Targeting RNA dynamics for HIV inhibition Dev P. Arya ([email protected]) Department of Chemistry, Clemson University

Targeting RNA dynamics for HIV inhibitionDev P. Arya ([email protected])Department of Chemistry, Clemson University

Nucleic Acid Recognition Organic Chemistry/Medicinal Chemistry/Biophysics/Molecular

Biology/Chemical Biology/Microbiology/Pathology

http://chemistry.clemson.edu/people/[email protected]

(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.

http://chemistry.clemson.edu/people/arya.html

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



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


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


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


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


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)


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]


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


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%


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


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


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 -- -- -- -- --


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



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



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 %


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 %


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 %


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

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



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

Documents

Targeting RNA dynamics for HIV inhibition Dev P. Arya ([email protected]) Department of Chemistry, Clemson University