Structure-Based Design of Ultrapotent Tricyclic Ligands for FK506-Binding Proteins.

Access to small, rigid, and sp3-rich molecules is a major limitation in the drug discovery for challenging protein targets. FK506-binding proteins hold high potential as drug targets or enablers of molecular glues but are fastidious in the chemotypes accepted as ligands. We here report an enantioselective synthesis of a highly rigidified pipecolate-mimicking tricyclic scaffold that precisely positions functional groups for interacting with FKBPs. This was enabled by a 14-step gram-scale synthesis featuring anodic oxidation, stereospecific vinylation, and N-acyl iminium cyclization. Structure-based optimization resulted in the discovery of FKBP inhibitors with picomolar biochemical and subnanomolar cellular activity that represent the most potent FKBP ligands known to date.

1,4-Pyrazolyl-Containing SAFit-Analogues are Selective FKBP51 Inhibitors With Improved Ligand Efficiency and Drug-Like Profile.

The FK506 binding protein 51 (FKBP51) is an appealing drug target due to its role in several diseases such as depression, anxiety, chronic pain and obesity. Towards this, selectivity versus the close homolog FKBP52 is essential. However, currently available FKBP51-selective ligands such as SAFit2 are too large and lack drug-like properties. Here, we present a structure activity relationship (SAR) analysis of the pipecolic ester moiety of SAFit1 and SAFit2, which culminated in the discovery of the 1,4-pyrazolyl derivative 23 d, displaying a binding affinity of 0.077 µM for FKBP51, reduced molecular weight (541.7 g/mol), lower hydrophobicity (cLogP=3.72) and higher ligand efficiency (LE=0.25). Cocrystal structures revealed the importance of the 1,4- and 1,3,4- substitution patterns of the pyrazole ring versus the 1,4,5 arrangement.

Automated Flow Peptide Synthesis Enables Engineering of Proteins with Stabilized Transient Binding Pockets.

Engineering at the amino acid level is key to enhancing the properties of existing proteins in a desired manner. So far, protein engineering has been dominated by genetic approaches, which have been extremely powerful but only allow for minimal variations beyond the canonical amino acids. Chemical peptide synthesis allows the unrestricted incorporation of a vast set of unnatural amino acids with much broader functionalities, including the incorporation of post-translational modifications or labels. Here we demonstrate the potential of chemical synthesis to generate proteins in a specific conformation, which would have been unattainable by recombinant protein expression. We use recently established rapid automated flow peptide synthesis combined with solid-phase late-stage modifications to rapidly generate a set of FK506-binding protein 51 constructs bearing defined intramolecular lactam bridges. This trapped an otherwise rarely populated transient pocket-as confirmed by crystal structures-which led to an up to 39-fold improved binding affinity for conformation-selective ligands and represents a unique system for the development of ligands for this rare conformation. Overall, our results show how rapid automated flow peptide synthesis can be applied to precision protein engineering.

Use of DNA forceps to measure receptor-ligand dissociation equilibrium constants in a single-molecule competition assay.

The ability of biophysicists to decipher the behavior of individual biomolecules has steadily improved over the past thirty years. However, it still remains unclear how an ensemble of data acquired at the single-molecule level compares with the data acquired on an ensemble of the same molecules. We here propose an assay to tackle this question in the context of dissociation equilibrium constant measurements. A sensor is built by engrafting a receptor and a ligand onto a flexible dsDNA scaffold and mounting this assembly on magnetic tweezers. This way, looking at the position of the magnetic bead enables one to determine in real-time if the two molecular partners are associated or not. Next, to quantify the affinity of the scrutinized single-receptor for a given competitor, various amounts of the latter molecule are introduced in solution and the equilibrium response of the sensor is monitored throughout the titration protocol. Proofs of concept are established for the binding of three rapamycin analogs to the FKBP12 cis-trans prolyl isomerase. For each of these drugs the mean affinity constant obtained on a ten of individual receptors agrees with the one previously determined in a bulk assay. Furthermore, experimental contingencies are sufficient to explain the dispersion observed over the single-molecule values.

Discovery of a Potent Proteolysis Targeting Chimera Enables Targeting the Scaffolding Functions of FK506-Binding Protein 51 (FKBP51).

The FK506-binding protein 51 (FKBP51) is a promising target in a variety of disorders including depression, chronic pain, and obesity. Previous FKBP51-targeting strategies were restricted to occupation of the FK506-binding site, which does not affect core functions of FKBP51. Here, we report the discovery of the first FKBP51 proteolysis targeting chimera (PROTAC) that enables degradation of FKBP51 abolishing its scaffolding function. Initial synthesis of 220 FKBP-focused PROTACs yielded a plethora of active PROTACs for FKBP12, six for FKBP51, and none for FKBP52. Structural analysis of a binary FKBP12:PROTAC complex revealed the molecular basis for negative cooperativity. Linker-based optimization of first generation FKBP51 PROTACs led to the PROTAC SelDeg51 with improved cellular activity, selectivity, and high cooperativity. The structure of the ternary FKBP51:SelDeg51:VCB complex revealed how SelDeg51 establishes cooperativity by dimerizing FKBP51 and the von Hippel-Lindau protein (VHL) in a glue-like fashion. SelDeg51 efficiently depletes FKBP51 and reactivates glucocorticoid receptor (GR)-signalling, highlighting the enhanced efficacy of full protein degradation compared to classical FKBP51 binding.

A novel synthetic inhibitor of polyamine utilization in Streptomyces coelicolor.

In this work, we present the first inhibitor of GlnA2Sc, a gamma-glutamylpolyamine synthetase, which allows Streptomyces coelicolor to detoxify high concentrations of polyamines and to utilize them as a carbon or nitrogen source. GlnA2 belongs to the class of glutamine synthetase-like (GS-like) enzymes that catalyze the glutamylation of different nitrogen-containing compounds. Whereas a number of inhibitors for GS are known, none of them are known to inhibit GlnA2. In this work, PPU268, an inhibitor for GlnA2 is presented that is structurally derived from the prototypic GS inhibitor-methionine sulfoximine (MSO). It combines two features: the binding mechanism of MSO and the amine substrate specificity of GlnA2Sc. This inhibitor is a novel compound to block the polyamine utilization in bacteria resulting in the inability to detoxify polyamines. This may offer a possibility to develop novel therapeutic strategies to combat actinobacterial human pathogens that encounter polyamines in the course of the infection processes.

A protein engineering approach toward understanding FKBP51 conformational dynamics and mechanisms of ligand binding.

Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51), a prospective target for stress-related diseases, metabolic disorders, some types of cancers and chronic pain. By randomizing selected regions of its ligand-binding domain and sorting yeast display libraries expressing these variants, mutants with high affinity to conformation-specific FKBP51 selective ligands were identified. These improved mutants are valuable tools for the discovery of novel selective ligands that preferentially and specifically bind the FKBP51 active site in its open conformation state. Moreover, they will help us understand the conformational dynamics and ligand binding mechanics of the FKBP51 binding pocket.

Deconstructing Protein Binding of Sulfonamides and Sulfonamide Analogues.

Sulfonamides are one of the most important pharmacophores in medicinal chemistry, and sulfonamide analogues have gained substantial interest in recent years. However, the protein interactions of sulfonamides and especially of their analogues are underexplored. Using FKBP12 as a model system, we describe the synthesis of optically pure sulfenamide, sulfinamide, and sulfonimidamide analogues of a well characterized sulfonamide ligand. This allowed us to precisely determine the binding contributions of each sulfonamide oxygen atom and the consequences of nitrogen replacements. We also present high-resolution cocrystal structures of sulfonamide analogues buried in the pocket of a protein target. This revealed intimate contacts with the protein including an unprecedented hydrogen bond acceptor of sulfonimidamides. The use of sulfonamide analogues enabled new exit vectors that allowed remodeling of a subpocket in FKBP12. Our results illuminate the protein interaction potential of sulfonamides/sulfonamide analogues and will aid in their rational design.

[4.3.1]Bicyclic FKBP Ligands Inhibit Legionella Pneumophila Infection by LpMip-Dependent and LpMip-Independent Mechanisms.

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.

Structure-Based Discovery of a New Selectivity-Enabling Motif for the FK506-Binding Protein 51.

In recent years, the selective inhibition of FKBP51 has emerged as a possible treatment for chronic pain, obesity-induced diabetes, or depression. All currently known advanced FKBP51-selective inhibitors, including the widely used SAFit2, contain a cyclohexyl residue as a key motif for enabling selectivity over the closest homologue and anti-target FKBP52. During a structure-based SAR exploration, we surprisingly discovered thiophenes as highly efficient cyclohexyl replacement moieties that retain the strong selectivity of SAFit-type inhibitors for FKBP51 over FKBP52. Cocrystal structures revealed that the thiophene-containing moieties enable selectivity by stabilizing a flipped-out conformation of Phe67 of FKBP51. Our best compound, 19b, potently binds to FKBP51 biochemically as well as in mammalian cells, desensitize TRPV1 in primary sensory neurons, and has an acceptable PK profile in mice, suggesting its use as a novel tool compound for studying FKBP51 in animal models of neuropathic pain.

Binding pocket stabilization by high-throughput screening of yeast display libraries.

Protein dynamics have a great influence on the binding pockets of some therapeutic targets. Flexible protein binding sites can result in transient binding pocket formation which might have a negative impact on drug screening efforts. Here, we describe a protein engineering strategy with FK506-binding protein 51 (FKBP51) as a model protein, which is a promising target for stress-related disorders. High-throughput screening of yeast display libraries of FKBP51 resulted in the identification of variants exhibiting higher affinity binding of conformation-specific FKBP51 selective inhibitors. The gene libraries of a random mutagenesis and site saturation mutagenesis of the FK1 domain of FKBP51 encoding sequence were used to create a yeast surface display library. Fluorescence-activated cell sorting for FKBP51 variants that bind conformation-specific fluorescently labeled ligands with high affinity allowed for the identification of 15 different protein variants with improved binding to either, or both FKBP51-specific ligands used in the screening, with improved affinities up to 34-fold compared to the wild type. These variants will pave the way to a better understanding of the conformational flexibility of the FKBP51 binding pocket and may enable the isolation of new selective ligands that preferably and selectively bind the active site of the protein in its open conformation state.

Mechanism-Based Design of the First GlnA4-Specific Inhibitors.

γ-Glutamylamine synthetases are an important class of enzymes that play a key role in glutamate-based metabolism. Methionine sulfoximine (MSO) is a well-established inhibitor for the archetypal glutamine synthetase (GS) but inhibitors for most GS-like enzymes are unknown. Assuming a conserved catalytic mechanism for GS and GS-like enzymes, we explored if subtype-selective inhibitors can be obtained by merging MSO with the cognate substrates of the respective GS-like enzymes. Using GlnA4Sc from Streptomyces coelicolor, an enzyme recently shown to produce γ-glutamylethanolamine, we demonstrate that MSO can be reengineered in a straightforward fashion into potent and selective GlnA4Sc inhibitors. Linkage chemistry as well as linker length between the MSO moiety and the terminal hydroxyl group derived from ethanolamine were in agreement with the postulated phosphorylated catalytic intermediate. The best GlnA4 inhibitor 7 b potently blocked S. coelicolor growth in the presence of ethanolamine as the sole nitrogen source. Our results provide the first GlnA4Sc -specific inhibitors and suggest a general strategy to develop mechanism-based inhibitors for GS-like enzymes.

Picomolar FKBP inhibitors enabled by a single water-displacing methyl group in bicyclic [4.3.1] aza-amides.

Methyl groups can have profound effects in drug discovery but the underlying mechanisms are diverse and incompletely understood. Here we report the stereospecific effect of a single, solvent-exposed methyl group in bicyclic [4.3.1] aza-amides, robustly leading to a 2 to 10-fold increase in binding affinity for FK506-binding proteins (FKBPs). This resulted in the most potent and efficient FKBP ligands known to date. By a combination of co-crystal structures, isothermal titration calorimetry (ITC), density-functional theory (DFT), and 3D reference interaction site model (3D-RISM) calculations we elucidated the origin of the observed affinity boost, which was purely entropically driven and relied on the displacement of a water molecule at the protein-ligand-bulk solvent interface. The best compounds potently occupied FKBPs in cells and enhanced bone morphogenic protein (BMP) signaling. Our results show how subtle manipulation of the solvent network can be used to design atom-efficient ligands for difficult, solvent-exposed binding pockets.

Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation.

In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the 'four-dimensional' protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the 'toolbox' of mass spectrometry researchers studying higher-order protein structure.

Med Chem Remote: The Frontiers in Medicinal Chemistry 2021.

Digital, but delicious! The Frontiers in Medicinal Chemistry 2021 meeting, originally intended to take place in Darmstadt, carried on as an online event from March 8-10 this year. Even with pandemic restrictions, the event co-presented by the Medicinal Chemistry Division of the German Chemical Society (GDCh), the German Pharmaceutical Society (DPhG), and the Swiss Chemical Society (SCS) proved to be a success, showcasing excellent speakers and facilitating participant interaction in an ingenious virtual setting. Over 350 participants from more than 10 countries gathered to discuss the latest trends and directions in medicinal chemistry, with sessions on molecular glues, covalent fragments, transient binding pockets and more. This report presents a summary of the key lectures and activities at the event.

Macrocyclic FKBP51 Ligands Define a Transient Binding Mode with Enhanced Selectivity.

Subtype selectivity represents a challenge in many drug discovery campaigns. A typical example is the FK506 binding protein 51 (FKBP51), which has emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs. FKBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During a macrocyclization pilot study, we observed 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 a new binding mode featuring a transient conformation, which is disfavored for the small FKBPs. Using a conformation-sensitive assay we show that this binding mode occurs in solution and is characteristic for this new class of compounds. The discovered macrocycles are non-immunosuppressive, engage FKBP51 in cells, and block the cellular effect of FKBP51 on IKKα. Our findings provide a new chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.

Structure-Based Design of High-Affinity Macrocyclic FKBP51 Inhibitors.

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.

Enantioselective Synthesis of a Tricyclic, sp3 -Rich Diazatetradecanedione: an Amino Acid-Based Natural Product-Like Scaffold.

6-, 7-, and 8-membered rings are assembled from a linear precursor by successive cyclisation reactions to construct a tricyclic diazatricyclo[6.5.1.04, 9 ]-tetradecanedione scaffold. Advanced building blocks based on d-aspartic acid and l-pyroglutamic acid were combined by a sp3 -sp2 Negishi coupling. A carbamate-guided syn-diastereoselective epoxidation followed by an intramolecular epoxide opening allowed the construction of the piperidine ring. An efficient one-pot hydroxyl-group protection twofold deprotection reaction prepared the ground for the cyclisation to the bicycle. A final deprotection of the orthogonal protecting groups and lactamisation led to the novel, sp3 -rich tricycle. The final compound is a substrate mimic of peptidyl-prolyl cis-trans isomerases featuring a locked trans-amide bond. Cheminformatic analysis of 179 virtual derivatives indicates favourable physicochemical properties and drug-like characteristics. As proof of concept we, show a low micromolar activity in a fluorescence polarisation assay towards the FK506-binding protein 12.

FKBP51 and FKBP12.6-Novel and tight interactors of Glomulin.

The protein factor Glomulin (Glmn) is a regulator of the SCF (Skp1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex. Mutations of Glmn lead to glomuvenous malformations. Glmn has been reported to be associated with FK506-binding proteins (FKBP). Here we present in vitro binding analyses of the FKBP-Glmn interaction. Interestingly, the previously described interaction of Glmn and FKBP12 was found to be comparatively weak. Instead, the closely related FKBP12.6 and FKBP51 emerged as novel binding partners. We show different binding affinities of full length and truncated FKBP51 and FKBP52 mutants. Using FKBP51 as a model system, we show that two amino acids lining the FK506-binding site are essential for binding Glmn and that the FKBP51-Glmn interaction is blocked by FKBP ligands. This data suggest FKBP inhibition as a pharmacological approach to regulate Glmn and Glmn-controlled processes.

Initial Metabolic Step of a Novel Ethanolamine Utilization Pathway and Its Regulation in Streptomyces coelicolor M145.

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.