Parkin Binding Assay Termination Memo, Controversial Binders, And Start Of PE I Imaging GBA Plan

20221215 — ADDP E000-25157 [parkin binding assay development]

(Ariba code: 0000475629) Cost center: Kamiguchi-san’s budget (2780002666).

Step	Description	Time	JPY	Compounds to use a trial	Note
1	Confirmation of a competition manner (goal: is to evaluate and find appropriate probe compound(s) for the competition assay) Sow: finding optimal probe ligand(s) for competition assay Start: 20230104	2-3w	940,000	4 cpds + Phospho-Ubiquitin (p-Ub) TR05561445-001, TR06763500-001 TR06125171-001 TR05804053-001 p-ub (considered to be a potential probe for a competition assay of Parkin protein)	Direct binding assay has been developed • Parkin protein has already provided by customer. If necessary, ADDP will request additional protein.
→ If we obtain an appropriate probe candidate for the competition assay → Go
2	Optimization of Competition assay: sow: optimizing an assay condition end: 20230228	2w	450,000	Eg. optimization of protein concentration using 6 test cpds
After 2	Iterative assay: competition assay using AS-MS		>400,000	22 or more cpds per one experimental batch, weekly or biweekly

20230331_ADDP — ASMS assay development result_E000-5157.pdf

An appropriate probe candidate for the competition assay were not obtained.

The results might be unique, in other words, unusual manners. However, it would be difficult to pursue further investigation for regular assay to optimize PET-probe.

Thus, ADDP would like to propose a termination at the step 1 of E000-25157 because all the ligands and phospho-Ub did not show clear competition manners among them.

Instead, ADDP proposes direct binding assay using AS-MS for PET-probe optimization iteratively (i.e. weekly, biweekly, or monthly). However, it should be discussed because there are the following issues:

• Only two compounds were detected as Parkin binders by Axcelead’s AS-MS method, and their bindings have not been successfully confirmed as any series of chemotype. According to this observation, it would be challenging to obtain the structure-activity relationships (SARs) of the compounds and to improve their potency.
• Direct binding assay by AS-MS is not appropriate for a regular assay because of limited throughput and costly assay system.

Regular/competitive assay 말고 iterative/direct binding assay 하자

summary and next actions

No Parkin binders with confidence

Some controversial binders

ID	Structure	Thermal shift assay (TSA) Max. ΔTm (°C)	NMR	MS	Note
00564 (T-4021920)		-2 (SGC)	No binding	No binding
97809 (T-4060063)		-0.48 (AXL)	Small perturbation	Binding or no binding	inconclusive
81829 (T-4056602)		-0.32 (AXL) ~+4 (Mayo patent)	Small perturbation	No binding or binding	Mayo patent

Next actions (to be discussed)

1. No go about Parkin PET discovery
2. Test these compounds in SPR assay (The binding should be weak according to the above data)
3. Others

RDDU’s PE I Imaging GBA — Plan

	Plan	Date	Candidate	Rodent	HDAC6	General (slide)	NLRP3
						Operational lead	Operational lead
	- Identify selective compound series - Target expression (Bmax>1nM) - regional distribution - (pk)	2022 01	PET lead compounds	Rodent NHP, human (homogenate vs ARG?), 기본적으로, 질환모델 아님.	NHP (homog, ARG), Mouse (homog, ARG), WT/HDAC6 KO rats (homog)	QTS (그럼 여기서 발표해야함?)	?
	Optimize characteristics (rational PET tracer design → Competition binding asay : Bmax/Kd ≥ 10		PET lead compounds			Medchem	Yamamoto, Takeshi: PET chemistry
	- Triage - ? (→ Target/reference region >2) - Specific binding		PET lead compounds	Rodent (consideration: 나중에 정상인군 필요할 텐데. 언제까지 결정-bmax 결과보고 human 과 유사종 고르면 안 되나?)	HDAC6 WT & WT mice	DMPK	Nishihara, Mitsuhiro: DMPK

Uncertain Spans

location	transcription	uncertainty
RDDU PE I plan, leftmost column	row label cells	The leftmost column of the planning table is cut by the photo edge in some rows and not transcribed; refer to body_full for the visible fragments.
RDDU PE I plan, NLRP3 column	? and trailing edge fragment InVicro	The ? cell content is a single visible glyph; the InVicro fragment near the photo edge may be a CRO/vendor name (Invicro) but is not clearly inside any one cell in this capture.
20221215 cost row	940,000, 450,000, >400,000	The numeric cells span split crop edges; values are reconstructed from the body_full image and the middle / lower binding-assay-development crops.