PET Imaging GBA history, Handover to Otake, Catalytic vs Allosteric binders, PET SC, GBA GT reporter table

Cf) Functional assay

purpose	To correlate with clinical efficacy - To measure biodistribution - To measure Bmax
caveat	ARG MAY not be feasible in acidic condition
Workflow?	우선 ii)로 Bmax 하고 나서 → optimization of compound → i) 을 achieve

History (Takeshi Wakabayashi / Otake / Makoto Fushimi)

20210425 Takeshi Wakabayashi

1. The development of FRET based binding assay with a new condition (pH4.7 and lower protein concentration) is ongoing in Axcelead. The Kd value of the FRET probe will be fixed soon (the flash result was reported to be ~100 nM). Then they will proceed to the competition assay (js: w/ what?) of the representative compounds and the data will be obtained in June.
2. MST based binding assay of the representative compounds is ongoing in TCAL. The timeline will be revised and shared later.
3. Because it was found that AT3375 was not commercially available at TSHO, the synthesis of AT3375 has been ordered to a chemistry CRO. The timeline will be shared soon.
4. The MDR1 data of representative compounds was obtained and summarized in the following slide.

20210714 Takeshi Wakabayashi (Otake: binder was identified, but will futher optimizing, and we will go to PET SC.)

1. The result of MST based binding assay of representative compounds was reported by TCAL IFG (isofagomine) showed 725 nM.
2. The result of TR-FRET based binding assay of ~100 compounds was reported by Axcelead. The greatest was 36 nM. The counter assay to validate the data is ongoing.
3. The synthesis of AT3375 and its derivatives is ongoing. A key intermediate was successfully prepared.
4. The next experimental plan to characterize the lead compounds in in vitro will be proposed by Yoshirou soon.

https://mytakedasharepoint.com/sites/InterACT-RAD-Pipeline/GBA%20Gene%20Therapy/Shared%20Documents/Forms/AllItems.aspx?RootFolder=…

20211006 RD-DDU joint meeting) binder was found. How to proceed will be discussed and proposed to PET SC.

Next milestone: to develop in vitro experimental plan to characterize the lead compound.

20211013 initial result for discovery of GCase binders (IC50: 36 nM, but poor membrane permeability and possible MDR1 substrate).

(Herrera Moro Chao, 2015 #1798) Cyclophellitol-epoxide ABP (Activity Based Probes) MDW933 administered i.c.v. labels specifically lysosomal GBA in the brain

Handover to Otake-san on GBA PET

Goal of current effort: Originally the hope (according to Paul McQuade) was to find a compound that binds the catalytic site (ie give a readout of active GBA expression and hence inform on tracking transgene expression following GT) and the allosteric site (ie to compete with GBA activator and hence inform on TO) at the same time. But now that GBA activator project has been terminated, we wouldn’t need the latter property and can focus on the former. Fyi, Two sites (allosteric site & catalytic site) are very close with each other on the GBA protein.

1. History

(20200825) A meeting ‘GBA activator PET discussion’ was held. Below were agreed.

Binding site investigation was planned (Takuto to lead below assessments):

• Evaluate competitiveness of Isofagomine and Ct-A by Ct-A derived TR-FRET probe (to see if 1) Ct-A also partially occupy catalytic site, 2) isofagomine (which is a catalytic binder) partially occupies allosteric site, or 3) binding of Ct-A and isofagomine induce conformation change of the another binding site), results available in end-Sep or early Oct
• Evaluate current front runners in MST assay, results will come soon

Makoto and Takuto to look at the TSHO cpds to see if there is any candidate (catalytic binder) suitable for tritiation

→ Makoto Fushimi: We’re actually checking availability of a REVERSIBLE compound for 3H labeling to measure Bmax. (TR06391037 (series C) and TR06391001 (series C) should be good, binding to the GBA catalytic site. MST results with these compounds to measure binding affinity will come soon.

20201027 Makoto Fushimi: the competition assay (FRET) was completed.

The catalytic site binders, TR06391037-001, TR06391001-001, TR06397573-001, and TR06058741-001, were found to be competitive with allosteric FRET probes, probe A and B (ref. No 34-37 in spreadsheets “probe A” and “probe B”).

This indicated that catalytic site binders would be promising PET probes to measure occupancy of allosteric binder drugs because of the competition. However, the data also showed a discrepancy about potency between catalytic site binders (IC50 < 100 nM) and allosteric binders (IC50 = ~1000 nM). I was wondering if the potent catalytic site binder might work as a PET probe to see occupancy of allosteric binders with moderate binding potency.

In addition, MST binding data should be reported in a couple of weeks.

20201201 GBA activator project is terminated.

20210118 Makoto Fushimi: Affinity selection mass-spectrometry (ASMS) was selected instead of MST after discussion with TCAL. The ASMS will allow us to get this binding affinity ranking data quicker and with more confidence. ASMS results will come soon.

20210121 Talakad Lohith: the variance in basal expression levels is important for the interpretation of the treated GT patients data, and hence we need to collect the GBA PET data in HVs.

20200204 Makoto Fushimi: ASMS data were reported as the attached pptx file.

Key points

• Allosteric site binders (Series A) TR06592162-002 with KD > 20 μM against wt GBA.
• Catalytic site binders Series C (TR06391001-001 & TR06391037-001): KD 0.89 - 1.38 μM affinity observed.
• Catalytic site binders Series D (TR06397573-001) with KD > 20 μM for wt GBA.
• Isofagomine has no signal in ASMS exps.

Caveat

• ASMS was found to need 500-2,000 nM GBA protein, and it would be difficult to measure lower KD values.

I would say that FRET seems to be fine as the 1st screening. Attached please find the latest FRET data shared before. Because of the potency of Series C and D (IC50 < 100 nM), these compounds should be tested in lower concentration to have precise IC50 values as the next actions.

2. Additional suggestion for GBA PET

I think below should be mapped out. For this planning, I am not sure who we should connect. Maybe Makoto-san or Akihiro Takano-san who is a clinical PET expert based in Osaka office?

[Clinical plan of PET study]

• Clinical plan of PET study: how and when to test in HV and Patients
• Preclinical and clinical Timeline: key question is ‘can it be ready before GT P1 clinical study (in PD patients)?‘

4. Resource estimation for PET (BUDGET and FTE)

5. Potential GBA PET competitor on our radar

Please see below description by Makoto san, but these two are not very important based on the assessment.

Irreversible PET probe (developed in University of Saskatchewan)

Perhaps, irreversible inhibitors could be used for GBA Gene Therapy PET because of its purpose to measure GBA expression level, not target occupancy. As for the attached patent from University of Saskatchewan, I’m not sure that they have a promising PET probe. There are no data on radioactive compounds or in vivo data as Talakad should also point out.

MDW933 (developed at RD DDU) seems to be a fluorescent BODIPY dye, not a PET probe.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587854/

Catalytic site binder vs Allosteric binder

Catalytic site binder	Allosteric binder	Two competitive (FRET), But different potency
TR06391037-001, TR06391001-001, TR06397573-001, and TR06058741-001 GBA functional IC50, N370S/4MUG [nM] 가 37-820 nM 이니 'catalytic'이구나 Binding potency: [ASMS] KD 0.89 - 1.38 μM [FRET] IC50이 낮기는 한데 (1037, probe A → 0.1 nM), 이건 ALLOSTERIC BINDER 와의 competiton 인데 relevant 한가? Because of the potency of Series C and D (IC50 < 100 nM, Fret 말하는듯), these compounds should be tested in lower concentration to have precise IC50 values as the next actions. Isofagomine	allosteric FRET probes, probe A and B	GBA activator

For meeting with Paul 20200825

• GBA activator: we continue to screen better molecules and determine physiocochemical properties. Medchem team will confirm binding affinity of current front runners by biophysics way once TCAL team will restart the assay.
• would it make sense that we start feasibility activity for GBA gene therapy first and the Bmax info of GBA derived from the feasibility activity will also be utilized for GBA activator when we will have better molecules with sufficient physicochemical properties.
• Fushimi-san is in charge of the chemistry effort of PET portfolio (he took over the role from Nomura-san), and Tomimatsu-san is in charge of the biology matter of PET

Goal: to determine the level, regional distribution and duration of gene expression

• PET Steering Committee (PETSC) chaired by QTS imaging: Decision body for PET feasibility, execution, and technical matter
• PET Steering Committee - will decide feasibility and oversee execution of PET Ligand development by PET Development Teams
• TDT to present rationale and impact of PET tracer to PET Steering Committee (PETSC)
• lead drug candidate (T-4273531) blocks binding of 3 PET chemotypes
• Does the stage of the GPR20 project warrant starting activities to estimate receptor density (Bmax)?

GBA GT reporter system

	GBA GT		Example	GBA activator
Principle	To monitor expression (over time)	PET Reporter (gene?) probe system: (target gene) therapeutic gene of interest 와 reporter gene 이 한 promoter 를 공유하면서 서로 연결되어 있다 (coupled) → transcribed into a single mRNA coding for both gene products. → Translation of this mRNA yields two distinct proteins in amounts that are directly correlated with each other. radiotracer 는 이 두 protein 중 reporter protein 만 detect한다. Endogenous target gene 은 안 detect 하니 결국 GT로 인한 증가분이 얼마인지 못 알아내겠는데. TE marker로서, GT의 Brain 내 regional 분포는 볼 수 있음.		TO에 쓰려면, 우리의 chemA와 같은 데 경쟁적으로 붙으면서도, GBA활성에 무영향이어야 할 걸
elements	Corresponding radiotracer (ie radiotracer도 따로 개발해야 하는구나) Reporter gene: 조건: shouldn't be present in CNS!, expression of the reporter gene should not significantly perturb underlying cell biology therapeutic gene of interest (target gene
principle	Use tracer that image the spatial distribution of target gene expression, Total 을 볼 수 있으니, GT 의 Before vs After 비교가 가능함. 그럼 우리도 이걸로 개발하면 되지 않나 (CBE 에 isotope 달아서, 아니지 human에서 GBA activity 저하시키면 안 되지.)? Direct 와 달리 total 을 보기 때문에 before & after comparison 가능함.
Example	둘다 gene 아니라 gene expression (ie protein 본다), radiolabeled DASA-23 is the only radiotracer for the PKM2 protein 2019 Haywood: PKM2 is not present in the healthy brain		(Bankiewicz, 2000 #641 AADC, before and after (8 wks) AAV administration 봤음. (fig 2)): NHP: 바로 앞으로부터 시간경과 AADC gene delivery to striatum. MPTP (unilateral) 3달후 PET

Uncertain Spans

location	transcription	uncertainty
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