Characterization and Inhibitor Development of the Mammalian N-end Rule Pathway

PACCON 2013Jan. 24, 2013

Min Jae Lee

Department of Applied Chemistry

College of Applied Science

Kyung Hee University, Korea

Protein Synthesis vs Degradation

Foreign vs Body Protein degradation

Extracellular vs Intracellular Protein degradation

Autophagy-Lysosomal System (ALS) vs

Ubiquitin-Proteasome System (UPS)

Ubiquitin (Ub)

C-terminal Gly-Gly-COO-

Ubiquitin (Ub) functions as a degradation signal

Lys-NH3+

Isopeptide bond

- Ub structure

Target Protein

Ubiquitin

Ubiquitins

(poly)ubiquitinated

Proteasome

(poly)ubiquitinated target substrate

peptide fragments

free Ub

The Ubiquitin-Proteasome System (UPS)

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

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

NH

HN B

OH

OH

O

O

N

N

OHN

NH

HN

H

O

O

O

O

Nature Review Cancer (2011)

O

CH3

NH

O

Screening for Small Molecule Inhibitors of Usp14

Ub-AMC

Ub

-AM

C h

ydro

lysi

s ac

tivi

ty

Ub Ub Usp14

Inhibitor hits

O

CH3

H2N O

free AMC

Usp14: endogenous Proteasome

inhibitor

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

Rhomboidal Structure of the Ubiquitin-Proteasome System

Met-

A Degradation Signal: the N-terminal Amino Acid

Rapid proteolysis(= short half-lives)

Ub

Ub

UbUBR

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

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

35S labeling

Autoradiography

Dipeptide inhibitors

Effect of D-form Dipeptides on UBR Protein Inhibition

with or without dipeptides

Functional Proteomic Screening for Mammalian N-end Rule Substrate

Lee et al, PNAS 2005

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

Lee et al, PNAS 2008Yanxialei 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

Classification of N-terminal Destabilizing Residues

Quantification of in vitro degradation assay

In silico Computational Docking Analysis (Type 1)

In silico Computational Docking Analysis (Type 2)

Rationally Designed Intraheterovalent Inhibitors

Chemical structures

Conceptual model of a bivalent inhibitor

Controls of RF-C11

Arg Phe

Lys

C11 UBR box ClpS box

In Vitro Effects of Bivalent Inhibitors

Comparison of IC50

RF-C11: 15.5 mMLys-Ala: 148 mM Arg-Ala: 283 mM

RF-C11: 2.74 mMTrp-Ala: 21.39 mM Phe-Ala: 72.37 mM

Type 1 Type 2

• 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

Optimizing RF-Cn Linker Length

Free a-Amine and Peptide Bond Are Essential for N-degron Recognition

Monomeric Phe-derivatives Inhibited N-end Rule Pathway

Chloro-Amphetamine Delays Phe-nsP4 Degradation

• 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

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

Thank you!