Pannexins, specifically PANX1, play an important role in apoptosis by regulating the formation and disassembly of apoptotic bodies. The study identified the antibiotic trovafloxacin as a small molecule that can inhibit PANX1 channel activity during apoptosis. Experiments showed trovafloxacin treatment and genetic deletion of PANX1 both promoted the formation of smaller apoptotic bodies. This suggests PANX1 inhibition could control the size of apoptotic structures expelled during cell death.

1. SALMAN UL ISLAM
19-JUNE-2014
Kyungpook National University

2. Contents
• Background information
• Aim of research
• Strategy
• Materials and methods
• Results
• Summary

3. BACKGROUND
INFORMATION

4. Pannexins are four-pass
transmembrane channels.
 PANX1 is the most widely
expressed among the
pannexin family members
(PANX1, PANX2, PANX3).
 PANX1 can conduct
molecules up to 1kDa in size
across the plasma
membrane (ATP and UTP
are 507 and 484 daltons,
respectively).
*UTP= Source of energy like
ATP.
PANNEXINS

5. PANX1 is a target of effector caspase cleavage during
apoptosis

6. Trovafloxacin
o It is a “Fourth Generation” flouroquinolone antibiotic. It is
bactericidal in nature.
Trovafloxacin

7. Generation Drug Names Spectrum
1st
Nalidixic acid
Cinoxacin
Gram- but not Pseudomonas
species
2nd
Norfloxacin
Ciprofloxacin
Enoxacin
Ofloxacin
Gram- (including Pseudomonas
species), some Gram+ (S.
aureus) and some atypicals
3rd
Levofloxacin
Sparfloxacin
Moxifloxacin
Gemifloxacin
Same as 2nd generation with
extended Gram+ and atypical
coverage
4th
*Trovafloxacin Same as 3rd generation with
broad anaerobic coverage
Classification of Flouroquinolones

8. Mode of action of Trovafloxacin
o Inhibition of bacterial DNA Gyrase (Topoisomerase II)
 Formation of quinolone-DNA-Gyrase complex
 Induced cleavage of DNA---rapid death of bacterial cells.It unwinds the
DNA supercoils
by cutting and
resealing the
single DNA
strand

9. AIM OF RESEARCH

10. Aim Of Research
o To identify small molecules that can modulate PANX1
function.

11. STRATEGY

12. Strategy
o To confirm that Trovafloxacin inhibits pannexin 1 activity during apoptosis:
 Induction of apoptosis by activating Fas Pathway using Anti-Fas antibody.
 Confirmation of opening of Pannexin 1 channels----- TO-PRO-3 take up by apoptotic cells.
 Measurement of apoptosis-induced plasma membrane PANX1 currents at the single cell level.
o To confirm that PANX1 inhibition by Trovafloxacin promotes formation of smaller apoptotic
bodies:
 Time lapse microscopic imaging of apoptotic cells.
 Flow cytometric analysis using fluorescent dyes.
o To confirm that PANX1 regulates disassembly of apoptotic thymocytes:
 Induction of apoptosis by Dexamethasone in thymocytes; ex vivo and in vivo.
 In vivo Testing of Trovafloxacin in thymic apoptosis.
 Generation of PANX1 global and conditional knockout mice.
 Time lapse microscopy & flow cytometry of panx1 knockout thymocytes.
o To confirm that apoptodia formation is correlated with apoptotic bodies formation:
 Induction of apoptosis.
 Pharmacological inhibition and genetic modification of PANX1.
 Time lapse microscopic and flowcytometric analysis of cells undergoing apoptosis.

13. MATERIALS
&
METHODS

14. Materials & methods
o LOPAC 1280: Product by Sigma Aldrich---having 1280
pharmacologically active molecules---collection of inhibitors,
receptor ligands, pharma-developed tools, and approved
drugs---impacts most signaling pathways and covers all major
drug target classes.

15. Materials & methods
o Anti-Fas antibody: The antibody demonstrates cytolytic
activity on human cells that express Fas. Cells undergo
apoptosis in response to this antibody.

16. Materials & methods
o TO-PRO-3: An excellent far red-fluorescent nuclear and chromosome
counterstain---impermeant to live cells but penetrates compromised
membranes characteristic of dead cells---useful indicator of dead cells
within a population
o ANNEXIN-V : A phosphatidylserine (PS) binding protein---used to detect PS
on the surface of cells undergoing apoptotis.

17. Materials & methods
o TEV(Tobacco Etch Virus) Protease: Cysteine protease
from Tobacco Etch Virus--- strict 7 amino acid cleavage
recognition sequence of Glu-Asn-Leu-Tyr-Phe-Gln↓Gly.
o Dexamethasone: Glucocorticoid---causes thymic and splenic
involution in rodents through apoptosis.
(Immunology and Cell Biology (2000) 78, 238–246; doi:10.1046/j.1440-1711.2000.00905.)

18. Materials & methods
o Patch Clamp Technique: Electrophysiological technique---
allows the study of single or multiple ion channels in cells.

19. Materials & methods

20. Materials & methods
o Time lapse microscopy: Live cell microscopy---to see the cells
in action.

21. Global knock out mice.

22. Cre-lox recombination
o Cre recombinase: Recombinase enzyme---derived from
Bacteriophage P1---catalyse the site specific recombination
event between two lox P sites.
o Lox P site: 34bp sequence---also derived from Bacteriophage
P1---specific site for cleavage by Cre recombinase enzyme.
o Floxing: It refers to the sandwiching of a Gene between
two lox P sites---it is then said to be floxed gene.

23. 34bp lox p site

24. Conditional knockout mice

25. RESULTS

26. Fig. 1 Trovafloxacin inhibits pannexin 1 activity during apoptosis.

27. Fig. 2 Trovafloxacin-mediated inhibition of PANX1 promotes
formation of smaller apoptotic bodies.

28. Fig. 3 Pannexin 1 regulates disassembly of apoptotic thymocytes.

29. Fig. 4 Formation of string-like apoptopodia after membrane blebbing
correlates with formation of apoptotic bodies.
http://www.nature.com/nature/journal/v507/n7492/full/nature13147.html#videos

30. SUMMARY

31. Summary
• During apoptosis, PANX1 channels are cleaved by caspases
which then relaese “find me’’ signal to recruit phagocytes.
• Trovafloxacin inhibits PANX1 channels leading to formation of
smaller apoptotic disassembled, less granular cells.
• Genetic modification of PANX1 is phenocopoying the
Trovafloxacin effects.
• Unique structure of Trovafloxacin may be responsible for its
unexpected effects, and further study on this may unlock the
clue which will lead to production of safer and more potent
antibiotics.

32. Best wishes for all of you!