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Students:DaliaAbdelSa+ar,MahtabNoorizadeh,OmaimaMahmoud

FacultyMembers:Dr.ShankarMunusamy,Dr.AshrafKhalilCollegeofPharmacy,QatarUniversity,Doha,Qatar

8thUREPCompeIIonQatarFoundaIonAnnualResearchConference,Doha,Qatar

21March2016

SynthesisandPharmacologicalScreeningofNovelChemicalChaperonesAgainstTunicamycin-InducedEndoplasmicReCculumStressandRenalCellDeath

Background: ER Stress & Kidney Disease

•  Endoplasmic reticulum (ER) plays a central role in protein folding and maturation.

•  Metabolic disturbances impair ER function and trigger ER stress in the kidney and leads to renal cell death1.

•  4-Phenylbutyric acid (4-PBA) - a drug to treat urea cycle disorders – also possess “chemical chaperone” properties2

–  Shown to protect the diabetic kidney against ER stress-induced renal damage3-4

•  Limitation: Low potency è Requires high doses (in grams) to exert therapeutic effects.

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Metabolic Alterations (ñ Glucose, Albumin, Free Fatty Acids, etc.)

Kidney Disease

Renal Cell Apoptosis

ER

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Goal and Objectives

Objectives

1.  Synthesize of novel and potent analogs of 4-PBA.

2.  Screen the synthesized 4-PBA analogs for chaperone activity using an in vitro model of tunicamycin-induced ER stress in renal cells.

3.  Investigate the cytoprotective role of 4-PBA analogs against tunicamycin-induced renal cell death.

Goal: To develop novel, potent derivatives of 4-PBA that would protect the kidney against ER stress and associated cell death.

1. Wash with DCM to remove unreacted material

2. Acidify with HCl

3. Extract with DCM, dry, and evaporate under reduced pressure

Methods: Synthesis of Analogs

Hydrolysis Separation Extraction Novel analogs

Methods: Pharmacological Screening Ø  NRK-52E (Normal Rat Kidney) cells

Western blotting MTT Assay

ER Stress Marker - GRP78

48 h

Cell Viability

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Plate cells Pre-treatment with 4-PBA or

Analogs

Tunicamycin (For 5 hours)

24 h 0 h - 24 h

Recovery Phase (For 19 hours)

Findings: Synthesis & Characterization

Figure 1 and 2. TLC of 4-PBA analogs A and B respectively

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Findings: Cell Viability – MTT Assay

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Figure 3: Cell Viability Assay. Cells were pre-treated with 4-PBA or analogs for 24h and then exposed to Tunicamycin (Tn) for 5 h. After 5 h, cells were washed and allowed to recover for 19 h. After 24 h of initial exposure to Tn, MTT assay was performed and absorbance was measured at 570 nm using a microplate reader. Values are expressed as Mean ± SEM (n = 3). *P < 0.05 compared to control and # P < 0.05 compared to Tn treated group.

Cell Viability - Pre-treatment Model

Control(D

MSO)

Tn 0.01

ug/ml

1mM 4-

PBA

2.5 m

M 4-PBA

Tn + 1m

M 4-PBA

Tn+ 2.5m

M 4-PBA

Analog A

25uM

Analog A

50uM

Tn+ Analo

g A 25

uM

Tn + Analo

g A 50

uM

Analog B

25uM

Analog B

50uM

Tn + Analo

g B 25

uM

Tn + Analo

g B 50

uM0

50

100

150

Figure 3: Cell Viabiilty Assay. Cells were pre-treated with 4-PBA or analogs for 24h and then exposed to Tunicamycin (Tn) for 5 h. After 5 h, cells were washed and allowed to recover for 19 h. After 24 h of initial exposure to Tn, MTT assay was performed and absorbance was measured at 570 nm using a microplate reader. Values are expressed as Mean±SEM (n = 3). *P < 0.05 compared to control and # P < 0.05 compared to Tn treated group.

% C

ontr

ol * * *

###

Findings: ER Stress – GRP78 Expression

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Figure 4. GRP78 Expression. Upper Panel – Western blot showing the expression of ER chaperone GRP78 in NRK-52E cells pre-treated for 24 h with 4-PBA or analogs and then subjected to Tunicamycin (Tn) mediated ER stress. Lower Panel – Densitometry analysis of the western blot with the GRP78 expression of treatment groups normalized to control group (set as 100%).

Control

Tn

Tn+ 1 m

M 4-PBA

Tn+Analo

g-A 50

uM

Tn+Analo

g-B 50

uM0

50

100

150

Densitometry-GRP78-24h Pre-treat.

GR

P78

Exp

ress

ion

(% C

ontr

ol)

GRP78

Summary of Findings & Significance ü  Two 4-PBA analogs - 4-methoxy-4-phenyl butyric acid (Analog-A) and

4-ethoxy-4-phenyl butyric acid (Analog-B) - were synthesized.

ü  Both analogs (A & B) possess cytoprotective and chemical chaperone properties to prevent ER stress and associated cell death.

ü  Analogs A & B are effective at 25 to 50 µM conc. (vs. 4-PBA used at 1 to 2.5 mM conc.) i.e., 50 to 100 times more potent than 4-PBA.

ü  Our study warrants further investigation of these prepared analogs in animal models to confirm their activity in vivo.

ü  If proven effective, the novel 4-PBA analogs could be potentially used to treat ER stress-induced kidney damage in patients with diabetes, obesity and other disorders associated with severe ER stress.

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Students’ roles and skills gained Students’ Role: As a team all 3 students played an active role in -

•  Synthesis and purification of two 4-PBA analogs

•  Optimization of in vitro model of tunicamycin-induced ER stress in NRK-52E cells

•  Standardization of the dose of analogs A and B for the cell culture model.

•  Conducting drug treatments in cell culture, cell viability assays and western blots

•  Compiling the raw data and writing the methods section of the UREP final report

•  Writing an abstract for submission to the DUPHAT conference

Skills Gained:

•  Organic drug synthesis, purification and interpretation of the spectral data

•  Aseptic cell culture techniques, cell viability assay and western blotting

•  Using equipment such as microplate reader and gel documentation system

•  Data interpretation, preparation of graphs & figures for dissemination

•  Scientific writing and oral communication skills

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Mentors’ role

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Post-project achievements/plans

•  The mentors have supervised and oriented students on the following.

1.  PFM: Cell culture studies, cell viability assay and western blotting; using microplate reader and gel documentation system; Preparation of graphs & figures; Data interpretation; Scientific writing and oral communication skills.

2.  Faculty Member: Organic drug synthesis, purification and interpretation of the spectral data; Scientific writing and oral communication skills.

•  To confirm the purity of the analogs using NMR and LC/MS techniques

•  To screen the analogs for other markers of ER stress (CHOP, JNK) and apoptosis (Caspase-12, Caspase-3, Annexin/propidium iodide staining) to unveil the mechanistic pathways that underlie their chaperone potential.

•  To publish a manuscript from the study in a peer-reviewed journal.

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Acknowledgements

Technical Support:

§  Vinitha Kuruvilla

§  Heba El-Gamal

§  Ayat Hammad

§  Shakila Afroz

Financial Support: This report was made possible by a UREP award [UREP14-008-3-006] from the Qatar National Research Fund (a member of The Qatar Foundation). The statements made herein are solely the responsibility of the author[s].

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References 1.  Inagi R. Endoplasmic reticulum stress as a progression factor for kidney

injury. Curr Opin Pharmacol. 2010;10(2):156-65.

2.  Carlisle RE, Brimble E, Werner KE, Cruz GL, Ask K, Ingram AJ, et al. 4-Phenylbutyrate inhibits tunicamycin-induced acute kidney injury via CHOP/GADD153 repression. PLoS One. 2014;9(1):e84663.

3.  Luo ZF, Feng B, Mu J, Qi W, Zeng W, Guo YH, et al. Effects of 4-phenylbutyric acid on the process and development of diabetic nephropathy induced in rats by streptozotocin: regulation of endoplasmic reticulum stress-oxidative activation. Toxicol Appl Pharmacol. 2010;246(1-2):49-57.

4.  Qi W, Mu J, Luo ZF, Zeng W, Guo YH, Pang Q, et al. Attenuation of diabetic nephropathy in diabetes rats induced by streptozotocin by regulating the endoplasmic reticulum stress inflammatory response. Metabolism. 2011;60(5):594-603.