Small molecule inhibitors of HIV RT Ribonuclease H

Article abstract of DOI:10.1016/j.bmcl.2009.10.043

Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity and were active in cell-based assays. In addition, sa

Full text of DOI:10.1016/j.bmcl.2009.10.043

Bioorganic & Medicinal Chemistry Letters 20 (2010) 398–402
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Small molecule inhibitors of HIV RT Ribonuclease H
à
§
*
Martin Di Grandi , Matthew Olson , Amar S. Prashad, Geraldine Bebernitz , Amara Luckay ,
Stanley Mullen, Yongbo Hu, Girija Krishnamurthy, Keith Pitts, John O’Connell
Wyeth Research, 401 North Middletown Road, Pearl River, NY, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H
inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity
and were active in cell-based assays. In addition, saturable, 1:1 stoichiometric binding to target was
established and time of addition studies were consistent with inhibition of RT-mediated HIV replication.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 22 July 2009
Revised 7 October 2009
Accepted 9 October 2009
Available online 15 October 2009
Keywords:
HIV
RT
RNase H
Antiviral
More than 20 years have elapsed since the discovery of the Hu-
man Immunodeficiency Virus (HIV) as the etiological agent for Ac-
quired Immune Deficiency Syndrome (AIDS). During this time,
progress in the treatment of this disease and its complications, as-
cribed largely to the 1996 introduction of combination therapy1 to
control viral load, has transformed AIDS into a chronic but manage-
able illness. While dramatic advances have slowed disease pro-
gression and reduced morbidity and mortality in patients, a
complete cure in the form of drug therapy or a vaccine remains
elusive. Drug resistance and persistent infection represent the ma-
jor obstacles to disease control and consequently, there is still a
need for new therapies with unique mechanisms of action.2
HIV reverse transcriptase (RT), one of three key encoded viral
proteins, provides two critical functions for viral replication.
Through RNA- and DNA-dependent DNA polymerization, it first
oversees the conversion of single stranded RNA to double stranded
DNA, followed by the degradation of the initial viral RNA strand.3
The latter is the responsibility of HIV RT Ribonuclease H (RNase
H), a non-specific, 15 kD endonuclease located at the C-terminal
subunit of the p66 strand of the RT dimer.4 To accomplish RNA
hydrolysis, RNase H employs several amino acids. In particular,
Asp443 and Asp498, in conjunction with Gly444, are believed to
coordinate a divalent cation (Mg+2 or Mn+2),5 which in turn posi-
tions another water molecule to serve as a general base. In addi-
tion, Asp549 forms
a critical hydrogen bond with a water
molecule which, via interaction with His539, serves as the source
of the nucleophile that ultimately cleaves the scissile phosphate
bond.
That HIV RT RNase H is a viable drug target has been demon-
strated, as point mutations within this domain have shown the
endonuclease activities of this enzyme are required for viral infec-
tivity.6 Despite this and the numerous reports in the literature
describing RNase H inhibitors,7 to date there are no approved drugs
with this mechanism of action.8
Several years ago, we embarked on a program to identify novel
HIV RT RNase H inhibitors and we now disclose the preliminary de-
tails of this research. The two structural classes of inhibitors to be
discussed, based on our initial HTS hits, are shown below (Fig. 1).
The thiocarbamates9 1 were synthesized by the convergent
route shown in Scheme 1. First anilines 3 were treated with
bromoacetyl bromide to give acetanilides 4, which upon treatment
O
R1
N
N N
R1
SR2
Ar
S
* Corresponding author.
N
H
R2
N
E-mail address: digranm@pfizer.com (M.Di Grandi).
Present address: JNJ PRD US RED, 665 Stockton Drive, Exton, PA, USA.
R3
S
à
Present address: Oncology BioScience, Astra Zeneca R&D Boston, 35 Gatehouse
Drive, Waltham, MA 02451, USA.
Thiocarbamate Core
1
Triazole Core
2
§
Present address: Novel Devices and Vaccines, Preclinical Evaluations Team,
Laboratory Branch, Div. of HIV/AIDS Prevention, Centers for Disease Control and
Prevention, 1600 Clifton Road NE, Atlanta, GA 30333, USA.
Figure 1. Two cores identified through HTS.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.043

M. Di Grandi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 398–402
399
with various carbamodithioic acids, 5, yielded the final products 1
in 25–55% overall yield. Intermediates 5 were prepared from com-
mercially available amines and carbon disulfide.
and 1536-well formats), is extremely sensitive, and allows for di-
rect determination of RNase H activity.
Compounds of interest from the HTS were required to have IC50
As shown in Scheme 2, the triazoles9 2, were prepared by first
treating aryl carbohydrazides 6 with various isothiocyanates in
hot acetonitrile to give crystalline hydrazine thiocarbamides 7.
Stirring compounds 7 with cesium carbonate in hot ethanol over-
night induced cyclization to the corresponding thiones 8, which
were then S-alkylated with an alkylating agent in the presence of
a suspension of sodium carbonate and sodium iodide in DMF at
room temperature overnight. The desired products 2 were afforded
in 30–65% yield, after reverse phase HPLC purification.
The thiocarbamate and triazole analogs 1a and 2c were identi-
fied in an HTS using a novel FRET-based assay (Fig. 2).10 In sum-
mary, an appropriate probe oligonucleotide substrate labeled
with a fluorophore at the 30 terminus and a quencher at the 50
end was annealed to complementary substrate RNA, thus minimiz-
ing the interaction between the two labels. In the absence of drug,
fully functional RNase H digests the RNA strand of the hybrid oli-
gonucleotide, releasing the tagged DNA strand. Upon liberation,
the designed DNA strand snaps into a hairpin loop, bringing the
50 and 30 ends in close proximity, and allowing the quencher and
fluorophore to interact. The resulting reduction in the fluorescent
signal is proportional to RNase H activity.11 The advantages of this
assay are that it is high throughput (readily amenable to 96-, 384-
610 lM in the primary assay, with at least 15-fold selectivity over
HIV RT (polymerase), Escherichia coli RNase H, and several human
DNA polymerases.12 Compounds meeting these criteria were next
screened for cell-based activity utilizing a cytopathic effect (CPE)
reduction assay in MT4 cells, where an IC50 610 lM and a thera-
peutic index of at least 20 was sought. Additionally, potential lead
compounds were further evaluated in a binding assay that utilizes
changes in the intrinsic fluorescence of the enzyme to confirm sat-
urable, stoichiometric binding to target.13 To further characterize
these compounds, some were assayed in time of addition studies.
A total of 500,000 compounds were screened from the Wyeth cor-
porate database and some 1500 hits were identified that then
underwent confirmation and secondary screening to yield putative
lead series for a discovery program.
The HTS hit 1a for the thiocarbamate series (Table 1) had
modest potency but exhibited good selectivity over the polymer-
ase activity of RT14 as well as the related E. coli RNase H, and
screened human polymerases.15 Newly prepared analogs dis-
played definable, albeit limited, SAR. For instance, replacing
one of the benzyl groups on the thiocarbamate nitrogen (i.e.,
R1 or R2) of 1b or 1c with a phenyl moiety resulted in com-
pounds with decreased potency against RNase H (1b vs 1g and
R1
HS
N
R2
S
O
R1
N
O
5
Ar
S
ArNH2
Ar
Br
N
H
R2
N
H
S
3
4
1
Scheme 1. The synthetic route for the preparation of thiocarbamates 1.
N NH
N N
O
O
H
N
Cs2CO3
Na2CO3
NaI, R2X
NHR3
R3NCS
R1
S
R1
SR2
R1
N
H
N
N
EtOH, heat
R1
NHNH2
R3
R3
S
6
7
8
2
Scheme 2. The synthetic route for the preparation of triazoles 2.
RNA:DNA Hybrid Substrate
A
T
G
G
20
A
A
A
T
A
A
A
C
T
T
5'-GGAA
C
T
A
T
C
10
..GAAAAUAUCAUCUUUGGUGUU.. AAGU-3'
RNase H
C
A
C
G
A
G
C
T
C
G
C
3'-CGCTCG CTTTTATAGTAGAAACCACAA CGAGCG
30
Q
F
G
C
G
Fluorescent
*
Q
F
Not Fluorescent
Figure 2. A schematic representation of the high-throughput FRET-based RNase H assay.

400
M. Di Grandi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 398–402
Table 1
In vitro IC50 data for thiocarbamates 1
O
R1
N
Ar
S
N
H
R2
S
Compound
HIV RT RNase H IC50
(lM)
E. coli RNase H IC50
(lM)
HIV RT IC50
(
lM)
Ar
R1
R2
1a
1b
1c
1d
1e
1f
1.9
3.3
4.7
5.8
8.3
>80
>80
>80
>168
>80
>80
>80
>80
>80
>80
>168.3
>80
2,4-(Me)2–Ph
2,3,4-Cl3–Ph
3-CN–Ph
2,4-Cl2–Ph
2-OMe-5-Me–Ph
3-CN–Ph
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
Ph
11.4
26.6
>80
>80
Ph
Ph
1g
2,3,4-Cl3–Ph
The HTS hit 2c from the triazole series (Table 3)18 was a potent
Table 2
EC50 data for selected thiocarbamates 1 in the CPE reduction assay
inhibitor of RNase H that was more than 100-fold selective over
both E. coli RNase H and HIV RT and was essentially devoid of activ-
ity in human polymerase assays.15 Modification of this core at R1–
R3 allowed for a diverse set of analogs that maintained a similar
selectivity profile to 2c (with a few exceptions) and had better de-
fined SAR than the thiocarbamate series. From these data, several
general trends were obvious. Thus, the preferred R3 group on the
triazole nitrogen was unsubstituted benzyl, and with rare excep-
tions (i.e., 2n), compounds that carried strongly electron donating,
electron withdrawing or essentially electron neutral groups
showed diminished activity relative to a simple benzyl group. For
R1, 2-thiophene appeared to be optimal, though in some cases, this
preference was marginal (compare 2a and 2h; 2b and 2e–g; 2c and
2i). Finally, aside from not tolerating strongly electron withdraw-
ing groups (2l is an exception), RNase H was very tolerant of sub-
stitution at R2 on sulfur.19
Compound HIV RT RNase
MTS
IC50
(lM)
MT4 cells
MT4 cells
TC50 M)
TI
H IC50
(l
M)
EC50
(l
M)
(l
1a
1b
1c
1d
1.9
3.3
4.7
5.8
>115
>100
>100
>120
3.7
24.8
6.5
115.1
>98.2
>116
105.2
31.3
4
18
1.3
80.9
1c vs 1f). Additionally, increasing the bulk on the amide aryl
group (1a vs 1d vs 1g) or incorporation of electron donating
groups was not well tolerated, and the thiocarbamate linkage
was required (data not shown).
Despite these limitations, several compounds had profiles that
matched our selection criteria and were studied further in a series
of cell-based assays. As shown in Table 2, compounds 1a–d were
non-cytotoxic in an MTS assay16 in Vero cells (entries 1–4). In a
CPE reduction assay in MT4 cells, 1a, 1c, and 1d had TIs of >18 in
Drilling down further, for the subseries where R3 is benzyl and
R1 is 2-thiophene, the most potent analogs prepared had either a
benzyl or picolyl group at R2 (2a and 2b). In all other cases, incor-
poration of substituents on R2 that either affected electron density
and/or lipophilicity diminished activity (compare 2a or 2b to en-
tries 2c or 2m). In some instances, lipophilicity had a greater neg-
ative impact on activity relative to increasing electron density
(compare 2d or 2e–2o).19 Finally, some compounds such as 2d,
MT4 cells and EC50s in the 1.3–6 l
M range.17 Encouraged by these
results, we sought to confirm binding to target via a fluorescent
binding assay using holoenzyme.13 Thus, when RT was treated
with increasing concentrations of compound 1a, the data indicated
a 1:1 binding stoichiometry and a Kd of 1 lM.
Table 3
In vitro IC50 data for triazoles 2
N
N
R1
SR2
N
R3
2
*
Compound
HIV RT RNase H IC50
(lM)
E. coli RNase H IC50
(lM)
HIV RT IC50
(
lM)
R1
R2
R3
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
0.2
<0.21
1
1
1
1.2
1.7
2.6
3.8
3.8
4
4.7
5.9
6.5
6.0
>99.8
105
11.6
27.3
103.2
4.5
46.2
12.4
30.4
21.9
56.8
57.0
25.2
80.0
>103.2
100.4
>103.2
2-Thio
2-Thio
2-Thio
Ph
4-PyrCH2
PhCH2
4-Cl-PhCH2
4-MeO–PhCH2
PhCH2
PhCH2
PhCH2
4-PyrCH2
4-Cl–PhCH2
4-MeO–PhCH2
4-PyrCH2
4-CF3–PhCH2
4-MeO–PhCH2
4-CF3–PhCH2
4-Cl–PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
Ph
>102.8
>103.6
>105.6
>106.4
>80
>64.4
>104
>104
>80
>80
>103.2
>100.4
>103.2
Ph
2-Fur
2-Pyr
2-Pyr
2-Fur
2-Fur
2-Fur
2-Fur
2-Thio
2-Fur
Ph
PhCH2
4-MeO–PhCH2
PhCH2
*
2-Thio = 2-thiophene; 2-Fur = 2-furan; 2-Pyr = 2-pyridine.

M. Di Grandi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 398–402
401
Table 4
were active and relatively non-cytotoxic in cell-based assays. In
addition, saturable, stoichiometric binding to target was estab-
lished and time of addition studies suggest these inhibitors are
operating at the level of RT-mediated HIV replication. Further re-
sults expanding on these preliminary data will be reported in
due course.
EC50 data for selected triazoles 2 in the CPE reduction assay
Compound HIV RT RNase
MTS
IC50
(lM)
MT4 cells
MT4 cells
TC50 M)
TI
H IC50
(l
M)
EC50
(
lM)
(l
2a
2b
2c
2d
2e
2f
2g
2h
2i
0.2
<0.21
1
1
1
1.2
1.7
2.6
3.8
3.8
4
109.7
41
16.9
33.9
28.9
46
4.0
4.4
1.4
1.1
5.8
18.5
3.6
3.9
3.3
5.9
4.7
18.7
4.3
>104.6
>131.5
27.6
7.9
26.0
30.0
19.9
7.3
0.6
3.2
8.8
22.1
4.7
0.2
Acknowledgements
3.1
59.5
32.0
>85.2
15.5
1.0
<0.005
16.9
2.5
We thank Drs. Tarek Mansour and Dave Shlaes for their support
of this work. We also thank the members of the Wyeth Chemical
Technologies group for analytical and spectral determination.
34
>80
29.4
35
78.6
33.8
24.6
2j
2k
2l
—
0.9
0.6
Supplementary data
4.7
5.9
2m
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.043.
2h, and 2k had IC50s against HIV RT (polymerase) that were within
10-fold of their corresponding RNase H IC50s. It is possible that
these select compounds bind to different or multiple sites on RT.20
As with the thiocarbamate series, the best triazoles analogs
were screened further in our cell-based assays and the results
are shown in Table 4. In general, this class appeared to have
References and notes
1. Palella, F. J., Jr.; Delaney, K. M.; Moorman, A. C.; Loveless, M. O.; Fuhrer, J.;
Satten, G. A.; Aschman, D. J.; Holmberg, S. D. N. Eng. J. Med. 1998, 338, 853.
2. Metzner, K. J. Future Virol. 2006, 1, 377.
3. Interestingly, the timing of DNA synthesis/RNA removal and whether the two
processes are linked is not well-understood; see: DeStefano, J. J.; Buiser, R. G.;
Mallaber, L. M.; Myers, T. W.; Bambara, R. A.; Fay, P. J. J. Biol. Chem. 1991, 266,
7423.
slightly higher inherent cellular toxicity (i.e., MTS values <50 lM)
relative to the thiocarbamate class.21 Still, several compounds that
were essentially inactive in the MTS assay were potent in our CPE
reduction assay and had TI >20 (see 2a–c and 2h). Of the analogs
that failed to meet our selection criterion of TI >20-fold, those that
had a 2-furan at R1 failed with the greatest frequency (2f and 2i–
2l), perhaps at least in part due to the lability of this group. And
whereas the thiocarbamates displayed a reasonable correlation be-
tween MTS and TC50 data, for some of the triazoles, especially for
compounds with TI <10, the correlation was poor. Moreover, these
compounds exhibited greater cellular toxicity in MT4 cells than
Vero.
4. For a recent review of the structure and function of HIV RNase H, see: Schultz,
S. J.; Champoux, J. Virus Res. 2008, 134, 86.
5. There has been some debate as to whether this particular RNase H requires one
or two divalent metal cations, see: Keck, J. L.; Goedken, E. R.; Marqusee, S. J. Biol.
Chem. 1998, 273, 34128. For an interesting proposal to resolve conflicting
published data, see: Cowan, J. A.; Ohyama, T.; Howard, K.; Rausch, J. W.; Cowan,
S. M.; Le Grice, S. F. J. Biol. Inorg. Chem. 2000, 5, 67. For evidence in support of a
two-metal ion mechanism, see: (a) Nowotny, M.; Gaidamakov, S. A.; Ghirlando,
R.; Cerritelli, S. M.; Crouch, R. J.; Yang, W. Mol. Cell 2007, 28, 264; Nowotny, M.;
Gaidamakov, S. A.; Ghirlando, R.; Cerritelli, S. M.; Crouch, R. J.; Yang, W.
[Erratum to document cited in CA148:072402]; Mol. Cell 2007, 28, 513; (b)
Klumpp, K.; Hang, J. Q.; Rajendran, S.; Yang, Y.; Derosier, A.; In, P. W. K.;
Overton, H.; Parkes, K. E. B.; Cammack, N.; Martin, J. A. Nucleic Acids Res. 2003,
31, 6852; (c) Pari, K.; Mueller, G. A.; DeRose, E. F.; Kirby, T. W.; London, R. E.
Biochemistry 2003, 42, 639.
6. Tisdale, M.; Schulze, T.; Larder, B. A.; Moelling, K. J. Gen. Virol. 1991, 72, 59.
7. Pyrimidinol carboxylic acids: Kirschberg, T. A.; Balakrishnan, M.; Squires, N. H.;
Barnes, T.; Brendza, K. M.; Chen, X.; Eisenberg, E. J.; Jin, W.; Kutty, N.; Leavitt, S.;
Liclican, A.; Liu, Q.; Liu, X.; Mak, J.; Perry, J. K.; Wang, M.; Watkins, W. J.;
Lansdon, E. B. J. Med. Chem. 2009, 52, 5781; N-Hydroxyimides: see Ref. 5b.
Diketoacids: Shaw-Reid, C. A.; Munshi, V.; Graham, P.; Wolfe, A.; Witmer, M.;
Danzeisen, R.; Olsen, D. B.; Carroll, S. S.; Embrey, M.; Wai, J. S.; Miller, M. D.;
Cole, J. L.; Hazuda, D. J. J. Biol. Chem. 2003, 278, 27770; N-Acyl hydrazones:
Borkow, G.; Fletcher, R. S.; Barnard, J.; Arion, D.; Motakis, D.; Dmitrienko, G. I.;
Parniak, M. A. Biochemistry 1997, 36, 3179; For a crystal structure of an N-acyl
hydrazone bound to HIV RT at a unique site, see: Himmel, D. M.; Sarafianos, S.
G.; Dharmasena, S.; Hossain, M. M.; McCoy-Simandle, K.; Ilina, T.; Clark, A. D.,
Jr.; Knight, J. L.; Julias, J. G.; Clark, P. K.; Krogh-Jespersen, K.; Levy, R. M.;
Hughes, S. H.; Parniak, M. A.; Arnold, E. Chem. Biol. 2006, 1, 702.
8. See Ref. 2 and Klumpp, K.; Mirzadegan, T. Curr. Pharm. Des. 2006, 1215, 1909.
9. The routes to the final compounds were optimized using a test case (i.e., a
single experiment) and all intermediates were characterized by NMR and MS at
a minimum. Once the chemistry was established, subsequent compounds were
prepared in parallel, evaluated by LC/MS, and were at least >85% pure based on
HPLC data. Compounds of interest were subsequently resynthesized and fully
characterized by HPLC, NMR and MS. (a) For a detailed description of the
preparation of the reported thiocarbamates, please see: Olson, M. W.; Di
Grandi, M.; Prashad, A. U.S. Pat. Appl. Publ. US 2005203176 A1; (b) for a
detailed description of the preparation of the reported triazoles, please see:
Olson, M.; Di Grandi, M. US Patent 7,563,905, July 21, 2009.
For compounds that met our requirements, we further assayed
them in our fluorescent binding assay, where we were able to con-
firm, for instance, saturable, 1:1 stoichiometric binding (Kd = 2
lM)
for compound 2c.13
In order to confirm these compounds were operating in cell at
the level of RT-associated HIV replication, time of addition studies
with compounds 1a and 2c were conducted.22 The data, shown in
Figure 3, is consistent with this hypothesis.
We have identified two classes of compounds, thiocarbamates 1
and triazoles 2 as HIV RT RNase H inhibitors using a novel FRET-
based HTS assay and demonstrated preliminary SAR for both ser-
ies. The potent analogs in each series exhibited selectivity against
the polymerase activity of HIV RT as well as several human poly-
merases and the related E. coli RNase H. Moreover, several analogs
HIV Time of Additon Experiment: Hela CD4 β gal assay
120
100
10. Olson, M.; O’Connell, J.F. PCT Int. Appl. 2004, WO 2004059012; Additionally,
since we initiated this program, several other reports of closely related FRET
RNase H assays have been published: (a) Parniak, M. A.; Min, H.-L.; Budihas, S.
R.; Le Grice, S. F. J.; Beutler, J. A. Anal. Biochem. 2003, 322, 33; (b) Nakayama, G.
R.; Bingham, P.; Tan, D.; Maegley, K. A. Anal. Biochem. 2006, 351, 260.
11. All compounds were evaluated under initial-rate conditions at or near the
empirically determined KM values for all substrates. DMSO concentration was
2% due to the addition of compound. HIV RT RNase H: Assays were carried out at
1 uM AZT
80
10 uM CSB
60
50 uM 1c
40
20
0
10 uM 2a
25 °C in a final volume of 25 lL in reaction buffer (50 mM HEPES (pH 8) with
0
2
4
6
8
10
12
2 mM MgOAc, 200 mM KOAc, 3% glycerol, 1 mM DTT, 0.02% NP-40 and 50 mg/
ml BSA), 100 nM RNA/DNA substrate and 2 nM HIV RT (KM for this substrate
was 33 nM). E. coli RNase H assay: E. coli RNase H (Ambion) assays were carried
using the same buffer and substrate conditions as HIV RT. Enzyme
Time (h)
Figure 3. Time of addition analysis of HIV for compounds 1a and 2c.

402
M. Di Grandi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 398–402
concentration was 150 pM (KM for this substrate was 150 nM). HIV RT assay:
assays were carried out at 25 °C in a final volume of 25 L in reaction buffer
(50 mM HEPES (pH 8.5) with 6 mM MgCl2, 80 mM KCl, 3% glycerol, 1.5 mM
DTT, 0.05% TX100 and 50 mg/ml BSA), 40 M poly rA-oligodT16 (Pharmacia/GE
BioTech) 20 mM TTP (Pharmacia/GE BioTech) 10 nM a-33P-TTP (0.03 Ci,
Perkin Elmer) and 1.6 nM HIV RT. Substrate hydrolysis was monitored as a
function of time using Wallac Victor II fluorescence microplate reader (Perkin
Elmer Life Sciences, Inc., Boston MA). For the FRET-based assays, excitation and
emission wavelengths were set at 585 and 615 nm, respectively, (10 nm band
pass). HIV RT was either purchased from Replidine, Inc. (Denver Colorado) or
expressed and purified in house. Instrument data collection was monitored
17. Primary screening for anti-HIV activity was performed in a human T-cell line
l
CEM-X174 infected with HIV-1 89.6 virus in the absence or presence of
increasing concentrations of the test compound as described previously (vide
infra). The toxicity of the compounds was determined simultaneously on the
same plate in uninfected CEM-X174; see: (a) Watson, K. M.; Buckheit, C. E.;
Buckheit, R. W., Jr. Antimicrob. Agents Chemother. 2008, 52, 2787; (b) Stephen, A.
G.; Worthy, K. M.; Towler, E.; Mikovits, J. A.; Sei, S.; Roberts, P.; Yang, Q.; Akee,
R. K.; Klausmeyer, P.; McCloud, T. G.; Henderson, L.; Rein, A.; Covell, D. G.;
Currens, M.; Shoemaker, R. H.; Fisher, R. J. Biochem. Biophys. Res. Commun. 2002,
296, 1228; (c) Weislow, O. S.; Kiser, R.; Fine, D. L.; Bader, J.; Shoemaker, R. H.;
Boyd, M. R. J. Natl. Cancer Inst. 1989, 81, 577. In a typical CPE reduction assay,
the EC50 is defined as the concentration of drug that reduced cell death in HIV-
infected MT4 cells by 50%; the TC50 is the concentration of drug that induced
l
l
with
a personal computer compatible with 32-bit Windows Workstation
software, designed to utilize the full capabilities of Windows 95/98/NT.
Fluorescent measurements were taken every 30 s.
50% cell death in uninfected MT4 cells; TI = TC50/EC50
.
12. Though human RNase
H
enzymes were not screened, current literature
18. Recently, Gilead scientists disclosed a series of triazoles based on Ref. [9b] as
HIV-1 RT inhibitors; see: Kirschberg, T. A.; Balakrishnan, M.; Huang, W.;
Hluhanich, R.; Kutty, N.; Liclican, A. C.; McColl, D. J.; Squires, N. H.; Lansdon, E. B.
Bioorg. Med. Chem. Lett. 2008, 18, 1131. Compounds 2a–2d in this Letter were
also prepared by the Gilead group and for at least compounds 2a–2c, our data
suggests these compounds to be selective RNase H inhibitors.
19. In addition to the biological data presented in Table 3, these conclusions are
based on data not shown.
20. Binding studies to determine stoichiometry were not conducted with these
compounds. However, in other series, not reported in this Letter, occasionally,
we observed greater than 1:1 binding stoichiometry. Alternatively, these
suggests selectivity over these enzymes should be attainable, see Ref. 5a.
13. These experiments were used to confirm the binding of compounds to the
target enzyme HIV RT. No inference as to the location of this binding with
respect to the RNase H domain is implied. Moreover in several instances, that
1:1 binding stoichiometry was demonstrated suggests binding is related to
enzyme inhibition. The intrinsic fluorescence of the enzyme was monitored at
an excitation wavelength of 295 nm and emission of 340 nm in the absence
and in the presence of increasing concentrations of the inhibitor. The
progressive decrease in the fluorescence of the enzyme upon interaction
with the compound was used to determine the binding affinity using
a
quadratic equation; see: Jamieson, E. R.; Jacobson, M. P.; Barnes, C. M.; Chow, C.
S.; Lippard, S. J. J. Biol. Chem. 1999, 274, 12346.
compounds could be weak polymerase inhibitors with residual RNase
activity (see Ref. 14).
H
14. Literature evidence suggests that inhibitors of the polymerase activity of HIV
RT can impact RNase H activity (see: Shaw-Reid, C. A.; Feuston, B.; Munshi, V.;
Getty, K.; Krueger, J.; Hazuda, D. J.; Parniak, M. A.; Miller, M. D.; Lewis, D.
Biochemistry 2005, 44, 1595) and in the assay described in this Letter, nNRTIs
do affect RNase H function (data not shown). However, most compounds
reported herein are substantially less active against the polymerase activity of
21. This result could reflect a higher inherent cytotoxicity for this series or could be
a reflection of the small number of thiocarbamates screened in this assay.
22. Yang, Q.; Stephen, A. G.; Adelsberger, J. W.; Roberts, P. E.; Zhu, W.; Currens, M.
J.; Feng, Y.; Crise, B. J.; Gorelick, R. J.; Rein, A. R.; Fisher, R. J.; Shoemaker, R. H.;
Sei, S. J. Virol. 2005, 79, 6122. In order to examine possible steps of viral
replication inhibited by the compounds, the addition of test compounds was
delayed for 0, 1, 2, 4, 6, 8, 10, and 12 h after MAGI cells were inoculated with
HIV-1NL4-3 (MAGI time-of-addition assay). In this time-of-addition assay
HIV RT and therefore, the observed RNase
compounds is independent of the inhibition of polymerase activity.
15. We screened against H DNA Pol
, H DNA Pol b, H DNA Pol , and H RNA Pol 2;
IC50s greater than 80 M were observed; data not shown.
H inhibitory activity of these
a
c
format, compounds were added at the following concentrations 10
lM Chicago
l
sky-blue 60, (CSB), 1 M AZT, 10 M 2c 50 M 1a.; See: Kimpton, J.; Emerman,
l
l
l
16. Barltrop, J. A.; Owen, T. C.; Cory, A. H.; Cory, J. G. Bioorg. Med. Chem. Lett. 1991, 1,
M. J. Virol. 1992, 66, 2232; Sei, S.; Yang, Q. E.; O’Neill, D.; Yoshimura, K.;
Nagashima, K.; Mitsuya, H. J. Virol. 2000, 74, 4621.
611.