Role of the conformational flexibility of evodiamine in its binding to protein hosts: a comparative spectroscopic and molecular modeling evaluation with rutaecarpine.

Spectroscopic studies combined with computational analysis indicate the inherent conformational flexibility of the ß-carbolin derivative evodiamine (EVD) featured with diverse pharmacological activities. Qualitative evaluation of the circular dichroism (CD) spectra of EVD enantiomers complemented with quantum chemical calculations reveals a chiral exciton signature that can be assigned to the folded, L-shaped conformation of the molecule. Changes of the exciton couplet measured in different solvents and the near-UV CD profile upon binding to human serum albumin (HSA) refer to the structural adaptability of EVD. The enantioselectivity of EVD-HSA interaction is demonstrated showing the binding preference of the (R)-enantiomer. Comparison of experimental and calculated CD spectra of various conformers of EVD as well as the results of molecular docking data suggest that the (R)-antipode is accomodated within the IIA subdomain of HSA in ridge-tile conformation. Rutaecarpine (RTC), the close congener of EVD, forms much tighter association complexes both with HSA and α1-acid glycoprotein. In contrast to EVD, the nearly planar geometry of the indoloquinazoline ring system of RTC allows its stacked dimeric binding to the HSA.

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

Harmane and norharmane are representative members of the large group of natural ß-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 ß-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.

mWTX-330, an IL-12 INDUKINE Molecule, Activates and Reshapes Tumor-Infiltrating CD8+ T and NK Cells to Generate Antitumor Immunity.

IL-12 is a pleotropic inflammatory cytokine, which has broad stimulatory effects on various immune cell populations, making it an attractive target for cancer immunotherapy. However, despite generating robust antitumor activity in syngeneic murine tumor models, clinical administration of IL-12 has been limited by severe toxicity. mWTX-330 is a selectively inducible INDUKINE molecule comprised of a half-life extension domain and an inactivation domain linked to chimeric IL-12 by tumor protease-sensitive linkers. Systemic administration of mWTX-330 in mice was well tolerated, resulted in robust antitumor immunity in multiple tumor models, and preferentially activated tumor-infiltrating immune cells rather than immune cells present in peripheral tissues. Antitumor activity was dependent on in vivo processing of the protease cleavable linkers and required CD8+ T cells for full efficacy. Within the tumor, mWTX-330 increased the frequency of cross-presenting dendritic cells (DC), activated natural killer (NK) cells, skewed conventional CD4+ T cells toward a T helper 1 (TH1) phenotype, drove regulatory T cells (Treg) fragility, and increased the frequency of polyfunctional CD8+ T cells. mWTX-330 treatment also increased the clonality of tumor-infiltrating T cells by expanding underrepresented T-cell receptor (TCR) clones, drove CD8+ T and NK cells towards increased mitochondrial respiration and fitness, and decreased the frequency of TOX+ exhausted CD8+ T cells within the tumor. A fully human version of this INDUKINE molecule was stable in human serum, was reliably and selectively processed by human tumor samples, and is currently in clinical development.

Cationic permethylated 6-monoamino-6-monodeoxy-ß-cyclodextrin as chiral selector of dansylated amino acids in capillary electrophoresis.

The resolution power of permethylated 6-monoamino-6-monodeoxy-ßCD (PMMABCD) - a single isomer, cationic CD derivative - developed previously for chiral analyses in capillary electrophoresis was further studied here. Dansylated amino acids (Dns-AA) were chosen as amphoteric chiral model compounds. Changes in the resolutions of Dns-AAs by varying pH and selector concentrations were investigated and correlated with their structures and chemical properties (isoelectric point and lipophilicity). Maximal resolutions could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of the selector. Baseline or substantially better resolution for 8 pairs of these Dns-AAs could be achieved. Low CD concentration was enough for the separation of the most apolar Dns-AAs. Chiral discrimination ability of PMMABCD was demonstrated by the separation of an artificial mixture of 8 Dns-AA pairs.

Stereoselective analysis of endomorphin diastereomers: resolution of biologically active analogues by capillary electrophoresis applying cyclodextrins as mobile phase additives.

Seven diastereomer pairs of tetrapeptide analogues (TP) of endomorphin-1 and -2 were synthesized. A stereoselective capillary electrophoretic method was developed for controlling stereoisomeric ratio or purity. The isoelectric points of the tetrapeptides were between 8.3 and 8.9 as predicted and measured. A few of the analytes could be resolved without selectors due to the difference in their mobility. Neutral and anionic cyclodextrins (CDs) were applied in order to improve resolution. Stability constants as well as the mobilities of complexes were determined. Contributions of differences in the mobilities of free analytes and in the mobilities and stabilities of their complexes formed by CDs were equally important in the efficient resolution and migration order of diastereomers. As a result of the optimization of the pH of buffers and the concentration of the CD derivatives each diastereomer pair was resolved at baseline at least or better.