Targeting RNA dynamics for HIV inhibition
Dev P. Arya (dparya@clemson.edu)
Department of Chemistry, Clemson University
Nucleic Acid Recognition
Organic Chemistry/Medicinal Chemistry/Biophysics/Molecular
Biology/Chemical Biology/Microbiology/Pathology
http://chemistry.clemson.edu/people/arya.html
dparya@clemson.edu
RNA targeting (HIV, Antimicrobial)
Charles, I.; Arya, D. P., Bioconjugate Chemistry, 2007
Xi, Arya, FEBS Letters, 2009
Biochemistry.51 (2012) 2331-2347.
(Triplex, HIV, bacteria)
Acc. Chem. Res. 44 (2011) 134-146
Chem. Commun. 2002, 70
J. Am. Chem. Soc. 2003, 125, 10148.
J. Am. Chem. Soc. 2003, 125, 8070
Biochemistry. 50 (2011) 2838-2849.
DNA duplex (TFs, cancer, bacteria,T.
Brucei).
J. Am. Chem. Soc. 133 (2011) 73617375 J. Am. Chem. Soc. 2003 125,
12398
Nat. Prod. Rep. 29 (2012) 134-143
Bioorg. Med. Chem. Lett. 19 (2009) 4974-4979
Biochemistry. 50 (2011) 9088-9113.
Biochemistry. 49 (2010) 452-469.
(DNA.RNA hybrids: HIV, Telomeres)
J. Am. Chem. Soc. 2001, 123, 5385.
Bioorg. Med. Chem. Lett. 18 (2008) 4142-4145
Biochimie. 90 (2008) 1026-1039
Outline
• Review of HIV lifecycle and replication
• Background on strategies utilized thus far to combat HIV
proliferation upon infection
• Summary of current knowledge on topic of ligand-RNA
interactions
• Role of RNA dynamics in targeting HIV
• Click chemistry as an ideal tool to target RNA dynamics
• Results
• Acknowledgements
The HIV replication cycle
Simon & Ho (2003) 1: 181-190
1.
Attachment of virus to
receptor (CD4) & co-receptor
(chemokine receptor CCR5 or
CXCR4)
2.
Fusion with target cell
membrane; virus entry
3.
Viral RNA genome undergoes
reverse transcription
4.
Proviral DNA integrates into
the host chromosome
5.
Viral proteins are translated
6.
Viral proteins assemble at the
cell membrane
7.
The immature virus particle
containing the RNA genome
egresses the cell
8.
Maturation of the viral
particle: the virion buds &
capsid proteins are processed,
leading to a structural
rearrangement of the virion
Current combative strategies
• Protease inhibitors: block replication at the end of the replication cycle
disallowing cleavage of nascent proteins necessary for assembly of
daughter virions
• Fusion inhibitors: disallow conformational changes between viral
envelope proteins and cell surface chemokine receptors
• Nucleoside- and non-nucleoside reverse transcriptase inhibitors (NRTI &
NNRTIs): bind RT and prevent reverse transcription and thus replication of
the viral genome
• Main problem with these therapeutics: single point mutations in viral
genome often result in emergence of resistant viral strains.
•
Targeting the HIV-1 Transactivation Response
Element with Therapeutics
The transactivation respose element (TAR) comprises
nt 1-59 of HIV-1 mRNA, and contains a stem loop
structure essential for transactivation.
C
The stem loop sequence, shown, is specifically
recognized by the Tat protein, and recruits RNA
polymerase II to the HIV-I mRNA transcripts for
transcription.
Advantages to targeting TAR:
• a novel target in the replication cycle
•TAR sequence is well-conserved within HIV-1 strains
• Only resistant strains will be those that contain mutations within the TAR stem-loop sequence
that arise simultaneously with a compensatory mutation(s) within the Tat gene
• Evidence shows that blocking the Tat/TAR interaction in infected cells prevents replication.
Sharp & Marciniak, (1989) Cell 59: 229, Johnston & Hoth, (1993) Science 260: 1286
The Tat-Tar interaction can be mimicked by
argininamide
• Binding of Tat to TAR is
mediated by a single arginine
residue
• Free arginine can bind in the
same manner, and
argininamide can be used to
substitute for this amino acid
• Argininamide binding occurs
within the 3-nt bulge region of
the TAR stem-loop
Calnan et al., (1991) Science 252: 1167; Tao & Frankel (1992) PNAS, 89: 2723; Puglisi, et al., (1992) Science.
257: 5066: 76-80.
Strategies used to target TAR
A number of strategies to
date center about targeting
the argininamide binding site.
Shown is one of the lowenergy NMR structures of
HIV-1 TAR and
acetylpromazine, a
nanomolar inhibitor identified
by computational screening.
Du, et al., Chemistry & Biology, Vol. 9, 707–712.
Baily;C., Colson;P. Nucleic Acids Res.,
1996, 24, 1460.
Baily;C., Colson;P. Nucleic Acids Res.,
1996, 24, 1460.
Hamy; F., et al. Biochemistry, 1998,
37, 5086.
Davis; B., et al. J. Mol. Biol. 2004, 336, 625.
Davis; B., et al. J. Mol. Biol. 2004, 336, 625.
Peytou; V., et.al. J. Med. Chem., 1999, 42, 4042
Mayer; M. et al. Methods Enzymology
2005, 394, 571.
Lind; K.E., et al. Chem. Biol. 2002, 9, 185.
Parolin; V. et al. Antimicrob. Agents Chemother. 2003, 47, 889.
HOECHST 33258
Hoechst binds HIV-1 TAR in a
relatively low affinity site, yet to
be specified precisely, but has
been localized by foot-printing to
the upper region of the
bulge/lower region of the upper
stem (AT selective DNA minor
groove binder, and is also a
nucleic
acid
intercalator),
although it will bind nonspecifically when present in
excess over TAR.
Dassonneville, et al., (1997) Nucleic Acids Research, 25: 4487–4492
Aminoglycosides as RNA binders
•
neomycin binds TAR with
only ~ 6 mM affinity
NH2
O
HO
HO
H 2N
NH 2 O
NH2
OH
HO
O
O
NH2
HO
O OH
O
OH
H 2N
C
Faber et al., (2000) J. Biol. Chem. 275: 20660–20666.
ITC Titration of TAR RNA with Neomycin
Time (min)
-10 0 10 20 30 40 50 60 70 80 90100110120130140150160
0.0
µcal/sec
-0.1
N
DH (kcal/mol)
K
-0.2
1.09+0.04
-0.3
(6.6+0.8)X10
5
-18.1+1.1
0
kcal/mole of injectant
-2
-4
NH2
-6
-8
O
HO
HO
-10
H 2N
NH 2 O
-12
-14
NH2
OH
HO
O
-16
O
NH2
-18
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
HO
O OH
O
Molar Ratio
OH
H 2N
ITC titration of TAR RNA with Neomycin. Neomycin (80mM) was serially injected to the TAR RNA
(4mM/ molecule) soltuion at 200C. Buffer 10mM Sodium Cacodylate,0.5mM EDTA, 100mM KCl at pH
7.0.
Neomycin does not perturb binding of Hoechst to TAR
Fluorescence Titration of TAR into
Hoechst in Absence of Neomycin
4
Fluorescence Titration of TAR into
Hoechst in Presence of Neomycin
4
4
5 10
4
4 10
5 10
4 10
4
4
4
4
4 10
4 10
4
4
3 10
3 10
4
4
2 10
2 10
4
4
1 10
1 10
0
Emission count (1/s)
Emission Count (1/s)
3.5 10
3.5 10
4
4
3 10
3 10
4
4
2.5 10
2.5 10
4
4
2 10
2 10
1.5 104
4
1.5 10
4
4
1 10
1 10
5000
5000
0
0
400
450
500
Wavelength
550
0
400
450
500
550
Wavelength
Titration of concentrated RNA or 1:1 RNA:neomycin solution (100 mM)
into 1.8 mL Hoechst 33258 2 mM up to 4 molar equivalents. In a 100 mM
NaCl, 10 mM cacodylate pH 6.8 buffer; excited at 338 nm.
Arresting TAR Dynamics
• TAR has inherent flexibility about its 3-nt bulge region
• Argininamide (Tat, and the RNA pol II complex/) binds via near-linear conformation
C
Our strategy:
•Not necessarily compete for Tat binding site, but arrest TAR motion trapping it in
a non-recognizable bent conformation: effect a deleterious conformational
change upon ligand binding.
•Design conjugates that take advantage of two modes of binding, increasing
specificity and affinity, and ideally bind the two different helices as well
Al-Hashimi (2005) Chem. Bio. Chem. 6: 1506 – 1519.
HOECHST-TAR NMR titrations
• Virtually all imino resonances shift slightly, indicating global
conformational changes and/or non-specific binding of
hoechst at higher concentrations of the drug.
• Resonances near the bulge have a steeper titration curve,
indicating specific binding of hoechst in the vicinity.
• Also, a bulge U resonance emerges upon addition of > 1
eq. concentrations of hoechst, indicating induced
conformational change in the region upon binding, and/or
protection by hoechst
G43
NH1
C
U38
NH3
Curves are fit according to a one-ligand per
site model
Meredith Newby
Identification of a HOECHST binding domain
within TAR
• The hoechst proton
(1,5)
resonances that shift the
most upon binding to TAR are
boxed in purple
(2,4)
(9)(16)
Parkinson et al., (1992) Mag. Res. Chem. 1064-1069.
Arresting TAR dynamics using click chemistry
G G
U
G
C
A
C=G
G=C
OH
A=U
N
NH
U G= C
C
NH2
O
HO
HO
NH2 O
HO
NH2
OH
H2N
O
NH2
O
OH
O OH
ng
ind i
B
n
i
yc
eom
O
OH
N
H2N
site
U23
A U40
G=C
A=U
C=G
C=G
G=C45
5'G=C 3'
N NH
HOECHST Binding site
N
N
Our strategy:
•Design conjugates that take advantage of two modes of binding, increasing
specificity and affinity, and ideally bind the two different helices as well
Synthesis of azide and alkyne functionalized neomycin
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
Part I- Aminosugar dimers
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target
19 HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
Structure of linkers used for dimer formation
Neo-Neo dimer
Linker length
DPA 51
7
Structure of the
compound
Neo
DPA 52
DPA 53
DPA 54
DPA 55
DPA 58
DPA 60
N
N
N
7
8
8
10
N
N N
O
N
N
NN
N N
N
N N
N
Neo
Neo
Neo
N
N
N
Neo
Neo
Neo
N
N
N
Neo
Neo
DPA 56
N
7
Neo
DPA 65
N
N N
N
N
N N
N N
N
Neo
N
N N
Neo
10
Neo
N N
N
Neo
N N
N
Neo
N N
N
16
20
O
O
4
O
O
N
N N
Neo
N
N N
Neo
N
N N
Neo
6
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target HIV TAR RNA Conformation,
Biochemistry.51 (2012) 2331-2347
Neomycin dimers significantly enhance the thermal stability of HIV-TAR RNA
0.62
0.6
0.59
0.6
0.58
0.57
0.56
Neo-Neo dimer Linker length ΔTm
0.58
0.54
260
0.56
A
65
70
75
78.2
80
T(0C)
0.52
0.5
HIV TAR RNA
DPA 52
0.48
20
30
40
50
60
70
80
90
0
9.3
7
7
7.57
8.22
9.3
10
9.62
T( C)
Neo
10.2
12
5.4
6.05
8
3.24
6
4
2
1
10.19
9.30
9.30
9.62
8.22
7.57
6.05
5.40
3.24
0.20
60
0.54
m
7
7
7
8
8
10
10
16
20
68.9
0.53
0.52
DT
DPA 51
DPA 52
DPA 65
DPA 53
DPA 54
DPA 55
DPA 56
DPA 58
DPA 60
Neomycin
0.55
0
0
7
8
8
10
10
16
20
Linker length
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
FRET competitive binding assay between TAR RNA and neomycin dimers
22
FRET competitive binding assay between TAR RNA and neomycin dimers
H2N
NH2
H2N
NH
HN
NH
H2N
HN
O
O
HO
HO2C
N
H
H
N
O
O
N
H
H
N
O
N
H
O
H
N
O
O
N
H
H
N
O
N
H
O
H
N
O
N
H
O
O
H
N
N
H
O
H
N
O
N
H
O
H
N
O
O
NH2
S
O
O
NH
HN
H2N
NH2
O
NH2
HN
HN
HN
HN
NH
NH
NH
NH2
NH2
N
HO2C
O
Fluorescein-labeled HIV-1 Tat peptide
N
50
Fluorescence Intensity
Fluorescence Intensity
80
IC50 = 86  9 nM
60
40
20
40
30
DPA 55
20
IC50 = 80  9 nM
10
0
0
0
0
1
2
log[TAR RNA], nM
3
1
2
3
log[DPA 55], nM
Saturation binding curve of fluorescein-labeled HIV-1
Tat peptide (100 nM) with HIV-1 TAR RNA (left); competition assay
with antagonist (right) in TK buffer at 25 °C.
IC50 values of dimers towards HIV-1 TAR RNA using FRET
713
600
500
400
300
8
67
7
61
7
59
7
80
100
47
128
200
56
10
10
16
20
700
60
DPA55
DPA56
DPA58
DPA60
7
7
7
8
8
Neo
77
DPA51
DPA52
DPA65
DPA53
DPA54
713 ±
165
77 ± 27
60 ± 8
56 ± 6
47 ± 6
128 ±
12
80 ± 9
59 ± 11
61 ± 13
67 ± 9
800
(nM)
Neomycin
IC50 (nM)
50
Linker
length
IC
Compound
10
16
20
0
0
8
10
Linker length
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
3 10
5
2.5 10
5
2 10
5
1.5 10
5
1 10
5
5 10
4
1
Fraction displaced
Fluroscence
Ethidium Bromide Displacement Assay between dimers and TAR RNA
0.8
0.6
0.4
0.2
0
0.5
560
600
640
680
Wavelength(nm)
720
1
1.5
2
2.5
log[DPA56 inmM]
3
25
IC50 values of dimers towards HIV-1 TAR RNA using FID titration (ethidium
bromide)
IC50 (nM)
417 ± 115
Neomycin
DPA51
DPA52
7
7
56 ± 1
52 ± 23
DPA65
DPA53
7
8
81 ± 2
36± 9
DPA54
8
67 ± 23
3 10
5
2.5 10
5
2 10
5
1.5 10
5
1 10
5
5 10
4
550
600
650
Wavelength(nm)
1.0
Fractional displacement
Linker
length
Fluroscence
Compound
0.8
0.6
0.4
0.2
0.0
0.8
10
99 ± 31
DPA56
10
97 ± 32
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
log[DPA 56 in mM]
500
Neo
417
DPA55
1.0
10
74
10
67
97
7
99
7
67
100
16
20
36
74 ± 21
81
20
52
DPA60
200
56
67 ± 23
300
50
16
IC (nM)
DPA58
400
0
0
7
8
8
Linker length
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
Compound Linker
length
Neomycin
DPA51
7
Comparison of IC50 values from two
methods.
IC50 (nM)
FRET
FID
713 ±
165
417 ±
115
800
56 ± 1
700
77 ± 27
Neo
FRET assay
Ethidium assay
7
60 ± 8
52 ± 23
DPA65
7
56 ± 6
DPA53
8
47 ± 6
81 ± 2
36±
9
67 ± 23
DPA54
8
128 ±
12
DPA55
10
80 ± 9
99 ± 31
DPA56
10
59 ± 11 97 ± 32
50
DPA52
IC (nM)
600
500
400
300
200
100
0
0
7
7
7
8
8
10
10
16
Linker length
DPA58
16
61 ± 13 67 ± 23
DPA60
20
67 ± 9
74 ± 21
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target HIV TAR RNA Conformation,
Biochemistry.51 (2012) 2331-2347
20
Maximum Protection from Cytopathic effects in MT-2 cells
Linker
Length
5%
Toxicity
(µM)
Maximum
protection
(conc.
Achieved in
µM)
7
>138
1% (9)
7
17
63% (8)
8
69
31% (17)
10
8
20% (4)
10
34
33% (17)
Neomycin
>206
9% (206)
Water
NA
2%
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target
28 HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
Inhibition of HIV antigen synthesis in cells
(In collaboration with W. Edward Robinson, Jr. at UC-Irvine)
Linker
length
Conc.
(µM)
Day 2
(%)
Day 4
(%)
Day 6
(%)
7
25
15
100
100
7
9
2
40
100
8
17
3-5
80
100
10
4
3-5
30
100
10
8
5-7
40
100
Control
NA
70
100
100
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target
29 HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
Decrease in the levels of reverse transcriptase in cells
Linker
Conc.
(µM)
Day 2
(cpm/ml)
Day 4
(cpm/ml)
Day 6
(cpm/ml)
7
25
21,485
347,845
268,357
7
9
8,800
45,539
221,445
8
17
14,805
165,301
427,475
10
4
20,072
107,933
305,277
10
8
15,989
105,704
412,475
Virus
Control
NA
46,029
928,112
1,078,741
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target
30 HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
Sl .#
Name
ΔTma
(0C)
IC50b
FRET
IC50c
EtBr
5%d
Toxicity(µM)
Maximum
protection from
HIV cytopathic
effectse
(concentration
achieved in µM)
Active Compounds
Inhibit HIV Antigen
Synthesis in Treated
Cellsf
Conc.
Day
Day
(µM)
2
4
Fluorescein
Labeled TATpeptide
--
86 ± 9 nM
--
--
--
--
--
--
Neomycin
0.40
713 ± 165 nM
417 ± 115 nM
--
--
--
--
--
1
DPA51
10.19
77 ± 27 nM
56 ± 1 nm
--
--
--
--
--
2
DPA52
9.30
60 ± 8 nM
52 ± 23 nm
>138
1% (9)
25
15%
100%
3
9.62
8.22
81 ± 2 nm
67 ± 23 nm
17
69
63% (8)
31% (17)
2%
40%
17
3-5%
80%
5
DPA55
7.57
56 ± 6 nM
128 ± 12
nM
80 ± 9 nM
9
4
DPA53
DPA54
99 ± 31 nm
8
20% (4)
4
3-5%
30%
6
DPA56
6.05 59 ± 11 nM 97 ± 32 nm
34
33% (17)
8
5-7%
40%
7
8
9
DPA58
DPA60
DPA65
----
----
----
----
----
5.40
3.24
9.30
61 ± 13 nM
67 ± 9 nM
47 ± 6 nM
67 ± 23 nm
74 ± 21 nm
36 ± 9 nm
S. Kumar, P. Kellish, W.E. Robinson Jr, D. Wang, D.H. Appella, D.P. Arya, Click Dimers To Target HIV
TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
Ligand
Linker Length
Wildtype
Bulgeless
Tetraloop
Bulgeless
U3 Bulge Mutant
DPA51
7
1.17x108
7.46x107
2.66x107
2.29x108
1.60x107
7
7
7
7
DPA52
7
7.08x10
8.89x10
1.39x10
7.50x10
6.93x106
8
7
7
7
DPA65
7
1.39x10
9.97x10
1.25x10
6.91x10
DPA53
8 (phenyl) 1.46x108
7
7
7
DPA54
8 (butyl)
2.61x10
2.17x10
1.23x10
2.11x106
7
7
6
7
DPA55
10
1.06x10
3.64x10
3.53x10
2.83x10
2.60x106
7
7
6
7
DPA56
10
6.60x10
6.33x10
4.97x10
5.87x10
3.84x106
6
7
7
7
DPA58
16
7.58x10
6.84x10
1.35x10
7.23x10
4.84x106
DPA60
20
2.53x107
4.36x107
1.97x106
2.68x107
1.52x106
7
7
Neomycin
N/A
2.99x10
1.58x10
Table representing the binding constants derived from scatchard analysis from the ethidium bromide displacement assay using the neomycin
dimers and neomycin with wildtype and mutant TAR RNA. Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8. [TAR RNA] =
200 nM/strand. [EtBr] = 5 µM.
Arresting TAR dynamics using click chemistry
Part II: Benzimidazole-aminosugars
G G
U
G
C
A
C=G
G=C
OH
A=U
N
NH
U G= C
C
NH2
O
HO
HO
NH2 O
HO
NH2
OH
H2N
O
NH2
O
OH
O OH
ng
ind i
B
n
i
yc
eom
O
OH
N
H2N
site
U23
A U40
G=C
A=U
C=G
C=G
G=C45
5'G=C 3'
N NH
HOECHST Binding site
N
N
Our strategy:
•Design conjugates that take advantage of two modes of binding, increasing
specificity and affinity, and ideally bind the two different helices as well
Synthesis of clickable Hoechst 33258 derived benzimidazole alkyne
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
Synthesis of clickable Hoechst 33258 derived bisbenzimidazole alkyne
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
Synthesis of azide functionalized benzimidazole
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
Synthesis of clickable Hoechst 33258 derived benzimidazoles
Benzimidazoles with a terminal azide
Benzimidazoles with a terminal alkyne
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
Synthesis of triazole linked neomycin-benzimidazoles
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
Scheme of Neomycin- Benzimidazole Conjugate Synthesis
NHR
O
N
A
O
N
H
N
N
HO
HO
N
N
N
N
O
N
+
n
N3
OH
N
O
OH
n
N N
N
Dioxane,
4M HCl in dioxane
HO
O
N
O
N
N
H
N
O
N
N
N
n
N N
N
N
N
H
N
N
N
NHR
O
HO
HO
n
N N
N
NHR O RHN
N N
NHR
N O
OH
O
OH
NHR
HO
O
O
RHN HO
B
N
N
H
N
N
N N
N
HO
HO
OH
N3
NHR
O
RHN
O
OH
OH
NHR
O
O
OH
RHN
N
OH
NHR
OH
R=Boc
O
N
O
N
n
Dioxane,
4M HCl in dioxane
N
N
NHR O RHN
N N
N O
O
NHR
OH
NHR
O
HO
HO
N N
N
RHN
O
O
OH
N
H
N
NHR O
+
n
EtOH, H2 O
CuSO4 , NaASc
NHR
O
NH 2
O
HO
HO
O
O
N
NHR
O
H2 N
NH2
O
N N
NH2
N O
OH
O
OH
NH2
HO
O
O
H2 N HO
N
B
HO
HO
NHR O RHN
N N
NHR
N O
OH
O
OH
NHR
R=Boc
HO
O
O
RHN HO
HO
N
N
N
N
NHR
O
NHR
O
RHN
CuSO4 , NaAsc
C 2H 5OH, H2 O
N
N
H
N
A
NHR O RHN
N N
N
H
N N
N
HO
HO
n
OH
RHN
N
NHR
O
NHR O RHN
N N
N
N N
N
NHR
O
OH
O
OH
R=Boc
H 2N
NH 2
O
HO
HO
n
OH
O
NHR
O
OH
N
H
NH 2 O H2 N
N N
N O
O
NH2
O
OH
O
NH 2
OH
OH
Yield= 50-63% for two steps
Scheme for protected NeomycinBenzimidazole synthesis
Scheme for deprotection of protected
Neomycin-Benzimidazole conjugates
4
2
0
0 0 4 11 11 12 12 14 16 19 20 22 24
Linker Length
0
100
50
0
81
150
140
180
200
DPA117
DPA116
DPA115
DPA114
184
DPA113
DPA114
DPA122
147
DPA121
200
140
DPA120
83
150
DPA119
285
250
78
50
IC (nm)
DPA117
DPA116
DPA115
DPA114
DPA113
DPA122
DPA121
DPA118
DPA120
DPA119
DPA123
Neomycin
300
DPA123
6
Neomycin
Benzimidazole Alkyne
8
350
33
m
DT
10
400
4 11 11 12 12 14 16 19 20 22 24
Linker Length
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
RNA/DNA
IC50 of DPA 123 (nm)
HIV TAR RNA
33
A-site RNA
38
polyrA.polyrU
4.7X103
Calf thymus DNA
6.0X103
UV Melting studies
Control
Neo-Ben
Benzimidazole
0.44
Table for DTm
0.42
TAR with
None
Neomycin
Benzimidazole
Neo-Benzimidazole 123
A
260
0.4
Tm(0C)
68
70
67
74
DTm(0C)
2
-1
6
0.38
0.36
0.34
20
30
40
50
60
70
80
90
100
0
T( C)
UV melting of TAR RNA without and without the presence of various ligands a)Neomycin Benzimidazole
Conjugate(purple) b)c Benzimidzole (red) c) None (blue) in the presence of buffer 10mM Sodium Cacodylate,
0.5mM EDTA,0.1 mM MgCl2. at pH 7.0. Heating rate0.30C/ min.
5% Toxicity concentrations and maximum
protection from HIV cytopathic effects in MT-2
cells
Compound
5% Toxicity
Maximum
concentration
protection
(mM)
(concentration in
mM)
DPA101
35
13% (17)
DPA113
176
6% (5)
DPA114
11
5% (10)
DPA116
83
3% (21)
DPA117
41
4% (20)
DPA118
>184
17% (184)
DPA119
94
25% (188) – 16%
at 24 microM
DPA120
94
6% (188)
DPA121
>186
0% (186)
DPA123
86
45% (83)
neomycin
>206
9% (206)
Hoechst
18
2% (2)
33258
Water
None
2%
Ed Robinson
Differential reactivity of mono and bisbenzmidazoles with 5’-azido-neomycin
Alternative synthesis of neomycin- Hoechst 33258 conjugate
Benzimdazole derived synthesis of neomycin- Hoechst 33258
Compoun
d
IC50
(nm)
ΔTm
(0C)
Neomycin
785
1
DPA165
60
6
DPA166
78
6
DPA165
DPA166
Buffer conditions: 10 mM sodium cacodylate, 0.5 mM EDTA, 100 mM KCl, pH 6.8.
UV denaturation experiment was done at a heating rate of 0.30C/min. The Tm values were
obtained from the first derivative plots.
5% Toxicity concentrations and maximum
protection from HIV cytopathic effects in MT-2
cells
Compound
5% Toxicity
Maximum
concentration
protection
(mM)
(concentration in
mM)
DPA165
>189
44% (189)
DPA166
11
18% (6)
neomycin
>206
9% (206)
Hoechst
18
2% (2)
33258
Water
None
2%
Ed Robinson
Future directions: DPA83 with TAR RNA
1.1
G G
U
G
C
A
C=G
G=C
OH
A=U
N
NH
U G= C
C
NH2
O
HO
HO
NH2 O
HO
NH2
OH
H2N
H2N
O
NH2
O
OH
O OH
sit
d ing
n
i
B
ycin
eom
O
OH
N
e
U23
A U40
G=C
A=U
C=G
C=G
G=C45
5'G=C 3'
N NH
HOECHST Binding site
Fraction displaced
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
1.0
1.2
N
IC50 (nM)
DPA83
13.1±7.1
1.6
1.8
log[DPA83 in mM]
N
Compound
1.4
DPA83
2.0
2.2
Conclusions
• We have devised a click chemistry based strategy for
the design of RNA conformation-targeted
therapeutics that are aimed at preventing virus
proliferation
• Dimeric aminsougars and benzimidazole-aminosugar
conjugates bind TAR with IC50 values in the nano
molar range, and show protection from HIV at nontoxic doses.
Acknowledgements
Meredith Newby, Dept of
Physics
Nihar Ranjan
Sunil Kumar
Patrick Kellish
Dr. Derrick Watkins
Ed Robinson, Department of
Pathology and Laboratory
Medicine, UC Irvine
Glaxo Smithkline (Raleigh,
NC)
Dr. Andy Norris
$ NIH