MRI GBA tail (Caminiti DATSCAN / Leocadi / Thaler / Agosta / Filippi), PET Imaging GBA progress

MRI GBA (continued)

DATSCAN longitudinal text

late-iPD. The GBA-PD group showed a significantly lower 123I-FP-CIT SUVr in the whole contralateral putamen, anterior and motor putamen, globus pallidus, hippocampus, and amygdala than the early-iPD group.

[longi] From baseline to “2-years” follow-up, we found that the GBA-PD showed a global SUVr rate of change (i.e., mean rate of change of all considered ROIs) equal to 0.11, early-iPD equal to 0.09, and late-iPD equal to 0.16 (Fig. 4). The late-iPD group showed the highest global SUVr rate of change. Of note, we found that the early-iPD and late-iPD groups significantly increased their SUVr rate of change, namely significant SUVr reductions over time, in the ventral striatum in comparison with the GBA-PD group at “2-years” (early-iPD vs. GBA-PD P = 0.024; late-iPD vs. GBA-PD P < 0.001). 더 빠른 진행 X

The linear regression model revealed that low SUVr values in the caudate nucleus (motor division) at baseline were associated with UPDRS-III motor scores worsening from “2-years” to “6-years” follow-up visits in the whole PD patients (B = 6.46; P = 0.021) and in the late-iPD group (B = 10.26; P = 0.009). In the whole PD group (385 patients).

Leocadi 2022 #2708 (Italy)

Ten GBA-positive and 20 GBA-negative PD patients,

UPDRS O, MMSE O, MOCA X, Digit Span test etc O

Baseline: GBA-PD = “cortical thinning of left temporal, parietal, and occipital gyri (vs. CTRL and iPD)

Longi: GBA-PD = “cortical thinning of posterior regions, frontal and orbitofrontal cortices; similar pattern of subcortical, hippocampal, and amygdala volume loss (vs. iPD), whereas the pattern of subcortical GM volume (caudate, pallidum, putamen, thalamus, hippo, amygdala) atrophy ( was similar in the two PD group

Js: MMSE 도 mri 도 모두 no longi change and no difference between GBA-D and Non-GBA-PD (early PD 라 그럴것임)

Cortical thickness and Sub-cortical Volume

	cohort	finding
Cross-sectional	(Thaler, 2018 #2709) Tel-Aviv Medical Center. 12 GBA-PD, 9 LRRK2-PD, 57 iPD, 14 GBA-NMC, 41 LRRK2-NMC, 49 CTRL	Cortical thickness and Sub-cortical Volume no differences in GM have been reported between GBA-PD, PD with LRRK2 mutations (LRRK2-PD), and iPD, although,, lower GM volumes were reported in bilateral hippocampus, nucleus accumbens, caudate, thalamus, putamen and amygdala, and the right pallidum in patients with PD (eg, GBA-PD, LRRK2-PD, and iPD) compared to unaffected participants (eg, GBA-NMC, LRRK2-NMC, and controls).
Cross-sectional	(Agosta, 2013 #2710) Christine Klein, Serbian PD patients a; 15 GBA-PD, 14 iPD, 16 CTRL	no differences in GM have been reported between GBA-PD, iPD, and controls20

(Filippi 2022 2nd) The greater involvement of the posterior cortical regions in GBA-PD compared to iPD could represent the neuroimaging counterpart of clinical findings reporting cognitive impairment as a

PET Imaging GBA

Progress so far

For drug activator (but, drug 자체를 label 하는 거 아님!) → TE, TO

For GBA protein

1. (active) GBA expression
2. Bmax

GBA protein 에 잘 붙으면서 & GBA activator drug 와 competition 해야 할 걸

GBA protein 에 잘 붙기만 하면 되는게 아니라, a chemical scaffold that binds to orthosteric active site similar to the fluorescent dye that could give a read out of active GBA expression (즉 catalytic site binder 이어야 함)

clinic에서는 irreversible binder 여도 됨 (competition 불 필요 하므로), 그러나 Bmax에서는 reversible 만 됨.

Allosteric vs Catalytic site

Activating allosteric site	Not promising because of low binding affinity probably derived from shallow binding pocket → → Continue medchem efforts to get high affinity compounds & Evaluate current front runners in MST assay
Catalytic site	Two sites (allosteric site & catalytic site) are very close, If our activator binds to both catalytic site and allosteric site, and if an allosteric site binder (our drug) and a catalytic site binder (eg. isofagomine) are competitive with each other, we can use the catalytic site binder as a PET ligand.
property	Doesn't need BBB penetrance

Isofagomine

Univ of Saskatchewen (patent):

F-18 labeled aziridine analog, irreversible type compound (forming covalent bond with the catalytic site), so that it’s useful to measure Bmax, but not applicable for our compound occupancy. No in vivo data.

• L444P (N370S 도 마찬가지일것) mutation 중에 혹시 ER degradation 안 당한 GBA protein 들은 catalytic site 온전하니 lysosome 내에서 온전히 활동하고 있을 것이며 (‘active GBA protein’), 여기에 이 ligand 붙을 것. 즉, 뇌내에 존재하는 GBA protein 들은 mutation 이건 아니건 active 하고, ligand에도 붙을 것.
• The ligand bind both mutant and WT gba protein
• The ligand bind both endogenous and exogenous gba
• the ligand signal nicely represents both amount and activity of GBA protein in both homoz and heteroz patients. Pre-Tx vs Post-Tx comparison will be possible.
• Paul: an irreversible binder is ok for in vivo TE (eg. LSD-1 PET tracer that we discovered which binds irreversible and was translated into the clinic and used to assess TO of the therapeutic candidate TAK-418.), but not ok for (in vitro) Bmax assessment because the system is required to reach equilibrium to assess level of specific binding, which would not be possible with a irreversible binder.) →

Binding site investigation (Takuto to lead)

• 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 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 available in 1-2 mons

Try obtain co-crystal for X-ray structure analysis

Paul: (competitive binder) 하나로써 이 둘 다 해결하는 것이 목적.

Makoto / Takuto TSHO

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 (GBA functional IC50, N370S/4MUG [nM]) 가 37-820 nM 이니 ‘catalytic’이구나), MST results (왼쪽의 activator 들의 MST와 별개임, 이전 reversible!) to come Sep/Oct)

→ The team will go to PET SC for presentation after suitable candidate identified for tritiation

Competition assay (FRET) — 2010 Makoto Fushimi

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. Should we have a meeting at this point or wait for the MST data? ⇒ yes.

Js) in the excel sheet, I see potency of 1037 (ie IC50), but how can I see competition? Where is potency info of the probe A & B?

→ MST 결과 나오면 Masato 에게도 알려수자.

Affinity selection mass-spectrometry (ASMS)

101

1. Affinity selection mass-spectrometry (ASMS)
   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.

2. Report in two or three weeks
   The assay will be started this week and completed in two or three weeks. The ASMS system was repaired by an engineer last week.

012
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.

02
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.

Current data and position

• To understand current data (ie ASMS and FRET)
  • Key question: Have we got catalytic site binders that have potentially sufficient binding affinity to move forward?

Position/background

• Primary goal: The development of a novel GBA PET tracer would demonstrate in vivo spatiotemporal distribution of the target gene expression at regional level in Phase 1 trial.
• Relationship with followings: Dose selection, relationship to other TE markers (CSF GBA protein, mRNA, activity, GlcCer/GlcSph)
• Focus of GBA GT PET: Originally the hope 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.

Next action?

• Should we do another run of binding assay to determine precise IC50 values?

Clinical plan

• Clinical plan of PET study: HV and Patients
• Preclinical and clinical Timeline: : ready before GT P1 clinical study (in PD patients)?

[meeting] Next action:

• Measure precise affinity using FRET assay. Check whether we can conduct it at lower pH 4.7.
• (Option) Measure affinity using MST assay to determine affinity at neutral pH

My question: do we need do assay for Selectivity before Bmax, and how?

Binder property comparison

	i) Ideally what we want a binder that bind to..	ii) Alternative
	Functional GBA: ie Lysosomal Orthosteric active site (ie catylitic) binder	Any (?) GBA
Required pH	4.7	neutral Or pH-independent