Targeting the UPS to Treat NDDs

Question 1

Areas to target the UPS for treatment of NDDs

Answer 1

• Stimulate Ubiquitylation
• inhibit DUB
• Increase proteasome proteolytic capacity

Question 2

PROTACS

Answer 2

AKA PROteolysis-Targeting Chimeras

– heterobifunctional molecules with a Protein binding site, E3 ligase binding site and flexible chemical linker

Question 3

Structure of PROTACS

Answer 3

Protein of interest (POI)-binding site and E3 ligase binding site bound together through flexible chemical linker

Question 4

Binding to Protacs

Answer 4

POI binding site binds target protein –> Binding induces induce artificial ubiquitination, leading to and degradation of the protein by the proteasome

Question 5

First vs. second gen PROTACs

Answer 5

• First generation Protacs too bulky to get into cells (therefore not super successful)
• Second generation of Protacs: non-peptide, small molecules membrane-permeable

Question 6

PROTACS in HD

Answer 6

• Under investigation in cellular models of HD (patient fibroblasts)
• Benefit: tissue specificity by developing molecules that recruit E3 ligases only expressed in a certain tissue

Question 7

PROTACS in AD

Answer 7

Patent for PROTAC of tau binding moieties linked to E3 ligase binding moiety (thalidomide)
– can induce ubiquitination of tau protein and promote its degradation in cells

Question 8

Targeting DUBs

Answer 8

Targeting DUBs is the easiest way to increase UPS activity

– DUBs are the most druggable enzymes within UPS

Question 9

DUB function

Answer 9

DUBs acting upstream of the proteasome may act as a “rescue crew”
– they salvage specific substrates by deubiquitination before they are engaged in degradation

Question 10

DUB inhibition

Answer 10

DUB inhibitors may accelerate the turnover of the specific substrate for proteasome-mediated degradation

Question 11

Inhibition of UPS14

Answer 11

Inhibiting the catalytic activity of USP14 may have therapeutic benefits in NDDs
as it increases protosomal degradation by preventing premature deubiquitination

Question 12

USP24 function

Answer 12

USP14 is responsible for trimming Ub chains on substrates destined for degradation and thus recycling mono-Ub

Question 13

USP14 binding area

Answer 13

USP14 and UCHL5 associate reversibly to the 19S lid

Question 14

Premature USP14 activation

Answer 14

Premature deubiquitination by USP14 can

prevent protein degradation

Question 15

USP14 Inhibitors in NDDs

Answer 15

• USP14 inhibitors are under development for the clearance of aggregation-prone proteins
• Extent of USP14 contribution to the clearance of proteins involved in neurodegeneration in vivo remains controversial
  – Inactivation or depletion of USP14 may not be beneficial in all NDDs

Question 16

Drawbacks of USP14 inhibitors

Answer 16

Potential drawbacks because USP14 loss causes severe morbidity and postnatal lethality in mice

Question 17

IU1

Answer 17

• USP14 inhibitor
• small molecule with remarkable specificity
  against USP14

Question 18

IU1 was discovered by

Answer 18

– identified by high-throughput screening

Question 19

IU1 function

Answer 19

Enhances clearance of disease-linked proteins in cultured cells in a proteasome- dependent manner:
– Tau, ATXN3

Question 20

T/F: IU1 has been shown to prevent ND in vivo

Answer 20

FALSE
It remains to be determined if IU1 can prevent
neurodegeneration in vivo

Question 21

Acute effects of USP inactivation

Answer 21

Acute inactivation of USP14 could

increase degradation of some disease proteins without perturbing Ub homeostasis

Question 22

Chronic effects of USP inactviation

Answer 22

Chronic inactivation of USP14 could cause an overall disturbance in mono-Ub availability that leads to compensatory mechanisms of protein turnover
lack of mono-Ub –> can’t tag for degradation –> new mechanism

Question 23

Nrf1 and Nrf2

Answer 23

• are transcription factors for
  proteasome genes
• Nrf1 and Nrf2 are also substrates of proteasomal degradation

Question 24

Nrf1 activity

Answer 24

Nrf1 only becomes active in the nucleus when increased UPS function is required

when only need low UPS –> Nrf1 isn’t active

Question 25

Nrf2 activity

Answer 25

regulates the antioxidant response • stimulates expression of proteasome subunits • drives the synthesis of lysosomal proteins is regulated by ox. stress

Question 26

Sulforaphane

Answer 26

small molecule that induces Nrf2 – Induces proteasomal genes – Increases proteasome levels and activity – enhances UPS function in vivo

Question 27

Sulforaphane for HD

Answer 27

proposed treatment | - reduces mutant huntingtin protein and mitigate polyQ toxicity in neuronal cells

Question 28

Sulforaphane Function

Answer 28

SFN increased Nrf2 accumulation and nuclear translocation --> goes to nuclues --> increase proteasomal genes --> enhances UPS function

Question 29

In short: stimulating ubquitylation is down by ____. ___ inhibits DUB. ___ increases proteolytic capacity

Answer 29

PROTAC IU1 (through USP14 inhibition) Sulforaphane (by increasing Nrf2)