Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase

Article abstract of DOI:10.1186/1743-422X-9-305

Background: Despite the effectiveness of highly active antiretroviral therapy (HAART), there remains an urgent need to develop new human immunodeficiency virus type 1 (HIV-1) inhibitors with better pharmacokinetic properties that are well tolerated, and t

Full text of DOI:10.1186/1743-422X-9-305

Elleder et al. Virology Journal 2012, 9:305
http://www.virologyj.com/content/9/1/305
RESEARCH
Open Access
Identification of a 3-aminoimidazo[1,2-a]pyridine
inhibitor of HIV-1 reverse transcriptase
Daniel Elleder1,4, Thomas J Baiga2,5, Rebecca L Russell2, John A Naughton1, Stephen H Hughes3,
Joseph P Noel2 and John AT Young1*
Abstract
Background: Despite the effectiveness of highly active antiretroviral therapy (HAART), there remains an urgent
need to develop new human immunodeficiency virus type 1 (HIV-1) inhibitors with better pharmacokinetic
properties that are well tolerated, and that block common drug resistant virus strains.
Methods: Here we screened an in-house small molecule library for novel inhibitors of HIV-1 replication.
Results: An active compound containing a 3-aminoimidazo[1,2-a]pyridine scaffold was identified and quantitatively
characterized as a non-nucleoside reverse transcriptase inhibitor (NNRTI).
Conclusions: The potency of this compound coupled with its inexpensive chemical synthesis and tractability for
downstream SAR analysis make this inhibitor a suitable lead candidate for further development as an antiviral drug.
Keywords: HIV-1, NNRTI, Inhibitor
Background
Here we screened a chemical library to identify novel
Despite more than 25 years of research, drug treatment inhibitors of HIV-1 replication. We report the identifica-
of HIV infection remains a major therapeutic challenge. tion of a lead compound containing a 3-aminoimidazo
The most effective regimen for treating HIV-1 infection [1,2-a]pyridine scaffold that acts as an NNRTI, with in-
is highly active antiretroviral therapy (HAART) that usu- herent features that render it attractive for further devel-
ally consists of a non-nucleoside reverse transcriptase in- opment as an antiviral drug.
hibitor (NNRTI) or protease inhibitor together with two
nucleoside (or nucleotide) reverse transcriptase inhibi-
tors (NRTIs).
Results
The Salk Institute in-house small molecule collection is a
chemically diverse set of synthetic compounds based on
divergent design principles. Fundamental to this com-
pound collection is a Diversity-Oriented Synthesis (DOS)
approach [2] that is pharmacophore or scaffold-centric,
with emphasis on the use of multi-component synthetic
reactions (MCRs) [3] and a variety of post-MCR transfor-
mations or reaction cascades [4]. The diversity criteria are
biased by Lipinski’s ‘Rule of 5’ [5], ADME-Tox filtering,
exclusion of reactive substituents [6] and incorporation of
masked functional groups within scaffold side chains to
enable additional chemical manipulations via forward or
reverse chemical genetic screening methodologies [7]. The
synthetic economy realized by such a library design per-
mits the inclusion of rare and exotic building blocks,
which further enhances the chemical diversity of the li-
brary and mitigates the limitations of library size, scope,
Over 50 NNRTIs have been described to date [1]. Five
of these NNRTIs have been approved by the United States
FDA for the clinical treatment of HIV infection and AIDS.
The first generation NNRTIs used are efavirenz, nevira-
pine and delavirdine. However, the rapid emergence of
virus drug-resistant virus limited the effectiveness of these
drugs. The second-generation NNRTIs, etravirine and ril-
pivirine, are more active against both drug-sensitive and
resistant virus strains [1]. However, despite this progress
there is a continued need for the development of novel
NNRTIs which have better pharmacokinetic properties
and inhibit common drug-resistant virus strains [1].
* Correspondence: jyoung@salk.edu
1The Salk Institute for Biological Studies, Nomis Center for Immunobiology
and Microbial Pathogenesis, 10010 North Torrey Pines Road, La Jolla, CA
92037, USA
Full list of author information is available at the end of the article
© 2012 Elleder et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Elleder et al. Virology Journal 2012, 9:305
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and scale. An additional attribute of the library design, due and purified at a larger scale for the subsequent experi-
in large part to the economics of syntheses, is the ability ments described in this report. Luciferase-based infectivity
to rapidly determine structure-to-activity relationships assays in the presence of increasing concentrations of
(SAR) early in the evaluation of lead compounds. Each F2 yielded dose response curve with a mean EC50 stand-
pharmacophore-based library subset can be readily ard deviation of 0.387
0.046 μM (n = 6) (Figure 1B).
expanded to further explore the relevant target space, en- Cytotoxicity for F2 was determined 24 hrs after the com-
abling both lead optimization and lead evolution (or scaf- pound was added to mock infected cells; the CC50 value
fold hopping) to proceed in parallel.
was 34.1 2.4 μM (n = 3), selectivity index = 88.1.
Four hundred and eighty compounds, constituting a
Because the initial screen was performed by scoring
cross-section of the Salk library, were screened in a firefly luciferase reporter gene expression from a VSVg
plate-based assay to identify small molecules that inhibit pseudotyped virus vector, it was possible that the F2
a single cycle of replication by a VSVg-pseudotyped compound inhibited either an early step of HIV-1 repli-
HIV-1 vector (pNL4-3LucR+E-) encoding firefly lucifer- cation, VSVg-mediated cellular entry, or firefly luciferase
ase [8]. This vector is competent for only the early steps reporter activity. To exclude possible effects on VSVg-
of retroviral replication leading up to viral DNA integra- specific cellular entry and firefly luciferase activity, F2
tion and gene expression. Human 293T cells were pre- was tested for its ability to block infection of CEM-GFP
treated for 1 hr with 1 μM final concentration of the lymphocytic indicator cells by a replication-competent
individual compounds and then challenged with the HIV-1 vector with a wild-type CXCR4-tropic HIV-1 enve-
VSVg pseudotyped HIV-1 vector in the continued pres- lope glycoprotein [10]. In this assay, infection by the wild-
ence of that compound. After 24 hrs, the luciferase ac- type virus leads to the expression of a GFP reporter gene in
tivity of each sample was determined.
a Tat-deficient HIV-1 provirus that is resident in the CEM-
A single compound, F2 (mol. wt. 325.83 g/mol), was GFP cell line. The measured EC50 in these experiments
identified that decreased luciferase activity by more than (0.862 0.088 μM; n = 2) (Figure 1C), was similar to the
50% under these conditions. F2 originated from a sub- value obtained with the VSVg-pseudotyped virus. The CC50
library representing the 3-aminoimidazo[1,2-a]pyridine value obtained with the CEM-GFP cells was 25.4 2.1 μM
scaffold (Figure 1A) derived from the 3-component (n = 2), selectivity index = 29.5. Similarly, F2 inhibited in-
Groebke condensation reaction [9]. F2 was re-synthesized fection of primary human peripheral blood mononuclear
Figure 1 The F2 compound blocks an early step of HIV-1 replication. A. Structure of compound F2. B. Dose–response curve of F2 in 293T
cells challenged with the VSVg pseudotyped pNL4-3lucR+E- vector. The effect of the compound on infection was determined by measurement
of virus-encoded firefly luciferase activity. The experiment shown, performed with triplicate samples, is representative of six independent
experiments. C. Dose–response curve of F2 in CEM-GFP cells challenged with the pLai3Luc2 HIV-1 vector [10].The numbers of GFP positive cells
at 2 days post-infection were determined by flow cytometry and the experiment shown, performed with duplicate samples, is representative of
two independent experiments. D. Dose–response curve of F2 in human PBMCs challenged with the NL4-3 Nef+ IRES rluc vector encoding renilla
luciferase activity, measured at 5 days post-infection. The experiment shown was performed with eight replicate samples. The error bars (panels
B-D) represent the standard errors of the mean.

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cells (PBMCs) by a replication-competent HIV-1 vector an in vitro assay was used to directly test the effect of F2
(NL4-3 Nef + IRES rluc) with a measured EC50 of 0.865 on recombinant purified HIV-1 reverse transcriptase (RT)
0.222 μM (Figure 1D) in the absence of cell toxicity, up to activity. F2 potently inhibited HIV-1 RT activity in vitro in
10μM of compound tested (data not shown, selectivity a dose-dependent manner with an IC50 = 2.554 0.365
index > 11.6). Taken together, these results suggested that μM (n = 2) (Figure 2B). Although the 50% inhibitory con-
F2 blocks an early step of HIV-1 replication.
centration of the F2 compound was higher in the in vitro
A quantitative real-time PCR-amplification approach was experiment with purified HIV-1 RT than in the cellular in-
used to determine whether F2 treatment blocks viral DNA fectivity assays, this type of result is seen frequently with
synthesis. Total DNA was isolated from cells 24 hrs post NNRTI inhibitors [12].
infection and quantified using primers and probes specific
The two main classes of existing HIV-1 RT inhibitors
for early and late HIV-1 reverse transcription products (the NRTIs and NNRTIs) act synergistically, especially
[11]. F2 (5 μM) added 1 hr before infection blocked the when used at high inhibitory concentrations [13,14]. To
synthesis of both early and late viral DNA products determine whether there is a similar synergy seen with F2,
(Figure 2A), suggesting that this compound might inhibit this compound was tested along with the NRTI 3’-azido-
HIV-1 reverse transcriptase. To directly test that possibility, 3’-deoxythymidine (AZT) and with the NNRTI nevirapine
(NVP) in combinations at several fixed molar ratios and
over a range of serial dilutions [13]. The resulting isobolo-
gram plots demonstrated that F2 exhibits synergy with
AZT and additivity with NVP in a cellular infectivity
assays, suggesting that F2 blocks HIV-1 reverse transcript-
ase activity by acting as a NNRTI (Figure 3A).
Since it is desirable for a candidate antiretroviral to be
effective against HIV-1 variants resistant to current
NNRTIs, the resistance profile of F2 was tested against
four commonly encountered NVP-resistant HIV-1 mu-
tant variants (K103N, V106A, Y188L, Y181C) [1]. The
mutant vectors used were based on the HIV-1 NL4-3
strain pseudotyped with VSVg and infection of 293T
cells was monitored using a standard single cycle
luciferase-based cellular infectivity assay. F2 inhibited in-
fection by the mutant viral vectors, albeit at reduced
levels compared to the wild-type virus (EC50 values were
reduced between 8- to more than 120-fold; Table 1).
The resistance profile of F2 was similar to NVP (Table 1),
reinforcing the idea that this compound acts as a
NNRTI.
Discussion
In this report we describe a new lead candidate NNRTI
inhibitor that has activity against wild-type and some of
the common drug-resistant variants of HIV-1 reverse
transcriptase. Serendipitously, this new lead scaffold is
structurally isosteric with THR-50, a benzimidazole-
Figure 2 The F2 compound inhibits HIV-1 reverse transcriptase.
based NNRTI [15] (Figure 3B). The imidazo[1,2-a]pyri-
dine pharmacophore of F2 can be realized by three
instances of bioistosteric replacement on the benzimida-
zole scaffold; (1) N1 nitrogen is converted to carbon, (2)
bridgehead carbon adjacent to C7 is converted to nitro-
gen, and (3) benzylic methylene (CH2) is converted to
NH. These subtle changes in the inhibitor core [15]
slightly enhance the physical properties, create an add-
itional H-bond donor interaction, and greatly simplify
the synthesis of analogs for optimization studies.
A. Effects of F2 (5 μM) treatment on the synthesis of early and late
viral DNA in human 293T cells challenged with the VSVg-
pseudotyped HIV-1 vector, measured at 24 hours post-infection. AZT
(5 μM) was used as a reference compound. The values represent
amounts of DNA relative to control, untreated cell populations, with
error bars showing standard deviations from three independent real-
time quantitative PCR assays. B. Effect of compound F2 on HIV-1 RT
activity in vitro, determined by measuring the [alpha32P]-dTTP
incorporation. The experiment shown, performed with duplicate
samples, is representative of two independent experiments. with
error bars representing standard errors of the mean.

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Figure 3 The F2 compound is a NNRTI. A. Isobologram analysis of the effect of combining F2 with either AZT or NVP in human 293T cells
challenged with the VSVg-pseudotyped HIV-1 vector. Depicted are isobologram plots for the 90% inhibitory level (EC90). The dashed line indicates
the values expected for an additive effect. Values below and to the left of the line indicate synergistic effects. FIC, fractional inhibitory
concentration. B. Structure and synthesis of compound F2 compared to THR-50 [15]. Reagents: (a) 0.1 M in 1,2-dichloroethane – 2,2,2-
trifluoroethanol (1:1), 5.0 mol% Sc(OTf)3, rt for 96h or microwaved at 140°C for 5min, >90% yield; (b) 2,6-F2BzCl, THF/pyridine (5:1), rt for 2h, 55%
yield; (c) Fe, AcOH, reflux, 97% yield; (d) 2,6-F2BnBr, NaH, THF, rt overnight, 75% yield.
While the THR-50 compound is the more potent inhibi- resulting in an overall yield of 40% and requiring purifica-
tor against wild-type and drug-resistant variants of HIV-1 tion operations after each reaction step. Furthermore, the
reverse transcriptase, the F2 compound has several distinct number of possible analogs to F2, based on commercially
advantages for downstream drug development. Specifically, available building blocks, is on the order of 106 compounds,
F2 features an imidazo[1,2-a]pyridine core scaffold that is versus 103 for THR-50. These two molecular structures are
generated in a simple, inexpensive, single chemical trans- further distinguished by the addition of an H-donating
formation that belongs to a special class of diversity gener- group (NH) for additional binding interactions as well as
ating chemistries known as Multi-Component Reactions lowering clog P and improving the pharmacological prop-
(MCR), leading to high product yield and purity. By con- erties. Therefore, the lead F2 compound is an excellent
trast, the benzimidazole core scaffold of the NNRTI THR- candidate for further development as an antiviral drug
50 results from a three-step chemical process (Figure 3B) through small molecule SAR and structure-guided design.
Table 1 Resistance profile against wild type and RT mutant VSV-G pseudotyped HIV-1 vectors
Inhibitor
Antiviral EC50 (μM)a, b
CC50 (μM)c
34.1 2.4
> 50
wt
Y188L
> 20
Y181C
V106A
1.275 0.357
[7.7]
K103N
3.233 0.127
[19.6]
F2
0.165 0.039
1.676 0.271
[10.2]
[>121.1]
> 20
NVP
0.019 0.002
0.014 0.003
2.879 0.006
[151.5]
1.260 0.159
[66.3]
0.841 0.049
[44.2]
[>1052.6]
0.011 0.005
[0.8]
AZT
0.006 0.0
[0.4]
0.015 0.001
[1.1]
0.014 0.002
[1.0]
> 50
a Values are averages standard deviations from two independent experiments performed in duplicate in 293T cells.
b The viruses were produced by cotransfection of the pNLNgoMIV R+E-.luc wild type or RT mutant plasmids and VSVg expressing plasmid. The values in brackets
indicate the fold change in EC50 for mutant relative to wild type.

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Methods
Chemistry
NL4-3 Nef+ IRES rluc vector encoding renilla luciferase
was derived from NL4-3 Nef+ IRES eGFP vector [16] by
The Salk Small Molecule Screening Collection was replacement of the eGFP open reading frame with renilla
designed and synthesized in-house using known meth- luciferase (kindly provided by the Chanda laboratory,
ods. The imidazo[1,2-a]pyridines were prepared in a 96 Sanford-Burnham Medical Institute, La Jolla). The set of
well microplate (Eppendorf, 2.2 ml deepwell, polypropyl- vectors harboring the RT mutations and corresponding
ene) as a combinatorial matrix where 2-amino pyridine wild type form (pNLNgoMIV R+E-.luc) were based on
were dispensed to all wells, aromatic aldehydes were dis- the HIV-1 NL4-3 strain; the RT region of these vectors
pensed as row reagents, isonitriles were dispensed as was derived from the BH10 isolate [17]. The vectors
column reagents, and lewis acid catalyst scadium (III) were pseudotyped with VSVg by cotransfection with
triflate was dispensed to all wells. The amine and isoni- pMD.G plasmid in 293T cells. To generate the cells that
triles were prepared as 0.5 M stock solutions in 1,2 express luciferase from established HIV proviral DNA,
dichloroethane (DCE), and the aldehydes and lewis acid 293T cells were infected with the VSVG pseudotyped
were prepared as 0.5 M and 0.025 M (respectively) stock HIV-1 pNL4-3LucR+E- vector and passaged for two
solutions in 2,2,2-trifluorothanol (TFE). For a given well: weeks to remove any remaining unintegrated viral DNA.
200 μl of 0.5M 2-amino pyridine in DCE, 200 μl of 0.5
M aromatic aldehyde in TFE and 200 μl of 0.025 M Tissue culture-based infectivity assays
scandium (III) triflate in TFE were added and mixed on Ten thousand human 293T cells were plated in 80 μl
a plate shaker for 15 minutes at room temperature. To medium in each well of 96-well tissue culture plate. Next
this solution was added 200 μl of 0.5 M isonitrile in TFE day, 10 μl of each diluted compound was added to reach
and 200 μl of (1:1 v/v) DCE-TFE solvent. Final reaction the desired concentration and incubated at 37°C for 1
volume was 1.0 ml and theoretical concentration was 0.1 hour. A 10 μl aliquot of medium containing the VSVG
M. The plate was sealed (heat seal, foil) and allowed to pseudotyped HIV-1 vector (multiplicity of infection of 0.1-
shake for 5 days at room temperature. After 5 days, the 0.5) was then added to each well. Twenty-four hours after
crude reaction mixture was evaporated to dryness and viral challenge, the medium was carefully removed and
resuspended in 1.0 ml dichloromethane (DCM). This 60 μl of the Bright-Glo reagent (Promega, Madison, WI)
crude reaction mixture was transferred to a filter plate diluted 1:1 in PBS was added to lyse the cells and provide
charged with a cocktail of scavenging resins (PS-Trisa- the luciferin substrate for virus-encoded firefly luciferase.
mine, PS-NCO, PS-TsNHNH2) and silica gel and After several minutes the luminescence associated with
allowed to gravity filter after 2 hr. Each well was rinsed each sample was measured, and served as readout to quan-
with 2.0ml DCM, the fractions combined and were con- tify virus infectivity in each well. The best fitted curves and
centrated to dryness. Resultant purity-enriched com- EC50 values were calculated using Prism 4 software
pound was resuspended in 1.0 ml DMSO (100%) for a (GraphPad Software, San Diego). The degree of synergism
theoretical stock solution of 100 mM. From the 100 mM between screen compounds and AZT (Sigma, St. Louis,
master (or mother) plate, 10 mM compound in DMSO MO) were determined by testing the compounds in the in-
daughter plates were generated and distributed for fectivity assay individually and in combinations at a fixed
assays. Analytical quality control was performed via molar ratio over a range of serial dilutions [13]. The data
high-throughput LCMS on an Agilent 1100 HPLC-LC/ were then analyzed by the isobologram technique, which
MSD Trap XCT MS employing ballistic gradients on evaluates the compound interactions by a dose-oriented
Synergi Fusion RP C18 (Phenomenex) columns and geometric method [13,14]. Cytotoxicity of the compounds
acetronitrile-water solvent system. Hits were resynthe- was measured 24 hours after treatment of mock-infected
sized on a 1.0 mmol scale, purified by preparative liquid cells by adding an equal volume of CellTiter-Glo (Promega,
chromatography and characterized by NMR and LCMS.
Madison, WI) and reading luminescence.
In the assays employing the replication-competent
pLai3Luc2 HIV-1 vector, CEM-GFP lymphocytic indica-
DNA constructs and virus production
The VSVg-pseudotyped HIV-1 vector was generated by tor cells [18] were pretreated with F2 for 1 hr in 24-well
transient transfection of human 293T cells (American plates, challenged with the virus by spinoculation at
Type Culture Collection No. CRL-11268), with plasmid 1,200 × g for 1 hr and the number of GFP positive cells
pNL4-3LucR+E- [8] and pMD.G plasmid that expresses was determined by flow cytometry two days later using
the VSVg glycoprotein. The titer was determined by FACScan (Becton-Dickinson, Franklin Lakes, NJ).
antibody staining for Gag (p24) expressing cells follow-
Human PBMCs from an uninfected individual were
ing infection. The LAI-based replication-competent obtained from the UCSD Center for AIDS Research.
HIV-1 vector was generated by transient transfection of Samples were collected with written informed consent
human 293T cells with plasmid pLai3Luc2 [10]. The under Salk Institutional Review Board Protocol # 10–

Elleder et al. Virology Journal 2012, 9:305
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004. PBMCs were activated in PBMC Growth Medium Sunnyvale, CA). The screens were scanned by a FLA-5100
(RPMI 1640 with 15% FBS, Pen/Strep, 25 mM HEPES, instrument (Fujifilm Life Science, Stamford, CT) and the
100 U/ml IL-2) supplemented with 5 μg/ml PHA-P. amount of incorporated labeled phosphate was used to
Two days after activation, the PBMC’s were maintained quantify the RT activity. The best fitted curves and IC50
in the same medium (without PHA-P) for 3 more days. values were calculated using Prism 4 software (GraphPad
Aliquots of 2x104 PBMCs were then plated in 80 μl Software, San Diego).
PBMC Growth Medium in each well of white 96 well
plates (CoStar) and returned to the incubator overnight.
Competing interests
The authors declare that they have no competing interests.
Serial 3-fold dilutions of compound F2, ranging from
100 μM to 1.37 μM in concentration, were made up in
10% DMSO. Eight replicate samples were then set up,
Authors’ contributions
DE, TB, RR, and JAN performed the experiments. DE, TB, SH, JPN, and JATY
conceived the study and designed the experiments. DE, TB, SH, and JATY
with 10 μl aliquots of the serially-diluted compounds
added to the cells, and preincubated at 37°C for one
hour prior to virus challenge. The cells were then chal-
lenged with 10 μl per well of the NL4-3 Nef+ IRES rluc
virus vector bringing the total volume in each well
to 100 μl final. The plates were incubated at 37°C for
five days and then viral infection was monitored by add-
ing 100 μl per well of Renilla-Glo Luciferase Reagent
(Promega) and measuring the resultant luciferase activ-
ities using a Topcount-HTS (PerkinElmer). Cell toxicity
was assessed on a duplicate plate of samples by adding
100 μl Cell-Titer Glo Reagent (Promega) and reading on
a Topcount-HTS.
analyzed the data and drafted the manuscript. All authors read and
approved the final manuscript.
Acknowledgements
This work was supported by the Nomis Foundation, the James B. Pendleton
Charitable Trust (JATY) and NIH Grant P30 CA014195-38 (JATY and JPN). JPN
is an investigator with the Howard Hughes Medical Institute. This research
was supported [in part] by the Intramural Research Program of NIH, National
Cancer Institute (SH). We thank members of the Noel and Young labs for
helpful discussions. We thank Justin Ramsey for assistance in preparing the
Salk in-house library and Kevin Olivieri, Lars Pache, and Sumit Chanda
(Sanford-Burnham Medical Research Institute) for providing the NL4-3 Nef+
IRES rluc virus construct.We thank Dr. David Smith and the UCSD Center for
AIDS Research for providing PBMC samples.
Author details
1The Salk Institute for Biological Studies, Nomis Center for Immunobiology
and Microbial Pathogenesis, 10010 North Torrey Pines Road, La Jolla, CA
92037, USA. 2The Salk Institute for Biological Studies, Howard Hughes
Medical Institute, Jack H. Skirball Center for Chemical Biology and
Proteomics, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. 3HIV
Drug Resistance Program, National Cancer Institute-Frederick, Frederick, MD
21702, USA. 4Current Address: Institute of Molecular Genetics, Academy of
Sciences of the Czech Republic, Videnska 1083, Prague, Czech Republic.
5EMD Millipore Corporation, Bioscience Business Unit, 10394 Pacific Center
Court, San Diego, CA 92121, USA.
Real-time PCR quantitation of viral DNA
Total cellular DNA was harvested 24 hours post infection
with the AccuPrep genomic DNA extraction kit (Bioneer
Life Science Corp., Rockville, MD). The amount of viral
DNA products was quantified by real-time PCR on ABI
Prism 7900 Sequence Detection System (Applied Biosys-
tems, Foster City, CA) with the following primers and
probes: early HIV-1 reverse transcripts with primers ert2f,
ert2r, probe ERT2, late HIV-1 reverse transcripts with pri-
mers MH531, MH532, probe LRT-P [11]. To normalize
for the number of cellular DNA equivalents in the sam-
ples, a single-copy locus in the PBGD gene was amplified
with primers PBGD1 (5’-AAGGGATTCACTCAGGC
TCTTTC), PBGD2 (5’-GGCATGTTCAAGCTCCTTGG)
and probe PBGD-P (5’-VIC-CCGGCAGATTGGAGAGA
AAAGCCTGT-MGBNFQ).
Received: 11 July 2012 Accepted: 28 November 2012
Published: 11 December 2012
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doi:10.1186/1743-422X-9-305
Cite this article as: Elleder et al.: Identification of a 3-aminoimidazo[1,2-
a]pyridine inhibitor of HIV-1 reverse transcriptase. Virology Journal 2012
9:305.
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