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 post-translational modification proteins such as PARP1, among many others.
Some cancer cells are thought to be especially sensitive to NAD+ levels. Nicotinamide
phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in the NAD+ salvage path-
way and its expression and activity is correlated to the concentration of NAD+ synthesized
in the body, making it a potential target for regulation by inhibition.
Nampt is a 55 kDa protein that forms a homodimer whose active site is located along the di-
merization plane. Nampt has several post-translation modification and phosphorylation
sites. The 188 Tyrosine site, located within the substrate channel outside of the active site,
was mutated to mimic phosphorylation Y-188D and mimic the non-phosphorylated state
Y-188F.
Nampt activity was monitored via conversion of its enzymatic product, Nicotinamide mono-
nucleoside NMN, to a fluorescence derivative. A linear relationship between fluorescence
intensity and NMN concentration will be presented along with experiments investigating
enzyme kinetics (including optimization of time course and preliminary Michaelis–Menten
kinetics). These analyses will be instrumental in evaluating Nampt kinetics, by matching cal-
culated Nampt Km and Kcat to literature values and comparing generated mutants and po-
tential inhibitors with this framework.
Kinetics Assay Calibration
Activity Studies of Nicotinamide Phosphoribosyltransferase (Nampt)
Christopher Funk, Cailyn Mather, Katelyn Pina, and Karen H. Almeida, Ph.D.
Physical Sciences Department, Rhode Island College, Providence, RI
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 375 nm and
emission from 410nm - 460nm.
Left Structure: X-ray structure of human Nampt complexed with FK866 inhibitor. Active site resi-
dues are highlighted in orange and yellow, FK866 in magenta and tyrosine188 is highlighted in red.
PDB ID: 2GVJ. Right Structure: Chemical structure of FK866 (APO-866).
Kinetics Tables
Inhibition of Nampt Wild Type with known inhibitor FK866.Panel A:Activity of Nampt Wild Type
(31ug/mL) with varying concentrations of FK866.Panel B:Extrapolated results from panel A using
theoretical controls showing the percent activity of Nampt Wild Type versus [FK866].The IC50 value was
determined to be 1.204 uM. The Zhang paper reported an IC50 value at 0.9+ or - 0.1nM.
Conclusions/Future Work
Assay standardization using Nampt Wild Type. Panel A : Maintaining Nampt at a constant
25ug/mL, while varying NAM concentrations to determine the appropriate [NAM] range for ki-
netics calculations. Panel B: Standard curve of fluorescence versus NMN derivative concentra-
tion. Panel C: Nampt Wild Type Assay (Michalelis-Menten Curve) with a R-squared value of
0.97 using calibrated assay parameters with a 15-minute incubation time at 37 degrees C.
FK866 Inhibition
Average Kcat/Km value is around 5,377 (M-1 sec-1) for Nampt Wild Type.
Preliminary data suggests no evidence in changing the tyrosine Y188D.
Y188F has no activity, Y188 residue is important for the activity of Nampt.
For FK866 the IC50 value is 1.204 uM. This preliminary data confirms that we can inhibit Nampt activity in our lab.
Previous fluorescence assay protocols were revised for incubation and [NAM] for improved kinetic data collection.
Calculated Km and Kcat values are specific to concentration and to protein used in each experiments. Establish a
range within our assays.
Preliminary data on Y188F activity suggests inhibited activity as previously hypothesized. Future assays will hope-
fully confirm these results.
A decrease in fluorescence was noted with the addition of FK866 inhibitor to Nampt Wild Type. Future work will in-
clude additional controls to confirm the theoretical percent activity. Include additional controls to confirm the theo-
retical percent activity.
A
B C
NAMPT WT
0 20 40
0.0
0.1
0.2
0.3
[NAM] uM
[NMN]generated
uM/min
NAMPT Y188D
0 20 40
0.0
0.1
0.2
0.3
[NAM] uM
[NMN]generated
uM/min
NAMPT Y188F
0 20 40
0.0
0.1
0.2
0.3
[NAM] uM
[NMN]generated
uM/min
A B C
Nampt Wild Type and Y188 mutant activity using 100% pure protein (coomassie stain
validation). Panel A: [NMN] generated per minute versus [NAM] using 25ug/mL con-
stant Nampt Wild Type with a 15 minute incubation time at 37 degrees C, with a R-
squared value of 0.4561. Panel B: [NMN] generated per minute versus [NAM] using
41ug/mL constant Nampt Y188D with a 15 minute incubation time at 37 degrees C,
and a R-squared value of 0.38. Panel C: [NMN] generated per minute versus [NAM]
using 25ug/mL constant Nampt Y188F with a 15 minute incubation time at 37 degrees
C, and a R-squared value of 0.267.
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
kcat,
(min-1)
Km,
(uM)
Kcat/Km,
(uM-1 min-
1)
Kcat/Km, (M-
1 sec-1) Reference R-squared time
Burgos and
Schramm 0.46 0.01 92.0000 1,533,333 Biochem 2008
Revollo 6.3 1.8 3.5000 58,333 Cell Metab 2007
Revollo 1.2 0.92 1.3043 21,739 JBC 2004
KHA 3/13 0.298 1.84 0.1620 2,699 0.99 60
CF 7/2 0.7314 6.45 0.1134 1,890 0.93 20
CF 7/3 0.5211 1.03 0.5059 8,432 0.75 20
CF 7/5 1.947 8.189 0.2378 3,963 0.98 15
KP 7/5 0.8008 2.18 0.3670 6,117 0.61 15
KP 7/3 0.6867 3.25 0.2113 3,522 0.79 20
CM 7/6 1.992 6.82 0.2923 4,872 0.97 15
CF 7/12 0.5676 0.821 0.6914 11,524 0.4561 15
NAMPT Y188D
kcat,
(min-1)
Km,
(uM)
Kcat/Km,
(uM-1 min-
1)
Kcat/Km, (M-
1 sec-1) Reference R-squared time
CF 71212 0.2097 0.275 0.7625 12,709 0.38 15
NAMPT Y188F
kcat,
(min-1)
Km,
(uM)
Kcat/Km,
(uM-1 min-
1)
Kcat/Km, (M-
1 sec-1) Reference R-squared time
CF 71212 0.1145 -0.331 -0.3459 -5,765 0.267 15
Calculated Kcat and Km values from fluorescence assays of Nampt Wild Type, Nampt Y188D,
and Nampt Y188F. Panel A: Collection of Kcat and Km values from literature and experimen-
tal data of Nampt Wild Type. The average Kcat/Km (M-1 sec-1) is 5,377 for Nampt Wild Type.
Panel B: Experimental values of Kcat and Km from Nampt mutants.
A
B
0 2 4 6
0
50
100
[FK866] uM
%Activity
B
Fluorescence Assay Mechanism
Nampt Mutations Kinetics
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
Activity %= F-Fco/ F100%-Fo
F= Sample Reactions containing buffer,NAM,Fk866
Fco= Buferr,no NAM,Fk866
Fo= Containing only buffer
F100%= Containing buffer and NAM
!
A
1.0
10-07
1.0
10-06
1.0
10-05
1.0
10-04
1.0
10-03
1.0
10-02
1.0
10-01
1.0
1000
1.0
1001
80
100
120
140
160
[FK866], uM
FluorescenceUnits