2.6.1 General aspects
Establishing a docking procedure and assessing the quality of a docking procedure for AtPARP is not straightforward because there is not much knowledge available. First, only NA and 3AB have been verified as AtPARP inhibitors so far218, and 4AN was only recently shown to inhibit AtPARP1, too.207,208 Furthermore, there are no decoy structures for any plant PARP known to this point. In addition to that, there is no X-ray structure of any AtPARP’s catalytic domain including a co-crystallised inhibitor in its active site deposited in the PDB.
These facts would be preconditions to apply a docking procedure or define a docking threshold to discriminate true inhibitors from decoys in a direct way. In contrast to AtPARP, these requirements are fulfilled for HsPARP1. To make use of this knowledge and establish a docking threshold for AtPARP inhibitors, the following steps as listed in Table 2.2 were performed:
Table 2.2: Steps to be performed to define an AtPARP docking procedure
Step Task
Definition of a docking threshold for human PARP1
1.1 definition of data sets for HsPARP1 ligands and HsPARP1 decoys 1.2 choosing the most suitable docking program for this purpose
1.3 establishing a docking procedure for selected data sets and verification
1.4 definition of criteria for discrimination of decoy and ligand structures and derivation of a docking threshold from these criteria
1.5 investigation of docking performance under these conditions Performing HsPARP1 docking procedure on AtPARP1 and AtPARP2
2.1 defining molecular, biological and statistical assumptions under which the HsPARP1 docking procedure can be transferred onto the AtPARP1/2 docking procedure
2.2 application of docking procedure, that was established for HsPARP1, by using analogue conditions as for the HsPARP1 procedure and incorporate underlying assumptions Definition of a new docking threshold for AtPARP1/2
3.1 investigation of differences of docking procedure of HsPARP1 and AtPARP1/2 3.2 derivation of new docking threshold for AtPARP1/2
Characteristics derived from the HsPARP1 docking procedure can be analysed after compounds from that database are bought and tested on AtPARP1 in validated in vitro assays.
The docking workflow starting from human PARP and resulting in selection of potential AtPARP inhibitors is shown in Figure 2.11
Figure 2.11: Docking workflow for establishing an AtPARP docking procedure
Step 1 identifies a docking threshold for HsPARP1, Step 2 applies all steps performed in steps 1 onto AtPARP1 and Step 3 transfers this into a development of a new threshold for AtPARP
2.6.2 Data sets
Two data sets for establishing a docking threshold were used. First, the Novikov data set described in 2.1.3 that contains a sample of HsPARP1 inhibitors (n = 142) and secondly, the decoy data set described in 2.1.4 which contains known human PARP structures which are known not to bind to HsPARP1 (n=1351).
2.6.3 Docking programs
The docking suite implemented in MOE, MOE dock, was used with five different placement routines: Alpha PMI, Alpha Triangle, Pharmacophore, Triangle Matcher and Proxy Triangle.
For each placement routine, three refinement strategies for docking poses were used. First, no refinement at all was performed for direct placement. Second, tethered refinement of all non-hydrogen side chain atoms with tethering factor 10 was performed allowing partial refinement of the active site during the ligand’s placement. As a third strategy during ligand placement, the active site’s amino acid side chain atoms were set free without any tethering. This allowed for more complex conformational changes during placement of a ligand in the active side.
docking program
+
HsPARP1 ligands HsPARP1 active site
+
discrimination criteria
HsPARP1 docking threshold
Step 1:
HsPARP1 DOCKING
assumptions
+
HsPARP1 ligands AtPARP2 active site
+
docking under same conditions
Step 3:
COMPARISON
HsPARP1 inhibitors docked into
AtPARP2 Step 2:
AtPARP2 DOCKING
docking threshold for new potential AtPARP2 inhibitors
results inference about results
+ HsPARP1 docking threshold
+
HsPARP1 inhibitors docked
into AtPARP2
=
docking score differences
Workflow for establishing an HsPARP1-derived AtPARP docking procedure
These settings result in 15 different docking routine combinations. In each routine, the reference ligand FRQ (24) was defined as the centre of the active site. Affinity dG was used as scoring function.
The scoring function extra precision glide (Glide XP) was used in the docking program Glide.188,189,192
The four scoring functions ASP, ChemPLP201, ChemScore191,199 and GOLDScore193,200 were used in GOLD200. Scoring functions PLP, PLP95 and ChemPLP were used in PLANTS.201,219
The binding site in which a ligand is placed during docking is defined differently in all docking programs. To define the binding site as similar as possible for all docking programs, the following settings have been used: FRQ was used as reference inhibitor in MOE (2.3.1), GOLD (2.3.7) and Glide (2.3.6). In PLANTS (2.3.8), the centre of the atomic coordinates of FRQ and a surrounding shell of 12 Å around this centre defined the active site. In GOLD and PLANTS, amino acid side chains, that participate in the known PARP pharmacophore, being Tyr907/ Tyr531/ Tyr878 (HsPARP1/ AtPARP1/ AtPARP2 numbering) respectively and Ser904/ Ser528/ Ser875 respectively, were defined as flexible. Also, upon inspection of the active sites, Glu763/ Glu388/ Lys735, respectively, were defined as flexible. The flexibility of side chains in GOLD was defined by not using rotamer libraries but by allowing full rotation about rotatable side chain bonds.
2.6.4 PARP pharmacophore-directed docking
To improve the identification of correct poses, the docking protocols have been adjusted. In Glide (2.3.6), GOLD (2.3.7), and PLANTS (2.3.8), the weights, wi, for rating hydrogen bonds between the protein and the inhibitor were changed from the default value of 1.
In HsPARP1, there is a hydrogen bond between the backbone nitrogen of Gly863 and the inhibitor, hb1, and another hydrogen bond between the carbonyl oxygen atom of Gly863 and the inhibitor, hb2. Both hydrogen both weights, whb1 and whb2, were increased. Furthermore, the weight of the hydrogen bond between the side chain atom Oγ of Ser904 and the inhibitor,
hb3, was increased 10-fold (such that whb1 = whb2 = whb3 = 10). In MOE, the hydrogen bond interactions hb1 and hb2 between the Gly863 and the inhibitor were modelled by incorporation of the pharmacophore features described in 2.5.
A view into the active site of HsPRAP1, including its inhibitor FRQ (24) and the corresponding hydrogen bonds, is displayed in Figure 2.12.These increased hydrogen bond weights were used to implement a pharmacophore-directed docking procedure. In an advanced setting (PLANTS protocol II, see 3.5.2), only the weights whb1 and whb3 were increased to 10, while whb2 was set to its default value of 1.
Figure 2.12: Hydrogen bond weights adjusted for pharmacophore-directed docking
View into active site of HsPARP1 showing inhibitor FRQ being hydrogen bonded to Gly863 and Ser904.
2.6.5 Definition of a correct docking pose
Based on the conformations of co-crystallised inhibitors from HsPARP, MmPARP (Mus musculus, mouse) and GgPARP complexes, a pose was defined as correct if the following features were satisfied:
• Existence of the two essential hydrogen bonds between the docked structure and the conserved glycine residue (e.g. Gly863, HsPARP1 numbering)
• Inhibitor core structure being able to exhibit π-π-interactions to the conserved tyrosine residue (e.g. Tyr907, HsPARP1 numbering)
• Tail of inhibitor structure does not point towards the protein surface but into the pocket of active site, similar to most HsPARP, MmPARP and GgPARP inhibitors
Figure 2.13: Definition of a correct docking pose
A-C: crystallized conformations of PARP inhibitors: A: FRQ in HsPARP1, B: KU8 in HsPARP3, C: GJW in HsPARP1. D: correct docking pose fulfilling all requirements E: incorrect docking pose since tail pointing towards protein surface, F: incorrect docking pose since tail points into active site but shows no hydrogen bonds to Gly863