Table 1. Predicted free energies of binding, VS rankings, chemical classification, occupation of the ANS1 and ANS2 pockets, selected physico–chemical properties, and biological properties of the 7 allosteric inhibitors discovered in the primary virtual screening (compounds 1-7) and of the nine analogs of compound 4 selected from hit expansion (compounds 4a-4i).
| Cpd # | Asinex # | ΔG’binda | Rank #b | Classc | ANS1d | ANS2d | MWe | PSAf | logPg oct/w | EC50 (μM)h | IC50 (μM)i MDA-MB231 | IC50 (μM)i ZR-75–1 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | BAS01060589 | −55.9 | 2 | carb. acid | x | x | 492.0 | 132.9 | 4.8 | 7 ± 3 | >50 | >50 |
| 2 | BAS00631909 | −51.6 | 14 | nitro | x | x | 515.4 | 102.8 | 5.6 | 3 ± 1 | 20.5 ± 4.4 | 16.7 ± 2.2 |
| 3 | BAS00590245 | −51.4 | 16 | carbonyl | x | 514.3 | 64.1 | 4.7 | 45 ± 16 | 18.6 ± 2.5 | 25.9 ± 1.0 | |
| 4 | BAS00380830 | −50.9 | 21 | nitro | x | 438.5 | 108.9 | 5.0 | 71 ± 28 | 4.0 ± 0.1 | 4.5 ± 0.2 | |
| 5 | BAS00434932 | −49.5 | 29 | nitro | x | 385.5 | 61.5 | 4.2 | 27 ± 6 | >50 | >50 | |
| 6 | BAS01060577 | −49.2 | 33 | nitro | x | x | 449.9 | 134.2 | 3.7 | 32 ± 7 | 49.2 ± 1.2 | >50 |
| 7 | BAS01123443 | −54.2 | 5 | carb. acid | x | x | 463.9 | 134.8 | 3.9 | 48 ± 18 | >50 | >50 |
| Hit expansion of compound 4 | ||||||||||||
| 4a | BAS02102259 | −50.8 | 2l | nitro | x | 506.6 | 127.1 | 5.6 | >100 | 21.1 ± 1.5 | 43.3 ± 9.5 | |
| 4b | BAS00619651 | −50.7 | 3 | nitro | x | 486.6 | 112.6 | 6.0 | 88 ± 57 | >50 | >50 | |
| 4c | BAS01404025 | −49.8 | 6 | nitro | x | 502.6 | 121.6 | 5.7 | 49 ± 21 | >50 | >50 | |
| 4d | BAS02102292 | −49.4 | 7 | nitro | x | 520.6 | 126.8 | 5.7 | 23 ± 12 | >50 | >50 | |
| 4e | BAS02102245 | −49.4 | 8 | nitro | x | 520.6 | 124.8 | 5.7 | 46 ± 14 | >50 | >50 | |
| 4f | BAS01547732 | −48.9 | 13 | nitro | x | 492.5 | 130.1 | 5.1 | 76 ± 39 | 19.4 ± 1.9 | 21.1 ± 1.2 | |
| 4g | BAS00111586 | −48.8 | 15 | nitro | x | 446.5 | 111.2 | 5.0 | 60 ± 37 | 6.5 ± 1.0 | 5.6 ± 0.5 | |
| 4h | BAS00916022 | −48.0 | 20 | nitro | x | 508.6 | 114.0 | 6.4 | >100 | >50 | >50 | |
| 4i | BAS00381203 | −47.2 | 24 | nitro | x | 398.5 | 110.2 | 4.3 | >100 | 23.4 ± 5.5 | 21.4 ± 0.3 | |
a Predicted free energy of binding of the ligand (kcal/mol) according to BEAR (MM-PBSA). bRanking position according to BEAR (MM-PBSA). cClassification of the ligand depending on the chemical group interacting with the conserved catalytic Lys33. dOccupation of the inner (ANS1) and outer (ANS2) pocket. eMolecular weight (range 95% of drugs 130/725). fPolar surface area (range 95% of drugs 7/200 Ǻ2). gLog of the octanol/water partition coefficient (range 95% of drugs -2/6.5). hEC50 values (μM) determined from binding competition with ANS, with standard deviation. iIC50 values (μM) obtained in cell-based assays performed with MDA-MB231 and ZR-75–1 breast cancer cell lines, with standard deviation. lRanking position within the focused library of 2217 analogs of compound 4.