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The first Job file was created using SPROUT version 3.4. The HIPPO file included target sites essential to ligand binding based on published information. The updated version of the SPROUT 4.0 and 4.11 was released during this study and it was found that the old files were not compatible with the new released version. Under these circumstances new Job files were created for the SPROUT 4.0 and work continued accordingly. The SPROUT was updated several times during this period, version 5.0 (commercially available) and 6.0 (in development use) being the latest ones. At the beginning some of the selected HIPPO files with starting templates from the ELEFANT did not give any proper results in the SPIDER module. Start templates as well as spacer templates seemed to be unsuitable for the selected target sites. As a result a new template library was created (Figure 58, page 120). It included templates such as phenol, naphthalene, biphenyl, pyrrole, furan, thiophene, pyridine, 2-aminopyridine, pyrimidine, indole and purine. Structures were generated and minimised in the MacroModel program. These files (.pdb) were converted into an MDL mol-file using the Babel program (version 1.6).278

Figure 58. New partial structures were added into the template library with the basic

selection of the templates.

Two of the crystal structure complexes included NADP+ as a cofactor. This molecule structure turned out to be problematic. The program could not recognise the cofactor structure and this caused problem in analysing boundary interactions in HIPPO. The program also viewed the molecule with incorrect bonds. Because of this only one file included NADP+ molecules with the corrected bond information (1EQU).

Some selected HIPPO files with the start templates gave either rather poor results or no results at all in the connection phase (SPIDER). This was mainly because the steric-, electrostatic- and/or hydrophobic properties were violated. Overall more than sixteen SPROUT structure generation simulations were made for this research. From all Job files, created for the 17βHSD/KSR1 complexes (two for E, three for EQU, two for DHT and two for DHEA), HIPPO files with the best skeletons were chosen to be presented in detail here.

In the ELEFANT module all selected target sites from HIPPO were labelled as a number. Here all target sites are labelled as in SPROUT, emboldened and italicised accordingly.

5.2.2.1 Structure generation for estradiol complex

In addition to the existing hydrogen bond interaction presented by SPROUT, some other essential amino acid residues were selected for the active site based on published information. Structure generation for 17βHSD/KSR1- E2 1 complex using known amino acid residues as a target site (Ser142, Tyr155, His221 and Glu282) and some hydrophobic spheric sites gave a result with poor predicted binding (score) values. Consequently, the HIPPO file including acceptor Asn152 (Nδ2) 4, acceptor His221 (Nε2) 5, donor Tyr218 6 and three hydrophobic spheric sites 1, 2 and 3 (Figure 59) have been described in detail here, since it generated plausible results with good scores.

5 4 6 1 2 3

Figure 59. Selected sites Asn152 4 (acceptor, red), His221 5 (acceptor) and Tyr218 6 (donor,

blue) for 17βHSD/KSR1 - E2 1 are presented with the three spheric sites (1, 2 and 3 green). Two of the selected target sites (Tyr218 and Asn152) were at positions 7 and 14 of the steroid skeleton (Figure 59 a, page 122). All target sites were identified at the ‘lower half’ of the binding pocket (indicating bottom half of Figure 59 a); His221 in the recognition end of the pocket and Tyr218 and Asn152 right of the pocket (Figure 59 b, page 122). Hydrophobic spheric sites were selected within regions, which were the most hydrophobic (yellow area in Figure 59 c, page 122).

(a) (b) (c)

Figure 60. a) Selected target sites and hydrophobic spheric sites (green) in the active site

with E2 1 ligand. b) Target sites and the boundary of the active site (red areas have acceptor and blue areas donor character while green and yellow is hydrophobic areas). c) Hydrophobic sites are placed in most hydrophobic areas (yellow surface) within the active site (hydrophobic surface area cut-off is here 60%).

The selected target sites were divided into five groups where His221 5 and one of the spheric sites 1 formed a combined target site (Figure 59, page 121). The starting templates used for the five groups were:

• Combined target site (His221 5 and spheric site 1) including phenol, aromatic 5- and 6-membered ring templates. Eight templates, differently positioned within the combined target site, have been accepted; three phenols, two aromatic 5-membered and three aromatic 6-membered ring templates.

• Spheric site 2 has five accepted templates; aromatic 5-membered ring, aromatic 6- membered ring, naphthalene and two biphenyls.

• Spheric site 3 has the same five start templates; aromatic 5-membered ring, aromatic 6-membered ring, naphthalene and two biphenyls.

• Asn152 4 has five accepted templates; C sp3

, amide, double bond, 5-membered ring and aromatic 6-membered ring.

• Tyr218 6 also has five templates; C sp3

, two amides and two amidines.

Usually aromatic 5- and 6-membered rings represent various heterocyclic structures with hydrogen donor properties (pyrrole, imidazole, pyridine etc.) in SPROUT. However, some of these specific templates were also added in the new template library.

In the SPIDER module the selected target sites with templates above were connected to each other. Originally this final stage of skeleton generation was the most time consuming part of design, although nowadays with more powerful computers and parallel versions of SPROUT

(v 4.0 and later versions) the linking is considerably expeditious. Presented here are the results of the three libraries of skeletons made in different connection orders, since the results were slightly dissimilar if connections were made in variable order. It is possible to change some options of the connections. The most common options are collected in the Table 13, the default values and the spacer templates used for these connections are shown in Figure 61.

Table 13. Option values for the connections in SPIDER module.

Maximum vertices 40

Minimum ring percentage 25

Max. 3-memebered ring 1

Max. 4-memebered ring 1

Max. spiro joins 1

Max. fused joins 2

Max. 5-memebered ring 2

Max. 6-memebered ring 3

Max. rotatable bonds 30

Seed vertex tolerance 1.7

Max. chain length 5

Max. new bond joins 20

O N

Figure 61. Spacer templates used for these connections. Dashed lines (so called dummies)

indicate direction and number of the possible interactions.

The first tree pair connections were made between combined target sites 1 and 5 and spheric site 2 (Figure 59, page 121). The result of these connections were 292 diverse structures. These partial skeletons were scored in the ALLIGATOR module and 223 of the skeletons were selected for further connections. All 69 pruned structures were partial skeletons with an unstable pentalene structure 23. The second tree pair connection was between the original set of skeletons 1, 2, 5 and Tyr218 6, which gave 64 skeletons. This amount of skeletons was moderate; there was no need to prune this set before the next tree pair connection. The third connection between 1, 2, 5, 6 and 3 (Figure 59, page 121) gave five structures all including an ester-alike partial skeleton 24 which was undesirable.

23 24

These tree pair connections led to the forest, which included skeletons unfit for use. However, the first connection was good and gave suitable structures, therefore the new forest was made connecting tree pairs in different orders as follows: The tree pair 1, 2, 5 was connected with spheric site 3 instead of Tyr218 6 (Figure 59, page 121). This connection led to a result including 480 skeletons. After this Tyr218 6 was linked with 1, 2, 3, 5 without pruning and 134 skeletons from this connection were scored. The last connection with Asn152 4 gave 48 skeletons. The skeletons with partial structure 24 were pruned and the group included 41 skeletons with good predicted binding values (-8.29 to -9.23 which is 5.13 nM to 0.59 nM). Linking this set with the target site 6 gave no result thus the final set 1, 2, 3,

4 and 5 included 41 skeletons. These were examined closely and inadequate structures were