Application of fragment-based drug discovery to membrane proteins: identification of ligands of the integral membrane enzyme DsbB

Abstract

Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here, we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The Escherichia coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1071 drug fragments for binding using target immobilized NMR Screening. Biochemical and biophysical validation of the eight most potent hits revealed an IC(50) range of 7-200 microM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins.

Figure 1

Stability of the DsbB in Micelles and NDs. The stability of DPC solubilized (a) and ND solubilized (b) DsbB after multiple cycles of compound application and washing was assessed by binding of a known ligand. Binding is displayed as the average ratio of peak heights for the compound in the presence of DsbB over that in the presence of the reference (T/R ratio). The reference in (a) was DPC solubilized OmpA and in (b) -/ND. Note the difference in vertical scale between parts a and b.

Figure 2

Detection of ligand binding to immobilized DsbB using TINS. The 1D ¹H NMR spectrum of 3 different fragments in solution (a – c) is shown for reference. The ¹H NMR spectrum of a mix of the 3 fragments in the presence of DsbB/DPC (red spectrum) or OmpA/DPC (blue spectrum) that have been immobilized on the sepharose support is shown in d. The spectra of the same mix recorded in the presence of DsbB/ND (green) or -/ND (magenta) is shown in e. The asterisk indicates the resonance from residual ¹H DMSO and the bracket shows residual sugar ¹H resonances from the sepharose media. The residual H₂O resonance at 4.7 ppm has been filtered out.

Figure 3

Comparison of TINS screening in micelles vs NDs. A total of 70 fragments were assayed for binding to DsbB solubilized in either detergent micelle or ND. a) The 70 fragments were screened for binding to DsbB/ND using either empty ND (-/ND) or OmpA/ND as a reference. The T/R (see text) for each compound is plotted for one screen versus the other. R² = 0.78. b) The T/R for each compound in the DsbB/ND vs -/ND screen is plotted against the value from the DsbB/DPC vs OmpA/DPC screen. Hits common to both screens are show in red. Hits found only in the ND screen are shown in blue while those found only in tehe DPC screen are in green.

Figure 4

Distribution of biological activity of the hits found in the TINS fragment screen of DsbB. Each fragment was assayed singly at 250 μM. The percentage of inhibition of each category is provided in the legend. The height of each bar represents the percentage of all hits found in the TINS screen within the given range of potencies. The three ranges of inhibitors mentioned in the text (low, medium and high) are indicated.

Figure 5

Potency determination of selected hits from the TINS screen. An example of an inhibition curve used to determine the IC₅₀ for compound 2. The curve represents the mean ± S.E.M. of three independent experiments performed in triplicate. The structures of the 8 most potent compounds are shown and the IC₅₀ values are provided in Table 3.

Figure 6

Mode of action determination for the most potent DsbB inhibitors. Fragment 2 was assayed in competition with synthetic UQ1 (a), the electron acceptor, or DsbA (b) the electron source. Fragment 8 was assayed in the same manner (panels c and d respectively). The k_cat and K_m apparent determined from the data are shown in Table 4 in the absence and presence of the indicated amount of each inhibitor. See supplementary information for data on the other 6 fragments.

Figure 7

NMR analysis of fragment binding to DsbB. The 8 most potent fragments were titrated into ¹⁵N DsbB[CSSC]. Data for the synthetic quinone UQ1 (a), competitive fragment 2 (b) and the mixed model fragment 8 (c) are shown. For each of these three compounds, the structure of the compound is shown in the left column and the characteristic peak perturbations in the [¹⁵N,¹H] HSQC spectrum (green 0 mM fragment, blue 5 mM fragment and red 10 mM fragment) are shown in the middle (Arginine 109 backbone amide) and right columns (Tryptophan 135 sidechain indole).