1. Acknowledgements:
This research is based in part upon work conducted using the Rhode Island Genomics and Sequencing Center which is supported in part by the National Science Foundation (MRI Grant No. DBI-0215393 and EPSCoR Grant Nos.
0554548 & EPS-1004057), the US Department of Agriculture (Grant Nos. 2002-34438-12688 and 2003-34438-13111), and the University of Rhode Island. The project described was supported by grants from the National Center for
Research Resources (5P20RR016457-11) and the National Institute for General Medical Science (8 P20 GM103430-11), components of the National Institutes of Health (NIH), and EPSCoR grants (Nos. 0554548 & EPS-1004057). Its
contents are solely the responsibility of the authors and do not necessarily represent the official views of the NSF, NIGMS, or the NIH.
Introduction
Background
Nicotinamide adenine dinucleotide (NAD+) is of paramount importance as a cellular me-
tabolite, and is a cofactor in more than 200 oxidation-reduction reactions. NAD+ serves as a
substrate for the DNA repair protein PARP1, among others. PARP1 hyperactivity can delete
cellular levels of NAD+. Nicotinamide phosphoribosyltransferase (NAMPT) is responsible for
replenishing cellular NAD+ pools, making it a potential target for regulation. NAMPT forms a
homodimer whose active site is located along the dimerization plane. Many studies have
been done on the inhibition of NAMPT through competition at the active site, but disruption
of the dimerization plane may also inhibit NAMPT activity. Molecular libraries of potential
inhibitors were screened for binding at these surfaces to investigate whether compounds
can disrupt the dimerization of NAMPT and will effectively inhibit NAMPT activity. NAMPT
activity was measured through organic conversion of the native product to a fluorescent de-
rivative. Potential inhibitors were screened and compared to uninhibited controls as well as
the activity of the enzyme in the presence of FK866, a potent NAMPT inhibitor. Compounds
that show less than 50% activity of NAMPT will be identified and investigated further. These
novel inhibitors could be potential chemotherapeutic agents individually or in combination
with other chemotherapy drugs.
Kinetics Assay Calibration
The Identification of Novel Inhibitors of Nicotinamide Phosphoribosyltransferase (NAMPT)
Katelyn Pina, Steven Berardinelli, Christopher Funk, Cailyn Mathers and Karen H. Almeida
Physical Sciences Department, Rhode Island College, Providence, Rhode Island
PARP-1 is an essential protein in the detection of metabolic, chemical or radiation-induced DNA
strand breaks. Upon binding, PARP catalyzes the production of long poly(ADP-ribose)polymers
called PAR. PAR chains act as a signal to recruit other DNA damage repair proteins. After suc-
cessful repair, PAR chains are degraded by PAR glycohydrolase (PARG) to allow for further
damage detection (right panel). ADP-ribose monomers are transferred from NAD+, yielding nico-
tinamide that is recycled into NAD+ via the NAD+ salvage pathway (left panel). Excessive DNA
damage can lead to hyper-activation of PARP and via the connection of these two processes, po-
tential depletion of NAD+ cellular levels. Nampt is the rate-limiting enzyme in the NAD+ salvage
pathway and therefore critical in maintaining sufficient cellular energy.
References
Role of Nicotinamide in DNA Damage, Mutagensis and DNA Repair. (2010) Journal of Nucleic
Acids. 2010: 157591. PMID: 20725615
Molecular basis for the inhibition of human NMPRTase, a novel target for anticancer agents.
(2006) Nat.Struct.Mol.Biol. 13: 582-588. PMID: 16826227
A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransfer-
ase. (2011) Anal. Biochem. 412:18-25. PMID: 21211508
Development of Fluorescent assay for NMN detection. Chemical transformation of nicotinamide
(NAM) to NAD+ via the NAD+ salvage pathway. The intermediate, nicotinamide mononucleotide
(NMN) is converted to a fluorescent derivative for quantitative detection, excitation 360/40 nm and
emission from 545/40 nm.
Top Structure: Chemical structure of FK866 (APO-866).
Bottom Structure: Structure of Nampt showing a dimerization plane and compounds being
docked into dimerizaton plane to determine inhibition. The yellow and red colors are post transla-
tional modifications, yellow are ubiquitination sites and red are phosphorylation sites. The blue is
the product bound to the active site.
Analyzed Data from Fluorimeter.Panel A: Table of average % activities from two independent trials each
trial containing three replicates for each data point.Panel B:Graphical representation of Panel A.This
shows that compound 10 is a potential inhibitor of Nampt.
Conclusions/Future Work
Assay standardization using Nampt Wild Type. Panel A: Standard curve of fluorescence
versus NMN derivative concentration. Panel B: Nampt Wild Type Assay (Michalelis-Menten
Curve) with a R-squared value of 0.97 using calibrated assay parameters with a 15-minute in-
cubation time at 37 degrees C.
A B
A
Preliminary Data of FK866. Panel A: Confirming that FK866 is a valid inhibitor of
Nampt in our lab through fluorometric assays. Panel B: Table shows the calculated
values of the IC50 value and 95% confidence interval compared to the Zhang paper.
The average IC50 for three trials is 23.65 nM compared to the Zhang paper which is
0.9 +/- 0.1 nM.
CM 70612
0 20 40 60 80 100
0.0
0.2
0.4
0.6
0.8
1.0
[NAM] uM
[NMN]generated
uM/min
A
Fluorescence Assay Mechanism
FK866 Preliminary Data
NAD+
N
O
NH2
N
NN
N
NH2
O
HOH
OP
O-
O
O
O
OHOH
OPO
O-
O
N+
O
NH2
O
OHOH
OP-
O
O-
O
PO
O-
O
O-
PO
O-
O
PRPP
Nicotinamide
O
OHOH
OP-
O
O-
O
N+
O
NH2
NMN
NAMPT
PPi
Nmnat
ATP PPi
O
OHOH
OP-
O
O-
O
N
NH
O
H
O
OH
O
KOH
Fluorescent
NMN derivative
+
NMN
PARP-1
Nicotinamide
NAD+
Nampt
Nmnat
Redox Reactions
NADH
ADP/ATP
DNA strand breaks
Poly-ADP-ribosylation
Reactions
Poly(ADP-ribose) polymers
PARG
Free ADP-ribose
PARP-1 (modified)
O
Structures
CM Trial 1% Activity
-4 -2 0 2 4
0
50
100
Log [FK866] nM
%Activity
KP trial 2 111512
-4 -2 0 2 4
-50
0
50
100
150
%Activity
Log [FK866] nM
KP Trial 3 113012
-4 -2 0 2 4
-50
0
50
100
150
FluorescenceUnits
Log [FK866] nM
Analyzed Data
Raw Data
1 2 3 4 5 6 7 8 9 10 11 12
A 164 172 165 939 913 991 159 151 172 173 177 165
B 827 771 865 149 168 239 822 806 868 166 182 153
C 1046 1024 996 154 163 152 742 772 825 362 222 188
D 625 633 528 162 141 141 134 131 145 130 147 150
E 129 139 150 1016 893 881 153 150 157 153 144 159
F 6511 6597 6160 5299 5927 6076 1236 1353 1182 150 152 147
G 795 879 743 143 146 144 911 846 768 209 246 249
H 726 866 881 250 250 234 155 144 156 142 127 153
B
FO A FO B F10O AFO C F10O B F10O C FFK A FFK B FFK C FCOFK A FCOFK B FCOFK C
F6 A F6 B F6 C FCO6 A FCO6 B FCO6 C F7 A F7 B F7 C FCO7 A FCO7 B FCO7 C
F8 A F8 B F8 C FCO8 A FCO8 B FCO8 C F9 A F9 B F9 C FCO9 A FCO9 B FCO9 C
F1O A F1O B F1O C FCO1O A FCO1O B FCO10 C
FO A FO B FO C F10O A F10O B F10O C FFK A FFK B FFK C FcOFK A FCOFK B FCOFK C
F1 A F1 B F1 C FCO1 A FCO1 B FCO1 C F2 A F2 B F2 C FCO2 A FCO2 B FCO2 C
F3 A F3 B F3 C FCO3 A FCO3 A FCO3 B F4 A F4 B F4 C FCO4 A FCO4 B FCO4 C
F5 A F5 B F5 C FCO5 A FCO5 B FCO5 C
A
Inhibition screen using compounds obtained from NCI.Compounds in Diversity Set III from NCI were pri-
oritized via Autodock Vina Molecular Docking (see K.Chauvin Poster).Top binding compounds were as-
sessed for inhibtion.Panel A:Raw fluorometric data of compounds (1-10) and controls.Panel B:Visual rep-
resentation of actual compounds and controls in each labeled well.Panel C:Summary of control and ex-
perimental wells. Panel D: Equation determining % activity for each individual compound normalized to
overall fluorescence.Inhibition of less than 50 % will be studied in more detail.
B
C D
Screen remaining in house NCI compounds and obtain more compounds for screening.
Any compound,including compound 10,that shows a 60% or less activity will be assessed further.
Evaluate compounds from additional target areas within Nampt to block dimerization internally.
B