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
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