Parkin Binding Assay Development Log (20210615 → 20220715)

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]

20211105 — ASMS results

Two compounds (TR03401305 and TR05804053) were confirmed as positive hits at reasonable, high concentrations of protein (7.5μM) a nd ligands.

20211118

Nov 18 2021 (Web 表示)

(From Cell free HTS: 11 representative compounds previously showing no binding in Parkin SPR assay)

• Solubility test (11 compounds) completed
• (initial) SAR test (=2nd ASMS) by Dec 2021? →

Parkin binding assay, (feasibility checking) routine assay to be considered in ADDP: kickoffmeeting with ADDP in 20211202, 2nd meeting in 202201

SBVC-2673:

• HIS-SUMO tags removed,
• full length (1-465) phosphorylated with PINK1 kinase
• was used 1st ASMS (from which 2 hits found), SAR study (ie ASMS), (Initial) routine binding assay (in ADDP)

20211217 — MAKOTO

The compounds for the 2nd Parkin ASMS have been finalized as follows.

[20211217_Parkin-GT-PET_2ndASMS_finalized.xlsb]

The plate will be prepared and sent out to Viva soon.

20220106 — makoto

the 2nd Parkin ASMS assay should be completed in 202201. (Mike would know the detailed timeline). After that, I’ll talk with ADDP about the assay development

20220106 — makoto (additional explorative effort: Biogen’s compound)

(additional explorative effort) Biogen’s compound

[https://www.sciencedirect.com/science/article/pii/S2589004221016205]

I’m planning to request WuXi to synthesize BIO-2008550, BIO-2007817, and BIO-2007818 in the following table → and evaluate them to check if they might be additional hits for Parkin PET discovery?

20220112 — makoto

The synthesis of the following 3 compounds have been requested.

20220228 — Michael Baksh

[the results of 2nd ASMS]

i) ASMS single point measurement using 10uM parkin protein (=SBMC-2673) & 10 uM compound → 10 potential hits were identified.

ii) ASMS dose response analysis (using increasing doses of compound & one concentration of protein) → Four compounds (TR05804053, TR05643502, TR06081020 and TR05561445) were confirmed as positive hits (근데 Kd값들이 2~10 uM로서 common criteria인 10nM보다 크잖아).

Summary: ASMS dose response confirms four hits with specific engagement to the full length phosphorylated Parkin construct (SBVC 2673)

20220310 — meeting

Next action

• To Identify additional hit

  • Select 6 compounds for 3rd evaluation (Makoto, by mid Apr)
  • After completion synthesis of 3 Biogen’s PAM compounds, initiate 3rd evaluation of 10 compounds (3 PAMs + 6 selected compounds with positive control) by AS-MS (Mike, Viva, around mid-Apr to May)

• To develop assay for compound optimization

  • Initiate the discussion with AXL to set up the binding assay such as SPR for compound selection (Makoto)

Update of ASMS binding data (Mike, Makoto)

• For 2nd AS-MS assay with full-length phosphorylated parkin protein, team selected 9 new compounds and a positive compound (TR05804053), and the obtained affinities demonstrate saturation binding (target engagement), but should not be used to rank relative compound affinity. The affinity tends to be weaker in AS-MS study because of the relatively large number of proteins used. Although AS-MS method can be utilized (for compound triage), to confirm the accurate affinity, other binding study is neeeded.
• Among 20 compounds, there are no correlation between functional activation and AS-MS affinity

Discussion about next step

• As sufficient amount of protein is already prepared by protein chemistry team, team can evaluate new three parkin PAM compounds and 6 compounds with one positive compound by AS-MS assay. The synthesis of 3 PAM compounds will be finished by mid-Apr. Extra 6 compounds will be selected by Makoto. Mike will prepare to start the 3rd AS-MS evaluation.
• Alternative binding assays with high throughput and accurate affinity, such as FRET and SPR, are needed; for FRET, appropriate probes are needed, and the competitiveness of the probe and the test compound should be considered.
• Makoto will discuss with AXL to set up an alternative binding assays. Especially, AXL is the best partner because AXL has strong capability for SPR. Mike would consult with AXL on what protein composition to use based on the binding method.

In silico analysis of Parkin reports difficulty in finding compounds with affinity below 10 nM.

20220317 — Makoto

AXL proposed to develop ASMS and SPR assays in parallel.

Draft plan

• ASMS: Replicate Viva’s data in April, Run competitive binding with phosphorylated ubiquitin and/or compounds
• SPR: Develop assay in May (using TR05561445-001 as a positive control).

@Baksh, Mike, would you share the experimental protocol of ASMS conducted by Viva? That would help Axcelead.

@Zou, Hua, would you send out the full-length phosphorylated Parkin construct (SBVC-2673) newly prepared by your team and share the detailed info (e.g. tag) of them? Axcelead would like to check if it could be used for SPR assay.

20220513 — Makoto

Biogen: As for the synthesis, BIO-2008550 is ready. BIO-2007817 and BIO-2007818 are pending because of the lockdown in Shanghai.

20220615 — ADDP

We have already started the development of this SPR assay.

20220706 — with ADDP

A) Takeda Chemistry

Synthesized 4 Parkin positive allosteric modulators (PAM)

B) Axcelead ASMS and SPR

1. ASMS in SPR buffer condition (0.05% Tween 20): TR05561445-001 (showed Kd=4.1 uM) and TR06763500-001 (showed Kd=76uM) showed specific binding. [https://mytakeda.sharepoint.com/:f:/r/sites/InterACT-RAD-Pipeline/Parkin%20Gene%20Therapy/Shared%20Documents/Subteam%20Translational/2022-07-05(6)%20Parkin%20GT%20PET%20discussion/AS-MS?csf=1&web=1]
2. SPR: TR06125171-001 showed specific binding. [https://mytakeda.sharepoint.com/:f:/r/sites/InterACT-RAD-Pipeline/Parkin%20Gene%20Therapy/Shared%20Documents/Subteam%20Translational/2022-07-05(6)%20Parkin%20GT%20PET%20discussion/SPR?csf=1&web=1]

C) Discussion and next actions

1. Assay: Further optimization should be required to obtain SAR data for compound optimization
   1. ASMS: Try to develop competitive assay
   2. SPR: Try other immobilization methods
   3. Try other methods (NMR, MST, ITC, TSA)
2. Compounds for further evaluation for the ASMS competition binding assay development at ADDP
   1. TR05561445-001 confirmed by Axcelead ASMS
   2. TR06763500-001 confirmed by Axcelead ASMS
   3. TR06125171-001 confirmed by Axcelead SPR
   4. TR05804053-001 confirmed by Viva ASMS

20220715 — ADDP E000-25157

[parkin binding assay development]

Uncertain Spans

location	transcription	uncertainty
20220228 row	(=SBMC-2673) and (SBVC 2673)	both spellings appear in the same paragraph; SBMC is likely an authoring typo for SBVC, but the visible glyph reads as SBMC in the first occurrence.
20220706 SharePoint URLs	long URLs with Parkin%20Gene%20Therapy	URLs are visually small in the source; verify before relying on the exact path.