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(PROTAC) Targeted Intracellular Protein Degradation

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(PROTAC) Targeted Intracellular Protein Degradation PROteolysis TArgeting Chimera (PROTAC) Targeted Intracellular Protein Degradation Literature presentation Junyong Kim May 23rd, 2019 Contemporary Drug Discovery Small molecule inhibition has been a sccessful approach for drug discovery Inhibition of drug Find high affinity Optimize In vivo test target affects inhibitor through phamacokinetics & & clinical trial disease phenotype SAR screening pharmacodynamics Only about 15% of the human proteome is ‘druggable’ Overexpression or gain/loss-of-function can lead to disease phenotypes Non-enzymatic components Russ, A.P.; Lampel, S. Drug Discov. Today 2005, 10, 1607–1610. Druggable vs. Undruggable Protein–Protein Protein–small molecule interaction interaction No dominant Dominant Amenable to Hot Segment Hot Segment small molecule inhibition Surface area < 2500 Å2 Peptides K < 200 nM d Small molecules Biological approach is needed for undruggable targets London, N.; Raveh, B.; Schueler-Furman, O. Curr. Opin. Chem. Biol. 2013, 17, 952–959. Biological Approaches for Undruggable Targets Chemical genetics CRISPR-Cas 9 RNAi PROTAC Fix defective genes by Prevent gene expression Degrade target proteins by insertion or removal by inhibiting translation ubiquitin–proteosome system First clinical trial started First FDA approval (2018) First clinical trial (2018) in 2016 (China), 2018 (EU) Onpattro™ (amyloidosis) ARV-110 (prostate cancer) Guo, J.; Liu, J.; Wei, W. Cell Res. 2019, 29, 179–180. Selective Estrogen Receptor Downregulator (SERD) Me OH Fulvestrant (2002 approval) HR+, HER2– breast cancer H O F F H H S F HO F F Decrease intracellular estrogen receptor (ERα) levels Exposure of hydrophobic domain Protein degradation Refolding Russ, A.P.; Lampel, S. Drug Discov. Today 2005, 10, 1607–1610. Selective Estrogen Receptor Downregulator (SERD) Me OH Fulvestrant (2002 approval) HR+, HER2– breast cancer H O F F H H S F HO F F Decrease intracellular estrogen receptor (ERα) levels Protein degradation Hydrophobic tag to mimic misfolded protein Russ, A.P.; Lampel, S. Drug Discov. Today 2005, 10, 1607–1610. Hydrophobic Tagging (HyT) Hydrophobic tags O NBoc O N NHBoc H NHBoc Adamantyl (Boc)3Arg Covalently attaching hydrophobic tags will induce protein degradation HaloTag reactive linker (covalently binds to HaloTag) O Cl O O N H Cells expressing EGFP–HaloTag Time-dependent protein degradation Dose-dependent protein degradation M M µ 0 h 1 h 2 h 4 h 8 h 16 h 24 h 24 h– µ 0 nM 10 nM 25 nM 50 nM 100 nM 200 nM 500 nM 1 10 HA-EGFP -HaloTag HA-EGFP -HaloTag β-actin β-actin Neklesa, T.K. et al. Nat. Chem. Biol. 2011, 7, 538–543. Hydrophobic Tagging (HyT) Injected HyT13 Mouse xenographed with NIH-3T3 cells expressing HA–HaloTag-Hras1 HyT suppresses HaloTag-HRas1 driven tumor by degradation Successful demontration of hydrophobic tagging leads to protein degradation Covalent tagging method requires high dose Protein degradation with alternative mechanism would have higher therapeutic potential Neklesa, T.K. et al. Nat. Chem. Biol. 2011, 7, 538–543. Proteolysis Targeting Chimera (PROTAC) Ubiquitin–proteasome system Target protein Ubiquitination by E1, E2, E3 ligases Ub Target protein E3 ligase Ubiquitinated protein Ubiquitin Polyubiquitinated protein Can we induce artificial interaction Proteasome Small peptide between E3 ligase and POI? Russ, A.P.; Lampel, S. Drug Discov. Today 2005, 10, 1607–1610. Proteolysis Targeting Chimera (PROTAC) Ubiquitin–proteasome system PROTAC linker Ubiquitination by E3 ligase Target protein E1, E2, E3 ligases ligand ligand Target protein Target protein Induced interaction Ubiquitinated protein Ub E3 ligase Ubiquitin Proteasome Small peptide Russ,Polyubiquitinated A.P.; Lampel, S. Drug Discov. protein Today 2005, 10, 1607–1610. Proof of Concept Me MetAP2 Me OH O β-TRCP(E3 ligase) ligand Me HO MeO O GGGGGGDRHDS*GLDS*M O NH N H O O Ovalicin MetAP2 covalent inhibitor Covalently bound ovalicin to MetAP2 (PDB:1B59) Sakamoto, K.M.; Kim, K.B.; Kumagai, A.; Mercurio, F.; Crews, C.M.; Deshaies, R.J. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 8554–8559. Proof of Concept Me MetAP2 Me OH O β-TRCP(E3 ligase) ligand Me HO MeO O GGGGGGDRHDS*GLDS*M O NH N H O O ATP, ubiquitin β-TRCP E1, E2 enzymes E3 ligase – + + + β-TRCP E3 ligase ligand – – + – Ovalicin – – – + Ubiquitinated MetAP2 Ubiquitinated MetAP2 MetAP2-PROTAC MetAP2 MetAP2-PROTAC MetAP2 Sakamoto, K.M.; Kim, K.B.; Kumagai, A.; Mercurio, F.; Crews, C.M.; Deshaies, R.J. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 8554–8559. Proof of Concept Me MetAP2 Me OH O β-TRCP(E3 ligase) ligand Me HO MeO O GGGGGGDRHDS*GLDS*M O NH N H O O Frog egg extract MetAP2 MetAP2-PROTAC +LLnL +epoxomicin MetAP2-PROTAC MetAP2 0 0 2 10 15 30 30 30 minutes Sakamoto, K.M.; Kim, K.B.; Kumagai, A.; Mercurio, F.; Crews, C.M.; Deshaies, R.J. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 8554–8559. Would it target therapeutically relevent targets? Would it operate with noncovalent ligands? Would it operate inside cells? Intracellular Validation of PROTAC O O O GGGGGGDRHDS*GLDS*M N N H 6 H O Me β-TRCP(E3 ligase) ligand Me H H H Microinjection O H Dihydrotestosterone Cells expressing GFP–AR Androgen receptor (AR) ligand >70% cells showed partial/complete loss of fluorescence Sakamoto, K.M.; Kim, K.B.; Verma, R.; Ransick, A.; Stein, B.; Crews, C.M.; Deshaies, R.J. Mol. Cell Proteomics 2003, 2, 1350–1358. Intracellular Validation of PROTAC O O O GGGGGGDRHDS*GLDS*M N N H 6 H O Me β-TRCP(E3 ligase) ligand Me H H H Microinjection O H Dihydrotestosterone Cells expressing GFP–AR Androgen receptor (AR) ligand PROTAC + E3 ligase ligand PROTAC + testosterone Sakamoto, K.M.; Kim, K.B.; Verma, R.; Ransick, A.; Stein, B.; Crews, C.M.; Deshaies, R.J. Mol. Cell Proteomics 2003, 2, 1350–1358. Intracellular Validation of PROTAC O O O GGGGGGDRHDS*GLDS*M N N H 6 H O Me β-TRCP(E3 ligase) ligand Me H H H Microinjection O H Dihydrotestosterone Cells expressing GFP–AR Androgen receptor (AR) ligand PROTAC + proteasome inhibitor Sakamoto, K.M.; Kim, K.B.; Verma, R.; Ransick, A.; Stein, B.; Crews, C.M.; Deshaies, R.J. Mol. Cell Proteomics 2003, 2, 1350–1358. Small Molecule PROTAC Cl Cl Peptide PROTAC Poor pharmacokinetics HN N N N Poor cell permeability O O OiPr Poor intracellular stability Not amenable for high-throughput screening OMe Nutlin-3 (MDM2 inhibitor) Science 2004, 844. Cl Cl H H O2N N N O O O 3 O F3C N O N H Me OH N N N O SARM (Androgen receptor ligand) O OiPr First cell permeable PROTAC demonstrating pharmaceutical utility OMe Nutlin-3 (MDM2 ligand) Schneekloth, A.R.; Pucheault, M.; Tae, H.S.; Crews, C.M. Bioorg. Med. Chem. Lett. 2008, 18, 5904–5908. Small Molecule Drugs vs. PROTAC Small molecule PROTAC Drug Drug Drug Drug PROTAC POI POI POI POI POI POI POI POI 1 drug molecule inhibits 1 protein 1 PROTAC degrades multiple proteins Catalytic mode of action can provide high potency and selectivity Bondeson, D.P.; Mares, A. et al. Nat. Chem. Biol. 2015, 11, 611–617. Small Molecule Drugs vs. PROTAC O O NH S H N O N N O 4 N NH2 O t-Bu DC50 = 1.4 nM S Me OH NH O O N DCmax = 10 nM VHL ligand (Kd = 320 nM) S RIPK2 ligand (IC50 = 660 nM) In general up to 1 µM of Kd is acceptable Only RIPK2 and MAPKAPK3 were degraded among 7640 proteins Bondeson, D.P.; Mares, A. et al. Nat. Chem. Biol. 2015, 11, 611–617. Small Molecule Drugs vs. PROTAC Small molecule PROTAC Drug Drug Drug Drug PROTAC POI POI POI POI POI POI POI POI 1 drug molecule inhibits 1 protein 1 PROTAC degrades multiple proteins Catalytic mode of action can provide high potency and selectivity Only affinity probes are required – no need to be inhibitors Removal of a protein instead of inhibition can provide additional therapeutic effect Bondeson, D.P.; Mares, A. et al. Nat. Chem. Biol. 2015, 11, 611–617. Potential Application of PROTAC in Neurodegenerative Diseases Accumulation of misfolded proteins (tau, β-amyloid) is suspected to cause neurodegenerative diseases Could misfolded proteins be removed via PROTAC? Harding, R.H.; Tong, Y. Acta Pharmacol. Sin. 2018, 39, 754–769. Potential Application of PROTAC in Neurodegenerative Diseases Direct injection of tau-directed PROTAC reduced tau levels by 50% http://arvinas.com/therapeutic-programs/next-generation-targets/ What Problems Are Left for PROTACs? PROTAC Efficient ternary structure formation and ubiquitin transfer Skaar, J.R.; Pagan, J.K.; Pagano, M. Nat. Rev. Drug Disc. 2014, 13, 889–903. What Problems Are Left for PROTACs? Binary complex Ternary complex Binarycomplex (%) Ternary complex (%) Ternary Ligand concentration Ligand concentration Hook effect – increasing the concentration of bisligand can decrease ternary complex concentration Douglass, E.F.; Miller, C.J.; Sparer, G.; Shapiro, H.; Spiegel, D.A. J. Am. Chem. Soc. 2013, 135, 6092–6099. What Problems Are Left for PROTACs? Unoccupied binding sites Saturated ternary complexes Binary complexes predominate Lower degradation Highest degradation Lower degradation Matching the right E3 ligase/ligand – target protein/ligand combination is crucial for efficient degradation Bondeson, D.P.; Mares, A. et al. Nat. Chem. Biol. 2015, 11, 611–617. What Problems Are Left for PROTACs? O O NH S N O H Linker N N N N OMe O t-Bu Me NH OH VHL ligand (K = 320 nM) Br F d VEGFR2 inhibitor (IC50 = 40 nM) What linker structure facilitate ternary complex formation and ubiquitination? PROTAC Linker # of atoms DC50 O Target Protein O O 10 2 µM O O O O 12 0.8 µM Ubiquitin O O O O O 16 No effect upto 3 µM O O N O O 18 Weak effect at 10 µM Bondeson, D.P.; Mares, A.
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