Parkin
| Brain | CSF | ||
|---|---|---|---|
| NSTM SMCxPro | 0.41 pg/mg | 0.06 pg/mL with 1 mL CSF | |
| DMPK LC-MS/MS | 32.5 pg/mg protein | ||
| DMPK SIMOA | 0.137-0.412 pg/mL (question: → 0.244 pg/mL 을 위해선 NSTM 은 250 ul CSF 필요한데 어떻게 dmpk 는 50? 이면 되는지?) |
pS65-Ub assays
| Detection Ab (for fluorescent label) | Capture Ab (for magnetic bead) | Recombinant Ps65Ub | ||
|---|---|---|---|---|
| Takeda | SMCxPro | Anti-Ub antibody (detection): P4D1 (Cell Signaling Technology, Cat# 3936, mouse monoclonal antibody) | Anti-pS65Ub antibody: E2J6T (Cell Signaling Technology, Cat# 62802, rabbit monoclonal antibody) |
|
| Mitokinin | SMCxPro | Ubiquitin: Ub P4D1 (many sources) | Phospho-ub: i) E2J6T clone (Cell Signaling) | K48 pS65-Ub (standard-Biotechne Catalog # UC-250) |
| Wolfdieter Springer | https://www.michaeljfox.org/grant/ps65-ub-biomarker-parkinsons-disease (Watzlawik, 2020 #1760) | Ubiquitin (including P4D1) | Phospho-ub (including E2J6T) | |
| Shalini Padmanabhan spadmanabhan@michaeljfox.org |
Different Ubiquitin linkage
Ubiquitination occurs through a linkage between the C-terminus of ubiquitin and the e-amino group of a lysine residue on the target substrate.
Ubiquitin itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63).
Ubiquitin to form polymers through various lysine
IVC
| Topic | Typical CSF volume per draw | Action |
|---|---|---|
| Rat | 100 uL | Very low feasibility, mitigation? 5 rats pooled to make 500 ul (ie minimum), For 100 ul, LLOQ is 0.6 pg/mL, so then window is only 2.2x. |
| NHP | 1 mL |
Parkin activity assay?
| Ps65-ub | Parkin protein assay | Parkin activity assay (this should mean E3 ligase activity assay) | |
|---|---|---|---|
| pros | Reflect the feedforward process to form polyubiquitinated chain which is a concerted effect of PINK1 and parkin, enabling lysosomal mitophagy. In the physiologic mitophagy process, the elongation of ps65-ub chain is the function of Parkin. (PINK1 is responsible for phosphorylation of ub) |
| |
| Cons | May not recognize certain truncated parkin depending on mutation (then only reflecting exogenous parkin when the endogenous parkin still has enzyme activity) | The assay is somewhat arbitrary because PINK1, ubiquitin E1, and E2 conjugating enzyme need to be added to enable the Parkin reaction. Eg. https://southbaybio.com/products/parkin-tr-fret-ubiquitin-kit.html (this is the one Takeshi considered for in vivo studies assay) TR-FRET detection of ubiquitin chain formation onto Parkin (js: this is not specific to phospho-ub!) | |
| question | What is the active site in the parkin protein? → it is catalytic cysteine residue (Cys431) on exon12. | Feasible in CSF? Probably yes |
TMQB
Oct 26 27 2021 Parkin GT TMQB draft.pptx
Objectives of TMQB meeting
Provide cross-functional translational leadership input to project teams on pre-CS TMPs
Align cross-functional translational input to DDU Strategy Investment Board (SIB) wherever possible
Proposed Schedule of PARKIN GT TMQB preparation schedule
Oct 12, 2021: 1st draft to Jaewon
Oct 12 - Oct 23, 2021: review and revisit (Team)
Oct 24, 2021: Meeting materials submission
TMQB meeting: Wednesday, October 27, 2021 8:00 AM-9:55 AM (Japan time)
| Topic | Contents | Responsible author | Presenter at TMQB |
|---|---|---|---|
| Translational Probability of Success (TPoS) | Slide template provided | Jaewon | Jaewon |
| Executive Summary | Jaewon | Jaewon | |
| Target validation | Human genetic/post-mortem data Animal models of disease mechs | Takeshi | Takeshi |
| Current status of asset generation | in vitro and in vivo pharmacology project timeline | Takeshi | Takeshi |
| Safety | TSR potential safety biomarkers, and necessary de-risking | Oak-san | Oak-san |
| Quantitative Pharmacology Targets | PK-PD relationships prediction of human response | Nishihara-san | Nishihara-san |
| Clinical biomarker feasibility | BMx development timeline slide Biomarker Usage Map (“Danhoff Slide”) | Jaewon | Jaewon |
| Topic | Presenter at TMQB | time (min) |
|---|---|---|
| Executive Summary | Jaewon | 2 |
| Target validation | Takeshi | 15 |
| Current status of asset generation | Takeshi | |
| Safety | Oak-san | 10 |
| QP/PD | Nishihara-san | 10 |
| Biomarkers | Jaewon | 15 |
| Translational Probability of Success (TPoS) | Jaewon | 8 |
| Total | 60 |
TPP/TCP
Working document of TCP slides
Working document Parkin GT Target Candidate Profile (TCP).pptx
Biodistribution: my rationale
- Gliosis widespread in the brain
- Lifelong progressing disorder, cortical involvement is matter of time.
- Even when limited in motor:
- When diagnosed, 60% of DA neuronal loss in SN, ie PD is a circuit disorder, before then, neuronal loss is compensated by other regional pathways in the direct/indirect pathway, so When motor Sx has developed, pathways outside of SN is also impaired.
- Gliosis
- Mild to moderate gliosis accompanied the neuronal loss previously described,
- also evidence of gliosis (in the absence of detectable neuronal loss) in raphe nucleus, NBM, striatum, GP, dentate nucleus, amygdala, hippocampus, and cerebral cortices
Unmet Needs
Unmet needs:
- Halting : one and only!,
- (restore) ↑ DA neurotransmission → no L-dopa → no LID, no fluctuation
- (partial) Neuroprotection / (partial) ↑ DA neurotransmission → ↓ L-DOPA dose → ↓ LID
Vector production
| 0602 | Sarah |
|
PINK-1
- codes for a mitochondrial membrane anchored-kinase protein,
Mutations
- AR (ie 대부분의 PINK-1 PD 환자는 Biallelic carrier이다. 드물게 heterozygous 도 발견된다.)
- Several different PINK1 mutations, primarily missense and nonsense mutations, have been identified in PD families. Mostly loss-of-function
-
60 mutations of different types (missense, nonsense, splicing, frameshift and deletions)
- missense mutations, the most frequent type, in 47.6%,
- The most frequent mutation of all was the missense mutation c.1040T>C, resulting in an amino acid change of leucine to proline at position 347
Uncertain Spans
- In the DMPK SIMOA CSF note, the value after
dmpk 는is visually small; recorded as50?. - The pS65-Ub assays reagent names/catalog strings are small; obvious OCR errors were corrected against the image, but catalog punctuation may need later confirmation.
- The lower TMQB timing table has one visible row with blank/cut-off time for
Current status of asset generation. - The PINK-1 section continues below the photo boundary; only visible lines are transcribed here.