Trp-cage miniprotein was used to investigate the role of a salt-bridge (Asp(9) -Arg(16) ) in protein formation, by mutating residues at both sides, we mapped its contribution to overall stability and its role in folding mechanism. We found that both of the above side-chains are also part of a dense interaction network composed of electrostatic, H-bonding, hydrophobic, etc. components. To elucidate the fold stabilizing effects, we compared and contrasted electronic circular dichroism and NMR data of miniproteins equipped with a salt-bridge with those of the salt-bridge deleted mutants. Data were acquired both in neutral and in acidic aqueous solutions to decipher the pH dependency of both fully and partially charged partners. Our results indicate that the folding of Trp-cage miniproteins is more complex than a simple two-state process as we detected an intermediate state that differs significantly from the native fold. The intermediate formation is related to the salt-bridge stabilization; in the miniprotein variants equipped with salt-bridge the population of the intermediate state at acidic pH is significantly higher than it is for the salt-bridge deleted mutants. In this molecular framework Arg(16) stabilizes more than Asp(9) does, because of its higher degree of 3D-fold cooperation. In conclusion, the Xxx(9) leftright arrow Xxx(16) salt-bridge is not an isolated entity of this fold; rather it is an integrated part of a complex interaction network.

The Ko family of fumitremorgin C analogs are potent and selective ABCG2 inhibitors. However, the most potent Ko compounds carry an ester linkage in their side-chain that makes them chemically and metabolically less stable. We have synthesized 16 tricyclic and 28 tetracyclic novel analogs devoid of ester linkages and tested them for ABCG2 inhibition potency and specificity. Unlike in the tricyclic analog group, potent ABCG2 inhibitory compounds were found among the tetracyclic analogs. The most potent compounds carried the 3S,6S,12aS configuration. We observed a marked stereospecificity as compounds with the 3S,6S,12aS configuration were at least 18-fold more potent inhibitors than their diastereoisomeric pairs with a 3S,6R,12aS configuration. This stereospecificity was not observed in ABCB1 and ABCC1 inhibition. Therefore, a single chiral center confers specificity for ABCG2 over ABCB1 and ABCC1. This is quite unexpected considering the large multivalent drug binding site these transporters harbor.

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Mono-, di- and trisaccharide representing the reducing terminal of the core structure of N-glycans were incorporated into Leu-Lys-Asn-Gly-Gly-Pro hexapeptide that is a partial structure of the Trp-cage mini-protein by linear assembly. These studies provide evidence that the used combination of Fmoc and Boc strategy and mild conditions result in glycopeptides in high purity and reasonable yield.

Transporters belonging in the ATP-binding cassette (ABC) family are crucially involved in the determination of the pharmacokinetic behavior of xenobiotics and in the development of drug resistance. The targeting of these transporters has been accepted as a rational option via which to modify the absorption or distribution of pharmaceutical agents and to combat ABC-related inefficiency. Inhibitors of the breast cancer resistance protein (BCRP/ABCG2) multidrug transporter are of interest as chemosensitizers for clinical drug resistance, for improving the pharmacokinetics of substrate chemotherapeutic drugs, and in functional assays of BCRP activity for tailoring chemotherapy. In this study, a reversephase high-performance liquid chromatographic method was developed for the determination of a simplified fumitremorgin C analog, Ko134, a potent ABCG2 inhibitor, in murine serum. The assay involves a simple sample preparation step followed by chromatographic separation on a C8 reversed-phase analytical column. The calibration plots were linear over the range 0.1 to10 μg/ml (r > 0.99). The limits of detection and quantification were 10 ng/ml and 50 ng/ml, respectively. The validation results demonstrated that the method is precise, accurate and selective for the determination of Ko134 in mouse serum. The method was successfully applied to evaluate the pharmacokinetic parameters of Ko134 after different modes of administration in mice.