Differential Inhibition of APOBEC3 DNA-Mutator Isozymes by Fluoro- and Non-Fluoro-Substituted 2′-Deoxyzebularine Embedded in Single-Stranded DNA

Article abstract of DOI:10.1002/cbic.201900505

The APOBEC3 (APOBEC3A-H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single-stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3-mediated mutagenesis of vi

Full text of DOI:10.1002/cbic.201900505

Accepted Article
Title: Differential inhibition of APOBEC3 DNA-mutator isozymes
by fluoro- versus non-fluoro-substituted 2'-deoxyzebularine
embedded in single-stranded DNA
Authors: Maksim V. Kvach, Fareeda M. Barzak, Stefan Harjes, Henry
A. M. Schares, Harikrishnan M. Kurup, Katherine F Jones,
Lorraine Sutton, John Donahue, Richard T. D’Aquila, Geoffrey
B. Jameson, Daniel A. Harki, Kurt L. Krause, Elena Harjes,
and Vyacheslav Filichev
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201900505
Link to VoR: http://dx.doi.org/10.1002/cbic.201900505
A Journal of
www.chembiochem.org

10.1002/cbic.201900505
ChemBioChem
Differential inhibition of APOBEC3 DNA-mutator isozymes by fluoro- versus non-fluoro-substituted 2'-
deoxyzebularine embedded in single-stranded DNA
Maksim V. Kvach,[a] Fareeda M. Barzak,[a] Stefan Harjes,[a] Henry A. M. Schares,[c] Harikrishnan M. Kurup,[a, b]
Katherine F. Jones,[d] Lorraine Sutton,[f] John Donahue,[f] Richard T. D’Aquila,[g] Geoffrey B. Jameson,[a, b]
Daniel A. Harki,[c, d] Kurt L. Krause,[b, e] Elena Harjes,[a, b]* Vyacheslav V. Filichev[a, b]
*
[a] Dr. M. V. Kvach, F. M. Barzak, Dr. S. Harjes, H. M. Kurup, Prof. Dr. G. B. Jameson, Dr. E. Harjes, Prof. Dr.
V. V. Filichev
School of Fundamental Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New
Zealand
E-mail: e.harjes@massey.ac.nz, v.filichev@massey.ac.nz
[b] H. M. Kurup, Prof. Dr. G. B. Jameson, Prof. K. L. Krause, Dr. E. Harjes, Prof. Dr. V. V. Filichev
Maurice Wilkins Centre for Molecular Biodiscovery, Auckland 1142, New Zealand;
[c] H. A. M. Schares, Prof. Dr. D. A. Harki
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA;
[d] K. F. Jones, Prof. Dr. D. A. Harki
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA;
[e] Prof. K. L. Krause
Department of Biochemistry, University of Otago, Dunedin 9016, New Zealand;
[f] L. Sutton, Dr. J. Donahue
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine,
Nashville, Tennessee, USA;
[g] Prof. Dr. R. T. D’Aquila
1
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Division of Infectious Diseases and Northwestern HIV Translational Research Center, Department of
Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
Twitter accounts:
Massey University: @MasseyUni
Maurice Wilkins Centre: @MWC_CoRE
Worldwide Cancer Research: @WorldwideCancer
Daniel Harki: @DanHarki
Katherine Jones: @_ChemKat
Henry Schares: @HenrySchares
University of Minnesota, Department of Medicinal Chemistry: @UMN_MedChem
University of Minnesota, Department of Chemistry: @UMNChemistry
Keywords: APOBEC3, cytidine deaminase, zebularine, enzyme activity, inhibitor, transition state analogue,
nucleoside, nucleotide, 5-fluoro-2'-deoxyzebularine.
Abstract
The APOBEC3 (APOBEC3A-H) enzyme family is part of the human innate immune system that restricts
pathogens by scrambling pathogenic single-stranded DNA (ssDNA) via deamination of cytosines to uracils.
However, APOBEC3-mediated mutagenesis of viral and cancer DNA promotes its evolution, enabling
disease progression and development of drug resistance. Therefore, APOBEC3 inhibition offers a new
strategy to complement existing anti-viral and anti-cancer therapies by making such therapies effective for
longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2'-
deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into
oligodeoxynucleotides (oligos) in place of 2'-deoxycytidine in the preferred substrates of APOBEC3A,
APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo with 5-
fluoro-2'-deoxyzebularine (5FdZ) exhibited a 3.5 times better inhibition constant in comparison with the
comparable 2'-deoxyzebularine (dZ) containing oligo against APOBEC3B. A similar trend of inhibition was
2
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
observed for wild-type APOBEC3A. In contrast, use of 5FdZ in an oligo designed for APOBEC3G inhibition
resulted in a less potent inhibitor than the dZ-containing oligo for both APOBEC3GCTD and full-length wild-
type APOBEC3G.
3
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Introduction
APOBEC3 (A3) enzymes are important components of the innate immune system that protect against
pathogens by deaminating cytosine residues to uracils from the single-stranded DNA (ssDNA) of the
invading viral genome (Figure 1A).[1] These A3 enzymes therefore restrict the spread of pathogenic genetic
information. Conversely, A3 enzymes, in particular A3B, can mutate genomic DNA, especially in cancer cells,
thereby contributing to cancer genome evolution, acquired drug resistance and poor survival prognosis in
multiple cancers (including breast, bladder, cervix, lung, head and neck).[2] A3B inhibition presents a
promising new strategy to complement existing anti-cancer therapies,[3] as A3B is a non-essential protein.[4]
Figure 1
A3 enzymes and cytidine deaminase (CDA) share a similar structural topology despite very low sequence
identity and, more importantly, a structurally homologous zinc-containing active site. This active site
includes a crucial Glu residue that functions as a general acid/base in the hydrolysis of cytosine.[5]
Consequently, A3s and CDA share a similar mechanism of cytosine deamination. However, CDA accepts
only individual nucleosides as substrates,[6] whereas A3 enzymes have a minimal ssDNA substrate of 2-3
nucleotides flanking the cytosine.[7]
To date, no selective small-molecule inhibitors of A3A or A3B have been reported. We recently developed
the first rationally designed competitive inhibitor of A3 by incorporating a known inhibitor of CDA, 2'-
deoxyzebularine (dZ, Figure 1B), into ssDNA oligonucleotides.[8] We demonstrated that dZ does not inhibit
A3 enzymes as the free nucleoside, but becomes a low µM inhibitor, if and only if, it is incorporated into
ssDNA. This key observation supports a mechanism in which ssDNA delivers dZ to the active site for
inhibition.
We propose that the inhibitory potential of ssDNAs can be further improved by incorporating potent
inhibitors of CDA (also an enzyme of pharmaceutical interest)[9] into ssDNA . Here, we consider several
cytidine derivatives known to inhibit CDA, which we have incorporated into ssDNA as possible inhibitors of
A3 enzymes (Figure 1B). 3-Deazauridine (the ribose analogue of 3dadU) has been reported as a weak
4
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
inhibitor of human liver CDA (Ki = 100 µM).[10] 5-Fluorozebularine has been shown to be a more potent
inhibitor of mouse kidney CDA in comparison with Zebularine (Ki = 0.3 µM versus 2.3 µM, respectively).[11]
However, RNA molecules are not preferred substrates of A3 enzymes.[12]
Here we report the first syntheses of the 2'-deoxy forms of 3-deazauridine and 5-fluorozebularine (3dadU
and 5FdZ, respectively). We report also the incorporation of these nucleosides into ssDNA and their
evaluation as A3 inhibitors using our previously described NMR-based[7a, 8, 13] and fluorescence-based[14]
enzymatic assays. 3-Deaza-2'-deoxyzebularine (3dadZ, Figure 1B) has a CH instead of the N3 atom in
comparison with dZ and in this way can be used to evaluate the importance of protonation of the N3 atom
in dZ in its inhibitory mechanism. Our results indicate subtleties in inhibition of the cytosine deamination
reaction among A3 enzymes, and support our general strategy of using known inhibitors of CDA to guide
the design of ssDNAs as inhibitors of A3 enzymes.
Results and discussion
Synthesis of modified nucleosides, their DMT-protected phosphoramidites and corresponding oligos
The synthesis of modified nucleosides started from heterocycles 1a-c and Hoffer’s chlorosugar[15] (Scheme
1). For the synthesis of 3dadZ (compounds 2-5a) and its incorporation into DNA, we followed previously
described procedures[16] with some modifications as described in the Supporting Information section.
Scheme 1
The pure β-anomer of 3dadU (compound 2b) was obtained using silyl modification of the classical Hilbert-
Johnson reaction[17] by reacting silylated 2,4-dihydroxypyridine with Hoffer’s chlorosugar in boiling CHCl3
(see Supporting Information). Double recrystallisation from EtOH provided 2b in 50% yield. Cleavage of the
toluoyl protecting groups was accomplished in MeOH/NH4OH providing nucleoside 3b,[18] which was then
converted into 4b by selective installation of the 4,4'-dimethoxytrityl (DMT) group on the 5'-end of the
nucleoside followed by benzoyl protection of the 4-hydroxyl group of the nucleobase (81% yield over three
steps from 2b). Phosphitylation of 4b was performed under standard conditions using N,N-
5
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
diisopropylamino-2-cyanoethoxychlorophosphine and Et3N in CH2Cl2 in 84% yield after silica gel column
chromatography.
The synthesis of 5FdZ as a free nucleoside has been performed in the past by enzymatic conversion of dC in
the presence of heterocycle 1c using trans-N-deoxyribosylase from Lactobacillus acidophilus.[19] Later, 5FdZ
was synthesised from 5-fluoro-2'-deoxyuridine in six steps.[20] We found both protocols to be unsatisfactory
in terms of potential scalability, complex procedures and overall yield. As with the synthesis of dZ[8] and
3dadU, we first used a Lewis acid free variant of the silyl-Hilbert-Johnson reaction for the preparation of
5FdZ from silylated heterocycle 1c and Hoffer’s chlorosugar; this procedure failed. Instead, it was necessary
to use freshly distilled SnCl4 and low temperatures (– 35 °C)[21] to obtain 3',5'-bis-O-toluoyl protected 5FdZ
(2c) in a good yield, although this product was contaminated with the α-anomer (β/α = 9:1). Use of a slow
stepwise gradient of acetone (0→20 %) in CH2Cl2 allowed isolation of pure β-anomer 2c in 45% yield.
Deprotection was performed using saturated NH3 in MeOH providing 5FdZ-nucleoside 3c, for which an
analytical sample was obtained after preparative TLC on silica gel. NMR analysis revealed that this
compound exists in two forms, i.e. as an “open” nucleoside and as a “cyclic” nucleoside formed after
intramolecular addition of 5'-OH to the double bond of the nucleobase (Figure 2A). Similar transformations
have been described for several pyrimidines.[22] Using 2D NMR techniques we assigned signals in 1H and 13C
NMR spectra for individual forms as reported in the Supporting Information and shown on Figure 2A.
Appearance of an NH signal at 8.69 ppm in 1H NMR and a significant shift in H6 and C6 signals in 1H and 13C
NMR spectra as a result of a change in hybridisation at C6 suggest formation of a “cyclic” nucleoside. The
1H-13C Heteronuclear Multiple Bond Correlation (HMBC) spectrum, which shows three-bond correlations,
was particularly helpful during the assignment (Figure 2B). The H6-C5' cross-peak, seen on the right-hand
upper corner of Figure 2B, confirms existence of a three-bond linkage between H6 of the nucleobase and
C5' of the sugar in the “cyclic” nucleoside. At the same time, the H6 proton cross-talks with all carbons of
the nucleobase (C2, C4, C5) and with C1' carbon of the sugar moiety, which is possible only for an O5'-C6
“cyclic” nucleoside. We observed that the ratio between “open” and “cyclic” forms changes in different
solvents. In D2O, the “open” form predominantly exists, whereas in d6-DMSO, CD3CN and d8-THF both forms
are present. This indicates that “open” and “cyclic” forms of 5FdZ-nucleoside are in dynamic equilibrium,
6
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
which complicates purification, but allows transformation of an equilibrium mixture of nucleosides to a
single 5'-DMT-modified product 4c. That is why “open” and “cyclic” forms of nucleoside 3c without any
purification after removal of toluoyl groups from 2c were treated with DMT-Cl in pyridine; compound 4c
was obtained in 60% yield over two steps from 2c. Finally, phosphitylation of 4c gave phosphoramidite 5c in
89% yield.
Figure 2
We incorporated the modified nucleosides at the location of dC in the preferred A3 substrate motifs. A3B
and A3A prefer the TCA motif (oligo1, Table 1) whereas A3G preferentially deaminates the CCCA motif
(oligo2, where the underlined C is deaminated first). Synthesis of DNA oligos was performed using an
automated DNA synthesiser with an increased coupling time for phosphoramidites 5a-c from 1.5 min for
standard phosphoramidites to 5 min. Cleavage from the solid support and deprotection of phosphates and
nucleobases were accomplished in concentrated aqueous NH4OH for oligos possessing 3dadU and 3dadZ.
Unfortunately, the same procedure led to degradation of 5FdZ-containing oligos as evident from the
reverse-phase HPLC profile in Figure 2C (red line). Deprotection by saturated NH3 in MeOH was also
unsuccessful (blue line, Figure 2C). We found that on-column deprotection of 5FdZ-oligo in organic
solvents[23] led to the least amount of by-products (black profile, Figure 2C). Here, 5FdZ-oligo on the CPG-
support was treated with 10% Et2NH in acetonitrile for 5 min, followed by incubation of the support in
ethylenediamine/toluene mixture for 2 hr at room temperature, allowing subsequent release of the
deprotected oligo in H2O. All oligos were purified by reverse-phase HPLC. Their compositions were
confirmed by ESI-MS (see Supporting Information).
Table 1
Evaluation of oligos as inhibitors of A3 enzymes using NMR-based activity assay
To directly assess the inhibition of A3 enzymes, we used a previously described NMR-based activity assay
wherein the DNA substrate deamination is monitored by 1H NMR spectroscopy in the presence of enzyme
with and without inhibitors.[7a, 8, 13] The NMR-based inhibition assay is a direct assay using just A3 enzymes;
7
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
it does not require a secondary enzyme, such as Uracil-DNA Glycosylase (UDG), which is used in many
indirect assays. By introducing different inhibitors of cytidine deamination into the A3 recognition motif
preferred by the particular A3 enzyme, we expect that the trend in inhibition for all A3 enzymes parallels to
a greater than lesser extent that observed for CDA inhibition, because the active site and therefore the
deamination mechanism are conserved. We evaluated the inhibitory activity of our modified DNAs using
active A3 enzymes with reliable expression and stability over time, which have also been previously
characterised in the NMR-based activity assays in our laboratory. This allows reliable determination of the
inhibitory potential of modified oligos by comparing Km values of the substrates to Ki values of inhibitors
determined under identical conditions (enzyme and substrate concentrations, buffer and ionic strength).
The enzymes chosen, A3BCTD-QM-∆L3-AL1swap, hereafter simplified to A3BCTD-AL1, and GST-fused A3GCTD
were recombinantly expressed and purified from Escherichia coli. To compare the inhibitory effect of
oligonucleotides between A3GCTD and full-length A3G (flA3G), we have used flA3G that was purified from
,
human cells grown planktonically[24] (see description of these enzymes and their purification in Supporting
Information).
Oligos containing 3dadZ and 3dadU instead of the target dC in the preferred TCA-recognition motif for
A3BCTD-AL1 had no effect on the initial speed of deamination by A3BCTD-AL1 (Figure 3). These oligos fail to
inhibit A3 enzymes under experimental conditions. These data are in line with previous results that 3dadU,
as an individual ribose-based single nucleoside, is a very weak inhibitor of human liver CDA (Ki = 100 µM).[10]
Although higher concentrations of 3dadU-oligo might result in inhibition of A3BCTD-AL1, such
concentrations would provide a weak basis for the development of modified 3dadU-oligos as inhibitors
compared to our current strategy. On the other hand, inhibition of A3BCTD-AL1 by 5FdZ-oligo was more
profound in comparison with dZ-oligo under identical conditions. Previously, we confirmed that dZ-oligo is
a competitive inhibitor of this enzyme.[8] By monitoring the reaction in the presence of inhibitor at various
concentrations, we obtained the inhibition constant (Ki) for 5FdZ-oligo (2.1 ± 0.8 µM, see Supporting
Information), which was 3.5 times lower than the Ki of dZ-oligo (7.5 ± 1.7 µM). The overall inhibition effect
was improved from 30-fold (dZ) to nearly 100-fold (5FdZ) if we compare the apparent inhibition constants
(Ki) of dZ and 5FdZ-containing oligos with Km of the ssDNA substrate 5'-ATTT-C-ATTT (Km = 200 µM). This
8
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
means that 5FdZ-containing oligos can potentially be used in cells in the low µM range to inhibit A3A and
A3B. Due to the presence of the electron withdrawing F, the heterocycle in 5FdZ is more activated towards
the nucleophilic addition of H2O than dZ (Figure 4), which is evident from existence of 5FdZ in equilibrium
between “open” and “closed” forms (see Figure 2A). This likely explains why 5FdZ is a better inhibitor than
dZ of A3BCTD-AL1 once placed in ssDNA. Formation of reversible covalent adducts with the enzyme is also
possible. Similar adducts have been described between zebularine and DNA methyltransferases.[25]
Our observations parallel those reported earlier for CDA that 5-fluorozebularine is a better inhibitor than
zebularine and 3-deazauridine (Ki = 0.3 µM, 2.3 µM[11] and 100 µM,[10] respectively). These results indicate
that the structure of the nucleoside used instead of dC in the preferred ssDNA substrate determines the
inhibitory potential of the oligos and that the trend of A3BCTD-AL1 inhibition correlates with Ki values
reported earlier for individual nucleosides against CDA. This result also opens the possibility to further
improve inhibition by introducing other inhibitors of cytidine deamination into ssDNA sequences.
Figure 3
The fact that 3dadZ does not inhibit A3BCTD-AL1 highlights the importance of protonation of N3 in dZ by the
conserved glutamic acid present in the active site of A3s (and CDA). This protonation makes C4 in dZ more
electrophilic and more susceptible to the nucleophilic attack by OH-/H2O coordinated to the Zn2+, which
converts dZ into a tetrahedral transition-state analogue of cytidine deamination (Figure 4).[26] This
mechanism is not realized in the case of 3dadZ, because the C=C double bond of 3dadZ is inactive towards
water addition. Moreover, the nucleobase of 3dadU is planar and does not mimic the tetrahedral geometry
of C4 in the transition state of cytidine deamination.
Figure 4
Next, having two active A3G enzymes, the C-terminal domain (A3GCTD) with the wild-type sequence and
full-length A3G (flA3G), we decided to test if inhibition of A3GCTD is a good model to investigate inhibition of
two-domain enzymes such as flA3G. Our studies were performed with two oligos, an A3G-preferred,
CC5FdZ-oligo in which the dC that is first deaminated by A3G was changed to 5FdZ, and the previously
9
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
reported inhibitor CCdZ-oligo.[8] Our data show that inhibition of A3G deaminase activity by targeting only
the catalytically active C-terminal domain, A3GCTD, accurately translates to the overall inhibition of flA3G
(Figure 5A). This is consistent with the fact that the N-terminal domain of A3G completely lacks deaminase
activity.[27]Accordingly, the specificity of ssDNA binding to the full-length A3B and A3G lies in the C-terminal
domains, and the inactive N-terminal domains enhance ssDNA deamination efficiency at the C-terminal
domain and regulate processivity of enzymes.[27a, 28]
Interestingly, the CC5FdZ-oligo (Ki = 71 ± 14 µM, Figure 5B) did not improve inhibition of A3GCTD in
comparison with CCdZ-oligo (Ki = 53 ± 10 µM),[8] which is in contrast to the trend observed above for
A3BCTD-AL1. Nonetheless, that both CCdZ-oligo and CC5FdZ-oligo are inhibitors supports our strategy of
targeting the catalytically active C-terminal domain of A3 enzymes using our DNA-based inhibitors as a
means to inhibit full-length enzymes.
Figure 5
Evaluation of oligos as inhibitors of A3A using a fluorescence-based activity assay
To investigate how a 5FdZ-containing oligo inhibits another wild-type A3 expressed in human cells, we
purified A3A from HEK293T and used it to perform our previously published fluorescence-based activity
assay.[14] The deamination of a fluorescently labeled substrate oligonucleotide was monitored in the
presence of dZ- and 5FdZ-containing oligo competitors (Figure 6). The assay was developed previously to
evaluate small molecule inhibitors of A3A, A3B and A3G under identical settings. In the current work, we
used an 18-mer oligo with 5'-…TATCCCA…-3' in the middle of the sequence as the enzyme substrate (see
details in the Supporting Information). The CCC-motif is a preferred sequence for deamination by A3G[28-29]
but this sequence is also readily deaminated by A3A and A3B;[30] therefore, the oligo is a pan-A3 substrate.
The results clearly show that for A3A, the 5FdZ-containing oligo is a more potent inhibitor with an IC50 of
0.16  0.01 µM than the equivalent dZ containing oligonucleotide sequence with an IC50 of 0.39  0.03 µM.
These data are consistent with the A3BCTD-AL1 data, as A3A and A3BCTD share all the residues surrounding
10
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
the target cytosine in x-ray structures.[31] Control assays with the dC- and dU-containing oligos can be found
in the Supporting Information.
Figure 6
Plausible explanation of differences in inhibition of A3A/A3BCTD and A3G by dZ and 5FdZ-containing
oligos
The fact that the trend of inhibition by dZ and 5FdZ-containing oligos is varied for A3A/A3BCTD and A3G
should not be completely unexpected as A3 family members differ strongly in their preferences to
deaminate methylated cytosine in ssDNA.[32] The selectivity of different A3 family members for non-
methylated against 5-methylated cytosine (5MedC) can be significantly changed by swapping loop 1 and
loop 7 between the enzymes.[5b, 33] This property speaks for subtle control of the active site specificity for
deamination of 5MedC, as the swapped amino acids are not in direct contact with the target cytosine.
Similar effects can be relevant to interaction of A3 enzymes with 5-fluoro-2'-deoxycytidine (5FdC) and 5FdZ.
Recently, we have also observed that 5-methyl-2'-deoxyzebularine is a worse inhibitor than dZ in the
[8]
context of an oligo designed to inhibit A3GCTD
.
Nevertheless, we have compared active sites of A3A, A3B and A3G to find possible differences in amino
acids in proximity to the target cytosine. Such differences may explain the preferences of A3 enzymes
towards various substrates and inhibitors.
Structural analysis of the A3BCTD-AL1 complex with ssDNA[31] and sequential alignment of A3A, A3BCTD, and
A3GCTD revealed that not only the zinc-coordinating residues, but most of the residues in the active site
close to the target cytosine are well conserved between these proteins. However, one residue in the
substrate-binding pocket differs: an isoleucine, Ile 279/Ile 96, of A3BCTD/A3A is threonine (Thr283) in the
corresponding position of A3GCTD sequence. The sidechain of Ile279 is ~4.4 Å distant from the NH2 group of
the target cytosine in the inactive, substrate-binding E255A mutant of A3BCTD (pdb-id: 5DT5). On the other
hand, the Thr283 hydroxyl group makes hydrogen bonds to a neighbouring Thr and to the main chain that
forms part of the substrate/inhibitor binding pocket (pdb-id: 3V4J). The latter interaction may reduce the
11
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
conformational flexibility needed to accommodate a substituent larger than hydrogen in the 5-position of
cytosine. We note that in the A3B-AL1 structure the bound cytidine is tightly supported by Tyr313 (Tyr315
in A3GCTD), which in turn is buttressed by a conserved Trp (Trp285 for A3GCTD) on the loop that for A3GCTD is
locked in place by hydrogen bonding to Thr283. Interestingly, mouse CDA (pdb-id: 2FR5) has Ile87 in a
similar position in the three-dimensional structure as Ile279 (Figure S3 in the Supporting Information).
Thus, the Ile versus Thr substitution may play a role in the differences seen for 5FdZ compared to dZ
inhibition of A3GCTD and A3BCTD-AL1. AID, mouse APOBEC1 and mouse APOBEC3 deaminate 5FdC as well as
5MedC with reduced speed compared to dC.[32b, 34] These results were explained by steric effects, as F and
Me are larger than H. As discussed above, the active site around the target cytosine is very similar for
A3A/A3BCTD and A3GCTD, with the exception of Ile/Thr. The dynamics of the active site may allow better
accommodation of 5FdZ in A3A/A3BCTD than in A3GCTD. In any event, the substrate and inhibitor binding and
the deamination mechanism vary subtly between A3s and CDAs. Examples of highly homologous enzymes
with significantly different transition states are well-established.[35]
Conclusion
The structure of modified nucleosides dZ and 5FdZ embedded in the otherwise identical DNA sequence
determines the inhibitory effects on human A3A, A3BCTD, and A3GCTD as well as full-length A3G. On the
other hand, the 2'-deoxyribose derivative of 3-deazauridine, a previously described weak inhibitor of CDA,
cannot inhibit A3 upon its incorporation into ssDNA under the conditions tested. Our results indicate that
some correlation exists between CDA and A3 inhibition, when CDA inhibitors replace the deamination-
susceptible cytidine in the ssDNA sequence. Our results also highlight the importance of protonation of the
N3 atom in dZ for its inhibitory mechanism. Noteworthy differences in inhibition profiles among different
A3 enzymes observed here point to possibilities of obtaining highly specific A3inhibitors, supporting our
approach towards developing oligonucleotide-based A3 inhibitors using chemically-modified nucleosides
whose structure can stall enzymatic cytosine deamination.[36] Future work will continue to focus on the
chemical optimisations of our ssDNA-based A3 inhibitors and their evaluation in vitro and in vivo.
12
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Nucleotides flanking the target dZ and 5FdZ can be further modified to improve inhibitory potential and
enhance the lifetime of oligonucleotides in biological media.
Supporting Information
Experimental details for the synthesis of 3dadZ, 3dadU and 5FdZ nucleosides, their DMT-protected
phosphoramidites and modified oligos, for protein expression and purification, and for the NMR-based
kinetic assays; examples of calculation of inhibition of A3-enzymes by FdZ-containing oligos; fluorescence-
based A3A deamination assays; and sequence alignment of proteins used in this study.
Author information
Corresponding authors
*E-mail: e.harjes@massey.ac.nz, v.filichev@massey.ac.nz
ORCID
Geoffrey B. Jameson 0000-0003-4839-0784
Vyacheslav V. Filichev 0000-0002-7383-3025
Elena Harjes 0000-0002-3643-9432
Daniel A. Harki 0000-0001-5950-931X
Maksim V. Kvach 0000-0003-3592-0336
Kurt L. Krause 0000-0002-3525-1112
Funding: VVF, EH, GBJ, MVK and SH are grateful for the financial support provided by the Worldwide
Cancer Research (grant 16-1197), Palmerston North Medical Research Foundation, Massey University
Research Fund (MURF 2015, 7003 and RM20734) and School of Fundamental Sciences, Massey University.
DAH acknowledges the United States National Institutes of Health (P01-CA234228, R01-GM110129 and
R01-GM118000) for financial support. KFJ acknowledges the National Science Foundation Graduate
13
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Research Fellowship Program for support. KLK thanks the Health Research Council, Otago University and
the Thrash Foundation.
Notes:
The authors declare the following competing financial interest(s): DAH is a co-founder, shareholder, and a
consultant of ApoGen Biotechnologies, Inc.
Acknowledgements:
NMR and mass-spectrometry facilities at Massey University and the assistance of Dr. P. J. B. Edwards and
Mr. D. Lun are gratefully acknowledged. We are also thankful for the assistance in preparation of full-length
A3G provided by Michele Krause and Chisu Song (Otago University). We thank Prof. Reuben S. Harris
(University of Minnesota, USA) and members of his cancer research program for helpful discussions. We
thank anonymous reviewers for the extensive and constructive feedback given to this manuscript.
References:
[1]
[2]
a) R. S. Harris, J. P. Dudley, Virology 2015, 479-480, 131-145; b) M. H. Malim, P. D. Bieniasz, Cold
Spring Harb. Perspect. Med. 2012, 2, a006940.
a) S. Venkatesan, R. Rosenthal, N. Kanu, N. McGranahan, J. Bartek, S. A. Quezada, J. Hare, R. S.
Harris, C. Swanton, Ann. Oncol. 2018, 29, 563-572; b) A. M. Sieuwerts, S. Willis, M. B. Burns, M. P.
Look, M. E. Meijer-Van Gelder, A. Schlicker, M. R. Heideman, H. Jacobs, L. Wessels, B. Leyland-
Jones, K. P. Gray, J. A. Foekens, R. S. Harris, J. W. M. Martens, Horm. Cancer 2014, 5, 405-413; c) E.
K. Law, A. M. Sieuwerts, K. LaPara, B. Leonard, G. J. Starrett, A. M. Molan, N. A. Temiz, R. I. Vogel,
M. E. Meijer-van Gelder, F. C. G. J. Sweep, P. N. Span, J. A. Foekens, J. W. M. Martens, D. Yee, R. S.
Harris, Sci. Adv. 2016, 2, e1601737; d) P. J. Vlachostergios, B. M. Faltas, Nat. Rev. Clin. Oncol. 2018,
15, 495-509; e) L. Galluzzi, I. Vitale, Trends Genet. 2017, 33, 491-492; f) J. Zou, C. Wang, X. Ma, E.
Wang, G. Peng, Cell Biosci. 2017, 7, 29.
[3]
[4]
[5]
M. E. Olson, R. S. Harris, D. A. Harki, Cell Chem. Biol. 2018, 25, 36-49.
J. M. Kidd, T. L. Newman, E. Tuzun, R. Kaul, E. E. Eichler, PLoS Genet 2007, 3, e63.
a) T. P. Ko, J. J. Lin, C. Y. Hu, Y. H. Hsu, A. H. Wang, S. H. Liaw, J. Biol. Chem. 2003, 278, 19111-19117;
b) Y. Fu, F. Ito, G. Zhang, B. Fernandez, H. Yang, X. S. Chen, Biochem. J. 2015, 471, 25-35.
C. B. Yoo, S. Jeong, G. Egger, G. Liang, P. Phiasivongsa, C. Tang, S. Redkar, P. A. Jones, Cancer Res.
2007, 67, 6400-6408.
a) S. Harjes, W. C. Solomon, M. Li, K.-M. Chen, E. Harjes, R. S. Harris, H. Matsuo, J. Virol. 2013, 87,
7008-7014; b) M. Liu, A. Mallinger, M. Tortorici, Y. Newbatt, M. Richards, A. Mirza, R. L. M. van
Montfort, R. Burke, J. Blagg, T. Kaserer, ACS Chem. Biol. 2018.
[6]
[7]
[8]
[9]
M. V. Kvach, F. M. Barzak, S. Harjes, H. A. M. Schares, G. B. Jameson, A. M. Ayoub, R. Moorthy, H.
Aihara, R. S. Harris, V. V. Filichev, D. A. Harki, E. Harjes, Biochemistry 2019, 58, 391-400.
a) R. Z. Mahfouz, A. Jankowska, Q. Ebrahem, X. Gu, V. Visconte, A. Tabarroki, P. Terse, J. Covey, K.
Chan, Y. Ling, K. J. Engelke, M. A. Sekeres, R. Tiu, J. Maciejewski, T. Radivoyevitch, Y.
Saunthararajah, Clin. Cancer Res. 2013, 19, 938-948; b) M. R. Savona, O. Odenike, P. C. Amrein, D.
14
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
P. Steensma, W. A. Harb, A. E. DeZern, L. C. Michaelis, S. Faderl, J. N. Lowder, P. Taverna, A.
Oganesian, R. Dua, R. Nawabi, M. Azab, G. Garcia-Manero, Blood 2015, 126, 1683-1683.
G. G. Chabot, J. Bouchard, R. L. Momparler, Biochem. Pharmacol. 1983, 32, 1327-1328.
J. J. Barchi, A. Haces, V. E. Marquez, J. J. McCormack, Nucleosides Nucleotides 1992, 11, 1781-1793.
C. S. Nabel, J. W. Lee, L. C. Wang, R. M. Kohli, Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 14225-14230.
A. Furukawa, T. Nagata, A. Matsugami, Y. Habu, R. Sugiyama, F. Hayashi, N. Kobayashi, S.
Yokoyama, H. Takaku, M. Katahira, EMBO J. 2009, 28, 440-451.
M. Li, S. M. D. Shandilya, M. A. Carpenter, A. Rathore, W. L. Brown, A. L. Perkins, D. A. Harki, J.
Solberg, D. J. Hook, K. K. Pandey, M. A. Parniak, J. R. Johnson, N. J. Krogan, M. Somasundaran, A. Ali,
C. A. Schiffer, R. S. Harris, ACS Chem. Biol. 2012, 7, 506-517.
a) M. Hoffer, Chem. Ber. 1960, 93, 2777-2781; b) V. Rolland, M. Kotera, J. Lhomme, Syn. Commun.
1997, 27, 3505-3511; c) M. Kögler, R. Busson, S. De Jonghe, J. Rozenski, K. Van Belle, T. Louat, H.
Munier-Lehmann, P. Herdewijn, Chem. Biodiv. 2012, 9, 536-556.
a) F. Seela, U. Bindig, Liebigs Ann. Chem. 1989, 895-901; b) R. H. Durland, T. S. Rao, R. Revankar, J.
H. Tinsley, M. A. Myrick, D. M. Seth, J. Rayford, P. Singh, K. Jayaraman, Nucleic Acids Res. 1994, 22,
3233-3240; c) M. Urban, N. Joubert, M. Hocek, R. E. Alexander, R. D. Kuchta, Biochemistry 2009, 48,
10866-10881.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
M. P. Kotick, C. Szantay, T. J. Bardos, J. Org. Chem. 1969, 34, 3806-3813.
B. L. Currie, R. K. Robins, M. J. Robins, J. Heterocycl. Chem. 1970, 7, 323-329.
T. B. Oeyen, S. G. Laland, Biochim. Biophys. Acta, Nucleic Acids Protein Synth. 1969, 182, 567-569.
D. Cech, A. Holy, Collect. Czech. Chem. Commun. 1977, 42, 2246-2260.
K. Ebenryter-Olbinska, J. Karolak-Wojciechowska, E. Sochacka, Carbohydr. Res. 2014, 392, 7-15.
a) P. K. Chang, J. Org. Chem. 1965, 30, 3913-3915; b) A. R. Katritzky, M. Kingsland, O. S. Tee, Chem.
Commun. 1968, 289-290; c) T. C. Thurber, L. B. Townsend, J. Org. Chem. 1976, 41, 1041-1051; d) J.
Cadet, L.-S. Kan, S. Y. Wang, J. Am. Chem. Soc. 1978, 100, 6715-6720; e) P. S. Liu, V. E. Marquez, J. S.
Driscoll, R. W. Fuller, J. J. McCormack, J. Med. Chem. 1981, 24, 662-666; f) S. M. N. Efange, E. M.
Alessi, H. C. Shih, Y. C. Cheng, T. J. Bardos, J. Med. Chem. 1985, 28, 904-910; g) G. W. M. Visser, A. T.
Bijma, J. A. R. Dijksman, G. C. M. Gorree, M. Van Walsum, J. D. M. Herscheid, Nucl. Med. Biol. 1989,
16, 351-357; h) V. E. Marquez, K. V. B. Rao, J. V. Silverton, J. A. Kelley, J. Org. Chem. 1984, 49, 912-
919.
[23]
Glen Research, 2013, Deprotection Supplement, Deprotection - Volumes 1-5,
http://www.glenresearch.com/Technical/Deprotection.pdf
[24]
[25]
Y. Durocher, S. Perret, A. Kamen, Nucleic Acids Res 2002, 30, E9.
L. Zhou, X. Cheng, B. A. Connolly, M. J. Dickman, P. J. Hurd, D. P. Hornby, J. Mol. Biol. 2002, 321,
591-599.
[26]
a) S. Xiang, S. A. Short, R. Wolfenden, C. W. Carter, Biochemistry 1995, 34, 4516-4523; b) C. H.
Borchers, V. E. Marquez, G. K. Schroeder, S. A. Short, M. J. Snider, J. P. Speir, R. Wolfenden, Proc.
Natl. Acad. Sci. U. S. A. 2004, 101, 15341-15345.
[27]
a) F. Navarro, B. Bollman, H. Chen, R. Konig, Q. Yu, K. Chiles, N. R. Landau, Virology 2005, 333, 374-
386; b) S. U. Siriwardena, T. A. Guruge, A. S. Bhagwat, J. Mol. Biol. 2015, 427, 3042-3055.
M. A. Carpenter, E. Rajagurubandara, P. Wijesinghe, A. S. Bhagwat, DNA repair 2010, 9, 579-587.
a) A. Rathore, M. A. Carpenter, O. Demir, T. Ikeda, M. Li, N. M. Shaban, E. K. Law, D. Anokhin, W. L.
Brown, R. E. Amaro, R. S. Harris, J. Mol. Biol. 2013, 425, 4442-4454; b) R. M. Kohli, S. R. Abrams, K.
S. Gajula, R. W. Maul, P. J. Gearhart, J. T. Stivers, J. Biol. Chem. 2009, 284, 22898-22904; c) R. M.
Kohli, R. W. Maul, A. F. Guminski, R. L. McClure, K. S. Gajula, H. Saribasak, M. A. McMahon, R. F.
Siliciano, P. J. Gearhart, J. T. Stivers, J. Biol. Chem. 2010, 285, 40956-40964; d) M. D. Stenglein, M. B.
Burns, M. Li, J. Lengyel, R. S. Harris, Nat. Struct. Mol. Biol. 2010, 17, 222-229.
I.-J. L. Byeon, C.-H. Byeon, T. Wu, M. Mitra, D. Singer, J. G. Levin, A. M. Gronenborn, Biochemistry
2016, 55, 2944-2959.
[28]
[29]
[30]
[31]
K. Shi, M. A. Carpenter, S. Banerjee, N. M. Shaban, K. Kurahashi, D. J. Salamango, J. L. McCann, G. J.
Starrett, J. V. Duffy, O. Demir, R. E. Amaro, D. A. Harki, R. S. Harris, H. Aihara, Nat Struct Mol Biol
2017, 24, 131-139.
15
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
[32]
a) M. A. Carpenter, M. Li, A. Rathore, L. Lackey, E. K. Law, A. M. Land, B. Leonard, S. M. D. Shandilya,
M.-F. Bohn, C. A. Schiffer, W. L. Brown, R. S. Harris, J. Biol. Chem. 2012, 287, 34801-34808; b) C. S.
Nabel, H. J. Jia, Y. Ye, L. Shen, H. L. Goldschmidt, J. T. Stivers, Y. Zhang, R. M. Kohli, Nat. Chem. Biol.
2012, 8, 751-758; c) R. Suspene, M.-M. Aynaud, J.-P. Vartanian, S. Wain-Hobson, PLoS One 2013, 8,
e63461; d) K. Kamba, T. Nagata, M. Katahira, PLoS One 2015, 10, e0124142; e) E. K. Schutsky, C. S.
Nabel, A. K. F. Davis, J. E. DeNizio, R. M. Kohli, Nucleic Acids Res 2017, 45, 7655-7665; f) F. Ito, Y. Fu,
S. A. Kao, H. Yang, X. S. Chen, J. Mol. Biol. 2017, 429, 1787-1799.
[33]
[34]
[35]
P. Wijesinghe, A. S. Bhagwat, Nucleic Acids Res 2012, 40, 9206-9217.
G. Rangam, K. M. Schmitz, A. J. Cobb, S. K. Petersen-Mahrt, PLoS One 2012, 7, e43279.
a) P. C. Kline, V. L. Schramm, Biochemistry 1993, 32, 13212-13219; b) P. C. Kline, V. L. Schramm,
Biochemistry 1995, 34, 1153-1162; c) A. Lewandowicz, V. L. Schramm, Biochemistry 2004, 43, 1458-
1468.
[36]
F. M. Barzak, S. Harjes, M. V. Kvach, H. M. Kurup, G. B. Jameson, V. V. Filichev, E. Harjes, Org.
Biomol. Chem. 2019, in press, doi: 10.1039/c9ob01781j.
16
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Figures:
Figure 1. A) Deamination of dC in ssDNA by APOBEC3 enzymes; B) Modified nucleotides as potential
inhibitors of cytidine deamination.
17
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Figure 2. A) NMR assignment of “open” (structure in black) and “cyclised” (structure in green) forms of
5FdZ. Chemical shifts (δ in ppm) are shown for 1H in blue and for 13C in red; B) 1H-13C HMBC spectrum
recorded in DMSO-d6 at rt showing 2-4 bond correlations and coexistence of “open” (black labels) and
“cyclised” (green labels) forms of 5FdZ. *, **, and *** are single bond correlations of H6-C6 (closed), H1'-
C1' (open) and H1'-C1' (closed), respectively. C) RP-HPLC profiles of 5FdZ-oligo cleaved from the support
and deprotected under different conditions. The major peak isolated after treatment with Et2NH/CH3CN
followed by ethylenediamine/toluene gave the expected mass in ESI-MS of 2680.6 Da (Calcd. for [M]:
2680.5 Da).
18
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Figure 3. Inhibition of A3BCTD-QM-ΔL3-AL1swap-catalysed deamination of the substrate (5'-ATTT-C-ATTT) by
oligos containing modified nucleosides. The bold C is the target 2'-deoxycytidine deaminated by the
enzyme. Determination of Ki for 5FdZ-oligo can be found in the Supporting information. Ki (mean and
standard deviations) for the dZ-oligo was reported previously[8] and is provided for comparison; all
experiments were repeated multiple times in the same laboratory and the same time interval. Plotted are
mean values. The error bars report SD.
19
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Figure 4. Proposed conversion of dZ into its hydrate and formation of a transition state analogue of
cytosine deamination.
20
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Figure 5. A) The speed of full-length A3G-catalysed deamination of the substrate (5'- ATTCCCAATT, 500 µM)
in the absence of inhibitor (no inhibitor) and in the presence of CCdZ- and CCFdZ-oligos (50 µM).
Conditions: 100 mM NaCl, 50 mM sodium phosphate buffer, pH 6.0, 10 % D2O, 1 mM citrate supplemented
with 50 µM TMSPS as standard. Error bars represent SD. B) The Dixon plot of inverse speed of deamination
against inhibitor concentration for A3GCTD catalysed deamination of 5'-ATTCCCAATT (320 µM, underlined C
is deaminated) in the presence of CC5FdZ-oligo. The green point was ignored by fitting as an outlier
according to the Q-test (with 95% confidence).
21
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Figure 6. A) Inhibition of human APOBEC3A-catalysed deamination of a fluorescently labelled
oligonucleotide reporter by dZ- and 5FdZ-containing oligonucleotide competitors. Representative graphical
data are shown. Individual replicates and the sequence of the fluorescent oligonucleotide reporter are
provided in the Supporting Information. IC50 values are the mean + SEM for four biological replicates.
22
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
#
X
Y
H
OH
H
Z
R1
R2
Yield
11%
50%
45%
2a
2b
2c
CH
CH
N
H
H
F
Tol
Tol
Tol
Tol
Tol
Tol
3a
3b
3c
CH
CH
N
H
OH
H
H
H
F
H
H
H
H
H
H
ND
quant.
ND
4a
4b
4c
CH
CH
N
H
OBz
H
H
H
F
DMT
DMT
DMT
H
H
H
58% from 2a
81% from 2b
60 % from 2c
5a
5b
5c
CH
CH
N
H
OBz
H
H
H
F
DMT
DMT
DMT
P(NiPr)2OCH2CH2CN
P(NiPr)2OCH2CH2CN
P(NiPr)2OCH2CH2CN
93%
84%
89%
Scheme 1. i) Hoffer’s chlorosugar, KOH, CH3CN, 15 min; ii) Hexamethyldisilazane (HMDS), (NH4)2SO4 (cat),
reflux 1h; iii) Hoffer’s chlorosugar, CHCl3, distill., 15 min; iv) HMDS, (NH4)2SO4 (cat), reflux 1h; v) Hoffer’s
chlorosugar, SnCl4, 1,2-dichloroethane, -35 °C; vi) for 3a and 3b: 28% aq. ammonia, MeOH, 48 h; for 3c:
satd. ammonia in MeOH, 48 h; vii) for 4a and 4c: 4,4′-dimethoxytritylchloride (DMTCl), pyridine, 0°C→r.t.,
overnight; for 4b: 4,4′-dimethoxytritylchloride, pyridine, 0°C→r.t., overnight then Bz2O, pyridine, 0°C→r.t.,
overnight viii), N,N-diisopropylamino-2-cyanoethoxychlorophosphine, Et3N, CH2Cl2; ix) DNA synthesis and
purification.
23
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Table 1. Oligonucleotides used in the current study.
Name
Sequence 5'3'
Oligos used in NMR-based activity assay:
oligo1
ATTT-C-ATTT
oligo2
dZ-oligoa
ATTCC-C-AATT
ATTT-dZ-ATTT
3dadZ-oligo
3dadU-oligo
5FdZ-oligo
CC5FdZ-oligo
ATTT-3dadZ-ATTT
ATTT-3dadU-ATTT
ATTT-5FdZ-ATTT
ATTCC-5FdZ-AATT
Oligos used in fluorescence based activity assay:
T4-dZ-oligoa
TTTT-dZ-AT
T4-5FdZ-oligo
TTTT-5FdZ-AT
a) Prepared as in reference [8].
24
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900505
ChemBioChem
Graphical abstract:
Table of content text:
In comparison with 2'-deoxyzebularine, incorporation of 5-fluoro-2'-deoxyzebularine into single-stranded
DNA instead of the target dC yielded improved inhibition of APOBEC3A and APOBEC3B but not of full-
length APOBEC3G.
25
This article is protected by copyright. All rights reserved.