RESUMO
In this research article, we report on the strengthening of a non-classical hydrogen bond (C-H···O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E-selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre-organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre-organization, the stabilization by a non-classical hydrogen bond between the H-C5 of l-fucose and the ring oxygen O5 of the neighboring d-galactose moiety is essential and yields 7.4 kJ mol-1. This effect could be further strengthened by replacing l-fucose by 6,6,6-trifluoro-l-fucose leading to an improved non-classical H-bond of 14.9 kJ mol-1, i.e., an improved pre-organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR-shifts of the H-C5Fuc, by X-ray diffraction analysis of glycomimetics co-crystallized with E-selectin as well as by isothermal titration calorimetry. Furthermore, the electron-withdrawing character of the CF3-group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid-stability a prerequisite for gastrointestinal stability could be substantially improved.
RESUMO
The d-GlcNAc moiety in sialyl Lewisx (sLex, 1) acts predominantly as a linker to position the d-Gal and the l-Fuc moieties in the bioactive spatial orientation. The hypothesis has been made that the NHAc group of GlcNAc pushes the fucose underneath the galactose and, thus, contributes to the stabilization of the bioactive conformation of the core of sLex (1). To test this hypothesis, GlcNAc mimetics consisting of (R,R)-1,2-cyclohexanediols substituted with alkyl and aryl substituents adjacent to the linking position of the fucose moiety were synthesized. To explore a broad range of extended and spatially demanding R-groups, an enzymatic approach for the synthesis of 3-alkyl/aryl-1,2-cyclohexanediols (3b-n) was applied. These cyclohexanediol derivatives were incorporated into the sLex mimetics 2b-n. For analyzing the relationship of affinity and core conformation, a 1H NMR structural-reporter-group concept was applied. Thus, the chemical shift of H-C5Fuc proved to be a sensitive indicator for the degree of pre-organization of the core of this class of sLex mimetics and therefore could be used to quantify the contribution of the R-groups.
Assuntos
Fucose , Oligossacarídeos , Antígeno Sialil Lewis X , Oligossacarídeos/química , Fucose/química , Conformação Molecular , Espectroscopia de Ressonância MagnéticaRESUMO
The selectin family consisting of E-, P- and L-selectin plays dominant roles in atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, and metastatic spreading of some cancers. An early goal in selectin-targeted drug discovery campaigns was to identify ligands binding to all three selectins, so-called pan-selectin antagonists. The physiological epitope, tetrasaccharide sialyl Lewisx (sLex, 1) binds to all selectins, albeit with very different affinities. Whereas P- and L-selectin require additional interactions contributed by sulfate groups for high binding affinity, E-selectin can functionally bind sLex-modified glycolipids and glycoproteins. Rivipansel (3) marked the first pan-selectin antagonist, which simultaneously interacted with both the sLex and the sulfate binding site. The aim of this contribution was to improve the pan-selectin affinity of rivipansel (3) by leveraging a new class of sLex mimetics in combination with an optimized linker length to the sulfate bearing group. As a result, the pan-selectin antagonist 11b exhibits an approximatively 5-fold improved affinity for E-, as well as P-selectin.
Assuntos
Selectinas , Humanos , Selectinas/metabolismo , Relação Estrutura-Atividade , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Oligossacarídeos/síntese química , Estrutura Molecular , Antígeno Sialil Lewis X , Relação Dose-Resposta a Droga , Selectina E/metabolismo , Selectina E/antagonistas & inibidores , GlicolipídeosRESUMO
The aim was to investigate the feasibility of using iontophoresis for the cutaneous delivery of the E-selectin antagonist CGP69669A, a sialyl Lewis(x) -glycomimetic with potential activity against inflammatory skin diseases. The effects of current density and formulation on iontophoretic transport were evaluated in porcine and human skin in vitro. Cumulative permeation of CGP69669A increased with current density (69.73±9.51, 113.97±26.80 and 160.44±13.79µg/cm(2) at 0.1, 0.3 and 0.5mA/cm(2) , respectively) and drug concentration (37.42±13.13, 78.96±23.13 and 160.44±13.79µg/cm(2) , at 1, 3 and 5mg/ml, respectively). In contrast, passive delivery was negligible. Although permeation from a 2% hydroxyethyl cellulose gel was lower than that from aqueous solution, skin deposition - more relevant for the local treatment of dermatological conditions - was 3-fold higher. The results demonstrated that although CGP69669A cannot be delivered passively into the skin it is an excellent candidate for transdermal iontophoresis, a technique that is ideally suited to the delivery of glycomimetics.
Assuntos
Iontoforese/métodos , Oligossacarídeos/administração & dosagem , Pele/metabolismo , Administração Cutânea , Animais , Selectina E/metabolismo , Estudos de Viabilidade , Humanos , Técnicas In Vitro , SuínosRESUMO
A new class of N-acetyl-D-glucosamine (GlcNAc) mimics for E-selectin antagonists was designed and synthesized. The mimic consists of a cyclohexane ring substituted with alkyl substituents adjacent to the linking position of the fucose moiety. Incorporation into E-selectin antagonists led to the test compounds 8 and the 2'-benzoylated analogues 21, which exhibit affinities in the low micromolar range. By using saturation transfer difference (STD)-NMR it could be shown that the increase in affinity does not result from an additional hydrophobic contact of the alkyl substituent with the target protein E-selectin, but rather from a steric effect stabilizing the antagonist in its bioactive conformation. The loss of affinity found for antagonists 10 and 35 containing a methyl substituent in a remote position (and therefore unable to support to the stabilization of the core) further supports this hypothesis. Finally, when a GlcNAc mimetic containing two methyl substituents (52 and 53) was used, in which one methyl was positioned adjacent to the fucose linking position and the other was in a remote position, the affinity was regained.
Assuntos
Acetilglucosamina/química , Selectina E/química , Selectina E/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A simple, rapid, precise and specific isocratic HPAE-PAD method for quantification of CGP69669A was developed and validated. CGP69669A is a glycomimetic of sialyl Lewis(x) and an antagonist of E-selectin with potential application in the treatment of inflammatory skin disease. Quantification was performed using a Dionex CarboPac(TM) PA-200 anion-exchange column (3 × 250 mm) with 100 mm NaOH solution as mobile phase, a flow rate of 0.50 mL/min and an injection volume of 10 µL. A quadruple potential waveform was used to detect the carbohydrate (+0.1 V from 0.00 to 0.40 s, -2.0 V from 0.41 to 0.42 s, +0.6 V at 0.43 s and -0.1 V from 0.44 to 0.50 s with current integrated between 0.20 and 0.40 s for detection) and rafinose was employed as an internal standard. The optimized conditions enabled rapid elution of CGP69669A (at 3.0 min) without interference from solvent peaks or substances present in the skin. The method showed good intra- and inter-day precision and accuracy and the response was linear from 1.0 to 25 µg/mL. This is the first validated direct method for the quantification of CGP69669A. It will now be employed in studies investigating the topical and transdermal delivery of CGP69669A in vitro and in vivo and it should also be of use for other applications of this molecule.
Assuntos
Cromatografia por Troca Iônica/métodos , Oligossacarídeos/farmacocinética , Pele/metabolismo , Animais , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/economia , Selectina E/metabolismo , Limite de Detecção , Modelos Lineares , Oligossacarídeos/análise , Absorção Cutânea , SuínosRESUMO
Because of their large polar surface area, carbohydrates often exhibit insufficient pharmacokinetic properties. Specifically, the carboxylic acid function of the tetrasaccharide sialyl Lewisx , a pharmacophore crucial for the formation of a salt bridge with selectins, prevents oral availability. A common approach is the transfer of carboxylic acid into ester prodrugs. Once the prodrug is either actively or passively absorbed, the active principle is released by hydrolysis. In the present study, ester prodrugs of selectin antagonists with aliphatic promoieties were synthesized and their potential for oral availability was investigated inâ vitro and inâ vivo. The addition of lipophilic ester moieties to overcome insufficient lipophilicity improved passive permeation into enterocytes, however at the same time supported efflux back to the small intestines as well as oxidation into non-hydrolysable metabolites. In summary, our examples demonstrate that different modifications of carbohydrates can result in opposing effects and have to be studied in their entirety.
Assuntos
Selectina E/antagonistas & inibidores , Ésteres/farmacologia , Pró-Fármacos/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Células CACO-2 , Relação Dose-Resposta a Droga , Selectina E/metabolismo , Ésteres/administração & dosagem , Ésteres/química , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Ratos , Relação Estrutura-AtividadeRESUMO
A highly convergent and efficient synthesis of a new sialyl Lewis(x) (sLe(x)) mimic, which was predicted by computational studies to fulfil the spacial requirements for a selectin antagonist, has been developed. With a beta(2,3)-amino acid residue l-galactose (bioisostere of the l-fucose moiety present in the natural sLe(x)) and succinate are linked, leading to a mimic of sLe(x) that contains all the required pharmacophores, namely the 3- and 4-hydroxy group of l-fucose, the 4- and 6-hydroxy group of d-galactose and the carboxylic acid of N-acetylneuraminic acid. The key step of the synthesis involves a tandem reaction consisting of a N-deprotection and a suitable O-->N intramolecular acyl migration reaction which is promoted by cerium ammonium nitrate (CAN). Finally, the new sialyl Lewis(x) mimic was biologically evaluated in a competitive binding assay.
Assuntos
Aminoácidos/química , Mimetismo Molecular , Oligossacarídeos/química , Ligação Competitiva , Reagentes de Ligações Cruzadas , Antígeno Sialil Lewis XRESUMO
The monoclonal antibody GSLA-2 recognizes the sialyl Lewis(a) (sLe(a)) epitope, which has an increased occurrence on mucin type glycoproteins of patients with colorectal carcinoma. GSLA-2 is therefore used in tumor diagnosis. To advance the understanding of this highly specific molecular recognition reaction, we have analyzed the binding epitope of sLe(a) at atomic resolution using saturation transfer difference NMR. To compare, the binding epitopes of sialyl Lewis(x) (sLe(x)) and of four synthetic derivatives of sLe(a) were explored. Surface plasmon resonance experiments furnished thermodynamic and kinetic data. It is observed that all pyranose rings of sLe(a) are in contact with the protein surface, with the neuramic acid residue receiving the largest fraction of saturation transfer. In contrast, sLe(x) binds very weakly, though specifically to GSLA-2, with a different binding epitope. This study provides a comprehensive picture of the recognition sLe(a) and explains the exquisite specificity of the antibody.
Assuntos
Anticorpos Monoclonais/química , Antígenos Glicosídicos Associados a Tumores/química , Gangliosídeos/química , Sítios de Ligação , Antígeno CA-19-9 , Sequência de Carboidratos , Mapeamento de Epitopos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Antígeno Sialil Lewis X , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
Investigation of ligand-protein interactions by the saturation transfer difference (STD) experiment has been well established in the drug discovery process through numerous examples. Thus, binding epitopes may be mapped by comparing signals of the ligand with and without saturation of the protein. Herein, it is shown that a less selective process allows more protons to assist in the saturation of the protein, thereby considerably enhancing the sensitivity of the STD experiment. Increasing the saturation power entails a greater risk of perturbing the ligand; however, an amplitude modulation of the waveform assists this procedure by distributing the applied energy in sidebands.
Assuntos
Glicoproteínas/química , Espectroscopia de Ressonância Magnética/métodos , Configuração de Carboidratos , Selectina E/química , Epitopos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética/estatística & dados numéricos , Glicoproteína Associada a Mielina/química , Albumina Sérica/químicaRESUMO
CONTEXT: Minimal residual disease (MRD) in patients treated for hairy cell (HC) leukemia as assessed by immunohistochemistry has not been included routinely in evaluation of treatment results. OBJECTIVE: To assess the presence of persistent HCs after treatment, as detected by immunohistochemistry, and to evaluate the correlation between the level of MRD and clinical outcome. DESIGN: Percentages of DBA.44-positive HCs were assessed on 116 biopsy specimens from 17 patients. The patients had a median follow-up of 55.4 months. RESULTS: Minimal residual disease was seen in 3 patterns. Group 1 (7 patients) had MRD levels ranging from "rare scattered suspicious HCs" to less than 1%. The MRD levels were stable throughout follow-up, and all patients remained in complete remission. Group 2 (6 patients) had MRD levels ranging from 1% to 5%, and 3 patients were in complete remission at 77.9, 63.8, and 108.0 months. Another patient showed evidence of disease activity (partial remission) at 47.6 months. Two other patients relapsed at 12.3 months and at 25.7 months, respectively, with greater than 1% HCs. Group 3 (4 patients) had MRD levels greater than 5%. Three patients relapsed at 11.3, 12.1, and 29.6 months, respectively, with greater than 5% HCs. The fourth patient had MRD levels of 5% at 14.6 months and 2% at 20.0 months but was subsequently lost to follow-up. CONCLUSIONS: Quantitative assessment of MRD may be of value in identifying patients at risk for relapse of hairy cell leukemia.