Aptamers: Cutting Edge Technology to Combat HIV/AIDS
Transcript of Aptamers: Cutting Edge Technology to Combat HIV/AIDS
Aptamers: Cutting Edge Technology to Combat HIV/AIDS
Makobetsa Khati (MScMed, DIC, DPhil, MPH)
27th September 2006
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South African Burden of Diseases
HIV/AIDS (38% YLLs) Diseases of poverty, e.g. TB (25%YLLs)
Chronic diseases, e.g. Heart diseases (21%YLLs) Injuries (16% YLLs)
Bradshaw et al. 2003. Initial Burden of disease estimates for Sou th Africa. SAMJ. 76(9): 682- 8
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Common Denominator in the African
burden of disease
HIV/AIDS is a common denominator in at least three o f the South African quadruple burden of diseases
� HIV/AIDS fuels the TB epidemic (disease of poverty)
� HIV/AIDS one of the underlying cause of some chroni c diseases (e.g. cardiomyopathy)
HIV/AIDS is the defining public health problem of ou r generation.
Greatest challenge facing South Africa and the entire African continent today.
The epidemic has attained a scale at which the impa ct on the economy and, even more broadly, on our society, is bot h evident and very serious
Now that we know the defining public health problem of our generation
What do we do?
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Do We Deny It?
AIDS DENIALISM!
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OrOrOrOr…………
““““Do we accept the diagnosis but Do we accept the diagnosis but Do we accept the diagnosis but Do we accept the diagnosis but defy the verdictdefy the verdictdefy the verdictdefy the verdict””””
Norman Cousins, 1989: The Biology of Hope
But How?
Aptamers: Aptamers: Aptamers: Aptamers: Part of the feasible solutionPart of the feasible solutionPart of the feasible solutionPart of the feasible solution
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Aptamers: An innovative technology platform
Aptamers: An innovative technology platform
Artificial nucleic acid ligands (ssRNA) selected in vitro for specific binding to a target.
Form well-defined 3-D shapes, allowing them to bind target molecules in a manner conceptually similar to antibodies (Abs)
Have molecular recognition properties of Abs
Small (6 kDa- 40 kDa) to probe protein structure and can penetrate viral defence mechanisms
Combine optimal characteristics of small molecules and Abs: High affinity & specificity
Functional products in their own right
Low immunogenicity
Resistant to nucleases
A new approach to drug discovery
N
N
NH2
O
OHOH
OOO-
O P
O
OO
PO
O
OH
O P
O-
O
O P
O-
O-
NH2
O
N
N
NH
N
F
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Structural lessons from aptamer-protein complexes
Aptamers are prone to bind to functional domains of t he target protein.
� E.g. substrate binding pockets or allosteric sites
� Modulate the biological function of the target mole cule
Aptamers are pre-existing molecules that have not bee n exploited during evolution
Bunka & Bunka & Bunka & Bunka & StockleyStockleyStockleyStockley 2006. Aptamers come of 2006. Aptamers come of 2006. Aptamers come of 2006. Aptamers come of ageageageage−−−−atatatat last. Nature Reviews 4: 588last. Nature Reviews 4: 588last. Nature Reviews 4: 588last. Nature Reviews 4: 588----596596596596
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AptamersAptamersAptamersAptamers: a Paradigm of Darwinian : a Paradigm of Darwinian : a Paradigm of Darwinian : a Paradigm of Darwinian Evolution in a Test TubeEvolution in a Test TubeEvolution in a Test TubeEvolution in a Test Tube
or
Made to fit? Off the peg?
�X
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Aptamers as therapeutics & multifunctional tools
Proske et al. 2005. Aptamers –basic research, drug development, a nd clinical applications. Appl Micr Biotech 69:367-74
Our Application of the Aptamer
Technology to Combat HIV/AIDS
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Problem Identified!
Entry inhibitorse.g. T20
RT inhibitorse.g. AZT &
NVP
PR inhibitorse.g. Saquinavir
Almost all of the ARV drugs currently in clinical use only act on the virus after it has infected target cells
Treatment of infected individuals is costly to our health service.
Drug resistance compounds problem
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More ProblemMore Problem
HIV cunningly shield itself from the immune system attack:
Occlusion of receptor binding sites
Hypervariable loops
Conformational shifts Extensive glycosylationSu gp120 is both a multiple lock system and the master keyLynchpin and Achilles' heelStrong and weak pointDesirable target for therapeutic intervention
gp120
CD4 d1-d2
17b Fab
Kwong et al., 1998. Nature 393:648-59
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BIASelection:
Isolation of anti-gp120 aptamers
BIASelection:
Isolation of anti-gp120 aptamersDNA Library
(1015)
Allow dissociationof weak binders
Elute bound RNA with 7M urea
discardunbound
RT-PCR
EnrichedDNA
library
gp120 (HIV-1Ba-L)
Transcribe 2´F RNA
SELEX protocol
Wash off weak binders
Khati et al. 2003. J. Virol. 77(23): 12692-98
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Neutralization of HIV-1Ba-L by aptamersNeutralization of HIV-1Ba-L by aptamers
Ctrl B1 B3 B4 B5 B9 B11 B19 B28 B30 B31 B33 B36 B380
1
2
3
4
5
6A
log
10IU
/ml
control
No neutralisation
1-2 log10
≥≥≥≥ 3 log10
≥≥≥≥ 4 log10
Virus input (log10 dilution)
Aptamer B4Control aptamer (SA19)
Limiting-dilution infectivity assay in human PBMC
LTR-PCR
β- globinPCR
Khati et al. 2003. J. Virol. 77(23): 12692-98
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Utility of aptamers against clinically
relevant HIV PIs
Isolated from a 29 year old woman from Cameroon
Group OBCF07
Isolated from a Senegalese woman living in the USA with full blown AIDS
A97USSN54
Isolated from seropositive individual in MalawiC93MW960
Isolated from seropositive individual in South Africa
C97ZA003
Isolated from seropositive individual in UgandaD92UG035
Derived from asymptomatic individual in Thailand
E92TH021
Isolated from seropositive individual in BrazilF93BR029
CharacteristicSubtypeHIV-1 Isolate
Khati et al. 2003. J. Virol. 77(23): 12692-98
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PanPanPanPan----clade neutralization of HIVclade neutralization of HIVclade neutralization of HIVclade neutralization of HIV----1 1 1 1 clinical isolates by aptamersclinical isolates by aptamersclinical isolates by aptamersclinical isolates by aptamers
92TH02
1
93BR02
9
92UG03
5
97ZA00
3
93MW
960
BCF07
97USSN54
0.0
0.5
1.0control aptamerB1B4B11B19B28B33B38B40B44B65B84
Rel
ativ
e R
T ou
tput
93MW
960
BCF0797
USSN54
0.0
0.5
1.0
AZT2G1217b IgGb12 IgG1
Rel
ativ
e R
Tou
tput
Khati et al. 2003. J. Virol. 77(23): 12692-98
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Mechanism of neutralization: Competition of
aptamers with NAbs for binding to gp120
0.01 0.1 1 10 100 1000-20
0
20
40
60
80
17b IgG17b IgG + CD4b12 IgG19b
[Aptamer B40] nM
% in
hibi
tion
ofan
tibod
y bi
ndin
g
C
N
CD4BS
CD4i
2G12
V1/V2
V3
Inner Outer
distal
proximal0
10
20
30
40
50
17b IgG
19b
17b IgG +CD4
1 10 1000
[Aptamer B4] nM
% in
hibi
tion
ofan
tibod
y bi
ndin
g
Khati et al. 2003. J. Virol. 77(23): 12692-98
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Aptamers interfere with gp120 binding to its Aptamers interfere with gp120 binding to its Aptamers interfere with gp120 binding to its Aptamers interfere with gp120 binding to its
natural receptorsnatural receptorsnatural receptorsnatural receptors
Aptamers interfere with gp120 binding to its Aptamers interfere with gp120 binding to its Aptamers interfere with gp120 binding to its Aptamers interfere with gp120 binding to its
natural receptorsnatural receptorsnatural receptorsnatural receptors
b12 IgG
-100 0 100 200 300 400 500 600 700-500
0
500
1000
1500
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2500
3000
3500
Time (seconds)
SP
R(R
U)
2G12 mAb
-100 0 100 200 300 400 500 600-500
0
500
1000
1500
2000
2500
3000
3500
4000
Time (seconds)
M D Y Q V S S P I Y D I N Y Y T S E G A G K Biotin
SO3 SO3
Aptamer B40
-100 0 100 200 300 400 500 600-500
0
500
1000
1500
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2500
Time (seconds)
SP
R (
RU
)
Aptamer B19
-100 0 100 200 300 400 500 600-500
0
500
1000
1500
2000
2500
Time (seconds)
gp 120 (HIV-1 Ba-L )
-100 0 100 200 300 400 500 600-500
0
500
1000
1500
2000
2500
Desulfated-CCR5 peptide
Sulfated-CCR5 peptideSHuCD4
Control
Time (seconds)
SP
R(R
U)
Aptamer B4
-100 0 100 200 300 400 500 600-500
0
500
1000
1500
2000
2500
Time (seconds)
CD4
Dey, Khati et al. (Nov 2005) J. Virol. 79 (21)
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Use aptamers for analysis and neutralization of endemic, South African strains of HIV-1 from adult and pediatric patients at various stages of disease.
Exploit aptamers to provide structural leads for the development of potent and even smaller molecules that can mimic their HIV neutralizing properties.
Determine if the structural mimetic would provide hope for salvage therapy for patients failing current ARV’s including HAART, as well as alternatives for initial therapy for newly infected individuals.
Bio2Biz: Bench to Bedside(Current Focus)
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Bio2Biz: Bench to Bedside(Future Focus)
Exploit aptamers to
elucidate and treat HIV
associated cardiomyopathy
Isolate aptamers against early
markers of active TB and develop
rapid and reliable diagnostics
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In the face of adversity It is not all gloom!
In the face of adversity It is not all gloom!