Effects of substitutions at the 4' and 2 positions on the bioactivity of 4'-ethynyl-2-fluoro-2'-deoxyadenosine

Article abstract of DOI:10.1128/AAC.01703-13

Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4'-ethynyl-2-fluoro-2'- deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of 4'-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2'-exo/C-3'-endo (north) conformation. Steady-state kinetic analysis of the incorporation of 4'-substituted NRTIs by RT reveals a correlation between the north conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4'-ethynyl and cyano substitutions decrease the susceptibility of adenosinebased compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pK_a of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites. Copyright

Full text of DOI:10.1128/AAC.01703-13

Effects of Substitutions at the 4′ and 2
Positions on the Bioactivity of 4 ′
-Ethynyl-2-Fluoro-2′-Deoxyadenosine
Karen A. Kirby, Eleftherios Michailidis, Tracy L. Fetterly,
Musetta A. Steinbach, Kamalendra Singh, Bruno Marchand,
Maxwell D. Leslie, Ariel N. Hagedorn, Eiichi N. Kodama,
Victor E. Marquez, Stephen H. Hughes, Hiroaki Mitsuya,
Michael A. Parniak and Stefan G. Sarafianos
Antimicrob. Agents Chemother. 2013, 57(12):6254. DOI:
1
0.1128/AAC.01703-13.
Published Ahead of Print 7 October 2013.
Updated information and services can be found at:
http://aac.asm.org/content/57/12/6254
These include:
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Effects of Substitutions at the 4= and 2 Positions on the Bioactivity of
=-Ethynyl-2-Fluoro-2=-Deoxyadenosine
4
a,b
a,b
a,b
a,b
a,b
a,b
Karen A. Kirby, Eleftherios Michailidis, Tracy L. Fetterly, Musetta A. Steinbach, Kamalendra Singh, Bruno Marchand,
a,b
a,b
c
d
e
f,g
Maxwell D. Leslie, Ariel N. Hagedorn, Eiichi N. Kodama, Victor E. Marquez, Stephen H. Hughes, Hiroaki Mitsuya,
Michael A. Parniak, Stefan G. Sarafianos
h
a,b,i
a
Christopher Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA ; Department of Molecular Microbiology & Immunology, University of Missouri
b
c
School of Medicine, Columbia, Missouri, USA ; Division of Emerging Infectious Diseases, Tohoku University School of Medicine, Sendai, Japan ; Chemical Biology
d
Laboratory, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, Maryland, USA ; HIV Drug Resistance Program, National Cancer Institute-Frederick,
e
f
Frederick, Maryland, USA ; Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto Japan ; Experimental Retrovirology Section, HIV/AIDS
g
Malignancy Branch, National Institutes of Health, Bethesda, Maryland, USA ; Department of Microbiology & Molecular Genetics, University of Pittsburgh School of
h
i
Medicine, Pittsburgh, Pennsylvania, USA ; Department of Biochemistry, University of Missouri, Columbia, Missouri, USA
Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4=-
ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of
EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine
how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and
their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of
4
=-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2=-exo/C-3=-endo (north) conforma-
tion. Steady-state kinetic analysis of the incorporation of 4=-substituted NRTIs by RT reveals a correlation between the north
conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the
kinetics of deamination by ADA demonstrate that 4=-ethynyl and cyano substitutions decrease the susceptibility of adenosine-
based compounds to ADA through steric interactions at the active site. However, the major determinant for decreased suscepti-
bility to ADA is the 2-halo substitution, which alters the pK of N1 on the adenine base. These results provide insight into how
a
NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites.
here are 10 nucleos(t)ide reverse transcriptase inhibitors delayed chain terminator, incorporating one incoming deoxy-
T
(NRTIs) that are currently approved for the treatment of hu- nucleoside triphosphate (dNTP) before DNA synthesis is blocked
man immunodeficiency virus type 1 (HIV-1) infections (1–5). (20). We previously showed that EFdA inhibits HIV replication in
Several more nucleoside analog drugs are approved or being stud- peripheral blood mononuclear cells (PBMCs) with a 50% effective
ied for the treatment of viruses, such as herpes simplex virus concentration (EC ) of 0.05 nM (20). Additionally, EFdA effec-
50
(HSV), hepatitis C virus (HCV), and hepatitis B virus (HBV), or as tively inhibits the replication of many common drug-resistant
anticancer agents (6–10). NRTIs are among the most effective strains of HIV, including strains that carry a substitution of R for
anti-HIV drugs. All approved anti-HIV NRTIs lack a 3=-hydroxyl K at position 65 (K65R) (a 0.2-fold change in EC ) (21, 22),
50
moiety and, thus, act as chain terminators following their incor- N348I (a 0.9-fold change in 50% inhibitory concentration [IC ])
50
poration by the viral reverse transcriptase (RT) into the nascent
DNA chain. However, the absence of a 3=-OH, while essential for
the inhibition of DNA synthesis, also imparts detrimental prop-
erties to these inhibitors, including reduced intracellular phos-
phorylation to the active triphosphate form and reduced RT bind-
ing affinity (11). Prolonged exposure to NRTI-based treatments
causes mitochondrial toxicity (12–14) and leads to the develop-
ment of NRTI resistance mutations (15–18), giving rise to com-
plications in the treatment of HIV-infected patients.
(
23), the excision-enhancing mutations M41L and T215Y (a 1.5-
fold change in EC ) (21), and multidrug resistance Q151M com-
plex mutations (A62V/V75I/F77L/F116Y/Q151M) (a 0.7-fold
change in EC ) (21). EFdA is also able to inhibit HIV containing
the M184V drug resistance mutation (with an EC50 of 8.3 nM, or
a 7.5-fold change) (21, 24). EFdA is generally a poor substrate for
human DNA polymerases in in vitro experiments (IC s of Ͼ100
50
50
50
␮
M for polymerase ␣ and ␤ and 10 ␮M for polymerase ␥; EFdA-
triphosphate [EFdA-TP] is incorporated by polymerase ␥ 4,300-
Ideally, an NRTI should have a strong binding affinity for the
RT target, a high barrier for the development of resistance, and
low toxicity. We have reported that a series of 4=-substituted
nucleosides in which the 3=-OH is retained has exceptional inhib-
itory activity against HIV-1 RT (19). Among these compounds,
Received 6 August 2013 Returned for modification 10 September 2013
Accepted 29 September 2013
Published ahead of print 7 October 2013
4
=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) is a highly active
Address correspondence to Stefan G. Sarafianos, sarafianoss@missouri.edu.
RT inhibitor that prevents translocation of the nucleic acid from
the nucleotide-binding or pretranslocation site to the primer-
binding or posttranslocation site on RT following its incorpora-
tion at the 3= primer terminus (20). Depending on the DNA tem-
plate sequence, EFdA can also act (albeit less frequently) as a
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/
AAC.01703-13.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01703-13
6254 aac.asm.org
Antimicrobial Agents and Chemotherapy p. 6254–6264
December 2013 Volume 57 Number 12

Structure-Activity Relationship for EFdA Analogs
fold less efficiently than dATP) and, thus, has a low potential for EFdA is poorly deaminated by ADA because of the 2-fluoro group
cytotoxicity (25, 26). Because the 50% cytotoxic concentration (21). However, the effects of specific 4= substitutions on sugar ring
(
CC ) for EFdA is more than 10 ␮M in MT4 cells or PBMCs, the conformation and how these substitutions would affect the recog-
50
selectivity of the compound is high. Moreover, in studies with nition of the compounds by HIV RT and ADA have not been
simian immunodeficiency virus (SIV)-infected macaques, no determined.
signs of clinical or pathological drug toxicity were observed after 6
In the present work, we carried out a series of structural, com-
months of continuous EFdA monotherapy (24). These data sug- putational, and biochemical studies using a variety of 2- and 4=-
gest that EFdA is a strong candidate for further development as a substituted dA and dT analogs (Fig. 1) to understand the specific
therapeutic agent.
effects of these different substitutions on sugar ring conformation,
The EC50s of NRTIs are also affected by cellular uptake, activa- recognition by HIV RT, and deamination by ADA. Our data show
tion to the active metabolite by host kinases, and catabolism by how the 2-fluoro and 4=-ethynyl moieties affect the antiviral po-
cellular enzymes. Previous studies have shown that EFdA has a tential of NRTIs and help determine the basis for the favorable
favorable cellular uptake profile and is efficiently phosphorylated inhibitory profile of EFdA.
to EFdA-TP (25). Additional reported data strongly suggest that,
in cells, EFdA is phosphorylated to EFdA-monophosphate
MATERIALS AND METHODS
(EFdA-MP) by deoxycytidine kinase (dCK) (21). The molecular
Chemicals. Compounds dA, FdA, and dT (compound abbreviations are
defined and their numbers specified in Results) were purchased from
Sigma-Aldrich (St. Louis, MO). Triphosphate forms of dA and dT were
details of the efficiency and specificity of this process are being
investigated in separate studies. The enzyme adenosine deaminase
(ADA), which is present at high concentrations in human serum, purchased from Fermentas (Glen Burnie, MD). Compounds 13 to 14 and
is involved in the catabolism of dA and its analogs (27, 28). ADA is the triphosphate forms of compounds 10, 11, 13, and 14 were synthesized
responsible for the deamination of adenosine or deoxyadenosine by Marquez and colleagues as previously described (49, 50). ADA (bovine
intestine, EC 3.5.4.4) was purchased from Calbiochem (San Diego, CA).
analogs to inosine- or deoxyinosine-based products (27, 29–33)
Deuterated dimethyl sulfoxide (DMSO-D ) was purchased from Cam-
and can therefore affect the activation pathway and intracellular
concentration of dA-based compounds. For example, ADA con-
verts dideoxyadenosine (ddA) to ddI, leading to a different and
indirect activation pathway for ddA (28, 34, 35). Thus, it is impor-
tant to study the interaction of dA-based compounds, such as
EFdA, with ADA to gain insights into their catabolism and subse-
quent activation pathways.
6
bridge Isotope Laboratories, Inc. (Andover, MA).
Nucleic acid substrates. DNA oligomers were synthesized by Inte-
grated DNA Technologies (Coralville, IA). In steady-state kinetics assays
of dATP and dATP analogs, a 26-nucleotide-long DNA template (Td26A
;
CCA TAG ATA GCA TTG GTG CTC GAA CA) was used. In steady-state
kinetics assays of dTTP and dTTP analogs, a 26-nucleotide-long template
(Td26T; CCA TAG TAA GCA TTG GTG CTC GAA CA) was used. An
We have also previously reported that small changes in the 18-nucleotide-long 5=-fluorescently labeled DNA primer (Pd18; TGT TCG
chemical composition of dA analogs can have a pronounced effect AGC ACC AAT GCT) was annealed to both Td26A and Td26T
.
1
NMR spectroscopy. One-dimensional H nuclear magnetic reso-
nance (NMR) spectra were collected in 10°C increments from 20 to 50°C
on a Bruker Avance DRX500 operating at 500 MHz and equipped with a
on their antiviral potency. These changes can be complex, because
they can have different and at times opposing effects on the rec-
ognition of NRTIs by the RT target and the various enzymes in-
volved in activation and catabolism. For example, the presence of
fluorine or another halogen at the 2 position on the adenine base
of 4=-substituted dA analogs significantly enhanced their antiviral
activity (21). Similarly, the nature of the 4= substitutions on the
5-mm HCN cryoprobe, as previously described (36). Samples were dis-
solved in DMSO-D to give concentrations of 2 to 4 mM. Spectra were
6
processed with resolution enhancement using negative line broadening
(lb ϭ Ϫ0.3). Coupling constants were analyzed by spectral simulation
using SpinWorks 3.0 (Kirk Marat, University of Manitoba, Winnipeg,
deoxyribose ring also affected the EC s of dA-based compounds Canada). Simulations were performed for the spin systems on the deoxy-
5
0
against both wild-type (WT) and NRTI-resistant HIV-1 variants ribose ring only. The root mean square (RMS) deviations of calculated
(
19).
and experimental coupling constants for all compounds were Ͻ0.15 Hz.
Pseudorotational analysis. The sugar ring conformations of the nu-
cleoside analogs were determined using the program PSEUROT 6.3 (DOS
version, purchased from Cornelis Altona, University of Leiden, Leiden,
The Netherlands) as previously described (36). PSEUROT assumes a two-
state approximation (equilibrium between north and south conforma-
tions) and utilizes a generalized Karplus equation combined with a
nonlinear Newton-Raphson minimization process to examine the con-
formational flexibility of five-membered rings (51). An iterative method-
Additionally, changes in the conformation of the sugar ring
and in the nature of the nucleobase can affect the biological activ-
ity of NRTIs. In solution, the structure of the deoxyribose ring of
nucleosides exists in a dynamic equilibrium between the C-2=-
exo/C-3=-endo (north) and C-2=-endo/C-3=-exo (south) confor-
mations. We have previously shown that EFdA exists primarily in
the north conformation (36) and that HIV-1 RT prefers the NRTI
or incoming dNTP sugar ring in the north conformation for op- ology was used to determine the optimal pseudorotational parameters PN
timal binding (37–40). In contrast, host cell kinases appear to and P (pseudorotational phase angle [P] for the north [N]or south [S]
S
prefer the sugar ring in the south conformation; compounds that conformation) and ␾
(N) and ␾
(S) (degree of maximum ring pucker
m
m
[
␾ ] for the north or south conformation), in which some of these pa-
favor the north conformation are generally phosphorylated inef-
ficiently (41, 42). Sugar ring conformation has also been reported
to play an important role in ADA recognition; the enzyme has
been reported to deaminate nucleosides biased toward the north
conformation up to 65-fold faster than those biased toward the
south conformation (43–46). Additionally, 2-fluoro-2=-deoxy-
adenosine (FdA) analogs are somewhat resistant to ADA-medi-
m
rameters were fixed during refinement in order to constrain the values
within the range commonly observed for nucleosides (average observed,
␾m ϭ 39°).
Molecular orbital calculations. In order to determine the role of mo-
lecular orbitals in the stability of the north and south sugar ring confor-
mations of EFdA, we carried out molecular orbital calculations using
semiempirical quantum chemical methods. All quantum chemical calcu-
ated catabolism due to the electron withdrawing properties of the lations were performed by the density functional theory (DFT) method
2
-fluoro group (47, 48), and we have previously reported that using the B3LYP approach with 6-31G** basis sets. The quantum chem-
December 2013 Volume 57 Number 12
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Kirby et al.
FIG 1 Deoxyadenosine and thymidine analogs used in the study. *, only the triphosphate forms of these compounds were used in this study.
istry program Jaguar (Schrödinger, Inc., NY) was used for all computa- Absorbance was measured every 20 s. Three determinations were made
tions. The structures of north and south conformations of EFdA were for each analog. Km and kcat for each compound were determined using
completely optimized by restricting the position of the 3=-C either in the Michaelis-Menten plots (GraphPad Prism 4).
north or south conformation. To obtain insight into the interaction of the
Because the reactions of ADA with FdA (compound 2), EFdA (com-
lone pairs of the O4 and 3=-O atoms with the antibonding (␲*) orbitals of pound 5), ECldA (compound 6), and CNFdA (compound 9) were very
the 4=-ethynyl group of EFdA, the natural bond orbitals (NBOs), natural slow under these conditions, a high-pressure liquid chromatography
(
semi-)localized molecular orbitals (NLMOs), and canonical molecular (HPLC) method was used to observe the hydrolytic deamination of these
orbitals (CMOs) were computed using the program NBO (version 5.0) compounds (54). In order to separate the substrate and product of the
52, 53). reaction, an elution gradient was developed using various combinations
HIV-1RTsteady-statekineticassays. Thesteady-statekineticparam- of 0.1 M phosphate, pH 7.4, and methanol. The following gradient was
(
eters, Km and kcat, for incorporation of dNTPs and dNTP analogs were run on an Agilent 1100 series HPLC system: 7% methanol for 3 min, 14%
determined using single-nucleotide incorporation in gel-based assays un- methanol for 2 min, 21% methanol for 2 min, 28% methanol for 2 min,
der saturating substrate conditions. The reactions were carried out in 50 35% methanol for 5 min, and 7% methanol for the final 2 min, returning
mM Tris-HCl, pH 7.8, 50 mM NaCl, 6 mM MgCl , 100 nM Td26A/Pd18 for the conditions to the original state. The gradient ran for a total of 16 min
2
dATP analogs, 100 nM Td26T/Pd18 for dTTP analogs, and 10 nM RT in a at a flow rate of 1 ml/min and an injection volume of 60 ␮l. The method
final volume of 20 ␮l and stopped at the reaction times of 1.5 to 3 min. The ran at room temperature, and the absorbance was measured at a wave-
products were resolved on 15% polyacrylamide 7 M urea gels, and the gels length of 260 nm. The solvents used for the HPLC gradient were vacuum
were scanned with a Typhoon FLA 9000 phosphorimager (GE Health- filtered prior to use.
care). The bands were quantified using Multi Gauge software (FujiFilm).
Km and kcat values were determined graphically using the Michaelis-Menten (compound 2), EdA (compound 3), EFdA (compound 5), ECldA (com-
equation in GraphPad Prism 4 (GraphPad Software, Inc.). pound 6), CNdA (compound 7), and CNFdA (compound 9) as ADA
Kinetic measurements were made using dA (compound 1), FdA
ADA kinetic assays. The deamination rates of dA (compound 1), EdA substrates. The reactions were carried out in 0.1 M phosphate buffer, pH
(
compound 3), and CNdA (compound 7) were determined by following 7.4. The FdA (compound 2) and EFdA (compound 5) analogs were tested
the disappearance of the substrate on a BioTek Synergy HT spectropho- using concentrations ranging from 10 ␮M to 9 mM. Compounds dA
tometer at 265 nm in 100-␮l solutions at 25°C with 0.1 M phosphate, pH (compound 1), EdA (compound 3), ECldA (compound 6), CNdA (com-
7.4. A change in molar extinction coefficients for all of the deaminated pound 7), and CNFdA (compound 9) were tested at 500 ␮M. Reactions
products was assumed to be ⌬ε ϭ Ϫ7,900 for all dA analogs (45). For each were started by the addition of 2.4 mU ADA. The reactions were run for 45
assay, the substrate concentrations varied between 15 ␮M and 240 ␮M. min at 25°C before being terminated by the addition of perchlorate at 0.3
6256 aac.asm.org
Antimicrobial Agents and Chemotherapy

Structure-Activity Relationship for EFdA Analogs
TABLE 1 Summary of pseudorotational analysis of the sugar ring of deoxyadenosine and thymidine analogs
a
Compound (no.)
PN (°)
PS (°)
␾ (N) (°)
␾ (S) (°)
% North
RMS error (Hz)
m
m
Deoxyadenosine analogs
dA (1)
FdA (2)
EdA (3)
b
c
18.7
3.4
169.1
169.1
152.1
152.1
146.5
148.0
152.8
39.0
31.1
29.2
23
24
64
63
75
73
61
56
59
0.03
0.01
0.40
0.49
0.20
0.21
0.12
0.12
0.23
38.7
c
c
37.3
39.7
38.7
38.4
47.8
45.0
41.6
39.0
39.0
c
c
EdDAP (4)
39.0
39.0
b
c
c
EFdA (5)
39.0
39.0
c
c
ECldA (6)
CNdA (7)
CNdDAP (8)
CNFdA (9)
39.0
39.0
38.0
c
c
38.0
c
c
154.0
154.2
37.2
38.0
38.0
c
c
38.0
Thymidine analogs
MedT (13)
EtdT (14)
a
c
24.3
23.3
132.5
150.3
38.0
31.5
30.2
43
39
0.05
0.18
c
38.0
Root mean square deviation between experimental and calculated coupling constants.
Values previously reported (36).
b
c
Value fixed during calculations.
M final concentration. Denatured protein was removed from the reaction conformations (see Fig. S1 in the supplemental material) of the
mixture by using centrifugation at 10,000 rpm for 5 min to pellet the nucleoside analogs in this study.
protein. The clear supernatant was then removed, and the final pH was
The pseudorotational parameters listed in Table 1 were calcu-
lated using PSEUROT 6.3 as described in Materials and Methods
and our previous report (36). The value of PN for each of the
adjusted to 7.4 by adding Na CO to reach a final concentration of 2.5
2
3
mM (54).
Structural analysis of predicted EFdA interactions at the active site
of ADA. Structural analysis of EFdA interactions at the ADA active site
were carried out using the coordinates of human ADA in complex with
4
=-ethynyl- or cyano-substituted deoxyadenosine compounds 3
to 9 (P ϭ 37.3 to 47.8°) lies in the northern hemisphere of the
N
dA obtained from the RCSB Protein Data Bank (PDB ID 3IAR). EFdA pseudorotational pathway (P ϭ 0 to 90°), although these values
N
was drawn and minimized using the ChemBioDraw Ultra 13.0 and are higher than what is typically observed for most nucleosides
ChemBio3D Ultra 13.0 software from the ChemBioOffice Suite (P ϭ 0 to 36°) (see Fig. S1 in the supplemental material). These
N
3
(
PerkinElmer, Waltham, MA). Using the Coot model building software
values correspond to a conformation between T (P ϭ 36°) and
4
N
package (55), the structure of EFdA was superposed to the dA molecule,
followed by the removal of dA.
E or 4=-exo (P ϭ 54°) in the pseudorotational pathway (see Fig.
4
N
S1) (56). A similar conformation was determined by Siddiqui et al.
from the crystal structure of 4=-ethynyl-2=,3=-dideoxycytidine
RESULTS
(
P ϭ 43.5°) (57). The P values of the 4=-cyano-substituted com-
N
N
Effect of 4= substitutions on the structure of dA and dT analogs.
To evaluate the structural effects of the 4= substitutions on the
sugar ring conformations of the dA and dT analogs, we collected
pounds 7 to 9 (P ϭ 41.6 to 47.8°) are slightly higher than those
N
observed for the 4=-ethynyl-substituted compounds 3 to 6 (P ϭ
N
1
37.3 to 39.7°). The values of P
substituted thymidine compounds (P ϭ 24.3° and 23.3°, respec-
N
for the 4=-methyl- and 4=-ethyl-
one-dimensional H NMR spectra over a range of physiologically
N
relevant temperatures for the following compounds: 2=-deoxy-
adenosine (dA, compound 1), 2-fluoro-2=-deoxyadenosine (FdA,
compound 2), 4=-ethynyl-2=-deoxyadenosine (EdA, compound 3),
tively) were also in the northern hemisphere but were much lower
than those observed for the 4=-ethynyl and 4=-cyano analogs.
3
These values correspond to a conformation between E or 3=-endo
4
=-ethynyl-2=-deoxyribofuranosyl-2,6-diaminopurine (EdDAP, also
3
(
(
P ϭ 18°) and T (P ϭ 36°) in the pseudorotational pathway
N
4
N
known as ENdA, compound 4), 4=-ethynyl-2-fluoro-2=-deoxy-
adenosine (EFdA, compound 5), 4=-ethynyl-2-chloro-2=-deoxy-
adenosine (ECldA, compound 6), 4=-cyano-2=-deoxyadenosine
see Fig. S1) (56).
The pseudorotational values determined for dA (compound 1)
are in agreement with previous reports, demonstrating that dA
strongly prefers the south conformation (36, 45). Comparison of
the conformational parameters of dA (compound 1) versus EdA
(
CNdA, compound 7), 4=-cyano-2=-deoxyribofuranosyl-2,6-di-
aminopurine (CNdDAP, compound 8), 4=-cyano-2-fluoro-2=-
deoxyadenosine (CNFdA, compound 9), thymidine (dT, com-
pound 12), 4=-methyl-thymidine (MedT, compound 13), and
(
compound 3) (23% versus 64% north) and FdA (compound 2)
versus EFdA (compound 5) (24% versus 75% north) showed that
4
=-ethyl-thymidine (EtdT, compound 14) (Fig. 1) (36). Resolving
the 4=-ethynyl strongly favors the north conformation of the sugar
the coupling constants between most of the hydrogen atoms on
the deoxyribose ring required spectral simulation (see Table S1
(
Table 1 and Fig. 2). Comparison of dA (compound 1) versus
in the supplemental material). The coupling constants deter- CNdA (compound 7) (23% versus 61% north) and FdA (com-
mined from each spectrum were used to calculate the structural pound 2) versus CNFdA (compound 9) (24% versus 59% north)
parameters of the deoxyribose ring of each compound. The showed that the 4=-cyano also has a large effect on sugar confor-
changes in the value of each coupling constant with temperature mation, albeit smaller than that of the 4=-ethynyl group (% north
were used to calculate the pseudorotational phase angle, P, and the in EdA [compound 3] Ͼ % north in CNdA [compound 7] and %
degree of maximum ring pucker, ␾ , for the C-2=-exo/C-3=-endo north in EFdA [compound 5] Ͼ % north in CNFdA [compound
m
(north or N) and C-2=-endo/C-3=-exo (south or S) sugar ring 9]) (Table 1).
December 2013 Volume 57 Number 12
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Kirby et al.
FIG 2 Dynamic equilibrium between the south and north sugar ring conformations of 4=-ethynyl-substituted compounds in solution (EFdA is shown). Natural
deoxynucleoside substrates favor the south conformation.
Evaluation of the conformational parameters of the thymidine (semi-)localized molecular orbitals (NLMOs). The NBOs are lo-
analogs dT (compound 12), MedT (compound 13), and EtdT calized orbitals that describe the Lewis-like molecular bonding
(
compound 14) demonstrated that 4=-methyl and ethyl substitu- pattern of electron pairs (59). The NLMOs have been described
tions have smaller effects on the sugar ring conformation than as semilocalized alternatives to canonical molecular orbitals
4
=-ethynyl and cyano groups. Although we were unable to accu- (CMOs) for representing the electron pairs of MO-type wave
rately determine the coupling constants for dT (compound 12) functions. Each NLMO consists of a parent NBO (strictly local-
due to overlap of one of the 2=-H signals with the DMSO solvent ized) and associated delocalizations needed to describe the density
peak, it has previously been determined that the deoxyribose ring of a full electron pair (59). The NBOs were used to compute
of dT favors the south conformation, which is similar to dA (56, CMOs. Both the north and south sugar ring conformations of
5
8). 4=-Methyl and 4=-ethyl substitutions shift the equilibrium EFdA have 375 NBOs (which include bonding orbitals, core or-
toward the north conformation relative to the equilibrium of thy- bitals, lone pairs, antibonding orbitals, and Rydberg orbitals), 76
midine (43% and 39% north, respectively), but this effect is NLMOs, and 375 CMOs. To assess the orbital interaction involv-
smaller than that of a 4=-ethynyl or 4=-cyano substitution in dA ing the lone pairs of O4 and 3=-O and the antibonding orbitals
analogs (64% and 61% north compared to 23% in dA) (Table 1). (␲*) of the 4=-ethynyl group, we examined the second-order per-
Hence, the 4=-ethynyl substitution imparts the largest effect on turbative estimates of donor-acceptor (bond-antibond) NBO in-
the conformation of the deoxyribose ring, followed by the 4=- teractions for both north and south conformations. These esti-
cyano, the 4=-methyl, and the 4=-ethyl groups.
mates provide the measure of delocalization due to the interaction
Effect of 2 position substitutions on the structure of dA ana- of the O4 or 3=-O lone pair with the ␲* antibonding orbital of the
logs. Using the same method, we also assessed the effect of substi- 4=-ethynyl group. Table 2 summarizes the energy of the donor-
tutions at the 2 position of the adenine base on the structure of dA acceptor NBO interactions. These results suggest that the interac-
analogs. Evaluation of the conformation parameters of dA (com- tion of the lone pairs of the O4 and 3=-O atoms with the antibo-
pound 1) versus FdA (compound 2) showed that substitutions at nding orbitals of the 4=-ethynyl moiety contributes to the greater
the 2 position of the adenine base have little to no effect on sugar stabilization energy (ϳ2-fold) of the north conformation. Further
ring conformation. Similarly, comparisons of the 4=-ethynyl-sub- NBO analyses show the reduced occupancy of the O4 and 3=-O
stituted EdA (compound 3), EdDAP (compound 4), EFdA (com- lone pairs (10% and 6%, respectively) and the remote interaction
pound 5), and ECldA (compound 6) showed that the 2-fluoro and of both lone pairs with the antibonding NBOs of the 4=-C§C in the
2
-chloro substitutions have only a modest effect on the sugar ring north conformation. In the south conformation, the O4 and 3=-O
conformation (Table 1). Finally, comparisons of CNdA (com- lone pairs have a reduced occupancy of 7% and 4%, respectively,
pound 7), CNdDAP (compound 8), and CNFdA (compound 9) and only the O4 lone pair has a remote interaction with the anti-
also confirmed that 2-fluoro and 2-amino substitutions on the bonding NBOs of the 4=-C§C. The NLMO analyses of the north
adenine ring have no significant impact on the sugar ring confor- conformation showed that the lone pairs of both O4 and 3=-O
mation (61%, 56%, and 59% north, respectively) (Table 1).
Molecular orbital calculations. Potential stabilizing orbital
interactions can occur between a lone pair of electrons in a p-like
a
TABLE 2 Summary of molecular orbital calculations
fully occupied orbital and an unoccupied antibonding orbital, al-
lowing the delocalization of electrons and, thus, increasing molec-
ular stability. To determine whether there are any favorable orbital
interactions in the north or south sugar ring conformations of
EFdA that could stabilize one conformation versus the other, we NBO donor (LP)
Interaction energy
(kcal/mol) for EFdA
conformation
NBO acceptor (␲*)
North
South
carried out quantum chemical computations to assess the orbital
O4
4=-C§C
4=-C§C
1.63
0.71
1.25
0.0
interactions of the lone pairs of the O4 and 3=-O atoms with the
3=-O
antibonding (␲*) orbitals of the 4=-ethynyl moiety. We first deter-
mined the electronic composition of natural bond orbitals Total energy
2.34
1.25
a
(NBOs) and their contribution in the formation of natural
LP, lone pair; ␲*, antibonding orbital.
6258 aac.asm.org
Antimicrobial Agents and Chemotherapy

Structure-Activity Relationship for EFdA Analogs
TABLE 3 Steady-state kinetic parameters for incorporation of the
triphosphate forms of the 4=-substituted compounds by HIV-1 RT
TABLE 4 Summary of steady-state kinetic data for the deamination of
dA, EdA, and CNdA by ADA
a
Fold change in
Mean Km Ϯ SD
(␮M)
kcat/Km
(s · ␮M )
Ϫ1
Ϫ1
Ϫ1
kcat
(min
kcat/Km
(min · ␮M
incorporation
efficiency
Compound (no.)
kcat (s
)
a
Ϫ1 a
)
Ϫ1
Ϫ1
b
dNTP
Km (␮M)
)
dA (1)
EdA (3)
CNdA (7)
a
42.6 Ϯ 15.0
145.9 Ϯ 37.6
156.9 Ϯ 84.2
13.5
11.3
8.9
0.32
0.08
0.06
dATP
EFdA-TP
2.35 Ϯ 0.06 0.66 Ϯ 0.13 0.3
0.27 Ϯ 0.01 3.47 Ϯ 0.04 12.9
1
45.9
60.7
15.3
13.9
1
EdDAP-TP 0.15 Ϯ 0.01 2.55 Ϯ 0.10 17.0
Data were determined spectrophotometrically.
MedA-TP
EtdA-TP
dTTP
MedT-TP
EtdT-TP
a
0.29 Ϯ 0.01 1.24 Ϯ 0.05 4.3
0.88 Ϯ 0.19 3.41 Ϯ 0.12 3.9
0.60 Ϯ 0.01 1.04 Ϯ 0.12 1.7
0.68 Ϯ 0.22 1.58 Ϯ 0.15 2.3
2.00 Ϯ 0.22 1.44 Ϯ 0.20 0.7
Effect of 2 position substitutions on susceptibility to ADA.
Because the 2 position-substituted compounds were refractory to
hydrolytic deamination by ADA, we were unable to obtain reliable
kinetic measurements using the spectrophotometric assay. In or-
der to at least obtain a relative order of susceptibility to ADA, we
used an HPLC-based assay (54), which allowed long incubation
times and a determination of the amount of product from the slow
hydrolytic deamination of compounds 2, 5, 6, and 9 by ADA.
These experiments showed that incubation of 500 ␮M substrate
1.3
0.4
Values are means Ϯ standard deviations of two independent experiments and were
determined from the Michaelis-Menten equation using GraphPad Prism 4.
b
Fold change in incorporation efficiency is the ratio of the incorporation efficiency
(
(
k
cat/K
m
) of NRTI-TP over that of dATP or dTTP [(kcat/K
/(kcat/K ) ].
m m
)NRTI-TP/(kcat/K )dATP or
kcat/K )
m NRTI-TP m dTTP
atoms are delocalized into the vicinal region (C-5= and 4=-C§C, for 45 min resulted in 15% conversion of FdA (compound 2),
ϳ5% and 2%, respectively). The delocalization of the O4 lone pair 1.3% of EFdA (compound 5), 2.8% of ECldA (compound 6), and
in the vicinal C-5= and 4=-C§C groups for the south conforma- Ͻ1% of CNFdA (compound 9) (Fig. 3). These data suggest that
tion is significantly less than ϳ1%, and there is almost no de- the 2-halo combined with 4= substitutions resulted in significantly
localization of the 3=-O lone pair in the C-5= and 4=-C§C vicin- decreased susceptibility of the dA compounds to ADA and that the
ity. Taken together, these data suggest (i) that the north sugar decreasing order of stability was CNFdA (compound 9) Ͼ EFdA
ring conformation is more stable than the south and (ii) that (compound 5) Ͼ ECldA (compound 6) Ͼ FdA (compound 2).
the north conformation is stabilized by the interactions of the This is consistent with our previous report that EFdA is not effi-
O4 and 3=-O lone pairs with the antibonding orbitals of the ciently deaminated by ADA (21).
4
=-ethynyl group.
Structural analysis of predicted EFdA interactions at the
Effect of 4= substitutions on the ability of dA and dT analogs ADA active site. Using the crystal structure coordinates of human
to block reverse transcription in vitro. In order to assess the ADA, we looked for potential interactions between the active-site
effect of various 4= substitutions on the ability of dA and dT ana- residues and the modeled EFdA that could lead to decreased sus-
logs to block reverse transcription in vitro, we tested the triphos- ceptibility of this inhibitor to ADA. As shown in Fig. 4, the place-
phate forms of compounds 1, 4, 5, and 10 to 14 for their ability to ment of EFdA at the expected position of a substrate would result
block DNA synthesis by HIV-1 RT (Table 3). Additional previ- in unfavorable steric interactions between the 4=-ethynyl group of
ously published in vitro and cell-based inhibition data for dA and EFdA and the sulfur and ε-carbon of Met155 (d ϭ 2.2 and 2.0 Å,
dT analogs are summarized in Table S2 in the supplemental ma- respectively). These potential steric effects could contribute to a
terial. Remarkably, almost all 4=-substituted analogs are excellent lower binding affinity for EdA than for dA, consistent with an
substrates of HIV-1 RT and are incorporated even more efficiently ϳ3.5-fold difference in the corresponding K values (K
ϭ
m-EdA
m
than the canonical substrate (higher k /K ). Specifically, the 4=- 146 ␮M versus Km-dA ϭ 43 ␮M), which is the primary contribut-
cat
m
ethynyl substitution resulted in the highest efficiencies of inhibitor ing factor in the observed decrease in the catalytic efficiency of
incorporation (EdDAP-TP and EFdA-TP have 60- and 45-fold ADA when EdA is the substrate.
higher k /K than dATP). The higher efficiency appears to be the
cat
m
DISCUSSION
result of both increased catalytic turnover rate (k ) and decreased
cat
Km (Table 3). Notably, the enhancement in incorporation effi- We have previously demonstrated that EFdA is more potent
ciency in vitro appears to be more pronounced for the dA than for against WT and drug-resistant HIV strains than other approved
the dT analogs (Table 3). Moreover, 4=-ethynyl dA analogs are 3 to NRTIs (19–24) in both biochemical and virological assays and
4
times more efficiently incorporated by HIV-1 RT than 4=-methyl that it has low cytotoxicity (24–26). This suggests that EFdA not
compounds. Inhibitors containing a 4=-ethyl modification are the only has a high affinity toward HIV-1 RT but is also likely to be
least efficiently incorporated by HIV-1 RT. efficiently activated by host kinases (21, 25) and is relatively stable
Effect of 4= substitutions on susceptibility to ADA. Bovine in cells (21). In fact, a favorable NRTI EC50 depends on multiple
ADA was chosen to study the hydrolytic deamination of com- desirable attributes that include (i) a strong binding affinity of
pounds 1 to 7 and 9 because it is readily available and because it is the active metabolite for the RT target, (ii) efficient cellular uptake
highly similar to human ADA (91% overall and 100% active-site and activation by host kinases, (iii) an ability to withstand deacti-
sequence identity) (60, 61), allowing us to use the biochemical vation by cellular enzymes, (iv) a high barrier to resistance, and (v)
data to infer what would happen with the human enzyme. We low cytotoxicity. The present study investigates how different sub-
found that 4= substitutions decreased the ability of ADA to use stitutions on the sugar ring and adenine base contribute to the
these nucleosides as substrates, primarily due to an increase in K
structural features of NRTIs and the effects of these features on the
m
(
Table 4). The 4=-ethynyl and 4=-cyano groups have similar effects first three of the factors listed above.
on ADA catabolism.
Sugar puckering affects several aspects of the activity of EFdA
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Kirby et al.
FIG 3 HPLC data taken after reaction of dA analogs (500 ␮M) with ADA for 45 min (260-nm signal is shown in blue). FdA (A), EFdA (B), ECldA (C), and
CNFdA (D) were converted to the corresponding deoxyinosine (dI) forms to different extents. Percentages of dA-based reactant and dI-based product were
calculated based on peak areas.
by influencing its interactions with HIV RT, dCK, and ADA. We
For a nucleoside analog to be a potent inhibitor of HIV repli-
have previously proposed that the extent to which an NRTI can cation, it must be converted to the triphosphate form in cells. The
exist in the north conformation affects its potency. In that respect, nucleoside substrate type or sugar ring conformation can affect
we have shown (Marquez and colleagues) that HIV-1 RT prefers the ability of cellular kinases to activate nucleoside analogs. The
as a substrate a version of zidovudine-triphosphate (AZT-TP) that type of nucleoside substrate can determine which kinase will carry
is locked in the north rather than in the south conformation (38), out the initial phosphorylation step. For example, dCK preferen-
suggesting that the HIV-1 RT active site prefers nucleosides or tially phosphorylates dC, dA, and dG analogs (62, 63), whereas
their analogs in the north conformation. Our NMR data and mo- thymidine kinase 1 phosphorylates dT analogs (64). Hence, the
lecular orbital calculations demonstrate that the 4=-ethynyl sub- differences in phosphorylation efficiency of various NRTIs likely
stitution causes the sugar ring conformation of EFdA to be pri- depend, at least in part, on the fact that different kinases are used
marily in the north conformation. This causes it (and presumably to convert the various analogs to the active metabolite (65). More-
the structure of its active metabolite EFdA-TP) to reside in a fa- over, the activation of NRTIs involves multiple enzymes whose
vorable conformation for binding at the polymerase active site of specificity may also impact the efficiency of activation. For exam-
HIV-1 RT (36). This hypothesis is further supported by our ple, AZT-MP is not phosphorylated efficiently to AZT-diphos-
steady-state kinetic assays and a previous report of in vitro HIV RT phate (AZT-DP) by thymidylate kinase, and this is the rate-limit-
inhibition by EFdA-TP (20).
ing step in AZT-TP formation (25, 66).
6260 aac.asm.org
Antimicrobial Agents and Chemotherapy

Structure-Activity Relationship for EFdA Analogs
zymes. For example, ADA can deaminate dA-based nucleosides
and change the pathway and efficiency of their activation. The
activity of ADA is affected by the structural determinants of its
substrates. While we have previously demonstrated (Marquez and
colleagues) that this enzyme efficiently deaminates nucleoside an-
alogs in the north conformation (43–46), the kinetic analysis of
ADA that we report here shows that the presence of a substituent
at the 2 position of the adenine base makes these derivatives poor
substrates for ADA. These results agree with previous studies re-
porting that 2-halo-substituted compounds are not efficiently
deaminated by ADA (29, 31–33).
The mechanism by which 2-halo substitutions affect ADA ac-
tivity does not appear to involve steric interactions in the ADA
binding pocket (Fig. 4). This suggests that they decrease suscepti-
bility through electronic rather than steric effects. The electronic
effect of 2-fluoro can be explained in terms of changes in pK and
EFdA (yellow sticks) on interactions with the binding pocket of ADA (gray charge distribution on the adenine base. The deamination mech-
surface). When EFdA binds in a manner similar to that of other substrates, the anism by ADA involves an addition-elimination reaction on the
FIG 4 Structural analysis of the predicted effect of the 4=-ethynyl group of
a
4
=-ethynyl group may clash with the sulfur and ε-carbon atoms of Met155 in
substrate to form an inosine-based product (Fig. 5A). The first
step involves electrophilic attack to protonate the N1 position of
the purine ring, followed by nucleophilic attack at the C-6 posi-
tion of the ring to form a tetrahedral intermediate (67–70). The
introduction of an electronegative fluorine atom at the 2 position
ADA. The figure was made using the PyMOL molecular graphics software
package (http://www.pymol.org/).
The sugar ring conformation can also affect the ability of host
kinases to activate nucleoside analogs. For example, dCK is known
to bind its natural nucleoside substrates in the south conforma-
tion (41, 42). Interestingly, although the EFdA sugar ring favors
the north conformation, it appears to be phosphorylated effi-
ciently by dCK in cells (25). This suggests that other structural
features of EFdA may contribute to its efficient phosphorylation,
has been reported to dramatically lower the pK of the N1 atom of
a
adenosine-based compounds (more than 100-fold) (Fig. 5B) (71).
This is the result of the highly electronegative fluorine atom in-
ϩ
Ϫ
creasing the positive charge at the C-2 position (C-2 ץ , F ץ ),
which would raise the energy barrier of N1 protonation. Hence,
the N1 protonation step becomes the rate-limiting step of the
such as the 4=-ethynyl, the 2-fluoro, or both. In fact, it has been hydrolytic deamination reaction, thus reducing the yield of the
previously reported that a 2-fluoro substitution on dA decreases 2-fluoro deoxyinosine product (Fig. 3). The presence of a 2-amino
the K value 33-fold compared to that of dA, making FdA a very instead of a 2-halo substitution has a different effect on the mech-
m
efficient substrate for dCK (63). Ongoing structural studies of anism and efficiency of the deamination reaction. In the case of
EFdA in complex with dCK should reveal the basis of efficient EdDAP, a resonance effect caused by the 2-amino substitution
activation of EFdA by dCK.
would distribute the positive charge of the protonated N1 to all
The EC50 of NRTIs can also be affected by catabolizing en- neighboring nitrogen atoms. Hence, differences in the deamina-
FIG 5 Reaction mechanism of hydrolytic deamination of dA (A) and FdA (B) by adenosine deaminase (67–70). The electronegative 2-fluoro group causes a
dramatic decrease in pK at the N1 position (71), resulting in a high-energy barrier for the N1 protonation step of the reaction. rxn, reaction.
a
December 2013 Volume 57 Number 12
aac.asm.org 6261

Kirby et al.
tion efficiency of EdDAP and EFdA can be accounted for in terms
of different charge distribution effects on the adenine base. This
explanation is consistent with experimental results that demon-
strated that EdDAP was catabolized more efficiently than EFdA
but less efficiently than either dA or EdA, suggesting that a
5. Sarafianos SG, Marchand B, Das K, Himmel DM, Parniak MA, Hughes
SH, Arnold E. 2009. Structure and function of HIV-1 reverse transcrip-
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2
-amino substitution has a smaller impact on deamination by
2
010. Tricyclic 2=-C-modified nucleosides as potential anti-HCV thera-
ADA than a 2-fluoro substitution (E. N. Kodama, personal com-
munication).
In addition to 2 position substitutions, the presence of a 4=-
ethynyl or cyano group also reduces deamination by ADA. For
example, ADA deaminates EdA or CNdA less efficiently than dA
peutics. Org. Lett. 12:4466–4469.
8
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(
Table 4) and EFdA or CNFdA less efficiently than FdA (Fig. 3).
Our structural analysis of the predicted interactions of EFdA at the
ADA active site suggests that the 4=-ethynyl group may sterically 10. Wauchope OR, Johnson C, Krishnamoorthy P, Andrei G, Snoeck R,
interact with the flexible Met155 side chain (Fig. 4), resulting in a
moderate decrease in the binding and reactivity of EFdA com-
pared to the binding and reactivity of dA (Table 4). These data
suggest that the effect of 4=-ethynyl on ADA activity is caused
primarily by steric interactions. Hence, the substrate specificity of
ADA is affected by both electronic and steric interactions from
different structural determinants of dA-based NRTIs (2-fluoro
and 4=-ethynyl, respectively).
Balzarini J, Seley-Radtke KL. 2012. Synthesis and biological evaluation of
a series of thieno-expanded tricyclic purine 2=-deoxy nucleoside ana-
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2. Brinkman K, ter Hofstede HJ, Burger DM, Smeitink JA, Koopmans PP.
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In conclusion, strategic substitutions have a pronounced and 13. Kakuda TN. 2000. Pharmacology of nucleoside and nucleotide reverse
complex effect on the ability of dA-based inhibitors to block
HIV-1 replication. In particular, substitutions at the 4= position of
the deoxyribose ring and the 2 position of the adenine base of
EFdA have separate effects that enhance its antiviral activity by
transcriptase inhibitor-induced mitochondrial toxicity. Clin. Ther. 22:
685–708.
1
4. Lewis W, Day BJ, Copeland WC. 2003. Mitochondrial toxicity of NRTI
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favoring structural conformations that improve its interactions 15. Sluis-Cremer N, Arion D, Parniak MA. 2000. Molecular mechanisms of
HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs).
Cell. Mol. Life Sci. 57:1408–1422.
6. Menendez-Arias L. 2008. Mechanisms of resistance to nucleoside ana-
logue inhibitors of HIV-1 reverse transcriptase. Virus Res. 134:124–146.
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Structural aspects of drug resistance and inhibition of HIV-1 reverse
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with the RT target and by decreasing its susceptibility to ADA.
Because EFdA is a poor substrate for ADA, it is likely to remain
intact for longer periods of time, thus reducing the dosage re-
quired to achieve an antiviral effect.
1
ACKNOWLEDGMENTS
1
8. Arts EJ, Hazuda DJ. 2012. HIV-1 antiretroviral drug therapy. Cold Spring
We thank Wei Wycoff for assistance with variable-temperature NMR data
collection and processing.
Harb. Perspect. Med. 2:a007161. doi:10.1101/cshperspect.a007161.
19. Kodama EI, Kohgo S, Kitano K, Machida H, Gatanaga H, Shigeta S,
Matsuoka M, Ohrui H, Mitsuya H. 2001. 4=-Ethynyl nucleoside analogs:
potent inhibitors of multidrug-resistant human immunodeficiency virus
variants in vitro. Antimicrob. Agents Chemother. 45:1539–1546.
0. Michailidis E, Marchand B, Kodama EN, Singh K, Matsuoka M, Kirby
KA, Ryan EM, Sawani AM, Nagy E, Ashida N, Mitsuya H, Parniak MA,
Sarafianos SG. 2009. Mechanism of inhibition of HIV-1 reverse transcrip-
tase by 4=-ethynyl-2-fluoro-2=-deoxyadenosine triphosphate, a transloca-
tion-defective reverse transcriptase inhibitor. J. Biol. Chem. 284:35681–
35691.
The 500-MHz NMR spectrometer is supported by NSF grant CHE-
8
9-08304 and NIH/NCRR grant S10 RR022341-01 (cold probe). This
work was supported, in whole or in part, by National Institutes of
Health grants AI076119, AI099284, AI100890, and GM103368 (S.G.S.)
and AI079801 (M.A.P.). This study was supported in part by the In-
tramural Research Program of the National Institutes of Health
2
(
NIH), National Cancer Institute, Center for Cancer Research. We also
acknowledge support from Ministry of Knowledge and Economy,
Bilateral International Collaborative R&D Program, Republic of 21. Kawamoto A, Kodama E, Sarafianos SG, Sakagami Y, Kohgo S, Kitano
Korea. B.M. is a recipient of the amfAR Mathilde Krim Fellowship and
a Canadian Institutes of Health Research (CIHR) Fellowship. E.N.K.
and H.M. are coinventors of EFdA.
K, Ashida N, Iwai Y, Hayakawa H, Nakata H, Mitsuya H, Arnold E,
Matsuoka M. 2008. 2=-Deoxy-4=-C-ethynyl-2-halo-adenosines active
against drug-resistant human immunodeficiency virus type 1 variants. Int.
J. Biochem. Cell Biol. 40:2410–2420.
2
2. Michailidis E, Ryan EM, Hachiya A, Kirby KA, Marchand B, Leslie MD,
Huber AD, Ong YT, Jackson JC, Singh K, Kodama EN, Mitsuya H,
Parniak MA, Sarafianos SG. 2013. Hypersusceptibility mechanism of
tenofovir-resistant HIV to EFdA. Retrovirology 10:65. doi:10.1186/1742
-4690-10-65.
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