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Rachel Botbyl
Erin Schuetz Lab
Pharmaceutical Sciences Department
About me
Cytochrome P450 (CYP) enzymes play
an important role in drug metabolism
CYP2C9/19
CYP2D6 CYP1A2
CYP2B6
CYP2C8
CYP2E1
CYP3A4/5
3A4WTseq(1>156)caagctTGAGAGCACAGTATTGAGAGCACAGtagacac
3A414bpDELseq(1>142)caagctTGAGAGCACAGtagacac
Promoter variant is correlated with higher
drug clearance in African American cohort
Midazolam clearance
Midazolam clearance
Higher level of drug clearance
suggests increased activity
Dr. Stephen D. Hall
Drug Disposition, Eli Lilly and Co.,
Indianapolis
Increased 3A4 activity decreases
clinical efficacy of drugs
Active Inactive
SV SV--OH
WT 3A4
Reductions in LDL Cholesterol
SV SV---OH
p=0.35 (additive)
WT VAR WT VAR
WT and VAR elements were cloned
into pGL4.23 vector
Is there a difference in basal luciferase
promoter activity in VAR compared to
WT?
14bpdeletion
VAR TGAGAGCACAG LUC
Promoter variant had a higher activity compared to WT
indicating deletion allele is an increased activity allele
LS180 HepG2
1.6
1.8 **
Fold Change over pGL4.23
1.2 1
1 0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0 0
WT VAR WT VAR
**p 0.005
Promoter variant is lacking GTF2
binding site
Matrix Common to
Family p-value Match Total #sequences TempSeq_LO5z_vH3
O$VTBP 0.14 2 1 2
V$DICE 0.05 1 1 1
V$FKHD 0.10 1 1 1
V$RBP2 0.02 1 1 1
V$RU49 0.04 2 1 2
V$ZF13 0.06 1 1 1
TempSeq_X8CP4Be
_
Matrix Match Common to
Family p-value Total #sequences
DEL allele does not have DICE so co- O$VTBP 0.14 2 1 2
repressors can not bind and hence V$FKHD 0.10 1 1 1
V$RBP2 0.02 1 1 1
could lead to increase in 3A4 V$RU49 0.04 2 1 2
transcription/Activity
Does GTF2 bind to 14 base pair
deletion sequence and suppress?
GTF2
2.5
LS180 HepG2
1.6
Fold Change over pGL 4.23
1.5 1
0.8 **
1
** 0.6
0.5 0.4
0.2
0 0
Control GTF2 Control GTF2
**p 0.005
Does GTF2 suppress luciferase
activity in WT but not in VAR?
GTF2
GTF2
14bpdeletion
VAR TGAGAGCACAG LUC
In Summary
Current Conclusions
The promoter variant is a high activity allele and is associated with
increased drug clearance
GTF2 binds to the 14-bp sequence which is absent in the variant
GTF2 plays an important role in suppressing CYP3A4 expression
Future Directions
Explore the effect of GTF2 in the context of the CYP3A4 promoter
POE Program
Suzanne Gronemeyer, PhD
James Marmion
Jenna Bencini
POE Class of 2016