.
Chemical reactions were performed using a combination of Schlenk
line and glovebox techniques under an inert atmosphere of dry argon.
NMR spectra were recorded on Bruker DRX and Avance
1
spectrometers for H at 300, 400, or 500 MHz, for 13C at 100 or
125 MHz, and for 11B at 160 MHz. Mass spectra were obtained using
Perseptive Biosystems Mariner API-Tof, Waters Q-Tof, or ABI
QSTAR using ESI or APCI. Data are reported as “m/z, calculated;
m/z, found”. The isotopic distribution of each carborane containing
ion matched that expected for the normal abundance of boron.
All cell lines were obtained from ATCC (Manassas, VA).
The MTT assay18 was carried out as follows: A549, DLD1, and
T47D cells were plated in 96-well plates at a density of 10 000 cells per
well overnight at 37 °C. Cells were then treated with different doses of
agents 1−7 for 72 h. MTT reagent was added to the cells for 3 h for
developing formazan crystals. Solubilization buffer was added to the
wells, and the absorbance was measured at 570 nm. 1 was tested
simultaneously in each 96-well plate to directly compare with the
results of the tested compounds. The experiments were repeated five
times, and the standard deviations are reported.
To further confirm Nampt inhibition, we measured the effect
of our most potent inhibitor, 6, on recombinant Nampt activity
using a commercially available colorimetric assay. In this assay,
6 exhibited ∼100-fold higher inhibitory activity than 1.
Cell rescue assays were conducted using NMN as described
previously.22 Briefly, A549 and DLD1 cells were plated at a density of
10 000 cells per well in 96-well plates overnight. Cells were then
treated with 10 nM concentrations of agents 1, 5−7. Simultaneously,
1, 10, or 100 μM NMN was added to the cultures. Cultures were then
continued for 72 h. After this time, MTT reagent was added to the
cells for a period of 3 h and the MTT crystals were solubilized using
solubilization buffer. Readings were taken at 570 nm wavelength.
Control cells were considered as 100% survival to plot the inhibition
and rescue graph. The experiments were repeated four times.
Before testing, each compound was repurified by RP-HPLC using a
Beckman System Gold liquid chromatograph equipped with a
CONCLUSION
■
Previous work has demonstrated that carboranes are potentially
valuable pharmacophores owing to their hydrophobicity,
extensively developed chemistry, and high biostability. In a
systematic manner, we have compared the three isomers of
carborane with similarly sized organic moieties in a single drug
structure, demonstrating that these clusters may be utilized in
drug design to produce simple, yet exceptionally potent new
molecules. To our knowledge, these new carborane-based
agents are now the most potent inhibitors reported to date for
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Journal of Medicinal Chemistry
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Phenomenex Luna C18(2) (250 mm × 4.6 mm) column using a 1
mL/min MeOH/H2O gradient (0.1% HCOOH) with analyte
detection by UV absorbance at 270 nm. After repurification, all
compounds were determined to be >95% pure by HPLC using UV
analysis.
Synthetic Methods. Compounds 2−7 were synthesized by the
coupling of trans-3-(3′-pyridyl)acrylic acid with the respective
amines.23 Scheme 1 outlines two synthetic routes that we followed
mixing thoroughly, followed by incubation at 30 °C for 20 min. After
this period, the absorbance at 450 nm was measured and compared
with the positive control.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures concerning the synthesis, spectral
characterization data, and testing of all compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
a
Scheme 1
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (573) 884-2424. Fax: (573) 882-2754. E-mail:
leemw@missouri.edu.
Notes
The authors declare no competing financial interest.
ABBREVIATION USED
■
amu, atomic mass unit; APCI, atmospheric pressure chemical
ionization; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide; NMN, nicotinamide mononucleotide
a
Reagents and conditions: (a) n-BuLi, −78 °C, 4 h; (b) t-BuMe2SiCl,
−78 °C, 1 h, rt, 12 h; (c) Cl(CH2)4Br, rt, 12 h; (d) 5% LiI, Cs2CO3,
NHBoc2, 2-butanone, 2 days, reflux; (e) 4 M HCl in dioxane, rt, 3.5 h;
(f) Et2S, toluene, reflux, 4 h; (g) HC2(CH2)4N(CO)2C6H4, reflux, 12
h; (h) NaBH4, 6:1 2-propanol/H2O, rt, 12 h; (i) HCLconc, reflux, 12 h;
(j) DMF, BOP, Et3N, C5H4(CH)2COOH, rt, 12 h.
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