Post on 13-Jan-2016
Targeting RNA dynamics for HIV inhibitionDev 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.htmldparya@clemson.edu
(Triplex, HIV, bacteria)Acc. Chem. Res. 44 (2011) 134-146 Chem. Commun. 2002, 70J. Am. Chem. Soc. 2003, 125, 10148.J. Am. Chem. Soc. 2003, 125, 8070Biochemistry. 50 (2011) 2838-2849.
RNA targeting (HIV, Antimicrobial)Charles, I.; Arya, D. P., Bioconjugate Chemistry, 2007Xi, Arya, FEBS Letters, 2009Biochemistry.51 (2012) 2331-2347.
(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
DNA duplex (TFs, cancer, bacteria,T. Brucei). J. Am. Chem. Soc. 133 (2011) 7361-7375 J. Am. Chem. Soc. 2003 125, 12398
Nat. Prod. Rep. 29 (2012) 134-143 Bioorg. Med. Chem. Lett. 19 (2009) 4974-4979Biochemistry. 50 (2011) 9088-9113.Biochemistry. 49 (2010) 452-469.
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 therapeuticsMain problem with these therapeutics: : single point mutations in viral single point mutations in viral genome often result in emergence of resistant viral strainsgenome often result in emergence of resistant viral strains ..
•
Targeting the HIV-1 Transactivation Response Element with Therapeutics
Advantages to targeting TAR:
• a novel target in the replication cyclea novel target in the replication cycle•TAR sequence is well-conserved within HIV-1 strainsTAR sequence is well-conserved within HIV-1 strains• Only resistant strains will be those that contain mutations within the TAR stem-loop sequence 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 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. Evidence shows that blocking the Tat/TAR interaction in infected cells prevents replication.
The transactivation respose element (TAR) comprises nt 1-59 of HIV-1 mRNA, and contains a stem loop structure essential for transactivation.
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.
Sharp & Marciniak, (1989) Cell 59: 229, Johnston & Hoth, (1993) Science 260: 1286
C
The Tat-Tar interaction can be mimicked by argininamide
Calnan et al., (1991) Science 252: 1167; Tao & Frankel (1992) PNAS, 89: 2723; Puglisi, et al., (1992) Science. 257: 5066: 76-80.
• 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
Strategies used to target TAR
A number of strategies to date center about targeting the argininamide binding site.
Shown is one of the low-energy 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.
Peytou; V., et.al. J. Med. Chem., 1999, 42, 4042
Davis; B., et al. J. Mol. Biol. 2004, 336, 625.Davis; B., et al. J. Mol. Biol. 2004, 336, 625.
Lind; K.E., et al. Chem. Biol. 2002, 9, 185.
Mayer; M. et al. Methods Enzymology2005, 394, 571.
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 non-specifically 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 M affinity
Faber et al., (2000) J. Biol. Chem. 275: 20660–20666.
C
OHO
HO
O
O
O
NH2
OH
HO
OH
H2N
NH2
NH2
NH2
H2N
O
O OH
HO
-0.3
-0.2
-0.1
0.0
-10 0 10 20 30 40 50 60 70 80 90100110120130140150160
Time (min)µ
cal/s
ec
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal
/mol
e of
inje
ctan
t
ITC Titration of TAR RNA with Neomycin
N K H (kcal/mol)
1.09+0.04 (6.6+0.8)X105
-18.1+1.1
ITC titration of TAR RNA with Neomycin. Neomycin (80M) was serially injected to the TAR RNA (4M/ molecule) soltuion at 200C. Buffer 10mM Sodium Cacodylate,0.5mM EDTA, 100mM KCl at pH 7.0.
OHO
HO
O
O
O
NH2
OH
HO
OH
H2N
NH2
NH2
NH2
H2N
O
O OH
HO
Neomycin does not perturb binding of Hoechst to TAR
0
5000
1 104
1.5 104
2 104
2.5 104
3 104
3.5 104
4 104
0
5000
1 104
1.5 104
2 104
2.5 104
3 104
3.5 104
4 104
400 450 500 550
Fluorescence Titration of TAR into Hoechst in Presence of Neomycin
Em
issi
on
co
un
t (1
/s)
Wavelength
0
1 104
2 104
3 104
4 104
5 104
0
1 104
2 104
3 104
4 104
5 104
400 450 500 550
Fluorescence Titration of TAR into Hoechst in Absence of Neomycin
Em
issi
on
Co
un
t (1
/s)
Wavelength
Titration of concentrated RNA or 1:1 RNA:neomycin solution (100 M) into 1.8 mL Hoechst 33258 2 M up to 4 molar equivalents. In a 100 mM NaCl, 10 mM cacodylate pH 6.8 buffer; excited at 338 nm.
Arresting TAR Dynamics
Our strategyOur strategy:
•Not necessarily compete for Tat binding site, but arrest TAR motion trapping it in Not necessarily compete for Tat binding site, but arrest TAR motion trapping it in a non-recognizable bent conformation: effect a deleterious conformational a non-recognizable bent conformation: effect a deleterious conformational change upon ligand binding.change upon ligand binding.
•Design conjugates that take advantage of two modes of binding, increasing Design conjugates that take advantage of two modes of binding, increasing specificity and affinity, and ideally bind the two different helices as well specificity and affinity, and ideally bind the two different helices as well
Al-Hashimi (2005) Chem. Bio. Chem. 6: 1506 – 1519.
• TAR has inherent flexibility about its 3-nt bulge region
• Argininamide (Tat, and the RNA pol II complex/) binds via near-linear conformation
C
HOECHST-TAR NMR titrations
U38 NH3
G43 NH1C
• 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
Curves are fit according to a one-ligand per site model Meredith Newby
Identification of a HOECHST binding domain within TAR
Parkinson et al., (1992) Mag. Res. Chem. 1064-1069.
(1,5)
(16)(9)
(2,4)
• The hoechst proton resonances that shift the most upon binding to TAR are boxed in purple
Arresting TAR dynamics using click chemistry
Our strategyOur strategy:
•Design conjugates that take advantage of two modes of binding, increasing Design conjugates that take advantage of two modes of binding, increasing specificity and affinity, and ideally bind the two different helices as well specificity and affinity, and ideally bind the two different helices as well
G GU GC AC=G
G=CA=U
U G=CC
U23
A U40
G=CA=UC=GC=GG=C45
5'G=C3'
NHN
OH
N
N
NHN
OHO
HO
O
O
O
NH2
OH
OH
OH
H2N
NH2
NH2
NH2
H2N
O
O OH
HO
Neomycin Binding site
HOECHST Binding site
Synthesis of azide and alkyne functionalized neomycin
OHO
HO
O
OO
O
O
NH2
OH
OH
H2N
NH2
OH
HO
NH2
NH2
H2N
OH
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
NHR
NHRRHN
OHSPTO(a) Boc2O, DMF, Et3N, H2O, 75 0C, 18h (b) 2,4,6 Triisopropyl-
benzenesulfonyl chloride, pyridine, rt
1,7 octadiyne, CuSO4, sodium ascorbate, H2O, C2H5OH
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
N3
NHR
NHRRHN
OH
NN
N
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
NHR
NHRRHN
OH
R= Boc
TPS= S
O
O
90%
90%
55 %
NaN3, DMF:H2O
(10:1),100 0C
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
19
O
O
O
HOHO
NHR
RHN
O
OH
RHN
OHRHN
OH NHR
N3 OH
O
O
NHR
X
2 mol eq. 1 mol eq.
1. CuI, DIPEA, Toluene, r.t.2. 4M HCl in dioxane, dioxane
O
O
O
HOHO
NH2
H2N
O
OH
H2N
OHH2N
OH NH2
N OH
O
O
NH2
NN
XO
O
O
OHOH
H2N
NH2
O
HO
NH2
HO NH2
OHNH2
NHO
O
O
H2N
NN
R = Boc
12 HCl
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
O
OO
n = 4, 5
Structure of linkers used for dimer formationNeo-Neo dimer Linker length Structure of the
compound
DPA 51 7
DPA 52 7
DPA 65 7
DPA 53 8
DPA 54 8
DPA 55 10
DPA 56 10
DPA 58 16
DPA 60 20
NN NNeo N
NN
Neo
N
N NNeo N N
N Neo
N
N NNeo N N
N Neo
O
N
N NNeoN
NNNeo
N
NN
NeoN
NNNeo
NN N
Neo NNN
Neo
NN N
Neo NNN
Neo
NN N
Neo NNN
NeoOO4
NN N
Neo NNN
NeoOO6
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
Neo-Neo dimer Linker length ΔTm
DPA 51 7 10.19
DPA 52 7 9.30
DPA 65 7 9.30
DPA 53 8 9.62
DPA 54 8 8.22
DPA 55 10 7.57
DPA 56 10 6.05
DPA 58 16 5.40
DPA 60 20 3.24
Neomycin 0.200
2
4
6
8
10
12
0 7 7 7 8 8 10 10 16 20
1
10
.2
9.3
9.3 9.6
2
8.2
2
7.5
7
6.0
5
5.4
3.2
4
T
m
Linker length
Neo
0.48
0.5
0.52
0.54
0.56
0.58
0.6
0.62
20 30 40 50 60 70 80 90
HIV TAR RNADPA 52
A2
60
T(0C)
0.52
0.53
0.54
0.55
0.56
0.57
0.58
0.59
0.6
60 65 70 75 80
T(0C)
68.9
78.2
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
0 1 2 30
20
40
60
80
IC50 = 86 9 nM
log[TAR RNA], nM
Flu
ore
sce
nce
In
ten
sity
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.
NH
HN
NH
HN
NH
HN
NH
HN
NH
HN
O
O
O
O
O
O
O
O
O
O
NH
HN
O
O
NH
NH2
HN
NH2
H2N
HN
H2NNH
NH
NH2
HN
H2NO
NH
NH2
HN
HN
H2NNH
NH
NH2
HN
HN
O
O
HO
O
HO2CNH
O
NH
HN
NH2
O
O
O
S
O
N
NHO2C
HN
O
Fluorescein-labeled HIV-1 Tat peptide
0 1 2 30
10
20
30
40
50
DPA 55
IC50 = 80 9 nM
log[DPA 55], nM
Flu
ore
scen
ce I
nte
nsi
ty
IC50 values of dimers towards HIV-1 TAR RNA using FRET
Compound Linker length
IC50
(nM)Neomycin 713 ±
165 DPA51 7 77 ± 27 DPA52 7 60 ± 8 DPA65 7 56 ± 6 DPA53 8 47 ± 6 DPA54 8 128 ± 12 DPA55 10 80 ± 9 DPA56 10 59 ± 11 DPA58 16 61 ± 13 DPA60 20 67 ± 9
0
100
200
300
400
500
600
700
800
0 7 7 7 8 8 10 10 16 20
713
77 60 56 47
128
80
59 61 67
IC50
(n
M)
Linker length
Neo
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
Ethidium Bromide Displacement Assay between dimers and TAR RNA
0
0.2
0.4
0.6
0.8
1
0.5 1 1.5 2 2.5 3
Fra
cti
on
dis
pla
ced
log[DPA56 inM]5 104
1 105
1.5 105
2 105
2.5 105
3 105
560 600 640 680 720
Flu
ros
ce
nc
e
Wavelength(nm)25
IC50 values of dimers towards HIV-1 TAR RNA using FID titration (ethidium bromide)
Compound Linker length
IC50 (nM)
Neomycin 417 ± 115
DPA51 7 56 ± 1 DPA52 7 52 ± 23
DPA65 7 81 ± 2DPA53 8 36± 9
DPA54 8 67 ± 23
DPA55 10 99 ± 31
DPA56 10 97 ± 32
DPA58 16 67 ± 23
DPA60 20 74 ± 21 0
100
200
300
400
500
0 7 7 7 8 8 10 10 16 2041
7
56
52 8
1
36 6
7 99
97
67 74
IC50 (
nM
)
Linker length
Neo
5 104
1 105
1.5 10 5
2 105
2.5 10 5
3 105
550 600 650
Flu
ro
sc
en
ce
Wavelength(nm)
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.00.0
0.2
0.4
0.6
0.8
1.0
Fra
ctio
na
l d
isp
lace
me
nt
log[DPA 56 in M]
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
Comparison of IC50 values from two methods.
0
100
200
300
400
500
600
700
800
0 7 7 7 8 8 10 10 16 20
FRET assayEthidium assay
IC50
(nM
)
Neo
Linker length
Compound Linker length
IC50 (nM)
FRET FID
Neomycin 713 ± 165
417 ± 115
DPA51 7 77 ± 27 56 ± 1
DPA52 7 60 ± 8 52 ± 23
DPA65 7 56 ± 6 81 ± 2
DPA53 8 47 ± 6 36± 9
DPA54 8 128 ± 12 67 ± 23
DPA55 10 80 ± 9 99 ± 31
DPA56 10 59 ± 11 97 ± 32
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
28
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 HIV TAR RNA Conformation, Biochemistry.51 (2012) 2331-2347
29
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 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)
30
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 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.
(µM)
Day
2
Day
4
Fluorescein Labeled TAT-
peptide
-- 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 DPA53 9.62 56 ± 6 nM 81 ± 2 nm 17 63% (8) 9 2% 40%
4 DPA54 8.22 128 ± 12 nM 67 ± 23 nm 69 31% (17) 17 3-5% 80%
5 DPA55 7.57 80 ± 9 nM 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 DPA58 5.40 61 ± 13 nM 67 ± 23 nm -- -- -- -- --
8 DPA60 3.24 67 ± 9 nM 74 ± 21 nm -- -- -- -- --
9 DPA65 9.30 47 ± 6 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
DPA52 7 7.08x107 8.89x107 1.39x107 7.50x107 6.93x106
DPA65 7 1.39x108 9.97x107 1.25x107 6.91x107 -
DPA53 8 (phenyl) 1.46x108 - - - -
DPA54 8 (butyl) 2.61x107 2.17x107 - 1.23x107 2.11x106
DPA55 10 1.06x107 3.64x107 3.53x106 2.83x107 2.60x106
DPA56 10 6.60x107 6.33x107 4.97x106 5.87x107 3.84x106
DPA58 16 7.58x106 6.84x107 1.35x107 7.23x107 4.84x106
DPA60 20 2.53x107 4.36x107 1.97x106 2.68x107 1.52x106
Neomycin N/A - 2.99x107 - 1.58x107 -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.
U
G
CU
G
UGCC
UC
A
G
GAC
G5` 3`
C
U
A
G
GCG
C
UC
AG
Wildtype TAR
GG
UGCC
UC
A
G
GAC
G5` 3`
C
U
A
G
GCG
C
UC
AG
Bulgeless TAR
U
G
U C
U
U
UGCC
UC
A
G
GAC
G5` 3`
GCG
C
UC
AG
C
Tetraloop TAR
G
U C
U
UGCC
UC
A
G
GAC
G5` 3`
GCG
C
UC
AG
Bulgeless Tetraloop TAR
UU
G
U
G
UGCC
UC
A
G
GAC
G5` 3`
C
U
A
G
GCG
C
UC
AG
U3 Bulge TAR
O
O
O
HOHO
NH2
H2N
OOH
H2N
OHH2N
OH NH2
NOH
O
O
NH2
N N
X
O
O
O
OHOH
H2N
NH2
O
HO
NH2
HO NH2
OHH2N
NHOO
O
H2N
NN
Variable Selectivity/Affinity
Variable Linker
Conformational Differences
Arresting TAR dynamics using click chemistry Part II: Benzimidazole-aminosugars
Our strategyOur strategy:
•Design conjugates that take advantage of two modes of binding, increasing Design conjugates that take advantage of two modes of binding, increasing specificity and affinity, and ideally bind the two different helices as well specificity and affinity, and ideally bind the two different helices as well
G GU GC AC=G
G=CA=U
U G=CC
U23
A U40
G=CA=UC=GC=GG=C45
5'G=C3'
NHN
OH
N
N
NHN
OHO
HO
O
O
O
NH2
OH
OH
OH
H2N
NH2
NH2
NH2
H2N
O
O OH
HO
Neomycin Binding site
HOECHST Binding site
Synthesis of clickable Hoechst 33258 derived benzimidazole alkyne
HO
CHO
O
OHC
1
N
N
NH2
NH2
Cl NH2
NO2
(i) N-methyl piperazine,
K2CO3, DMF, 80 0C
(ii) Pd-C, H2, EtOH
O
OHC
1
NH
NO
N
N 2
Na2S2O5, H2O, C2H5OH, reflux
Propargyl bromide,
K2CO3, acetone, 60 0C
55%
75 %
90 %
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
Synthesis of clickable Hoechst 33258 derived bisbenzimidazole alkyne
NO2
NO2
O OH(i) SOCl2, reflux (ii) MeNHOMe.HCl, pyridine, DCM,rt (iii) Pd-C,H2,EtOH (iv) 4-(prop-2-ynyloxy)benzaldehyde, Na2S2O5, C2H5OH H2O, reflux (v) LAH, THF-Ether
NH
NO
OHC
N
N
NH2
NH2
Na2S2O5, H2O, C2H5OH reflux
NH
N
N
NNH
N
O
NH
NO
OHC+
74 %
65 %
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
Synthesis of azide functionalized benzimidazole
HO
CHO
O
OHC(i) 2-bromo ethanol, PPh3, DIAD,
dioxane (ii) NaN3 , DMF, 90 0C
1N3
N
N
NH2
NH2
Cl NH2
NO2
N-methyl piperazine, K2CO3, DMF, 80 0C,55 %, (ii) Pd-C, H2, EtOH
NH
NO
N
N
Na2S2O5, H2O, C2H5OH, reflux
2
N3
O
OHC
1N3
69 %
55 %
65 %
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
N
NH
NO
N3N3
n
N
N
NH
NO
N N
NN3
n
Sodium ascorbate, CuSO4,C2H5OH, H2O, rt n = 1, 4, 5, 7, 9
65-80 %
N
N
NH
NO
n
N
N
NH
NO
N3
N
NN n
Sodium ascorbate, CuSO4, C2H5OH, H2O, rt
n = 0, 2, 4
75-90 %
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
Synthesis of triazole linked neomycin-benzimidazoles
+
N
N
NH
NO
N
NN
N
N
N OHOHO
O
OO
O
O
NH2
HO
OH
H2N
NH2
HO
NH2
NH2 H2N
OHN
NN
n
N
N
NH
NO
N
NN
N3
n
OHOHO
O
OO
O
O
NHR
HO
OH
RHN
NHR
HO
NHR
NHR RHN
OHN
NN
(a) Sodium ascorbate, EtOH, H2O, CuSO4 (b) Dioxane, 4M HCl, rt 55-75 %
n = 1, 4, 5, 7, 9
R=Boc
N. Ranjan, P. Kellish, D.P. Arya, 2013, submitted
+
N
N
NH
NO
N
NN
N
N
N OHOHO
O
O
OO
O
NHR
HO
OH
RHN
NHR
HO
NHR
NHR RHN
OHN
NN
n
N
N
NH
NO
N
NN
N3
n
OHOHO
O
O
OO
O
NHR
HO
OH
RHN
NHR
HO
NHR
NHR RHN
OHN
NN
CuSO4, NaAscC2H5OH, H2O
N
N
NH
NO
N
N
N OHOHO
O
O
OO
ONHR
OH
OH
RHN
NHR
OH
N3
NHR
NHRRHN
OH+
EtOH, H2OCuSO4, NaASc
N
N
NH
NO
N
N
NOHO
HO
O
O
OO
ONHR
OH
OH
RHN
NHR
OH
NHR
NHR RHN
OHN
NN
R=Boc
n
n
A
B
N
N
NH
NO
N
NN
N
N
N OHOHO
O
O
OO
O
NHR
HO
OH
RHN
NHR
HO
NHR
NHR RHN
OHN
NN
n
N
N
NH
NO
N
N
NOHO
HO
O
O
OO
ONH2
OH
OH
H2N
NH2
OH
NH2
NH2H2N
OHN
NN
n
A
B
N
N
NH
NO
N
NN
N
N
N OHOHO
O
O
OO
O
NH2
HO
OH
H2N
NH2
HO
NH2
NH2 H2N
OHNNN
n
R=Boc
N
N
NH
NO
N
NN
OHOHO
O
O
OO
ONHR
OH
OH
RHN
NHR
OH
NHR
NHR RHN
OHN
NN
R=Boc
n
Dioxane,4M HCl in dioxane
Dioxane,4M HCl in dioxane
Scheme of Neomycin- Benzimidazole Conjugate Synthesis
Scheme for protected Neomycin-Benzimidazole synthesis
Scheme for deprotection of protected Neomycin-Benzimidazole conjugates
Yield= 50-63% for two steps
0
2
4
6
8
10
0 0 4 11 11 12 12 14 16 19 20 22 24
T
m
Linker Length
Ne
om
yc
in
Be
nzi
mid
azo
le A
lky
ne
DP
A1
23
DP
A1
15
DP
A1
14
DP
A1
13
DP
A1
22
DP
A1
21
DP
A1
18
DP
A1
20
DP
A1
19
DP
A1
17
DP
A1
16
0
50
100
150
200
250
300
350
400
0 4 11 11 12 12 14 16 19 20 22 24
285
33
78 83
140
147
81
184
140 15
0
200
180
IC50
(nm
)
Linker LengthN
eo
my
cin
DP
A1
23
DP
A1
17
DP
A1
16
DP
A1
15
DP
A1
14
DP
A1
13
DP
A1
22
DP
A1
21
DP
A1
20
DP
A1
19 D
PA
11
4N. 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
0.34
0.36
0.38
0.4
0.42
0.44
20 30 40 50 60 70 80 90 100
ControlNeo-BenBenzimidazole
A2
60
T(0C)
TAR with Tm(0C) Tm(0C)
None 68
Neomycin 70 2
Benzimidazole 67 -1
Neo-Benzimidazole 123 74 6
Table forTm
UV Melting studies
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.
Ed Robinson
5% Toxicity concentrations and maximum protection from HIV cytopathic effects in
MT-2 cellsCompound 5% Toxicity
concentration (M)
Maximum protection
(concentration in M)
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 33258
18 2% (2)
Water None 2%
Differential reactivity of mono and bisbenzmidazoles with 5’-azido-neomycin
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
N3
NHR
NHR RHN
OH
HN
NO
N
N
NH
N
NN N
H
N
O
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
N
NHR
NHR RHN
OH
NN
O
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
N
NHR
NHR RHN
OH
NN
NH
N
N
N
HN
N
NH
N
NN
O
CuSO4, Ethanol-H2O
CuSO4, Ethanol-H2O
~20% reaction based on azide
65-85 %
R= Boc
Alternative synthesis of neomycin- Hoechst 33258 conjugate
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
N3
NHR
NHR RHN
OHn =1, 4, 9
n
O
HN
NOHC +
O
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
N
NHR
NHR RHN
OH
NNHN
N
OHC
n
n =1, 4, 9
CuSO4,sodium ascorbateH2O,DMSO, heat
60-75 %
R=Boc
Benzimdazole derived synthesis of neomycin- Hoechst 33258
O
OHO
HO
O
OO
O
O
NHR
OH
OH
RHN
NHR
OH
N
NHR
NHR RHN
OH
NNHN
N
OHC
nn =1, 4, 9N
N
NH2
NH2
(i) Na2S2O5, H2O, C2H5OH reflux(ii) Dioxane, 4M HCl
60-75 %
O
OHO
HO
O
OO
O
O
NH2
OH
OH
H2N
NH2
OH
N
NH2
NH2 H2N
OH
NN
NH
N
N
N
HN
N
n =1-9
n
+
Compound
IC50
(nm)
ΔTm
(0C)
Neomycin 785 1
DPA165 60 6
DPA166 78 6
OHO
HO
O
O
O
NH2
OH
HO
OH
H2N
NH2
NH2
NH2
H2N
O
O OH
NH
N
ON
N
NH
N
NN
N
6Cl
OHO
HO
O
O
O
NH2
OH
HO
OH
H2N
NH2
NH2
NH2
H2N
O
O OH
NN
NNH
N
ON
N
NH
N
.6HCl
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 cellsCompound 5% Toxicity
concentration (M)
Maximum protection
(concentration in M)
DPA165 >189 44% (189)DPA166 11 18% (6)
neomycin >206 9% (206)Hoechst 33258
18 2% (2)
Water None 2%
Ed Robinson
Future directions: DPA83 with TAR RNA
1.0 1.2 1.4 1.6 1.8 2.0 2.2
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Fra
ctio
n d
isp
lace
d
log[DPA83 in M]
Compound IC50 (nM)
DPA83 13.1±7.1
O
O
O
HOHO
NH2
H2N
O
OH
H2N
OHH2N
OH NH2
N OH
O
O
NH2
NN
O
NN
N
N
O
O
O
HOHO
NH2
H2N
OOH
H2N
HO
H2N
OHNH2
N
OH
O
O
NH2
NN
O
N
N
N
O
NH
N
O
N
N
NN
N
G GU GC AC=G
G=CA=U
U G=CC
U23
A U40
G=CA=UC=GC=GG=C45
5'G=C3'
NHN
OH
N
N
NHN
OHO
HO
O
O
O
NH2
OH
OH
OH
H2N
NH2
NH2
NH2
H2N
O
O OH
HO
Neomycin Binding site
HOECHST Binding site
DPA83
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 non-toxic doses.
Meredith Newby, Dept of Physics
Ed Robinson, Department of Pathology and Laboratory Medicine, UC Irvine
Glaxo Smithkline (Raleigh, NC)
$ NIH
Acknowledgements
Nihar RanjanSunil KumarPatrick Kellish
Dr. Derrick Watkins
Dr. Andy Norris