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

Page 2 © CSIR 2006 www.csir.co.za

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


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?


Do We Deny It?

AIDS DENIALISM!


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


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


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


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


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


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


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


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


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



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



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



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



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

2000

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

2000

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)


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)


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


In the face of adversity It is not all gloom!

In the face of adversity It is not all gloom!

Aptamers: Cutting Edge Technology to Combat HIV/AIDS

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