20240722_184142

		Cell	Note	Date	Plate
concentration 3H-BCPP-EF	Protein	CC-2511	Healthy fibroblast	220530	plate1
		ND30171	PD-PARK2 (ARG42PRO EX3DEL) fibroblasts	220530	plate2
with the protein amount that we determine is usable, will cluster radioligand concentration around the literature reported Ki value of ~2nM with 9 concentrations in total that will try to capture both the linear portion of the curve as well as the plateau. → 20220518: nine ( 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.000) → 20220523 Masato & AK: 10 concentration; 75 nM, 50 nM, 25nM, 12.5 nM, 6.25nM, 3.125nM, 1.563 nM, 0.781 nM, 0.391 nM, 0 nM or 20220526 AK (Nine): 1,3,4,7,9,11, 15, 30, 60→ nine: 75.000 40.000 15.000 11.000 9.000 7.000 4.000 3.000 1.000		ND40078	PD-PARK2 (ARG275TRP/ARG275GLN Compound) fibroblasts	220530	plate3
		ND29369 (heteroz)	PD-PARK2 (ARG275TRP) fibroblasts	220530	plate4
		ND30159	Fibroblast of sporadic Parkinson's disease patient	220530	plate5
			Input count	220530	plate6
Rotenone block	안 하는듯
Self block (cold probe)	BCPP-EF 10 uM
protocol	plate1 / [3H] BCPP-EF [nM]: 75.000 60.000 15.000 11.000 9.000 7.000 4.000 3.000 1.000 0.000 — columns 1-12, rows m=1, m=2 (green band) and m=1, m=2 (orange bands) for DMSO / BCPP-EF 10 uM / Filterblank. plate2 / Input count 750 nM — 3H BCPP-EF: 750.000 400.000 150.000 110.000 90.000 70.000 40.000 30.000 10.000 0.000 — columns 1-12, rows m=1, m=2 (orange bands) for the same DMSO / BCPP-EF 10 uM / Filterblank conditions. タンパク使用量 16 mg Protein amount 400ug x (duplicates), x (blocking +/-) = 14.4mg (no pooling) Or 400ug x 10 conc x (triplicate), x (blocking +/-) = 24mg 20220523: 400ug x (zero 포함) x (duplicate) x (blocking +/-) = 16mg
Result	1) TNTechnos did not test the ligand concentrations prior to the assays to make sure they were applying the concentrations we wanted, so the higher concentrations did not reach 75nM     a. The average radioligand concentrations between the two assays:         0.551657, 1.798633 2.671347 6.69197 8.525766 10.56962 12.25172 33.148 55.18 2) The highest 2 concentrations were greatly driven by nonspecific binding, specifically filter binding, which Nakashima-san had pointed out. I made the decision to exclude those 2 concentrations from all the patient samples because of this and to remain consistent across all. I did include a slide with the graph and estimated Kd and Bmax with those values included. 3) The autoradiography curves do not reach saturation as the highest concentration I used at the time was only 40 nM. I would feel more comfortable reporting these values with the assays having been repeated due to the lack of saturation and observed variability, so I have not included those analyses here. I have included a representative saturation isotherm from ARG in the slide deck. 4) No significant difference was shown by one-way ANOVA between groups, however the study is low powered as we are aware. 5) No Significant difference in Kd values were observed between groups. Averaged Saturation Binding Isotherms (Bound (fmol/mg) 0-100 vs Free Radioligand (nM) 0-13, with Healthy / sPD / PARK2-PD curves) — Individual Bmax values dot plot (Bmax (fmol/mg) 0-200, F (2,6) = 2.98, p = 0.13, groups Healthy / sPD / PARK2-PD). Individual Patient-derived Binding Parameters Sample Group Kd (nM) Bmax (fmol/mg) GM01650 Healthy 4.3 62.6 106-05n Healthy 4.0 60.1 CC-2511 Healthy 3.1 58.6 Mean ± SD n = 3 3.8 ± 0.6 60.4 ± 2.0 ND30159 Sporadic PD 2 43.9 ND37132 Sporadic PD 1.6 21.8 Mean ± SD n = 2 1.8 ± 0.3 32.9 ± 15.6 ND30171 PARK2 PD 8.3 155.7 ND40078 PARK2 PD 4.6 74.5 ND29369 PARK2 PD 2.9 54.2 ND31618 PARK2 PD 5.3 106.1 Mean ± SD n = 4 5.3 ± 2.3 97.6 ± 44.2 Cf) Internal data: Elevated NDUFS2, NDUFS4, NDUFA10)

[Scatchard plot]

The most common linear transformation of binding data is the Scatchard plot.

• the x-axis is specific binding (usually labeled ‘bound’) and
• the y-axis is the ratio of specific binding to concentration of free radioligand (usually labeled ‘bound or free’).

B = Bound (LR)

    Concentration of ligand that is specifically bound to the receptor at equilibrium.

F = Free (L)

    Concentration of free ligand present in the incubation at equilibrium. As discussed in Section 2.03.3.6, this is often estimated by the concentration of the drug added to the incubation.

K_d

    Equilibrium dissociation constant. In the Scatchard plot, the slope of the line is equal to K_d^-1. K_d is expressed in the same units as the drug concentration (i.e., molarity).

B_max (R_tot)

    Maximum number of binding sites in the incubation at equilibrium, or total receptor concentration. B_max is expressed in the same units as the specific binding, usually as a concentration relative to amount of protein in the incubation (i.e., fmoles per mg protein).

The reason for plotting B/F as the y-axis and B as the x-axis comes from rearranging eqn [5] (and substituting B for LR, F for L, and B_max for R_tot):

B = F·B_max/(K_d+F) becomes B·K_d+B·F = F·B_max

Dividing by F, and rearranging results in eqn [6] in the form of y = mx + b:

B/F·K_d + B = B_max

B/F = B_max/K_d − B/K_d

(6) B/F = −1/K_d · B + B_max/K_d

When making a Scatchard plot, you have to choose units for the y-axis. One choice is to express both free ligand and specific binding in counts per minute (cpm), so the ratio of bound to free is a unitless fraction. The advantage of this choice is that you can interpret y-values as the fraction of radioligand bound to receptors. If the highest y-value is large (greater than 0.10), then the free concentration will be substantially less than the added concentration of radioligand, and the standard analyses are not appropriate. As such, one should either revise the experimental protocol or use special analysis methods that deal with ligand depletion (see Section 2.03.3.6). The disadvantage is that the experimenter cannot interpret the slope of the line without performing unit conversions. An alternative is to express the y-axis as fmol ligand bound per mg protein per concentration (n mol l^-1). While these values are hard to interpret, they simplify calculation of the K_d which equals the reciprocal of the slope. The specific binding units cancel when the slope is calculated. The negative reciprocal of the slope is expressed in units of concentration (n mol l^-1) which equals the K_d (see Figure 5).

(Scatchard plot graph: Slope = −K_a = −1/K_d, B-axis 0 to 120, x-intercept = B_max.)

ARG

0220308 AK

I informed Jaewon that I started the first ARG assay with [3H]BCPP-EF blocked with rotenone at ~Kd concentration and ~saturating concentration to see the counts and specific binding yielded I should have these data be end of next week or early the following to see if assay conditions must be optimized or if saturation studies can move forward 2. If ARG results yielded allow for saturations to move forward, we may submit only post-mortem tissue results and make a "no-go" decision on continued fibroblast studies

0220420

(left figure) dpm bar chart, dpm 0-15000, clusters Total / BCPP-EF / Rotenone repeated for WT Rat, HC, HC, sPD, sPD groups; (right figure) dpm dot plot, dpm 0-20000, clusters Total / BCPP-EF / Rotenone for WT Rat (n=1) ▪, Healthy Control (n=2) ▪, sporadic Parkinson's disease (n=2) ▴. • [3H]BCPP-EF: 9.4 nM, blocker: BCPP-EF 10 uM, or rotenone 10 uM → results: BCPP-EF (10mM) displaced [3H]BCPP-EF (9.4 nM) with a much greater signal-to-noise window (94%) than Rotenone (10mM; 8%), an MC1 inhibitor [Next step] • Section all post mortem tissue blocks for saturation binding analysis ○ 2 weeks to section 20 additional blocks → 3 weeks to carry out saturation (9 point min.) and expose tissue autoradiography

{Tsukada, 2014 #883} bovine cardiomyocyte submitochondrial particles (SMP), Inhibition of specific binding of 3H-dihydrorotenone (fixed at 4.5 nM,) by BCPP-EF. → results: (18F BCPP-EF; IC50=2.68nM; Ki =2.31 nM, 즉 Kd 없고 이 Ki 값에서 AK 가 원용한 것임. AK 가 ARG optimization 할 때 이 값의 양쪽 벌어진 값인 1 & 10 nM 을 test 하고, 이 중 10 nM 을 선택하여 moved forward.

(Inhibition ratio (%) versus Concentration (nM) plot, 0.01 to 100, with curves for triangular open BMS, filled-circle BCPP-EF, and open-circle BCPP-BF. Inset:)

	IC50 (nM)	Ki (nM)
BMS	1.10	0.95
BCPP-EF	2.68	2.31
BCPP-BF	0.81	0.70

Meeting 20211203 schedule

Christina Murphy 202202 Update to MJF with 2-3 (in vitro rsults) 20220311: invite for RFA 20220512 Full proposal due

Meeting 20220307 Christina Murphy (program officer: 212.509.0995 Ext. 464)

2022 May: update to MJF (in vitro rsults), 2022, June 10: invite for RFP (nov 18th) 2022 Aug 11: full proposal due (Jan ...) 2022 June 6 - Send total time for project and total approximate budget (보통 24m, $500k) 환자수도 넣자. 2022 June 10: Receive Invitation from System to submit RFP 2022 June 13th Letter of Intent (1-2 page proposal): preliminary data, rationale, (계획) → send to clinical department for ...

Uncertain Spans

• “75.000 60.000 15.000 11.000 9.000 7.000 4.000 3.000 1.000” — the second column header in the upper protocol grid is “60.000” by visual reading; the OCR draft had “63.000”. The image aliasing makes the digit ambiguous.
• “B/F·K_d + B = B_max” — the page renders the equation as BKd+BF=FBmax then B/FKd+B=Bmax; the typesetting collapses operators and I have re-spaced them per algebraic intent. The original glyph spacing is preserved as evidence.
• “ND37132 / Sporadic PD / 1.6 / 21.8” — the Bmax value 21.8 reads clearly but the table-row banding alternates so the row identity is confirmed only by adjacent CC-2511 / 106-05n alignment.
• The 2022 Aug 11 bullet ends with “(Jan …” and is cut at the right column boundary; the rest of the parenthetical continues on photo 20240722_184156.