Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules

Figures & data

Figure 1. Structure of the open state of hVDAC1 in LDAO micelles (PDBID:2JK4)¹⁵. Left: side (membrane) view. Right: top (cytoplasm) view. The barrel pore is coloured in blue and the alpha helix in yellow.

Table 1. List of selected compounds including experimental techniques used to show direct interaction as well as observed effect(s) on VDAC1 and corresponding references.

Compound	Technique(s) showing direct interaction	Effect(s) on activity	Reference
Aspirin	PLB	Reduces conductance Induces VDAC-HK II detachment	^24
Cannabidiol	PLB, MST	Reduces conductance Increases mitochondrial Ca^2+	^34
Catechin	Affinity chromatography		^33
Curcumin	PLB	Reduces conductance by promoting closed state(s)	^25
DCCD	PLB, NMR [^14C] DCCD-labelled VDAC1	Reduces conductance Inhibits HK-VDAC1 binding	^44^,^46^,^47^,^48
DIDS	PLB, MST	Reduces conductance by complete closure of the channel Inhibits oligomerization	^48^,^49
DPC	PLB, MST	Reduces conductance Prevents oligomerization	^49
Emodin	Affinity chromatography		^33
Fluoxetine	PLB	Reduces conductance Prevents Cyto C release	^50^,^51
Itraconazole	PLB, Photoaffinity labelling MST	Reduces conductance	^35^,^26
Metformin	PLB, MST	Reduces conductance No binding observed by MST	^30
NADH	PLB, NMR	Affects gating without reducing the conductance Sterically closes the channel	^38^,^52^,^14
Olesoxime	PLB	Reduces conductance	^53
Propofol	PLB	Promotes closure when a negative voltage is applied No effects on open state conductance	^54
Quinidine	PLB	Reduces conductance by steric blockage	^55
Ubiquinone 0	Photoaffinity labelling	Induces over-expression of VDAC	^56^,^57
VBIT4	PLB, MST	Reduces conductance Inhibits oligomerization Inhibits HK detachment	^29

Structures of compounds are shown in supplementary Figure S3.

Figure 2. Thermal stability of hVDAC1 in LDAO micelles determined by NanoDSF in presence or absence of 5% DMSO. The S-shaped transition of the ratio of tryptophan fluorescence at 350 and 330 nm is displayed in the top figure while corresponding first derivatives are shown in the bottom figure. Inflection points, which correspond to the T_m, are indicated by dashed lines. T_m values were 57.60 ± 0.05 °C and 56.00 ± 0.07 °C in the absence or in the presence of 5% DMSO, respectively. Standard deviations were obtained by performing triplicates of each experiment.

Figure 3. First derivative of the 350/330 nm ratio obtained from NanoDSF as a function of the temperature. Only compounds displaying a significant T_m shift are shown. (A) Compounds solubilised in buffer. (B) Compounds solubilised in DMSO. Reference T_m, obtained for hVDAC1 (in absence (A) or in presence of 5% DMSO (B)) is indicated.

Figure 4. MST binding check performed on 100 nM hVDAC1 with (green), or without (blue) 250 µM VBIT4.

Figure 5. MST dose-response curve of the eight compounds displaying precise or minimum K_d values (cannabidiol, curcumin, DIDS, emodin, itraconazole, VBIT4, propofol and fluoxetine). Error bars show standard deviation obtained from three replicas of the same measurement.

Table 2. Results obtained by NanoDSF and MST including T_m shift, MST binding check and dissociation constant evaluated for each tested compounds and published affinity if available. A thermal shift was considered significant when its variation exceeded ± 0.22 °C. (N.D., not determined, when experiments were not performed due to inconclusive binding check assay; N.A., not available, when experiments were performed but were inconclusive). hVDAC1 = human recombinant VDAC1 and rVDAC1 = VDAC1 extracted from rat.

Compound	ΔTm °C	MST Binding Check	Kd (µM)	Published Kd (µM)
Aspirin	+1.01	Positive	N.A.
Cannabidiol	+0.94	Positive	6 ± 2	11.2 [rVDAC1]
Catechin	+0.91	N.A.	N.D.
Curcumin	−0.76	Positive	6 ± 1
DCCD	+8.19	Positive	N.A.
DIDS	−0.53	Positive	0.5 ± 0.2	70 [rVDAC1]
DPC	−0.45	Positive	N.A.	150 [rVDAC1]
Emodin	−0.21	Positive	10 ± 5
Fluoxetine	+0.81	Positive	>7 700
Itraconazole	−0.22	Positive	5 ± 2	120 [rVDAC1]
Metformin	+0.12	Positive	N.A.
NADH	+0.18	Positive	N.A.	9000 (hVDAC1)
Olesoxime	−0.56	Positive	N.A.
Propofol	+1.31	Positive	>650
Quinidine	−0.82	Positive	N.A.
Ubiquinone 0	−0.94	Negative	N.D.
VBIT4	−3.51	Positive	3 ± 1	53 [hVDAC1]

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