Solvent Selection for Insoluble Ligands, a Challenge for Biological Assay Development: A TNF-α/SPD304 Study

Abstract

Many active compounds may be excluded from biological assays due to their low aqueous solubility. In this study, a simple method for the determination of the solubility of compounds containing aromatic rings is proposed. In addition to DMSO, five organic solvents for screening experiments of TNF-α inhibitors were explored. DMSO and PEG3350 were the most suitable for both protein stability and ligand-binding experiments. In addition, glycerol is a promising solvent for the screening of other compounds for which it might provide acceptable solubilization, due to its strong tendency to preserve the protein. Moreover, a fluorescence binding assay was developed using the TNF-α/SPD304 system, and a K d of 5.36 ± 0.21 μM was determined. The results of this study could be used for the future screening of potential TNF-α inhibitors, while the protocols developed in this work could be applied to other proteins.

Rationally Designed Less Toxic SPD‐304 Analogs and Preliminary Evaluation of Their TNF Inhibitory Effects

“…Initial evaluation of the synthesized analogs 2a – b and 3a – d was performed in terms of their potency to inhibit TNF trimerization by utilizing an in vitro fluorescence binding assay . Specifically, the change of the fluorescence signal of three tyrosine residues located at the ligand‐binding interface (Tyr59, Tyr119, and Tyr151, Fig.…”

“…Specifically, the change of the fluorescence signal of three tyrosine residues located at the ligand‐binding interface (Tyr59, Tyr119, and Tyr151, Fig. A) is monitored upon titration of the ligands . In this way, the dissociation constant of the parent compound 1 was calculated to be K d = 5.4 µM.…”

Identification of an in vivo orally active dual-binding protein-protein interaction inhibitor targeting TNFα through combined in silico/in vitro/in vivo screening

“…On the TNFα/SPD304 complex, the phase shift variations (ΔΦ) suggested a “one-site binding” with a corresponding K d constant of 9.1 ± 1.1 µM which is consistent with the Kd values obtained by Papaneophytou et al . in the literature using a fluorescence binding assay ( K d  = 5.4 ± 0.2 μM) 33 . On the TNFα/compound 1 complex, the phase shift variations (ΔΦ) suggested a “two-site binding” (Fig.…”

Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones

“…Differences in fluorescence intensity at 345 nm between the complex (cyclin A2/5921) and free protein (excitation at 295 nm) were analysed as previously described in [31] (Eq. 2), in order to determine the dissociation constant (K d ) of recombinant cyclin A2 with 5921: $$F_{\mathit{\text{obs}}}=F_{BG}+M{F}_{P_F}false[P_{F}false]+FR·M{F}_{P_F}·\frac{false(false[L_{T}false]+false[P_{T}false]+K_{d}false]false)\pm \sqrt{{([L_T]+[P_T]+K_d])}^2-4[P_L][L_T]}}{2}$$F obs is the observed fluorescence intensity; F BG is the fluorescence background signal; MF P F and P F are the molar fluorescence and concentration of unbound protein, respectively; FR is the fluorescence ratio of bound protein; L T and P T are total concentrations of ligand and protein, respectively.…”

“…The slits were set at 5 and 20 nm in the excitation and emission respectively. For the binding assay of 6xHis-CyclinA 2 with the 5921 ligand, measured intensities were corrected for blank signals, as previously explained [31]. Briefly, 1.5 ml of protein solution in fluorescence buffer (0.1 to 0.65 µM) was placed in a cuvette.…”