Structural Adaptation of the Single-Stranded DNA-Binding Protein C-Terminal to DNA Metabolizing Partners Guides Inhibitor Design.

Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide-protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy-entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands.

Routing Nanomolar Protein Cargoes to Lipid Raft-Mediated/Caveolar Endocytosis through a Ganglioside GM1-Specific Recognition Tag.

There is a pressing need to develop ways to deliver therapeutic macromolecules to their intracellular targets. Certain viral and bacterial proteins are readily internalized in functional form through lipid raft-mediated/caveolar endocytosis, but mimicking this process with protein cargoes at therapeutically relevant concentrations is a great challenge. Targeting ganglioside GM1 in the caveolar pits triggers endocytosis. A pentapeptide sequence WYKYW is presented, which specifically captures the glycan moiety of GM1 (K D = 24 nm). The WYKYW-tag facilitates the GM1-dependent endocytosis of proteins in which the cargo-loaded caveosomes do not fuse with lysosomes. A structurally intact immunoglobulin G complex (580 kDa) is successfully delivered into live HeLa cells at extracellular concentrations ranging from 20 to 160 nm, and escape of the cargo proteins to the cytosol is observed. The short peptidic WYKYW-tag is an advantageous endocytosis routing sequence for lipid raft-mediated/caveolar cell delivery of therapeutic macromolecules, especially for cancer cells that overexpress GM1.

Synthesis and in vitro investigation of potential antiproliferative monosaccharide-d-secoestrone bioconjugates.

The syntheses of monosaccharide-d-secoestrone conjugates are reported. They were prepared from 3-(prop-2-inyloxy)-d-secoestrone alcohol or oxime and monosaccharide azides via Cu(I)-catalyzed azide-alkyne cycloaddition reactions (CuAAC). The antiproliferative activities of the conjugates were investigated in vitro against a panel of human adherent cancer cell lines (HeLa, A2780 and MCF-7) by means of MTT assays. The protected d-glucose-containing d-secoestrone oxime bioconjugate (24b) proved to be the most effective with an IC50 value in the low micromolar range against A2780 cell line.

Synthesis and Biological Evaluation of Triazolyl 13α-Estrone-Nucleoside Bioconjugates.

2'-Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne-azide click reaction (CuAAC). For the introduction of the azido group the 5'-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields were realized in our hands when the 3'-hydroxy groups of the nucleosides were protected by acetyl groups and the 5'-hydroxy groups were modified by the tosyl-azide exchange method. The commonly used conditions for click reaction between the protected-5'-azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and the potential inhibitory activity of the new conjugates on human 17ß-hydroxysteroid dehydrogenase 1 (17ß-HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines (the protected cytidine conjugate proved to be the most potent with IC50 value of 9 µM). The thymidine conjugate displayed considerable 17ß-HSD1 inhibitory activity (IC50 = 19 µM).

Synthesis and in vitro antiproliferative evaluation of d-secooxime derivatives of 13ß- and 13α-estrone.

d-Secooximes were synthesized from the d-secoaldehydes in the 13ß- and 13α-estrone series. The oximes were modified at three sites in the molecule: the oxime function was transformed into an oxime ether, oxime ester or nitrile group, the propenyl side-chain was saturated and the 3-benzyl ether was removed in order to obtain a phenolic hydroxy function. Triazoles were formed via Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) from 3-(prop-2-yniloxy)-d-secooximes and benzyl azides. All the products were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7, A2780 and A431). Some of them exhibited activities with submicromolar IC50 values, better than that of the reference agent cisplatin. The structural modifications led to significant differences in the cytostatic properties. Flow cytometry indicated that one of the most potent agents resulted in a cell cycle blockade.