Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

Benaki, Dimitra; Paxinou, Eugenia; Magafa, Vassiliki; Pairas, George; Manessi‐Zoupa, Evy; Cordopatis, Paul; Mikros, Emmanuel

doi:10.1007/bf02443608

Cited by 4 publications

(4 citation statements)

References 45 publications

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“…The Fmoc group was removed with 25% piperidine in DMF. Activation was performed in situ using DIC/HOAt in DMF [21,22]. Couplings were performed with 3/3.3/4.5 molar excesses of Fmoc-amino acid/DIC/HOAt, respectively.…”

Section: Synthesis Of Leuprolidementioning

confidence: 99%

“…Liposomes are non-toxic carrier systems, which have been studied for intravenous delivery of, inter alia, lipophilic compounds. It has been shown that the physicochemical characteristics of liposomes, including lipid composition, surface charge and size, can modulate their in vivo stability and improve the pharmacokinetic properties of the encapsulated drugs [6][7][8][9][10][11][12][13], reducing side effects and enhancing activity: in recent years, liposomes have been successfully applied for drug delivery in numerous cases [14][15][16][17][18][19][20][21][22][23][24][25]. The liposomal formulation LILs was achieved using the thin film hydration method, following reversed phase evaporation methodology.…”

Section: Introductionmentioning

confidence: 99%

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Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide

Saroglou

Hatziantoniou

Smyrniotakis

et al. 2005

Journal of Peptide Science

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A novel liposomal formulation was developed for the encapsulation of the oligopeptide leuprolide (GlpHisTrpSerTyr-D-LeuLeuArgProNHEt), a potent analogue of gonadotropin releasing hormone used in the treatment of advanced prostate cancer, endometriosis and precocious puberty. Leuprolide was synthesized using solid phase methodology on a {3-[(ethyl-Fmoc-amino)-methyl]-1-indol-1-yl}-acetyl AM resin and Fmoc/tBu chemistry. The new liposomal formulation, called 'liposomes in liposomes' is composed of egg phosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 98.91:1.09 (internal liposomes) and egg phosphatidylcholine:dipalmitoylphosphatidylglycerol:cholesterol in a molar ratio of 68.71:0.76:30.53 (external liposomes). It offers high encapsulation efficiency (73.8% for leuprolide); it can provide new delivery characteristics and it may have possible advantages in future applications regarding the encapsulation and delivery of bioactive peptides to target tissues. Furthermore, the physicochemical characteristics (size distribution and zeta-potential) of the liposomal formulations and the thermal effects on leuprolide in model lipidic bilayers composed of dipalmitoylphosphatidylcholine were studied using differential scanning calorimetry. Finally, the dynamic effects of leuprolide in an egg phosphatidylcholine/cholesterol system were examined using solid state 13C MAS NMR spectroscopy.

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Section: Synthesis Of Leuprolidementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide

Saroglou

Hatziantoniou

Smyrniotakis

et al. 2005

Journal of Peptide Science

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“…As shown earlier for Leuprolide synthesis (32) and confirmed in this study, the use of DIC/HOAt as coupling reagent and the [3‐((ethyl‐Fmoc‐amino)‐methyl)‐1‐indol‐1‐yl]‐acetyl AM resin as solid support leads to high overall yield (55%). From our previous experience (33), the use of the Sieber Ethylamide resin (ethylamino‐xanthen‐3‐yloxy‐Merrifield resin) for the synthesis of Leuprolide afforded lower yield (∼29%) and purity. Besides, the use of the [3‐((ethyl‐Fmoc‐amino)‐methyl)‐1‐indol‐1‐yl]‐acetyl AM resin makes the synthesis of the ethylamide analogues less complicated avoiding the extra step of aminolysis with ethylamine.…”

Section: Resultsmentioning

confidence: 99%

GnRH analogues containing conformationally restricted amino acids in positions 3 and 6: differential impact on pituitary binding affinity and direct antiproliferative effect on breast cancer cells^†

Zompra

Magafa

Lamari

et al. 2005

The Journal of Peptide Research

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Analogues of GnRH have been widely used in oncology and gynaecology to induce reversible chemical castration. In addition to the classic hypophysiotropic action of GnRH, it has been shown that many malignant cells, such as breast cancer cells, secrete GnRH and express the GnRH receptor/s. In order to study the effect of modifications in position 3 and 6 of GnRH on both pituitary binding affinity and breast cancer cell proliferation, we synthesized eight new GnRH analogues. All GnRH analogues lacked the carboxy–terminal Gly10–amide of GnRH and an ethylamide residue was added to Pro9. Gly6 was substituted by α,α‐dialkyl amino acids (Aib: α‐aminoisobutyric acid, Deg: diethylglycine) and Trp3 by D–Trp, D‐ and L‐1,2,3,4,‐tetrahydro‐isoquinoline‐3‐carboxylic acid (Tic). During competition binding experiments in mouse anterior pituitary αT3‐1 cells, [Aib6,desGly10]GnRH‐NHEt bound to the GnRH receptor with IC50 values comparable to those of parent hormone in contrast to the analogues substituted at position 3. However, [L‐Tic3,Deg6,desGly10]GnRH‐NHEt had high pituitary binding affinity. With the exception of GnRH and [Aib6,desGly10]GnRH‐NHEt, all GnRH analogues significantly inhibited the proliferation of human breast cancer cells (MCF‐7); higher inhibitory effect was observed for analogues modified at position 3. Results show differential impact of the modifications on the binding affinity to the GnRH receptor in mouse pituitary cells and on the inhibition of human breast cancer cell proliferation and provide insight into structure‐activity relationship of GnRH in different biological systems.

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“…Although some sporadic attempts have been done to study the solution conformation of leuprolide, there is no NMR three-dimensional model available. [37][38][39] In this study, the solution structure of leuprolide was determined through NMR spectroscopy. Furthermore, structural differentiation imposed by the substitution of DLeu in position 6 by D/L Glu, an acidic amino acid, was also investigated.…”

Section: Antiproliferative Effect Of Leuprolide and Gnrh Analogs 261mentioning

confidence: 99%

Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin-releasing hormone peptide analogs

et al. 2011

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Analogs of GnRH, including [DLeu6, desGly1o]-GnRH-NHEt (leuprolide, commercial product), have been widely used in oncology to induce reversible chemical castration. Several studies have provided evidence that, besides their pituitary effects, GnRH analogs may exert direct antiproliferative effects on tumor cells. To study the effect of modifications in positions 4 and 6 of leuprolide on prostate cancer cell proliferation, we synthesized 12 new leuprolide analogs. All GnRH analogs lacked the carboxy-terminal Gly10-amide of GnRH, and an ethylamide residue was added to Pro9. Gly6 was substituted by DLys, Nepsilon-modified DLys, Glu, and DGlu. To improve the enzymatic stability, NMeSer was incorporated in position 4, and the rate of hydrolysis by alpha-chymotrypsin and subtilisin was investigated. Our results demonstrate that this incorporation increases enzymatic stability in all analogs of GnRH, whereas the antiproliferative effect on PC3 and LNCaP prostate cancer cells is similar to that of leuprolide. Conformational studies were performed to elucidate structural changes occurring on substitution of native residues and to study structure-activity relationship for these analogs. The solution models of [DLeu6, desGly10]-GnRH-NHEt (leuprolide), [NMeSer4, DGlu6, desGly10]-GnRH-NHEt, [Glu6, desGly10]-GnRH-NHEt, and [DGIu6, desGly10]-GnRH-NHEt peptides were determined through two-dimensional nuclear magnetic resonance spectroscopy in dimethylsulfoxide. Nuclear magnetic resonance data provide experimental evidence for the U-turn-like structure appeared in all four analogs, which could be characterized as beta-hairpin conformation. The most stable analog [NMeSer4, DGlu6, desGly10]-GnRH-NHEt against proteolytic cleavage forms a second extra backbone turn observed for residues 1-4.

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Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

Cited by 4 publications

References 45 publications

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide

GnRH analogues containing conformationally restricted amino acids in positions 3 and 6: differential impact on pituitary binding affinity and direct antiproliferative effect on breast cancer cells^†

Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin-releasing hormone peptide analogs

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Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

Cited by 4 publications

References 45 publications

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide

GnRH analogues containing conformationally restricted amino acids in positions 3 and 6: differential impact on pituitary binding affinity and direct antiproliferative effect on breast cancer cells†

Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin-releasing hormone peptide analogs

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Product

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GnRH analogues containing conformationally restricted amino acids in positions 3 and 6: differential impact on pituitary binding affinity and direct antiproliferative effect on breast cancer cells^†