The FK506-binding protein 51 (FKBP51) has emerged as a key regulator of endocrine stress responses in mammals and as a potential therapeutic target for stress-related disorders (depression, post-traumatic stress disorder), metabolic disorders (obesity and diabetes) and chronic pain. Recently, FKBP51 has been implicated in several cellular pathways and numerous interacting protein partners have been reported. However, no consensus on the underlying molecular mechanisms has yet emerged. Here, we review the protein interaction partners reported for FKBP51, the proposed pathways involved, their relevance to FKBP51’s physiological function(s), the interplay with other FKBPs, and implications for the development of FKBP51-directed drugs.

Enhancement by displacement. A single methyl group displaces a water molecule from the binding site of FKBPs, resulting in the most potent binders known, outperforming the natural products FK506 and rapamycin in biochemical and cellular assays.

Streptomyces coelicolor is a Gram-positive soil bacterium with a high metabolic and adaptive potential that is able to utilize a variety of nitrogen sources. However, little is known about the utilization of the alternative nitrogen source ethanolamine. Our study revealed that S. coelicolor can utilize ethanolamine as a sole nitrogen or carbon (N/C) source, although it grows poorly on this nitrogen source due to the absence of a specific ethanolamine permease. Heterologous expression of a putative ethanolamine permease (SPRI_5940) from Streptomycespristinaespiralis positively influenced the biomass accumulation of the overexpression strain grown in defined medium with ethanolamine. In this study, we demonstrated that a glutamine synthetase-like protein, GlnA4 (SCO1613), is involved in the initial metabolic step of a novel ethanolamine utilization pathway in S. coelicolor M145. GlnA4 acts as a gamma-glutamylethanolamide synthetase. Transcriptional analysis revealed that expression of glnA4 was induced by ethanolamine and repressed in the presence of ammonium. Regulation of glnA4 is governed by the transcriptional repressor EpuRI (SCO1614). The ΔglnA4 mutant strain was unable to grow on defined liquid Evans medium supplemented with ethanolamine. High-performance liquid chromatography (HPLC) analysis demonstrated that strain ΔglnA4 is unable to utilize ethanolamine. GlnA4-catalyzed glutamylation of ethanolamine was confirmed in an enzymatic in vitro assay, and the GlnA4 reaction product, gamma-glutamylethanolamide, was detected by HPLC/electrospray ionization-mass spectrometry (HPLC/ESI-MS). In this work, the first step of ethanolamine utilization in S. coelicolor M145 was elucidated, and a putative ethanolamine utilization pathway was deduced based on the sequence similarity and genomic localization of homologous genes. IMPORTANCE Until now, knowledge of the utilization of ethanolamine in Streptomyces was limited. Our work represents the first attempt to reveal a novel ethanolamine utilization pathway in the actinobacterial model organism S. coelicolor through the characterization of the key enzyme gamma-glutamylethanolamide synthetase GlnA4, which is absolutely required for growth in the presence of ethanolamine. The novel ethanolamine utilization pathway is dissimilar to the currently known ethanolamine utilization pathway, which occurs in metabolome. The novel ethanolamine utilization pathway does not result in the production of toxic by-products (such as acetaldehyde); thus, it is not encapsulated. We believe that this contribution is a milestone in understanding the ecology of Streptomyces and the utilization of alternative nitrogen sources. Our report provides new insight into bacterial primary metabolism, which remains complex and partially unexplored.

High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N'-phenyloctandiamide.

Enzymes from the histone deacetylase (HDAC) family are highly regulated by different mechanisms. However, only very limited knowledge exists about the regulation of HDAC8, an established target in multiple types of cancer. A previous dedicated study of HDAC class I enzymes identified no redox-sensitive cysteinyl thiol in HDAC8. This is in contrast to the observation that HDAC8 preparations show different enzyme activities depending on the addition of reducing agents. In the light of the importance of HDAC8 in tumorigenesis a possible regulation by redox signaling was investigated using biochemical and biophysical methods combined with site directed mutagenesis. The occurrence of a characteristic disulfide bond under oxidizing conditions is associated with a complete but reversible loss of enzyme activity. Cysteines 102 and 153 are the integral components of the redox-switch. A possible regulation of HDAC8 by redox signal transduction is suggested by the observed relationship between inhibition of reactive oxygen species generating NOX and concomitant increased HDAC8 activity in neuroblastoma tumor cells. The slow kinetics for direct oxidation of HDAC8 by hydrogen peroxide suggests that transmitters of oxidative equivalents are required to transfer the H2O2 signal to HDAC8.

The FK506-binding protein 51 (FKBP51) emerged as a key player in several diseases like stress-related disorders, chronic pain, and obesity. Linear analogues of FK506 called SAFit were shown to be highly selective for FKBP51 over its closest homologue FKBP52, allowing the proof-of-concept studies in animal models. Here, we designed and synthesized the first macrocyclic FKBP51-selective ligands to stabilize the active conformation. All macrocycles retained full FKBP51 affinity and selectivity over FKBP52 and the incorporation of polar functionalities further enhanced affinity. Six high-resolution crystal structures of macrocyclic inhibitors in complex with FKBP51 confirmed the desired selectivity-enabling binding mode. Our results show that macrocyclization is a viable strategy to target the shallow FKBP51 binding site selectively.

Subtype selectivity represents ac hallenge in many drug discovery campaigns.Atypical example is the FK506 binding protein 51 (FKBP51), whichh as emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs.F KBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During am acrocyclization pilot study,w eo bserved that many of these macrocyclic analogs have unanticipated and unprecedented preference for FKBP51 over FKBP12 and FKBP12.6. Structural studies revealed that these macrocycles bind with anew binding mode featuring atransient conformation, which is disfavored for the small FKBPs.Using aconformation-sensitive assayweshow that this binding mode occurs in solution and is characteristic for this new class of compounds.T he discovered macrocycles are non-immunosuppressive,e ngage FKBP51 in cells,a nd blockt he cellular effect of FKBP51 on IKKa.O ur findings provide an ew chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.

Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH 0 /ΔG 0 . The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design.