Identification and optimization of thienopyridine carboxamides as inhibitors of HIV regulatory complexes

Article abstract of DOI:10.1128/AAC.02366-16

Viral regulatory complexes perform critical functions during virus replication and are important targets for therapeutic intervention. In HIV, the Tat and Rev proteins form complexes with multiple viral and cellular factors to direct transcription and exp

Full text of DOI:10.1128/AAC.02366-16

AAC Accepted Manuscript Posted Online 17 April 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.02366-16
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
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Identification and optimization of thienopyridine carboxamides as
inhibitors of HIV regulatory complexes
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Robert L. Nakamura,a,d,† Mark A. Burlingame,c,† Shumin Yang,b,d David C. Crosby,d
11 Dale J. Talbot,a,d Kitty Chui,a Alan D. Frankel,d and Adam R. Rensloc*
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aAdvanced Genetic Systems, San Francisco, CA
bSchool of Medicine, Tsinghua University, Beijing, 100084, China
15 cSmall Molecule Discovery Center and Department of Pharmaceutical Chemistry,
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dDepartment of Biochemistry and Biophysics, University of California, San Francisco,
94158, USA
†contributed equally
*Corresponding Author: adam.renslo@ucsf.edu
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ABSTRACT
Viral regulatory complexes perform critical functions during virus replication and
are important targets for therapeutic intervention. In HIV, the Tat and Rev proteins form
complexes with multiple viral and cellular factors to direct transcription and export of the
viral RNA. These complexes are composed of many proteins and are dynamic, making
them difficult to fully recapitulate in vitro. Therefore we developed a cell-based reporter
assay to monitor the assembly of viral complexes for inhibitor screening. We screened a
small molecule library and identified multiple hits that inhibit the activity of the viral
33 complexes. A subsequent chemistry effort was focused on a thieno[2,3-b]pyridine
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scaffold, examples of which inhibited HIV replication and the emergence from viral
latency. Notable aspects of the SAR effort include migration to the regioisomeric
36 thieno[2,3-c]pyridine ring system and the identification of analogs with single-digit
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nanomolar activity in both reporter and HIV infectivity assays, an improvement of >100-
fold in potency over the original hits. These results validate the screening strategy
employed and reveal a promising lead series for the development of a new class of HIV
therapeutics.
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INTRODUCTION
RNA-protein complexes are essential to the assembly and activity of many viral
regulatory systems and represent an important target class for antiviral drug discovery. In
45 HIV, the Rev-RRE protein-RNA complex is one such target due to its essential activity in
46 mediating the export of unspliced and partially spliced RNAs from the nucleus to the
47 cytoplasm (1, 2). Disrupting the Rev-RRE interaction prevents the expression of late
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viral proteins and the packaging of viral RNA, thus inhibiting virus replication.
Rev is a 116-amino acid RNA-binding protein that is expressed from fully spliced
mRNAs early in the virus life cycle (3). Rev binds the Rev Response Element (RRE), a
highly structured ~350nt RNA element encoded within the env gene (reviewed (2, 4)).
Current models suggest that about six Rev molecules bind the RNA in order to properly
position two of the nuclear export sequences on Rev for binding to a dimer of the Crm1-
RanGTP export complex (5, 6). This complex is then exported through the nuclear pore,
after which it disassembles in the cytoplasm to allow translation of the late HIV proteins
and packaging of the viral genome (2, 7).
The formation of the export complex is driven by several critical intermolecular
interactions. Rev binds the RNA primarily through its arginine rich motif (ARM), an
alpha-helical domain that forms several important hydrogen bonds with the RNA (8, 9).
The NMR structures of the Rev peptide complexed to RRE IIB or to an RNA aptamer,
and a recent co-crystal structure of a Rev-RRE dimer (10), show that the α-helix binds in
the widened major groove of the high-affinity IIB site, primarily using arginine and
asparagine side chains for base-specific recognition (11-13). The co-crystal structure also
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shows that a glutamine at position 51 interacts symmetrically across the dimer interface
to help stabilize the complex (10). Flanking the ARM of Rev are hydrophobic
oligomerization domains that drive the formation of a stable Rev dimer, and Rev dimers
in turn form higher order structures through hydrophobic multimerization domains that
are located opposite of the oligomerization domain, ultimately assembling a very high
affinity (picomolar) complex (14-16). Given the essential nature of this assembly and the
requirement for high-affinity binding, small molecules that interfere with any of these
interfaces are expected to block formation of competent export complexes and be of
potential therapeutic benefit.
To identify small molecule inhibitors of the Rev-RRE complex, we developed a
high-throughput assay based upon the established Tat-hybrid assay platform for
characterizing RNA-protein or RNA-peptide interactions in mammalian cells (17, 18).
76 This system takes advantage of the function of the HIV transcription activator Tat, which
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binds to the TAR RNA hairpin located at the 5' end of the viral transcripts (19, 20) and
enhances the processivity of RNA polymerase II transcription complexes initiated at the
HIV LTR (21-23). The Tat activation domain can be used to activate reporter expression
via heterologous RNA-protein interactions (24, 25) (Figure 1A). Reporter activity is
proportional to RNA-binding affinity and thus the assay has been used extensively to
characterize RNA-protein interactions, including HIV Tat-TAR, bovine
immunodeficiency virus (BIV) Tat-TAR, HIV Rev-RRE, and to screen for novel proteins
that bind the Mason Pfizer Monkey Virus (MPMV) CTE (17, 26-32).
We engineered the Tat-hybrid platform with the Rev-RRE interaction and used it
to screen a targeted small molecule compound library. We hypothesized that compounds
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containing a carboxamide moiety might mimic the essential asparagine or glutamine side
chains and compete for Rev-Rev dimerization or Rev binding to the RRE RNA and thus
constructed a library of ~4500 carboxamide-containing small molecules selected from a
larger diversity library. Herein we describe the discovery and structure-activity studies of
a thienopyridine inhibitor scaffold in which an unsubstituted carboxamide function was
92 found to be essential for activity. Optimized thienopyridine analogs exhibited low
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nanomolar potencies in multiple reporter-based assays as well as in HIV replication
assays. Interestingly, related thienopyridine analogs were identified independently as
Rev inhibitors using an orthogonal screening approach (33).
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MATERIALS AND METHODS
Small molecule screen. RNA reporter plasmids were constructed using the pHIV LTR
99 vector (18). To express HIV Tat fusion proteins, we constructed a mammalian codon-
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optimized pcDNA-Tat vector containing the first 48 amino acids of HIV Tat with a
fusion to HIV Rev (residues 3-70). HIV tat-fusion proteins have been previously
described (17, 34). A HeLa cell line expressing the pcDNA3 HIV-1 LTR-RREIIB-FFL
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103 reporter was generated to obtain a consistent background for library screens. The
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plasmid was transfected into HeLa cells and stable integrants were selected using
neomycin (G418) (800 µg/ml) for 10 days. Resistant cells were transfected with the
106 pcDNA3 Tat-Rev3-70 plasmid harboring a hygromycin resistance marker. Individual
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clonal cell lines were assayed for luciferase signal and 3,6 diaminoacridine, a nonspecific
small molecule inhibitor of the reporter, was used to identify cell lines with high signal-
109 to-noise (35). A Z’ value of 0.75 was obtained for the cell line used for the small
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molecule screen (Figure S1).
A library of approximately 4500 carboxamide-containing compounds was
identified and purchased from ChemDiv. Approximately 5000 cells were plated in a
white 384-well plate and compounds were added to the cells to a final concentration of
30 uM using a Biomek FX robotic liquid handler (Beckman Coulter). The cells were
incubated with test compounds for 48 hours and assayed for luciferase activity (Promega
116 Bright Glo). The cytotoxicity of each compound was determined in parallel via 3-(4,5-
117 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assays
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(36). Additional compounds structurally related to screening hits were purchased from
Hit2Lead/Chembridge, Asinex, and Specs.
The test compounds that displayed activity were re-confirmed in duplicate or
triplicate at multiple concentrations using the Tat-hybrid reporter cell lines or by transient
transfection. Previously characterized thienopyridines and 3,6 diaminoacridine (35) were
used as controls. Cells were counted and plated into white 384-well or 96-well cell-
culture treated assay plates and compounds were added. After 48 hours, the cells were
prepared for luminescence assays that were performed on a plate reader (Tecan Evolution
or MD Analyst). The cytotoxicity of each compound was determined in parallel via
MTT-based cell viability assays.
Rev reporter assay. 293T cells were plated in 96-well plates and co-transfected with an
HIV RRE (Rev-dependent) reporter plasmid (37) and a pSV2 Rev expression plasmid.
Compounds were incubated with the cells for 48 hours and reporter activity was assessed
by p24 ELISA. Leptomycin B, an inhibitor of the Crm1 pathway that inhibits Rev-
dependent export, was included as a positive control. The cytotoxicity of each compound
was determined in parallel via MTT-based cell viability assays.
U1 activation assay. U1 cells obtained from the AIDS Research and Reference Reagent
Program were maintained under standard culture conditions in RPMI 1640 supplemented
138 with 10% fetal bovine serum (heat inactivated), 2 mM L-glutamine, 100U/mL penicillin
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and 100 µg/mL streptomycin. U1 cells contain an integrated copy of a phorbol ester
inducible HIV-1 provirus and addition of phytohemagglutinin (PHA) to the cell culture
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was used to induce virus production. On the day of the assay, the U1 cells were activated
142 with PHA and 2.5x105 cells were plated in 96-well plates. Test compounds diluted in
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media were immediately applied to the cells at final concentrations from 1 to 10,000 nM.
Cultures were incubated for 3 days and supernatants harvested. Cell-free virus
production was measured by p24 ELISA in culture supernatants. Compound toxicity was
determined by MTT cell-viability assay in parallel assays.
148 Virus spreading assay. Jurkat (E6-1) cells (NIH AIDS Reagent Program) were cultured
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in RPMI media supplemented with 25 mM HEPES pH 7.4, 10% heat-inactivated fetal
calf serum (Hyclone, Waltham MA), and 1% penicillin/ streptomycin. 2.5x105 Jurkat
cells were inoculated with 250 pg p24 in 250 μl media in a 96-well flat bottom
polystyrene cell culture microplate. Input virus was removed after 18 hours via washing
cells in phosphate-buffered saline (PBS). Cells were resuspended in 250 μl of media
containing compounds at the described concentrations. Cells were incubated at 37°C, 5%
155 CO2 and supernatant samples were removed every 48 hours and replaced with fresh
156 media containing the compounds. Viral replication was determined using ELISA to
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quantify p24 viral capsid protein in the culture supernatant. Mouse monoclonal and
rabbit polyclonal α-p24 antibodies used in the ELISA were obtained from the NIH AIDS
Reagent Program. Toxicity was monitored using the MTT cell viability assay.
HIV-1 replication assay in PBMCs. Virus isolates were obtained from the NIH AIDS
Reagent Program. Human PBMCs, seronegative for HIV and hepatitis B virus (Astarte
163 Biologics, LLC) were stimulated in R-3 medium (RPMI 1640 medium with 25 mM
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HEPES and L-glutamine, 20% heat-inactivated fetal bovine serum, 1%
penicillin/streptomycin, 20IU/mL IL-2) with 5 μg/mL phytohemagglutinin (PHA-P) for
48 to 72 h. Stimulated PBMCs were resuspended and diluted in fresh R-3 medium and
added to 96-well plates at 5×104 cells/well. The 50% tissue culture infective dose
(TCID50) of each virus stock was measured by end-point dilution assay described by
Reed and Muench (38). Spinoculation (1,200×g, 2 hrs) was applied to improve the
efficiency of infection.
Cells were infected with 40 TCID50 of virus stock under spinoculation in the
presence of different concentrations of test compounds (biological quintuplicate
wells/concentration) and incubated at 37C, 5% CO2 for 7 days. On day 4, half of the
supernatant was removed and replaced with fresh R-3 medium containing the appropriate
concentration of test compounds. On day 7, cell-free supernatant samples were collected
for analysis of p24 antigen expression measured by ELISA as described above. Antiviral
activity was assessed by the inhibition of p24 expression.
in vitro ADME assays. Aqueous solubility studies at pH 7.4 in phosphate buffer, Caco-2
permeability studies, and human liver microsome stability studies were performed by
Absorption Systems (Exton, PA) using their standard methods.
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RESULTS
We developed a Tat-hybrid based screening assay to identify small molecules that
target RNA-protein interactions in general and the Rev-RRE interaction in particular
(Figure 1B). To screen small molecules, we engineered a stable Tat-hybrid reporter cell
line encoding the RRE IIB RNA target and driving a firefly luciferase reporter. The
188 reporter exhibited good signal-to-noise and was compatible with multi-well plates,
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robotic liquid handling, and standard luminescence plate readers. The Tat-Rev
expression plasmid was then integrated into the reporter cell line and clonal cell lines
were selected that constitutively expressed luciferase to high levels, indicative of delivery
of Tat via the heterologous RNA-protein interaction. We validated the assay using
siRNA knockdowns, targeting the Tat activation domain, and with 3,6 diaminoacridine, a
known small molecule inhibitor of the Rev-RRE interaction that served as a positive
control (Figure S1) (35).
The RRE IIB-Rev screening cell line was plated in 384-well assay plates and
compounds were added to the cells to a final concentration of 30 µM. Compounds were
screened in duplicate and the luciferase activity of each compound was highly consistent
(typically <10% deviation in activity, Figure S2). In parallel, MTT cell viability assays
were performed to assess the toxicity of compounds and minimize the level of false
positives. Screening data for the initial library screen is shown in Figure 1C.
From the primary screen, we obtained 11 validated hits with confirmed dose-
dependent activity and a lack of toxicity in the MTT assay. Of these 11 compounds, two
were benzopyrans, one was a tetrasubstituted thiophene, and the remaining eight hits
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possessed a common thieno[2,3-b]pyridine core (Figure S3). To further validate the hits,
we used an electrophoretic mobility shift assay (EMSA) to evaluate which of the three hit
207 scaffolds were capable of disrupting the Rev-RRE IIB interaction in vitro. Only the
208 thiophene scaffold disrupted the complex as determined by EMSA (Figure S4). This
209 result demonstrated that the Tat-hybrid screening method could identify bona fide
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inhibitors of RNA-protein interactions, though our experience with EMSA raised
questions about this method. We observed that compounds that disrupted the RNA-
protein complex by EMSA, 3,6 diaminoacridine, neomycin B, and thiophenes, were toxic
213 to cells. These types of relatively non-specific intercalating agents (eg. acridines) and
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RNA-binding compounds (eg. aminoglycosides) can be readily picked up as binding
inhibitors in EMSA assays but may be missed in cell-based assay because of their high
toxicity. Conversely, the lack of EMSA inhibition by other types of compounds that
scored well in the cell-based assay does not necessarily rule out Rev-RRE as their target
218 since the in vitro EMSA lacks other proteins required for Rev function (eg. Crm1 and
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Ran) and potential accessory factors (eg. hnRNPs and chaperones) that may alter the
nature of Rev-RRE complexes for inhibitors to act on. Other experimental evidence
221 supporting Rev-RRE as a plausible target of thienopyridines is provided below. Because
222 of their superior toxicity profile and a nascent structure-activity relationship (SAR)
223 profile, we focused our efforts on the thienopyridine scaffold.
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Compounds 1a-c (Figure 2) are representative of the original thienopyridine hits,
which varied in terms of substitution on the pyridine ring but universally possessed
amino and carboxamide substitution on the thiophene ring. Commercially available
analogs of 1a bearing a carboxylic acid (1d) or methyl ketone (1e) in place of the
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228 carboxamide were purchased and found not to be significantly active in the Tat-hybrid
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assay. This suggested the importance of the carboxamide function for activity in the Tat-
hybrid reporter cell line (Figures 2 and 3, Table 1). Evaluation of additional commercial
analogs suggested that modification of the 4 or 5 position of the thienopyridine ring was
generally well tolerated and this afforded some confidence that improvements in potency
could be realized with the synthesis of additional analogs (Table S1).
With reasonable activity in the Tat-hybrid reporter assay, low toxicity in the MTT
assay, and a nascent SAR profile, we embarked on structure-activity studies of the
236 thienopyridine series. One of the first analogs synthesized was compound 2a derived
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from a thieno[2,3-c]pyridine ring system that is regioisomeric with the original hit
compounds (Figure 2). Gratifyingly, 2a exhibited sub-μM potency in both the Tat-hybrid
assay and in a viral replication assay in U1 cells (Table 1). The U1 cells contain an
integrated and inducible HIV-1 provirus, meaning that inhibition of viral replication in
theses cells is indicative of compound action subsequent to retroviral genome integration,
as expected for a Rev-RRE inhibitor. As the potency of analog 2a was similar to its
directly analogous thieno[2,3-b]thionopyridine congener 1f in both assays (Figure 2,
Table 1), we focused our SAR studies on the comparatively unexplored thieno[2,3-
c]thionopyridine scaffold represented by 2a.
All thieno[2,3-c]pyridine analogs described herein were prepared by one of the
three synthetic approaches described below (Figure 4) (39). Thus, the preparation of
amine-bearing analogs 2 involved SNAr reaction of secondary amines with 3,5-dichloro-
4-pyridinecarbonitrile, followed by reaction with 2-mercaptoacetamide in the presence of
sodium methoxide. Aryl and heteroaryl-substituted analogs 3 were prepared similarly,
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prepared from 3,5-dihalopyridinecarboxaldehyde via initial formation of the thieno[2,3-
c]pyridine ring as before, followed by Suzuki coupling (40).
New analogs were evaluated in both the Tat-hybrid assay, which reports on the
Rev-RRE interaction, and in the viral activation assay in U1 cells (Table 1).
Representative dose-response curves in the U1 assay are shown for analogs 1a, 2b, and
4e (Figure 5, Table S2). We found that the activities of ~50 synthetic thienopyridine
analogs were well correlated in the Tat-hybrid and U1 assays (Figure 6), suggesting that
compound action remained “on-target” during optimization, and consistent with the post-
integration pharmacology expected of a Rev-RRE inhibitor.
To further explore amine substitution as in analog 2a, additional analogs such as
the piperidine 2b, morpholine 2d, and N-Me piperazine analog 2e were prepared and
evaluated (Figure 7 and Table 1). Piperidine analog 2b was nearly 10-fold more potent
than the dimethylamino analog 2a, an effect that appears to derive from the aliphatic
nature of the piperidine ring, substitution of which (2c) or replacement with morpholine
(2d) or piperazine (2e) rings resulted in less potent analogs. Spirocyclic (2g) and
267 geminal-diflouro (2h) substitutions at the 4-position proved inferior to the parent
268 piperidine 2b. Tetrahydroquinoline (2i) and phenyl ether (2j) analogs were equipotent to
269 2b but with their additional mass and lipophilicity produced inferior LipE values (LipE =
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pKi – clogP) (41). Finally, introduction of a heteroaryl ring at the 3-position of the
piperidine ring as in 2k was poorly tolerated. Although N-acylpiperazine analog 2f was
notably more potent than the basic N-methylpiperazine analog 2e, the exploration of
273 additional amide variants did not yield useful gains in potency.
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Having identified analogs like 2b and 2i that were up to 10-fold more potent than
2a, we next explored 4-aryl-substituted thienopyridine analogs either with (3a-j) or
without (4a-j) a 3-amino group (Figure 8 and Table 2). In the case of substituted phenyl
rings, we found substitution at the para and/or meta position(s) was favored over ortho
substitution (compare 4c and 4d). Moderately electron-rich analogs like 3a/4a and 3e/4e
were more potent than either electron-deficient congeners (4c) or more electron-rich
analogs like 3f/4f. Consistent with substitution effects in the piperazine series (2k),
bulky substitution at the 3-position (meta) as in 3j/4j was poorly tolerated. The most
282 consistent effect in this series was the favorable effect of removing the 3-amino group (as
283 in analogs 4, X = H). Particularly potent were des-amino analogs bearing a moderately
284 electron-rich aryl ring, as in the analogs 4a and 4e, which exhibited low nanomolar
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potency in both the Tat-hybrid and U1 assays and were the most potent analogs evaluated
(Table 2).
Analogs 4a and 4e combine exceptional potencies with reasonable calculated
lipophilicities, thus affording LipE values of 5.0 and 5.3, respectively. These analogs,
along with the potent but more lipophilic analogs 4g and 4h were evaluated in an HIV-1
replication assay. Jurkat cells were infected with HIV-1 laboratory isolate NL4-3 in the
presence of the test compounds and viral production was monitored by collecting viral
supernatants and measuring p24 levels by ELISA assay (Figure 9, Table S3). All four
analogs displayed low nanomolar efficacy in this assay and moreover, their rank-order
294 potencies in the infectivity assay were correlated with IC50 values in the U1 activation
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assay (Table 2). Replication curves for the des-amino analogs 4e and 4h, are shown in
Figure 9B and 9C. These compounds exhibited IC50 values of 3.9 nM and 3.4 nM
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respectively in the HIV-1 replication assay as compared to TC50 values of 19,200 and
20,500 nM in the MTT cell viability assay. For comparison, AZT exhibited an IC50 of 3
299 nM and TC50 of >100,000 nM in these same assays. The piperazine analog 2b was also
300 evaluated in the HIV-1 replication assay and exhibited activities in the mid-high
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nanomolar range (77 nM) and modest toxicity (41,400 nM) (Figure 9A).
We next tested compound 4e in replication assays using primary cells and clinical
isolates of HIV-1. Peripheral blood monocyte cells (PBMC) were stimulated with PHA
and infected with several clinical isolates of HIV-1 in the presence of the compounds.
IC50 values for the compounds were determined by collecting viral supernatants and
measuring p24 levels by ELISA (Table 3). Results of this experiment are shown in
Figure 10 where the dose response and toxicity curves for isolate 93BR021 (CCR5-
tropic, group M, subtype B) are shown. Here we observed an IC50 value of 55 nM and
TC50 of 32,300 nM. AZT was again used as a positive control and exhibited an IC50 of
310 18.5 nM and the TC50 was determined to be >50,000 nM by MTT assay.
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We next evaluated analogs 2b, 2f, 4a, and 4e in a panel of standard in vitro
ADME assays to assess drug-like properties and the potential for efficacy in animals.
All four analogs are “rule-of-five” compliant and exhibited moderate to high permeability
across Caco-2 monolayers, a model of intestinal absorption (Table 4). Analogs 4a and
4e, with only two hydrogen bond donors, were more permeable but also notably less
316 soluble than 2b and 2f. The more lipophilic analogs 4a and 4e also appear more prone to
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oxidative metabolism in the presence of human liver microsomes. This is perhaps
unsurprising given the presence of methoxyphenyl (4a) and benzylic methylene (4e)
functions, both potential sites of oxidative metabolism.
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DISCUSSION
Here we used a screening approach based on the HIV LTR Tat-hybrid system to
323 identify small molecules putatively targeting a protein-RNA interaction. A focused
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library of carboxamide-containing compounds was screened based on the expectation that
such molecules can interact with RNA bases and could potentially disrupt or alter Rev-
RNA binding interactions. Of the various hits identified in the screen, a series of
thienopyridine carboxamides emerged as the most promising hit scaffold. We then used
an HIV reporter cell line and the U1 replication assay to validate the screening hits and to
evaluate an additional ~100 commercial analogs. These analyses revealed that an
unsubstituted carboxamide group at the 2-position of the thienopyridine ring was
essential for activity. Furthermore, substitution on the thienopyridine ring was well
tolerated, particularly at the 4 and 5 positions. This initial survey of commercially
available analogs provided a preliminary SAR profile that encouraged further synthetic
efforts on the scaffold.
A variety of analogs was prepared based on a regioisomeric thieno[2,3-c]pyridine
ring system. Substitution of the 4 position with N-linked heteroaliphatic rings (2) or C-
linked aryl and heteroaryl rings (3 and 4) was well tolerated, with the C-linked aryl
338 analogs in general possessing superior potencies. Most significantly, we found that the 3-
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amino group is not essential for activity and in fact des-amino analogs in possess notably
superior potencies in both the Tat-hybrid reporter assay and the U1 assay. The
341 elimination of two hydrogen bond donors in such analogs likely improves intrinsic cell
342 permeability, which may wholly or partly explain the enhanced potency of analogs 4 in
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cell-based assays. Of the des-amino analogs, compound 4e showed the best potencies
and therapeutic indices in the reporter and U1 assays and the HIV replication assays, with
345 an EC50 of 8 nM in the reporter assay and IC50 values of 9 nM and 3.9 nM in the U1 and
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HIV replication assays, respectively (Table 2). By comparison, the most potent of the N-
linked analogs, piperazine 2b, displayed an EC50 of 65 nM in the reporter assay and IC50
values of 28 nM and 77 nM in the U1 and HIV replication assays respectively (Table 1).
Overall, our structure activity studies improved potencies from the low μM to low nM
regime, likely by improved target-level binding affinity and improved cellular
permeability. While further optimization of in vivo drug-like properties will be required,
leads like 4e appear promising as a starting point for such efforts.
Thienopyridine analogs that are closely related to our initial screening hits were
described as inhibitors of Rev-RRE by another group (33) using a reporter assay that is
dependent upon the activity of Rev (42). We therefore tested selected compounds from
356 our SAR studies (1f, 2b, 4a and 4e) in a similar Rev-dependent reporter assay (Tables 1
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and 2). Several additional experiments were described strongly suggesting that Rev is the
target of the thienopyridines analogs, including additional reporter experiments that
showed inhibition of a Rev-dependent, but not a CTE- dependent reporter, as well as
experiments that showed that the protein composition of inhibitor-treated samples was
consistent with an inhibitor of Rev function (33).
Biochemical assays including EMSA did not show inhibition of the Rev-RRE
363 interaction by the thienopyridine class in vitro. These assays generally capture a snapshot
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of Rev activity and lack critical interactions with cellular cofactors such as Crm1 and
Ran. We have found that compounds that inhibit formation of RNA-protein complexes
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366 are often toxic, suggesting that EMSA or other in vitro assays may lack the sensitivity to
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reveal subtle effects of small molecule inhibitors. In some cases, such as with 3,6
diaminoacridine and neomycin B, inhibition of Rev-RRE formation is observed by
369 EMSA, but both are toxic at therapeutic concentrations against HIV. Similarly, the
370 thiophene class identified by the small molecule screen also inhibits the Rev-RRE
371 interaction by EMSA (Figure S4), but several of the commercially available thiophene
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analogs showed substantial toxicity and thus were not pursued further. Interestingly,
SAR analysis of the thiophenes and thienopyridines identified the thieonpyridine as a
better scaffold that likely maintained similar binding characteristics (43).
Although our studies and the previous work implicate Rev as the likely target of
thienopyridine carboxamides, other targets cannot be ruled out. For example, our screen
is dependent upon the activity of the HIV LTR, and thus an inhibitor of HIV transcription
would also produce a positive response in the assay. However, this would appear
unlikely because we showed that the thienopyridine compound 4a specifically targets the
Rev-RRE reporter, but not the HIV Tat-TAR reporter, using the Tat-hybrid assay (Figure
S5). The previous screen did not use the HIV LTR and these investigators further
uncovered a thienopyridine resistance mutation in the RRE, consistent with the Rev-RRE
interaction as the target of thienopyridines (44). We have also generated this RRE-
defective virus and shown that it is resistant to compound 4e (Figure S6). Though
strongly supportive that Rev is the target of the compounds, these data do not explicitly
rule out the possibility of alternative targets, for example that thienopyridines directly
target structured RNA, or that multiple regions of the HIV genome may be targeted by
the compounds in addition to the RRE. In summary, we identified and optimized a class
18

389
390
391
392
393
394
of thienopyridine carboxamides that potently inhibits HIV replication in cells. This work
represents an early but important step toward the development of a new class of HIV
therapeutics that could eventually find a place in future HIV combination therapies.
While the weight of evidence suggests these compounds act by inhibition of the Rev-
RRE interaction, further studies will be required to definitively establish their molecular
pharmacology.
19

395
396
397
398
399
400
FIGURE LEGENDS
Figure 1. Screening strategy to identify inhibitors of RNA-protein complexes. A.
Schematic diagram of the Tat-hybrid system. A plasmid encoding the HIV LTR, RRE
RNA target, and a reporter gene is co-expressed with a plasmid encoding a fusion protein
of the HIV Tat activation domain and a heterologous RNA-binding domain from the HIV
401 Rev protein. A productive interaction between the RNA and RNA-binding protein
402 confers activation of the reporter gene. B. Schematic showing that inhibitors of the
403
404
405
406
407
408
409
410
411
412
413
414
415
RNA-protein interaction are identified by a decrease in reporter signal. C. Screening data
from a small molecule screen for inhibitors of Rev-RRE complexes.
Figure 2. Structures of hits 1a, 1b, 1c, and 1f identified in the primary screen.
Commercially available analogs 1d and 1e are congeners of compounds 1a lacking the
carboxamide function and were found to be inactive in the reporter assay. Compound 2a
is a regioisomer of hit 1f and the heterocyclic scaffold upon which additional synthetic
analogs were based.
Figure 3. Dose response curves for the activity and toxicity of thienopyridine analogs 1a
(A) and 1d (B). The Tat-hybrid reporter cells were plated in 96 well plates and test
compounds were added to the cells at doses ranging from 1 nM to 50,000 nM in
duplicate. Cell lysates were collected after 48 hours and activity values were determined
416 by luciferase assay and toxicity by MTT assay. Average activity values for 1a and 1d
417 (filled symbols) and toxicity values (open symbols) are shown.
20

418
419
420
421
Figure 4. Synthesis of thieno[2,3-c]pyridine analogs 2-4. Conditions: (a) 1-2 equiv.
R1R2NH, Et3N, DMF, 25-40 oC, 12-24 hrs; (b) 2 equiv. 2-mercaptoacetamide, 10 wt% in
MeOH/NH3, 2 equiv. NaOMe, DMF, μW, 80 oC, 10-60 min; (c) 1-2 equiv. Ar-B(OH)2,
422 0.025 equiv PCy3, 0.01 equiv. Pd2(dba)3, 1.7 equiv. K2CO3, dioxane, μW, 150 oC, 30
423
424
425
min; (d) 0.75 equiv. 2-mercaptoacetamide, 10 wt% in MeOH/NH3, 1.2 eq NaOMe, DMF,
μW, 80 oC, 10-60 min.
426 Figure 5. Activity of Thienopyridine analogs 1a, 2b, and 4e in the U1 activation assay.
427 U1 cells were plated in 96 well plates and activated by the addition of
428 phytohemagglutinin. The test compounds were added to the cells at doses ranging from 1
429
430
431
432
433
434
435
436
437
438
439
440
nM to 3160 nM in triplicate and supernatants were collected 72 hours later. p24 values
were determined by ELISA and average values for 1a (blue), 2b (red), and 4e (green) are
shown.
Figure 6. Comparison of activities for thienopyridine analogs in the Tat-hybrid and U1
viral activation assays. Each data point is for one compound and the relative size
indicates the LipE value for that compound.
Figure 7. Structures of thieno[2,3-c]pyridine analogs 2b-k.
Figure 8. Structures of thieno[2,3-c]pyridine analogs 3 and 4.
21

441
442
443
444
445
446
447
448
449
450
451
452
Figure 9. Replication assays with HIV-1 isolate NL4-3 in Jurkat cells. Replication
spreading assays were performed by infecting Jurkat cells with NL4-3 in the presence of
compound at doses ranging from 3.16 nM to 1000 nM in triplicate. Supernatants were
collected every 48-72 hours and p24 values were determined by ELISA. Average
replication curves are shown for (A) 2b, (B) 4e, and (C) 4h.
Figure 10. Replication assay with HIV-1 primary isolate 93BR021 in PBMC.
Replication assays were performed in PBMC by stimulating cells with
phytohemagglutinin then infecting with clinical isolates of HIV. Compound 4e was
added to PBMC at doses ranging from 1 nM to 1000 nM in triplicate or more, and
supernatants were collected every 48-72 hours. p24 values were determined by ELISA
and average replication curves are shown for 4e in blue and toxicity is shown in green.
22

453
TABLES
454
455
456
Table 1. Activity of thienopyridine analogs 1 and 2
in reporter and antiviral assays.
Compound Tat-hybrid U1 cell
Rev
HIV-1
LipEa
EC50
(nM)
IC50
reporter
replication
(nM)
EC50 (nM) IC50 (nM)
1a
1b
1c
1d
1e
1f
2,170
1,160
700
1473
528
750
---
---
---
---
---
---
530
---
680
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
77
---
---
---
634
---
---
---
---
---
4.7
5.2
4.5
---
>100,000
31,200
210
---
2.4
6.2
5.8
5.9
6.5
6.2
5.3
5.2
5.75
5.0
5.3
4.6
4.8
659
450
28
2a
2b
2c
2d
2e
2f
710
65
330
391
---
930
7,620
140
---
219
173
219
13
2g
2h
2i
370
350
70
2j
100
34
2k
730
1087
a LipE = pKi – clogP; pKi estimated from Tat-hybrid
EC50; clogP values were determined in Vortex
(Dotmatics) using the calculated property XlogP.
457
458
23

459
460
Table 2. Activity of thienopyridine analogs 3 and 4 in
461 reporter and antiviral assays.
462
Compound Tat-hybrid U1 cell
Rev
HIV-1
LipEa
EC50
(nM)
IC50
(nM)
reporter
replication
EC50 (nM) IC50 (nM)
3a
3b
3e
3f
70
140
200
330
4,700
10
57
38
---
---
---
---
---
115
---
---
150
---
---
---
---
---
---
---
---
---
---
4.5
---
---
3.9
---
6.4
3.4
---
---
4.9
4.6
4.6
4.5
1.15
5.0
4.0
3.5
5.3
4.5
4.2
3.2
2.2
1.9
91
277
---
3j
4a
4c
4d
4e
4f
11
140
560
8
108
946
9
70
65
4g
4h
4i
41
66
34
8.9
7.7
368
160
4j
150
a LipE = pKi – clogP; pKi estimated from Tat-hybrid
EC50; clogP values were determined in Vortex using the
calculated property XlogP.
463
464
24

465
466
467
Table 3. Activity of thienopyridine
analog 4e in replication assays in PBMC.
Viral
Isolate
Coreceptor
IC50
(nM)
Ba-L
CCR5
CCR5
48.8
54.8
9.2
93BR021
93BR028
92TH014
92UG005
92HT599
LAI
CCR5
CCR5
19.6
70.8
25.9
291
CXCR4
CXCR4
CXCR4
92HT593
CXCR4,
CCR5
13.4
468
469
470
471
472
473
474 Table 4. In vitro ADME properties of selected thienopyridine analogs.
475
Compound
Tat-hybrid
EC50
Aqueous
solubility
pH 7.4 (nM)
Caco-2
Papp
(nm/sec)
HLM
T1/2
(min)
(nM)
2b
2f
65
140
10
8
34,000
258,000
440
505
97
28
>60
8
4a
4e
594
666
280
18
476
477
478
25

479
480
ACKNOWLEDGEMENTS
481
482
483
484
485
486
487
488
We thank David Rekosh and Marie-Louise Hammarskjold of the University of
Virginia for providing the Rev reporter construct and control small molecules, Kip Guy
for critical help in designing the library screen, and members of the Frankel Lab for
providing critical feedback and comments.
This work was supported by the National Institutes of Health grants CA103407 to
A.F. and R.N., AI075143 to R.N. and A.F., AI076087 to A.R. and R.N., P50GM082250
to A.F., the United States Treasury Department Qualified Therapeutic Discovery Grant to
AGS, and the China Scholarship Council for the financial support of S.Y.
26

489
490
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29

A
Rev
RRE
Tat
Reporter
HIV LTR
B
Tat
Rev
RRE
X
Reporter
HIV LTR
C
200
150
100
50
0
0
1000
2000
3000
4000
Compound Number