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The non-natural amino acid mesitylalanine (2,4,6-trimethyl-L-phenylalanine; Msa) has an electron-richer and a more conformationally restricted side-chain than that of its natural phenylalanine counterpart. Taking these properties into account, we have synthesized ten somatostatin analogs containing Msa residues in different key positions to modify...
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... modifications normally include changes in hydrophobicity, stability, conformation and biological activity of the new molecules [1][2][3][4]. Somatostatin [5,6], also known as somatotropin release-inhibiting factor (SRIF-14) (Figure 1), is one of the most studied peptides due to its important biological properties. This hormone is produced in the hypothalamus and is involved in multiple biological functions, mediated by its direct interaction with at least five different G-protein coupled receptors, named SSTR1-5 [7,8]. ...
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Regulating the formation of nanostructural morphology of synthetic hybrid peptides through external stimuli is still a great challenge. Here, we report the synthesis of constrained amino acid building block gabapentin (Gpn) based hybrid peptides and their structural and morphological diversity in different conditions. The synthesized three hybrid p...
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... Obtained results showed that peptides 1 and 2 are relatively stable in human plasma (t 1/2 ~ 8 h and 2 h, respectively) compared to many natural peptides. 81,82 . ...
... Other examples include insulin [58] and modifications of native hormones to optimize analogs with improved stability and specificity, like somatostatin, octreotide, leuprolide, and icatibant, to name a few. Some of these improvements have been made possible by exploring the conformational space sampled by the native peptides in solution [30,32,59,60]. ...
Tumor necrosis factor (TNF) is a homotrimer that has two spatially distinct binding regions, three lectin-like domains (LLD) at the TIP of the protein and three basolaterally located receptor-binding sites, the latter of which are responsible for the inflammatory and cell death-inducing properties of the cytokine. Solnatide (a.k.a. TIP peptide, AP301) is a 17-mer cyclic peptide that mimics the LLD of human TNF which activates the amiloride-sensitive epithelial sodium channel (ENaC) and as such recapitulates the capacity of TNF to enhance alveolar fluid clearance, as demonstrated in numerous preclinical studies. TNF and solnatide interact with glycoproteins and these interactions are necessary for their trypanolytic and ENaC-activating activities. In view of the crucial role of ENaC in lung liquid clearance, solnatide is currently being evaluated as a novel therapeutic agent to treat pulmonary edema in patients with moderate-to-severe acute respiratory distress syndrome (ARDS), as well as severe COVID-19 patients with ARDS. To facilitate the description of the functional properties of solnatide in detail, as well as to further target-docking studies, we have studied its folding properties by NMR. In solution, solnatide populates a set of conformations characterized by a small hydrophobic core and two electrostatically charged poles. Using the structural information determined here and also that available for the ENaC protein, we propose a model to describe solnatide interaction with the C-terminal domain of the ENaCα subunit. This model may serve to guide future experiments to validate specific interactions with ENaCα and the design of new solnatide analogs with unexplored functionalities.
... These modifications stabilize a characteristic pharmacophoric -turn motif, provide increased resistance to proteolytic degradation, and confer subtype selectivity (31). For example, the in vivo half-life of octreotide (trade name Sandostatin), an SS drug analog approved for the treatment of GH-producing tumors, is 90 min following intravenous infusion compared to 1 to 3 min for native human SS (32,33). ...
Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.
... As the mono-acylation probably requires less energy than the dilactoyl addition, the same trend may have also occurred for Oct-Ac and Oct-Pam but could not be observed due to their higher acylation rates compared to Oct-SDS. The acylation patterns were similar between the different octreotide salt complexes, the major impurity generated being a di-lactoyl addition on octreotide N-terminal -NH 2. Encouragingly, this degradation product may preserve octreotide activity as it is widely accepted that the Phe-Trp-Lys-Thr sequence constitute the pharmacophore [47]. On the contrary, following the same rationale, one should expect that lysine acylation would affect octreotide activity. ...
The generation of acylated impurities has represented an important hurdle in the development of long acting injectables for therapeutic peptides using biocompatible polymers with a polyester moiety. We investigated here an in situ forming depot (ISFD) technology that uses polyethylene glycol - polyester copolymers and a solvent exchange mechanism to promote depot formation. This technology has shown promises in formulating small molecules as well as therapeutic proteins. In the present work, using the well-known somatostatin analog octreotide acetate (OctAc) as a model molecule, we evaluated this delivery platform to release therapeutic peptides. Peptide acylation was found to be pronounced in the formulation, while it was very limited once the depot was formed and during the release process. The octreotide acylation pattern was fully characterized by LC-MS/MS. Moreover, it was demonstrated that exchanging the acetate anion with more hydrophobic counterions like pamoate or lauryl sulfate allowed to greatly improve the peptide stability profile, as well as the formulation release performance. Finally, the in vivo evaluation through pharmacokinetics studies in rat of these new octreotide salts in ISFD formulations showed that octreotide was quantifiable up to four weeks post-administration with a high bioavailability and an acceptable initial burst.
... 1-month sustained release subcutaneous depot formulations (Somatuline Autogel® and Somatuline Depot®) were developed to avoid daily injections (Bronstein et al. 2005;Cendros et al. 2005;Castinetti et al. 2009;Gomez et al. 2012). Lanreotide contains 2 unnatural amino acid residues at positions 1 and 4 followed by an amidated Thr8 (Fig. 16), increasing its elimination half-life to 23 to 30 days compared to 3 min for endogenous somatostatin (Martin-Gago et al. 2013). Less than 5% and 0.5% of the drug is excreted unchanged in the urine and faeces respectively after subcutaneous administration, suggesting the peptide undergoes extensive metabolism in humans (Somatuline package insert 2014). ...
Although peptide drugs make up only about 2% of all drugs approved by the United States Food and Drug Administration (FDA), they play important roles in the treatment of certain diseases where no small molecule drugs or therapeutic antibodies can fulfil. A primary reason for the strong preference of small molecules over peptides is the latter’s susceptibility to degradation by human proteases, resulting in very short systemic half-lives. To circumvent this, peptide chemists have resorted to introducing unnatural amino acids and chemical modifications to enhance their metabolic stabilities. Hence, understanding how peptide drugs are metabolized by the human body will help in the design of more stable peptide drugs. This review covers 25 FDA-approved peptide drugs up to 20 residues in length and details their metabolic and degradation pathways in the human body after administration.
... In contrast to the wealth of conformational studies available for SST and its analogues [16][17][18][19][20][21][22] , there is no information on the conformations of CST in solution to date. To bridge this gap of knowledge, we synthesised CST (r,mCST-14, Fig. 1a) using standard Fmoc/ t Bu solid-phase peptide synthesis (as described in the Methods section and in Supplementary Fig. 1a) 23 , and studied its folding properties in solution by NMR. ...
... For instance, it is well known that substitution of L-Trp to D-Trp increases the population of the native β-turn and of and the analogue half-life in serum 27 . Also, substitutions of Phe by L-mesityl alanine (2,4,6-trimethylphenylalanine, Msa) favour π-π interactions between aromatic rings and increase the overall conformational stability of SST derivatives 19,20 . For these reasons, in all the CST analogues, L-Trp was replaced by D-Trp in position 7 and Ser11 was replaced by Thr11 to stabilise the β-turn; and either Phe6 or Phe5 were replaced by Msa (analogues 2 and 4; Fig. 1c). ...
... Since CST binds in vitro to the five known Somatostatin receptors 19,20,22,30,31 with nanomolar affinity, we examined whether these analogues maintained similar profiles. Analogues 2-3 had a high preference towards receptor 2, like all SST analogues currently on the market. ...
Ulcerative colitis and Crohn’s disease are forms of inflammatory bowel disease whose incidence and prevalence are increasing worldwide. These diseases lead to chronic inflammation of the gastrointestinal tract as a result of an abnormal response of the immune system. Recent studies positioned Cortistatin, which shows low stability in plasma, as a candidate for IBD treatment. Here, using NMR structural information, we design five Cortistatin analogues adopting selected native Cortistatin conformations in solution. One of them, A5, preserves the anti-inflammatory and immunomodulatory activities of Cortistatin in vitro and in mouse models of the disease. Additionally, A5 displays an increased half-life in serum and a unique receptor binding profile, thereby overcoming the limitations of the native Cortistatin as a therapeutic agent. This study provides an efficient approach to the rational design of Cortistatin analogues and opens up new possibilities for the treatment of patients that fail to respond to other therapies.
... In contrast to the wealth amount of conformational studies available for somatostatin and its analogues [16][17][18][19][20][21][22] , there is no information describing the conformations of cortistatin in solution to date. To bridge this gap, we synthesized cortistatin (peptide 1, Fig. 1b,c) using standard Fmoc/ t Bu solid-phase peptide synthesis (as described in the methods and in Supplementary Fig. 1) 23 , and studied its folding properties in solution by NMR. ...
... For instance, it is well known that substitution of L-Trp to D-Trp increases the population of the native β-turn, as well as the hormone activity and its half-life in serum 27 . It has also been shown that substitutions of Phe, by L-mesityl alanine (3,5-dimethylphenylalanine, Msa) favored π-π interactions between aromatic rings and increased the overall conformational stability of SST derivatives 19,20 . For these reasons, in all CST analogs L-Trp was replaced by D-Trp in position 7 and Ser11 was replaced by Thr11 to stabilize the β-turn; and either Phe6 or Phe5 were replaced by Msa (analogs 2 and 4) (Fig. 1b,c). ...
... Since cortistatin binds in vitro with nanomolar affinity to the five known Somatostatin receptors, we set to investigate if the new analogs maintained similar profiles 19,20,22,30,31 . ...
Ulcerative colitis and Crohn’s disease are inflammatory bowel diseases (IBD) that lead to chronic inflammations of the gastrointestinal tract due to an abnormal response of the immune system. Finding new effective drugs to tackle IBD represents a major therapeutic concern since IBD incidence and prevalence is increasing worldwide. Recent studies positioned Cortistatin (CST) as a candidate for IBD treatment due to its anti-inflammatory and immunomodulatory activity. Here, we studied the structural properties of CST using NMR and synthesized and characterized new analogs displaying enriched populations of some native conformations. One of them, Analog 5, preserved the activity against IBD with an increased half-life in serum, overcoming the native hormone limitation and opening the door for the use of CST analogs as therapeutic agents. This work represents a new approach to the rational design of molecules to treat IBD and a possibility for patients that fail to respond to other therapies.
Graphical abstract
... peptides showed high conformational stability (particularly those that also had a D-Trp in position 8), thereby allowing the determination of their 3D structures by NMR for the first time, in a similar way as previous studies devoted to short-ring analogs, including Octreotide. These structures confirmed the relevance of non-covalent interactions between aromatic residues in positions 6, 7 and 11 in the modulation of the conformation and binding behavior of the correspondent Somatostatin analogs [22][23][24] . ...
... Fmoc-L-Dfp-OH was obtained in excellent yield and optical purity after acid hydrolysis of (3) (to give 4) and final Fmoc-protection 22 . Peptides 1-6 ( Fig. 1) were prepared by standard Fmoc/tBu solid-phase peptide synthesis (SPPS) 35 using 2-chlorotrityl chloride resin [22][23][24] . The schematic representation of the synthetic strategy used for the six peptides is shown in Fig. 2b. ...
... The affinity for SSTR3 dropped one order of magnitude compared to peptide 1, whereas that towards SSTR2 increased. Overall, the geometry of peptide 4 was similar to that of Somatostatin analogs with Msa in position 7, previously described by our group [22][23][24] . The main difference was that the π − π interaction was now offset-stacked instead of edge-to-face. ...
Somatostatin is a 14-residue peptide hormone that regulates the endocrine system by binding to five G-protein-coupled receptors (SSTR1–5). We have designed six new Somatostatin analogs with L-3-(3′,5′-difluorophenyl)-alanine (Dfp) as a substitute of Phe and studied the effect of an electron-poor aromatic ring in the network of aromatic interactions present in Somatostatin. Replacement of each of the Phe residues (positions 6, 7 and 11) by Dfp and use of a D-Trp8 yielded peptides whose main conformations could be characterized in aqueous solution by NMR. Receptor binding studies revealed that the analog with Dfp at position 7 displayed a remarkable affinity to SSTR2 and SSTR3. Analogs with Dfp at positions 6 or 11 displayed a π-π interaction with the Phe present at 11 or 6, respectively. Interestingly, these analogs, particularly [D-Trp8,L-Dfp11]-SRIF, showed high selectivity towards SSTR2, with a higher value than that of Octreotide and a similar one to that of native Somatostatin.
... [L-Mas 7 ]-SRIF is SSTR2selective, but [L-Mas 7 -D-Trp 8 ]-SRIF binds to SSTR1 and SSTR2. [L-Mas 11 ]-SRIF is also SSTR2-selective, but [L-Mas 11 -D-Trp 8 ]-SRIF switchs its selectivity to SSTR5 [65]. In a study by Reubi et al., the N-Imidazolebenzyl-histidine (imBzl-his) was used to replace Trp 8 . ...
Somatostatin (SST) is a cyclic hormone-release inhibitory peptide that has high binding affinity to all of its five SST receptors (SSTRs). SST negatively regulates cell proliferation and the release of multiple hormones via activation of its cognate receptors. A variety of SST analogs, some with high affinity and selectivity of receptor subtypes, have been synthesized and developed. Certain long-acting SST analogs such as octreotide, lanreotide and pasireotide have been clinically applied to the treatment of human diseases caused by excessive release of growth hormone (acromegaly), or adrenocorticotropic hormone (Cushing's syndrome), and for the treatment of carcinoid syndrome. Investigations into new biological activities of these long-acting SSTs and their possible clinical applications are also still ongoing. Also, novel SST analogs are being designed and developed to target different receptor subtype(s) or mimic natural SST's multiple biological properties. Additionally, since SSTRs, especially SSTR2, are aberrantly expressed in many cancer cells and tumor blood vessels, internalizing SST analogs is currently being used as drug-delivery vehicle for the application of receptor-targeted therapeutics. This review will discuss recent advances in the development and applications of SST and its analogs.
Intratumorale Heterogenität (ITH) kommt bei allen Tumorentitäten vor und resultiert aus der für Tumoren charakteristischen genomischen Instabilität. Diese genomische Instabilität ist einer der Hauptgründe für den krebsassoziierten Tod von Patienten und ist zudem generelles Charakteristikum für malignes (bösartiges) Wachstum von Tumoren. Die ITH kann sowohl bei „low stage“ Primärtumoren als auch bei hoch differenzierten metastasierenden „high stage“ Tumoren beobachtet werden.
Aus dieser intratumoralen Heterogenität resultieren ferner massive Schwierigkeiten bei der Behandlung von Tumorerkrankungen durch die Entstehung von Therapieresistenzen. Im Falle der neuroendokrinen Tumoren konnte festgestellt werden, dass diese ITH im Zusammenhang mit der spezifischen Expression unterschiedlicher Rezeptoren steht und dabei abhängig vom Tumorstadium die Rezeptorexpression stark variiert. In diesem Fall handelt es sich um den humanen Somatostatinrezeptor (Subtyp-2) (hSst2-R) und den humanen glukoseabhängigen insulinotropen Peptidrezeptor (engl. gastric inhibitory polypetide receptor, kurz, hGIP-R). In der Literatur wird beschrieben, dass in 88 % der Fälle eines Somatostatinrezeptor-negativen (hSst2-R-negativen) Tumors hingegen der hGIP-Rezeptor in überexprimierter Form vorliegt.
Ziel dieser Dissertation war es daher, im Rahmen der Tumorentität der neuroendokrinen Tumoren ein Radiopharmakon zu entwickeln, mit welchem sich der hSst2- und zugleich der hGIP-Rezeptor adressieren lässt. Hierfür wurden zunächst zwei stabil transfizierte Zelllinien etabliert, welche zum einen den Somatostatin- und zum anderen den hGIP-Rezeptor überexprimieren. Für die Herstellung der peptidbasierten Radiopharmaka wurde ein vollautomatischer Peptidsynthesizer qualifiziert, mit welchem die unterschiedlichen Somatostatin- und hGIP-R-Analoga synthetisiert wurden. Dadurch, dass im heterobivalenten Konstrukt sowohl das hSst2-R- als auch das hGIP-R-Bindemotiv enthalten sein sollen, wurden im ersten Schritt unterschiedliche hSst2-R-Analoga synthetisiert. Durch deren spezifischen Modifikationen sollten die pharmakologischen Eigenschaften sowohl des Monomers (im Vergleich zu [68Ga]Ga-DOTA-TATE(Tyr3)), als auch als Bestandteil des heterobivalenten Endprodukts verbessert werden. Im zweiten Schritt wurden anhand eines Alanine Walk die für die Rezeptorbindung kritischen Aminosäuren des GIP(1-30)-NH2-Analogon ausfindig gemacht. Die daraus gewonnenen Erkenntnisse sollten dafür verwendet werden, die pharmakologischen Eigenschaften des GIP(1-30)-NH2 in weiterführenden Versuchen, sowohl für das Monomer, als auch als Bestandteil des heterobivalenten Konstrukts zu verbessern. Im letzten Schritt der Arbeit sollten beide Rezeptorbindemotive (für hSst2-R und hGIP-R) miteinander verbunden werden, mit dem Ziel beide Rezeptoren spezifisch zu binden.
