PDEStrIAn: A Phosphodiesterase Structure and Ligand Interaction

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PDEStrIAn: A phosphodiesterase structure and ligand interaction annotated database as a tool for structure-based drug design Chimed Jansen, Albert J. Kooistra, Georgi K. Kanev, Rob Leurs, Iwan J. P. De Esch, and Chris de Graaf J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01813 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 28, 2016

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Journal of Medicinal Chemistry

PDEStrIAn: A phosphodiesterase structure and ligand interaction annotated database as a tool for structurebased drug design Chimed Jansen, Albert J. Kooistra, Georgi K. Kanev, Rob Leurs, Iwan J.P. de Esch, Chris de Graaf*

Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute of Molecules, Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

ACS Paragon Plus Environment


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ABSTRACT A systematic analysis is presented of the 220 phosphodiesterase (PDE) catalytic domain crystal structures present in the Protein Data Bank (PDB) with a focus on PDE-ligand interactions. The consistent structural alignment of 57 PDE ligand binding site residues enables the systematic analysis of PDE-ligand Interaction FingerPrints (IFPs), the identification of subtype-specific PDEligand interaction features, and the classification of ligands according to their binding modes. We illustrate how systematic mining of this phosphodiesterase structure and ligand interaction annotated (PDEStrIAn) database provides 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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1. INTRODUCTION

1.1 Phosphodiesterases (PDE) structure and function. 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; 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. During this reaction the substrate is held in place by interaction with a key conserved glutamine residue (e.g. Q369 in PDE4D), a “hydrophobic clamp” formed by a conserved phenylalanine (F340 in PDE4D) and an aliphatic valine, leucine or isoleucine residue (I336 in PDE4D), and ionic bonds between the phosphate group and metal ions (Figure 1A-B). Hydrolysis of the cyclic phosphate ester bond occurs through attack by a water molecule that is activated by the metal ions.8, 10



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1.2 PDEs as drug targets. The pervasive and tissue specific expression of PDE isoforms allows isoform-specific PDE inhibitors to be applied in a wide range of therapeutic areas.11 To date thirteen selective PDE inhibitors have been approved for use as pharmaceuticals, of which five have been published crystalized in complex with their respective PDE targets (Figure 1C). The first of these to reach blockbuster status was sildenafil (Viagra®, crystallized in complex with PDE4B (PDB: 1XOS12), PDE5A (PDB: 1TBF13, 1UDT14, 2H4215), and PDE5A/6C (PDB: 3JWQ16)), approved for the treatment of erectile dysfunction and pulmonary hypertension, thereby establishing PDE5 as a highly successful drug target. Three PDE5 inhibitors have followed, vardenafil (Levitra®, PDB: 1XP012, 1UHO14, 3B2R17; additionally crystallized with PDE4B (PDB: 1XOT12)), tadalafil (Cialis®, PDB: 1UDU14, 1XOZ12) and most recently avanafil (Stendra®), bringing improvements in selectivity.18 Following considerable effort including the late stage failure of rolipram (PDB: 1RO619, 1XN012, 1XMY12, 4X0F, 1TBB13, 3G4K20, 1Q9M21, 1OYN21), three PDE4 drugs have entered the market, drotaverine (No-Spa®) as an antispasmodic and recently roflumilast (Daxas®, PDB: 1XMU12, 1XOQ12, 3G4L20) as a treatment for COPD and apremilast as a treatment for psoriatic arthritis (Otezla®).22-24 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®). The development of PDE3 and PDE4 drugs has been hampered by problematic contraindications, notably nausea and cardiovascular side effects.25, 26 There are several marketed non-selective PDE inhibitors that inhibit both PDE3 and PDE4 such as theophylline27 and several other drugs that inhibit a broad range of PDE families such as dipyridamole.28 One of the oldest prescribed PDE drugs is papaverine (crystallized in complex with both PDE4 (PDB: 3IAK), and PDE10 (PDB: 2WEY29)), which has long been used as a vasodilator. Recently papaverine was


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found to be a potent and selective PDE10 inhibitor and it has played a role in generating interest in PDE10 as a drug target.30 The development of selective inhibitors of PDE10 continues to receive considerable interest, in spite of the recent failure of PF-02545920 (1, PDB: 3HR131) to reach the market (ClinicalTrials.gov identifier: NCT01939548). With many PDE drug discovery programs ongoing, including the targeting of parasite PDE families,32 the potential for new PDE drugs is evident.

Figure 1. The binding modes of: (A) the substrate cAMP (PDB: 2PW333), and (B) the product AMP (PDB: 1PTW10) bound to PDE4D. The key interacting residues are shown colored by the pocket region, H160 (H160MB.02), N321 (N321HC1.25), I336 (I336HC.32), F340 (F340S.35), Q369 (Q369Q.50) and F372 (F372HC.52), and named according to the nomenclature presented in Figure 4. (C) Molecular structures and indications of the five marketed PDE inhibitors crystalized with PDEs: sildenafil, vardenafil, tadalafil, roflumilast, papaverine two PDE inhibitors which failed to reach the



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market: rolipram and 1 and the PDE substrates cAMP and cGMP with arrows indicating the bond broken during hydrolysis by PDEs to form AMP and GMP.

1.3 From PDE structure analysis to inhibitor design. The drugs targeting PDEs described in 1.2 were developed using classical medicinal chemistry approaches with the exception of papaverine which is a natural product (Figure 1). Increasingly however, PDEs are being specifically targeted and the discovery of new inhibitors is now supported by a growing understanding of PDE crystal structures. The information from the crystal structures of PDE–inhibitor complexes can be used to guide the discovery and design of new PDE inhibitors.11, 12, 20, 34-38 Selective inhibitors have been developed and optimized by an iterative process of biochemical and/or (structure-based) virtual screening, determination of crystal structures of PDE targets with validated hits, structure-based ligand optimization, and chemical synthesis for several PDE targets, including PDE239, PDE440, 41, PDE542, PDE943-45 and PDE10A31,

46-49

. The current review will describe how an integrative

chemo/bioinformatics analysis of the increasing number of PDE crystal structures offers new insights into the molecular determinants of PDE ligand selectivity, and provide a medicinal chemistry toolbox to target well defined (combinations of) PDE isoforms. Four previous reviews of PDE crystal structures have been published.11, 12, 34, 35 The first described the binding of 10 inhibitors to PDE4B, PDE4D and PDE5A in 15 crystal structures.12 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 determine substrate and inhibitor binding.11 In another


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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.34 The most comprehensive previous analysis of PDE structures reviewed the first 60 PDE structures published in the PDB, covering PDE families 1, 2, 3, 4, 5, 7 and 9.35 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 160 PDE crystal structures added to the PDB since the previous analysis by Ke and Wang35 and with new cheminformatics and structural chemogenomics tools at hand, the question of the drivers of PDE selectivity can now be revisited. The current review presents a systematic structural chemogenomics analysis of the 220 phosphodiesterase (PDE) catalytic domain crystal structures present in the Protein Data Bank (PDB) with a focus on PDE-ligand interactions. The consistent structural alignment of 57 PDE ligand binding site residues enables the systematic analysis of PDE-ligand Interaction FingerPrints (IFPs), the identification of subtype-specific PDE-ligand interaction features, and the 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 ChEMBL50 database provides a toolbox for scaffold hopping and ligand design. The structure-based PDE-ligand interaction analysis provides a comprehensive map to guide future PDE focused drug discovery efforts.

2. THE STRUCTURE OF PHOSPHODIESTERASES



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2.1 Phosphodiesterase isoforms in cyclic nucleotide signaling. The role of PDEs in signaling cascades begins when an extracellular stimulus is detected by a transmembrane receptor that upon activation stimulates the activity of adenylyl cyclase (AC) or guanylyl cyclase (GC). While active, AC continuously catalyzes the conversion of ATP into cAMP and GC continuously catalyzes the conversion of GTP into cGMP. This results in high concentrations of cAMP or cGMP and a strong amplification of the signal. The cyclic nucleotides then pass the signal on, usually to protein kinase A (PKA) for cAMP or protein kinase G (PKG) for cGMP. In turn these protein kinases activate proteins further down the signaling cascade. The specific roles of cAMP and cGMP are dependent on, amongst others, the signaling cascade, the cell type and the location of the cyclic nucleotides in the cell. The concentrations of the cyclic nucleotides are reduced by proximal PDEs with the correct cyclic nucleotide selectivity. There are 21 genes that encode human PDEs (Figure 2A) and these are divided into 11 gene families that encode structurally related PDE isoforms. This diversity allows PDEs to regulate a diverse range of signaling outcomes.3, 51, 52

2.2 Structural organization of PDEs. Each PDE contains a catalytic domain, a lengthy Nterminus that may contain one or more structured domains and an unstructured C-terminus (Figure 2).53 The structured regions found in the N-termini of PDEs have diverse roles and the role of the same domain family may vary between PDE families. The GAF domains have been shown to modulate PDE activity, play a role in PDE dimerization and are able to bind the cyclic nucleotides cAMP and cGMP.54 Differences in the GAF-A and GAF-B domains of PDE2, PDE5, PDE6, PDE10, PDE11 and parasite PDEB families influence the specific substrate binding and dimerization roles of the GAF domains in each case.55, 56 The UCR domains regulate PDE4 activity and crystallography has shown direct contact between UCR and inhibitors bound to the active sites


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of PDE4B57 (PDB: 4WZI and 4X0F) and PDE4D20 (PDB: 3IAD, 3G4G and 3G45). The activation of PDE1 by Ca2+-calmodulin through interaction with the CaM binding domain regulates PDE1 activity.58 In PDE9A the Pat7 nuclear localization domain was thought to keep PDE9A localized in the nucleus, however splice variants have been found to localize in a variety of organelles.59 The TM domain in PDE3 associates PDE3A and PDE3B with the endoplasmic reticulum and other membrane rich regions of cells much like the TD domains of PDE4 and PDE7 target membranes.60 The signal regulatory domain (REC) of PDE8 is not the site of PKA phosphorylation, but is thought to affect signaling, while the PAS domain allows PDE8 to associate with IkappaB proteins.61, 62 The PDE amino-terminal domains influence PDE localization, oligomerization and activity, or the interaction with protein partners and alternative splicing of the N-terminus further diversifies the influence of PDEs on signaling pathways.20, 53, 55, 63 Crystal structures are available of N-terminal GAF domains of PDE2 (complete dimer), PDE5, (GAF-A & GAF-B), PDE6 (complete dimer with PDE5A/6C chimera catalytic domain) and PDE10 (GAF-B). Inhibitor binding has been observed for GAF domains and this may result in stabilization of an open conformation of the catalytic domain.64 The full length PDE structures of PDE2 and PDE5A/6C (Figure 2B-C) show a dimer formation with the helix between GAF-B and the catalytic domain intertwining the two proteins. The orientation of the catalytic domain differs greatly between PDE2A and PDE5A/6C possibly reflecting differences in the mode of regulation.65 Multiple isoforms (resulting from splice variants) of the PDE genes result in differences in the PDE sequence, or truncation of the PDE sequence when compared to the canonical sequence. The truncations typically occur at the carboxy-terminal unstructured region or at the amino-terminal region where one or more structured domain may be absent. Differences in the sequence of the catalytic domain are not seen and isoforms without a catalytic domain will be inactive. However



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PDEs resulting from alternative spicing may show altered catalytic activity due to the influence of the amino-terminal and carboxy-terminal regions on the substrate access to the catalytic site. Sequence differences in, or truncation of, amino-terminal domains may also affect the localization and activation mechanism of a PDE and generating inhibitors selective for specific isoforms is of interest for drug discovery.66


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Figure 2. (A) The structures and domains of the 11 human PDE families are shown along with the 3 parasite PDEs that have been crystalized. The amino-terminal domains may regulate the activity of



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the conserved catalytic domain, play a role in the localization of PDEs or the interaction with protein partners. The amino-terminal domains include the CaM-binding domain (CaM), GAF domains, transmembrane domain (TM domain), targeting domain (TD), upstream conserved regions (UCRs), signal regulatory domain (REC), PAS domain, Pat7 nuclear localization, FYVE-type domains and coiled coil regions. The names of PDE subtypes are given in blue in cases where crystal structures of the PDE have been published and the specific domains that have been crystalized are shown in blue. The PDE subtypes and domains for which crystal structures have not been published are shown in gray. The structures of two domains are partially resolved: PDE6C catalytic domain (12%, residues of the M-loop, part of H14 and H15) and PDE4 UCR2 (90%, residues in engineered construct) and these are shown in blue/gray stripes. (B) A schematic diagram of the PDE5A/6C dimer showing one PDE in pink and the other in blue. (C) A full length dimer of PDE5A/6C obtained using cryo-electron microscopy (PDB: 3JAB), colored as (B). The surface of the binding site is shown in gray and the bound IBMX in green.

2.3 Conserved structural architecture of PDEs. 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 3A). 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 3B). 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


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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 3C), and forms the catalytic site which includes a metal binding region at the apex of H6 – H13 and a substrate binding region formed between H13 – H16.

2.4 PDE Crystal Structures. There are 220 PDE crystal structures in the Protein Databank at the time of writing (25-04-2015, Figure 3D). The first crystal structure published of the PDE catalytic domain was an unliganded structure of PDE4B 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 (PDB: 1MKD67) bound to PDE4D, provided insights into the role of the catechol scaffold during binding and into PDE dimerization.67 A subsequent study in PDE4 showed the structural basis for selectivity between the (R)-rolipram (PDB: 1Q9M) and (S)-rolipram (PDB: 1OYN) enantiomers.21 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. The “glutamine switch” hypothesis has since been brought into doubt by structural33,

68-70

, site-directed mutation71,

72

and

computational studies73 and is no longer thought to be the driver of substrate selectivity. Also the term “hydrophobic clamp” was introduced to describe a hallmark ligand-PDE interaction.13 The number of PDE crystal structures has continued to climb at a rate of about 20 per year and there are now structures of the catalytic domains of 9 of the 11 human PDE families (a structures of PDE6 and PDE11 are still lacking). At the subtype level only 12 of the 21 subtypes have been crystalized (Figure 3D, structures of PDE1A, PDE1B, PDE3A, PDE6A, PDE6B, PDE6C, PDE7B, PDE8B and



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PDE11A 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, 74, 75 The growth in novel PDE inhibitors included in the ChEMBL database50 since the year 2000 provides a different view on which PDE targets are of greatest interest (Figure 3E). 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 publication of large numbers of novel inhibitors since 2010 coincides with the release of a significant number of PDE10 crystal structures. 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, PDE3 and PDE7 there are a significant number of novel inhibitors published, 76, 77 despite the fact that just three PDE1 (PDB: 4NPV, 4NPW, 1TAZ)13, 77 two PDE3 (PDB: 1SO2, 1SOJ),78 and four PDE7 (PDB: 1ZKL, 4PM0, 4Y2B, 3G3N)79-82 crystal structures having has been published (Figure 3D-E). 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.38, 83 Along with PDE6, 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 targets.


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Figure 3. Structural overviews of PDEs. (A) 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. (B) An overlay of the backbone ribbons of all PDE crystal structures. (C) 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 4. (D) 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



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of the graph. The two crystal structures marked PDE5A/6C (*) are partial binding pocket chimeras of PDE6C (residues 787-826) in PDE5A (residues 746-786 and 827-859) constructs. (E) An overview of novel active PDE inhibitors published in the ChEMBL database50 (pIC50 ≥5) 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.

2.5 PDE binding site topology. In order to describe the interactions between the ligands and the substrate binding pocket in a consistent manner, we propose 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 4A). The 10 regions consist of; the invariant glutamine (Q) regions Q, Q1 and Q2; 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 4B). Of the 57 pocket residues, 13 are conserved across all PDEs, 11 of which are in metal binding regions MB, MB1, and MB2 (Figure 5). 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 5. 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


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in a consistent manner across the PDE families, the novel nomenclature combines the amino acid sequence reference with the pocket region name and the position of amino acid in the pocket sequence (Figure 4D). 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.84, 85 Through processing the available PDE crystallographic data in a systematic manner, studying the protein fold, molecular interactions with (subpockets in) the PDE-ligand binding site, ligand substructures and decorations, and combining this data in an accessible format, we constructed the PhosphoDiEsterase Structure and Ligand Interaction Annotated (PDEStrIAn) database for medicinal chemists that links structure-based PDE-ligand interaction maps to PDE ligand topology. The utility of this chemical toolbox is enhanced by the difficulty in achieving ligand selectivity between the PDE families. To facilitate this and future studies of the PDE super family, we introduce a novel standardized nomenclature for PDE binding site residues. The PDEStrIAn database contains the most comprehensive structural chemogenomics analysis of PDEs to date and the key findings of this analysis are presented here.



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Figure 4. 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: 1OYN21). 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 as aligned in Figure 5. The color coding and pocket region names below the WebLogo


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figure are those defined in panel A. (D) A PDE ligand binding site 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 conserved PDE pocket residues of different PDE subtypes 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).



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Figure 5. 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. The PDE binding site residue nomenclature is defined in Figure 4. Conserved residues are highlighted in green. PDE6C residues in the PDE5A/6C chimera are highlighted beige.


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3. Mining structural PDE-ligand interaction data: Phosphodiesterase Structure and ligand Interaction Annotated (PDEStrIAn) database

3.1 Systematic analysis of structural PDE-ligand interactions. To map the structural PDEligand interaction space, the structures and protein-ligand Interaction FingerPrints (IFPs)85,

86

of

PDE crystal structures published in the PDB were systematically analyzed and annotated in the PDEStrIAn database. An overview of the construction of the PDEStrIAn database is provided in Figure 6A 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 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 (10). The PDEStrIAn database



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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enables the systematic protein-based (PDE binding site region) and ligand-based (PDE ligand substructure) analysis of PDE structural chemogenomics data, providing a comprehensive analysis of the molecular and structural determinants of PDE-ligand interactions.

3.2 Interaction FingerPrint (IFP) Generation. A key step in mapping out structural PDE-ligand interaction space in the PDEStrIAn database was to derive IFPs from the crystal structure complexes. The binding mode and IFP of sildenafil (Figure 1C) to PDE5A illustrates the use of IFPs to encode protein-ligand interactions (Figure 6B-D). The binding mode of sildenafil is presented from the pocket opening showing all interacting residues (Figure 6B). Figure 6C shows a top view projection of sildenafil, the residues forming specific interactions with sildenafil, and the metal ions over the surface of the pocket, this visualization method is used throughout the remainder of the article. The IFP of the binding mode of sildenafil is presented in Figure 6D. 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.86 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-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, Q817Q.50 additionally shows hydrogen bond acceptor and donor interactions and F820HC.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.


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Figure 6. Systematic structure-based analysis of PDE-ligand interactions. (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. (BC) Sildenafil (2D structure presented in C) bound to PDE5A (PDB: 1UDT14) 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.



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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.

3.3 PDE-ligand Binding Mode Analysis. Protein-ligand interaction fingerprints were derived from the 203 available PDE crystal structures containing ligands, providing an overview of PDEligand interactions in the different PDE subpockets (Table 1). The 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 7. 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 occur in all but two structures with I/V/LHC.32 (PDE4B: 3O0J and PDE10: 4WN1) and F/YS.35 (PDE9: 3DYS and 4GH6). Aromatic interactions with F/YS.35 occur in 59% of structures and with FHC.52 in all PDE crystal structures. Hydrogen bond donor or acceptor interactions with QQ.50 are found in 86% 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 (see Table 1).

Table 1: Schematic representation of the ligand binding modes in 203 ligand bound PDE crystal structures in the PDEStrIAn database. The top row reports: top line: four symbol PDB code and literature references (in superscript), second line: compound name or number (in bold), compound ID and ChEMBL ID number (between brackets), and IC50 values of the co-crystallized ligands are


Page 25 of 123


1 2 3 provided. The name of the co-crystallized ligand corresponds to the conventional name, if this is 4 5 unavailable, the name or number as used in the accompanying publication is used or the 3-letter 6 7 8 PDB-code. Chemical structures of the co-crystallized ligands are oriented in a similar way to the 9 10 three-dimensional binding modes presented throughout the manuscript, and the ligand scaffolds that 11 12 interact with the conserved QQ.50 residue and hydrophobic clamp (HC) are highlighting in red. The 13 14 15 numbers given below each figure list the residues from each pocket that form interactions with the 16 17 ligands. The color coding of the pockets is given in the key at the end of the table (from left to right: 18 19 20 Q (red), Q1 (pink), Q2 (red brown), HC (yellow), HC1 (light yellow), HC2 (light brown), MB (dark 21 22 blue), MB1 (marine blue), MB2 (purple), S (green)), the ligand binding pocket regions are defined 23 24 in Figure 4. At the end of the Table an overview of 17 unliganded PDE crystal structures is 25 26 27 provided. 28 29 30 31 32 33 34 35 PDE1 PDE2A 36 4NPV77 4NPW77 3ITU64 4C1I87 4D0839 (+)-EHNA (296435): 635 nM 37 2 (7A, -): 35 nM 3 (19A, -): N.D. 4 (12, -): 10.1 nM IBMX (275084): 4 µM 38 39 40 41 42 43 1 1 1 2 3 2 1 2 1 1 1 2 3 1 1 3 1 2 2 2 1 1 2 1 1 2 2 1 1 3 1 1 2 2 1 1 2 3 1 4 44 PDE2A PDE3B 45 4D0939 4HTX88 4JIB89 1SO278 1SOJ78 46 6 (BAY60-7550, 370962): 4.7 nM 8 (MERCK1, 131355): 0.27 nM 5 (3, -): 1.7 nM 7 (22, 2387140): 45 nM IBMX (275084): 242 nM HO 47 HC1/MB/Q1 48 O N N Q 49 HN 50 N HC1/HC2/MB/MB1/S 51 52 HC2/Q2/S N 53 N 54 1 2 2 1 1 2 2 1 2 1 1 2 2 2 1 1 4 1 2 1 2 2 2 2 1 1 2 1 4 1 2 3 2 1 1 1 2 1 1 1 2 3 1 1 1 55 56 57 58 59 60 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 123

PDE4A 3TVX90 pentoxifylline (628): 168 µM

PDE4B 2QYK91 10 (NVP, 74078): 3.3 µM

3I8V 9 (RO-201724, 18701): 4.3 µM

2QYL91 10 (NVP, 74078): 650 nM

3G4520 11 (PMNPQ, 127944): 70 pM

O

O

HN

Q

NH

O

HC1/MB/MB1/S Q2/S

1 3 1 2 3

1

2

1 3 2 2 3

1

2

1 2 1 2 2

1

2 1 2 1 2 2

1 1

2 1 3 2 2 3

1 1

2

PDE4B 3FRG92

3GWT93

3WD994

3W5E95

4MYQ96

12 (2, 462150): 8.4 nM

13 (4, 570015): 7.94 pM

14 (10f, 2442472): 8.3 nM

15 (31e, 2385758): 11 nM

16 (A-33, 1782306): 32 nM

H2N

Q

O HC1 H N

N

N

HN Q2/S

O

MB1/MB2/S

1

2 3 2 1 4NW741 17 (8, -): 237 nM

2

1

2 2 3 2 1 1 2 4KP640 18 (44, 2402509): 870 pM

1

1 2 3 2 1 3 3 1 3 1 2 2 1 1 1 1 1 2 1 2 3 1 1 1 2 3O0J97 3LY298 1RO619 rac-rolipram (63): 550 nm 19 (AN2898, -): 240 nM 20 (9, 1096812): 21 pM

1 4 1 2 1 1 1 1 2 1XN012 rac-rolipram (63): 550 nm

1 3 1 2 3 1 1 2 1XMY12 (R)-rolipram (430893): 231 nM

1 3 2 2 3 1 1 2 1XLX12 cilomilast (511115): 25 nM

1 3 2 2 3 1 1 3KKT atizoram (1229569): -

2

1 3 2 2 3 1 1 2 1 3 2 2 3 1 1 2 1 3 2 2 3 1 2 1XM412 1XMU12 1Y2H36 21 (20, 519827): 56 nM piclamilast (42126): 41 pM roflumilast (193240): 84 nM

1 3 2 2 3 1 1 1Y2J36 22 (21, -): 33 nM

1 3 2 2 3 1 1 3HMV99 23 (6, -): 200 nM

1

1 3 2 2 3 2 1 1XOS12 sildenafil (192): 20 µM

2

1 1 2 1 4X0F57

2 1 3 2 2 3 2 2 2 1 3 2 2 3 2 2 1XM612 1XLZ12 (R)-rolipram (430893): 231 nM (R)-mesopram (603830): 420nM filaminast (590754): 960 nM

2 1 4 2 2 3 2 1 2 1 3 2 3 1 1 2 1XOT12 3D3P100 24 (20a, 521203): 3.2 nM vardenafil (1520): 3.8 µM HC1/Q1

HC1/Q1 H2N

HC1/MB/MB1/S N N O

O Q

HC1/MB/MB1/S NH

Q

N N H

S Q1/Q2/S

1 3

2 3

1 1

2

1 2 2 2 2

O O N+ O-

HC2/Q2/S

1 1

2

1 1 2 2 3

2 1

4 1 1 2 2 2


1 1

3 1 4

HN

O

2 2 1 1 1

2

Page 27 of 123

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3O56101 25 (10d, -): 794 pM

1 4

2 3

1 1 1

3O57101 26 (16, -): 79.4 pM

1

1 4

1TB513 AMP (752) -

2 2 2 1 1 1 2

1

2 1

1ROR19 AMP (752) -

2 1

1 1

2 3

1 1

1RO919 8-Br-AMP (1230617): -

1 1

2 3

1 1

2

PDE4D 2QYN91 10 (NVP, 74078): 570 nM

3G5820 11 (PMNPQ, 127944): 5 nM

1 2 1 2 2 1 1 2 3G4K20 (R)-rolipram (63): 288 nM

1 3 2 2 3 1 1 2 1Q9M21 (R)-rolipram (430893): 231 nM

1 3 2 2 3 1 1 2 1 3 2 2 3 1 1 2 1 3 2 2 3 1 1 2 1OYN21 3K4S 1XOM12 Cilomilast (511115): 11 nM 9 (RO-201724, 18701): 631 nM rac-rolipram (63): 550 nm

1 3 2 2 3 1 1 1XOR12

1 3 2 2 3 1 2 1MKD67 zardaverine (313842): 160 nM

1 3 2 2 3 1 2 1 3 2 2 3 1 1 2 1 3 2 2 3 1 1 2 1XON12 1XOQ12 3G4L20 roflumilast (193240): 680 pM roflumilast (193240): 5.8 nM piclamilast (42126): 21 pM

1 4

2

1 3 2 2 3 2 1 4WCU102 30 (15, -): 3 nM

1 3 1 2 3 1 2 1 3IAD20 33 (D159153, -): 1 µM

2CHM113 53 (2, - ): 5.5 nM

1 1 4 2 3 1 1 1 3HC8114 54 (1, 564374): 2.9 nM

1 1 3 2 2 2 3 3TGE115 55 (1, 551052): 70 pM

1

1 2 4 2 3 1 1 1 1 2 3 2 3 1 3TGG115 3HDZ116 56 (17, 1916290): 80 pM 57 (1, -): 51 nM

1 1 3 4 2 2 1 2 2 2H4415 58 (Icarisid II): 1.7 µM

1 4 3 2 2

1 4 3 2 1 1

2

1 4 3 2 2 1

2 1 4 3 2 2

2

2

2

1

2 1 3 3 2 2

PDE5A 1UDU14 tadalafil (779): 1.2 nM

1XOZ12 tadalafil (779): 1.2 nM

Q O

1 2 4 2 1

2

2

4MD6117 59 (5R, -): 17 nM

3JWQ16 sildenafil (192): 25 nM (Ki)

3JWR16 IBMX (275084): 8.5 µM (Ki)

HN

O Q2/S

N O

1 4 4 2 2

O N

MB1/S

1

2

1 4 4 2 1

1

1 1

2 2 3

1 1

2 1

PDE7A 1ZKL79 IBMX (275084): -

1

1 2 1

1

4PM080 60 (24, -): 45 nM

1

1

1 1 1 2

PDE5A/6C

2

1

1

2

PDE8A 4Y2B81 61 (22, -): 4.4 nM

1 2 3 1 2 1

2 2

1 2 3 1 1 1

3G3N82 62 (15: 1236215): 510 nM

2

2 1 1 1 1

3ECN118 IBMX (275084): 700 µM

2 1

2 1 1

1

2

PDE9A 3N3Z71 IBMX (275084): >200 µM

2YY2 IBMX (275084): >200 µM

3QI471 IBMX (275084): 117 µM

2HD1119 IBMX (275084): >200 µM

1

1 2 1 1 3DYS8 GMP (283807): -

2

1 1 1 2 1 3DYL8 cGMP (395336): -

1

1

1 2 1 3DYN8 cGMP (395336): -

2 1

1

1 2 2

1

1

1

1

1 2 2

1 1

2 1

1 2 2

2 1

2 1


1 2 1 1 3DYQ8 cGMP (395336): -

1 2 2

2 1

3DY88 GMP (283807): -

2 1

1 2 2 2 1 1 3JSI44 63 (572934): 2.1 nM

1 1 1 3 2 2

1 1

2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3K3E120 3QI371 3K3H120 64 (R-BAY73-6691, 1513993): 64 (R-BAY73-6691, 1513993): 22 65 (S-BAY73-6691, 094374): 22 nM 88 nM nM

Page 30 of 123

3JSW44 66 (7, 572517): 66 nM

4E9043 67 (PF-04447943, 2179105): 8 nM

O Q

N N

HN

F

Cl

N

Q1/Q2/S F F

HC1/MB/MB1/S

1 1 2 2 2

1 1

2

1

3 2 2

1 1

2

1 1 3 2 2

1 1

2 1 1 2 2 2 1 1 1

2 1 1 2 2 2

PDE9A 4G2L43 68 (19, 2180070): 32 nM

4G2J43 69 (R-3h, 2180064): 986 nM

1 1

2

PDE10A 4QGE121 70 (3r, -): 0.60 nM

4GH645 71 (28, 2178115): 21 nM

3QPO122 72 (13, 1819128): 247 nM

x

1 0 2 2 1

1 1

2

1

2 2 2 2 1 1

2

1

1 2 2 1 1 1

2 1

1 2 2 1 1 1

2 1 1 3 2 1 3

2

3

PDE10A 3QPP122 73 (16, 1819131):12 nM

3QPN122 74 (24, 1738857): 17 nM

2O8H123 75 (1, 219121): 25 nM (Ki)

2OVY123 76 (29, 219445): 6 nM

2OVV123 77 (21, 410834): 12 nM (Ki) O

N

Q

N

O

HC2/MB1/S

N O

1 1 5 2 1 3 2 3 4DDL124 78 (43, 1956250): 4.9 nM

1 1 5 2 1 1 3 4DFF49 79 (7, 2017087): 35 nM (Ki)

1 1 2 2

1 1

1 1 1 2 1 1 2 3LXG46 83 (49, 1086110): 7.3 nM

1 1 1 2 2 1 1 1 3 1 4 2 1 1 3 4 2 2 3 3SNI47 3SN747 3SNL47 84 (74, 1916091): 11 nM 85 (96, 1916113): 0.7 nM 86 (115, 1916132): 0.7 nM

1 1

2

1 1

2

1 1 2 2 2

2 1 1 4FCB48 82 (48, -): 0.28 nM

3

1 1 4 2 2 3 4FCD48 87 (4, 2069321): 2.9 nM

2 3 1 1 2Y0J126 88 (42, 1641615): 12 nM

1 2 2 1 1 1 2 1 3 2 2 1 1 1 2 1 3 2 2 2WEY29 4MSC37 4MSE37 125 80 (ZT1595, -) 1.1 µM (Ki) 81 (ZT1598, -): 5.5 µM (Ki) papaverine (19224): 40 nM

2 3 1 1 2 1 1 1 2 3 1 1 2 1 1 2 2 3 1 1 2 3UI7127 3UUO128 3WI2129 89 (19, 1939914): 1 nM (Ki) 90 (1, 1939782): 11 nM (Ki) 91 (7g, 3091492): 92 nM

HC1/Q1 N N

Q

N

Cl

N

O Q2/S

1 1 1 2 2

HC1/MB/MB1/S O MB1/S

1 1

2

1 1

2 1 2

2

1 1

2 1


2 2 2

1

2 1 2 4 2 2

3

Page 31 of 123

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4WN1130 92 (42b, -): 5.1 nM

1 4 1 3 1 3WYK131 96 (1, -): 23 nM

3HQY31 93 (2, 562317): 11.5 nM

3

1

5 2 3 1 3WYL131 97 (20a, -): 1.4 nM Q

3HQZ31 94 (3, 560377): 0.42 nM

3

3HR131 1 (PF-02545920): 0.37 nM

2 5 2 3 3 3WYM131 98 (TAK-063, -): 0.3 nM

2 5 2 3 1 4AJM132 99 (3A6, -): -

3HQW31 95 (1, 1235237): 35 nM

3 1 1 5 2 3 3 4AEL133 100 (5,1957361): 12 nM

N HC1/Q1/MB/MB1 N

O O

N N

S/Q2 F F

1 1

HC2/Q2/S F

2 2 1 1 2 2 4HEU134 101 (7, 2206214): 0.097 nM

1 1 2 2 2 1 1 4HF4134 102 (16, -): 2.4 nM

2

1 1 2 2 2 1 1 1 2 1 1 5 2 2 3 1 1 4 2 1 1 3 4PHW135 4MVH136 4MUW 136 103 (Cpd 4, -): 0.1 nM 104 (26, 3086091): 4.5 nM 105 (1, 3086086): 9.7 nM

4 5 2 3 1 3 4TPM137 106 (8, 2180767): 2.4 nM

1 1 4 2 2 1 1 4TPP137 107 (47, -): 2.6 nM

3

1 1 5 2 2 1 1 3 1 1 4 2 2 1 1 3 1 1 5 2 2 1 1 3 4P0N135 4P1R138 3WS8139 108 (24, 3287905): 2.2 nM 109 (7, 3287662): 4.1 nM 110 (14c, 3288410): 210 nM

1 4 4 2 3 1 3 3WS9139 111 (14d, 3288411): 92 nM

1 1 4 2

1 2OUU68 cGMP (395336): -

1

2 2

1 1

1 5 2 1 1 3 4BBX140 112 (26, 3262043): 60 nM

1 1 4 2 2 2OUY68 cAMP (316966): -

3

1 3 1 2 3 2 2OUQ68 cGMP (395336): -

2

1

2

1

1

1

2 2

2 1

3 1 1 5 2 1 1 2OUR68 cAMP (316966): -

3 1 1 4 2 1 2OUN68 AMP (752): -

3

2 1 1 1 1 1 1 2 1 1 1 2 1 2 2 2 1 2 4LLJ37 4LKQ37 4MSA37 115 (ZT0449, 164921): 500 µM 113 (ZT0214, -): 380 µM 114 (ZT0017, -): 450 µM

2 1

1

1 1 1


2

2 1 1

2 2

2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4LM037 116 (ZT0448, 165372): 560 µM

1

2

1 4AJF37 121 (F03, 6804): -

4MSN37 117 (ZT0451, 167727): 94 µM

2 1 1 1 2 1 1 4AJD37 122 (F04, -): -

Page 32 of 123

4LM137 4LM437 4LLK37 118 (ZT0450, 164449): 590 µM 119 (ZT0902, 266540): 910 µM 120 (ZT0217, 395092): 990 µM

2 1 1

2

2 1 1 4MRW 37 123 (ZT0120, 1409793): 500 µM

2 3 4AJG37 124 (F07, -): -

1 1 1

2 1 2 4LM337 125 (ZT0464, -): 1200 µM

O Q

N N

1 1 1 2 2 2 1 2 1 2 1 1 2 1 1 2 3 2 1 1 2 1 2 1 3 2 2 2 4LM237 4LLP37 4MRZ37 4LLX37 4MS037 126 (ZT0462, -): 940 µM 127 (ZT0401, 3092391): 16 µM 128 (ZT0429, -): 300 µM 129 (ZT0434, -): 1300µM 130 (ZT0443, -): 410 µM

1 1 1 2 3

2 1

2 1

2

1

TcrPDEC

4MSH37

2R8Q75 IBMX (275084): 580 µM

3V945 132 (wyq16, -): 0.23 µM

2 1

1

2 1

2 2

1

2 1

1 2 2 1

2 1

2 2

2 1

2 1

2 *

LmjPDEB1

131 (ZT0143, 1413383): 210 µM

1 1

2

PDE10A

Unliganded PDE crystal structures

1 1 2

PDE1: 1TAZ13 PDE2: 1Z1L70, 3IBJ64, 3ITM64, 4HTZ88 PDE4B: 1F0J7, 4WZI57 PDE4C: 2QYM91 PDE4D: 3SL3107 PDE5: 1T9R13, 2H4015 PDE8: 3ECM118 PDE10: 2OUP68, 2OUS68, 2OUV68 TbrPDEB1: 4I1574 TcPDEC: 3V935

Q

Q1

Q2

HC

HC1

HC2

MB

MB1

MB2

S

# residues with contacts










*)

No ligand bound to the PDE catalytic site.


Page 33 of 123

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Figure 7. Frequency of interactions between pocket residues and ligands in 203 ligand bound PDE crystal structures in the PDEStrIAn database. A heat map of the IFP results for PDE crystal



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 123

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%. Schematic representations of all individual PDE-ligand complexes are presented in Table 1 and the complete set of PDE-ligand IFPs available as Supplementary Information.

The heat map in Figure 7 could potentially 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 with ligands bound (PDE3, PDE7, PDE8 and parasite PDEs). Still, this data can be applied to quickly identify interactions of interest when designing selectivity into PDE inhibitors.

3.4 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 1.4Å (Figure 8). From this study, one particular cluster (D) was shown to contain 71 water molecules from crystal structures of PDE1PDE7 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 (PDB: 1XOR).12 The structural water molecules in cluster D are bound by the residues DMB.22 and F/YHC1.1 stabilizing


Page 35 of 123

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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.

Figure 8. Water molecules involved in PDE ligand binding. (A) Crystallographic water molecules forming interactions with PDE ligands in 203 ligand-bound crystal structures in the PDEStrIAn database. 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 AI, with A-D and F-G forming interactions with ligands and residues, E forming interactions with ligands, metals and the protein and H-I forming interactions with just the ligand. The water molecules are shown over the pocket surface of PDE4D (PDB: 1OYN21). (B) The water molecules that interact with PDE inhibitors in crystal structures. Water molecules are colored according to the interactions they form; cyan form interactions with the ligand, magenta form interactions with ligands and protein residues, and red form interactions with ligands, metals and the protein residues. (C) Overlay of all PDE ligands and metal ions in all 220 phosphodiesterase (PDE) catalytic domain



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crystal structures present in the PDB (for an overview of the chemical structures of co-crystallized ligands, see Table 1).

The other clusters of water molecules are less general for PDEs. For example cluster B containing 12 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 E contains 19 PDE4 waters, and 4 PDE10, 1 PDE2 and 1 PDE9 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 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.

4. PDE LIGAND SCAFFOLD ANALYSIS. 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 (Table 1, Figure 9). 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 9 shows that 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. Another common scaffold type found particularly in PDE4


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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 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 scaffolds in Figure 9 were 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 multiple scaffolds being identified in a single ligand crystalized with a PDE. 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 (pIC50 ≥5) were gathered into a database for analysis. This PDE specific Markush library containing a scaffold and all R-group variations for a vector can potentially be used in combination with docking to efficiently probe a particular sub-pocket with relevant chemical diversity.



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Figure 9. Scaffold analysis of the ligands in PDE crystal structures. The core scaffolds from each ligand crystalized with a PDE are oriented as presented in Figures 6C, 8C, 10, 12, 14, 16-18, 20, 21 and Table 1. The points of attachment around the scaffold of the crystalized PDE ligands are named according to the vector of the bond broken using the R group reference scheme shown on the bottom right. 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. Scaffolds were defined such that only one scaffold would be identified in each ligand. The scaffolds were also screened against active PDE inhibitors published in the ChEMBL database using ChemAxon’s R-group decomposition tool (https://www.chemaxon.com) and the numbers 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 Rgroups 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). 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.

5. PDE SUBPOCKET INTERACTIONS 5.1 PDE Subpocket Based Ligand Scaffold Annotation. Building from the systematic analysis of the core scaffold of PDE ligands (section 4) an analysis was made of the moieties of ligands that



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interact with regions of the substrate binding pocket in PDE crystal structures (Figures 10-15). In this case the interactions identified by IFP analysis were the starting point for identification of fragments of the ligands involved in interactions.

5.2 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 primarily hydrophobic (91% of ligands that interact with Q1 only form hydrophobic interactions). However, two exceptions are shown in Figures 10A-B: PDE3 inhibitor 8 (PDB: 1SO278) and PDE5 inhibitor 53 (PDB: 2CHM113). The Q1 pocket shows significant water molecule occupancy in PDE crystal structures (Figure 8), indicating the potential to accommodate polar functional groups in this region of the binding site. 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 PDE5,141 can be attributed to the hydrogen bond interaction that the dihydropyridazinone carbonyl oxygen can form with HQ1.27, as illustrated for the PDE3 inhibitor 8 (IC50 = 0.27 nM) in Figure 10A (PDB: 1SO2).78 The corresponding YQ1.27 residue in PDE4 forms a 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 53 (IC50 = 5.5 nM) shown in Figure 10B (PDB: 2CHM), forms a hydrogen bond with QQ1.27 in a PDE5 crystal structure.113


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Figure 10. Examples of compounds that interact with the Q1 and Q2 pockets. The binding modes are shown of: (A) 8 to PDE3B (PDB: 1SO278) (B) 53 to PDE5A (PDB: 2CHM,113 superposed over



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the binding site surface of PDB: 1UDT14), (C) 74 to PDE10A (PDB: 3QPN,122 superposed over the binding site surface of PDB: 3HR131), and (D) 1 to PDE10A (PDB: 3HR131). (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.

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.142 The key Q2 residues involved in ligand interactions are located at positions Q2.33, Q2.46 and Q2.49, that form hydrophobic interactions in 55%, 25% and 66% of structures respectively. The Q2 pocket plays a particularly important role in inhibitor design for PDE10, where 36% of ligands address the Q2 pocket, forming both π-π interactions and hydrogen bonds with Y683Q2.33. The selective PDE10 inhibitor 74 (IC50 = 17 nM) in Figure 10C (PDB: 3QPN) was discovered using molecular docking of a virtual combinatorial library with the specific aim of improving selectivity by addressing the Q2 pocket.122 Interestingly, while 86% of ligands in PDE crystal structures form a hydrogen bond with QQ.50, 25% of PDE10 inhibitors do not form this hydrogen bond, including 1 shown in Figure 10D (PDB: 3HR131). 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 1 (IC50 = 0.37nM). Hydrogen bonds to the Q2 pocket are rare except in PDE10 structures, where 39% of inhibitors form hydrogen bonds with YQ2.33 further stabilizing occupation of the Q2 pocket.


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Figure 11 shows that the fragments that target Q1 are small aliphatic or aromatic moieties in most cases. Methoxy fragments, particularly from catechol scaffolds, are the most common groups making up 23% of the fragments that interact with Q1. In PDE4 π-π interactions are seen between YQ1.27 and pyridine, thiophene or benzene rings. Besides the carbonyl in 8 and 53 (Figure 10), alcohol can also act as a hydrogen bond acceptor in Q1, but only one inhibitor ((+)-EHNA, PDE2) forms a hydrogen bond donating interaction with Q1 (Table 1, Figure 11). Q2 is larger and able to accommodate a more diverse set of chemical moieties, ranging from alkoxyl and alkyl groups of varying size to various (hetero)aromatic moieties (Figure 11). The formation of specific interactions occurs primarily with fragments targeting Q2 in PDE10 (26 PDE inhibitors: 1, 72 – 74, 80 – 82, 91 – 95, 99 – 111, 117, see Table 1), with a range of ring systems forming both hydrogen bonds and ππ interactions. A few fragments targeting PDE5 (59, Table 1) and PDE9 (cGMP, GMP, 70, Table 1) also form specific interactions with Q2 (Figure 11).



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Figure 11. Substructures of crystalized PDE ligands interacting with the Q1 and Q2 regions of the PDE binding pocket. Fragments are arranged with the PDE subtype and number of instances a fragment occurs in a crystal structure of that subtype listed below. Fragments that form interactions other than hydrophobic interactions are positioned above a figure indicating the interactions formed


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by the fragment with the PDE subtypes listed below. The interactions with the pocket region are color coded to indicate the presence of; a hydrophobic interaction (blue); a face-to-face π-π interaction (green); an edge-to-face π-π interaction (teal); a hydrogen bond acceptor (red); and a hydrogen bond donor (purple); no interaction (white).

5.3 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 (96%, Figure 7, Figure 13). Although most ligands only form hydrophobic interactions with HC1, several also form hydrogen bonds with the residue at position HC1.25, for example inhibitor 23 (IC50 = 50 nM) shown in Figure 12A (PDB: 3HMV) forms a hydrogen bond to N567HC1.25 of PDE4B via an amide group.99 The binding of 23 also shows a unique displacement of QQ.50 away from the pocket, despite its potent inhibition of PDE4B. The chloro substituent in the PDE10A inhibitor 86 (IC50 = 0.7 nM), shown in Figure 12B (PDB: 3SNL), slightly shifts the ligand within the binding pocket compared to analogue 85 (IC50 = 0.7 nM; PDB: 3SN7), resulting in the formation of a hydrogen bond interaction between the pyridine nitrogen of 86 with Y514HC1.01.47



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Figure 12. Examples of compounds that interact with the HC1, HC2 and S pockets. The binding modes are shown of; (A) 23 bound to PDE4B (PDB: 3HMV,99 superposed over the PDE4 binding


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site surface of PDB: 1OYN21), (B) 86 bound to PDE10A (PDB: 3SNL,47 superposed over the binding site surface of PDB: 3HR131), (C) 76 to PDE10A (PDB: 2OVY,123 superposed over the binding site surface of PDB: 3HR131), and (D) 68 to PDE9A (PDB: 4G2L,43 superposed over the binding site surface of PDB: 3K3E120) 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 ligands are shown.

By contrast, the HC2 region interacts with just 23% of ligands, and exclusively through hydrophobic interactions. In Figure 12C (PDB: 2OVY) the quinoxaline moiety of the potent PDE10A inhibitor 76 (IC50 = 6 nM) targets HC2, forming hydrophobic interactions with G718HC2.51, A722HC2.54 and V723HC2.55.123 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 PDE10 over PDE3. The S pocket is primarily addressed by interactions between the core scaffolds of ligands and the residue at position F/YS.35 (hydrophobic: 99%, π-π interactions: 59%, hydrogen bonds: 5%), a phenylalanine in all PDEs except PDE9 where a tyrosine is found at this position. The presence of tyrosine at position YS.35 in the S pocket has been used in the structure-based design of PDE9A inhibitors. In Figure 12D (PDB: 4G2L), the protonated nitrogen atom in 68 (IC50 = 32 nM) forms a hydrogen bond with Y484S.35.43 The compound forms part of a structure-based effort to improve brain penetration of the closely related Alzheimer drug candidate 133, which completed a phase II trial in 2010. The water mediated hydrogen bond between 133 and Y484S.35 was replaced by a direct hydrogen bond in the binding of 68 reducing potency and selectivity, but improving brain penetration. Figure 13 shows that the fragments targeting HC1 include a large number of moieties (43) that form specific interactions (hydrogen bonds: 15; π-π interactions: 33). Due to the proximity of HC1



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to the hydrophobic clamp, many interactions are formed by part of core scaffold of the ligand, or small polar substituents extending from the scaffold. This holds true for fragments forming both hydrophobic and polar interactions. With the most conserved PDE water cluster found on the surface of HC1 (cluster D, Figure 8A), many fragments are clearly interacting with HC1 indirectly. HC2 extends further from the PDE ligand binding pocket and is largely solvent exposed, perhaps this explains the lack of polar interactions with HC2 residues. Although few fragments reach HC2, those that do include aliphatic, polar and aromatic moieties.


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Figure 13. Substructures of crystalized PDE ligands interacting with the HC1 and HC2 regions of the PDE binding pocket. Fragments are arranged and labeled as described below Figure 11.



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5.4 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 14A (PDB: 1TB7) showing AMP bound to PDE4D and Figure 14B (PDB: 1T9S) showing GMP bound to PDE5A.13 The cyclic nucleotides adopt very similar binding modes with each making ionic bonds to both the metal ions (most often Zn2+ and Mg2+) that are coordinated by conserved residues HMB.03, HMB.04, DMB.05, and DMB.22. 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.69


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Figure 14. Examples of compounds which interact with the MB, MB1 and MB2 pockets. The binding modes are shown of: (A) AMP to PDE4D (PDB: 1TB7,13 superposed over the binding site



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surface of PDB: 1OYN21), (B) GMP to PDE5A (PDB: 1T9S,13 superposed over the binding site surface of 1UDT14), (C) 19 to PDE4B (PDB: 3O0J97), and (D) 32 to PDE4D (PDB: 3V9B,104 superposed over the binding site surface of PDB: 1OYN21). (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 ligands are shown.

Just two of the crystalized PDE inhibitors form tight interactions with the catalytic metal ions in PDEs, PDE4 inhibitors zardaverine (IC50 = 0.39 µM, PDB: 1XOR12) and 19 (IC50 = 0.24 µM, PDB: 3O0J97), shown in Figure 14C. 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 19 that binds to the metal ions and H406MB.02 is unique amongst crystalized PDE inhibitors, as is the catechol like dicyanophenoxy ring that interacts with Q615Q.50. Few ligands form specific interactions with the MB1 and MB2 pockets, one of those is 32 (IC50 = 0.5 nM) bound to PDE10A (PDB: 3V9B) shown in Figure 14D that forms a hydrogen bond with M575MB1.17.104 In Figure 12C the PDE10A inhibitor 76 is shown to form π-π interactions with F629MB1.20 (PDB: 2OVY), an interaction shared by 75 and 77.123 Figure 15 shows that the metal binding regions of the pocket (MB, MB1, and MB2) are targeted by a variety of functional groups. Interactions between ligands and HMB.02 are common (hydrophobic: 56%, π-π interactions: 16%, hydrogen bonds: 9%) and make up most of the interactions with the MB region. Due to the proximity of the metal ions, polar interactions with MB residues may include groups interacting directly or indirectly with the metal ions. The interactions with MB1 occur primarily with aliphatic residue I/L/MMB1.17 as hydrophobic interactions (80% of


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ligands). Positioned at the gateway to the PDE binding site, the fragments interacting with I/L/MMB1.17 result primarily from ligands extending out towards the solvent. The MB2 region extends from the far end of the MB region, towards the solvent and few ligands extend far enough to reach it. One exception is the PDE2A inhibitor 2 (4D0839) that forms a hydrogen bond between a morpholine group and N704MB.09.




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Figure 15. Substructures of crystalized PDE ligands interacting with the MB, MB1 and MB2 regions of the PDE binding pocket. Fragments are arranged and labeled as described below Figure 11.

5.5 Interactions with H-loop, UCR2 domain, C-terminus. Ligands can be designed to not only target the PDE catalytic site as roflumilast does (PDE4D, PDB: 1XOQ12, Figure 16A), 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 (58, Figure 16B, PDB: 2H4415) crystal structures. Moreover in PDE4 structures the C-terminus can fold over to close the pocket as shown for atizoram bound to PDE4D in Figure 16C (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 interactions between PDE4 inhibitors and the UCR2 domain. This interaction has been utilized to modulate inhibition and achieve selectivity for PDE4D over PDE4B (3IAD, 3G4G, 3G45, 4X0F and 4WZI were crystalized from constructs containing portions of UCR2).20,

143

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 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



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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.20 One of these inhibitors, 33 (PDE4D7 IC50 < 1 nM), is shown in Figure 16D (PDE4D, PDB: 3IAD).


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Figure 16. Interactions between ligands bound to the PDE catalytic site and regions that are able to fold over the pocket. (A) In the structure of roflumilast bound to the catalytic domain of PDE4D (PDB: 1XOQ12, superposed over the binding site surface of PDB: 1OYN21) the catalytic pocket is open. (B) In the structure of inhibitor 58 bound to PDE5A (PDB: 2H44,15 superposed over a the binding site surface of PDB: 1UDT14) the H-loop folds over the pocket entrance enclosing the ligand. (C) The structure of atizoram bound to the catalytic domain of PDE4B shows closing of the pocket by the C-terminal residues (PDB: 3KKT, superposed over the binding site surface of 1OYN). (D) In the case of 33 bound to a PDE4D construct which includes the UCR2 regulatory domain, the pocket is closed off by a helix of UCR2 (PDB: 3IAD,20 superposed over the binding site surface of PDB: 1OYN21). (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 ligands are shown.

6. PDE STRUCTURE-BASED MEDICINAL CHEMISTRY TOOLBOX. The superposition of all PDE crystal structures combined with the annotation of the binding pockets and water clusters (Figures 3, 4 and 8), and the generated PDE-ligand interaction and ligand scaffold analysis data (Figures 6, 7 and 9-16), make the PDEStrIAn database especially suited for ligand design and structure-based optimization. The consistent manner of the structural PDE-ligand interaction data set creation allows for an easy comparison of multiple structures, co-crystallized inhibitors and their interactions. The fragmentation of crystalized PDE ligands into scaffolds and Rgroups taking the binding conformation into account (Figures 9, 11, 13 and 15) and applying this to generate libraries of orientation specific scaffolds and R-groups provides a PDE structure-based medicinal chemistry toolbox for addressing different pocket regions and scaffold hopping. Together


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these tools can support future structure-based PDE ligand discovery and design, including structurebased virtual screening,74 fragment growing,36, 37 and modulation of PDE activity and selectivity.20 Section 6 provides complementary examples to the structure-based ligand interaction analyses and ligand design examples to target different PDE subpockets described in section 5, including: (6.1) an example of fragment-based ligand discovery and design guided by PDE4D crystal structures; (6.2) the investigation and utilization of halogen bonding in the design of PDE5 ligands; the combination of IFP, chemical similarity, and SAR analyses for (6.3) the identification of dissimilar ligand scaffolds that can adopt similar binding modes in PDE4A and PDE10, and (6.4) the structure-based design of high affinity ligands that target subpockets of PDE4B simultaneously.

6.1 PDE Structure-Guided Fragment-Based Ligand Discovery and Design. An example of fragment growing in PDE4D is shown in Figures 17A-D. An initial fragment screening yielded 34 as an initial hit for further optimization (IC50 = 82 µM).36 Structure-guided fragment growing starting from 34 (PDB: 1Y2B; Figure 17A) and guided with six further PDE4B and PDE4D cocrystal structures enabled the design of a series potent of PDE4 inhibitors.36 The addition of a phenyl group that forms a π-π interaction with H462MB.02 brought a 400-fold increase in potency (35, IC50 = 0.27 µM; PDB: 1Y2C; Figure 17B). The addition of a para-methoxy group resulted in a binding mode switch of 36 out of the pocket (PDB: 1Y2D, Figure 17C) and a 10-fold reduction in potency (IC50 = 2.0 µM). Placing a nitro group in the meta position instead resulted in inhibitor 37 coordinating with the magnesium ion (PDB: 1Y2K, Figure 17D) and a 4000-fold increase in potency over the initial screening hit in just two rounds of synthesis (IC50 = 0.021 µM).36




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Figure 17. The growing of (A) the PDE4D fragment screening hit 34 (PDB: 1Y2B), with (B) an Nphenyl (35, PDB: 1Y2C), (C) an additional para-methoxy group (36, PDB: 1Y2D) and (D) replacing this with a meta-nitro group (37, PDB: 1Y2K).36 Each structure is shown superposed over the PDE4 binding site surface of 1OYN. (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 ligands are shown.

6.2 Halogen bonding in structure-based PDE ligand design. A series of halogenated pyrimidinone PDE5A inhibitor analogues were designed, including fluorinated 46 (IC50

=

91 nM,

PDB: 3SHY; Figure 18B); chlorinated 47 (IC50 = 36 nM; PDB: 3SHZ), and the brominated 48 (IC50 = 13 nM, PDB: 3SIE; Figure 18C), that target Y612H1.01 in PDE5A via a putative halogen bond.42 Note that conformational differences seen at the piperazine ring side of 48 in Figure 18C, result from a reorientation of the H-loop towards the ligand in the absence of metal ions under the particular crystallization conditions used. 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 computer-aided design of PDE inhibitors. Moreover, recently solved higher resolution PDE5A structures bound to non-substituted 45 (IC50 = 52 nM, PDB: 4OEX; Figure 18A) and iodinated 49 (IC50 = 7 nM, PDB: 4OEW) pyrimidinone analogues111 indicated that this halogen bond interaction with Y612H1.01 is in fact stabilized by a conserved water molecule (i.e. water cluster C, Figure 8). Interestingly, ITC studies revealed that replacing the hydrogen in 45 with a fluorine (46) results in entropically driven binding, while replacing it with a bromine (48) results in purely enthalpically driven binding (Figure 18D). Quantum mechanical calculations were also performed on the series giving halogen bond strengths of -1.57 (47, Cl), -3.09



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(48, Br) and -5.59 (49, I) kJ/mol providing further evidence of the contribution of the halogen bond to binding.111


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Figure 18. Halogenated monocyclic pyrimidones form halogen bonds with Y612HC1.01 in PDE5A (A) non-substituted pyrimidone 45 (PDB: 4OEX)111 (B) fluorinated pyrimidone 46 (PDB: 3SHY)42;



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(C) brominated pyrimidine 48 (PDB: 3SIE)42; (D) the thermodynamic signatures of 45, 46 and 48 showing significant changes to the thermodynamic profile of binding. The changes to the free energy on binding of the inhibitors are shown in kJ/mol (∆G (Gibbs free energy) = ∆H (enthalpy) T∆S (product of temperature and entropy)).42 (E) The IFP bit strings of the proteins and ligands shown in A-C. Only those residues which form at least one interaction with one of the four ligands are shown.

6.3 Chemically dissimilar PDE ligands that share similar structural PDE-ligand interaction fingerprints. To assess the relationships between the binding mode similarity and chemical similarity of PDE ligands we performed an all-against-all comparison of 203 crystallized PDE-ligand complexes using the protein-ligand interaction fingerprint Tanomito similarity and ECFP-4144 fingerprint Tanimito similarity of the co-crystallized ligand (Figure 19). We determined that 3% of all co-crystallized PDE ligands are chemically similar (ECFP-4 Tanimoto similarity > 0.4145), illustrating the high chemical diversity of combinations of different central scaffolds (targeting the hydrophobic clamp and/or conserved QQ.50 residue) and R-groups (targeting the other different PDE subpockets) presented in Figures 9, 11, 13 and 15. Chemical diversity for ligands bound to the same PDE isoform is however variable for PDE10 (4% similar pairs), PDE4 (8% similar pairs) PDE9 (22% similar pairs), and PDE5 (60% similar pairs) ligand sets (Figure 19B). Interestingly, 19% and 4% of the chemically dissimilar pairs of co-crystallized PDE ligands adopt similar binding modes based on previously defined IFP Tanimoto similarity cutoffs used for binding mode prediction (≥0.686,

146

)

and structure-based virtual screening studies (≥0.7574, 147), respectively. The percentage of dissimilar ligands with similar interaction fingerprints is even higher within PDE4 (55%), PDE9 (24%), and


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PDE10 (24%) ligand sets (Figure 19B), and examples of such ligand pairs are presented in Figure 20.

Figure 19. (A) Relationships between ligand similarity (ECFP-4144) and protein-ligand interaction pattern (IFP86) similarity for all 203 crystallized PDE-ligand complexes in the PDEStrIAn database. Data are colored according to plot density to identify hotspots. Ligand (ECFP-4 Tanimoto similarity > 0.4145) and interaction fingerprint (IFP Tanimoto similarity > 0.686, 146) similarity cutoffs, indicated by dashed black lines, are used to define four different classes containing ligand pairs with: I) similar IFP and dissimilar ECFP-4 fingerprint, II) similar IFP and similar ECFP-4 fingerprint, III) dissimilar IFP and dissimilar ECFP-4 fingerprint, IV) dissimilar IFP and similar ECFP-4 fingerprint; (B) Distribution of ligand pairs for the different ECFP-4 and IFP similarity quadrants defined in panel A are shown for all PDE subtypes. Distributions for those PDE families with over 10 PDEligand crystal structures are shown separately for pairs within each of the families, PDE4 (5625), PDE5 (961), PDE9 (361), and PDE10 (4900).



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Figure 20 shows that for example the equipotent compounds 9 (PDB: 3I8V) and 10 (PDB: 2QYK)91 have similar structural PDE interaction fingerprints (IFP-Tc: 0.82) and bind with Q, Q1, Q2, HC, HC1, MB1, and S pockets in human PDE4A while sharing almost no structural similarity (ECFP-4: 0.06). Compounds 9 and 10: i) accept an hydrogen bond from the sidechain amide group of QQ.50 in the Q pocket and target Q1, HC, and HC1 pockets with a naphthyridine (9) vs. di-alkoxybenzene (10) group, ii) interact with Q2 and S pockets via a butane alkyl group (9) and nitrobenzene ring (10), and iii) and target the MB1 pocket with an imidazolidinone (9) and nitrobenzene group (10). Figures 20C-D show how compounds 94 (PDB: 3HQZ)31 and 101 (PDB: 4HEU)134 share similar PDE interaction patterns with human and rat PDE10, respectively (IFP-Tc: 0.72), by i) targeting HC and HC1 pockets with a phenylpyrazole (94) vs. a phenoxypyridine (101) moiety, ii) accepting a hydrogen bond from the sidechain hydroxyl group of YQ2.33 in the Q2 pocket with a quinoline (94) vs. benzimidazole (101) group, and iii) and binding the Q1 pocket with a pyridine (94) and piperidine-methanol (101) group. Interestingly, structure-activity relationship studies around 94 and 101 show similar trends, but also provide complementary information regarding the optimal ways to target the Q1 pocket.31, 134 Insertion of an carbon atom linker between the pyridine and pyrazole ring systems of 94 results in compound 93 (PDB: 3HQY, Table 1) which has a flipped binding mode of the pyridine into the MB1 pocket and a lower PDE4A affinity (IC50 = 11.5 nM), compared to the water-mediated hydrogen bond stabilized favorable fit of 94 (IC50 = 0.42 nM) in the Q1 pocket (Figure 20C).31 The methanol group of compound 101 forms a direct hydrogen bond with the backbone of TQ1.27 and binds PDE10 with high affinity (IC50 = 0.097 nM). Interestingly, the ethanol piperidine group of the chemically similar PDE10 inhibitor 102 (PDB: 4HF4, Table 1)134 adopts a flipped binding mode in the MB1 pocket compared to the methanol piperidine group of 101 (Figure


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20D), and has a 12 fold higher IC50 compared to its smaller methanol analogue.134 The similar binding mode flips of 93 versus 94 and 102 versus 103 suggest that steric compatibility with the Q1 pocket is an important determinant for PDE10 binding affinity and illustrate the potential of integrated structural protein-ligand interaction and SAR analysis to guide PDE ligand design.



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Figure 20. (A-B) Dissimilar ligands 9 (PDB: 3IV8), and 10 (PDB: 2QYK)91 adopt similar binding modes in human PDE4A crystal structures; (C-D) Dissimilar ligands 94 (PDB: 3HQZ)31, and 101


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(PDB: 4HEU)134 adopt similar binding modes in rat and human PDE10 crystal structures, respectively. (E) The IFP bit strings of the proteins and ligands shown in C-D.

6.4 Structure-based design of ligands that target different PDE subpockets simultaneously. Figure 21 illustrates how the structural alignment of PDE-ligand complexes and IFP analysis allows the rational incorporation and combination of fragments that address specific PDE pockets simultaneously. Compound 24 (PDB: 3D3P)100 and 25 (PDB: 3O56)101 share pyrazolopyridine scaffolds and adopt similar binding modes in PDE4B targeting Q, Q1, HC1 and MB1. The IFPs of 24 and 25 are distinguished by compound specific interactions in HC2 pocket (phenyl group of 24, Figure 21A) and MB2 (pyrrolidine of 25, Figure 21B). Compound 24 (IC50 = 3.2 nM) and 25 (IC50 = 0.8 nM) inhibit PDE4B with high potency which is further improved by combining the phenyl substituent of 24 and pyrrolidine substituent of 25 in inhibitor 26 (PDB: 3O57)101, which targets MB2 and HC2 simultaneously with 40 and 10 fold improvements in potency (IC50 = 0.08 nM) compared to 25 and 26 respectively (Figure 21C). This structure-activity-relationship suggests that targeting MB2 in combination with the HC2 pocket can be an attractive strategy to design PDE inhibitors with improved affinity. PDE3 inhibitor 8 (PDB: 1SO2, IC50 = 0.27 nM, Figure 10A)78 and PDE4B inhibitor 32 (PDB: 3V9B, IC50 = 0.5 nM, Figure 14D)104 are examples of other high affinity PDE inhibitors that also target HC2 and MB2 subpockets simultaneously.



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Figure 21. Merging of PDE4B inhibitors 24 (PDB: 3D3P)100 (A) and 25 (PDB: 3O56)101 (B) lead to high affinity inhibitor 26 (PDB: 3O57)101 (C). Chemical fingerprint (ECFP-4144) and interaction fingerprint (IFP86) based Tanimoto similarities between 24, 25, and 26 are determined and reported


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IC50 values of 24, 25, and 26 for PDE4B are reported between brackets. (D) The IFP bit strings of the proteins and ligands shown in A-C.

The integrated analysis of PDE ligand scaffolds, substituents, and structural interaction patterns (Figures 6-21, Table 1) enable systematic, structure-based design of ligands that target specific (combinations of) PDE binding subpockets, including PDE isoform specific interaction sites (Figure 7), for example by fragment growing (Figure 17), linking, or merging (Figure 21) design strategies.147,

148

IFPs can be used to predict protein-ligand binding modes based on molecular

docking and molecular dynamics simulations, and identify new ligands147, 149-151, even in the absence of a crystallized protein-ligand complex, as for example demonstrated in structure-based virtual screening studies against the apo TbrPDEB1 crystal structure (PDB: 4I15).74 The combination of such computational methods and the presented PDE structure-based medicinal chemistry toolbox can be used to extend PDE-ligand structure information and provide new starting points for PDE ligand design.

7. CONCLUSIONS AND PERSPECTIVES The presented comparative analysis of crystal structures across the PDE superfamily provides a comprehensive structure-based PDE-ligand interaction map highlighting conserved and PDE subtype specific interactions in the PDEStrIAn database. The conserved structural architecture around the PDE substrate binding pocket allows the definition of a structure-based nomenclature for PDE binding site residues that enables systematic cross family comparisons of PDE structures and PDE-ligand interactions. The current review demonstrates that the structure-based annotation of



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PDE subpockets and binding site water molecules combined with PDE-ligand interaction fingerprint analyses facilitates the structural classification of PDE-ligand binding modes and detailed comparison of PDE interaction features. We have integrated the comparative structural chemogenomics analysis with an extensive chemical scaffold analysis of crystalized (and not yet crystalized) PDE ligands. This provides a versatile chemical toolbox to design new small molecule ligands that target specific (combinations of) PDE binding sites. The potential of structure-based PDE ligand design has been illustrated by various examples of structure-based ligand optimization customized to the chemical and structural properties of different PDE isoforms, including the application of fragment-based ligand design, halogen bonding, and the targeting of regions that are able to fold over the PDE pocket. The presented PDE-customized chemical toolbox can in principle be implemented in various (computer-aided) PDE ligand design approaches, ranging from Markush R-group substitution along well-defined vectors in the intended PDE binding site to scaffold hopping approaches. Combination of the high resolution structural PDE-ligand interaction data and ligand scaffolds annotated by binding site offers new opportunities for the development of novel PDE inhibitors with improved selectivity and potency profiles.

SUPPORTING INFORMATION The PDE-ligand interaction fingerprints are included in the Supplementary Material file that is available free of charge via the Internet at http://pubs.acs.org. The structure-based PDE-ligand interaction data described, including the processed PDB collection, PDE binding pocket sequence alignment, structurally aligned binding site mol2 files, and the PDE-ligand IFPs, are available via http://pdestrian.vu-compmedchem.nl and from the ZENODO repository (DOI: 10.5281/zenodo.45774).


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ACKNOWLEDGMENTS This work was supported by TI Pharma grant T4-302 and the European Commission 7th Framework Programme FP7-HEALTH-2013-INNOVATION-1, PDE4NPD (No. 602666). Moira Rachmann is acknowledged for assistance in the PDE-ligand structure and interaction analysis work.

AUTHOR INFORMATION Corresponding Author * C. de Graaf, phone: +31-20598-7553; e-mail: [email protected].

ABBREVIATIONS AC, adenylyl cyclase; GC, guanylyl cyclase; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CaM, CaM-binding domain; IFP, PDE-ligand Interaction FingerPrint; REC, signal regulatory domain; TD, targeting domain; TM domain, transmembrane domain; UCRs, upstream conserved regions; Q, glutamine region; HC, hydrophobic clamp region; MB, metal binding region; S, solvent filled region.

BIOGRAPHIES Chimed Jansen received a B.A. from the University of Sunderland and a B.Sc. in Pharmaceutical Sciences from the VU University Amsterdam where his M.Sc. in Drug Design and Synthesis was awarded cum laude and his thesis on Thermodynamics in Medicinal Chemistry received the Taeke Bultsma Award. He obtained his PhD from the VU University Amsterdam for medicinal chemistry research on Trypanosoma brucei phosphodiesterase B1 as a drug target for Human African



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Trypanosomiasis. In 2014 Dr. Jansen joined the Drug Discovery Unit at the University of Dundee as a computational chemist where he forms part of a team headed by Prof. Dr. Ian Gilbert which targets neglected diseases and focuses on the identification and optimization of antimalarial leads.

Albert J. Kooistra obtained his B.Sc. degree in Telematics at the University of Twente, his M.Sc. degree (cum laude) in Bioinformatics, and his Ph.D. in computational Medicinal Chemistry at the Vrije Universiteit Amsterdam and worked for several years as a software designer and developer. He carried out his PhD research from 2010-2014 in the Division of Medicinal Chemistry on the development of virtual screening tools and protocols for mainly GPCRs, as well as performing chemogenomic and structural analyses covering several protein families. As a postdoctoral researcher in the same division, he is currently developing novel in silico approaches for knowledgebased steering of medicinal chemistry efforts. Dr. Kooistra is also one of the founders of the structural kinase database KLIFS (http://klifs.vu-compmedchem.nl).

Georgi. K. Kanev obtained his M.Sc. degree in Bioinformatics at the Vrije Universiteit Amsterdam. Since 2015 he is carrying out his PhD under the supervision of Prof. Dr. Iwan de Esch at Division of Medicinal Chemistry, Vrije Universiteit Amsterdam and Prof. Dr. Tom Würdinger at the Cancer Center Amsterdam (CCA). His research focuses on integrating phenotypic and chemogenomics data, machine learning, database design and algorithms development with the aim to develop a reliable synergy prediction platform.

Rob Leurs performed his PhD research on G-protein Coupled Receptors (GPCRs) at Vrije Universiteit Amsterdam. After a postdoctoral fellowship at INSERM in Paris, he was awarded with


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a Royal Netherlands Academy of Arts and Sciences fellowship. He was awarded the Galenus Research Prize (1997), the Organon Award for Pharmacology (2000), a Pfizer Academic Award (2001), and a STW/NWO Pionieer grant (2001). Rob Leurs is full professor and head of the Division of Medicinal Chemistry at Vrije Universiteit Amsterdam, and co-founder of Griffin Discoveries, a company that valorizes the GPCR expertise and is currently involved in the discovery and development of histamine receptor ligands. Prof. Dr. Leurs is project leader of the EU FP7funded project PDE4NPD, Phosphodiesterase inhibitors for the treatment of neglected parasitic diseases (www.PDE4NPD.eu).

Iwan J.P. de Esch performed his PhD research at the Department of Pharmacochemistry at Vrije Universiteit Amsterdam. He became a research associate in the drug design group at the University of Cambridge in 1998 and co-founded De Novo Pharmaceuticals in 2000. In 2003 Iwan de Esch returned to academia and is now full professor Biocomputational Chemistry for Drug Innovation at Vrije Universiteit Amsterdam. Prof. Dr. De Esch is co-founder of IOTA Pharmaceuticals and also co-founder of Griffin Pharmaceuticals, thereby contributing to the valorization of the fragmentbased drug discovery and the GPCR research line of the academic group. In 2011, he was awarded the Galenus Research Price for his work on fragment-based drug discovery.

Chris de Graaf performed his PhD research at Vrije Universiteit Amsterdam on computational ligand binding mode and affinity predictions in cytochrome P450 enzymes (2002-2006). As postdoctoral fellow in the Structural Chemogenomics group (Dr. Rognan) at Université de Strasbourg he worked in collaboration with AstraZeneca Pharmaceuticals on the development and application of novel G Protein-Coupled Receptor modeling techniques (2006-2008). In 2009 Chris de Graaf



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obtained an NWO Veni grant to develop a research line in the computational prediction of structural protein-ligand interactions and was appointed assistant professor in the Division Medicinal Chemistry at Vrije Universiteit Amsterdam. In this interdisciplinary research environment Dr. De Graaf is developing chemo/bioinformatics methods to complement synthetic medicinal chemistry and molecular pharmacology programs, including e-science technologies for structure-based polypharmacology prediction (https://www.esciencecenter.nl/project/3d-e-chem).

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Methoxybenzyl)Aminophthalazines:

Synthesis

and

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Inhibitory

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Yang, S.-W.; Smotryski, J.; McElroy, W. T.; Tan, Z.; Ho, G.; Tulshian, D.; Greenlee, W. J.; Guzzi, M.; Zhang, X.; Mullins, D.; Xiao, L.; Hruza, A.; Chan, T.-M.; Rindgen, D.; Bleickardt, C.; Hodgson, R. Discovery of Orally Active Pyrazoloquinolines as Potent PDE10



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Table Of Contents (TOC) Graphic



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Figure 1. The binding modes of: (A) the substrate cAMP (PDB: 2PW333), and (B) the product AMP (PDB: 1PTW10) bound to PDE4D. The key interacting residues are shown colored by the pocket region, H160 (H160MB.02), N321 (N321HC1.25), I336 (I336HC.32), F340 (F340S.35), Q369 (Q369Q.50) and F372 (F372HC.52), and named according to the nomenclature presented in Figure 4. (C) Molecular structures and indications of the five marketed PDE inhibitors crystalized with PDEs: sildenafil, vardenafil, tadalafil, roflumilast, papaverine two PDE inhibitors which failed to reach the market: rolipram and 1 and the PDE substrates cAMP and cGMP with arrows indicating the bond broken during hydrolysis by PDEs to form AMP and GMP. 101x60mm (300 x 300 DPI)


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Figure 2. (A) The structures and domains of the 11 human PDE families are shown along with the 3 parasite PDEs that have been crystalized. The amino-terminal domains may regulate the activity of the conserved catalytic domain, play a role in the localization of PDEs or the interaction with protein partners. The amino-terminal domains include the CaM-binding domain (CaM), GAF domains, transmembrane domain (TM domain), targeting domain (TD), upstream conserved regions (UCRs), signal regulatory domain (REC), PAS domain, Pat7 nuclear localization, FYVE-type domains and coiled coil regions. The names of PDE subtypes are given in blue in cases where crystal structures of the PDE have been published and the specific domains that have been crystalized are shown in blue. The PDE subtypes and domains for which crystal structures have not been published are shown in gray. The structures of two domains are partially resolved: PDE6C catalytic domain (12% of the residues of the M-loop, part of H14 and H15) and PDE4 UCR2 (90% of the residues in engineered construct) and these are shown in blue/gray stripes. (B) A schematic diagram of the PDE5A/6C dimer showing one PDE in pink and the other in blue. (C) A full length dimer of PDE5A/6C obtained using cryo-electron microscopy (PDB: 3JAB), colored as (B). The surface of the binding site is shown in gray and the bound IBMX in green.



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201x239mm (300 x 300 DPI)


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Figure 3. Structural overviews of PDEs. (A) 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. (B) An overlay of the backbone ribbons of all PDE crystal structures. (C) 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 4. (D) 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 crystal structures marked PDE5A/6C (*) are partial binding pocket chimeras of PDE6C (residues 787-826) in PDE5A (residues 746-786 and 827-859) constructs. (E) An overview of novel active PDE inhibitors published in the ChEMBL database (pIC50 ≥5) 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. 113x76mm (300 x 300 DPI)



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Figure 4. 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: 1OYN21). 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 as aligned in Figure 5. The color coding and pocket region names below the WebLogo figure are those defined in panel A. (D) A PDE ligand binding site 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 conserved PDE pocket residues of different PDE subtypes 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). 113x75mm (300 x 300 DPI)


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Figure 5. 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. The PDE binding site residue nomenclature is defined in Figure 4. Conserved residues are highlighted in green. PDE6C residues in the PDE5A/6C chimera are highlighted beige. 143x121mm (300 x 300 DPI)



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Figure 6. Systematic structure-based analysis of PDE-ligand interactions. (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. (B-C) Sildenafil (2D structure presented in C) bound to PDE5A (PDB: 1UDT14) 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. 148x129mm (300 x 300 DPI)


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Figure 7. Frequency of interactions between pocket residues and ligands in 203 ligand bound PDE crystal structures in the PDEStrIAn database. 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%. Schematic representations of all individual PDE-ligand complexes are presented in Table 1 and the complete set of PDE-ligand IFPs available as Supplementary Information. 200x237mm (300 x 300 DPI)



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Figure 8. Water molecules involved in PDE ligand binding. (A) Crystallographic water molecules forming interactions with PDE ligands in 203 ligand-bound crystal structures in the PDEStrIAn database. 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-D and F-G forming interactions with ligands and residues, E forming interactions with ligands, metals and the protein and H-I forming interactions with just the ligand. The water molecules are shown over the pocket surface of PDE4D (PDB: 1OYN21). (B) The water molecules that interact with PDE inhibitors in crystal structures. Water molecules are colored according to the interactions they form; cyan form interactions with the ligand, magenta form interactions with ligands and protein residues, and red form interactions with ligands, metals and the protein residues. (C) Overlay of all PDE ligands and metal ions in all 220 phosphodiesterase (PDE) catalytic domain crystal structures present in the PDB (for an overview of the chemical structures of co-crystallized ligands, see Table 1). 69x28mm (300 x 300 DPI)


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Figure 9. Scaffold analysis of the ligands in PDE crystal structures. The core scaffolds from each ligand crystalized with a PDE are oriented as presented in 6C, 8C, 10, 12, 14, 16-18, 20, 21 and Table 1. The points of attachment around the scaffold of the crystalized PDE ligands are named according to the vector of the bond broken using the R group reference scheme shown on the bottom right. 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. Scaffolds were defined such that only one scaffold would be identified in each ligand. The scaffolds were also screened against active PDE inhibitors published in the ChEMBL database using ChemAxon’s R-group decomposition tool and the numbers 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 Rgroups 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



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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). 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. 211x262mm (300 x 300 DPI)


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Figure 10. Examples of compounds that interact with the Q1 and Q2 pockets. The binding modes are shown of: (A) 8 to PDE3B (PDB: 1SO278) (B) 53 to PDE5A (PDB: 2CHM,113 superposed over the binding site surface of PDB: 1UDT14), (C) 74 to PDE10A (PDB: 3QPN,122 superposed over the binding site surface of PDB: 3HR131), and (D) 1 to PDE10A (PDB: 3HR131). (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. 200x236mm (300 x 300 DPI)



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Figure 11. Substructures of crystalized PDE ligands interacting with the Q1 and Q2 regions of the PDE binding pocket. Fragments are arranged with the PDE subtype and number of instances a fragment occurs in a crystal structure of that subtype listed below. Fragments that form interactions other than hydrophobic interactions are positioned above a figure indicating the interactions formed by the fragment with the PDE subtypes listed below. The interactions with the pocket region are color coded to indicate the presence of; a hydrophobic interaction (blue); a face-to-face π-π interaction (green); an edge-to-face π-π interaction (teal); a hydrogen bond acceptor (red); and a hydrogen bond donor (purple); no interaction (white). 181x193mm (300 x 300 DPI)


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Figure 12. Examples of compounds that interact with the HC1, HC2 and S pockets. The binding modes are shown of; (A) 23 bound to PDE4B (PDB: 3HMV,99 superposed over the PDE4 binding site surface of PDB: 1OYN21), (B) 86 bound to PDE10A (PDB: 3SNL,47 superposed over the binding site surface of PDB: 3HR131), (C) 76 to PDE10A (PDB: 2OVY,123 superposed over the binding site surface of PDB: 3HR131), and (D) 68 to PDE9A (PDB: 4G2L,43 superposed over the binding site surface of PDB: 3K3E120) 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. 203x244mm (300 x 300 DPI)



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Figure 13. Substructures of crystalized PDE ligands interacting with the HC1 and HC2 regions of the PDE binding pocket. Fragments are arranged and labeled as described below Figure 11. 200x236mm (300 x 300 DPI)


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Figure 14. Examples of compounds which interact with the MB, MB1 and MB2 pockets. The binding modes are shown of: (A) AMP to PDE4D (PDB: 1TB7,13 superposed over the binding site surface of PDB: 1OYN21), (B) GMP to PDE5A (PDB: 1T9S,13 superposed over the binding site surface of 1UDT14), (C) 19 to PDE4B (PDB: 3O0J97), and (D) 32 to PDE4D (PDB: 3V9B,104 superposed over the binding site surface of PDB: 1OYN21). (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 ligands are shown. 205x248mm (300 x 300 DPI)



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Figure 15. Substructures of crystalized PDE ligands interacting with the MB, MB1 and MB2 regions of the PDE binding pocket. Fragments are arranged and labeled as described below Figure 11. 235x325mm (300 x 300 DPI)


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Figure 16. Interactions between ligands bound to the PDE catalytic site and regions that are able to fold over the pocket. (A) In the structure of roflumilast bound to the catalytic domain of PDE4D (PDB: 1XOQ12, superposed over the binding site surface of PDB: 1OYN21) the catalytic pocket is open. (B) In the structure of inhibitor 58 bound to PDE5A (PDB: 2H44,15 superposed over a the binding site surface of PDB: 1UDT14) the H-loop folds over the pocket entrance enclosing the ligand. (C) The structure of atizoram bound to the catalytic domain of PDE4B shows closing of the pocket by the C-terminal residues (PDB: 3KKT, superposed over the binding site surface of 1OYN). (D) In the case of 33 bound to a PDE4D construct which includes the UCR2 regulatory domain, the pocket is closed off by a helix of UCR2 (PDB: 3IAD,20 superposed over the binding site surface of PDB: 1OYN21). (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 ligands are shown. 220x287mm (300 x 300 DPI)



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Figure 17. The growing of (A) the PDE4D fragment screening hit 34 (PDB: 1Y2B), with (B) an N-phenyl (35, PDB: 1Y2C), (C) an additional para-methoxy group (36, PDB: 1Y2D) and (D) replacing this with a meta-nitro group (37, PDB: 1Y2K). Each structure is shown superposed over the PDE4 binding site surface of 1OYN. (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 ligands are shown. 210x261mm (300 x 300 DPI)


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Figure 18. Halogenated monocyclic pyrimidones form halogen bonds with Y612HC1.01 in PDE5A (A) nonsubstituted pyrimidone 45 (PDB: 4OEX)95 (B) fluorinated pyrimidone 46 (PDB: 3SHY)42; (C) brominated pyrimidone 48 (PDB: 3SIE)42; (D) the thermodynamic signatures of 45, 46 and 47 showing significant changes to the thermodynamic profile of binding. The changes to the free energy on binding of the inhibitors are shown in kJ/mol (∆G (Gibbs free energy) = ∆H (enthalpy) -T∆S (product of temperature and entropy)).42 (E) The IFP bit strings of the proteins and ligands shown in A-C. Only those residues which form at least one interaction with one of the four ligands are shown. 204x245mm (300 x 300 DPI)



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Figure 19. A) Relationships between ligand similarity (ECFP-4145) and protein-ligand interaction pattern (IFP86) similarity for all 205 crystallized PDE-ligand complexes in the PDEStrIAn database. Data are colored according to plot density to identify hotspots. Ligand (ECFP-4 Tanimoto similarity > 0.4146) and interaction fingerprint (IFP Tanimoto similarity > 0.686, 147) similarity cutoffs, indicated by dashed black lines, are used to define four different classes containing ligand pairs with: I) similar IFP and dissimilar ECFP-4 fingerprint, II) similar IFP and similar ECFP-4 fingerprint, III) dissimilar IFP and dissimilar ECFP-4 fingerprint, IV) dissimilar IFP and similar ECFP-4 fingerprint; B) Distribution of ligand pairs for the different ECFP-4 and IFP similarity quadrants defined in panel A are shown for all PDE subtypes. Distributions for those PDE families with over 10 PDE-ligand crystal structures are shown separately for pairs within each of the families, PDE4 (5625), PDE5 (961), PDE9 (361), and PDE10 (4900). 107x60mm (300 x 300 DPI)


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Figure 20. A-B) Dissimilar ligands 9 (PDB: 3IV8), and 10 (PDB: 2QYK)91 adopt similar binding modes in human PDE4A crystal structures; C-D) Dissimilar ligands 94 (PDB: 3HQZ)31, and 101 (PDB: 4HEU)134 adopt similar binding modes in rat and human PDE10 crystal structures, respectively. E) The IFP bit strings of the proteins and ligands shown in C-D. 194x223mm (300 x 300 DPI)



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Figure 21. Merging of PDE4B inhibitors 24 (PDB: 3D3P)100 (A) and 25 (PDB: 3O56)101 (B) lead to high affinity inhibitor 26 (PDB: 3O57)101 (C). Chemical fingerprint (ECFP-4144) and interaction fingerprint (IFP86) based Tanimoto similarities between 24, 25, and 26 are determined and reported IC50 values of 24, 25, and 26 for PDE4B are reported between brackets. (D) The IFP bit strings of the proteins and ligands shown in A-C. 185x202mm (300 x 300 DPI)


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TOC graphic (high resolution) 169x84mm (300 x 300 DPI)


PDEStrIAn: A Phosphodiesterase Structure and Ligand Interaction

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