Cytidine deaminase can deaminate fused pyrimidine ribonucleosides

Article abstract of DOI:10.1039/d1ob00705j

The tolerance of cytidine deaminase (CDA) to expanded heterocycles is exploredviathree fluorescent cytidine analogues, where the pyrimidine core is fused to three distinct five-membered heterocycles at the 5/6 positions. The reaction between CDA and each analogue is followed by absorption and emission spectroscopy, revealing shorter reaction times for all analogues than the native substrate. Pseudo-first order and Michaelis-Menten kinetic analyses provide insight into the enzymatic deamination reactions and assist in drawing comparison to established structure activity relationships. Finally, inhibitor screening modalities are created for each analogue and validated with zebularine and tetrahydrouridine, two known CDA inhibitors.

Full text of DOI:10.1039/d1ob00705j

Volume 19
Number 28
28 July 2021
Pages 6173-6370
Organic &
Biomolecular
Chemistry
rsc.li/obc
ISSN 1477-0520
PAPER
Yitzhak Tor et al.
Cytidine deaminase can deaminate fused pyrimidine
ribonucleosides

Organic &
Biomolecular Chemistry
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PAPER
Cytidine deaminase can deaminate fused
pyrimidine ribonucleosides†
Cite this: Org. Biomol. Chem., 2021,
9, 6237
1
Paul T. Ludford, III, Yao Li, Shenghua Yang and Yitzhak Tor
*
The tolerance of cytidine deaminase (CDA) to expanded heterocycles is explored via three fluorescent
cytidine analogues, where the pyrimidine core is fused to three distinct five-membered heterocycles at
the 5/6 positions. The reaction between CDA and each analogue is followed by absorption and emission
spectroscopy, revealing shorter reaction times for all analogues than the native substrate. Pseudo-first
order and Michaelis–Menten kinetic analyses provide insight into the enzymatic deamination reactions
and assist in drawing comparison to established structure activity relationships. Finally, inhibitor screening
modalities are created for each analogue and validated with zebularine and tetrahydrouridine, two known
CDA inhibitors.
Received 12th April 2021,
Accepted 5th May 2021
DOI: 10.1039/d1ob00705j
rsc.li/obc
windows and potential interference by the inhibitors or the
protein. As such they are fundamentally unfit for real-time and
Introduction
Cytidine deaminase (CDA) is a key enzyme of the pyrimidine high throughput operations.
salvage pathway that deaminates cytidine and 2′-deoxycytidine In light of the emerging role of CDA in human disease,
to uridine and 2′-deoxyuridine, respectively (Fig. 1a). It has the search for new inhibitors of CDA will likely continue, and
been shown to also deaminate several chemotherapeutic new rapid methods of monitoring CDA activity and its sub-
agents, including the anti-cancer agents cytarabine, gemcita- strate/inhibitor tolerance are needed. While numerous fluo-
bine and 5′-azadeoxycytidine, thus diminishing their rescent C analogues have been described, to the best of our
2
3
1
2
–5
potency.
co-administration and mitigation of this detrimental process.
Since deamination involves a nucleophilic attack at the C4 pinone riboside suggests the binding pocket might be roomier
Inhibitors of CDA are thus highly sought after for knowledge, there are none reported that undergo deamination
1
24
by CDA. That CDA binds the bulkier 7-member ring of diaze-
6
–8
8,17
position by a Zn-activated water molecule,
a common inhi- than previously thought.
Larger two ring heterocycles, such
bition strategy is to employ transition state analogues, which as purine-related analogues, might therefore be amenable to
8
–14
mimic the tetrahedral intermediate formed.
Three such deamination and augment this repertoire. We therefore set out
inhibitors are diazepinone riboside, phosphapyrimidine ribo- to explore expanded fluorescent C heterocycles and their CDA
1
3,15–17
side, and tetrahydrouridine (THU) (Fig. 1b).
Separate
from this approach, zebularine, a cytidine analogue, has been
1
6,17
found to be a potent inhibitor as well (Fig. 1b).
Of these,
THU is the only one to have been co-administered with the pre-
2
–5
viously mentioned chemotherapeutic agents.
In the search for new inhibitors and substrates, several
approaches have been used to monitor CDA activity. These
include a colorimetric assay that monitors ammonia pro-
1
8
duction, assays using high performance liquid chromato-
1
9,20
graphy to monitor cytidine and uridine concentrations,
and spectrophotometric assays monitoring changes in absorp-
1
,21,22
tion spectra.
These assays suffer from lengthy time
Department of Chemistry and Biochemistry, University of California, San Diego,
9
500 Gilman Drive, La Jolla, CA 92093-0358, USA. E-mail: ytor@ucsd.edu
Fig. 1 (a) Top: Enzymatic deamination of cytidine to uridine by CDA;
tz
th
mth
tz
th
†
Electronic supplementary information (ESI) available. CCDC 2075449. For ESI
Bottom: enzymatic deamination of C, C, and
C to U, U, and
mth
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
d1ob00705j
U, respectively; (b) CDA inhibitors diazepinone riboside, phosphapyri-
midine riboside, tetrahydrouridine, and zebularine.
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tolerance as substrates and potential tools for studying the
enzyme and its inhibition.
In recent advances by Shin et al. and Rovira et al., two fluo-
rescent fused cytidine and uridine analogues have been
tz
th
25–27
reported (Fig. 1a; C and C).
These fluorescent ribonu-
cleosides absorb above 300 nm and emit in the visible
2
5–27
spectrum.
The emission range and maxima of each C and
U analogue pair is distinct, providing a plethora of wave-
lengths with which to monitor their interconversions. In
addition, the spectra are sufficiently red-shifted to minimize
potential background signals from proteins and canonical pyr-
imidine-based inhibitors. We hypothesized that these ana-
logues may thus provide a simple pathway for monitoring CDA
activity using fluorescence spectroscopy.
Herein we report three new emissive substrates of CDA and
exploit them to provide insight into the enzyme pocket and for
fabricating three screening assays. This is facilitated first by
2
5–27
supplementing our previously reported emissive C analogs
mth
mth
Scheme 1 Synthetic pathways to
U and
C. Reagents and con-
and synthesizing a third fluorescent ribonucleoside analogue ditions: (a) (i) piperidine, 50 °C, 2 h; (ii) NaH (60% in mineral oil), THF,
mth
mth
pair,
C and
U (Fig. 1a). We then monitor the reactions 70 °C, overnight, 19% over two steps. (b) (i) 1, BaCO
3
, hydroxylamine
hydrochloride, MeOH, 70 °C, overnight; (ii) 2 M HCl in OEt , OEt
MeOH, RT, 24 h, 78% over two steps. (c) Potassium cyanate, acetic acid
2
2
,
through absorption and emission spectroscopy and compare
the reaction rates to establish a structure activity relationship.
Finally, we demonstrate the utility of these newfound sub-
(
(
30%), RT, overnight, 94%. (d) Sodium methoxide, MeOH, RT, 15 h, 91%.
e) N,O-Bis(trimethylsilyl)acetamide, β-D-ribofuranose 1-acetate 2,4,5-
strates by assembling a screening assay template with each tribenzoate, TMS Triflate, ACN, 85 °C, 3 h, 85%. (f) (i) Phosphoryl(V)
substrate and validating them using zebularine and THU as chloride, 1,2,4-triazole, pyridine, RT, 1 h; (ii) saturated ammonium
hydroxide, RT, 3 h, 43% over two steps. (g) Ammonia saturated MeOH,
model inhibitors.
6
5 °C, overnight, 53%. (h) Ammonia saturated MeOH, 65 °C, overnight,
85%. Inset: Crystal structure of 5.
Results
Synthesis and photophysical analysis of
mth
tz
mth
ence to 2-aminopurine in water) of 0.24 and 0.30, respectively.
The resulting Stokes shifts calculated from the absorption and
C and
U
tz
th
th
The synthesis of C and C, as well as U and U, have been
−1
mth
emission maxima were 8930 and 9260 cm for
C and
2
5–27
mth
mth
reported.
The pathway to
C and
U began with a mth
U, respectively.
Michael addition of methyl thioglycolate to methyl crotonate
(
Scheme 1). The resulting thioether was cyclized under Claisen
2
8
Reactions of cytidine analogues with CDA
condensation conditions to yield 1.
added to 1 to yield an oxime, which was subsequently treated To determine whether CDA accepted C, C and
with HCl in ether, yielding 2 as a precipitate. Compound 2 strates, HPLC analysis of their enzymatic deamination reac-
Hydroxylamine was
tz
th
mth
C as sub-
2
8
was treated with potassium cyanate under mildly acidic con- tions was performed and compared to the reaction of the
2
8
ditions to produce the urea 3. Compound 3 was then cyclized native substrate. Chromatograms taken after 60 minutes con-
2
8
to the nucleobase 4 with sodium methoxide, which was firmed complete conversion to uridine or the corresponding U
treated with N,O-bis(trimethylsilyl)acetamide and then glycosy- analogue (Fig. S1–4†). The reactions were then monitored via
lated with a benzoyl protected ribose precursor in the presence changes in absorbance (Fig. 2a–c). Initially, steady state traces
mth
28
TMS triflate to give the fully protected
U (5). The reaction were taken various time points during the reaction revealing
tz
tz
th
th
mth
produced only one diastereomer and crystal structure determi- isosbestic points for C and U, C and U, and
nation confirmed the proposed structure, revealing the desired
C and
U at 293, 292, and 305 nm respectively. For each analogue
mth
beta anomeric configuration (Scheme 1). A portion of nucleo- pair, the absorption intensity decreased and blue-shifted as
side 5 was deprotected with methanolic ammonia to provide the reaction progressed (Fig. 2a–c). The reactions were further
mth 28
mth
U. The remaining was converted to
C via 6, by treat- monitored by changes in emission upon excitation at the
ment with phosphoryl chloride and triazole in pyridine, fol- respective isosbestic point (Fig. 2d–f). Steady state traces were
2
8
tz
lowed by ammonolysis in methanolic ammonia (Scheme 1).
taken at various time points over 30 minutes. As C converted
mth
mth
tz
Absorption spectra of
C and
U were taken in water, to U, emission intensity decreased and blue-shifted (Fig. 2d).
displaying maxima at 323 and 306 nm, and extinction coeffi- For the conversion of C to U, emission intensity slightly
cients of 3350 and 2550 M cm , respectively. Excitation at increased and slightly blue shifted revealing an isoemissive
these maxima yielded visible emission, with emission maxima point at 430 nm (Fig. 2e). As
th
th
−
1
−1
mth
mth
C converted to
U, emission
at 455 and 427 nm and quantum yields (determined with refer- intensity increased and blue-shifted (Fig. 2f). The absorption
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tz
th
Fig. 3 Enzymatic conversion of C (black), C (green), C (blue), and
mth
tz
th
mth
C (red) to U, U, U, and
U by CDA. Reactions were monitored by
absorbance (a, c) and emission (b, d) and fit to a first order curve (a, b) or
to an integrated Michaelis–Menten set of differential equations (c, d).
d½Sꢀ
¼
ꢁk1½Eꢀ½Sꢀ þ kꢁ1½ESꢀ
ð3Þ
dt
tz
th
mth
Fig. 2 Steady state absorption traces of (a) C, (b) C, and (c)
C
tz
th
mth
CDA-mediated conversion to U, U, and U respectively, over a time
range from 0 to 30 min; steady state emission traces of (d) C, (e) C,
d½Eꢀ
dt
tz
th
¼ ꢁk1½Eꢀ½Sꢀ þ kꢁ1½ESꢀ þ k2½ESꢀ
ð4Þ
ð5Þ
ð6Þ
ð7Þ
mth
tz
th
mth
and (f)
C CDA-mediated conversion to U, U, and
U respect-
ively, over a time range from 0 to 30 min.
d½ESꢀ
dt
¼
k1½Eꢀ½Sꢀ ꢁ kꢁ1½ESꢀ ꢁ k2½ESꢀ
d½Pꢀ
tz
th
mth
of C, C, and
C reactions with CDA was then monitored at
3
40, 330, and 330 nm respectively (Fig. 3a and c). Finally, the
tz
th
mth
¼ k ½ESꢀ
2
emission of C, C, and
C reactions with CDA were
dt
recorded at 408, 400, and 427 nm, respectively, upon excitation
at the corresponding isosbestic point (Fig. 3b and d).
IC50
KI ¼
Sꢀ
1
þ ½
KM
Analysis of the reactions of cytidine analogues with CDA
Analysis of the absorption curves via the Michaelis–Menten
differential equations revealed k values of 0.91 ± 0.42, 1.04 ±
The data from the aforementioned reactions monitored by
absorption and emission were analyzed using two kinetic
models, a pseudo-first order model (eqn (1) and (2)) and a set of
1
−1 −1
0
±
.01, 1.01 ± 0.47, and 0.75 ± 0.16 µM
2.1, 3.2 ± 0.3, 34 ± 7.2, and 56 ± 5.9 s for C, C, C and
C, respectively. Analysis of the emission curves via the same
s
and k values of 3.2
2
−1
tz
th
differential equations based on Michaelis–Menten kinetics (eqn mth
(3)–(6)). The pseudo-first order model fits to the absorption
method revealed k values of 1.06 ± 0.32, 0.95 ± 0.14 and 0.71 ±
1
curves revealed kapp values of 3.1 ± 0.1, 3.3 ± 0.1, 7.2 ± 0.2, and
−1 −1
0
s
.18 µM
s
2
and k values of 4.6 ± 1.3, 46 ± 5.3, and 45 ± 2.3
for C, C and
6
1
.1 ± 0.2 s− and t values of 220 ± 12, 210 ± 16, 92 ± 5, and
1
−1
tz
th
mth
1
/2
th
C, respectively. k−1 values were assumed
tz
mth
10 ± 2 s for C, C, C, and
C, respectively. The pseudo-first
to be 10% of k values. K values calculated from the k , k−1,
1
M
1
order model fits to the emission curves revealed kapp values of
and k
2
values of the absorption curve fits were 3.6, 3.1, 34, and
−
1
4
9
.1 ± 0.1, 7.4 ± 0.1, and 5.7 ± 0.1 s and t values of 170 ± 10,
tz
th
mth
1/2
75 µM for C, C, C, and
M
C, respectively. The K of C pre-
tz
th
mth
4 ± 2, and 120 ± 8 s for C, C, and
C, respectively.
viously reported for the Q27/A70 variant of CDA, which was
Sꢀ ¼ ½Sꢀ eꢁ
kappt
used in this work, was 17.6 µM. KM values calculated from the
½
ð1Þ
0
k
1
, k−1, and k
2
values obtained from the emission curve fits
kappt
tz
th
mth
½
Pꢀ ¼ ½Sꢀ ð1 ꢁ eꢁ
Þ
ð2Þ were 4.4, 49, and 63 µM for C, C and
C, respectively.
0
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mth
mth
Inhibition by zebularine and THU
the absorption and emission spectra of
C and
U.
tz
Extinction coefficients, Stokes shifts, and quantum yields were
Testing the potency of zebularine as a CDA inhibitor using C,
th
mth
calculated indicating decreases in absorption at wavelengths
C and
.7 ± 0.2, and 1.5 ± 0.1 µM, respectively (Table 2). Using the
Cheng–Prusoff equation (eqn (7)), K values were calculated to
be 1.5 ± 0.1, 2.2 ± 0.1, and 1.3 ± 0.1 µM, respectively (Table 2).
C as C surrogates revealed IC50 values of 5.0 ± 0.4,
above 305 nm, and increases in emission intensity at wave-
2
mth
lengths between 360 and 470 nm would occur if
C was con-
I
mth 28
verted to
U.
tz
th
With the new and previously reported analogues in hand,
we incubated cytidine and each C analogue with CDA for
Similarly, evaluating THU as an inhibitor employing C, C,
mth
and
C revealed IC50 values of 3.1 ± 0.5, 1.7 ± 0.3, and 0.61 ±
.11 µM, respectively (Table 2). K values were found to be 0.95
0.16, 1.4 ± 0.2, and 0.53 ± 0.10 µM, respectively. Reassessing
6
0 minutes. The reactions were HPLC analyzed, confirming all
0
±
I
2
8
three substrates were deaminated to completion (Fig. S1–4†).
mth
To explore whether the unique spectral features of the ana-
logues could be used to monitor the deamination reactions,
we first monitored the steady state absorption spectra of a reac-
tion of CDA with each C analogue over 30 minutes (Fig. 2a–c).
zebularine using
C as the emissive surrogate in the pres-
ence of 100 µM adenosine revealed IC50 and K values of 2.34 ±
I
0
.59 and 2.02 ± 0.51 µM, respectively.
tz
The spectra revealed isosbestic points of 293 nm for C and
tz
th
th
mth
mth
U, 292 nm for C and U, and 305 nm for
C and
U
Discussion
(
Fig. 2a–c). Steady state emission spectra were then taken at
Over the years, highly isomorphic fluorescent ribonucleosides the same time points during the reaction by exciting at the
have provided insight into enzyme kinetics and substrate respective isoabsorptive wavelengths (Fig. 2d–f). The resulting
2
9–33
specificity.
take range anywhere from immune response and signalling to the visible spectrum with which to spectroscopically observe
The diverse cellular pathways these enzymes spectra indicated a range of wavelengths from the near UV to
2
9–33
purine degradation and general metabolism.
of adenosine deaminase (ADA), comparing two adenosine ana- the reaction of cytidine, C, C, and
logues as substrates illustrated the importance of N7 on dea- absorption at 260 nm, 340 nm, 330 nm, and 330 nm respect-
mination kinetics.
further exploring other enzymes including cytidine deaminase.
In the case their enzymatic interconversion (Fig. 2). We chose to monitor
tz
th
mth
C with CDA via
2
6,27,29,32
We took this case as inspiration for ively (Fig. 3a and c). We also chose to monitor the reaction of
tz
th
mth
C, C, and
C via emission at 408 nm upon excitation at
We suspected that, as in the case of ADA, synthetic cytidine 293 nm, 400 nm upon excitation at 292 nm, and 427 nm upon
analogues might be able to provide indirect insight into CDA excitation at 305 nm respectively (Fig. 3b and d). The reaction
substrate scope. We thus explored the ability of CDA to deami- times to completion of all three C analogues were found to be
nate increasingly perturbed C analogues, with distinct levels of shorter than cytidine in both absorption and emission
tz
steric bulk and heteroatom composition, ranging from C, (Table 1).
th
mth
C, to
C (Fig. 1a). We already had previously synthesized
To quantify our observations, curves assuming pseudo-first
C had yet to be reported so we began by order conditions were fit to the data yielding kapp values (eqn
tz
th
mth
C and C, but
developing a synthetic route to it.
(1), (2) and Table 1). The kapp values were converted to t1/2
mth
Synthesis of
C started with cheap, commercially avail- values for further comparison (Table 1). The reaction t1/2 value
tz
able starting materials methyl thioglycolate and methyl croto- for C was found to be slightly shorter than C while the t1/2
th
mth
nate and was completed over 7 steps (Scheme 1). The corres- values for C and
C were found to be two- to three-fold
U, the potential product of a reaction shorter with C being the shortest (Table 1). The kapp and t1/2
C with CDA, was synthesized so that it could be used as values determined by curve fitting the observed changes in
a reference to confirm successful deamination by CDA absorption and emission were found to be in good agreement
Scheme 1). In anticipation of the spectral changes that would with each other (Fig. 3a, b and Table 1). Intriguingly, the reac-
mth
th
ponding U analogue
of
mth
(
mth
be observed during a reaction of
C with CDA, we compared tion rates suggested the addition of a thiophene ring off of the
Table 1 Reaction rate constants for CDA mediated deamination
Pseudo-first order
Michaelis–Menten
(µM−1 s−1
k
app (×10− s−1
3
)
t
1/2 (s)
R
2
k
1
)
k
a
−1 (s−1
)
k
2
(s−1
)
M
K (µM)
b
Abs
Em
C
3.1 ± 0.1
3.3 ± 0.1
7.2 ± 0.2
6.1 ± 0.2
4.1 ± 0.1
7.4 ± 0.1
5.7 ± 0.1
220 ± 12
210 ± 16
92 ± 5
110 ± 2
170 ± 10
94 ± 2
0.981
0.979
0.992
0.987
0.988
0.999
0.999
0.91 ± 0.42
1.04 ± 0.01
1.01 ± 0.47
0.75 ± 0.16
1.06 ± 0.32
0.95 ± 0.14
0.71 ± 0.18
0.091
0.104
0.101
0.075
0.106
0.095
0.071
3.2 ± 2.1
3.2 ± 0.3
34 ± 7.2
56 ± 5.9
4.6 ± 1.3
46 ± 5.3
45 ± 2.3
3.6
3.1
34
75
4.4
49
tz
C
th
C
mth
C
C
tz
C
th
C
mth
120 ± 8
63
a
b
Data are presented as mean ± SD. Values were assumed to be ≤10% of k
1
values. Values calculated from k
1
, k−1, and k
2
.
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and 6 positions of the pyrimidine ring improved deamina-
Paper
5
tion kinetics by CDA.
To further clarify these observations, a set of differential
equations corresponding to the Michaelis–Menten kinetics
were solved setting initial concentrations to those used in the
experiments to yield time dependent substrate and product
3
4–36
concentration curves.
experimental data and optimized by maximizing R , yielding
, k , and K values (eqn (3)–(6), Fig. 3c, d and Table 1).
Those curves were fitted to the
2
k
1
2
M
While perhaps less accurate than a thorough Michaelis–
Menten analyses, this streamlined approach was taken as our
goal was to only compare several related substrates to one
another and gain insight into their interactions with the
enzyme. k− values below 10% of k values had minimal effect
1
1
2
on R (varying less than 0.0001). K
M
values were only slightly
affected at this range (3.3% at most), thus we restricted k−1
values to 10% of k values, effectively setting them as an upper
1
limit to what they could be. K
M
values of CDA with various sub-
strates have been previously reported indicating the enzyme
1
,2,6,12,17,18,20–23
adheres to Michaelis–Menten kinetics.
model revealed a slightly larger binding rate constant (k
The
) for
th
th
mth
mth
Fig. 4 (a, b) Conversion of C to U (a) and
C to
U (b) in the
1
presence of various concentrations of zebularine ([I] = 0 µM (black),
.1 µM (red), 1 µM (blue), 10 µM (green), 100 µM (grey), 1 M (light blue).
tz
th
C and C when compared to cytidine, suggesting the fusion
of a five-membered isothiazole or thiophene ring improved (c, d) Semi-log plot of % inhibition in decimal form after 4 ( C) or 5 ( C,
binding. The slightly smaller binding rate constant for
0
th
tz
mth
mth
C) minutes versus [Zebularine] (c) or [THU] (d) fit to a sigmoidal Hill
C
tz
th
mth
mth
curve: C (green), C (blue),
light blue).
C (red),
C with 100 µM adenosine
indicated the bulky methyl group diminishes binding, but not
detrimentally, suggesting some tolerance for steric pertur-
bation. The model further showed the deamination and
(
th
mth
unbinding rate constant (k
2
) for C and
C was faster than
tz
Table 2 Experimentally determined IC50 and K
I
values of zebularine
cytidine, but about the same for C as compared to cytidine
Table 1). This indicated the shorter t1/2 observed for
mth
and THU
(
C was
dominated by the deamination rate rather than binding. This
further confirmed the expansion of the ring system in cytidine
could potentially improve substrate reception by the enzyme.
With a number of new substrates to monitor CDA activity
effectively and in real time, we chose two known inhibitors,
zebularine and THU, to validate these emissive nucleosides as
tools for screening assays. Reactions of each C analogue with
2
Inh.
IC50 (µM)
K
I
(µM)
R
tz
C
C
Zeb
Zeb
Zeb
THU
THU
THU
5.0 ± 0.4
2.7 ± 0.2
1.5 ± 0.1
3.1 ± 0.5
1.7 ± 0.3
0.61 ± 0.11
1.5 ± 0.1
2.2 ± 0.1
1.3 ± 0.1
0.95 ± 0.16
1.4 ± 0.2
0.53 ± 0.10
0.999
0.999
0.999
0.948
0.995
0.995
th
mth
C
C
tz
C
C
th
mth
CDA were monitored via emission under the same conditions Data are presented as mean ± SD.
as described above, but with varying concentrations of either
inhibitor (Fig. 4a and b). A selected time point for each reac-
tion was compared to a reference reaction without inhibitor analogue was an effective alternative for characterizing inhibi-
and plotted against inhibitor concentration (Fig. 4c and d). tors of CDA. Furthermore, the similarity of the IC50 and KI
mth
IC50 values were calculated from Hill curves fit to the resulting values obtained with and without excess adenosine using
C
plots (Fig. 4c and d). The IC50 values were then converted to KI to monitor CDA activity indicated that background signal from
values via the Cheng–Prusoff equation (eqn (7) and Table 2). similar molecules was less of a factor.
The K values of zebularine and THU show slight variance
I
between the three substrates used. While its origin is unclear
at present, we note that Laliberte et al. have also reported dis-
Conclusions
crepancies in K
I
values for zebularine and THU depending on
1
7
the substrates used. In addition, zebularine was analyzed in To explore the binding pocket of CDA, a new fluorescent C and
mth
mth
mth
the presence of 100 μM adenosine with
C by the same U analogue pair,
C and
U, was synthesized (Scheme 1).
method as described without adenosine (Fig. 4c). The IC50 and The absorption and emission spectra were analyzed yielding
KI values resulting from the analysis with 100 μM adenosine extinction coefficients, Stokes shifts, and emission quantum
mth
are 2.34 ± 0.59 and 2.02 ± 0.51 µM, respectively, in good agree- yields. Upon reacting
C and two previously synthesized C
tz
th
ment with the values calculated without adenosine (Table 2). analogues, C and C, with CDA we discovered that all three
The consistency in IC50 and K values confirmed that each C serve as viable substrates and, in fact, have shorter reaction
I
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half-lives than the native cytidine (Table 1). This confirmed
our hypothesis that fusing additional aromatic rings onto the
pyrimidine core may retain substrate viability by CDA. Further
analysis indicated that the thieno- and isothiazolo-based
analogs improved binding affinity and accelerated deamina-
tion rates while a methyl substitution onto the thiophene
slightly diminished binding affinity (Table 1). We were also
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tz
th
mth
able to use C, C, and
C to establish three new templates
for spectroscopy-based screening assays with zebularine and
THU as model inhibitors (Fig. 4). As all three C and U ana-
logues have red shifted absorption and emission spectra rela-
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attractive alternatives for monitoring CDA activity and its inhi-
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Author contributions
Paul T. Ludford III: data curation, formal analysis, investi-
gation, methodology, software, validation, visualization,
writing – original draft; Yao Li: data curation, formal analysis,
investigation, methodology; Shenghua Yang: investigation;
Yitzhak Tor: conceptualization, funding acquisition, project
administration, resources, supervision, writing – review and
editing.
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We thank the National Institutes of Health for generous
support (through grant GM 069773) and the Chemistry &
Biochemistry MS Facility. We thank Dr Milan Gembicky the
UCSD X-ray Crystallography Facility for determining the crystal
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