Synthesis and serum protein binding of novel ring-substituted harmine derivatives

A series of new derivatives of the natural β-carboline alkaloid harmine, introducing hydrophobic substituents into positions 7 and 9 were synthetized as potential anticancer agents. Their binding affinities for human serum albumin (HSA) and α1-acid glycoprotein (AAG) were investigated by affinity chromatography combined with fluorescence, circular dichroism (CD) and UV absorption spectroscopy. The weak binding of harmine to both proteins (Ka ~ 3 × 104 M-1) was highly increased by aromatic substitutions (Ka ~ 105-106 M-1). Derivatives having a substituted benzyl group in the N9-position of the β-carboline nucleus showed about tenfold and hundredfold affinity enhancement for HSA and AAG, respectively. Such a strong plasma protein interaction would be of pharmacokinetic relevance for these potential drug candidates. Induced CD spectra indicated the variant selective, dimeric binding of the 7-pyridylethoxy derivative to AAG. Absorbance and fluorescence spectra refer to the binding preference of the neutral form of the studied β-carbolines for both proteins

Biológiailag jelentős nem-kovalens kölcsönhatások tanulmányozása: fehérje-kötődés, nukleinsav-kötődés, önszerveződés = Investigations on non-covalent interactions of biological importance: protein binding, nucleic acid binding, self-assembly

Nem-kovalens kölcsönhatások (fehérjekötődés, aggregáció) kisérleti vizsgálatára UV-Vis, fluoreszcencia és kiroptikai spektroszkópiai módszereket alkalmaztak, a vizsgálati minták tisztítását kromatográfiás (HPLC, kapilláris elektroforézis) technikákkal végezték. A humán vérplazma minor fehérje komponensének (alfa-1 savas glikoprotein, AGP) részletes vizsgálata kimutatta, hogy az AGP genetikai variánsai eltérő, a kötődési erősségtől függő módon csökkentik a myeloid leukémia gyógyszerének (Imatinib, Glivec', Novartis) hatásosságát. Daganatos betegek klinikai vérmintáiból kidolgozták nagy tisztaságú AGP kinyerését és kapilláris elektroforézissel igazolták, hogy az AGP cukorláncának változatossága (glycoform heterogeneity) különféle daganatos megbetegedések érzékeny markere. Enantiomerek elválasztására egy ciklodextrin származékot tartalmazó új királis állófázist fejlesztettek ki. Megfigyelték, hogy AGP kötődésben királis inverzió következhet be. A kiroptikai spektrumokban megjelenő exciton sávpár intenzitását nem-kovalens kölcsönhatások esetére elméleti alapon indokolták. | UV-Vis, fluorescence and chiroptical spectroscopic methods have been applied for experimental studies of non-covalent interactions, the purification of the samples were done by chromatographic (HPLC, capillary electrophoresis) techniques. Detailed studies of the minor protein component of human blood plasma, alpha-1 acid glycoprotein (AGP) revealed that the decrease in the efficiency of the medication against chronic myelogenous leukaemia, Imatinib (Glivec', Novartis) brought about by the genetic variants of AGP was in correlation with their binding strength. Highly purified AGP samples were prepared from clinical blood samples of cancer patients and their capillary electrophoretic analysis proved the variability of sugar-chain of AGP (glycoform heterogeneity) to be a sensitive marker of several malignant diseases. New chiral stationary phase containing a cyclodextrin derivative has been developed for enantiomer separation. Chiral inversion has been demonstrated in AGP binding. The intensity of exciton couplets appearing in chiroptical spectra has been explained on theoretical grounds for non-covalent interactions

Fatty Acid Modulated Human Serum Albumin Binding of the β‑Carboline Alkaloids Norharmane and Harmane

Harmane and norharmane are representative members of the large group of natural β-carboline alkaloids featured with diverse pharmacological activities. In blood, these agents are transported by human serum albumin (HSA) which has a profound impact on the pharmacokinetic and pharmacodynamic properties of many therapeutic drugs and xenobiotics. By combination of various spectroscopic methods, the present contribution is aimed to elucidate how nonesterified fatty acids (FAs), the primary endogenous ligands of HSA, affect the binding properties of harmane and norharmane. Analysis of induced circular dichroism (CD) and fluorescence spectroscopic data indicates the inclusion of the neutral form of both molecules into the binding pocket of subdomain IIIA, which hosts two FA binding sites, too. The induced CD and UV absorption spectra of harmane and norharmane exhibit peculiar changes upon addition of FAs, suggesting the formation of ternary complexes in which the lipid ligands significantly alter the binding mode of the alkaloids via cooperative allosteric mechanism. To our knowledge, it is the first instance of the demonstration of drug-FA cobinding at site IIIA. In line with these results, molecular docking calculations showed two distinct binding positions of norharmane within subdomain IIIA. The profound increase in the affinity constants of β-carbolines estimated in the presence of FAs predicts that the unbound, pharmacologically active serum fraction of these compounds strongly depends on the actual lipid binding profile of HSA

Fatty acid modulated human serum albumin binding of the β-carboline alkaloids norharmane and harmane

Harmane and norharmane are representative members of the large group of natural β-carboline alkaloids featured with diverse pharmacological activities. In blood, these agents are transported by human serum albumin (HSA) which has a profound impact on the pharmacokinetic and pharmacodynamic properties of many therapeutic drugs and xenobiotics. By combination of various spectroscopic methods, the present contribution is aimed to elucidate how non-esterified fatty acids (FAs), the primary endogenous ligands of HSA, affect the binding properties of harmane and norharmane. Analysis of induced circular dichroism (CD) and fluorescence spectroscopic data indicates the inclusion of the neutral form of both molecules into the binding pocket of subdomain IIIA, which hosts two FA binding sites, too. The induced CD and UV absorption spectra of harmane and norharmane exhibit peculiar changes upon addition of FAs suggesting the formation of ternary complexes in which the lipid ligands significantly alter the binding mode of the alkaloids via cooperative allosteric mechanism. To our knowledge, it is the first instance of the demonstration of drug-FA co-binding at site IIIA. In line with these results, molecular docking calculations showed two distinct binding positions of norharmane within subdomain IIIA. The profound increase in the affinity constants of β-carbolines estimated in the presence of FAs predicts that the unbound, pharmacologically active serum fraction of these compounds strongly depends on the actual lipid binding profile of HSA