Target Validation (continued), PE I Planning, Parkin Binding Assay HTS History, And 20210709 11-Candidate List

doesn’t help → we need a study. (then sse?) (사전정보가 없가 없을 때) Proportion 에 대한 sample size 방법?,

제출기술: Postmortem brain analysis of 12 idiopathic PD patients revealed soluble Parkin protein reduction by ~50% in the striatum in ~36% PD patients compared to control⁸. As the sample number published is low, the team is considering analyzing additional samples to reach effect size. A study using postmortem PD brains would require a sample size of 38 for estimating the expected proportion (36%) with 10% margin of error and 80% confidence.

i) 이 proportion 이 맞는지는 38명 필요. ii) 군간차이 확인은 8+8명 필요.

아래에 GE 가 설자리가 없네?

Questions / Needs / criteria / timeline

Type	Questions/Needs	How to address	criteria	timeline
TV	- Disease impact: PD patients have less Parkin protein? (ie 군간차이), need n=8 each	PM Brains: 5 sPD SNs and 5 HC SNs → this is pilot and can inform effect size, if we see a sign then we may increase n.	- Group difference of brain Parkin protein level (between sPD and HC) d>0.65 -군전체를 대상으로 임상할게 아니니까 큰 group differnce 가 필요한 게 아니지. 어차피 subgroup 이 필요한 거니까. 대신 statistically significant difference 필요?	-
TV	- Phenotype impact: relationship between parkin protein level – mt function		- Correlation coefficient between brain parkin protein level and brain mitochondrial function (? r>0.6	-
TV	다음은 n 부족으로 못함. (frequency) Proportion of 36%: need n=38		다음으로 충당되나? Correlation of parkin protein level between brain and plasma → if r is very large, it should, but if not, it shoundlt,
BM	a correlation of the Parkin protein level between in the target brain region (ie NS) and plasma	PM brain and plasma from PD patients and HC: 11 SNs and 11 matched plasma (1 parkin-PD, 5 sPD, 5 HC) → this is pilot and can inform effect size, if we see a sign then we may increase n.	Correlation coefficient of parkin protein level between brain and plasma: r>0.6
BM	The normal reference value of the plasma parkin protein level	a cross-sectional observational reference cohort study in healthy individuals.

PE I

Candidate selection	Affinity		In vivo PET
checking availability of a REVERSIBLE compound for tritiation (3H labeling) to measure Bmax	MST study	go to PET SC for presentation after suitable candidate identified for tritiation	Bind endogenous & exogenous Activated & inactivated endogenous parkin Activated & inactivated transgene expression an irreversible binder is ok

Parkin Binding Assay / HTS history

Date	Note
20210608	ENZYME-BASED HTS vs Cell-based HTS
20210609	Makoto Fushimi: Misaki and I reviewed the data about enzyme-based HTS hits. Biophysical validation (e.g. NMR, ASMS, native MS) might be a plausible action to check their direct binding to Parkin. However, I'm afraid that there might be a small chance because of NO SPR binding of these compounds.
20210610	Misaki Homma: These representative hits are from enzyme HTS and have NOT been filtered by cell-based mitophagy assay. We had about 2 months medchem activity on enzyme hits. - Enzyme-based HTS hits (20170523_Medchem.pptx, representative 8 compounds on page 1 and 2) (여러 clusters but 총 몇 물질인지 불명) [`https://mytakeda.sharepoint.com/sites/InterACT-RAD-Pipeline/NS%20DDU_Parkin%20act/PRDWideDocument/MedChem/Medchem%20meeting/2017/0523_Medchem.pptx`] - SPR data (ppt, 20170609_Parkin_SPR_Kamada.pptx) (shows priority 1 & 2, but don't show how many compounds) [`https://mytakeda.sharepoint.com/sites/InterACT-RAD-Pipeline/NS%20DDU_Parkin%20act/PRDWideDocument/BMRL/170614 Biophysics SPR%20results/170609_Parkin_SPR_Kamada.pptx`] - Native HTS hit report is the following. (excel, 686 compounds, no 'SPR' or 'BINDING' other than Herg binding, so) [`\\polaris2.takeda.co.jp\DavWWWRoot\sites\37\3757\PRDWideDocument\BMRL\170301 Parkin HTS hit\170301 Parkin HTS hit report.xlsx`] Takeshi: If my memory serves me well, almost all of HTS hits didn't show mitophagy induction in cells. But representative hits showed somewhat mitophagy induction activity. Can we review hits without result of mitophagy assay?
20210611	Misaki Homma: Some clusters from enzyme assay showed mitophagy activity. The compounds list is prepared based on enzyme assays, not mitophagy. We added the data of mitophagy to the list. I review the raw hit data yesterday. I think that all promising clusters were included into this list. A two-month medchem study from the hit compounds showed little improvement in enzyme activity. The only cluster improved enzyme activity slightly was named chemotype A (T-213). Our medchem history was shared with Makoto. Takeshi: We just want to find candidates of Parkin binder in current GT project. I wonder if we can obtain these compounds from previous enzymatic HTS hits.
20210614	Hit Clusters: Low priority Makoto: I revisited the original hit report for enzymatic Parkin Activator HTS and → found the following two hits as additional candidates thanks to Misaki. Just to be clear, no other clusters were identified by the HTS. As a result, we have 11 candidates for biophysical validation Takeshi: Thank you so much for reviewing the past result several times. It is really good news for me and I look forward to seeing biophysics result in the near future. Makoto: Is there any resource to characterize these compounds? If not, perhaps we should make a presentation at PET Steering Committee to get endorsement to start Parkin GT PET activities. Hopefully, the formal start might give us a chance to have some resource. 686 enzymatic HTS hits 중에서 몇 개 biophysical tested? Relaunch: enzymatic activity 고려 없이, binding 만 보는 relaunch 안 되나? Question: how do you select a candidate? Ie why not 686 all? 작년엔 누가 했나? 올해 돈 쓸 명분: 작년과 차이 Xin: rep 이 team meeting에 발표, pet steering committee에서 발표, plan, report to team, external monitor, external opportunity.

20210615 row

HTS 당시	작년	올해	Plan B
20210615 Homma: Our contact person in TCAL was Doug (doug.dougan@takeda.com). He is a structural biologist. I think it seems better to access Pedro (Pedro.Serrano@takeda.com) and Mack.Flinspach@takeda.com)	Cell based hits 몇 개 representative comp 봄. TCAL (일걸)	Enzyme based hits 봤더니, 10개(?) Representative compound만 SPR DATA 있는데, 다 negative 더라 - 10 cluster로부터 10 compounds 정해서 (1더해서) 11를 test 하겠다. SPR말고 MS, NMR하겠다. TCAL 이 무보수로 할 수도, nstm? TCAL or AXCELEAD Small discussion with Paul → pet sc → Tcal Paul에 논의사항: project background,, bm plan, timeline, progenra?	plan B,: AAV PET, Progenra, binding assay (ie ASMS)-HTS (Xin: 대만에 CRO (Will cost 30 mil yen))

20210708 — Quick memo for the today’s meeting

Three imaging approaches for target engagement are considering by the team:

• Small molecule PET tracer
• Parkin immuno-PET (Sehlin, 2019 #1799, Review)
• AAV PET

all the approaches will be beneficial for the project, although detecting the protein expression meets the project needs better than detecting capsid distribution. We’ll prioritize the approaches in next meeting (about 1 mon later), and a solid plan is needed before PE. Makoto’ll talk to TCAL on the resource/FTE requirement on biophysical evaluation of 11 candidate compounds (to identify if there is any binder or not).

Others:

• AAV PET development work can only be started after capsid selection finalization in project team (근데 Paul 은 AAV5, 9 이라면 지금부터 parallel로 진행가능하다고).
• It’s estimated that the success rate on small molecule PET is low by computational work by using Parkin crystal structure, but there is still a chance if we can identity any Parkin binder from current 11 compounds.

20210709: 11 (below list) was sent to TCAL

TNO	TRNUM	SOURCE	Cell free Parkin activation EC50 (uM)	Cell free Parkin activation (% at 3 uM)	Cell free Parkin activation (% at 30 uM)
T-1649016	TR05804053	SRC	8.8
T-1282725	TR03581292	SRC	1.1
T-1205130	TR04830952	SRC	6.8
T-0105929	TR03941892	SRC	11
T-1418243	TR01255840 TR05532678	TCAL SRC	1.6
T-1811390	TR03410305	SRC	1.9
T-1815930	TR05059770	SRC	0.45
T-1808773	TR04453293 TR01908161 TR03261509	SRC TCAL TBOS	0.39
T-1598257	TR04432533	SRC	10
T-1046777	TR04752190	SRC		8	186
T-3777213	TR06125171 TR06125171 TR06125171	SRC TBOS TCAL	0.38

SPR was tried but no compounds showed binding. For your detailed information, please check the following slides. [https://mytakeda.sharepoint.com/sites/InterACT-RAD-Pipeline/NS%20DDU_Parkin%20act/PRDWideDocument/BMRL/Biophysics/170609_Parkin_SPR_Kamada.pptx] Two compounds (TR03401305 and TR05804053) were confirmed as positive hits at reasonable, high concentrations of protein (7.5μM) a nd ligands.