Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

The small molecule activator S3969 stimulates the epithelial sodium channel by interacting with a specific binding pocket in the channel's ß-subunit.

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969-binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of ß-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics simulations predicted critical residues in the thumb domain of ß-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the ß-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. Molecular dynamics simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of ß-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (ßR437C - γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.

Structure-guided discovery of orexin receptor-binding PET ligands.

Molecular imaging using positron emission tomography (PET) can serve as a promising tool for visualizing biological targets in the brain. Insights into the expression pattern and the in vivo imaging of the G protein-coupled orexin receptors OX1R and OX2R will further our understanding of the orexin system and its role in various physiological and pathophysiological processes. Guided by crystal structures of our lead compound JH112 and the approved hypnotic drug suvorexant bound to OX1R and OX2R, respectively, we herein describe the design and synthesis of two novel radioligands, [18F]KD23 and [18F]KD10. Key to the success of our structural modifications was a bioisosteric replacement of the triazole moiety with a fluorophenyl group. The 19F-substituted analog KD23 showed high affinity for the OX1R and selectivity over OX2R, while the high affinity ligand KD10 displayed similar Ki values for both subtypes. Radiolabeling starting from the respective pinacol ester precursors resulted in excellent radiochemical yields of 93% and 88% for [18F]KD23 and [18F]KD10, respectively, within 20 min. The new compounds will be useful in PET studies aimed at subtype-selective imaging of orexin receptors in brain tissue.

Visualization of ß-adrenergic receptor dynamics and differential localization in cardiomyocytes.

A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two ß-adrenergic receptor (ß-AR) subtypes, ß1 and ß2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for ß1-AR but not for ß2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent ß-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the ß2-AR is confined to and diffuses within the T-tubular network, as opposed to the ß1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the ß2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the ß2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.

Development of disulfide-functionalized peptides covalently binding G protein-coupled receptors.

A broadly applicable synthesis of peptides incorporating mixed disulfides between cysteine and homocysteine and cysteamine was developed. The method was established using pharmacologically relevant G protein-coupled receptor (GPCR) ligands including the µ-receptor agonist Dmt-DALDA and extended to the orexin derivative Oxa(17-33) and NT(8-13), the C-terminal hexapeptide of neurotensin. The newly developed NT(8-13) analog 6b incorporating an S-functionalized homocysteine revealed covalent binding of the neurotensin receptor 1 (NTSR1) in a radioligand depletion study.

Sensitization to amphetamine psychostimulant effect: A key role for ventral tegmental area neurotensin type 2 receptors and MAP kinase pathway.

Neurotensin is an endogenous neuropeptide that acts as a potent modulator of ventral tegmental area (VTA) neurotransmission. The present study was aimed at determining VTA cell population and neurotensin receptor subtype responsible for the initiation of amphetamine-induced psychomotor activity and extracellular signal-regulated kinases (ERK1/2) sensitization. During an induction phase, rats were injected intra-VTA on two occasions, every second day, with [D-Tyr11 ]-neurotensin (D-Tyr-NT), SR142948 (a mix Ntsr1/Ntsr2 receptor subtype antagonist), SR48692 (a Ntsr1 antagonist), D-Tyr-NT + SR142498, D-Tyr-NT + SR48692, or the vehicle. Effects of intra-VTA drugs were evaluated at locomotor activity and ERK1/2 phosphorylation. Five days after the last VTA microinjection, the effect of a systemic injection of amphetamine was tested (sensitization test). Results show that D-Tyr-NT stimulated locomotor activity during the induction phase, an effect that was blocked by SR142948, but not SR48692. Amphetamine also induced significantly higher ambulatory activity in rats preinjected with D-Tyr-NT than in rats preinjected with the vehicle. This sensitization effect was again attenuated by SR142948, but not SR48692, hence suggesting that this effect is mediated by Ntsr2 receptors. To confirm this, we tested a highly selective Ntsr2 peptide-peptoid hybrid ligand, NT150. At the concentration tested, NT150 stimulated locomotor activity and lead to sensitized locomotor activity and a selective neurochemical (pERK1/2) response in tyrosine hydroxylase-positive neurons of the VTA. Both effects were prevented by SR142948. Taken together, these results show that neurotensin, acting on Ntsr2 receptor subtypes, stimulates locomotor activity and initiates neural changes (ERK1/2 phosphorylation) that lead to amphetamine-induced sensitization.

NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids.

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the µ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8-13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr11 in NT(8-13). Additionally, Arg8, Arg9, and Ile12 of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (Ki(NTS2): 29nM, Ki(NTS1): 35,000nM) was observed for the peptide analog 17Rtrans.

Improved radiosynthesis and preliminary in vivo evaluation of a (18)F-labeled glycopeptide-peptoid hybrid for PET imaging of neurotensin receptor 2.

The neurotensin receptor 2 (NTS2) is an attractive target for cancer imaging, as it is overexpressed in a variety of tumor types including prostate, pancreas and breast carcinoma. The aim of this study was the development of the first NTS2 subtype selective (18)F-labeled radioligand for imaging NTS2 expression in vivo by positron emission tomography (PET). The radiosynthesis of glycopeptoid (18)F-4 was realized by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), applying the prosthetic group 6-deoxy-6-[(18)F]fluoroglucosyl azide for (18)F-fluoroglycosylation of the alkyne-terminated NT(8-13) analog Pra-N-Me-Arg-Arg-Pro-N-homo-Tyr-Ile-Leu-OH. The binding affinity of the peptide-peptoid 4 for NTS2 was 7nM with excellent subtype selectivity over NTS1 (260-fold). In vitro autoradiography studies of rat brain slices confirmed the high selectivity of (18)F-4 for NTS2. Biodistribution experiments using HT29 and PC3 tumor-bearing nude mice revealed high renal and only moderate tumor uptake, while PET imaging experiments revealed specific binding of (18)F-4 in NTS2-positive tumors. As (18)F-4 displayed high stability in vitro but fast degradation in vivo, future work will focus on the development of metabolically more stable NT(8-13) analogs.

Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, while its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify novel CFTR modulators. We docked ~155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered novel mid-nanomolar potentiators as well as inhibitors that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Radical arylation of tyrosine and its application in the synthesis of a highly selective neurotensin receptor 2 ligand.

A small library of Fmoc-protected 3-arylated tyrosines was created by radical arylation. The new building blocks were successfully applied in the synthesis of two novel neurotensin receptor ligands. Both isomers showed high affinity for the human NTS2 receptor with K(i) values in the nanomolar range. Interestingly, subtype selectivity strongly depends on the configuration of the peptide in position 11. Isomer (11R)-3 displayed an excellent preference for NTS2 compared to NTS1.

Functional characterization of a partial loss-of-function mutation of the epithelial sodium channel (ENaC) associated with atypical cystic fibrosis.

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the alpha-subunit of ENaC (alphaF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type alpha beta gamma-ENaC or mutant alpha(F61L) beta gamma-ENaC in Xenopus laevis oocytes. The alphaF61L mutation reduced the ENaC mediated whole-cell currents by approximately 90%. In contrast, the mutation decreased channel surface expression only by approximately 40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (P(o)). This was confirmed by experiments using the betaS520C mutant ENaC which can be converted to a channel with a P(o) of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average P(o) of ENaC by approximately 75%. Na(+) self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel P(o). The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the alphaF61L mutation. We conclude that the alphaF61L mutation may contribute to respiratory symptoms in atypical CF patients.

Click-chemistry-derived tetracycline-amino acid conjugates exhibiting exceptional potency and exclusive recognition of the reverse tet repressor.

A click-chemistry-based synthesis of biologically active doxycycline-amino acid conjugates is described. Starting from 9-aminodoxycycline derivatives and complementary functionalized amino acids, ligation was accomplished by copper(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC). The final products were tested in a variety of TetR and revTetR systems, and the C-terminally linked phenylalanine conjugate 12 c exhibited high selectivity for revTetR over TetR. Besides the unique property of the specific effector 12 c to effectively differentiate TetR and its reverse phenotype, the test compound proved to be almost devoid of any antibacterial activity; this will be highly beneficial for future applications to control gene expression in bacterial systems.

Structure-based exploration of an allosteric binding pocket in the NTS1 receptor using bitopic NT(8-13) derivatives and molecular dynamics simulations.

Crystal structures of neurotensin receptor subtype 1 (NTS1) allowed us to visualize the binding mode of the endogenous peptide hormone neurotensin and its pharmacologically active C-terminal fragment NT(8-13) within the orthosteric binding pocket of NTS1. Beneath the orthosteric binding pocket, we detected a cavity that exhibits different sequences in the neurotensin receptor subtypes NTS1 and NTS2. In this study, we explored this allosteric binding pocket using bitopic test peptides of type NT(8-13)-Xaa, in which the C-terminal part of NT(8-13) is connected to different amino acids that extend into the newly discovered pocket. Our test compounds showed nanomolar affinities for NTS1, a measurable increase in subtype selectivity compared to the parent peptide NT(8-13), and the capacity to activate the receptor in an IP accumulation assay. Computational investigation of the selected test compounds at NTS1 showed a conserved binding mode within the orthosteric binding pocket, whereas the allosteric cavity was able to adapt to different residues, which suggests a high degree of structural plasticity within that cavity of NTS1.

Peptide backbone modifications on the C-terminal hexapeptide of neurotensin.

To compare backbone-induced susceptibilities with affinity changes that are caused by side-chain modifications in the respective positions, structure activity relationship studies on a series of NT(8-13) analogues were performed providing valuable insights into the major requirement for neurotensin receptor recognition and activation. The data led us to highly potent NTR1 ligands and the generation of a pharmacophore model that will be helpful for the discovery of therapeutically relevant non-peptidic NTR1 agonists.

Novel insights into GPCR-peptide interactions: mutations in extracellular loop 1, ligand backbone methylations and molecular modeling of neurotensin receptor 1.

Investigating prototypical interactions between NT(8-13) and the human neurotensin receptor 1 (hNTR1), we created a receptor-ligand model that was validated by site-directed mutagenesis and structure-activity relationship studies. Stabilization of the extracellular loop 1 (EL1) by pi-stacking clusters proved to be important for agonist binding when substitution of six conserved amino acids by alanine resulted in an agonist specific loss of maximal binding capacity. In agreement with our modeling studies, EL1 seems to adopt a clamp-type border area controlling the shape of the binding site crevice. Employing chemically manipulated peptide analogs as molecular probes, the impact of backbone modifications on receptor-ligand interaction, especially the influence on ligand conformation, was examined in binding studies and explained by in silico analysis.

Theranostic Value of Multimers: Lessons Learned from Trimerization of Neurotensin Receptor Ligands and Other Targeting Vectors.

Neurotensin receptor 1 (NTS1) is overexpressed on a variety of cancer entities; for example, prostate cancer, ductal pancreatic adenocarcinoma, and breast cancer. Therefore, it represents an interesting target for the diagnosis of these cancers types by positron emission tomography (PET) [...].

(18)F- and (68)Ga-Labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1.

The neurotensin (NT) receptor-1 (NTS1) is overexpressed in a variety of carcinomas and is therefore an interesting target for imaging with positron emission tomography (PET). The aim of this study was the development of new NT derivatives based on the metabolically stable peptide sequence NLys-Lys-Pro-Tyr-Tle-Leu suitable for PET imaging. The NT peptides were synthesized by solid-phase supported peptide synthesis and elongated with respective chelators (NODA-GA, DOTA) for (68)Ga-labeling or propargylglycine for (18)F-labeling via copper-catalyzed azide-alkyne cycloaddition. Receptor affinities of the peptides for NTS1 were in the range of 19-110 nM. Biodistribution studies using HT29 tumor-bearing mice showed highest tumor uptake for [(68)Ga]6 and [(68)Ga]8 and specific binding in small-animal PET studies. The tumor uptake of (68)Ga-labeled peptides in vivo significantly correlated with the in vitro Ki values for NTS1. [(68)Ga]8 displayed an excellent tumor-to-background ratio and could therefore be considered as an appropriate molecular probe for NTS1 imaging by PET.

Discovery and Characterization of Biased Allosteric Agonists of the Chemokine Receptor CXCR3.

In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the ß-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or ß-arrestin 2 recruitment. Concomitant structure-activity relationship studies indicated very steep structure-activity relationships, which steer the ligand bias between the ß-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further development and rational design of strongly biased allosteric agonists of CXCR3.

Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.