Docking studies

Methods which dock a ligand into the protein binding site are useful for predicting the conformations that a ligand can take. Docking simulation algorithms include many approximations therefore the best result conformation is not always the only and right solution. Instead the method is a good way to check if the molecule is structurally possible and the right size to fit into the active site. A good 17βHSD/KSR1 inhibitor molecule should be deficient in activity for ER’s. It was important to examine the molecule dockings of the

112-116 into the ERα and ERβ substrate binding sites. In silico docking simulations were

also applied to study how well possible inhibitor molecules fit into the 17βHSD/KSR1 active site. The AutoDock and eHiTS docking simulation programs were used for this study. The SPA-Docking program information was also used for the purpose of support. The AutoDock results were more reliable than the eHiTS results since it has been widely used and there are many examples of its successful application in the literature. Hundreds of publications have cited the AutoDock methods papers.280 The eHiTS-program seems to have problems especially when the ligand is flexible.

The docking algorithm of the AutoDock program that was used was GA-LS, which is a hybrid of the genetic algorithm with local search. This algorithm is also known as a Lamarckian genetic algorithm or LGA. A systematic algorithm is used in eHiTS with no random, stochastic or evolutionary element. The eHiTS system generates all major docking modes that are compatible with the steric and chemistry constraints of the target cavity for each candidate structure. In the SPA-docking program the algorithm is a combination of simulated annealing, evolutionary and local search methods.

5.2.4.1 Docking simulations into the 17βHSD/KSR1 active site

AutoDock simulations were carried out in a 40*40*40 Å3 grid box (Figure 71, page 153). During the docking simulation the receptor was rigid and it was ensured that important amino acid residues were within the grid box. The results of the best fitted conformation of molecules 112, 113, 114, 115 and 116 into the 17βHSD/KSR1 are shown in the Table 31 (page 153). The AutoDock value in the first row of the table is the docking energy. It is impossible to compare the docking energy results of the different programs therefore the HIPPO score is also examined for these docking results.

Figure 71. The used grid box volume for AutoDock docking simulations was 40Å*40Å*40Å

(green-blue-red squares). The grid box surrounding active site of the 17βHSD/KSR1.

Table 31. The results of the AutoDock program for molecules 112, 113, 114, 115 and 116

docked into the 17βHSD/KSR1 with HIPPO score values for the molecule in question.

112 113 114 115 116 Autodock

(Docking Energy) -11.5 -13.1 -12.4 -12.8 -12.8 HIPPO

(Scoring value) -16.4 -11.9 -8.9 -11.4 -10.1

The HIPPO scoring value for the molecule 112 is high after docking simulation. Alignment of the SPROUT generated molecule (brown) and AutoDock result conformation (blue) are shown in Figure 72 (page 154). Molecules 113 and 115 also have rather good scoring values. On the other hand the scoring value for molecule 116 is slightly poorer than after minimisation and the scoring value for 114 is rather moderate compare to the original scoring value. According to ALLIGATOR ‘Vertex score table’ there is no any van der Waals clashes observed for either of these docked molecules.

Figure 72. Alignment of SPROUT generated molecule 112 (brown, scoring value -15.0) and

the best AutoDock result for molecules 112 (blue, scoring value -16.4).

For most of these molecules the eHiTS program resulted in zero solutions. It is difficult to draw any conclusions as to why this program was not capable of fitting the molecules into the active site. However, it is probable that the molecule was discarded from the active site because of the steric clashes between molecule and receptor. The earlier docking simulation gave reasonable docking result for molecules 80a, 83a and 94a, using the SPA-docking program. The HIPPO score values for the best results were also rather good. Unlike the eHiTS results these results supported the AutoDock results in the sense that it was possible to dock these molecules into the active site according to the SPA-dock program. It was possible to observe consistency between SPA-dock docking energy values and HIPPO score values. However there was not consistency between docking energy values and HIPPO score values (Table 32) nor yet between AutoDock and SPA-dock results.

Table 32. Docking simulations made for 80a, 83a and 94a using the AutoDock and SPA-

dock programs.

80a 83a 94a Autodock (DE) -12,0 -13,5 -14,1

HIPPO score -13,6 -10,2 -13,1

SPA-dock (DE) -62,9 -47,7 -42,4

5.2.4.2 Docking simulations into the estrogen receptor α and β active sites

The AutoDock program was also applied to study how the molecule fit in silico into the estrogen receptor substrate binding site. A good inhibitor molecule is selective for 17βHSD/KSR1 and binding into the ER substrate binding site is undesirable. All five molecules were docked into the ERα and ERβ using same docking algorithm. Docking energies and HIPPO scoring values of the molecules 112, 113, 114, 115 and 116 are shown in the Table 33.

Table 33. Docking results of the five molecules docked into the ERα and ERβ.

ERα 112 ERα 113 ERα 114 ERα 115 ERα 116 ERβ 112 ERβ 113 ERβ 114 ERβ 115 ERβ 116 Auto dock

(Docking Ene rgy) -8.3 -10.7 -6.8 -6.1 1.4 -8.8 -11.0 -9.2 -12.4 -12.1 HIP PO

(Scoring value) -9.2 -9.5 -10.1 -9.4 -9.4 -8.6 -9.9 -10.0 -10.5 -10.4

Originally de novo generated conformations 112, 113 and 115 had better scoring values than docked molecules conformations in the ERα and ERβ substrate binding sites. The original scoring value for molecule 116 was slightly better than docked molecule conformation in the ERα substrate binding site. Instead conformation in the ERβ had same scoring value as original molecule 116. Molecule 114 seemed to have a rather good original scoring value (Table 28, page 150), although after docking simulation the scoring value was rather poor (Table 31). Both ERα and ERβ bound molecule 114 well and the HIPPO scoring values after docking simulation were good (Table 33). Due to this possible activity for estrogen receptors,

114 is not a good inhibitor for the 17βHSD/KSR1. Molecule 116 also indicated equal activity

for estrogen receptors as 17βHSD/KSR1, thus this molecule is not a good inhibitor molecule for the 17βHSD/KSR1 enzyme.

Molecules 112, 113 and 115 had good predicted binding activity values and were worthy of synthesising for a more detailed biological evaluation. Noteworthy is that the scoring value after the docking of molecules 112, 113 and 115 into the ERα and ERβ were poorer than the original conformations (Table 28, page 150), however the values are better than the original natural ligand scores in every case (EQU 2 -8.27, DHT 3 -8.62, DHEA 4 -8.83, respectively).