Design, evaluation, and comparison of ghrelin receptor agonists and inverse agonists as suitable radiotracers for PET imaging.

Ghrelin agonist and inverse agonist radiotracers, suitable for positron emission tomography (PET), were developed to study the behavior of ghrelin receptor ligands in vivo and for further design of druggable peptides. The target peptides were synthesized on solid support and conjugated to the bifunctional chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), which is known to form a stable complex with Ga(3+). Complexation with (68)Ga could be achieved under mild conditions and led to radiotracers with high radiochemical purity and specific activity. The biological activity of the radiotracers was evaluated in vitro by an inositol phosphate turnover assay. Pharmacokinetic profile and metabolic stability of the (68)Ga-NODAGA-radiotracers were investigated by small animal PET in rodent. Ghrelin derived agonists presented very high kidney accumulation, negligible tissue distribution, fast blood clearance, and poor stability in blood. Contrarily, the inverse agonist radiotracer exhibited very high stability in blood, large diffusion in tissues, reasonable kidney and liver metabolism, and slow blood clearance. This pharmacokinetic profile makes the ghrelin inverse agonist motif KwFwLL-CONH(2) suitable for further development of radiotracers and a promising lead to design peptide-based therapeutics against obesity.

Development of a ghrelin receptor inverse agonist for positron emission tomography.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Targeted delivery of compounds to Trypanosoma brucei using the melamine motif.

There is an urgent need for the development of new drugs for the treatment of human African trypanosomiasis. The causative organism, Trypanosoma brucei, has been shown to have some unusual plasma membrane transporters, in particular the P2 aminopurine transporter and related permeases, which have been used for the selective targeting of trypanocidal compounds to the organism. In this paper, we report the addition of melamine-based P2-targeting motifs to three different classes of compound in order to try and improve activity through increased selective uptake. The classes reported here are fluoroquinolones, difluoromethylornithine and artesunate derivatives.

Ghrelin--a novel generation of anti-obesity drug: design, pharmacomodulation and biological activity of ghrelin analogues.

Ghrelin is a unique bioactive peptide with respect to both the structure and its biological function. This 28-amino acid peptide is modified with an n-octanoyl group at serine-3, and accordingly is the only lipidated biologically active peptide hormone known so far. Ghrelin binds to the so-called ghrelin or GHS receptor, a member of the class A of G-protein coupled receptors, which leads to Ca(2+) release intracellularly due to the activation of the Gq-system. Interestingly, the ghrelin receptor shows a significant constitutive activity which means that in addition to agonists and antagonists, inverse agonists play an important role in receptor modulation. In this review, the major activities of ghrelin are summarized with a strong focus on the regulation of food intake. So far reported agonists, antagonists and inverse agonists are shown and structure activitiy relationships are discussed. Furthermore, the application of ghrelin ligands as novel anti-obesity drugs is outlined and the state of the art in this field is summarized.

In vitro antileishmanial activity of fluoro-artemisinin derivatives against Leishmania donovani.

The antileishmanial activity of 19 fluoro-artemisinin derivatives was evaluated in vitro against the promastigote forms of Leishmania donovani. The most active compound BB 201, an amino derivative, exhibited an IC50 at about 1microM and no cross-resistance was found on miltefosine-resistant and sitamaquine-resistant lines. Despite these promising data, no activity was observed on intramacrophage amastigote stage. Although the membranes that have to be crossed by the compounds and pH conditions between intraerythrocyte Plasmodium and intramacrophage Leishmania have similarities, the targets affected by artemisinin derivatives in promastigotes could be differentially expressed in amastigotes.

Development of Substrate-Derived Sirtuin Inhibitors with Potential Anticancer Activity.

RhoGDIα is a key regulator of Rho proteins, coordinating their GTP/GDP and membrane/cytosol cycle. Recently, it was demonstrated by quantitative mass spectrometry that RhoGDIα is heavily targeted by post-translational lysine acetylation. For one site in its N-terminal domain, namely K52, we reported earlier that acetylation completely switches off RhoGDIα function. Herein we show that K52-acetylated RhoGDIα is specifically deacetylated by the sirtuin deacetylase Sirt2. We show that acetylation at K52 decelerates cervical cancer cell proliferation, suggesting RhoGDIα acetylation to be a promising therapeutic target. We demonstrate that treatment of cervical cancer cells with a RhoGDIα-derived K52-trifluoroacetylated, substrate-derived peptidic sirtuin inhibitor severely impairs cell proliferation. Finally, we conclude that the potency of substrate-derived sirtuin inhibitors depends on structural features, the substrate-derived amino acid sequence as a determinant for selectivity, as well as the presence of an acetyl-lysine analogue to increase its potency. These data reveal a prospective therapeutic potential for novel substrate-derived sirtuin inhibitors.

Integrating solid-state NMR and computational modeling to investigate the structure and dynamics of membrane-associated ghrelin.

The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide's secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide's positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8-17 form an α-helix, while residues 21-23 and 26-27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane.

Rational design of dual peptides targeting ghrelin and Y2 receptors to regulate food intake and body weight.

Ghrelin and Y2 receptors play a central role in appetite regulation inducing opposite effects. The Y2 receptor induces satiety, while the ghrelin receptor promotes hunger and weight gain. However, the food regulating system is tightly controlled by interconnected pathways where redundancies can lead to poor efficacy and drug tolerance when addressing a single molecule. We developed a multitarget strategy to synthesize dual peptides simultaneously inhibiting the ghrelin receptor and stimulating the Y2 receptor. Dual peptides showed dual activity in vitro, and one compound induced a slight diminution of food intake in a rodent model of obesity. In addition, stability studies in rats revealed different behaviors between the dual peptide and its corresponding monomers. The Y2 receptor agonist was unstable in blood, while the dual peptide showed an intermediate stability compared to that of the highly stable ghrelin receptor inverse agonist.

An aromatic region to induce a switch between agonism and inverse agonism at the ghrelin receptor.

The ghrelin receptor displays a high constitutive activity suggested to be involved in the regulation of appetite and food intake. Here, we have created peptides with small changes in the core binding motif -wFw- of the hexapeptide KwFwLL-NH(2) that can swap the peptide behavior from inverse agonism to agonism, indicating the importance of this sequence. Introduction of ß-(3-benzothienyl)-d-alanine (d-Bth), 3,3-diphenyl-d-alanine (d-Dip) and 1-naphthyl-d-alanine (d-1-Nal) at position 2 resulted in highly potent and efficient inverse agonists, whereas the substitution of d-tryptophane at position 4 with 1-naphthyl-d-alanine (d-1-Nal) and 2-naphthyl-d-alanine (d-2-Nal) induces agonism in functional assays. Competitive binding studies showed a high affinity of the inverse agonist K-(d-1-Nal)-FwLL-NH(2) at the ghrelin receptor. Moreover, mutagenesis studies of the receptor revealed key positions for the switch between inverse agonist and agonist response. Hence, only minor changes in the peptide sequence can decide between agonism and inverse agonism and have a major impact on the biological activity.

Ghrelin receptor: high constitutive activity and methods for developing inverse agonists.

The ghrelin receptor is a G protein-coupled receptor (GPCR) mainly distributed in the brain, and also expressed in peripheral tissues. Remarkably, the ghrelin receptor possesses a naturally high constitutive activity representing 50% of its maximal activity. Its endogenous ligand ghrelin is the only known orexigenic gastrointestinal peptide and plays a central role in the regulation of appetite, food intake, and energy homeostasis. Reducing the constitutive activity of the ghrelin receptor by inverse agonists is the strategy adopted by our group to develop anti-obesity drugs. Therefore, short peptides were synthesized and showed high inverse agonist potency toward the ghrelin receptor. This review describes the methods used to synthesize the peptides and to evaluate their biological activity. Peptide synthesis was performed on solid phase using a Fmoc/tBu-strategy. Peptide potency was measured with a signal transduction assay, the inositol trisphosphate turnover assay, adapted to a receptor expressing constitutive activity.

S(N)/S(N)' competition: selective access to new 10-fluoro artemisinins.

In this paper, we report a simple route to accede to a new family of C-10 fluorinated derivatives of artemisinin 7. We demonstrated that nucleophilic substitution of the allylic bromide 6 with alcohols can occur at carbon 10 (compounds 7) under solvolytic conditions (S(N)'/S(N) ratio, 87:13). Furthermore, using the particular properties of hexafluoroisopropanol (HFIP), we are able to increase the selectivity of the substitution. Primary alcohols are completely selective for allylic substitution. With amines as nucleophiles, selectivity of substitution is dependent on their nucleophilicity, but attack at carbon 16 was always favored. However, the S(N)'/S(N) ratio could be slightly increased by adding HFIP, which is able to modulate their nucleophilicity through hydrogen bonding. In preliminary in vitro assessments, these new compounds, 7, exhibited a satisfying activity against malaria.