Targeting FKBP isoforms with small-molecule ligands

Available online at www.sciencedirect.com

Targeting FKBP isoforms with small-molecule ligands Elizabeth A Blackburn and Malcolm D Walkinshaw The FK506 binding protein (FKBP) family of proteins provide an interesting series of drug targets since different isoforms modulate diverse cellular pathways. There are therapeutic opportunities in the fields of cancer therapy, neurodegenerative conditions and psychiatric disorders. X-ray crystallographic or NMR data are available for eight of fourteen human FKBPs covering ten of the twenty-two different FKBP domains. We have made a detailed sequence and structural comparison of human FKBP domains. These data show that the chemical scaffolds common to the immunosuppressive inhibitors FK506 and rapamycin bind to the most conserved region of the binding site. This observation opens the way to the design of isoform specific inhibitors. Address The Centre for Translational and Chemical Biology (CTCB), ISMB, University of Edinburgh, Edinburgh EH9 3JR, UK Corresponding author: Walkinshaw, Malcolm D ([email protected])

Current Opinion in Pharmacology 2011, 11:365–371 This review comes from a themed issue on Cancer Edited by Maria Fiammetta Romano

1471-4892/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2011.04.007

Introduction The FK506 binding proteins (FKBPs) form a subset of the immunophilin family that includes the structurally dissimilar cyclophilins [1]. FKBPs are present in almost all tissues, often with high levels of expression [2]. They have regulatory or chaperone function and are characterized by the presence of at least one peptidyl-prolyl isomerase (PPIase) domain [3]. FKBP12 contains a single PPIase domain, which binds the immunosuppressive drug FK506, and can be considered the archetypal FKBP. In this review, we focus on the 14 human FKBP proteins that exhibit significant sequence identity to FKBP12 (Figure 1). Mammalian FKBPs can be divided into four groups: cytoplasmic, TPR domain, endoplasmic reticulum (ER) or secretory pathway and nuclear [4]. The cytoplasmic FKBP isoforms FKBP12 and 12.6 and the nuclear FKBP25 and 133 contain a single PPIase domain. FKBP36, 38, 51 and 52 contain multiple TPR domains. www.sciencedirect.com

The ER FKBPs: FKBP13, 19, 22, 23, 60 and 65 all contain an N-terminal ER signal peptide. The biological roles of the human FKBPs appear to be diverse and frequently involve the domains flanking the PPIase domain [5]. Early interest in the family centred on the ability of FKBP12 to bind the natural product immunosuppressive drugs FK506 and rapamycin [6]. Although these drugs bind to the catalytic site of FKBP12 they do not exert their effect through inhibition of PPIase activity. FKBP12 forms a complex with the drug that facilitates binding of calcineurin (CN) in the case of FK506 and mammalian target of rapamycin (mTOR) with rapamycin. Inhibition of CN or mTOR pathways reduces T cell activation and proliferation [7]. FK506 has also been found to have neuroprotective and neuroregenerative effects that do not rely on immunosuppression and is moderated by FKBP12 and 52 [11]. It has proved hard to pin-down which specific isoform is implicated in the action of a given ligand, as multiple FKBPs are often expressed within a cell. A large number of non-immunosuppressive ligands of FKBP12 were discovered between 1995 and 2005. More recently it has been reported that inhibition of FKBP12 and FKBP52 by FK506 reduces the aggregation of alpha-synuclein and cell death in a model of Parkinson’s disease [12].

The human genome has 22 PPIase domains in 14 proteins Structural data and predictions from sequence strongly suggest that the 22 human PPIase domains maintain a similar fold. Amino acid sequences were aligned to examine residue conservation within the domain (Figures 2 and 3(a)). PPIase domain identity with FKBP12 is within the range of 17–83%. These data show the N-terminal domain of a protein containing multiple PPIase domains is always the most similar to FKBP12 (Figure 1(b), full alignment data is shown in Supplementary Table 2). The cytoplasmic FKBP isoforms 12 and 12.6 show very close identity (83%). The secretory pathway FKBPs fall into a single cluster (Figure 1(b)). The proteins FKBP60 and 65 contain four PPIase domains and there is a correlation between identity and the position of the domain in the chain. For example, FKBP60_FK1 is nearest neighbour to FKBP65_FK1. The TPR FKBPs show greater complexity. The FKBP51 and 52 N-terminal PPIase domains have 51 and 53% identity to FKBP12. However, FKBP51_FK2 and FKBP52_FK2 show greater similarity to FKBP38 than their respective FK1 domains. Biochemical data support this observation in that FKBP52_FK1 provides the majority of the PPIase activity of FKBP52 [15]. The nuclear FKBPs 25 and 133 Current Opinion in Pharmacology 2011, 11:365–371

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Figure 1

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FKBP19 1

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FKBP22 1

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FKBP23 142 166

254 278

365 389

477

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262 286

374 399

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Ca2+ binding Current Opinion in Pharmacology

The domain structure of the human FKBPs. (a) The domain structure of the 14 human FKBPs retrieved from a BLAST search of sequences from The Reference Sequence collection, using E-value cut-off of less than 1  106 against human FKBP12 [8,9]. Human PPIase domains with crystallographic data deposited in the PDB are annotated with a blue star and the PDB accession code of a representative structure. (b) Dendrogram showing sequence identity of the human PPIase domains (UPGMA clustering of the ClustalW pairwise alignment scores from sequences in Figure 2 [10]). Multiple PPIase domains are labelled sequentially from N-terminus (FK1) to C-terminus (FKn).

show relatively low identity (36%). FKBP25 is closest to FKBP12, 12.6 and the N-terminal domains of 51 and 52. FKBP133 is a near neighbour of FKBP13 (40%), a member of the ER FKBPs.

Binding-pocket differences: the possibility of designing domain-specific ligands Figure 3(a) illustrates the differences in amino acids forming the FKBP binding site. A red surface denotes highly conserved amino acids. The floor of the PPIase binding site is formed by tryptophan in FKBP12 (Figure 3(b)) and 16 X-ray/NMR structures of FKBP12 in complex with 12 different small-molecule inhibitors all show the ligand making hydrophobic contacts with Trp59 of FKBP12 Current Opinion in Pharmacology 2011, 11:365–371

(see Figure 2 for numbering scheme and Supplementary Table 3 for PDB ID). Interestingly Trp59 is not particularly well conserved and is frequently replaced by an alternative hydrophobic residue, most commonly methionine (Figure 2). There is a strong preference for methionine in the ER FKBPs. The most conserved residues form a crescent that makes hydrophobic contacts with the most buried portion of FK506 and rapamycin; these include Tyr26, Phe 46 and Phe99 (Figures 2 and 3(c)). Asp37 is well conserved, as are Ile56 and Tyr82; these residues make hydrogen bonding interactions with FK506 and rapamycin (Figure 2). The exposed loop formed by His87 to Ile91 in FKBP12 is www.sciencedirect.com

Targeting FKBP isoforms with small-molecule ligands Blackburn and Walkinshaw 367

Figure 2

FKBP12 numbering

Y26

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FKBP12 FKBP12.6

GQTCVVHYTGMLE----DGKK-FDS-----SRDRN--KPFKFMLGKQE---VIRGWEEGVAQMSVGQRAKLTIS GQTCVVHYTGMLQ----NGKK-FDS-----SRDRN--KPFKFRIGKQE---VIKGFEEGAAQMSLGQRAKLTCT

FKBP36 FKBP38 FKBP51_FK1 FKBP51_FK2 FKBP52_FK1 FKBP52_FK2

DASVLVKYSGYLE----HMDRPFDS-----NYFRK--TPRLMKLGEDI---TLWGMELGLLSMRRGELARFLFK GQVVTVHLQTSLE----NGTR-VQEE-----------PELVFTLGDCD---VIQALDLSVPLMDVGETAMVTAD GDKVYVHYKGKLS----NGKK-FDS-----SHDRN--EPFVFSLGKGQ---VIKAWDIGVATMKKGEICHLLCK GATVEIHLEGRCG-----GRM-FDC------------RDVAFTVGEGEDHDIPIGIDKALEKMQREEQCILYLG GDRVFVHYTGWLL----DGTK-FDS-----SLDRK--DKFSFDLGKGE---VIKAWDIAIATMKVGEVCHITCK GAIVEVALEGYYK-----DKL-FDQ------------RELRFEIGEGENLDLPYGLERAIQRMEKGEHSIVYLK

FKBP13 FKBP19 FKBP22 FKBP23 FKBP60_FK1 FKBP60_FK2 FKBP60_FK3 FKBP60_FK4 FKBP65_FK1 FKBP65_FK2 FKBP65_FK3 FKBP65_FK4

GDVLHMHYTGKLE----DGTE-FDS-----SLPQN--QPFVFSLGTGQ---VIKGWDQGLLGMCEGEKRKLVIP GDTLHIHYTGSLV----DGRI-IDT-----SLTR---DPLVIELGQKQ---VIPGLEQSLLDMCVGEKRRAIIP GDLMLVHYEGYLEK---DGSL-FHS-----THKHNNGQPIWFTLGILE---ALKGWDQGLKGMCVGEKRKLIIP GDLLNAHYDGYLAK---DGSK-FYC-----SRTQNEGHPKWFVLGVGQ---VIKGLDIAMTDMCPGEKRKVVIP GDFVRYHYVGTFP----DGQK-FDS-----SYDRD--STFNVFVGKGQ---LITGMDQALVGMCVNERRFVKIP SDFVRYHYNGTFL----DGTL-FDS-----SHNRM--KTYDTYVGIGW---LIPGMDKGLLGMCVGEKRIITIP GDFLRYHYNGTLL----DGTL-FDS-----SYSRN--RTFDTYIGQGY---VIPGMDEGLLGVCIGEKRRIVVP GDYLKYHYNASLL----DGTL-LDS-----TWNLG--KTYNIVLGSGQ---VVLGMDMGLREMCVGEKRTVIIP GDFVRYHYNGTFE----DGKK-FDS-----SYDRN--TLVAIVVGVGR---LITGMDRGLMGMCVNERRRLIVP GDFVRYHYNGTLL----DGTS-FDT-----SYSKG--GTYDTYVGSGW---LIKGMDQGLLGMCPGERRKIIIP GDFMRYHYNGSLM----DGTL-FDS-----SYSRN--HTYNTYIGQGY---IIPGMDQGLQGACMGERRRITIP GDFVRYHYNCSLL----DGTQ-LFT-----SHDYG--APQEATLGANK---VIEGLDTGLQGMCVGERRQLIVP

FKBP25 FKBP133

GDVVHCWYTGTLQ----DGTV-FDTNIQTSAKKKKNAKPLSFKVGVGK---VIRGWDEALLTMSKGEKARLEIE GDSLEVAYTGWLFQNHVLGQV-FDS-----TANKD--KLLRLKLGSGK---VIKGWEDGMLGMKKGGKRLLIVP

FKBP12 numbering

Y82

H87

I91

F99

FKBP12 FKBP12.6

PDYAYGATG-HPGIIPPHATLVFDVELLKLE PDVAYGATG-HPGVIPPNATLIFDVELLNLE

FKBP36 FKBP38 FKBP51_FK1 FKBP51_FK2 FKBP52_FK1 FKBP52_FK2

PNYAYGTLG-CPPLIPPNTTVLFEIELLDFL SKYCYGPQGRSPY-IPPHAALCLEVTLKTAV PEYAYGSAG-SLPKIPSNATLFFEIELLDFK PRYGFGEAGKPKFGIEPNAELIYEVTLKSFE PEYAYGSAG-SPPKIPPNATLVFEVELFEFK PSYAFGSVGKEKFQIPPNAELKYELHLKSFE

FKBP13 FKBP19 FKBP22 FKBP23 FKBP60_FK1 FKBP60_FK2 FKBP60_FK3 FKBP60_FK4 FKBP65_FK1 FKBP65_FK2 FKBP65_FK3 FKBP65_FK4

SELGYGERG-APPKIPGGATLVFEVELLKIE SHLAYGKRG-FPPSVPADAVVQYDVELIALI PALGYGKEG--KGKIPPESTLIFNIDLLEIR PSFAYGKEGYAEGKIPPDATLIFEIELYAVT PKLAYGNEG-VSGVIPPNSVLHFDVLLMDIW PFLAYGEDG-DGKDIPGQASLVFDVALLDLH PHLGYGEEG-RGN-IPGSAVLVFDIHVIDFH PHLGYGEAG-VDGEVPGSAVLVFDIELLELV PHLGYGSIG-LAGLIPPDATLYFDVVLLDVW PFLAYGEKG-YGTVIPPQASLVFHVLLIDVH PHLAYGENG-TGDKIPGSAVLIFNVHVIDFH PHLAHGESG-ARG-VPGSAVLLFEVELVSRE

FKBP25 FKBP133

PEWAYGKKGQPDAKIPPNAKLTFEVELVDID PACAVGSEGVIGWTQATDSILVFEVEVRRVK Current Opinion in Pharmacology

Amino acid sequences of human PPIase domains. PPIase domains from the 14 human FKBPs were aligned with FKBP12 [9] (red box). The hydrophobic amino acids forming the base of the PPIase domain are labelled with a grey background and those forming hydrogen bonds between FKBP12 and FK506 with black backgrounds. Key residues of FKBP12 have a numerical label (crystallographic numbering convention, 2ppn.pdb). The secondary structure of FKBP12, from crystallographic data, is shown above the sequences [13]. b-Sheet is denoted by blue boxes and a-helices by red cylinders. Protein sequences retrieved from the UniProtKB database [14].

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Current Opinion in Pharmacology 2011, 11:365–371

368 Cancer

Figure 3

Residue conservation

(a)

FKBP12

(b)

(c)

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F36 H87 D37 F99

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Y26 F46

E54 F48 Q53

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7

8

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Current Opinion in Pharmacology

(a) The 22 FKBP domains show remarkable diversity in the catalytic site. Surface representation of FKBP12, 2DG3.pdb was coloured according to sequence conservation, ranging from white (variable residues) through yellow and orange to red (conserved residues). Sequence conservation was calculated from the alignment of the 22 domains in Figures 1 and 2 [16]. (b) FKBP12 represented as secondary structure elements. Amino acids making key interactions with FK506 and rapamycin are highlighted as sticks [2]. (c) Electrostatic surface representations of FKBP12, 2DG3.pdb Red areas show negative charge, blue positive charge and white hydrophobic surface (PyMOL). Rapamycin is shown in black sticks.

poorly conserved (Figure 3(b)). Ile91 is replaced by lysine in many FKBPs, for example, FKBP51. The solvent exposed moiety of FK506 and rapamycin make contacts with an acidic patch on FKBP12. This surface region shows considerable variation in charge between different PPIase domains (see Figure 3(c) and Supplementary Figure 1). Exploiting these differences is likely to be central to targeting specific FKBPs.

Small-molecules inhibitors of FKBP12 based on the FK506/rapamycin scaffold Small-molecule inhibitors of FKBP12 have been reviewed in some detail by Babine et al., Dornan et al. and Wang et al. [17–19]. Many synthetic ligands have been based on the chemically similar FKBP12 binding domains of FK506 and rapamycin; many are potent isomerase inhibitors with Ki values for FKBP12 in the low nanomolar range. Acyclic ligands have been developed by replacing the non-FKBP binding loop with a variety of aliphatic chains, rings and aromatic groups. Figure 4 illustrates the main scaffolds; a more comprehensive list is available in Supplementary Table 4. A pharmacophore binding model has been developed [20] that provides flexibility between a five and six member ring and emphasizes the importance of the a-keto amide functionality (highlighted in Figure 4). Key groups within the FKBP12 binding domain of FK506 have also been substituted with bioisosteres. Table 4 of the Supplementary Material illustrates a compound (M4 core) where the aketo amide linker is replaced with a sulphonamide [21]. Pfizer have developed a reaction scheme for synthesizing a sulfamide linker to replace the metabolically unstable Current Opinion in Pharmacology 2011, 11:365–371

ketoamide in neuroimmunophilin ligands [22]. GPI-1046 (Guilford Pharmaceuticals and Amgen) and V10367 (Vertex Pharmaceuticals) are examples of compounds that exhibit neurotrophic action in both animal models and in the clinic (Figure 4). These compounds do not show immunosuppressive effects.

Screening strategies for discovering novel molecular inhibitors The recent literature cites new assays to detect FKBP inhibitors; however, relatively few new compounds have been reported. One assay has been developed where the increased stability of FKBP in complex with a smallmolecule ligand is detected by unfolding with chemical denaturants [23,24]. Another method describes how changes to the charge-state distribution of a protein in electrospray ionization mass spectrometry can provide an estimate the affinity of a complex [25]. Juli et al. performed an NMR screen for small-molecule inhibitors of the legionella MIP protein that is similar to FKBP12 [26]. The authors report affinity data for a series of N-sulfonyl pipecolyl amides binding to FKBP12. These compounds have similar structures to previously reported compounds [27–30] (see Supplementary Table 4).

Investigating specificity for protein substrates FKBPs have greater sequence specificity for peptide substrates than the cyclophilins showing higher activity towards substrates with a hydrophobic amino acid in the position preceding proline [35,36,37]. The groups of Fischer and Schmid have developed a library of fluorescent peptides to examine the substrate specificity of prolyl www.sciencedirect.com

Targeting FKBP isoforms with small-molecule ligands Blackburn and Walkinshaw 369

Figure 4 Me

Me

Me H

Me

O

OMe

O

O

OH

Me

OH O

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Compound 3

Compound 2

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12.6

YES 0.2 0.75 306 29500

YES 4.4 0.8 392 21400

38 with calcineurin n.d. 499 48 85

Ki (nM) 51 YES 104 14.7 447 155000

52

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YES 23 6.12 936 259000

YES 166 1.14 290 29500

YES n.d. 0.9 631 169

Reference

[31] [32] [32] [32]

Current Opinion in Pharmacology

Structures of selected compounds with nanomolar affinity for FKBP; these are representative of the key inhibitor scaffolds [6,21,31,32–34]. The a-keto amide functionality of FK506 is emphasized by the shaded region. A more complete selection of inhibitors is shown in Supplementary Table 4. The inset table shows a compilation of affinity data from Edlich et al. [32] and Kozany et al. [31]. The data illustrates the specificity of certain inhibitors for different family members. n.d. not determined.

isomerases [36]. Sensitive monitoring of PPIase activity is achieved by labelling the peptides with fluorescent groups at the N-terminus and C-terminus. Fluorescent quenching is dependent on cis–trans geometry. This assay has the advantage over the traditional PPIase assay, as isomerisation is not coupled to proteolysis by a-chymotrypsin [38]. Another interesting high-throughput PPIase assay has been developed where 7-amino-4-methylcoumarin is conjugated to a tetrapeptide substrate rather than p-nitroaniline [39,40]. This assay has the advantage of being performed at room temperature.

Structure-based drug discovery using FKBP12 as a template Over 20 NMR or X-ray structures are available for FKBP12 free and in complex with small molecules (Figure 1 and Supplementary Table 3) [18]. These structures have www.sciencedirect.com

provided templates for structure-based drug design. An NMR fragment-linking approach was an early example of designing ligands with nanomolar affinity from lower affinity fragments binding to specific sites on the protein [41]. A more recent high-throughput NMR study by Stebbins et al. identified various fragment-like micromolar affinity ligands with novel scaffolds [42]. NMR has the advantage of highlighting amino acids that form important interactions at the binding interface and confirms binding at the PPIase active site. Structural data were utilised by Rohrig et al., to improve the potency of a known fragment-like inhibitor by changing the link to a second-fragment in the binding groove bounded by Gln53 and Glu54 (Figure 3(b)) [43]. High-resolution X-ray structures of FKBP12 have been used in virtual screening to discover chemical scaffolds that differ from the FK506 binding moiety (Supplementary Table 4) [27,44,30]. Current Opinion in Pharmacology 2011, 11:365–371

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FKBP specificity for inhibitors

Appendix A. Supplementary data

FK506 and rapamycin show affinity in the high picomolar to high nanomolar range for the majority of PPIase domains [20,31,32]. The inset table in Figure 4 shows a compilation of selected affinity data from Edlich et al. [32] and Kozany et al. [31]. Kozany et al. have developed a series of labelled probes for use in a fluorescence polarisation assay. The equilibrium dissociation constant for fluorescently labelled rapamycin and two small-molecule analogues of the FKBP12 binding domain of FK506/rapamycin were determined for a range of FKBPs (compound (1) Figure 4). These data can be used in competition assays to measure ligand affinity with some precision.

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.coph.2011. 04.007.

Rather surprisingly, FKBP38 and FKBP13 show a marked preference for rapamycin over FK506. FKBP38 is interesting as it has a much deeper and narrower active site than FKBP12 (Supplemental Figure 1(f)). In the PPIase domain of FKBP38, Trp59 is replaced with Leu, Phe36 with Val and His87 with Arg (Figure 2). Many of the aromatic hydrophobic residues lining the domain in FKBP12 are replaced with Leu in FKBP38. FKBP38 only binds FK506 in the presence of calmodulin and Ca2+ because of a proposed allosteric rearrangement. Rapamycin shows a 500-fold lower affinity for FKBP38 than FKBP12. In contrast, compounds GPI-1046 and DM-CHX show much higher affinity for FKBP38. Compound DM-CHX also shows a preference for FKBP25 which has been shown to be regulator of the p53 pathway. FKBP51 and 52 have received attention as anti-tumour targets [45]; they have high overall sequence homology but still show some specificity for ligands. The N-terminal PPIase domains of FKBP51 and 52 have 70% sequence identity but despite their high similarity, FKBP51 has a two-fold higher affinity for GPI-1046 and FKBP52 a twofold higher affinity for Compound (1) (Figure 4).

Conclusions Designing small-molecule ligands with specificity between different FKBPs is still in its infancy. The structural data show that FK506 and rapamycin, and their close analogues, all bind in a mode where the common motif makes non-covalent interactions with the most highly conserved residues in the PPIase domain. This means that scaffolds based on FK506 and rapamycin are likely to hit multiple FKBP targets and make it difficult to tease out which cellular pathway the drug is targeting. Compounds GPI-1046 and DM-CHX show that achieving specificity is possible for FKBP38. However, FKBP38 only shows identity to other FKBPs of between 10 and 37%. Even after many years of interest in FKBPs there is still a requirement for novel drug scaffolds. There is also a lack of structural and biochemical data for the ER FKBPs. The biggest advances in recent years have been in the development of fluorescent probes to examine ligand specificity. Current Opinion in Pharmacology 2011, 11:365–371

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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6.

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