The α7 nicotinic acetylcholine receptor (α7-nAChR) is assumed to be implicated in a variety of neurological disorders, such as schizophrenia and Alzheimer's disease (AD). The progress of these disorders can be studied through imaging α7-nAChR with positron emission tomography (PET). [18F]ASEM is a novel and potent α7-nAChR PET radioligand showing great promise in recent tests. However, the mechanism of the molecular interaction between [18F]ASEM and α7-nAChR is still unclear. In this paper, the binding profile of [18F]ASEM to a chimera structure of α7-nAChR was investigated with molecular docking, molecular dynamics, and metadynamics simulation methods. We found that [18F]ASEM binds at the same site as the crystallized agonist epibatidine but with a different binding mode. The dibenzo[b,d]thiophene ring has a different orientation compared to the pyridine ring of epibatidine and has van der Waals interactions with residues from loop C on one side and π-π stacking interaction with Trp53 on the other side. The conformation of Trp53 was found to have a great impact on the binding of [18F]ASEM. Six binding modes in terms of the side chain dihedral angles χ1 and χ2 of Trp53 were discovered by metadynamics simulation. In the most stable binding mode, Trp53 adopts a different conformation from that in the crystalline structure and has a rather favorable π-π stacking interaction with [18F]ASEM. We believe that these discoveries can be valuable for the development of novel PET radioligands.
Peptide drugs have been difficult to translate into effective therapies due to their low in vivo stability. Here, we report a strategy to develop peptide-based therapeutic nanoparticles by screening a peptide library differing by single-site amino acid mutations of lysine-modified cholesterol. Certain cholesterol-modified peptides are found to promote and stabilize peptide α-helix formation, resulting in selectively cell-permeable peptides. One cholesterol-modified peptide self-assembles into stable nanoparticles with considerable α-helix propensity stabilized by intermolecular van der Waals interactions between inter-peptide cholesterol molecules, and shows 68.3% stability after incubation with serum for 16 h. The nanoparticles in turn interact with cell membrane cholesterols that are disproportionately present in cancer cell membranes, inducing lipid raft-mediated endocytosis and cancer cell death. Our results introduce a strategy to identify peptide nanoparticles that can effectively reduce tumor volumes when administered to in in vivo mice models. Our results also provide a simple platform for developing peptide-based anticancer drugs.
Hypoxia is a common feature of tumor cells. Nitroreductase (NTR), a common biomarker of hypoxia, has been widely used to evaluate the extent of tumor hypoxia. In this study, three fluorescent probes (FBN-1-3) were synthesized to monitor the extent of hypoxia in cancer cells in real time. FBN-1-3 were composed of a fluorescein analogue and one of three different aromatic nitro groups. Of these probes, FBN-1 showed excellent sensitivity and selectivity in detecting hypoxia via a reduction in O-2 concentration. Confocal fluorescence imaging and flow cytometry demonstrated that HepG-2, A549, and SKOV-3 cells incubated with FBN-1 under reduced oxygen conditions showed significantly enhanced fluorescence. A mouse HepG-2 tumor model confirmed that FBN-1 responds rapidly to NTR and can be used to evaluate the degree of tumor hypoxia. The changes in intra- and extracellular NTR in tumor cells were also concurrently monitored, which did not reveal a link between NTR concentration and degree of hypoxia. Our work provides a functional probe for tumor hypoxia, and our results suggest the fluorescent response of our probe is due to a decrease in O-2 concentration, and not NTR concentration.
Various squaraine dyes have been developed for biological imaging. Nevertheless, squaraine dyes with emission in the second window (NIR-II, 1000-1700 nm) have few reports largely due to the short of a simple and universal design strategy. In this contribution, molecular engineering strategy is explored to develop squaraine dyes with NIR-II emission. First, NIR-I squaraine dye SQ2 is constructed by the ethyl-grafted 1,8-naphtholactam as donor units and square acid as acceptor unit in a donor-acceptor-donor (D-A-D) structure. To red-shift the fluorescence emission into NIR-II window, malonitrile, as a forceful electron-withdrawing group, is introduced to strengthen square acid acceptor. As a result, the fluorescence spectrum of acceptor-engineered squaraine dye SQ1 exhibits a significant red- shift into NIR-II window. To translate NIR-II fluorophores SQ1 into effective theranostic agents, fibronectin-targeting SQ1 nanoprobe was constructed and showed excellent NIR-II imaging performance in angiography and tumor imaging, including lung metastatic foci in deep tissue. Furthermore, SQ1 nanoprobe can be used for photoacoustic imaging and photothermal ablation of tumors. This research demonstrates that the donor-acceptor engineering strategy is feasible and effective to develop NIR-II squaraine dyes.
This report describes all key steps in the O-O bond formation from two separated [Ru-V=O(bda)L-2](+) cations to form the dinuclear [(bda)L2RuIV-O-Ru-IV(bda)L-2](2+) in explicit solvent. The three steps involve the diffusion of the catalysts in the water phase, formation of the prereactive dimer, and the bond formation between the two catalysts. On the basis of the calculated parameters, we compute the rate constant of two catalysts with different L-ligands, isoquinoline and picoline, and the computed values are in excellent agreement with the experimental ones. The interaction of the axial ligands is key to the improved rates of the larger ligand, mainly by facilitating the formation of the prereactive dimer from the solvated monomer. By comparing the binding free energy of hydrophilic Ru-IV-OH and hydrophobic Ru-V=O, the hydrophobic driving force of Ru-V=O in this system has been estimated to 1 kcal mol(-1).
Understanding unbinding kinetics of protein-ligand systems is of great importance for the design of ligands with desired specificity and safety. In recent years, enhanced sampling techniques have emerged as effective tools for studying unbinding kinetics of protein-ligand systems at the atomistic level. However, in many protein-ligand systems, the ligand unbinding processes are strongly coupled to protein conformational changes and the disclosure of the hidden degrees of freedom closely related to the protein conformational changes so that sampling is enhanced over these degrees of freedom remains a great challenge. Here, we show how potential-scaled molecular dynamics (sMD) and infrequent metadynamics (InMetaD) simulation techniques can be combined to successfully reveal the unbinding mechanism of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[F-18]fluorodibenzo[b,d]thiophen e 5,5-dioxide ([F-18]ASEM) from a chimera structure of the alpha 7-nicotinic acetylcholine receptor. By using sMD simulations, we disclosed that the "close to "open" conformational change of loop C plays a key role in the ASEM unbinding process. By carrying out InMetaD simulations with this conformational change taken into account as an additional collective variable, we further captured the key states in the unbinding process and clarified the unbinding mechanism of ASEM from the protein. Our work indicates that combining sMD and InMetaD simulation techniques can be an effective approach for revealing the unbinding mechanism of a protein-ligand system where protein conformational changes control the unbinding process.
A terpyridine- and guanine-functionalized peptide was developed that could form different morphologies by self-assembly or coordination with Fe2+ in dimethyl sulfoxide. The self-assembly of the peptide is attributed to the G-quartet formation of a guanine moiety and intermolecular terpyridine π-π stacking. Upon the addition of Fe2+, a Fe2+-(terpyridine)2 complex is formed that turns the square-planar self-assembly to a three-dimensional self-assembly. As a consequence, a variety of interesting morphologies and chemical properties were observed. The self-assembled polymers were studied by nuclear magnetic resonance spectroscopy, ultraviolet-visible and fluorescence spectroscopy, viscosity measurement, circular dichroism, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, dynamic light scattering, and atomic force microscopy. This external stimuli driven self-assembly of a peptide may be further applied to drug delivery applications.
The amyloid beta (A beta) fibril is a hallmark of Alzheimer's disease (AD) and has therefore served as an important target for early diagnosis of AD. The Pittsburgh Compound-B (PiB) is one of the most famous positron emission tomography (PET) tracers commonly used for in vivo detection of A beta fibrils. Many theoretical studies have predicted the existence of various core binding sites with different microenvironments for probes binding to the A beta fibril. However, little attention has been devoted to how the probes actually penetrate into the different core binding sites. In this study, an integrated molecular modeling scheme is used to study the penetration of PiB into the core binding sites of the A beta(1-42) fibril structure recently obtained by cryogenic electron microscopy. We find that there are two core binding sites for PiB with dramatic differences in cavity size and microenvironment properties, and furthermore that the penetration of PiB into site-1 is energetically prohibitive, whereas the penetration into site 2 is much more favorable. Therefore, the binding capacity at site-2 may be larger than that at site-1 despite its lower binding affinity. Our results thus suggest that site-2 may be a major binding site for PiB binding to A beta fibril and emphasize the importance to adopt a full dynamical picture when studying tracer fibril binding problems in general, something that in turn can be used to guide the development of tracers with higher affinity and selectivity for the A beta fibril.
Intrinsically disordered proteins (IDPs) exert their functions by binding to partner proteins via a complex process that includes coupled folding and binding. Because inhibiting the binding of the IDP p53 to its partner MDM2 has become a promising strategy for the design of anticancer drugs, we carried out metadynamics simulations to study the coupled folding and binding process linking the IDP p53 to MDM2 in atomic detail. Using bias-exchange metadynamics (BE-MetaD) and infrequent metadynamics (InMetaD), we estimated the binding free energy, the unbinding rate, and the binding rate. By analyzing the stable intermediates, we uncovered the role non-native interactions played in the p53-MDM2 binding/unbinding process. We used a three-state model to describe the whole binding/unbinding process and to obtain the corresponding rate constants. Our work shows that the binding of p53 favors an induced-fit mechanism which proceeds in a stepwise fashion. Our results can be helpful for gaining an in-depth understanding of the coupled folding and binding process needed for the design of MDM2 inhibitors.