Heteroaryl derivatives of suvorexant as OX1R selective PET ligand candidates: Cu-mediated 18F-fluorination of boroxines, in vitro and initial in vivo evaluation.

BACKGROUND: The orexin receptor (OXR) plays a role in drug addiction and is aberrantly expressed in colorectal tumors. Subtype-selective OXR PET ligands suitable for in vivo use have not yet been reported. This work reports the development of 18F-labeled OXR PET ligand candidates derived from the OXR antagonist suvorexant and the OX1R-selective antagonist JH112. RESULTS: Computational analysis predicted that fluorine substitution (1e) and introduction of the fluorobenzothiazole scaffold (1f) would be suitable for maintaining high OX1R affinity. After multi-step synthesis of 1a-1f, in vitro OXR binding studies confirmed the molecular dynamics calculations and revealed single-digit nanomolar OX1R affinities for 1a-f, ranging from 0.69 to 2.5 nM. The benzothiazole 1f showed high OX1R affinity (Ki = 0.69 nM), along with 77-fold subtype selectivity over OX2R. Cu-mediated 18F-fluorination of boroxine precursors allowed for a shortened reaction time of 5 min to provide the non-selective OXR ligand [18F]1c and its selective OX1R congener [18F]1f in activity yields of 14% and 22%, respectively, within a total synthesis time of 52-76 min. [18F]1c and [18F]1f were stable in plasma and serum in vitro, with logD7.4 of 2.28 ([18F]1c) and 2.37 ([18F]1f), and high plasma protein binding of 66% and 77%, respectively. Dynamic PET imaging in rats showed similar brain uptake of [18F]1c (0.17%ID/g) and [18F]1f (0.15%ID/g). However, preinjection of suvorexant did not significantly block [18F]1c or [18F]1f uptake in the rat brain. Pretreatment with cyclosporine A to study the role of P-glycoprotein (P-gp) in limiting brain accumulation moderately increased brain uptake of [18F]1c and [18F]1f. Accordingly, in vitro experiments demonstrated that the P-gp inhibitor zosuquidar only moderately inhibited polarized, basal to apical transport of 1c (p < 0.05) and had no effect on the transport of 1f, indicating that P-gp does not play a relevant role in brain accumulation of [18F]1c and [18F]1f in vivo. CONCLUSIONS: The in vitro and in vivo results of [18F]1c and [18F]1f provide a solid basis for further development of suitable OXR PET ligands for brain imaging.

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-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.

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.

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.

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.

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.

The broad-spectrum antiinfective drug artesunate interferes with the canonical nuclear factor kappa B (NF-κB) pathway by targeting RelA/p65.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of standard antiviral therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy is based on the exploitation of cell-directed signaling inhibitors. The broad antiinfective drug artesunate (ART) offers additional therapeutic options such as oral bioavailability and low levels of toxic side-effects. Here, novel ART-derived compounds including dimers and trimers were synthesized showing further improvements over the parental drug. Antiviral activity and mechanistic aspects were determined leading to the following statements: (i) ART exerts antiviral activity towards human and animal herpesviruses, (ii) no induction of ART-resistant HCMV mutants occurred in vitro, (iii) chemically modified derivatives of ART showed strongly enhanced anti-HCMV efficacy, (iv) NF-κB reporter constructs, upregulated during HCMV replication, could be partially blocked by ART treatment, (v) ART activity analyzed in stable reporter cell clones indicated an inhibition of stimulated NF-κB but not CREB pathway, (vi) solid-phase immobilized ART was able to bind to NF-κB RelA/p65, and (vii) peptides within NF-κB RelA/p65 represent candidates of ART binding as analyzed by in silico docking and mass spectrometry. These novel findings open new prospects for the future medical use of ART and ART-related drug candidates.

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.

H-CRRETAWAC-OH, a lead structure for the development of radiotracer targeting integrin α5ß1?

Imaging of angiogenic processes is of great interest in preclinical research as well as in clinical settings. The most commonly addressed target structure for imaging angiogenesis is the integrin α(v)ß(3). Here we describe the synthesis and evaluation of [(18)F]FProp-Cys(*)-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys(*)-OH, a radiolabelled peptide designed to selectively target the integrin α(5)ß(1). Conjugation of 4-nitrophenyl-(RS)-2-[(18)F]fluoropropionate provided [(18)F]FProp-Cys(*)-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys(*)-OH in high radiochemical purity (>95%) and a radiochemical yield of approx. 55%. In vitro evaluation showed α(5)ß(1) binding affinity in the nanomolar range, whereas affinity to α(v)ß(3) and α(IIb)ß(3) was >50 µM. Cell uptake studies using human melanoma M21 (α(v)ß(3)-positive and α(5)ß(1)-negative), human melanoma M21-L (α(v)ß(3)-negative and α(5)ß(1)-negative), and human prostate carcinoma DU145 (α(v)ß(3)-negative and α(5)ß(1)-positive) confirmed receptor-specific binding. The radiotracer was stable in human serum and showed low protein binding. Biodistribution studies showed tumour uptake ranging from 2.5 to 3.5% ID/g between 30 and 120 min post-injection. However, blocking studies and studies using mice bearing α(5)ß(1)-negative M21 tumours did not confirm receptor-specific uptake of [(18)F]FProp-Cys(*)-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys(*)-OH, although this radiopeptide revealed high affinity and substantial selectivity to α(5)ß(1) in vitro. Further experiments are needed to study the in vivo metabolism of this peptide and to develop improved radiopeptide candidates suitable for PET imaging of α(5)ß(1) expression in vivo.

In vivo monitoring of the antiangiogenic effect of neurotensin receptor-mediated radiotherapy by small-animal positron emission tomography: a pilot study.

The neurotensin receptor (NTS1) has emerged as an interesting target for molecular imaging and radiotherapy of NTS-positive tumors due to the overexpression in a range of tumors. The aim of this study was to develop a 177Lu-labeled NTS1 radioligand, its application for radiotherapy in a preclinical model and the imaging of therapy success by small-animal positron emission tomography (µPET) using [68Ga]DOTA-RGD as a specific tracer for imaging angiogenesis. The 177Lu-labeled peptide was subjected to studies on HT29-tumor-bearing nude mice in vivo, defining four groups of animals (single dose, two fractionated doses, four fractionated doses and sham-treated animals). Body weight and tumor diameters were determined three times per week. Up to day 28 after treatment, µPET studies were performed with [68Ga]DOTA-RGD. At days 7-10 after treatment with four fractionated doses of 11-14 MBq (each at days 0, 3, 6 and 10), the tumor growth was slightly decreased in comparison with untreated animals. Using a single high dose of 51 MBq, a significantly decreased tumor diameter of about 50% was observed with the beginning of treatment. Our preliminary PET imaging data suggested decreased tumor uptake values of [68Ga]DOTA-RGD in treated animals compared to controls at day 7 after treatment. This pilot study suggests that early PET imaging with [68Ga]DOTA-RGD in radiotherapy studies to monitor integrin expression could be a promising tool to predict therapy success in vivo. Further successive PET experiments are needed to confirm the significance and predictive value of RGD-PET for NTS-mediated radiotherapy.

The therapeutically anti-prion active antibody-fragment scFv-W226: paramagnetic relaxation-enhanced NMR spectroscopy aided structure elucidation of the paratope-epitope interface.

Antibodies have become indispensable reagents with numerous applications in biological and biotechnical analysis, in diagnostics as well as in therapy. In all cases, selective interaction with an epitope is crucial and depends on the conformation of the paratope. While epitopes are routinely mapped at high throughput, methods revealing structural insights on a rather short timescale are rare. We here demonstrate paramagnetic relaxation-enhanced (PRE) NMR spectroscopy to be a powerful tool unraveling structural information about epitope-orientation in a groove spanned by the complementary determining regions. In particular, we utilize the spin label TOAC, which is fused to the peptidic epitope using standard solid-phase chemistry and which is characterized by a reduced mobility compared to, e.g., spin labels attached to the side-chain functionalities of cysteine or lysine residues. We apply the method to determine the orientation of helix 1 of the prion protein, which is the epitope for the therapeutically anti-prion active scF(v) fragment W226.

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.

Synthesis of a (68)ga-labeled peptoid-Peptide hybrid for imaging of neurotensin receptor expression in vivo.

The neurotensin receptor subtype 1 (NTS1) represents an attractive molecular target for imaging various tumors. Positron emission tomography (PET) gained widespread importance due to its sensitivity. We combined the design of a metabolically stable neurotensin analogue with a (68)Ga-radiolabeling approach. The (68)Ga-labeled peptoid-peptide hybrid [(68)Ga]3 revealed high stability, specific tumor uptake (0.7%ID/g, 65 min p.i.), and advantageous biokinetics in vivo using HT29 tumor-bearing nude mice. Because of the ability to internalize into NTS1-expressing tumor cells, [(68)Ga]3 proved to be highly suitable for a reliable and practical visualization of NTS1-expressing tumors in vivo by small animal PET.

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.

3,4,6-Tri-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranosyl phenylthiosulfonate: a thiol-reactive agent for the chemoselective 18F-glycosylation of peptides.

3,4,5-Tri-O-acetyl-2-[18F]fluoro-2-deoxy-d-glucopyranosyl 1-phenylthiosulfonate (Ac3-[18F]FGlc-PTS) was developed as a thiol-reactive labeling reagent for the site-specific 18F-glycosylation of peptides. Taking advantage of highly accessible 1,3,4,6-tetra-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranose, a three-step radiochemical pathway was investigated and optimized, providing Ac3-[18F]FGlc-PTS in a radiochemical yield of about 33% in 90 min (decay-corrected and based on starting [18F]fluoride). Ac3-[18F]FGlc-PTS was reacted with the model pentapeptide CAKAY, confirming chemoselectivity and excellent conjugation yields of >90% under mild reaction conditions. The optimized method was adopted to the 18F-glycosylation of the alphavbeta3-affine peptide c(RGDfC), achieving high conjugation yields (95%, decay-corrected). The alphavbeta3 binding affinity of the glycosylated c(RGDfC) remained uninfluenced as determined by competition binding studies versus 125I-echistatin using both isolated alphavbeta3 and human umbilical vein endothelial cells (Ki = 68 +/- 10 nM (alphavbeta3) versus Ki = 77 +/- 4 nM (HUVEC)). The whole radiosynthetic procedure, including the preparation of the 18F-glycosylating reagent Ac3-[18F]FGlc-PTS, peptide ligation, and final HPLC purification, provided a decay-uncorrected radiochemical yield of 13% after a total synthesis time of 130 min. Ac3-[18F]FGlc-PTS represents a novel 18F-labeling reagent for the mild chemoselective 18F-glycosylation of peptides indicating its potential for the design and development of 18F-labeled bioactive S-glycopeptides suitable to study their pharmacokinetics in vivo by positron emission tomography (PET).