1. Characterization and Inhibitor Development of the Mammalian N-end Rule Pathway PACCON 2013 Jan. 24, 2013 Min Jae Lee Department of Applied Chemistry College of Applied Science Kyung Hee University, Korea

2. Protein Synthesis vs Degradation Foreign vs Body Protein degradation Extracellular vs Intracellular Protein degradation Autophagy-Lysosomal System (ALS) vs Ubiquitin-Proteasome System (UPS)

3. Ubiquitin (Ub) C-terminal Gly-Gly-COO - Ubiquitin (Ub) functions as a degradation signal Lys-NH 3 + Isopeptide bond - Ub structure

4. Target Protein Ubiquitin Ubiquitins (poly)ubiquitinated Proteasome (poly)ubiquitinated target substrate peptide fragments free Ub The Ubiquitin-Proteasome System (UPS)

5. Conjugation of poly-Ub chain to the substrates UPS (A Little More Details) E3 ubiquitin ligases Deubiquitinating enzymes Degradation of the tagged protein by the 26S proteasome

6. A Case Study: Proteasome Inhibitor as Cancer Drug - MG132 (Z-Leu-Leu-Leu-al), a proteasome inhibitor - Bortezomib, Velcade® FDA approved for multiple myeloma in 2003; survival from 10% to 40~50% First new therapy for MM since the early 60s Sales ~$2 billion/yr; >100,000 treated Price: $1548.0 per 3.5 mg vial

7. Nature Review Cancer (2011)

8. Screening for Small Molecule Inhibitors of Usp14 Ub-AMC Ub-AMC hydrolysis activity Ub Usp14 Inhibitor hits free AMC Usp14: endogenous Proteasome inhibitor

9. Where is the decision to degrade a protein made? rate- determinant “Passive” - Traditional view - Our view “Active” role of proteasome on Ub chain dynamics Lee et al, Nature 2010 Inhibitor (IU1) of inhibitor (Usp14) enhanced the activity of proteasome IU1 can potentially be used to eliminate toxic proteins

10. Rhomboidal Structure of the Ubiquitin-Proteasome System

11. Met- A Degradation Signal: the N-terminal Amino Acid Rapid proteolysis (= short half-lives) Ub UBR protein X substrate … Stable (= long half-lives) UBR proteins: N-end rule E3 ubiquitin ligases Start codon vs N-terminal methionine Some N-terminal residues

12. In Vitro Model System of the N-end Rule Pathway Ubiuqitin / Protein / Reference (UPR) technique Stabilization by dipeptide inhibitors Two types of primary destabilizing residues 35 S labeling Autoradiography Dipeptide inhibitors

13. Effect of D -form Dipeptides on UBR Protein Inhibition

14. with or without dipeptides Functional Proteomic Screening for Mammalian N-end Rule Substrate Lee et al, PNAS 2005

15. Regulator of G-protein Signaling proteins (RGS4, RGS5, and RGS16) High-Throughput Screening (HTS) from 18,000 cDNAs Collaboration with “Meso Scale Discovery” 42 candidates

16. Lee et al, PNAS 2008 Yanxialei et al, submitted Physiological Functions of the N-end Rule Pathway Lee et al, JBC 2012 Lee et al, PNAS 2005 Inhibitor development Cardiomyocyte proliferation Endogenous substrates

17. Classification of N-terminal Destabilizing Residues Quantification of in vitro degradation assay

18. In silico Computational Docking Analysis (Type 1)

19. In silico Computational Docking Analysis (Type 2)

20. Rationally Designed Intraheterovalent Inhibitors Chemical structures Conceptual model of a bivalent inhibitor Controls of RF-C11 Arg Phe Lys C11 UBR boxClpS box

21. In Vitro Effects of Bivalent Inhibitors Comparison of IC 50 RF-C11: 15.5  M Lys-Ala: 148  M Arg-Ala: 283  M RF-C11: 2.74  M Trp-Ala: 21.39  M Phe-Ala: 72.37  M Type 1Type 2

22. Bivalent inhibitors can be used for the study of biochemical mechanisms of UBR box proteins and physiological function of the N- end rule pathway RF-C11 provides chemical insight for developing better rationally designed N-end rule inhibitors RF-C11, a Rationally-designed Intraheterovalent Inhibitior RF-C11 showed ~ 20 times better inhibitory effects Lee et al, PNAS 2008 Optimization is on-going

23. Optimizing RF-Cn Linker Length

24. Free  -Amine and Peptide Bond Are Essential for N-degron Recognition

25. Monomeric Phe-derivatives Inhibited N-end Rule Pathway

26. Chloro-Amphetamine Delays Phe-nsP4 Degradation

27. UBR1 and its evolutionarily descendent UBR2 may have different specificity to N-degrons Recognition of N-degron is stereo-specific The optimal linker length of RF-Cn is n=5 Heterovalent N-end rule inhibitors are successfully applied to mammalian cells. alpha-Amine should be intact for effective inhibition Peptide bonds are not essential for inhibition (Phe-amide exhibits similar activity to Phe-Ala) Chloro-amphetamine has been identified as an effective N- end rule inhibitor Summary of Biochemical Understanding of the N-end Rule Pathway

28. Funded by - AHAF (American Health Assistant Foundation) postdoctoral grant, - Korea NRF (National Research Foundation) New Researcher Grant, - Kyung Hee University Fellow Grant - Korea NRF International Collaboration Grant - Korea Ministry of Health and Welfare (MHW) Alzheimer’s Disease Research Grant

29. Thank you!