Bifunctional inhibition of HIV-1 reverse transcriptase: A first step in designing a bifunctional triphosphate

Article abstract of DOI:10.1016/j.bmcl.2012.12.015

The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof of concept has so far been elusive. As a first step, we describe molecular modeling to guide focused chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T-6PEG-TIBO conjugate was successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-stranded DNA.

Full text of DOI:10.1016/j.bmcl.2012.12.015

Accepted Manuscript
Bifunctional Inhibition of HIV-1 Reverse Transcriptase: A First Step in De‐
signing a Bifunctional Triphosphate
Dongyuan Piao, Aravind Basavapathruni, Pinar Iyidogan, Guangxiu Dai,
Wolfgang Hinz, Adrian S. Ray, Eisuke Murakami, Joy Y. Feng, Fei You, Ginger
E. Dutschman, David J. Austin, Kathlyn A. Parker, Karen S. Anderson
PII:
S0960-894X(12)01599-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2012.12.015
BMCL 19900
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
3 December 2011
4 December 2012
10 December 2012
Please cite this article as: Piao, D., Basavapathruni, A., Iyidogan, P., Dai, G., Hinz, W., Ray, A.S., Murakami, E.,
Feng, J.Y., You, F., Dutschman, G.E., Austin, D.J., Parker, K.A., Anderson, K.S., Bifunctional Inhibition of HIV-1
Reverse Transcriptase: A First Step in Designing a Bifunctional Triphosphate, Bioorganic & Medicinal Chemistry
Letters (2012), doi: http://dx.doi.org/10.1016/j.bmcl.2012.12.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bifunctional Inhibition of HIV-1 Reverse Transcriptase: A First Step in
‡
Designing a Bifunctional Triphosphate
†
a
†b
†b
†b,c
b,c
Dongyuan Piao , Aravind Basavapathruni , Pinar Iyidogan , Guangxiu Dai , Wolfgang Hinz ,
b
b
b
a
b
c
Adrian S. Ray , Eisuke Murakami , Joy Y. Feng , Fei You , Ginger E. Dutschman , David J. Austin ,
a
b*
Kathlyn A. Parker , Karen S. Anderson
†
These authors contributed equally to this work.
‡
This work was supported by National Institutes of Health Grant 1R21GM060913-01A1 (to K.A.P.) and
GM49551 (to K.S.A.)
a
Brown University, Department of Chemistry, Providence, RI 02912
b
Yale University School of Medicine, Department of Pharmacology, New Haven, CT 06520
c
Yale University, Department of Chemistry, New Haven, CT 06520
*
To whom correspondence should be addressed:
Yale University School of Medicine
Dept. of Pharmacology, SHM B350B
3
33 Cedar St.
New Haven, CT 06520-8066.
Tel.: 203-785-4526
Fax: 203-785-7670
E-mail: karen.anderson@yale.edu
TITLE RUNNING HEAD: HIV-1 RT Bifunctional Inhibition
1

CURRENT ADDRESSES:
Dongyuan Piao
Nomadics, Inc. (an ICx company)
Eisuke Murakami
Gilead Sciences
2
15 First Street, Suite 104
333 Lakeside Drive
Foster City, CA 94404
Cambridge, MA 02142
Aravind Basavapathruni
Epizyme, Inc
Joy Y. Feng
Gilead Sciences
Cambridge, MA 02139
333 Lakeside Drive
Foster City, CA 94404
Guangxiu Dai
Fei You
Hutchison MediPharma Limited
Endocyte, Inc
7
20 Cailun Rd.
3000 Kent Ave.
Suite A1-100
Z.J.Hi-Tech Park
Shanghai, China 201203
West Lafayette, IN 47906
Wolfgang Hinz
David J. Austin
The Rothberg Institute For Childhood Diseases Darby & Darby
5
30 Whitfield Street
805 Third Avenue
Guilford, CT 06437
New York, NY 10022
Adrian S. Ray
Kathlyn A. Parker
Gilead Sciences
State University of New York
Department of Chemistry
Stony Brook, NY 11794
3
33 Lakeside Drive
Foster City, CA 94404
Keywords
HIV-1, Reverse Transcriptase, Bifunctional, NRTI, NNRTI, PEG
2

ABBREVIATIONS
HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; NRTIs, nucleoside RT
inhibitors; NNRTIs, nonnucleoside RT inhibitors; dNTP, deoxynucleoside triphosphate; dNMP, 3-
deoxynucleoside monophosphate; PP , pyrophosphate; AZT, -D-(+)-3-azido-3-deoxythymidine; d4T,
i

-D-(+)-2,3-didehydro-3-deoxythymidine; K , Dissociation constant; WT, wild type; TEAB,
d
triethylammonium bicarbonate; MOI, multiplicity of infection; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PEG, polyethylene glycol; 8-Cl-TIBO, 8-chloro
tetrahydroimidazobenzodiazepinone; SAR, structure-activity relationship; MW, molecular weight;
TBDMS, tert-butyldimethylsilyl; Tris, tris(hydroxymethyl)aminomethane
3

ABSTRACT
The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel
inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently
occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof-of-
concept has so far been elusive. As a first step, we describe molecular modeling to guide focused
chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered
by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T-6PEG-TIBO conjugate was
successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-
stranded DNA.
4

HIV reverse transcriptase (RT) plays a pivotal role in the viral life cycle, whose multiple functions
include RNA-directed and DNA-directed DNA polymerization. HIV-1 RT inhibitors, critical components
of the highly active anti-retroviral therapy (HAART) against AIDS, are designed to target either the active
site or a nearby hydrophobic site of the viral polymerase, but not both. The nucleoside analog RT
inhibitors (NRTIs) are prodrug nucleosides which are transported across cellular host membranes and
phosphorylated to the metabolically active nucleotides. These nucleotides serve as substrates for RT and
lacking a 3 hydroxyl group, they serve to chain-terminate DNA synthesis. This chain termination and
1
their competition with natural deoxynucleoside triphosphates (dNTPs) yield their clinical utility .
Structural evidence shows that the allosteric nonnucleoside RT inhibitors (NNRTIs) bind to an inducible
2
,3
pocket approximately 10 Å away from the polymerase site , and steady-state kinetic studies suggest they
4
-8
are noncompetitive with respect to the binding of dNTP and oligonucleotides . While NRTIs and
NNRTIs are potent inhibitors of reverse transcription, resulting in an array of FDA-approved
formulations, resistance at the level of RT proves to hinder their full utility – this phenomenon calls for
new and improved antiviral inhibitors.
9
In light of viral resistance, RT has been targeted with using other strategies including dual NRTIs ,
1
0
interference of RT and DNA binding , among others. One approach that has previously received some
attention is the concept of a “bifunctional inhibitor”. Pursuit of a “bifunctional inhibitor” for HIV-1
reverse transcriptase (a molecule which simultaneously targets both inhibitory pockets) has been both an
1
1-15
intriguing and challenging endeavor, despite more than a decade of global research efforts
.
The genesis of the idea, in part, was inspired by kinetic observations of HIV-1 RT. Biochemical
1
6-19
studies from our lab and others
suggest that the natural dNTP and the NNRTI can simultaneously
occupy their respective sites. Moreover, these studies also indicated that communication occurs between
the active and the NNRTI binding sites since the inhibition of RT by NNRTIs is manifested through a
remote effect on the chemical step of polymerization. At the molecular level, transient kinetic studies
5

have shown that nonnucleoside inhibitors change the rate-limiting step of incorporation, resulting in
1
6,17
chemistry as the slowest step
. This crosstalk between the two binding sites sets a hypothesis for
designing a single molecular “bifunctional” inhibitor including an NRTI and NNRTI component tethered
together to target both sites simultenaously. This innovative approach has been successful in yielding
2
0,21
inhibitors of other enzyme systems, FKBP and stromelysin
.
The attraction of a bifunctional inhibitor against HIV-1 RT is many fold. First, combining two
inhibitory moieties within one molecule can have synergistic effects that may improve potency. Second,
the onset of resistance may decrease as a result of simultaneous mutations that must be produced at both
binding pockets. Lastly, potential therapeutic agents based on the bifunctional inhibition designed
specifically for HIV-1 RT would presumably not interfere with human polymerases such as the human
mitochondrial DNA polymerase  and thus result in fewer side effects. Unfortunately, in practice, the
1
1,13,14, 22-24
development of an HIV-1 RT bifunctional inhibitor has been extremely challenging
with d4T, AZT, and various NNRTI scaffolds have thus far not been successful to illustrate the
bifunctional proof of concept. Renoud-Grappin et al. showed that a linkage to d4T did not reduce its
– attempts
1
4
antiviral inhibition, but further addition of the NNRTI decreased the antiviral activity 100-fold . This loss
of activity can be attributed to a lack of binding of the NNRTI, as this group of inhibitors showed activity
against HIV-2 RT, for which no NNRTI pocket exists. Other compounds utilizing AZT and ddC as NRTI
1
3
parents illustrated NRTI-linkage is detrimental at both the C5, N3 (of AZT) and C4 positions (of ddC) ,
though the linkers chosen in this study may have influenced activity. Along with others, these two studies
depict the complex structure-activity relationship that exists for putative bifunctional inhibitors, but a
promising finding demonstrated that a linkage at the 5-position of d4T with a methylamino linker did not
1
4
perturb activity of the NRTI in cell culture . This contrasts with data from Gavriliu et al., which argues
2
5
attachment of this methylamino linker indeed disrupts d4T’s antiviral activity in culture . Comparison of
these two studies yields significant differences in the linker terminus, further supporting the complexity in
6

addressing linkage position and linker design for an RT bifunctional inhibitor. Work from our lab
preparing a bifunctional inhibitor prodrug based on phenylethylthiazolylthiourea (PETT) compounds such
as HI-236 as an NNRTI showed antiviral activity in cell culture however structural data with PETT-2
26,27
NNRTIs suggested the choice of position for linkage from the NNRTI was not optimal
.
In order to circumvent issues raised above and to fully understand the potential benefits of the
bifunctional concept, several basic questions were addressed for the rational design. First is the choice of
NRTI and the point of connection. To date, eight NRTIs have been approved for clinical use. The
considerations in choosing one of these are two fold: (1) the chemical transformation to introduce
properly substituted derivatives and (2) the in vivo transformation of the latter to its triphosphate by
human kinases. N4-substitution of ddC (zalcitabine) has afforded active compounds against HIV
replication, but the modifications made at this position substantially reduced potency relative to the parent
1
3
compound ddC . Substitution at both N-3 and C-5 of AZT (Retrovir) has lead to inactive antiviral
1
1
compounds . The substitutions at C-5 in d4T (stavudine, Figure 1) yield mixed results, but mostly result
1
4,25,28-30
in inactive analogs
. Attempted modifications to AZT and d4T suggest the difficulty for human
thymidine kinase (hTK) to phosphorylate 2,3-dideoxythymidine analogs, but there is evidence that C-5
3
1-33
substituted thymidine can be phosphorylated in vivo by thymidine kinase
. Guided by these previous
3
4-
studies, we chose d4T as our nucleoside because of the observed slight resistance in cell culture studies
3
6
,
and at the enzymatic level, it is incorporated as efficiently as the natural substrate (dTTP) with both
3
7
DNA/DNA and DNA/RNA primer-templates .
Likewise, linkage to the NNRTI end should not interfere with the desired interactions between the
inhibitor and the NNRTI pocket, namely the hydrogen bonding and aryl-aryl interactions. This is evident
in the case of HEPT, an early generation NNRTI – substitution at N3 caused the loss of hydrogen bonding
1
1
to the backbone carbonyl of lysine 101, diminishing its affinity for RT , while derivatives of HEPT
3
8
modified at position N1 preserved key interactions and activity . The connection to N3 of TSAO-T also
7

resulted in active compounds, presumably because of preserved hydrogen bonding between the 4-amino
1
1
group and the backbone carbonyl of lysine 101 . At the time that initial compounds were developed for
our study, only a few NNRTI structures were available and thus choosing an NNRTI depended on this
limited structural data. Structures that were available gave insight into using 8-Cl-TIBO as our NNRTI of
3
9,40
choice, since a plausible linkage could be attached
.
Design of a bifunctional HIV-1 RT inhibitor was guided by molecular modeling that benefitted
from the availability of emerging x-ray crystallographic and biochemical data for RT. Based on the
rational design, an iterative and systematic synthetic effort was undertaken to prepare, purify and
characterize several generations of small molecule compounds. These compounds were then tested in
biochemical and cellular assays to develop a bifunctional structure-activity relationship. With d4T and 8-
Cl-TIBO as our initial building blocks to illustrate our “proof of concept,” here we present the first
evidence of DNA incorporation of a rationally designed and fully synthesized bifunctional nucleoside
triphosphate by HIV-1 RT.
9
Thymidine-PEG-TIBO Inhibitors. To begin our design, we chose polyethylene glycol as our
linker (Figure 1), for its low toxicity in biological venues and for its amphiphilic nature, thus giving it
favorable solubility in the cellular environment. We also chose to begin using a thymidine analog as our
NRTI, to avoid potential synthetic pitfalls with other NRTIs. And at the time this study was initiated in
our lab, structural evidence suggested that a bifunctional concept could be achieved with the NNRTI 8-Cl-
3
9,40
TIBO
, with linkage stemming from the 8-position (Figure 1). To aid in the design of the inhibitor,
modeling was performed to not only to verify that linkage could be achieved on position 8 of TIBO, but
to also estimate the number of PEG units needed to bridge the two pockets. 8-Cl-TIBO was modeled into
2
4
the ternary RT structure (solved with natural substrate dTTP) , which suggested that the 8-position of
TIBO was directed towards the dNTP pocket, and that approximately six PEG units could connect the
NRTI to the NNRTI as shown in Figure 2.
8

Though the 5-position is a debatable site for derivatization (as stated earlier), linkage precedence
1
2,14,33,41
guided the attachment of linker to this position
, as thymidine kinase can phosphorylate C-5
substituted pyrimidines and that d4T with long linkers remained active against HIV. We thus set out to
synthesize the thymidine-6PEG-TIBO bifunctional inhibitor. As illustrated in Scheme 1, we chose a
modular strategy for linker attachment to allow a variation in linker length and composition. Shown in
Scheme 1 is the synthesis of the bifunctional molecule, where an ester linkage was introduced into the
linker region for chemical feasibility. Molecules with four, five, or six PEG monomers were synthesized
to develop a structure-activity relationship (SAR) for this group of inhibitors.
d4T-PEG-TIBO Inhibitors. We moved to synthetic efforts employing d4T as our NRTI moiety
(Scheme 2). As mentioned earlier, d4T was chosen as our NRTI because of its similar kinetics of
incorporation relative to natural thymidine and the low-level resistance developed against this NRTI.
Modeling also suggested that we could pursue attachment of PEG to the 5-position of d4T, with the
assumption that d4TTP bound in a similar manner to natural dTTP – a reasonable supposition based on
the incorporation kinetics of d4TTP. An initial evaluation of antiviral activities of these compounds was
carried out by examining their ability to inhibit HIV-1 replication in MT-2 cell culture. Table 1 shows the
antiviral potency (EC ) and general cellular toxicity (IC ) for the bifunctional compounds 16a-c,
5
0
50
containing 4, 5, and 6 PEG linkers. As illustrated in Table 1, these compounds showed some modest
antiviral activity in the mid-micromolar range and the bifunctional molecule linked with four PEG units
(16a) was most inhibitory, though the bifunctional inhibitors altogether fell short in illustrating a proof of
concept. In trying to rationalize the poor activity a number of questions arise: (1) Was the nucleoside
moiety in the bifunctional compounds phosphorylated as would be required for activity at the cellular
level?; (2) was the ester functionality cleaved by cellular proteases to yield a cleaved inhibitor; and (3)
was the linker length not optimal and/or did linker attachment perturb binding? As an initial step, we
addressed the question of phosphorylation by synthesizing the protected monophosphate of d4T-4PEG-
9

4
2
TIBO, with protecting groups utilized in stampidine, a prodrug formulation of d4T . This approach did
not yield an improvement in activity (data not shown). Synthesis of the corresponding triphosphate of
d4T-4PEG-TIBO as a substrate was attempted to address the phosphorylation requirement for in vitro
incorporation assays catalyzed by HIV-1 RT. However, a triphosphate product could not be synthesized
(data not shown).
4
3
Though N3 substituted derivatives of d4T have been shown to be less active than d4T itself , we
attempted to synthesize a bifunctional inhibitor with N3 substitution (Scheme 3). Chemistry at this
position was less problematic than C5 derivatization and a bifunctional inhibitor that displayed “proof of
concept” might overcome the loss of activity that might be observed with N3 linkage. This includes the
caveat that disrupted Watson-Crick base pairing may be a potential obstacle for activity with N3
substitution. The first compound of this series is shown in Figure 3A, where the PEG linker was directly
attached to the N3 position of d4U without use of an ester moiety. This was deliberately planned through
chemical synthesis to avoid ester cleavage in the cell as this may have been one problem associated with
the lack of activity with the d4T-PEG-series. d4U was initially chosen for chemical feasibility and
because the substitution now resides on N3, the linkage was lengthened by 2 PEG units. Estimation for
PEG linker length was provided by modeling studies. In cell culture, this bifunctional compound, 22, did
not illustrate a “proof of concept”, and we observed a minor increase in cell toxicity that could be
generated by potential off-target interactions through the bifunctional inhibitor (Table 1). For nucleoside
4
4
analogs, the first phosphorylation limits the rate of triphosphate formation , and thus we again attempted
to synthesize the prodrug version of N3-d4U-6PEG-TIBO (using stampidine formulation, see above).
Again, this modification did not improve activity (data not shown) – suggesting that subsequent
phosphorylations might also pose an obstacle to antiviral activity. Since our two initial prototypes were
not showing significant activity, we asked if linkage at either the NRTI or NNRTI end might be affecting
activity. The TIBO-6PEG fragment (Figure 3B) was tested in our cell culture assay. As shown in Table 1,
1
0

the addition of linker significantly affects the activity of TIBO, decreasing activity by ~ 2000 fold. The
perturbation upon TIBO’s activity certainly may account for our large hit on bifunctional activity.
The MT-2 cell culture inhibition assay illustrated the pitfalls with our strategy, but it does not
directly address potential interactions of inhibitors with RT. To focus on RT interactions, the triphosphate
of N3-d4U-6PEG-TIBO (Figure 4A) was successfully synthesized and tested for incorporation with RT.
Figure 4B shows a gel comparing the incorporation of the triphosphate of N3-d4U-6PEG-TIBO with that
of natural dTTP. While the reaction using N3-d4U-6PEG-TIBO triphosphate showed a higher migrating
product band, the migration appeared to be similar to that for a *D24-mer, which corresponds to a natural
single deoxynucleotide incorporation band size (Figure 4B). This observation indicates the decomposition
of the N3-d4U-6PEG-TIBO triphosphate into d4UTP serving as a substrate for RT, since the expected
product mobility of the incorporated bifunctional inhibitor band size might be expected to be higher than
the incorporated dTTP band size. We believe that the cleavage of the PEG linker along with the TIBO
from the nucleotide part could occur due to the thermal instability of the bifunctional inhibitor either
during the reaction time at 37C or after the incorporation process when the samples were running on a
denaturing gel. Because the temperature of the gel apparatus goes up to 55C. Addition to this thermal
denaturation, attaching the linker to the N3 position compels the nucleotide part to be in the unfavourable
syn conformation that could impart additional strain on the inhibitor structure, and thus result in a higher
propensity towards decomposition. This is supported by the observation that a very faint higher migrating
band can be observed (Supplementary figure 1).
In the light of these findings, we synthesized a new bifunctional inhibitor (a 4PEG linker attached
to the C5 position of d4T and a different NNRTI drug) in order to compare the incorporation efficiency
with the TIBO bifunctional (Supplementary figure 2). The incorporation reaction of N3-d4U-6PEG-TIBO
triphosphate at the room temperature resulted in a major denaturation band of the bifunctional product due
to its intrinsic chemical and physical properties (Supplementary figure 1). However, the new bifunctional
1
1

inhibitor (d4TTP-4PEG-TMC) was successfully incorporated by RT showing a much higher migration
band corresponding to the size of the inhibitor with trace amounts of degredation product band likely
owing to increased compound stability.
Implications for Future Research. Molecular modeling, as demonstrated in this study, should
become an indispensable tool in the design of bifunctional RT inhibitors, so the probability of problems,
such as wrong connection sites or inadequate linker length, can be minimized, and downstream synthetic
efforts can be more focused. It should be appreciated, however, that the inhibitors designed from
molecular modeling are “rough sketches”, not exact molecular “blueprints” to be taken literally. Rather,
they should serve as the starting point from which structures can be refined based on experimental tests.
For example, though structural and modeling studies suggested that the 8-position of 8-Cl-TIBO would be
a favorable site for linkage, they should not preclude other positions, such as C-9, as feasible, even
preferable, anchoring points. The ultimate support of the dual-binding of the bifunctional inhibitor relies
on the availability of x-ray structural evidence of RT complexed with the inhibitor.
The effect of linker to the binding of the bifunctional inhibitor is underappreciated. More than an
inert, weightless tether, the linker itself can significantly alter the way the bifunctional inhibitors bind to
RT, and derail the intended interaction altogether. The 2000-fold decrease in activity of TIBO-6PEG vs.
TIBO is one such example. On the other hand, the linker can be a source of positive contribution to
binding. Creative use of diverse structures as linker that can bind to RT can add an unexplored dimension
to bifunctional inhibitor design.
Synthetic difficulties remain a significant bottleneck in the preparation of the triphosphates of
bifunctional inhibitor, as we encountered in numerous occurrences in our endeavors. Our successful
synthesis of the triphosphate of N3-d4U-6PEG-TIBO and its small degree of incorporation by RT due to
its thermal instability, however, illustrate a first step toward a successful RT bifunctional strategy.
1
2

Acknowledgement
WT
We thank Stephen Hughes, Paul Boyer, and Andrea Ferris for the HIV-1 RT clone; Marina
Udier-Blagovic, Theresa Lyons and William L. Jorgensen for their help in modeling studies. This work
was supported by NIH GM49551 to K.S.A. and NIH R21GM060913 to K.A.P..
Supplementary data
Details on synthetic procedures, spectral characterization, molecular modeling, and biological
assays are contained in Supporting Information.
1
3

References
1
.
Goody, R. S.; Muller, B.; Restle, T., Factors contributing to the inhibition of HIV reverse
transcriptase by chain-terminating nucleotides in vitro and in vivo. FEBS Lett 1991, 291, (1), 1-5.
2
.
Tantillo, C.; Ding, J.; Jacobo-Molina, A.; Nanni, R. G.; Boyer, P. L.; Hughes, S. H.; Pauwels, R.;
Andries, K.; Janssen, P. A.; Arnold, E., Locations of anti-AIDS drug binding sites and resistance
mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms
of drug inhibition and resistance. J Mol Biol 1994, 243, (3), 369-87.
3
.
Kohlstaedt, L. A.; Wang, J.; Friedman, J. M.; Rice, P. A.; Steitz, T. A., Crystal structure at 3.5 A
resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science 1992, 256, (5065), 1783-
9
4
0.
.
Merluzzi, V. J.; Hargrave, K. D.; Labadia, M.; Grozinger, K.; Skoog, M.; Wu, J. C.; Shih, C. K.;
Eckner, K.; Hattox, S.; Adams, J.; et al., Inhibition of HIV-1 replication by a nonnucleoside reverse
transcriptase inhibitor. Science 1990, 250, (4986), 1411-3.
5
.
Carroll, S. S.; Olsen, D. B.; Bennett, C. D.; Gotlib, L.; Graham, D. J.; Condra, J. H.; Stern, A. M.;
Shafer, J. A.; Kuo, L. C., Inhibition of HIV-1 reverse transcriptase by pyridinone derivatives. Potency,
binding characteristics, and effect of template sequence. J Biol Chem 1993, 268, (1), 276-81.
6
.
Althaus, I. W.; Chou, J. J.; Gonzales, A. J.; Deibel, M. R.; Chou, K. C.; Kezdy, F. J.; Romero, D.
L.; Aristoff, P. A.; Tarpley, W. G.; Reusser, F., Steady-state kinetic studies with the non-nucleoside HIV-
1
7
reverse transcriptase inhibitor U-87201E. J Biol Chem 1993, 268, (9), 6119-24.
.
Frank, K. B.; Noll, G. J.; Connell, E. V.; Sim, I. S., Kinetic interaction of human
immunodeficiency virus type 1 reverse transcriptase with the antiviral tetrahydroimidazo[4,5,1-jk]-[1,4]-
benzodiazepine-2-(1H)-thione compound, R82150. J Biol Chem 1991, 266, (22), 14232-6.
8
.
Debyser, Z.; Pauwels, R.; Andries, K.; Desmyter, J.; Kukla, M.; Janssen, P. A.; De Clercq, E., An
antiviral target on reverse transcriptase of human immunodeficiency virus type 1 revealed by
1
4

tetrahydroimidazo-[4,5,1-jk] [1,4]benzodiazepin-2 (1H)-one and -thione derivatives. Proc Natl Acad Sci
U S A 1991, 88, (4), 1451-5.
9
.
Peghini, P. A.; Zahner, R.; Kuster, H.; Schott, H.; Schwendener, R. A., In vitro anti-human
immunodeficiency virus and anti-hepatitis B virus activities and pharmacokinetic properties of
heterodinucleoside phosphates containing AZT or ddC. Antivir Chem Chemother 1998, 9, (2), 117-26.
1
0.
Skillman, A. G.; Maurer, K. W.; Roe, D. C.; Stauber, M. J.; Eargle, D.; Ewing, T. J.; Muscate, A.;
Davioud-Charvet, E.; Medaglia, M. V.; Fisher, R. J.; Arnold, E.; Gao, H. Q.; Buckheit, R.; Boyer, P. L.;
Hughes, S. H.; Kuntz, I. D.; Kenyon, G. L., A novel mechanism for inhibition of HIV-1 reverse
transcriptase. Bioorg Chem 2002, 30, (6), 443-58.
1
1.
Velazquez, S.; Alvarez, R.; San-Felix, A.; Jimeno, M. L.; De Clercq, E.; Balzarini, J.; Camarasa,
M. J., Synthesis and anti-HIV activity of [AZT]-[TSAO-T] and [AZT]-[HEPT] dimers as potential
multifunctional inhibitors of HIV-1 reverse transcriptase. J Med Chem 1995, 38, (10), 1641-9.
1
2.
Ijichi, K.; Fujiwara, M.; Mori, K.; Morozumi, M.; Machida, H.; Shigeta, S.; Konno, K.; Yokota,
T.; Baba, M., Antiviral activities of nucleotide heterodimers against human immunodeficiency virus type
1
1
in vitro. Antiviral Res 1996, 31, (1-2), 115-20.
3. Pontikis, R.; Dolle, V.; Guillaumel, J.; Dechaux, E.; Note, R.; Nguyen, C. H.; Legraverend, M.;
Bisagni, E.; Aubertin, A. M.; Grierson, D. S.; Monneret, C., Synthesis and evaluation of "AZT-HEPT",
AZT-pyridinone", and "ddC-HEPT" conjugates as inhibitors of HIV reverse transcriptase. J Med Chem
"
2
000, 43, (10), 1927-39.
4. Renoud-Grappin, M.; Fossey, C.; Fontaine, G.; Laduree, D.; Aubertin, A. M.; Kirn, A.,
1
Imidazo[1,5-b]pyridazine-d4T conjugates: synthesis and anti-human immunodeficiency virus evaluation.
Antivir Chem Chemother 1998, 9, (3), 205-23.
1
5.
Nanni, R. E.; J., D.; Jacobo-Molina, A.; Hughes, S. H.; Arnold, E., Review of HIV-1 reverse
transcriptase three-dimensional structure: Implications for drug design. Perspectives in Drug Discovery
and Design 1993, 1, 129-150.
1
5

1
6.
Rittinger, K.; Divita, G.; Goody, R. S., Human immunodeficiency virus reverse transcriptase
substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors.
Proc Natl Acad Sci U S A 1995, 92, (17), 8046-9.
1
7.
reverse transcriptase by nonnucleoside inhibitors. Science 1995, 267, (5200), 988-93.
8. Basavapathruni, A.; Bailey, C. M.; Anderson, K. S., Defining a molecular mechanism of synergy
between nucleoside and nonnucleoside AIDS drugs. J Biol Chem 2004, 279, (8), 6221-6224.
9. Basavapathruni, A.; Vingerhoets, J.; de Bethune, M. P.; Chung, R.; Bailey, C. M.; Kim, J.;
Spence, R. A.; Kati, W. M.; Anderson, K. S.; Johnson, K. A., Mechanism of inhibition of HIV-1
1
1
Anderson, K. S., Modulation of human immunodeficiency virus type 1 synergistic inhibition by reverse
transcriptase mutations. Biochemistry 2006, 45, (23), 7334-40.
2
0.
proteins: SAR by NMR. Science 1996, 274, (5292), 1531-4.
1. Hajduk, P. J.; Meadows, R. P.; Fesik, S. W., Discovering high-affinity ligands for proteins.
Science 1997, 278, (5337), 497,499.
2. Laduree, D.; Sugeac, E.; Fossey, C.; Schmidt, S.; Laumond, G.; Aubertin, A. M., Synthesis of
Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W., Discovering high-affinity ligands for
2
2
certain heterodimers expected as HIV-1 reverse transcriptase inhibitors. Nucleosides, Nucleotides &
Nucleic Acids. 2003, 22, (5-8), 873-875.
2
3.
Laumond, G.; Aubertin, A. M., Synthesis and anti-HIV activity of [d4U]-[Trovirdine analogue] and
d4T]-[Trovirdine analogue] heterodimers as inhibitors of HIV-1 reverse transcriptase. Nucleosides
Nucleotides Nucleic Acids 2002, 21, (8-9), 505-33.
4. Huang, H.; Chopra, R.; Verdine, G. L.; Harrison, S. C., Structure of a covalently trapped catalytic
complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 1998, 282, (5394),
669-75.
Gavriliu, D.; Fossey, C.; Ciurea, A.; Delbederi, Z.; Sugeac, E.; Laduree, D.; Schmidt, S.;
[
2
1
1
6

2
5.
Gavriliu, D.; Fossey, C.; Fontaine, G.; Benzaria, S.; Ciurea, A.; Delbederi, Z.; Lelong, B.;
Laduree, D.; Aubertin, A. M.; Kirn, A., Synthesis and antiviral activity of C-5 substituted analogues of
d4T bearing methylamino- or methyldiamino-linker arms. Nucleosides Nucleotides Nucleic Acids 2000,
1
2
9, 1017-1031.
6. Ren, J.; Diprose, J.; Warren, J.; Esnouf, R. M.; Bird, L. E.; Ikemizu, S.; Slater, M.; Milton, J.;
Balzarini, J.; Stuart, D. I.; Stammers, D. K., Phenylethylthiazolylthiourea (PETT) non-nucleoside
inhibitors of HIV-1 and HIV-2 reverse transcriptases. Structural and biochemical analyses. J Biol Chem
2
000, 275, (8), 5633-9.
7. Younis, Y.; Hunter, R.; Muhanji, C. I.; Hale, I.; Singh, R.; Bailey, C. M.; Sullivan, T. J.;
2
Anderson, K. S., [d4U]-spacer-[HI-236] double-drug inhibitors of HIV-1 reverse-transcriptase. Bioorg
Med Chem 2010, 18, (13), 4661-73.
2
8.
Ciurea, A.; Fossey, C.; Benzaria, S.; Gavriliu, D.; Delbederi, Z.; Lelong, B.; Laduree, D.;
Aubertin, A. M.; Kirn, A., Synthesis of 5-alkenylated D4T analogues via the Pd-catalyzed cross-coupling
reaction. Nucleosides Nucleotides Nucleic Acids 2001, 20, 1655-1670.
2
9.
Delbederi, Z.; Fossey, C.; Fontaine, G.; Benzaria, S.; Gavriliu, D.; Ciurea, A.; Lelong, B.;
Laduree, D.; Aubertin, A. M.; Kirn, A., Synthesis and antiviral activity of C-5 substituted beta-D- and
beta-L-D4T analogues. Nucleosides Nucleotides Nucleic Acids 2000, 19, 1441-1461.
3
0.
Laduree, D.; Fossey, C.; Renoud-Grappin, M.; Fontaine, G.; Camara, F.; Gavriliu, D.; Ciurea, A.;
Aubertin, A. M.; Kirn, A., Synthesis of novel C-5 substituted d4T analogues bearing linker arms as
potential anti-HIV agents. Nucleosides Nucleotides 1999, 18, 883-884.
3
1.
Rong, F.-G.; Soloway, A. H.; Ikeda, S.; Ives, D. H., Synthesis and Biochemical Activity of
Hydrophilic Carborane-containing Pyrimidine Nucleosides as Potential Agents for DNA Incorporation
and BNCT. Nucleosides & Nucleotides 1997, 16, 379-401.
1
7

3
2.
Soloway, A. H.; Zhuo, J.-C.; Rong, F.-G.; Lunato, A. J.; Ives, D. H.; Barth, R. F.; Anisuzzaman,
A. K. M.; Barth, C. D.; Barnum, B. A., Identification, development, synthesis and evaluation of boron-
containing nucleosides for neutron capture therapy. J. Organometallic Chem. 1999, 581, 150-155.
3
3.
Rong, F.-G.; Soloway, A. H.; Ikeda, S.; Ives, D. H., Synthesis and biochemical activity of 5-
tethered carborane-containing pyrimidine nucleosides as potential agents for DNA incorporation.
Nucleosides & Nucleotides 1995, 14, 1873-1887.
3
4.
Am J Med 1997, 102, (5B), 70-5.
5. Lacey, S. F.; Larder, B. A., Novel mutation (V75T) in human immunodeficiency virus type 1
Mayers, D. L., Prevalence and incidence of resistance to zidovudine and other antiretroviral drugs.
3
reverse transcriptase confers resistance to 2',3'-didehydro-2',3'-dideoxythymidine in cell culture.
Antimicrob Agents Chemother 1994, 38, (6), 1428-32.
3
6.
Lin, P. F.; Samanta, H.; Rose, R. E.; Patick, A. K.; Trimble, J.; Bechtold, C. M.; Revie, D. R.;
Khan, N. C.; Federici, M. E.; Li, H.; et al., Genotypic and phenotypic analysis of human
immunodeficiency virus type 1 isolates from patients on prolonged stavudine therapy. J Infect Dis 1994,
1
3
70, (5), 1157-64.
7. Vaccaro, J. A.; Parnell, K. M.; Terezakis, S. A.; Anderson, K. S., Mechanism of inhibition of the
human immunodeficiency virus type 1 reverse transcriptase by d4TTP: an equivalent incorporation
efficiency relative to the natural substrate dTTP. Antimicrob Agents Chemother 2000, 44, (1), 217-21.
3
8.
activity of novel N-1 side chain-modified analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine
HEPT). J Med Chem 1997, 40, (12), 1845-54.
9. Ding, J.; Das, K.; Moereels, H.; Koymans, L.; Andries, K.; Janssen, P. A.; Hughes, S. H.; Arnold,
Pontikis, R.; Benhida, R.; Aubertin, A. M.; Grierson, D. S.; Monneret, C., Synthesis and anti-HIV
(
3
E., Structure of HIV-1 RT/TIBO R 86183 complex reveals similarity in the binding of diverse
nonnucleoside inhibitors. Nat Struct Biol 1995, 2, (5), 407-15.
1
8

4
0.
Das, K.; Ding, J.; Hsiou, Y.; Clark, A. D., Jr.; Moereels, H.; Koymans, L.; Andries, K.; Pauwels,
R.; Janssen, P. A.; Boyer, P. L.; Clark, P.; Smith, R. H., Jr.; Kroeger Smith, M. B.; Michejda, C. J.;
Hughes, S. H.; Arnold, E., Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT
and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant. J Mol Biol 1996, 264,
(
5), 1085-100.
1. Rong, F.-G., Soloway, A.H., Synthesis of 5-tethered carborane-containing pyrimidine nucleosides
as potential agents for DNA incorporation. Nucleosides & Nucleotides 1994, 13, 2021-2034.
2. Uckun, F. M.; Pendergrass, S.; Venkatachalam, T. K.; Qazi, S.; Richman, D., Stampidine is a
4
4
potent inhibitor of Zidovudine- and nucleoside analog reverse transcriptase inhibitor-resistant primary
clinical human immunodeficiency virus type 1 isolates with thymidine analog mutations. Antimicrob
Agents Chemother 2002, 46, (11), 3613-6.
4
3.
Maillard, M.; Florent, J.-C.; Lemaitre, M.; Begassat, F.; Bugnicourt, A.; Ferrieux, C.; Rombi, C.;
Pacaud, E. D.; Thierry, D. E.; Zerial, A.; Monneret, C.; Grierson, D. S., Preparation and anti-HIV activity
of N-3 amino substituted thymidine nucleoside analogs. Bioorg Med Chem Lett 1992, 2, 1469-1474.
4
4.
nucleoside analog active against human immunodeficiency virus. Antimicrob Agents Chemother 1989, 33,
6), 844-9.
Ho, H. T.; Hitchcock, M. J., Cellular pharmacology of 2',3'-dideoxy-2',3'-didehydrothymidine, a
(
1
9

Scheme captions
Scheme 1. Synthesis of Thymidine-PEG-TIBO conjugates.
Scheme 2. Synthesis of d4T-PEG-TIBO bifunctional RT inhibitors.
Scheme 3. Synthesis of N3-d4U-6PEG-TIBO.
Figure legends
Figure 1. Bifunctional Building Blocks.
Structures of d4T (1), 8-Cl-TIBO (2), and PEG (3). The 8-position of 8-Cl-TIBO is indicated with a ( ).
*
Figure 2. Modeled Fit of dTTP-PEG-TIBO.
Using coordinates from the previously published ternary RT structure (RT, primer/template, and dTTP;
2
4
40
pdb 1RTD) and the RT structure solved with 8-Cl TIBO (pdb 1HNV) , the NNRTI pocket from 1HNV
was aligned and superimposed onto 1RTD (see Experimental Section). In this hybrid modeled structure,
the chlorine at position 8 of TIBO is clearly oriented towards the dTTP in the active site. The straight-line
distance between 8-Cl of TIBO and 5-methyl of dTTP is 17.10 Å. Shown in blue and yellow are the p66
and p51 subunits of RT, respectively, and the green and red curved lines represent primer and template,
respectively. The modeled bifunctional inhibitor is shown in ball and stick representation, and the grey
spheres symbolize the active site magnesium ions.
Figure 3. Structures of the synthesized bifunctional inhibitors.
(A) Structure of N3-d4U-6PEG-TIBO. (B) Structure of TIBO-6PEG.
Figure 4. Incorporation of triphosphate of N3-d4U-6PEG-TIBO.
2
0

(A) Structure of triphosphate of N3-d4U-6PEG-TIBO (23). (B) Acrylamide gel showing incorporation of
dTTP (100 M) and triphosphate of N3-d4U-6PEG-TIBO (150 M) over a time course under single
turnover conditions (250 nM RT, 50 nM D23/D36 primer/template). Time points are t = 0, 30 sec, 5 min,
1
0 min, 30 min, and 60 min. Note the misincorporation of a second molecule of dTTP during this time
course.
2
1

Table 1. Activity of Potential Bifunctional Inhibitors in HIV-infected Cell Culture.
Compounds were tested for their in vitro effect on viral replication. EC – antiviral
5
0
potency; IC – cellular toxicity.
5
0
Compound
EC50
IC50
d4T
2.2 M
14 nM
> 100 M
> 100 nM
> 100 M
> 100 M
> 100 M
8
-Cl-TIBO
d4T-4PEG-TIBO (16a)
d4T-5PEG-TIBO (16b)
d4T-6PEG-TIBO (16c)
45 M
100 M
120 M
N3-d4U-6PEG-TIBO (22)
TIBO-6PEG (2c)
20 M
27 M
38 M
> 100 M

Figure(1)

Figure(2)

Figure(3)

Figure(4)

Scheme(1)

Scheme(2)

Scheme(3)