HRMS (ESI) calcd. act as a new leading compound for further PP121 optimization. with PGAM1 To further understand the molecular mechanism of the anthraquinone derivatives interacting with PGAM1, we identified the X-ray structure of PGAM1 in complex with compound 9i at resolution of 1 1.98 ? (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate binding site with good electron denseness (Number 3A,B). The allosteric pocket was surrounded from the residues of F22, R90, K100, R116 and R191. In detail, the anthraquinone scaffold and sulfonamide of compound 9i interacted with the main chain carbonyl of K100 through water bridges (Number 3C). In addition, a hydrophobic connection was observed between F22 and chlorine-substituted phenyl ring of compound 9i (Number 3C). Compound 9i also engaged in a -cation connection with R116 (Number 3C), which explains why modifications of the hydroxyl group led to decreased potency [39]. To validate the binding mode revealed from the co-crystal structure, we tested the activity of PGAM1 mutants PP121 (Supplementary Data, Number S1) and the inhibition activity of compound 9i on different mutations of PGAM1. Compound 9i failed to inhibit mutations of PGAM1 (F22A, R116H and R191H) as efficiently as the crazy type at concentration of 5 M which agreed with the results from crystal structure. Furthermore, a substrate competitive assay shown that compound 9i held a noncompetitive home with substrate 3PG which was also consistent with the binding mode exposed by X-ray structure. The co-crystal structure together with the molecular biological assays illustrated the binding mode of the anthraquinone inhibitor with PGAM1 and offered useful information for further optimization. Open in a separate window Number 3 Binding mode of anthraquinone inhibitor 9i with PGAM1. (a) Chemical structure of compound 9i and FoCFc electron denseness of compound 9i contoured at 2.0; (b) Overlay of compound 9i (PBD: 6ISN) and 3PG (PBD:2F90) in PGAM1; (c) Relationships of compound 9i and the crucial residues of PGAM1 in the co-crystal structure; (d) Inhibition of compounds 9i on wild-type and mutations of PGAM1 at concentration of 5 M; (e) Noncompetitive property of compound 9i with substrate 3PG. The data are offered as mean s.d. Table 5 Data collection and refinement statistics. = 8.8 Hz, 1H), 7.93C7.84 (m, 2H), 7.53 (d, = 8.8 Hz, 1H), 5.07 (s, 2H), 4.74 (s, 2H), 4.20 (qd, = 4.0, 7.2 Hz, PP121 4H), 1.23 (td, = 2.4, 7.2 Hz, 6H). 13C-NMR (151 MHz, DMSO) 181.69, 181.42, 168.27, 167.97, 156.49, 146.23, 134.58, 134.32, 133.94, 132.26, 127.15, 126.94, 126.67, 126.18, 124.64, 118.20, 68.72, Bmp7 65.22, 60.99, 60.44, 14.07, 13.98. MS (ESI) (= 7.6 Hz, 2H), 8.01 (d, = 8.4 Hz, 1H), 7.93C7.85 (m, 2H), 7.51 (d, = 8.8 Hz, 1H), 4.98 (s, 2H), 4.67 (s, 2H). 13C-NMR (151 MHz, DMSO) 181.95, 181.45, 169.75, 169.45, 156.76, 146.32, 134.62, 134.31, 133.98, 132.34, 126.97, 126.82, 126.71, 126.20, 124.61, 118.12, 68.62, 65.05. MS (ESI) (= 8.4 Hz, 1H), 7.31 (d, = 8.8 Hz, 1H), 5.09 (s, 2H), 3.02 (s, 3H), 2.87 (s, 3H). 13C-NMR (151 MHz, DMSO) 188.67, 180.76, 166.18, 152.60, 151.79, 135.20, 134.26, 133.48, 132.95, 126.81, 126.60, 124.89, 120.20, 118.29, 115.94, 66.12, 35.46, 35.01. MS (ESI) ((9a). Yellow solid, 25% yield. 1H-NMR (400 MHz, DMSO-= 8.4 Hz, 2H), 8.01 (d, = 8.4 Hz, 2H), 7.95C7.86 (m, 2H), PP121 7.73 (s, 1H). 13C-NMR (151 MHz, DMSO) 187.78, 180.56, 150.37, 144.20, 143.23, 135.02, 134.22, 133.28, 132.79, 132.68 (q, = 31.7 Hz), 130.34, 127.60 (2C), 126.77, 126.61, 126.59, 126.39, 123.71, 123.38 (q, = 273.3 Hz), 113.49, 113.35. MS (ESI) ((9b). Orange solid, 50% yield. 1H-NMR (400 MHz, DMSO-= 8.0 Hz, 2H), 8.25C8.06 (m, 4H), 7.97C7.86 (m, 2H), 7.73 (s, 1H). 13C-NMR (151 MHz, DMSO) 187.79, 180.53, 150.41, 149.90, 145.71, 143.61, 135.05, 134.24, 133.27, 132.79, 130.11, 128.22(2C), 126.77, 126.41, 124.65(2C), 123.70, 114.04, 113.50. MS (ESI) ((9c). Yellow solid, 41% yield. 1H-NMR (400 MHz, DMSO-= 1.2, 9.2 Hz, 2H). 13C-NMR (151 MHz, DMSO) 187.78, 180.60, 151.20, 150.35, 142.96, 139.19, 135.02, 134.23, 133.30, 132.82, 130.63, 129.29, 126.78, 126.40, 123.74, 121.47, 119.80 (q, = 259.7 Hz), 113.22, 113.12. MS (ESI) ((9d). Yellow solid, 40% yield. 1H-NMR (400 MHz,.