RESUMO
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.
Assuntos
Proteínas de Ligação a Tacrolimo , Ligantes , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Humanos , Estereoisomerismo , Desenho de Fármacos , Ciclização , Relação Estrutura-Atividade , Oxirredução , Ligação ProteicaRESUMO
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.
Assuntos
Quimera de Direcionamento de Proteólise , Proteína 1A de Ligação a Tacrolimo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Proteínas de Ligação a Tacrolimo/química , Domínios Proteicos , Sítios de Ligação , Proteólise , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Doença dos Legionários/tratamento farmacológico , Doença dos Legionários/microbiologia , Proteínas de Ligação a Tacrolimo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Legionella/metabolismoRESUMO
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.
Assuntos
Glutamato-Amônia Ligase , Metionina Sulfoximina , Inibidores Enzimáticos/farmacologia , Etanolamina , Glutamato-Amônia Ligase/metabolismo , Glutamatos , Metionina Sulfoximina/farmacologia , Nitrogênio/metabolismoRESUMO
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.
Assuntos
Peróxido de Hidrogênio/química , Ferro/química , Proteínas/química , Álcool Desidrogenase/química , Sítios de Ligação , Heme/química , Modelos Moleculares , Mioglobina/química , Oxirredução , Peptídeos/química , Conformação Proteica , Estabilidade Proteica , Proteína 1A de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/químicaRESUMO
Subtype selectivity represents a challenge in many drug discovery campaigns. A typical example is the FK506 binding proteinâ 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.
Assuntos
Ligantes , Proteínas de Ligação a Tacrolimo/metabolismo , Sítios de Ligação , Ciclização , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Rodaminas/química , Rodaminas/metabolismo , Especificidade por Substrato , Tacrolimo/química , Tacrolimo/metabolismo , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Proteínas de Ligação a Tacrolimo/químicaRESUMO
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 179â 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.
Assuntos
Aminoácidos Básicos/química , Produtos Biológicos/síntese química , Piperidinas/química , Produtos Biológicos/química , Ciclização , Compostos de Epóxi/química , EstereoisomerismoRESUMO
Classâ IIa histone deacetylases (HDACs) show extremely low enzymatic activity and no commonly accepted endogenous substrate is known today. Increasing evidence suggests that these enzymes exert their effect rather through molecular recognition of acetylated proteins and recruiting other proteins like HDAC3 to the desired target location. Accordingly, classâ IIa HDACs like bromodomains have been suggested to act as "Readers" of acetyl marks, whereas enzymatically active HDACs of classâ I or IIb are called "Erasers" to highlight their capability to remove acetyl groups from acetylated histones or other proteins. Small-molecule ligands of classâ IIa histone deacetylases (HDACs) have gained tremendous attention during the last decade and have been suggested as pharmaceutical targets in several indication areas such as cancer, Huntington's disease and muscular atrophy. Up to now, only enzyme activity assays with artificial chemically activated trifluoroacetylated substrates are in use for the identification and characterization of new active compounds against classâ IIa HDACs. Here, we describe the first binding assay for this class of HDAC enzymes that involves a simple mix-and-measure procedure and an extraordinarily robust fluorescence lifetime readout based on [1,3]dioxolo[4,5-f]benzodioxole-based ligand probes. The principle of the assay is generic and can also be transferred to classâ I HDAC8.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos/métodos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Espectrometria de Fluorescência/métodos , Benzodioxóis/química , Benzodioxóis/metabolismo , Sítios de Ligação , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Histona Desacetilases/química , Humanos , LigantesRESUMO
BACKGROUND: The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied. METHODS: Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases. RESULTS: The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation. CONCLUSIONS: The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes. GENERAL SIGNIFICANCE: The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness.
Assuntos
Amidoidrolases/química , Histona Desacetilases/química , Termodinâmica , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Ligação de Hidrogênio , Ligantes , Multimerização Proteica , Estabilidade ProteicaRESUMO
A series of perfluorinated SAHA (PFSAHA) was prepared and profiled against a panel of human and bacterial members of the Histone deacetylase (HDAC) family. Some of the active substances show nanomolar inhibitory activity and several hundred fold selectivity for the HDAC like enzyme PA3774 from P. aeruginosa. The extraordinary selectivity against human HDACs results from the distinct oligomeric state of PA3774 which consists of two head-to-head dimers. The binding pocket is defined by the surface of both opposite monomers confining the access of ligands to the active site. In addition, the aromatic cap group of PFSAHA undergoes an edge-to-face aromatic interaction with phenylalanine from the opposite monomer.
Assuntos
Fluorocarbonos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Sítios de Ligação , Fluorocarbonos/síntese química , Fluorocarbonos/química , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Modelos Moleculares , Relação Quantitativa Estrutura-AtividadeRESUMO
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.
Assuntos
Alcaligenes/química , Proteínas de Bactérias/química , Bordetella/química , Histona Desacetilase 1/química , Alcaligenes/enzimologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ligação Competitiva , Bioensaio , Bordetella/enzimologia , Compostos de Dansil/química , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência , Histona Desacetilase 1/metabolismo , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Performing kinetic studies on protein ligand interactions provides important information on complex formation and dissociation. Beside kinetic parameters such as association rates and residence times, kinetic experiments also reveal insights into reaction mechanisms. Exploiting intrinsic tryptophan fluorescence a parallelized high-throughput Förster resonance energy transfer (FRET)-based reporter displacement assay with very low protein consumption was developed to enable the large-scale kinetic characterization of the binding of ligands to recombinant human histone deacetylases (HDACs) and a bacterial histone deacetylase-like amidohydrolase (HDAH) from Bordetella/Alcaligenes. For the binding of trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and two other SAHA derivatives to HDAH, two different modes of action, simple one-step binding and a two-step mechanism comprising initial binding and induced fit, were verified. In contrast to HDAH, all compounds bound to human HDAC1, HDAC6, and HDAC8 through a two-step mechanism. A quantitative view on the inhibitor-HDAC systems revealed two types of interaction, fast binding and slow dissociation. We provide arguments for the thesis that the relationship between quantitative kinetic and mechanistic information and chemical structures of compounds will serve as a valuable tool for drug optimization.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Alcaligenes/enzimologia , Bordetella/enzimologia , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Ligantes , Ligação ProteicaRESUMO
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.
Assuntos
Aminoidrolases/metabolismo , Bioensaio , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Pseudomonas aeruginosa/enzimologia , Aminoidrolases/antagonistas & inibidores , Ligação Competitiva , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Cinética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimentoRESUMO
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.
RESUMO
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.
Assuntos
DNA , Nanotecnologia , Ligantes , Ligação Proteica , DNA/químicaRESUMO
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 0.077â µM for FKBP51, reduced molecular weight (541.7â 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.
RESUMO
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.
Assuntos
Engenharia de Proteínas , Proteínas de Ligação a Tacrolimo , Proteínas de Ligação a Tacrolimo/química , Ligantes , Sequência de Aminoácidos , Domínio Catalítico , Conformação Proteica , Ligação ProteicaRESUMO
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.
Assuntos
Streptomyces coelicolor , Humanos , Streptomyces coelicolor/metabolismo , Poliaminas/metabolismo , Glutamato-Amônia Ligase/metabolismo , Nitrogênio/metabolismoRESUMO
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.
Assuntos
Mamíferos , Proteínas de Ligação a Tacrolimo , Camundongos , Animais , Proteínas de Ligação a Tacrolimo/metabolismo , Conformação Molecular , Mamíferos/metabolismoRESUMO
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.