20240722_183528

In vitro & Rat	(Tsukada, 2014 #883)	Vs rotenone In vitro binding assay (in bovine cardiomyocyte SMP): competition, BCPP-EF (variable concentrations) with ³H-dihydrorotenone (concentration was fixed at 4.5 nMr), n not said, PET: After continuous infusion of vehicle or rotenone at dose of 0.1 mg/kg/h for 1 h, PET scanning was conducted for 60 min after injection of each PET probe		In vitro binding assay results (suppl fig)
NHP	(Tsukada, 2014 #884)	Vs rotenone - PET: After infusion of vehicle or rotenone (was converted to digitalized imaging data), 결과: Fig 4. 2. 2. (미취주) PET: 결고 fig5
In vitro	(Kazami, 2019 #1082)	In vitro binding assay (in bovine cardiomyocyte SMP (sub-mitochondrial particles)): competition, BCPP-EF (variable concentrations) with ³H-dihydrorotenone (concentration was fixed at 4.5 nMr), js: the same description as Tsukada 2014 #883 above. n not said. Protein-bound radioactivities remaining in the filter paper were then measured using a liquid scintillation counter (3100, PerkinElmer, Shelton, CT). Specific binding was plotted against the concentration of each testing compound in order to obtain 50% inhibitory concentration (IC50) values, which were converted to the inhibition constant (Ki) using the Cheng and Prusoff equation (Cheng and Prusoff, 1973). → IC50 values were 5.2, 2.4, 1.9, 1.0, and 0.7 nM for BCPP-EM, BCPP-EF, rotenone, BMS, and BCPP-BF, respectively (Table 1). Lipophilicity of BCPP-EF: logP of 3.0, which is within appropriate range for BBB penetration

• SMP: A submitochondrial particle is a compartmentalized membranous product of exposing mitochondria to ultrasound.^[1] This causes the cristae to pinch off, forcing the inner mitochondrial membrane inside out. As a consequence, the F₁ particle becomes exposed and on the outside. Using chaotropic agents such as urea, these particles can be removed, dissociating the related ATPase activity from the membrane. However, the electron transport complexes remain on the membrane. If F₁ particles are removed from submitochondrial particles, they can be reconstituted by carefully removing the chaotropic agent.^[2]

Radioligand binding study
A potent inhibitor is radiolabeled, → incubated with a potential source of the target site, the bound and free radioligand separated by centrifuguation or filtration. → specific binding is defined .... non-specific binding: is measured by incubating sections, cells, or homogenates under identical conditions but in the presence of an unlabeled ligand at a concentration saturating the target receptors. (a competing ligand is included in the assay, usually at a minimum concentration of 100 times the KD, as this will occupy 99% of the receptors) → The equilibrium is then broken, radiolabeled ligand bound to receptor separated from free, and the results analyzed. By definition, specific binding is saturable and reversible. Total binding − non-specific binding = specific binding			Photoaffinity labelling study
Ideally, NSB binding is only 10% to 20% of the total radioligand binding. If NSB makes up more than half of the total binding, it will be hard to get quality data. If the system exhibits a great deal of NSB, use a different kind of filter, wash with a larger volume of buffer or a different temperature buffer, or use a different radioligand.
	Self-blocking (homologous) Hetterologus blocking (structurally dissimilar) To assess Total Specigic binding 붙어야 할 곳에 붙는지 Total SELECTIVE BINDING 안 붙어야 할 곳에 붙는지 응용 ¹⁸F-AV-1451, self-block in healthy NHP showed a large decrease in signal. Healthy NHPs are devoid of NFT. Therefore, all displaceable signal from a self-block experiment represents specific binding to off-target sites. The large decrease observed for ¹⁸F-AV-1451 in healthy NHP suggests substantial off-target binding, which may perturb signal quantification in disease groups [22]. 단점: both the labeled and unlabeled forms of the drug will bind to the same speciƥc and nonspeciƥc sites. This means that the unlabeled drug will reduce binding purely by isotopic dilution. When ¹⁸FTHK5351 In vivo imaging in (MCI) and AD patients after 10 mg of selegiline, used clinically as an irreversible (MAO) inhibitor, reduced brain uptake of ¹⁸F-THK1351 by 37–52% compared with baseline. The greatest decrease was observed in regions expected to have high MAO-B concentrations, and signal loss was maintained during the third scans 9–28 days later [48]. A substantial portion of signal in AD and MCI patients therefore appears to be specific off-target binding to MAO. concent useful rule of thumb is to use the unlabeled compound at a concentration at least 100 times its Kd for the receptor
4. Validation to confirm that the SB site observed is the inhibition site for enzyme activity. two types of experiments are ... A ligand is designed → labelled → bind to the site → on exposure to UV light, it covalently derivatize the site. Commercial suppliers of radiolabeled ligands include the following: PerkinElmer ( http://www.perkinelmer.com ), American Radiolabeled Chemicals Inc. ( http://www.arcincusa.com ), Sigma-Aldrich ( http://www.sigmaaldrich.com/chemistry/stable-isotopes-isotec.html ), ViTrax Radiochemicals ( http://www.vitrax.com ), Quotient Bioresearch ( http://www.quotientbioresearch.com ), IsoFlex ( http://www.isoflex.com ).
resource	Maguire 2012 (book), Bylund (book)
Eg	{Harada, 2013 #878}
Cons	이 실험자체에서 binding site 특정은 불가. Informs nothing on functionality

	Saturation assay	Competition binding assay	Kinetic assay
scheme	increasing amounts of radioligands	Radioligand: fixed concentration increasing concentrations of unlabeled compound (eg 우리약)	time course of association/dissociation and reverse (k−1) rate constants (forward (k+1) and reverse (k−1) rate constants for radioligand binding)
	Total binding: Increasing concentration of radioligand in absence of cold ligand. Measures both specific binding to receptor, as well as non-specific binding. Non-specific binding: Increasing concentration of radioligand in presence of cold ligand. Measures binding of the radioligand to non-receptor components. (other proteins & lipids, filter paper). Specific binding: Total minus non-specific binding. Measures binding to the receptor, specifically. An assay is considered barely adequate if 50% of the total binding is specific, 70% is good, and 90% is excellent.
Measured parameter	Bmax (receptor expression level, the amount of ligand bound/mg protein present in the assay) Bmax는 all binding sites가 saturation될때의 ligand농도이므로, ligand의 function이 아니라 (실험에 사용된 protein농도는 단일하므로) receptor의 amount를 반영함. Kd (binding affinity of hot ligand, = the concentration of ligand that occupies 50% of the binding sites. Expressed in units of mol/liter or molar (M), is the concentration of ligand that occupies half of the receptors at equilibrium. corresponding to 50% of Bmax, 아래 그림(Bylund p136)에서 Bmax가 10이니, 그 반인 5에 해당하는 x죽값, 수: y죽값 아님!)	IC50: Concentration of a competing ligand that displaces half of the radioactive ligand Ki: affinity of a cold ligand for the receptor 즉, (cold가 여러 dose니까) 기본적으로 cold의 능력을 보는 것이네. Target density (?) (once Kd of a radiolabeled ligand for a target has been determined from saturation assay)
	[ligand]/Kd Fractional occupancy at equilibrium 0 0% 1 50% 4 80% 9 90% 99 99% 'saturation curve': x죽: concentrations of radioligand = total free concentration of radioligand = total added − specific bound y죽: Bound" (the amount of radioactive ligand that is bound to the receptor) Fig. 1. Typical saturation binding experiment. In this simulation the Bmax (receptor density) is 10 pM and the Kd (the dissociation constant or the free concentration which gives half maximal binding) is 100 pM. Figure 1: SATURATION BINDING ASSAY CURVE (FILTRATION). 96-well saturation binding assay (10 µg membranes/well, TopCount™).	Below picture: x죽은 increasing amounts of cold ligands Figure 2: COMPETITION BINDING ASSAY CURVE (FILTRATION). 96-well competition binding assay curve (10 µg membranes/well, TopCount™). Recommended radioligand concentration = 1.7 nM.
matrix	Tissue sections, cultured cells, homogenates, (Bylund, 2011 #1244) protocol: cell: membrane preparation, intact cells in suspension (Basu, 1978 #1243) fibroblast, but (plasma) membrane preparation (n: 'normal subjects' 복수표현, pt것은 한명(M.C라는 사람)) Incubate with radiolabeled ligand for a defined time and temperature to reach equilibrium.
Question?	Cf) Specific binding: if 50% of the total binding is specific: barely adequate, 70% is good, and 90% is excellent.	radiolabeled ligand 의 농도를 어떻게 정하나? Kc미리 구해야 하나? 그러러면, saturation analysis미리 해야 하는데... Self block: unlabeled ligand 의 충분한 농도를 어떻게 미리 아나? Use Kd? Or concentration to label ?% of the target Multiple binding site시 ok? Predicted Bmax unknown시 ok?

Questions	Comment
Does the calculated curve go near the data points?	If the curve doesn't go near the data, then something went wrong with the curve fit, and the "best-fit" values of Bmax and Kd should be ignored.
Were sufficient concentrations of radioligand used?	Ideally, the highest concentration should be at least 10 times the Kd. Calculate the ratio of the highest radioligand concentration used divided by the Kd reported by the program (both in nM or pM). The ratio should be greater than 10.
Is the Bmax reasonable?	Typical values for Bmax are 10 to 1000 fmol binding sites per milligram of membrane protein, 1000 sites per cell, or 1 receptor per square micron of membrane. If using cells transfected with receptor genes, then the Bmax may be 10 to 100 times larger than these values.

Table 7.5.2 Evaluating the Assumptions of Saturation Binding Analysis

Assumption	Comment
Binding has reached equilibrium.	It takes longest for the lower concentrations to equilibrate, so test equilibration time with the lowest concentration of radioligand.
There is only one population of receptors.	See Theory: Comparing One- and Two-Site Models, below.

Uncertain Spans

location	transcription	uncertainty
Tsukada 2014 #884 row	결과: Fig 4. 2. 2. / (미취주) PET: 결고 fig5	Korean shorthand annotations near the figure are partially abbreviated; final tokens (결고 / 결과) are read as written but may be intended as 결과.
Self-blocking column 단점	speciƥc and nonspeciƥc sites	Source uses a non-standard ƥ glyph (likely a font/ligature artefact for fi); preserved as seen.
Saturation cell	[3H]-Cyclopentyl-1,3-dipropylxanthine axis	Embedded plot legend; legend entries 3 and 4 (1,3-Dipropyl-8-phenyl..., 2-Chloro-N6-Cyclopent...) are truncated by the embedded figure crop and not fully readable.
Bottom-left review-table row labels	ficient ? tions of? d used? reasonable?	The leftmost column of the bottom comment-style table is sliced by the page edge; only the right portion of the row labels is visible. They are inferred to be coefficient ? / concentrations of radioligand used? / reasonable? from context but kept as unverified text in the body.