Solid-Phase-Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides.

Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80%. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.

Binding of Per- and Polyfluoroalkyl Substances (PFAS) to the PPARγ/RXRα-DNA Complex.

Nuclear receptors are the fundamental building blocks of gene expression regulation and the focus of many drug targets. While binding to DNA, nuclear receptors act as transcription factors, governing a multitude of functions in the human body. Peroxisome proliferator-activator receptor γ (PPARγ) and the retinoid X receptor α (RXRα) form heterodimers with unique properties and have a primordial role in insulin sensitization. This PPARγ/RXRα heterodimer has been shown to be impacted by per- and polyfluoroalkyl substances (PFAS) and linked to a variety of significant health conditions in humans. Herein, a selection of the most common PFAS (legacy and emerging) was studied utilizing molecular dynamics simulations for PPARγ/RXRα. The local and global structural effects of PFAS binding on the known ligand binding pockets of PPARγ and RXRα as well as the DNA binding domain (DBD) of RXRα were inspected. The binding free energies were predicted computationally and were compared between the different binding pockets. In addition, two electronic structure approaches were utilized to model the interaction of PFAS within the DNA binding domain, density functional theory (DFT) and domain-based pair natural orbital coupled cluster with perturbative triples (DLPNO-CCSD(T)) approaches, with implicit solvation. Residue decomposition and hydrogen-bonding analysis were also performed, detailing the role of prominent residues in molecular recognition. The role of l-carnitine is explored as a potential in vivo remediation strategy for PFAS interaction with the PPARγ/RXRα heterodimer. In this work, it was found that PFAS can bind and act as agonists for all of the investigated pockets. For the first time in the literature, PFAS are postulated to bind to the DNA binding domain in a nonspecific manner. In addition, for the PPARγ ligand binding domain, l-carnitine shows promise in replacing smaller PFAS from the pocket.

DNA preference of indenoisoquinolines: a computational approach.

The human topoisomerase IB (hTopoIB) enzyme is a monomeric protein that relaxes the supercoils on double-stranded DNA by forming a covalent DNA/hTopoIB complex by introducing a nick on the DNA strand. Inhibition of hTopoIB results in cell death, which makes this protein a strong target for the treatment of various cancer types, including small-cell lung cancers and ovarian cancers. Camptothecin (CPT) and indenoisoquinoline (IQN) classes of compounds inhibit the hTopoIB activity by intercalating to nicked DNA pairs; however, these inhibitors show different preferences towards DNA bases when bound to the DNA/hTopoIB complex. Here, we investigated the affinities of CPT and one IQN derivative towards different DNA base pairs. The two inhibitors showed different stacking behaviors in the intercalation site and interaction pattern with binding pocket residues, indicating that they have different inhibition mechanisms in the binding pocket that affects the base-pair selectivity. The results obtained from this study are expected to guide researchers in designing gene-specific and more potent compounds to fight cancer through hTopoIB poisoning.

Molecular Insights for Alzheimer's Disease: An Unexplored Storyline on the Nanoscale Impact of Nascent Aß1-42 toward the Lipid Membrane.

Deciphering the mechanism of Alzheimer's disease is a key element for designing an efficient therapeutic strategy. Molecular dynamics (MD) calculations, atomic force microscopy, and infrared spectroscopy were combined to investigate ß-amyloid (Aß1-42) peptide interactions with supported lipid bilayers (SLBs). The MD simulations showed that nascent Aß1-42 monomers remain anchored within a model phospholipid bilayer's hydrophobic core, which suggests their stability in their native environment. We tested this prediction experimentally by studying the behavior of Aß1-42 monomers and oligomers when interacting with SLBs. When Aß1-42 monomers and oligomers were self-assembled with a lipid bilayer and deposited as an SLB, they remain within the bilayers. Their presence in the bilayers induces destabilization of the model membranes. No specific interactions between Aß1-42 and the SLBs were detected when SLBs free of Aß1-42 were exposed to Aß1-42. This study suggests that Aß can remain in the membrane after cleavage by γ-secretase and cause severe damage to the membrane.

Investigation of iron release from the N- and C-lobes of human serum transferrin by quantum chemical calculations.

Human serum transferrin binds ferric ions with high affinity and delivers them into cells via receptor-mediated endocytosis upon a decrease in pH in the endosome. Protonation events and conformational changes are known to play an important role in iron-release though the release is not yet fully understood. Human serum transferrin consists of two similar lobes which release iron at different rates. In this study, we investigate the iron binding sites of N- and C-lobes using quantum mechanical tools, particularly, the quantum chemical cluster approach. This study supports the inevitable role of axial tyrosine for the release of iron in quantum chemical models and provides valuable information about the proton transfer pathways for the protonation of Tyr188 and Tyr517 in N- and C-lobes, respectively. The calculations show that the release process is similar in both lobes; however, the energetic differences of the release process in N- and C-lobes, demonstrated for the first time, indicated that the release of iron in the N-lobe is thermodynamically favorable, in contrast to the one in the C-lobe.

Activity of Topotecan toward the DNA/Topoisomerase I Complex: A Theoretical Rationalization.

Topotecan (TPT) is a nontoxic anticancer drug characterized by a pH-dependent lactone/carboxyl equilibrium. TPT acts on the covalently bonded DNA/topoisomerase I (DNA/TopoI) complex by intercalating between two DNA bases at the active site. This turns TopoI into a DNA-damaging agent and inhibits supercoil relaxation. Although only the lactone form of the drug is active and effectively inhibits TopoI, both forms have been co-crystallized at the same location within the DNA/TopoI complex. To gain further insights into the pH-dependent activity of TPT, the differences between two TPT:DNA/TopoI complexes presenting either the lactone (acidic pH) or the carboxyl (basic pH) form of TPT were studied by means of molecular dynamic simulations, quantum mechanical/molecular mechanical calculations, and topological analysis. We identified two specific amino acids that have a direct relationship with the activity of the drug, i.e., lysine 532 (K532) and asparagine 722 (N722). K532 forms a stable hydrogen bond bridge between TPT and DNA only when the drug is in its active lactone form. The presence of the active drug triggers the formation of an additional stable interaction between DNA and protein residues, where N722 acts as a bridge between the two fragments, thus increasing the binding affinity of DNA for TopoI and further slowing the release of DNA. Overall, our results provide a clear understanding of the activity of the TPT-like class of molecules and can help in the future design of new anticancer drugs targeting topoisomerase enzymes.