PDEStrIAn: PDE Structure and
Ligand Interaction Annotated database
Chapter 2
Chimed Jansen, Albert J. Kooistra, Iwan J. P. de Esch, Rob Leurs and Chris de Graaf
Manuscript in preparation
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Abstract
A systematic analysis is presented of the 168 phosphodiesterase (PDE) catalytic domain
crystal structures present in the Protein Data Bank (PDB) with a focus on PDE-ligand
interactions. The PDE Structure and ligand Interaction Annotated (PDEStrIAn) database
contains a consistent alignment of 57 PDE ligand binding site residues, that enables the
identification of subtype-specific PDE-ligand interaction features and classification of
ligands according to their binding modes. We illustrate how systematic mining of PDE-
ligand interaction space gives new insights into how conserved and selective PDE
interaction hot spots can accommodate the large diversity of chemical scaffolds in PDE
ligands. A substructure analysis of the co-crystalized PDE ligands in combination with
those in the ChEMBL database provides a toolbox for scaffold hopping and ligand design.
These analyses lead to an improved understanding of the structural requirements of PDE
binding that will be useful in future drug discovery studies.
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2.1 Phosphodiesterases as Drug Targets
Cyclic nucleotide Phosphodiesterases (PDEs) play a key role in regulating the levels of the
ubiquitous second messengers, cyclic adenosine monophosphate (cAMP) and cyclic
guanosine monophosphate (cGMP). PDEs therefore provide a handle for control of an array
of biochemical pathways throughout the body,1, 2 and have proven to be effective drug
targets.3 There are eleven human PDE families; PDE1, PDE2, PDE3, PDE10 and PDE11
hydrolyze both cAMP and cGMP; PDE4, PDE7 and PDE8 selectively hydrolyze cAMP;
and PDE5, PDE6 and PDE9 selectively hydrolyze cGMP.4 Kinetoplastid parasite PDEs are
also of interest as potential drug targets, the PDEA and PDEB families selectively hydrolyze
cAMP, while the PDEC family hydrolyzes both cAMP and cGMP.5, 6 The hydrolysis of the
cyclic nucleotides occurs in the substrate binding pocket of the PDE catalytic domain and
is catalyzed by two metal ions that occupy the adjacent metal binding region (Figure 1A-
B). The identity of one of the metal ions is required to be Zn2+ in order to maintain activity,
the identity of the second may vary, though in most cases it is Mg2+.7-9 X-ray co-crystal
structures of both the substrate and product give insight into the mechanism of the
hydrolysis, in which the substrate is held in place by interaction with a key conserved
glutamine residue, QQ.50, a “hydrophobic clamp” formed by IHC.32 and FHC.52, and ionic
bonds between the phosphate group and metal ions (Figure 1A-B, Figure 2). Hydrolysis of
the cyclic phosphate ester bond occurs through attack by a water molecule activated by the
metal ions.8, 10
The pervasive and tissue specific expression of PDEs, allows PDE inhibitors to be applied
in a wide range of therapeutic areas.11 To date twelve selective PDE inhibitors have been
approved for use as pharmaceuticals, of which five have been crystalized in complex with
a PDE (Figure 1C). The first of these to reach blockbuster status was sildenafil (Viagra®),
approved for the treatment of erectile dysfunction and pulmonary hypertension, thereby
establishing PDEs as highly successful drug targets. Three PDE5 inhibitors have followed,
tadalafil (Cialis®), vardenafil (Levitra®) and most recently avanafil (Stendra®), bringing
improvements in selectivity.12 Following considerable effort including the late stage failure
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of rolipram, two PDE4 drugs have entered the market, drotaverine (No-Spa®) as an
antispasmodic and recently roflumilast (Daxas®) as a treatment for COPD.13, 14 Five PDE3
drugs have reached the market; cilostazol (Pletal®) for the treatment of intermittent
claudication, anagrelide (Agrylin®) for the treatement of thrombocythemia, and the
cardiotonic vasodilators amrinone (Inocor®), enoximone (Perfan®) and milrinone
(Primacor®). Like the PDE4 drugs they display problematic contraindications, notably
nausea and cardiovascular side effects.15, 16 There are several marketed non-selective PDE
inhibitors targeting both PDE3 and PDE4 such as theophylline and several which inhibit a
broad range of PDE families such as dipyridamole. One of the oldest prescribed PDE drugs
is papaverine, which has long been used as a vasodilator. Recently papaverine was found
to be a potent and selective PDE10 inhibitor and it has played a role in generating interest
in PDE10 as a drug target.17 The development of selective inhibitors of PDE10 continues
to receive considerable interest, in spite of the recent failure of PF-02545920 to reach the
market.18 With only 4 of the 11 human PDE families targeted by selective drugs so far, and
drugs targeting PDEs present in parasitic organisms for the treatment of neglected diseases
yet to be developed, the potential for new drugs targeting PDEs is evident.
2.2 The Structure of Phosphodiesterases
All PDE inhibitors crystalized to date have been found to bind to the substrate binding
pocket of the PDE catalytic domain. The catalytic domain consists of up to 16 helices (H1-
H16) and 16 loops (A-N) that fold to form a substrate binding pocket (Figure 1D). When
all published PDE crystal structures are overlaid, it is clear that PDEs share a highly
conserved fold, with an overall RMSD of C-alpha atoms of 1.2Å (Figure 1E). The
conformation of the H-loop, which includes H8 and H9, is conserved across most PDE
crystal structures and borders the metal binding region. However significant differences
occur in certain crystal structures, for example in PDE5 the H-loop folds over the substrate
binding pocket in crystal structures containing a bound ligand. Similarly the M-loop which
also borders the substrate binding pocket shows some flexibility, including cases of induced
fit for bulky ligands. The most highly conserved fold is made up of 10 helices (H5-H7 and
H10-H16), with an RMSD across all PDE structures of 1.0Å (Figure 1F) and fold forms the
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catalytic site which includes a metal binding region at the apex of H6 – H13 and a substrate
binding region formed between H13 – H16.
The PDE catalytic domain is adjoined by a lengthy variable N-terminus containing one or
more highly ordered structural elements or domains, and a usually short unstructured C-
terminus. The amino-terminal domains include the CaM-binding domain (CaM), GAF
domains, transmembrane domain (TM domain), upstream conserved regions (UCRs) and
PAS domain and may regulate the activity of the catalytic domain, play a role in the
localization of PDEs, or the interaction with protein partners.19-21 Crystal structures are
available of N-terminal GAF domains of PDE2 (complete dimer), PDE5, (GAF-A & GAF-
B), PDE6 (GAF-A) and PDE10 (GAF-B). Ligand binding has been observed for GAF
domains and this may result in ordering the catalytic domain in an open conformation.22
There are 168 PDE crystal structures in the Protein Databank at the time of writing (Figure
1G). The first crystal structure published of the PDE catalytic domain was an unliganded
structure of PDE4B2B containing metal ions, the structure established the 16 helix
nomenclature (H1-H16) for PDE structures.7 The first ligand bound PDE crystal structure,
containing zardaverine bound to PDE4D, provided insights into the role of the catechol
scaffold during binding and into PDE dimerization.23 A subsequent study in PDE4 showed
the structural basis for selectivity between the rolipram enantiomers.24 The first (catalyzed)
substrate bound structure soon followed with AMP bound to PDE4D providing insights into
the catalytic process.10 This was followed by an in depth study that involved PDE1B,
PDE4B, PDE4D and PDE5. As a result, the“glutamine switch” was identified as the
probable mechanism that controls substrate selectivity. Also the term the term “hydrophobic
clamp” was introduced to describe a hallmark ligand-PDE interaction .25 The number of
PDE crystal structures has continued to climb at a rate of about 15 per year and there are
now structures of the catalytic domains of 10 of the 11 human PDE families (a structure of
PDE11 is still lacking). At the subtype level only 13 of the 21 subtypes have been
crystalized (Figure 1G, structures of 1A, 1B, 3A, 6A, 6B, 7B, 8B and 11A are still lacking).
Beyond human PDEs, efforts to control parasite proliferation by means of PDE inhibitors
have shown great promise, yet only a small fraction of these PDE targets have been
crystalized.5, 26, 27
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The growth in novel PDE inhibitors included in the ChEMBL database since the year 2000
provides a different view on which PDE targets are of greatest interest (Figure 1H). As
might be expected PDE4 and PDE5 inhibitors make up the largest contribution, however
few PDE9 inhibitors have been published despite the publication of 18 PDE9 crystal
structures. In the case of PDE10, the discovery of novel inhibitors follows the release of a
significant number of crystal structures and in 2012 over half of the novel PDE inhibitors
published in ChEMBL targeted PDE10. Parasite PDEs have also gained significant interest
as targets to treat neglected diseases with growing numbers of both novel inhibitors and
crystal structures published. In the cases of PDE1 and PDE3 there are a significant number
of novel inhibitors published, despite the fact that just one PDE1 and two PDE3 crystal
structures having has been published.28, 29 In the case of PDE3, inhibitors of both PDE3 and
PDE4 may be an added factor in the number of active compounds registered in ChEMBL.30
Along with PDE7, PDE8 and PDE11, these are targets with limited crystallographic data to
support drug discovery, although it can be anticipated that pharmaceutical interest will lead
to structural biology activities for these new targets.
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Figure 1: The growing structural understanding of PDEs and PDE inhibition
(A-B) The binding modes of cAMP (A, PDB: 2PW3) and AMP (B, PDB: 1PTW) bound to
PDE4D. The key interacting residues are shown colored by the pocket region, HMB.02
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(H160), NHC1.25 (N321), IHC.32 (I336), FS.35 (F340), QQ.50 (Q369) and FHC.52 (F372), and
named according to the nomenclature presented in Figure 2.
(C) Molecular structures and indications of the five marketed PDE inhibitors crystalized
with PDEs (1-5), two PDE inhibitors which failed to reach the market (6-7) and the PDE
substrates cAMP and cGMP with arrows indicating the bond broken during hydrolysis by
PDEs to form AMP and GMP (8-11).
(D-F) Structural overviews of PDEs. (C) A schematic diagram of the structure of PDEs.
Loops are lettered A-N (green) and helices are numbered H1-H16 (blue), the region of the
substrate binding pocket is highlighted (yellow). Two loops have been emphasized, the H-
loop (purple) which borders the substrate binding region and the M-loop (red) which
borders the metal binding region. The faded region, including H1 – H7, has been moved
from behind the protein to the side for clarity. (D) An overlay of the backbone ribbons of
all PDE crystal structures. (E) An overview of the conserved helices in PDE structures
showing the position of the substrate binding site as a surface. The colors of the surface
denote regions of the binding pocket as described in detail in Figure 2A.
(G) An overview of PDE crystal structure publications by year. Details of the number of
crystal structures published for each of the 21 subtypes spread over 11 PDE families are
shown to the right of the graph. The two PDE6C (*) crystal structures published are binding
pocket chimeras of PDE6C in PDE5A constructs.
(H) An overview of novel active PDE inhibitors published in the ChEMBL database by year
starting with the year 2000 and excluding earlier PDE inhibitors. This table provides an
indication of the influence of PDE crystal structures on the discovery of novel PDE
inhibitors.
2.3 A Novel PDE Binding Site Nomenclature
In order to describe the interactions between the ligands and the substrate binding pocket in
a consistent manner, we suggest a systematic nomenclature for the residues of the binding
pocket. To identify regions of the pocket involved in ligand binding and to identify
differences in binding across the PDE super family, the pocket was divided into 10 regions
(Figure 2A). The 10 regions consist of; the invariant glutamine (Q) regions Q, Q1 and Q2;
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the hydrophobic clamp (HC) regions HC, HC1 and HC2; the metal binding (MB) regions
MB, MB1 and MB2; and the solvent filled (S) region S. A total of 57 residues were selected
as pocket residues to allow for variation of the pocket conformation and the range of binding
modes adopted by ligands (Figure 2B). Of the 57 pocket residues, 13 are conserved across
all PDEs, 11 of which are involved in metal binding (Figure 2C). The two other conserved
amino acids play a key role in substrate binding, these are the glutamine residue, QQ.50, and
the phenylalanine residue, FHC.52. The identities of the PDE substrate binding pocket amino
acids at each position can be compared across the PDE subtypes using the alignment
provided in Figure 2D. The only gaps in the alignment are found at Q2.44, as a result of the
variable length of the M-loop, and at Q2.31 where H14 tightens for one turn in TcPDEC.
Multiple splice variants of the PDE subtypes are expressed which affects the numbering of
amino acids, the amino acids in the alignment are numbered according to the canonical
sequence of each PDE subtype. In order to identify PDE pocket residues in a consistent
manner across the PDE families, novel nomenclature combines the amino acid sequence
reference with the pocket region name and the position of amino acid in the pocket sequence
(Figure 2E). Recently, a similar nomenclature was applied to enable the construction of an
automated database of kinase structures for public access in which protein-ligand
interactions are stored as IFPs.31
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Figure 2: The PDE ligand binding pocket of the catalytic domain
(A) The PDE ligand binding site shown as a surface over a representation of the protein
backbone (PDE4D, PDB: 1OYN). The surface is labeled and colored to show the 10 defined
regions of the binding site. The substrate binding site includes the Q, Q1 and Q2 regions
that surround the important invariant glutamine residue QQ.50, the HC, HC1 and HC2
regions that surround the hydrophobic clamp (I/V/LHC.32 and FHC.52) and the S region
solvent filled sub-pockets. The metal binding site is divided into the MB, MB1 and MB2
regions.
(B) The positions of the C-alpha atoms of pocket residues are shown as spheres in the color
of the regions to which they belong. The pocket residues are labeled according to their
position in the pocket and their position in the PDE sequence.
(C) A WebLogo representation of the conservation of the 57 amino acids of the PDE binding
pocket across the PDE subtypes listed in panel D (color coding as defined in panel A).
(D) An alignment of the pocket residues in each of the PDE subtypes of which crystal
structures have been published. Residue numbers are taken from the canonical sequence of
each PDE subtype. A color bar above the residues indicates the pocket region in which the
residues are found.
(E) A nomenclature is presented that combines the standard amino acid reference
containing the single letter amino acid code (red) and isoform specific residue number
(purple) with the PDE pocket residue region name (blue) and the PDE pocket residue
number (green). When referencing PDE pocket residues of a subtype the isoform number
may be omitted (YHC1.01) and when referencing PDE pocket residues across the families the
amino acid code and isoform number may be omitted (HC1.01).
Four previous analyses of PDE crystal structures have been published. The first described
the binding of 10 inhibitors to PDE4B, PDE4D and PDE5A in 15 crystal structures.32 An
analysis of ligand interactions is provided with key interactions across multiple PDEs
identified. Methods of improving selectivity and potency are discussed in detail with several
examples provided that show the effect of addressing regions of the pocket. An overview
of the therapeutic importance of PDE targets, which included the structural analysis of 20
crystal structures from PDE families 1, 3, 4, 5 and 9, found similar binding motifs to
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determine substrate and inhibitor binding.11 In another analysis of the same PDE structures,
in depth descriptions of the binding modes taken by several representative ligands, cAMP,
cGMP, theophylline, IBMX, zaprinast, sildenafil and rolipram are provided.33 The most
comprehensive previous analyses of PDE structures reviewed the first 60 PDE structures
published in the PDB, covering PDE families 1, 2, 3, 4, 5, 7 and 9.34 In that analysis the
core interactions between inhibitors and the invariant glutamine and hydrophobic clamp
were described as essential and multiple recognition elements were used to explain
selectivity in detail for PDE4 and PDE7. With over 100 PDE crystal structures added to the
PDB since the previous analysis by Ke and Wang and with new cheminformatics tools at
hand, the question of the drivers of PDE selectivity can now be revisited.
Through processing the available PDE crystallographic data in a systematic manner,
studying the protein fold, binding site interactions, ligand substructures and decorations,
and combing this data is an accessible format, we constructed the PDEStrIAn database as a
toolbox for drug designers. The utility of this toolbox is enhanced by the difficulty in
achieving selectivity between the PDE families and the potential PDE inhibitors have
shown as drugs indicate its relevance. To facilitate this and future studies of the PDE super
family a novel standardized nomenclature for PDE binding site residues is introduced here.
The PDEStrIAn database contains the most comprehensive structural analysis of PDEs to
date and the key findings of this analysis are presented here.
2.4 Building PDEStrIAn
The PDEStrIAn database was constructed to aid the systematic analysis of PDE crystal
structures published in the PDB. An overview of the method used to construct the
PDEStrIAn database is provided in Figure 3A and a detailed description follows below. The
canonical sequences of human and parasite PDE subtypes were collected from the UniProt
database and aligned using ClustalW (1). The PDE crystal structures containing a catalytic
domain were gathered from the Protein DataBank and a chain was selected for further
analysis according to their B-factor, ligand placement, the presence of gaps, solvent
molecules and Ramachandran plots (2). The structures were aligned together with the
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canonical sequences using a combined sequence and structural alignment (3). The pocket
residues were defined and the sequence alignment was manually optimized to improve the
alignment of pocket residues (4). The definitive set of pocket residues received a new
consistent nomenclature including the name of the pocket region (5). The ligands, water
molecules, metal ions and pocket residues were isolated and processed using an interaction
fingerprint (IFP) generation protocol (6) forming the basis of the PDEStrIAn database (7).
The interactions were systematically analyzed by pocket region and according to the
substructures of ligands involved in interaction with specific regions (8). Interactions
between the ligand and water molecules and metal ions found in the crystal structures were
processed to assess their role in ligand binding (9). The scaffolds present in crystalized PDE
inhibitors were identified and the occurrence of the scaffolds in crystalized ligands and PDE
inhibitors found in the ChEMBL database was analyzed. The data can be arranged by PDE
pocket region and several example structures for the regions are provided. The PDEStrIAn
database can be applied to aid the design of novel PDE inhibitors by presenting knowledge
from known PDE inhibitors in an accessible format.
2.5 IFP Generation
A key step in the construction of the PDEStrIAn database was the preparation of IFPs from
the crystal structure complexes. The binding mode and IFP of sildenafil (Figure 1C) to
PDE5A (PDB: 1UDT) illustrates the use of IFPs to encode protein-ligand interactions
(Figure 3B-D). The binding mode of sildenafil is presented from the pocket opening
showing all residues interacting with sildenafil (Figure 3B). Figure 3C shows a top view
projection in which sildenafil, the residues forming specific interactions with sildenafil and
the metal ions are shown projected over the surface of the pocket, this is the visualization
method used throughout the remainder of the article. The IFP of the binding mode of
sildenafil is presented in Figure 3D. The IFP is a bit string in which each bit encodes the
presence (1) or absence (0) of a particular interaction type between a protein residue and a
ligand 35. The bit string is made up of five bits per binding site residue; a hydrophobic
interaction bit; a face-to-face π-π interaction bit; an edge-to-face π-π interaction bit; a
hydrogen bond acceptor bit; and a hydrogen bond donor bit. Ionic interactions and cation-
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π interactions were excluded from the bit string because these interactions were not found.
In the example of sildenafil bound to PDE5A, 13 residues show the presence of hydrophobic
interactions, QQ.50 additionally shows hydrogen bond acceptor and donor interactions and
FHC.52 additionally shows face-to-face and edge-to-face π-π interactions. The IFP of a
protein-ligand complex can be rapidly compared to those of other ligands bound to the same
or similar proteins, to identify key residues involved in ligand binding, or to find similarities
in the binding modes of multiple ligands.
Figure 3: Building the PDEStrIAn database
(A) An overview of the steps undertaken to build the PDEStrIAn database. For those steps
represented in figures, the relevant figure names are provided. Examples of structures
which show specific interactions with pocket regions have been retrieved from the database
as indicated at the bottom of the flowchart.
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(B-C) Sildenafil (2D structure presented in Figure 1C) bound to PDE5A (PDB: 1UDT)
shown from the opening of the ligand binding pocket and from above. Residues and areas
of the pocket are color coded according to pocket region.
(D) The IFP bit string for residues interacting with sildenafil. Each residue displays five bit
positions that can either be on (1), indicating the presence of an interaction, or off (0),
indicating the absence of an interaction. The bits that are on are color coded according to
the type of interaction made with the ligand. For clarity only the 13 of the 57 binding site
residues that have at least one interaction with sildenafil are shown in the bit string.
2.6 Ligand-PDE Interaction Analysis
The presence of interactions between ligands and residues in the PDE crystal structures can
be aggregated for each PDE family and plotted into a heat map as shown in Figure 4.
Comparing the frequency of interactions made by each pocket residue across all PDE-ligand
complexes four residues stand out, I/V/LHC.32, F/YS.35, QQ.50 and FHC.52. Interactions with
these residues are seen across all PDE families and in almost all crystal structures.
Hydrophobic interactions with I/V/LHC.32 occur in all but one structure, and with F/YS.35 in
all but two structures. Aromatic interactions with F/YS.35 occur in 57% of structures and
with FHC.52 in all PDE crystal structures. Hydrogen bond donor or acceptor interactions with
QQ.50 are found in over 90% PDE crystal structures. These key interactions are driven by
the core scaffolds of the bound ligands, which consistently include a flat aromatic or fully
conjugated ring system and one or more hydrogen bond donors or acceptors.
The heat map in Figure 4 could potentially also be used to identify interactions that are
specific to particular PDE subtypes. These PDE subtype specific interaction hotspots may
be important drivers for PDE ligand selectivity, although one should be aware that for
several subtypes relatively few structures have been solved (PDE3, PDE3, PDE6, PDE7,
PDE8, parasite PDEs). Still, this data can be applied to quickly identify interactions of
interest when designing selectivity into PDE inhibitors. Examples of selective polar
interactions will be discussed in the paragraphs addressing the pocket regions, Q2 (Figure
8C; PFK interacts with YQ2.33 in PDE10), HC1 (Figure 9A; HBT interacts with NHC1.25 in
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PDE4B), S (Figure 9D; 0WL (CHEMBL2180070) interacts with YS.35 in PDE9) and MB1
(Figure 10D; IHM interacts with MMB1.17 in PDE4D).
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Figure 4: Frequency of interactions between pocket residues and ligands in crystal
structures of PDEs
A heat map of the IFP results for PDE crystal structures showing the frequency of
interaction types between the bound ligands and pocket residues for each PDE family. The
colors indicate the interaction type and the intensity of colors indicates the percentage of
crystal structures of a PDE family in which a specific interaction takes place, with white
representing 0% and solid color indicating 100%.
2.7 Ligand-Water Interactions
Water molecules play an important role in protein ligand binding and an analysis of
interactions between crystallographic water molecules and ligands in PDE crystal structures
was performed to identify key water molecules. The superposed PDE structures allow the
identification of areas in the binding sites in which a significant number of water molecules
have been found within a proximity of 1.5A (Figure 5). From this study, one particular
cluster (D) was shown to contain 49 water molecules from crystal structures of PDE2-PDE7
and PDE9-PDE10. In one crystal structure the water molecule at this particular position has
been displaced by a ligand, one of two conformations observed for zardaverine.32 The
structural water molecules in cluster D are bound by the residues DMB.22 and F/YHC1.1
stabilizing their position. Water at this position does not appear to form hydrogen bonds
with the cyclic nucleotides, but seems to form hydrogen bonds with AMP and GMP, thereby
stabilizing the catalytic product of PDEs. This consistent placement of a water molecule
indicates that water at this position should be considered during molecular modeling and
virtual screening studies.
The other clusters of water molecules are less general for PDEs. For example cluster B
containing 9 water molecules is only found in PDE9 structures, and occurs in a unique sub-
pocket that has not yet been addressed by PDE9 inhibitors. Cluster G contains 10 PDE4
waters and 3 PDE10 waters. However, given the proximity of this cluster to the metal ions,
this may have more to do with the types of inhibitors being developed for these targets than
a particular specificity of water molecules to those PDEs. Two clusters are found at the
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solvent exposed side of the pocket and they are dependent on the particular ligand present
in the crystal structure. Cluster I contains water molecules bound to nucleotide-like
moieties, while in structures containing cluster H water molecules, catechols are the most
common ligand scaffolds.
Figure 5: Water molecules involved in PDE ligand binding
(A) Crystalographic water molecules forming interactions with PDE ligands. The oxygen
atom of each water is shown, colored by the PDE family of the crystal structure from which
it was extracted. Nine clusters were identified, these are circled and labeled A-I, with A-F
forming interactions with ligands and residues (magenta), G forming interactions with
ligands, metals and the protein (red) and H-I forming interactions with just the ligand
(cyan). The water molecules are shown over a 1OYN (PDB:) pocket surface.
(B) The water molecules that interact with PDE inhibitors in crystal structures shown with
an overlay of all PDE ligands and metal ions. Water molecules are colored according to
the interactions they form, cyan form interactions with the ligand, magenta form
interactions with ligands and protein residues, purple form interactions with the ligand and
metals and red form interactions with ligands, metals and the protein residues.
2.8 Ligand Scaffold Analysis
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The structural motifs of PDE ligands can be assessed according to the sub-pockets with
which they interact. This provides a way to quickly identify those functional groups that
have successfully been used to address a given sub-pocket. To aid such efforts a scaffold
analysis was performed using the core scaffold of each ligand crystalized with a PDE
(Figure 6). The information about the binding mode of the scaffold and vectors of the side-
chains was retained by superposing the crystal structures and systematically extracting data
(Figure 7).
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Figure 6: Scaffold analysis of the ligands in PDE crystal structures
The core scaffold from each ligand crystalised with a PDE was identified. Scaffolds were
defined such that only one scaffold would be identified in each ligand. The scaffolds were
screened against active PDE inhibitors published in the ChEMBL database and the number
of hits are shown in green. The number of unique hits among ligands crystalized with PDEs
are shown, as well as the total number of hits and the PDB code of each structure. The R-
groups were collected for each hit molecule and the number of unique R-groups is provided
at each attachment point, ChEMBL R-groups are in green and crystal structure R-groups
are in black. The R-groups indicate points of attachment and have been numbered
according to the vector of the attachment in each crystal structure. Numbers run
sequentially as the angle of the vector changes from 16 (0º back of the pocket) to 4 (90º
towards metal ions) to 8 (180º towards solvent) to 12 (270º towards QQ.50) as described in
Figure 7. In Figure 6 alternate binding modes of scaffolds have been left out and the R-
groups from alternate binding modes are included with the R-groups of the most common
binding mode of the scaffold.
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A B
C D
Figure 7: The steps involved in defining the scaffolds of crystalized ligands. A In this step
the crystal structure is oriented in a consistent manner following alignment. B) A schematic
structure of the ligand is drawn using the orientation of the crystal structure as a guide. C)
The points of attachment around the scaffold of the crystalized PDE ligands were named
according to the vector of the bond broken using the chart shown. D) The placement of the
scaffolds was consistent with the orientation of the ligand in the pocket allowing the vector
to provide information about the placement of R-groups in the PDE binding pocket.
The most common scaffold type among crystalized ligands bound to PDEs are purines, with
IBMX, cAMP/AMP and cGMP/GMP accounting for most cases. The purines are
remarkable in that they often act as both hydrogen bond acceptors and donors to the
conserved glutamine, QQ.50, whereas most scaffolds only act as hydrogen bond acceptors.
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Another common scaffold type found particularly in PDE4 inhibitors is the catechol-like
moiety. While ethers are not the strongest hydrogen bond acceptors, the combination of two
acceptors targeting QQ.50 has resulted in highly potent PDE4 inhibitors. The quinolines are
another scaffold type commonly deployed in PDE inhibitor design. The nitrogen atoms in
quinolines usually act as acceptors where hydrogen bonds are formed with QQ.50. A common
theme across PDE ligands is the presence of a fused ring system in the scaffold. These fused
ring systems can be optimally accommodated in the narrow hydrophobic clamp between
the aliphatic residues at position I/V/LHC.32 and the conserved aromatic residues at position
FHC.52.
The scaffold was defined as the smallest fragment of each ligand involved in interactions
with the invariant glutamine and hydrophobic clamp, that retains the character of the
moieties involved in those interactions and prevents redundancy across ligands crystalized
with PDEs. A count of the scaffolds in ligands crystalized with PDEs and active PDE
inhibitors found in the ChEMBL database was also made. The bonds broken to isolate the
scaffolds were assigned R numbers according to a cyclic scheme based on the vector of the
bond broken using the orientation of the ligand in the crystal structure as a reference. In this
way R-groups with particular vectors in the pocket could be grouped together to allow the
crossing of R-groups from multiple ligands in a for example a Markush enumeration. The
R-groups found in ligands crystalized with PDEs and in PDE inhibitors found in the
ChEMBL database were gathered into a database for analysis. A Markush library containing
a scaffold and all R-group variations for a vector can be used in combination with docking
to efficiently probe a particular sub-pocket with relevant chemical diversity.
An analysis was also made of the moieties of ligands that interact with regions of the
substrate binding pocket in PDE crystal structures. In this case the interactions identified by
IFP analysis were the starting point for identification of fragments of the ligands involved
in interactions. For example 84 ligands across most PDE families only address the Q1
pocket with fragments that form hydrophobic interactions. Six ligands bound to PDE4 form
aromatic interactions with Q1 and those fragments are found in 13 PDE4 and PDE5 ligands
forming interactions with the Q1 pocket. Hydrogen bonding with the Q1 pocket is seen in
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5 crystal structures, 2 in PDE2, 1 in PDE3, PDE5 and PDE10. In each of these cases these
fragments were only found in compounds forming hydrogen bonds with the Q1 pocket
suggesting these fragments could be utilized in more ligands to improve binding.
2.9 Interactions with the Q1 and Q2 Pockets
The Q1 pocket flanks the invariant glutamine QQ.50 forming a sub-pocket deep in the
substrate binding site. Interactions with the Q1 pocket are nearly exclusively hydrophobic,
however two exceptions are shown in Figure 8. The Q1 pocket shows significant water
molecule occupancy in PDE crystal structures, indicating the potential to accommodate
polar functional groups. However, few ligands exploit this potential. Ligands that form
polar interactions with Q1 are among the most potent for each family, showing that the
potential of addressing the Q1 pocket is underutilized in PDE drug discovery efforts. The
high selectivity of the dihydropyridazinones for PDE3 over for example PDE4 and PDE5,36
can be attributed to the hydrogen bond interaction that the pyridazinone carbonyl oxygen
can form with HQ1.27, as illustrated for the PDE3 inhibitor MERCK1 (IC50 = 0.11 nM) in
Figure 8A (PDB: 1SO237). The corresponding YQ1.27 residue in PDE4 forms an hydrogen
bond with the conserved QQ.50 residue in most crystal structures. In PDE5 the QQ1.27 residue
also forms hydrogen bonds with QQ.50 in most crystal structures. However of the ligands
only 3P4 (IC50 = 5.5 nM) shown in Figure 8B (PDB: 2CHM), forms a hydrogen bond with
QQ1.27 in a PDE5 crystal structure.38
The Q2 pocket lies adjacent to QQ.50 towards the opening of the PDE substrate pocket. The
size of the Q2 pocket is family dependent, with significant Q2 pockets seen in structures of
PDE1, PDE10 and the parasite PDEs (LmjPDEB1, TcrPBEC and TbrPDB1). In the parasite
PDEs a sub-pocket of the Q2 pocket, dubbed the P-pocket, has been targeted in attempts to
achieve selectivity over human PDEs.39 The key residues involved in ligand interactions are
located at positions Q2.33, Q2.46 and Q2.49, that form hydrophobic interactions in 55%,
20% and 67% of structures respectively. The Q2 pocket plays a particularly important role
in inhibitor design for PDE10, where 30% of ligands address the Q2 pocket, forming both
π-π interactions and hydrogen bonds with YQ2.33. The selective PDE10 inhibitor PFK (IC50
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= 17 nM) in Figure 8C (PDB: 3QPN) was discovered using molecular docking of a virtual
combinatorial library with the specific aim of improving selectivity by addressing the Q2
pocket.40 Interestingly, while 90% of ligands in PDE crystal structures form a hydrogen
bond with QQ.50, 24% of PDE10 inhibitors do not form this hydrogen bond, including PF-
02545920 shown in Figures 8D (PDB: 3HR141). A traditional hydrogen bond may be
compensated by the presence of an N-H···π hydrogen bond between QQ.50 and a phenyl ring
in the ligand. The contribution of the N-H···π hydrogen bonds may be significant as
indicated by the potency of PF-02545920 (IC50 = 0.37nM). Additionally, hydrogen bonds
to the Q2 pocket are unique to PDE10, where ligands form hydrogen bonds with YQ2.33
further stabilizing occupation of the Q2 pocket.
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Figure 8: Examples of ligands interacting with the Q1 and Q2 pockets.
(A-D) Examples of compounds that interact with the Q1 and Q2 pockets. The binding
modes are shown of; (A) MERCK1 to PDE3A (PDB: 1SO2) (B) 3P4 to PDE5A (PDB:
2CHM), superposed over a 1UDT pocket surface, (C) PFK to PDE10A (PDB: 3QPN),
superposed over a 3HR1 (PDB:) pocket surface, and (D) PF-02545920 to PDE10A (PDB:
3HR1), superposed over a 3HR1 pocket surface).
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(E) The IFP bit strings of the proteins and ligands shown in A-D. Only those residues that
form at least one interaction with one of the four ligands are shown.
2.10 Interactions with the HC1, HC2 and S Pockets
Almost all ligands in PDE co-crystal structures interact with residues located in the HC1
region around the hydrophobic clamp (98%). Although most ligands only form hydrophobic
interactions with HC1, several also form hydrogen bonds with the residue at position
HC1.25, for example HBT (CID 1021494, IC50 = 50 nM) shown in Figure 9A (PDB:
3HMV) forms a hydrogen bond to NHC1.25 of PDE4B via an amide group.42 The binding of
HBT also shows a unique displacement of QQ.50 away from the pocket, despite its potent
inhibition of PDE4B. A series of halogenated pyrimidinone PDE5A inhibitor analogues
were designed, including fluorinated 5FO (PDB: 3SHY), chlorinated 5CO (PDB: 3SHZ),
and the brominated 5BO (IC50 = 13 nM) shown in Figure 9B (PDB: 3SIE), that target YH1.01
in PDE5A via a putative halogen bond.43 Interestingly, experimentally determined IC50
values showed a good correlation with the calculated halogen bond energies, demonstrating
that the halogen bond is an applicable tool in the design of PDE inhibitors. By contrast, the
HC2 region interacts with just 21% of ligands, and exclusively through hydrophobic
interactions. In Figure 9C (PDB: 2OVY) the quinoxaline scaffold of the potent PDE10A
inhibitor PFJ (CHEMBL219445, IC50 = 6 nM) targets HC2 44, forming hydrophobic
interactions with GHC2.51, AHC2.54 and VHC2.55 in PDE10A. In SAR studies it was found that
this bulky group does not fit the smaller HC2 sub-pocket of PDE3A/B, explaining its high
selectivity for PDE10A over PDE3. The S pocket is primarily addressed by interactions
between the core scaffolds of ligands and the residue at position F/YS.35, a phenylalanine in
all PDEs except PDE9 where a tyrosine is found at this position. The presence of tyrosine
at position YS.35 in S pocket has been used in the structure-based design of PDE9A
inhibitors. In Figure 9D (PDB: 4G2L), the protonated nitrogen atom in 0WL
(CHEMBL2180070, IC50 = 32 nM) forms a hydrogen bond with YS.35.45 The compound
forms part of a structure-based effort to improve brain penetration of the closely related
Alzheimer drug candidate PF-04447943, which completed a phase II trial in 2010. The
water mediated hydrogen bond between PF-04447943 and YS.35 was replaced by a direct
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hydrogen bond in the binding of 0WL (CHEMBL2180070) reducing potency and
selectivity, but improving brain penetration.
Figure 9: Examples of ligands interacting with the HC1, HC2 and S pockets.
(A-D) Examples of compounds that interact with the HC1, HC2 and S pockets. The binding
modes of are shown of; (A) HBT bound to PDE4B (PDB: 3HMV), superposed over a 1OYN
72
(PDB:) pocket surface, (B) 5BO bound to PDE5A (PDB: 3SIE) superposed over a 1UDT
(PDB:) pocket surface, (C) PFJ to PDE10A (PDB: 2OVY) superposed over a 3HR1 (PDB:)
pocket surface, and (D) 0WL to PDE9A (PDB: 4G2L) superposed over a 3K3E (PDB:)
pocket surface.
(E) The IFP bit strings of the proteins and ligands shown in A-D. Only those residues that
form at least one interaction with one of the four residues are shown.
2.11 Interactions with the MB, MB1 and MB2 Pockets and the Metal
Ions
The MB pocket contains the metal ions responsible for the hydrolysis of the cyclic
nucleotides cAMP and cGMP to AMP and GMP respectively. The binding modes of the
products are shown in Figure 10A (PDB: 1TB7) showing AMP bound to PDE4D and Figure
10B (PDB: 1T9S) showing GMP bound to PDE5A 25. The cyclic nucleotides adopt very
similar binding modes with each making ionic bonds to both Zn2+ and Mg2+. The pattern of
hydrogen bond donors and acceptors surrounding the adenine ring of AMP and guanine
ring of GMP differ. The PDEs match these differing hydrogen bond patterns by flipping
QQ.50. Although this “glutamine switch” was proposed to drive cyclic nucleotide selectivity,
multiple differences are now thought to control substrate specificity with no individual
residue playing a dominant role.46
Just two of the crystalized PDE inhibitors form tight interactions with the catalytic metal
ions in PDEs, zardaverine (IC50 = 0.39 µM, PDE4D, PDB: 1XOR32) and AN2898 (IC50 =
0.24 µM, PDE4B, PDB: 3O0J47), shown in Figure 10C. These compounds do not display
unusual potency as PDE inhibitors, suggesting that addressing the metal ions directly
provides little additional interaction energy if any, most likely due to the displacement of
structural water molecules during binding. The oxaborole of AN2898 that binds to the metal
ions and HMB.02 is unique amongst crystalized PDE inhibitors, as is the catechol like
dicyanophenoxy ring that interacts with QQ.50. The MB1 and MB2 pockets form few
specific interactions with ligands, only IHM (CID 78225170, IC50 = 0.5 nM) bound to
PDE10A (PDB: 3V9B) shown in Figure 10D forms a hydrogen bond with MMB1.17.48 In
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Figure 9C PFJ (CHEMBL219445) is shown to form π-π interactions with FMB1.20, an
interaction shared by two analogues and the recent PDE2 crystal structure containing
BAY60-7550 (PDB: 4HTX). All other inhibitor interactions with MB1 and MB2 are
hydrophobic.
74
Figure 10: Functional groups interacting with the MB, MB1 and MB2 pockets illustrated
with examples.
(A-D) Examples of compounds which interact with the MB, MB1 and MB2 pockets. The
binding modes of are shown of; (A) AMP to PDE4D (PDB: 1TB7), superposed over a 1OYN
(PDB:) pocket surface, (B) GMP to PDE5A (PDB: 1T9S) superposed over a 1UDT (PDB:)
pocket surface, (C) AN2898 to PDE4B (PDB: 3O0J) superposed over a 1OYN (PDB:)
pocket surface, and (D) IHM to PDE4D (PDB: 3V9B) superposed over a 1OYN (PDB:)
pocket surface.
(E) The IFP bit strings of the proteins and ligands shown in A-D. Only those residues which
form at least one interaction with one of the four residues are shown.
2.12 Structure-Based PDE Ligand Discovery and Design
The superposition of all PDE crystal structures combined with the annotation of the binding
pockets, water clusters, and the generated PDE-ligand interaction data, make the
PDEStrIAn database especially suited for ligand design and structure-based optimization.
The consistent manner of the database creation allows for an easy comparison of multiple
structures, co-crystallized inhibitors and their interactions. The fragmentation of crystalized
PDE ligands into scaffolds and R-groups taking the binding conformation into account and
applying this to generate libraries of orientation specific R-groups and scaffolds provides a
toolbox for addressing different pocket regions and scaffold hopping. Together these tools
can support future structure-based PDE ligand discovery and design, including structure-
based virtual screening,26 fragment growing,49, 50 and modulation of PDE activity and
selectivity.19
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Figure 11: Drug design case studies: fragment growing and allosteric modulations
(A-D) The growing of the PDE4D fragment screening hit, 3,5-dimethyl-1H-pyrazole-4-
carboxylic acid ethyl ester (A, PDB: 1Y2B), with an N-phenyl (B, PDB: 1Y2C), an
additional para-methoxy group (C, PDB: 1Y2D) and replacing this with a meta-nitro group
(D, PDB: 1Y2K). Each structure is shown superposed over a 1OYN (PDB:) pocket surface.
(E/J) The IFP bit strings of the proteins and ligands shown in A-D. Only those residues
which form at least one interaction with one of the four residues are shown.
(F-I) Interactions between ligands bound to the PDE catalytic site and regions that are able
to fold over the pocket. In the structure of roflumilast bound to the catalytic domain of
PDE4D (F, PDB: 1XOQ, superposed over a PDB: 1OYN pocket surface) the catalytic
pocket is open. In the structure of Icarisid II bound to PDE5A (G, PDB: 2H44, superposed
over a PDB: 1UDT pocket surface) the H-loop folds over the pocket entrance enclosing the
ligand. The structure of atizoram bound to the catalytic domain of PDE4B shows closing of
the pocket by the C-terminal residues (H, PDB: 3KKT, superposed over a surface of PDB:
76
1OYN). In the case of D159153 bound to a PDE4D construct which includes the UCR2
regulatory domain, the pocket is closed off by a helix of UCR2 (I, PDB: 3IAD, superposed
over a PDB: 1OYN pocket surface).
An example of fragment growing in PDE4D is shown in Figures 11A-D. An initial fragment
screening yielded 3,5-dimethyl-1H-pyrazole-4-carboxylic acid ethyl ester as an initial hit
for further optimization (PDE4D IC50 = 82 µM49). Structure-guided fragment growing
starting from the pyrozole hit (Figure 11A, PDB: 1Y2B) and guided with six further PDE4B
and PDE4D co-crystal structures enabled the design of a series potent of PDE4 inhibitors.
The addition of a phenyl group in a first round of synthesis that forms a π-π interaction with
HMB.02 brought a 400-fold increase in potency (PDE4D IC50 = 0.27 µM, Figure 11B, PDB:
1Y2C). The addition of a para-methoxy group (Figure 11C, PDB: 1Y2D) resulted in a
binding mode switch out of the pocket and a 10-fold reduction in potency (PDE4D IC50 =
2.0 µM). Placing a nitro group in the meta position instead resulted in the inhibitor
coordinating with the magnesium ion (Figure 11D, PDB: 1Y2K) and a 4000-fold increase
in potency over the initial screening hit in just two rounds of synthesis (PDE4D IC50 = 0.021
µM49).
Ligands can be designed to not only target the PDE catalytic site (PDE4D, PDB: 1XOQ,
Figure 11F), but also protein regions that fold over the opening of the catalytic site. The
catalytic site can be closed off by the H-loop extending over the pocket, examples of this
fold are seen in PDE2A and PDE5 (Figure 11G, PDB: 2H44) crystal structures. Moreover
in PDE4 structures the C-Terminus can fold over to close the pocket as shown in Figure
11H (PDE4D, PDB: 3KKT). The extent to which a ligand is encapsulated by a PDE pocket
can be influenced by the design of the ligand and this will impact the selectivity and kinetics
of binding. An example of the structure-based design of interactions with residues that close
the PDE pocket is that of UCR2 interactions with PDE4 inhibitors. This interaction has been
utilized to modulate inhibition and achieve selectivity for PDE4D over PDE4B.19, 51 The
proposed mechanism of regulation, following kinetic and structural studies of PDE4
inhibitor binding, is a two-site model with negative cooperativity. In this model PDE4 forms
a dimer and the binding of UCR2 to one monomer reduces the affinity of UCR2 to the other
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monomer. Inhibitors which form strong interactions with UCR2 while bound to PDE4 are
therefore likely to stabilize a partial inhibition of PDE4, where one monomer is full and
closed, while the other is empty and open. Through modulation of the interactions between
the ligand and UCR2 it is possible to design partial inhibitors of PDE4 that are less emetic
than full inhibitors. Additionally by forming interactions specific to UCR2 in one PDE4
subtype, PDE4 subtype selectivity becomes possible. Through the application of this
knowledge the authors were able to design a series of PDE4 inhibitors with improved side-
effect profiles based on mouse model studies. One of these inhibitors, D159153 (PDE4D7
IC50 < 1 nM, 89% Imax), is shown in Figure 11I (PDE4D, PDB: 3IAD).
2.13 Conclusion
Crystal structures of PDEs were extensively analyzed across the PDE superfamily resulting
in database of PDE-ligand interactions complemented with an analysis of ligand-water and
ligand-metal interactions. Through the aggregation of this data into a thorough overview,
key interactions and PDE subtype specific interactions are easily identifiable. A novel
nomenclature for PDE pocket residues is proposed that enables cross family comparisons
of PDE crystal structures to be performed systematically. A scaffold analysis of crystalized
PDE ligands provides a toolbox for computational chemists to perform scaffold hopping or
Markush R-group substitution studies when targeting PDEs. By relating R-groups to vectors
in the pocket and by additional analysis of ligand substructures interacting with specific
regions of the PDE catalytic pocket, support will be provided for the design of novel PDE
inhibitors with improved selectivity and potency profiles.
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