Crystal Structures in Complex with the Enzyme
Chapter 6 interaction with the backbone carbonyl group of Tyr253’ from the symmetry equivalent
crystal mate being part of the functional tetramer (Figure 6.2 B).
Fragment optimization applying fragment growing
After analysis of the crystal structures, we initiated a structure-based optimization of the discovered fragment hits with the goal to design a new lead inhibitor scaffold. The discovered fragments were decorated with different substituents in order to achieve additional interactions with the binding site and thereby increase binding affinity.
As a first step to optimizeJ15(Chart 6.1), we eliminated the hydroxyl group since the crystal structure revealed that this moiety does not establish any interaction to the protein.
Furthermore, the hydroxyl group can potentially present a steric impediment for further synthetically optimization of the fragment hit. As a next step, moieties were attached to 6.1 with the aim to address the hydrophobic binding pocket of 17β-HSD14 that is composed by Leu191, Trp192 and Leu195 (Figure 6.2). Two of the designed inhibitors (the pyridine 6.4 and the quinoline 6.6) were modeled with MOE [173] into the crystal structure of 17β-HSD14 applying the crystallographically determined binding mode of the scaffold ofJ15 as a starting point. According to the derived models these compounds should theoretically fit nicely into the binding pocket (Figure 6.3). Therefore,6.4,6.6 and the other four compounds (Chart 6.1) were synthesized.
Figure 6.2: Crystal structures of 17β-HSD14 in complex with cofactor NAD+ and (A) fragmentJ6 and (B) fragmentJ15. Close-up view of the binding pocket. Carbon atoms ofJ6are shown in light blue, ofJ15in light green and of the cofactor NAD+ in blue. The inhibitors and cofactor NAD+are shown as stick models. The amino acids within a distance of 4.6 Å are shown as thin stick models with carbon atom in white. H-bonds are depicted as dashed lines.
The strategy for fragment growing of J6 is displayed in Chart 6.2. The N-methyl-cyclopentanamine tail was removed from the compound since, according to the crystal structure, this moiety should not contribute significantly to the affinity as no pronounced interaction could be identified. Furthermore, removal of the moiety facilitated the synthesis of follow-up derivatives. Similar as in the case of J15, the designed inhibitors were first modeled into the crystal structure obtained with J6 (Figure 6.4), and, since the modeled binding mode looked promising, compound6.9 was subsequently synthesized.
Chart 6.1: Fragment growing strategy for fragmentJ15.
Figure 6.3: Modeled structures of 17β-HSD14 in complex with compounds6.4 (A) and 6.6(B). The surface of the protein is displayed in gray. The inhibitors are shown as magenta stick models (hetero-atoms color-coded). Amino acids (white) and cofactor NAD+(beige) are shown as thin stick models. H-bonds are depicted as black dotted lines.
Chapter 6
Fragment optimization applying fragment linking
Since fragments J6 and J15 occupy positions in the binding cleft of 17β-HSD14 directly adjacent to each other, covalent linking of both fragments appeared as a feasible strategy to generate a new ligand with improved potency. However, covalent linking to two fragments is known to be a non-trivial and risky endeavor, since already small reinforced special changes either of the valence and torsion angles or even distances of the two fragments with respect to each other can partly or entirely diminish affinity of the generated compound. The trimmed fragmentsJ15 and J6, also used in our fragment growing strategy (6.1 and6.8, respectively) were covalently connected via an ethyl linker to give compound 6.10 (Chart 6.3). Prior to synthesis, 6.10 was validated by modelling. Since the original fragment portions of the Chart 6.2: Fragment growing strategy for fragmentJ6.
Figure 6.4: Modeled structure of 17β-HSD14 in complex with compound6.9. The surface of the protein is displayed in gray. The inhibitor is shown in magenta as stick model with color-coded heteroatoms. Protein residues (white) and the cofactor NAD+(beige) are shown as thin sticks. H-bonds are depicted as black dotted lines.
generated supermolecule maintained binding modes (Figure 6.5), the ligand was subsequently synthesized.
Inhibitory Activity validation
The original fragment hits J6 and J15 and the building blocks 6.1 and 6.8 were tested for their inhibitory activity against 17β-HSD14. Even at high concentration of 250 μM, all four molecules did not show any significant affinity to block the protein (Table 6.2). Most likely it would have been difficult to identify them in a biophysical screening cascade.
Chart 6.3: Fragment linking strategy for fragmentJ6andJ15.
Figure 6.5: Modeled structure of 17β-HSD14 in complex with compound6.10. The surface of the protein is displayed in gray. The inhibitor is shown in magenta as stick model with color-coded heteroatoms. Protein residues (white) and the cofactor NAD+(beige) are shown as thin sticks. H-bonds are depicted as black dotted lines.
Chapter 6
cmpd.
17β-HSD14 (% Inh. @ 250mM)a
J15 16
J6 n.i.
6.1 n.i.
6.8 n.i.
Subsequently, also the newly synthesized, size-increased compounds were tested for their inhibitory activity against 17β-HSD14. All compounds that resulted from fragment growing showed an improved affinity relative to the starting fragments. From the optimized fragments, compound6.4 (derived from fragmentJ15) showed the highest affinity with aKi
of 1.9 µM. The optimized compound starting from fragment J6, ligand 6.9,also showed an improved in affinity toward the enzyme with 63% @ 100 µM (Table 6.3). However, due to its poor solubility, it was impossible to determine itsKi value.
Table 6.3: 17β-HSD14 binding constants (Ki) of the design compounds.
cmpd. R
Pos.
CO2H
17β-HSD14
(% Inh. @ 250mM)a Ki (µM)a
6.2 H 3 76 8.7 ± 0.8
6.4 OH 3 93 1.9 ± 0.1
6.3 H 4 80 6.8 ± 1.2
6.5 OH 4 83 4.8 ± 1.0
6.6 - 3 86 8.1 ± 0.8
6.7 - 4 92 7.0 ± 0.5
6.9 - - 63 @ 100mM
-The generated compound 6.10 from the fragment linking strategy was identified to have an increased affinity compared to the individual starting fragments (Table 6.4). This is a very encouraging result since there are only a few examples of successful fragment linking in literature.
Table 6.4: 17β-HSD14 binding constant (Ki) of the ligand designed by employing the fragment linking strategy.
cmpd.
17β-HSD14
(% Inh. @ 250mM)a Ki(µM)a
6.10 67% 17.6 ± 0.5
Binding mode confirmation of the optimized compounds
To further validate the best compounds from each fragment optimization strategies, we co-crystalized them in complex with 17β-HSD14 in order to confirm the predicted binding mode. Two crystal structures were determined and the data collection and refinment statistic are reported in Table 6.5.
Table 6.5.Data collection and refinement statistics.
a Complex with6.4, Complex with 6.10,
(A) Data collection and processing
space group I422 I422
unit cell parameters a, b, c (Å) 91.4, 91.4, 133.1 91.0, 91.0, 133.0
Matthews coefficientb (Å3/Da) 2.4 2.4
solvent contentb (%) 49.2 49.4
(B) Diffraction data
resolution range (Å) 50-1.62 (1.72–1.62) 50-2.08 (2.21–2.08)
unique reflections 36077 (5762) 17131 (2714)
R(I)sym (%) 7.1 (49.8) 10.2 (50.9)
completeness (%) 99.9 (99.7) 99.9 (99.9)
redundancy 8.5 (8.5) 8.8 (9.2)
<I/σ(I)> 18.9 (3.5) 17.0(4.2)
(C) Refinement*
resolution range (Å) 39.90-1.62 46.22-2.08
reflections used in refinement (work/free) 36077 (34273/1804) 17131 (16274/857) final R value for all reflections (work/free) (%) 0.13/0.16 0.19/0.21
protein residues 254 251
water molecules 126 48
RMSD from ideality: bond lengths (Å) 0.008 0.007
RMSD from ideality: bond angles (°) 0.994 0.818
a Values in parenthesis describe the highest resolution shell.b Calculated with Matthews_coef program from CCP4 suite version 6.4.0.[132]*Preliminary data.
The superimposition of the crystal structures of the enzyme in complex with fragment J15 and ligand 6.4 reveals that the benzoic acid moiety of the optimized ligand establishes the same interaction as fragment J15 (Figure 6.6 A). In addition, the central pyridine moiety addresses the hydrophobic pocket composed by Leu191, Trp192 and Leu195 and its 3-OH
Chapter 6