Unexpected link between an antibiotic, pannexin channels and apoptosis.

SALMAN UL ISLAM19-JUNE-2014

KyungpookNational University

Contents

• Background information

• Aim of research

• Strategy

• Materials and methods

• Results

• Summary

BACKGROUND INFORMATION

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 UTPare 507 and 484 daltons, respectively).*UTP= Source of energy like ATP.

PANNEXINS

PANX1 is a target of effector caspase cleavage during apoptosis

Trovafloxacin

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

Trovafloxacin

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

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 supercoilsby cutting and resealing the single DNA

strand

AIM OF RESEARCH

Aim Of Research

o To identify small molecules that can modulate PANX1 function.

STRATEGY

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.

MATERIALS&

METHODS

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.

Materials & methods

o Anti-Fas antibody: The antibody demonstrates cytolyticactivity on human cells that express Fas. Cells undergo apoptosis in response to this antibody.

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.

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 splenicinvolution in rodents through apoptosis.(Immunology and Cell Biology (2000) 78, 238–246; doi:10.1046/j.1440-1711.2000.00905.)

Materials & methods

o Patch Clamp Technique: Electrophysiological technique---

allows the study of single or multiple ion channels in cells.

Materials & methods

Materials & methods

o Time lapse microscopy: Live cell microscopy---to see the cells in action.

Global knock out mice.

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 BacteriophageP1---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.

34bp lox p site

Conditional knockout mice

RESULTS

Fig. 1 Trovafloxacin inhibits pannexin 1 activity during apoptosis.

Fig. 2 Trovafloxacin-mediated inhibition of PANX1 promotesformation of smaller apoptotic bodies.

Fig. 3 Pannexin 1 regulates disassembly of apoptotic thymocytes.

Fig. 4 Formation of string-like apoptopodia after membrane blebbingcorrelates with formation of apoptotic bodies.

http://www.nature.com/nature/journal/v507/n7492/full/nature13147.html#videos

SUMMARY

Summary

• During apoptosis, PANX1 channels are cleaved by caspaseswhich 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.

Best wishes for all of you!