2022 Undergraduate Research Symposium: Justin Ahrens Graduate co-author: Maddy Denison The main goal of this project is to use IrIII complexes to monitor cytochrome P450 (CYP) activity. CYP P450 metabolism determines how quickly drugs are excreted from the body and what kind of dose is required for pharmaceuticals to be effective. Existing probes for CYPs are non-fluorescent, but when CYPs metabolize them, they are “turned on” and fluoresce. Even though these probes are able to monitor metabolism, once they are metabolized and “turned on” they cannot be reversed or turned off. Therefore, this is no current way to monitor dynamic changes in CYP activity continuously over time. The Kodanko laboratory is addressing this unmet need by developing IrIII complexes that are reversible sensors of CYP enzymes. A chiral resolution strategy with enantioenriched ligands was used to prepare four analogs of IrIII complexes with different stereochemistry. These complexes were evaluated as emissive sensors in human liver hepatocytes to establish which IrIII complex is the lead compound for sensing CYP in living cells. Results from this multi-disciplinary study will be reported.

1. Using Iridium (III) Complexes to
Sensor the Activity of Cytochrome
P450 3A4 Enzyme to Monitor Drug
Metabolism
JUSTIN AHRENS
NCUR PRESENTATION
3-23-22
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2. 2
Kodanko Lab Workflow
Conceptualization
and compound
design
Chemical synthesis Structural characterization
and purity assessment
Photochemical
characterization
Extracellular
biological properties
In cellulo
experiments
Draw Conclusions
(and hopefully publish papers)

3. Talk Overview
Background
Barber Summer Project
UROP Project
Current Project
Conclusions and Future Directions
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4. What Is Cytochrome P450 (CYP)
A superfamily of enzymes that is normally found in hepatocytes
The primary function of CYPs is to aid in drug metabolism for the body.
The goal of CYPs is make drugs easier to excrete.
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cytochrome P450 enzyme system. (2015). National Cancer Institute. Retrieved March 26, 2022, from
https://www.cancer.gov/publications/dictionaries/cancer-terms/def/cytochrome-p450-enzyme-system.

5. Cytochrome P450
5
Zanger, U. M.; Schwab, M. Cytochrome P450 Enzymes in Drug Metabolism: Regulation of Gene Expression, Enzyme Activities, and
Impact of Genetic Variation. Pharmacology & Therapeutics 2013, 138 (1), 103–141. https://doi.org/10.1016/j.pharmthera.2012.12.007

6. Main Goal
Develop iridium(III) probes that will be able to monitor
CYP3A4 activity and active site occupancy during drug
metabolism in hepatocytes
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7. Barber Project Goals
Build a library of Ir(III) probes to learn more about how cytochrome P450 3A4 binds to
probes to learn more about the drug metabolism process
Synthesize lead Ir(III) probes that will be able to monitor cytochrome P450 3A4 enzymes
activation to sensor drug metabolism in hepatocytes
Building a new library of Ir(III) probes that will be analogs of the lead probe but instead
changing the ligands to try and make more lead Ir(III) probes
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8. 8
Barber
Project
Structures

9. Barber Project Results
 Synthesis and characterization of all the iridium(III) probes was very easy to make and
purify at high yields
Iridium(III) probe #1 was deemed as the lead complex because it inhibited CYP3A4 the
best out of all the probes
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0
10000
20000
30000
CYP3A4 Activity
Chemiluminescence
HEPG2-CYP3A4 / DMSO
1 μM of Complex 5
3 μM of Complex 5
10 μM of Complex 5
0.3 μM of Complex 1
1 μM of Complex 1
3 μM of Complex 1
10 μM of Complex 1
A
AB
B
C
AB
AC
D
E

10. Barber Project Conclusions
Reason for why iridium(III) probe #1 has the best inhibition because it’s the only probe
that has interactions between the Boc ligand and the serine residue of CYP3A4
There is still a lot of space in the CYP3A4 active site which we
could fill better with different ligands with different stereochemistry
Kodanko group hypothesizes that the L stereochemistry will have
better interactions then the D orientation
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11. UROP Project Goals
Synthesize better lead Ir(III) probes that will be able to monitor Cytochrome P450 3A4
enzymes activation to sensor drug metabolism in hepatocytes
Find out if the L or D Enantiomer has better binding interactions with Cytochrome P450
3A4 in its active site
Synthesize and find out if the Δ or Λ Enantiomer is creating background noise which
would interfere with binding interactions in the Cytochrome P450 3A4 active site
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12. UROP Project Results
Synthesis and characterization of new chiral iridium(III) probes was simple to produce
and purify at high yields
Proved by CD that my iridium(III) probes are diastereomers
All the new chiral iridium(III) probes showed that they can
absorb light better than iridium probes from Barber Project
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-6
-4
-2
0
2
4
6
200 300 400 500 600
CD
[mdeg]
λ (nm)
3
6

13. 13
UROP
Project
Structures

14. UROP Project Conclusions
New chiral iridium(III) probes optical properties that are ideal for monitoring CYP activity
and inhibiting the CYP3A4 enzyme
Data showed that the Lambda chirality has weaker bonding interactions with CYP3A4
compared to the Delta orientation
Proved that the L stereochemistry has better binding
interactions with CYP3A4 active site then the D
orientation
14
-50000
0
50000
100000
150000
200000
250000
300000
200 300 400 500 600 700 800
Δε
λ/nm
3 4 5 6

15. Goals For Current Project
Synthesize more chiral Ir(III) probes with different ligands to be able to better inhibit
CYP3A4 enzymes to sensor drug metabolism in hepatocytes
Find out if the Δ or Λ Enantiomer has better binding interactions with the active site of
CYP3A4 than chiral Ir(III) probes from the UROP project
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16. 16
Current
Project
Structures

17. The Future Of The Ir Project
Recent data from the Kodanko lab has shown that iridium sensors can detect drug
binding to CYP3A4 in hepatocytes
Future goals are to continue to optimize the structure and biological properties of these
compounds.
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18. Acknowledgments
• Undergraduate Research Opportunities Program (UROP)
• Richard Barber Fellowship
• Wayne State University
• Wayne State Department of Chemistry
• Current Group Members
• Maddy Denison
• Sayak Gupta
• Former Group Members
• Nick Toupin
• Principal Investigator
• Dr. Jeremy Kodanko
• Collaborators
• Dr. Irina Sevrioukova
• Dr. Thomas Kocarek
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19. 19
Thank You!