1. 1. Potential Pharmacological Chaperones Targeting Cancer-Associated MCL-1 And Parkinson Disease-Associated -Synuclein Misook Oh, et. al., Proc. Natl. Acad. Sci. U.S.A (2014) 111:11007-11012 Presented by: Rajni Msc 2nd Year
2. 2. Molecular Chaperones Molecular Cell Biology, 6th Edition W.H. Freeman & Company
3. 3. Pharmacological Chaperones (PC) Structural genomics consortium (www.sgc.org)
4. 4. Enzymes inhibitors v/s Pharmacological chaperones as drugs (PC) Most drugs are enzymes inhibitors. These drugs compete with the natural substrates for binding with the target protein. e.g. Amprenavir-is a protease inhibitor which is used to treat HIV infection. PC are bind to the protein and stabilize them against thermal denaturation, aggregation, and prevent certain protein- protein interaction. Effective PC are-cofactor mimetics,ligand mimetics,secondary structure mimetics bind to target protein and stabilized them.
5. 5. Challenges in Discovering PC Discovering Pharmacological chaperones for specific target is challenging because 1. featureless target protein surface 2. lack of suitable chemical libraries 3. shortage of efficient High-Throughput-Screening methods In this study, authors have addressed all these challenges and discovered a potential pharmacological chaperones against- 1. Cancer-associated , myeloid cell leukemia1(MCL-1) protein 2. Parkinson disease-associated,-synuclein protein
6. 6. Cellpress , www.cell.com MCL-1 interaction with proapoptotic proteins (BAK) leads to cancer [e.g. BAK]
7. 7. -Synuclein misfolding and aggregation is associated with Parkinsons Irwin et. al., Nat. Rev. Neurosci. (2013)
8. 8. Outline Design of an scaffold as -helix mimetics Construction of a peptoid-encoded one-bead-one-compound (OBOC) combinatorial library of -helix mimetics High throughput screening (HTS) against MCL-1 protein and -synuclein MCL-1 binding assay and cellular assays -synuclein binding and aggregation assay
9. 9. Outline Design of an scaffold as -helix mimetics Construction of a peptoid-encoded one-bead-one-compound (OBOC) combinatorial library of -helix mimetics High throughput screening (HTS) against MCL-1 protein and -synuclein MCL-1 binding assay and cellular assays -synuclein binding and aggregation assay
10. 10. Design of Triazine-Piperazine-Triazine scaffold as -Helix Mimetic
11. 11. Outline Design of an scaffold as -helix mimetics Construction of a peptoid-encoded one-bead-one-compound (OBOC) combinatorial library of -helix mimetics High throughput screening (HTS) against MCL-1 protein and -synuclein MCL-1 binding assay and cellular assays -synuclein binding and aggregation assay
12. 12. General strategy for identification of -helix mimetics that bind to the target protein
13. 13. Advantages of using peptoid-encoded OBOC library Large number of library molecules can be screened simultaneously in single tube Peptide encoding helps in identification of hit compound Many proteins alter surface upon binding of partner and create new binding pockets The dynamic pockets can also be identified using this unbiased OBOC library.
14. 14. Structure of MCL-1 binding hit-compounds
15. 15. Outline Design of an scaffold as -helix mimetics Construction of a peptoid-encoded one-bead-one-compound (OBOC) combinatorial library of -helix mimetics High throughput screening (HTS) against MCL-1 protein and -synuclein MCL-1 binding assay and cellular assays -synuclein binding and aggregation assay
16. 16. Binding assay of hit compounds to MCL-1 9c has highest binding affinity for MCL-1 and can antagonize the interaction between MCL-1 and BH3 (domain of BAK)
17. 17. 9c selectively binds to MCL-1 BCL-XL (member of BCL-2 family) gets inhibited by BH3 peptide, but not by 9c
18. 18. Predicted binding mode of 9c Computer docking predicts that BH3 and 9c, both binds in the hydrophobic groove of MCL-1 BH3-peptide 9c compound
19. 19. MCL-1 Cellular assay 9c is cell permeable and competitively bind to MCL-1 therefore liberating BAK protein and confirmed the cellular uptake of 9c by using confocal microscopy
20. 20. Effect of 9c on cell viability Jurkat T-leukemia cells normal fibroblast cells 9c has selective toxicity to malignant cell with MCL-1 overexpression over normal cells
21. 21. Outline Design of an scaffold as -helix mimetics Construction of a peptoid-encoded one-bead-one-compound (OBOC) combinatorial library of -helix mimetics High throughput screening (HTS) against MCL-1 protein and -synuclein MCL-1 binding assay and cellular assays -synuclein binding and aggregation assay
22. 22. General strategy for identification of -helix mimetics that bind to the target protein
23. 23. Binding assay of -Synuclein Q1 has highest binding affinity for WT -synuclein and increase their thermal stability
24. 24. Aggregation assay of -Synuclein Q compounds having strong binding to mutant-type -synuclein and also delayed the -synuclein aggregation
25. 25. Summary Designed novel -Helix Mimetics (Triazine-Piperazine- Triazine) scaffold. Constructed OBOC library of this -Helix Mimetics by using different building blocks. Designed novel High-Throughput-Screening of OBOC library of -Helix Mimetics to identify compounds against cancer associated-MCL-1 and parkinson-associated -Synuclein. Binding assays with MCL-1 shows that PC can prevent MCL-1 interaction with natural binding partner BAK. Binding assays with -Synuclein shows that PC can enhance its stability and prevent aggregation. Present work is proof-of-concept that pharmacological chaperones can be important in diseases involving protein- protein interaction.