Addressing the global HIV/AIDS epidemic: non-

subtype B HIV-1 protease

C. Taylor Gilliland

Mentor: Ben M. Dunn, Ph.D.Dept. of Biochemistry and Molecular Biology

College of MedicineUniversity of Florida

HIV/AIDS Epidemic Status

Source: 2006 Report on the Global AIDS Epidemic, UNAIDS, WHO

A global view of HIV infectionA global view of HIV infection38.6 million people [33.4_46.0 million] living with HIV, 2005

2.4

HIV/AIDS Epidemic Status

Source: 2006 Report on the Global AIDS Epidemic, UNAIDS, WHO

Impact of AIDS on life expectancy in five African countries, 1970Impact of AIDS on life expectancy in five African countries, 1970 ––20102010

Life expectancy at birth (years)

Source: United Nations Population Division (2004). World Population Prospects: The 2004 Revision, database.

Botswana

South Africa

Swaziland

Zambia

Zimbabwe

1970–1975 1975–1980

1980–19851985–1990

1990–19951995–2000

2000–20052005–2010

70

65

60

55

50

45

40

35

30

25

20

4.1

HIV Genetic Epidemiology

Takebe et al. (2004) Pediatrics International. 46. 236-244.

Spira et al. (2003) J of Antimicrobial Chemotherapy. 51. 229-340.

Geographic Distribution of HIV-1 Subtypes and RFs

CC

CC

A,AGA,AG

FF

BFBF

BB BB

DD

Role of HIV-1 Protease

LTR LTR

VPR VPUENV

TAT

NEFGAG POLVIF

REV

MA CA p2 NC

MA

A B C D’ D E

CA p2 NC p6

p6*

GF H

PR RT INRNaseH

1MUI

QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.

1MUI

URF A/C PR

Subtype F PR

Subtype A2 PR

Subtype H PR

CRF12_BF PR

CRF02_AG PR

CRF01_AE PR

Subtype C PR

Non-Subtype B HIV-1 Proteases Studied in Dunn Lab

AtazanavirTipranavir

Clinically Approved HIV-1 Protease Inhibitors

MethodologySub-cloning protease gene in pET23a vector

Transformation and expression in E. coli BL21(DE3) pLysS

1mM IPTG @ OD 0.8French press/ 27% Sucrose bed

Inclusion bodies

Unfold in 8M urea, 0.2 %BME

Dialysis in 50mM Na2HPO4 pH 7.3 buffer for ~16h

30, 50% Ammonium sulfate precipitation

Size exclusion column

Thermodynamics Kinetics Crystallography

SDS-PAGE gel

Activity test

DNAsequencing

Stored @ -800C

SDS-PAGE gel

Inhibition of HIV-1 PrInhibitor

RTV IDV SQV NFV APV LPV ATV TPV

Subtype

Ki (nM)

B (LAI)

0.7 ± 0.1 1.8 ± 0.1 1.7 ± 0.4 2.2 ± 0.3 0.4 ± 0.10.11 ± 0.03

0.07 ± 0.01

0.4 ± 0.04

C 0.27 ± 0.06

3.3 ± 0.3 2.7 ± 0.3 2.6 ± 0.20.29 ± 0.03

0.2 ± 0.02

0.13 ± 0.01

0.13 ± 0.03

A 0.25 ± 0.03

2.1 ± 0.20.80 ± 0.06

2.0 ± 0.30.26 ± 0.03

0.03 ± 0.01

0.10 ± 0.02

0.56 ± 0.04

H 0.18 ± 0.03

1.7 ± 0.20.65 ± 0.10

1.5 ± 0.20.35 ± 0.07

1.1 ± 0.26

0.03 ± 0.004

0.22 ± 0.04

D 0.18 ± 0.03

2.1 ± 0.2 2.1 ± 0.2 3.1 ± 0.40.35 ± 0.06

0.07 ± 0.02

0.05 ± 0.01

0.43 ± 0.07

AC P81S

1.5 ± 0.2 10.2 ± 0.4 18.7 ± 0.7 5.1 ± 0.90.79 ± 0.09

2.3 ± 1.10.54 ± 0.06

0.9 ± 0.1

CRF-BF

0.07 ± 0.02

1.7 ± 0.20.74 ± 0.08

1.3 ± 0.20.46 ± 0.08

0.1± 0.20.06 ± 0.01

0.56 ± 0.05

F 0.77 ± 0.05

3.1 ± 0.5 1.3 ± 0.30.48 ± 0.02

0.41 ± 0.04

1.0 ± 0.16

0.81 ± 0.07

Inhibitor

Rela

tive F

old

Ch

an

ge

Relative Fold Change in Ki Values of Non-Subtype B Pr

Inhibitor

Rela

tive F

old

Change

Effect of P81S Mutation in URF-AC and Subtype B

Inhibitor

RTV IDV SQV NFV APV LPV ATV TPV

Subtype Ki (nM)

URF-AC 0.48 ± 0.1 0.97 ± 0.1 1.2 ± 0.3 0.51 ± 0.20.089 ±

0.020.15 ± 0.02

0.1 ± 0.06

URF-AC P81S 1.46 ± 0.210.23 ±

0.3718.7 ± 0.7

5.08 ± 0.89

0.79 ± 0.09

2.3 ± 1.10.54 ± 0.06

0.9 ± 0.1

B P81S 2.7 ± 0.2 0.81 ± 0.04 1.2 ± 0.2 0.52 ± 0.06 0.25 ± 0.03

B PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFAC ------------V------R---------------IN---K---------------------------K-----------S-------M----------

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

LPV

P81 P81’

PDB: 1MUI

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

LPV

S81

S81’

Conclusions- tight binding of inhibitors to all subtypes and recombinant forms

- effectiveness of tipranavir (TPV)

- P81S as possible secondary drug-resistance mutation

- continuing efforts to obtain first structure of non-subtype B HIV-1 protease

AcknowledgmentsBen M. Dunn, Ph.D., College of Medicine, University of FloridaRoxana M. Coman, M.D., Dunn LabMelissa Marzahn, Dunn LabMarty Fernandez, Dunn LabKaran Desai, Dunn Lab

Alex Wlodawer, Ph.D., MCL, NCI-Frederick, NIHTinoush Moulaei, Ph.D., Wlodawer Lab

Arnold and Mabel Beckman Foundation