Structure–Affinity Relationship Analysis of Selective FKBP51 Ligands

The FK506-binding protein 51 (FKBP51) is a promising drug target for the treatment of stress-related psychiatric or metabolic disorders. Just recently, the first selective ligands for FKBP51 were reported based on an induced fit mechanism, but they are too large for a further drug development process. We therefore designed and synthesized a novel series of selective ligands to explore the requirements necessary for binding to the induced-fit conformation. All ligands of this series show no binding toward the structurally very similar antitarget FKBP52. With the cocrystal structure of the best ligand in this novel series we confirmed the induced fit mechanism. Furthermore, the structure–affinity relationship provides information about beneficial structural features, which is valuable for the development of improved FKBP51-directed drugs

Rapid, Structure-Based Exploration of Pipecolic Acid Amides as Novel Selective Antagonists of the FK506-Binding Protein 51

The FK506-binding protein 51 (FKBP51) is a key regulator of stress hormone receptors and an established risk factor for stress-related disorders. Drug development for FKBP51 has been impaired by the structurally similar but functionally opposing homologue FKBP52. High selectivity between FKBP51 and FKBP52 can be achieved by ligands that stabilize a recently discovered FKBP51-favoring conformation. However, drug-like parameters for these ligands remained unfavorable. In the present study, we replaced the potentially labile pipecolic ester group of previous FKBP51 ligands by various low molecular weight amides. This resulted in the first series of pipecolic acid amides, which had much lower molecular weights without affecting FKBP51 selectivity. We discovered a geminally substituted cyclopentyl amide as a preferred FKBP51-binding motif and elucidated its binding mode to provide a new lead structure for future drug optimization

Rapid, Structure-Based Exploration of Pipecolic Acid Amides as Novel Selective Antagonists of the FK506-Binding Protein 51

The FK506-binding protein 51 (FKBP51) is a key regulator of stress hormone receptors and an established risk factor for stress-related disorders. Drug development for FKBP51 has been impaired by the structurally similar but functionally opposing homologue FKBP52. High selectivity between FKBP51 and FKBP52 can be achieved by ligands that stabilize a recently discovered FKBP51-favoring conformation. However, drug-like parameters for these ligands remained unfavorable. In the present study, we replaced the potentially labile pipecolic ester group of previous FKBP51 ligands by various low molecular weight amides. This resulted in the first series of pipecolic acid amides, which had much lower molecular weights without affecting FKBP51 selectivity. We discovered a geminally substituted cyclopentyl amide as a preferred FKBP51-binding motif and elucidated its binding mode to provide a new lead structure for future drug optimization

Rapid, Structure-Based Exploration of Pipecolic Acid Amides as Novel Selective Antagonists of the FK506-Binding Protein 51

The FK506-binding protein 51 (FKBP51) is a key regulator of stress hormone receptors and an established risk factor for stress-related disorders. Drug development for FKBP51 has been impaired by the structurally similar but functionally opposing homologue FKBP52. High selectivity between FKBP51 and FKBP52 can be achieved by ligands that stabilize a recently discovered FKBP51-favoring conformation. However, drug-like parameters for these ligands remained unfavorable. In the present study, we replaced the potentially labile pipecolic ester group of previous FKBP51 ligands by various low molecular weight amides. This resulted in the first series of pipecolic acid amides, which had much lower molecular weights without affecting FKBP51 selectivity. We discovered a geminally substituted cyclopentyl amide as a preferred FKBP51-binding motif and elucidated its binding mode to provide a new lead structure for future drug optimization

Influence of the PH domain and the activation status of Akt.

<p><b>A</b> Domain structure of Akt <b>B</b> Purified GST-tagged active Akt or a mutant lacking the PH domain were incubated with purified FLAG-tagged FKBP51. After 3 h, the interaction was tested by GST pulldown and Western blotting. <b>C</b> and <b>D</b> HEK293T cells were transfected with the indicated HA-tagged Akt1 phosphorylation site mutants with or without co-transfected FLAG-tagged FKBP51^K352A/R356A (TPR_mut). After 2 days cells were collected, lysed, immunoprecipitated and analyzed by Western blotting (duplicates were analyzed for D). <b>E</b> HEK293T cells were starved for 16 h, stimulated with FCS for 45 min or treated with wortmannin. Controls were incubated in the presence of 10% FCS and treated with DMSO. Cells were lysed, immunoprecipitated and analyzed by Western blotting.</p

The FKBP51-Akt interaction depends on the conformation of Akt.

<p><b>A</b> HEK293T cells were transfected with FLAG-tagged FKBP51^K352A/R356A (TPR_mut) and HA-tagged Akt1. After 2 days cells were treated with 10 µM inhibitor VIII, AT7867 or DMSO for 1 h. Cell lysates and immunoprecipitates were analyzed in duplicates by Western blotting. <b>B</b> GSH beads loaded with purified activated GST_Akt1ΔPH were incubated with FKBP51 with or without AMP-PNP. Eluates were analyzed by Western blotting.</p

Schematic model of possible Hsp90-Akt-FKBP51 complexes.

<p><b>A</b> FKBP51 can bind directly to Akt via its FK1 domain, but not with its FK506-binding pocket. Several other FK1-possessing FKBP homologs may bind to Akt in a similar mode. <b>B</b> Akt and several other kinases can bind to FKBP51 indirectly via Hsp90. <b>C</b> FKBP51 could assist the chaperoning of Akt by binding to Hsp90 via its TPR domain and by interacting with Akt via its FK1 domain.</p

Other AGC kinases can also bind to FKBP51.

<p><b>A</b> HEK293T cells were co-transfected in duplicates with GST-tagged Akt1 or ΔN_SGK^S422D and FLAG-tagged FKBP51. After 48 h, the lysates were immunoprecipitated and analyzed in duplicates by Western blotting. <b>B</b> HeLa cells were co-transfected with FLAG-tagged FKBPs and HA-tagged S6K, treated with rapamycin (25 nM) or DMSO for 60 min and lysed. Lysates were immunoprecipitated and analyzed by Western blotting.</p

Influence of FKBP inhibitors.

<p><b>A</b> Purified proteins were mixed and treated with DMSO or rapamycin (1 µM). After 3 h a GST-pull-down was performed followed by Western blotting. <b>B</b> HEK293T cells were transfected with the indicated constructs. After 48 h 1 µM FK1706 was added for 1 h. The lysates were immunoprecipitated and a Western blot was performed. <b>C and D</b> HEK293T cells were transfected with FLAG-FKBP51 and HA-tagged PHLPP1 or HA-tagged PHLPP2. After 48 hFKBP inhibitors (1 µM) or DMSO were added for 1h. Lysates were immunoprecipitated and analyzed by Western blotting. <b>E</b> HEK293T cells were stimulated with FCS for 1 h in the presence of the indicated compounds. After cell lysis, cellular Akt phosphorylation was determined using a homogeneous time- resolved FRET assay. <b>F</b> SHSY-5Y cells were stimulated with FCS for 1 h in the presence of the indicated compounds. After cell lysis, cellular Akt and mTOR phosphorylation was determined using a homogeneous time resolved FRET assay. <b>G</b> SU.86.86 cells were plated, treated with Gemcitabine in the absence or presence of FK1706. Cell survival relative to DMSO treated controls was determined.</p

Evaluation of Synthetic FK506 Analogues as Ligands for the FK506-Binding Proteins 51 and 52

The FK506-binding proteins (FKBP) 51 and 52 are cochaperones that modulate the signal transduction of steroid hormone receptors. Both proteins have been implicated in prostate cancer. Furthermore, single nucleotide polymorphisms in the gene encoding FKBP51 have been associated with a variety of psychiatric disorders. Rapamycin and FK506 are two macrocyclic natural products that bind to these proteins indiscriminately but with nanomolar affinity. We here report the cocrystal structure of FKBP51 with a simplified α-ketoamide analogue derived from FK506 and the first structure–activity relationship analysis for FKBP51 and FKBP52 based on this compound. In particular, the <i>tert</i>-pentyl group of this ligand was systematically replaced by a cyclohexyl ring system, which more closely resembles the pyranose ring in the high-affinity ligands rapamycin and FK506. The interaction with FKBPs was found to be surprisingly tolerant to the stereochemistry of the attached cyclohexyl substituents. The molecular basis for this tolerance was elucidated by X-ray cocrystallography