Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. Herein, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-β (Aβ) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand–peptide adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimer’s disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aβ forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Overall, our in vitro and in vivo studies of DMPD toward Aβ with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.
The vibrational theory of olfaction assumes that electron transfer occurs across odorants at the active sites of odorant receptors (ORs), serving as a sensitive measure of odorant vibrational frequencies, ultimately leading to olfactory perception. A previous study reported that human subjects differentiated hydrogen/ deuterium isotopomers (isomers with isotopic atoms) of the musk compound cyclopentadecanone as evidence supporting the theory. Here, we find no evidence for such differentiation at the molecular level. In fact, we find that the human musk-recognizing receptor, OR5AN1, identified using a heterologous OR expression system and robustly responding to cyclopentadecanone and muscone, fails to distinguish isotopomers of these compounds in vitro. Furthermore, the mouse (methylthio)methanethiol-recognizing receptor, MOR244-3, as well as other selected human and mouse ORs, responded similarly to normal, deuterated, and 13 C isotopomers of their respective ligands, paralleling our results with the musk receptor OR5AN1. These findings suggest that the proposed vibration theory does not apply to the human musk receptor OR5AN1, mouse thiol receptor MOR244-3, or other ORs examined. Also, contrary to the vibration theory predictions, muscone-d 30 lacks the 1,380-to 1,550-cm −1 IR bands claimed to be essential for musk odor. Furthermore, our theoretical analysis shows that the proposed electron transfer mechanism of the vibrational frequencies of odorants could be easily suppressed by quantum effects of nonodorant molecular vibrational modes. These and other concerns about electron transfer at ORs, together with our extensive experimental data, argue against the plausibility of the vibration theory.olfaction | isotopomers | cyclopentadecanone | muscone | electron transfer I n 1870, the British physician William Ogle wrote: "As in the eye and the ear the sensory impression is known to result not from the contact of material particles given off by the object seen or heard, but from waves or undulations of the ether or the air, one cannot but suspect that the same may be true in the remaining sense, and that the undulatory theory of smell. . . [may be] the true one" (1, 2). Of the 29 different "theories of odour" listed in the 1967 edition of The Chemical Senses (3), nine associate odor with vibrations, particularly those theories championed by Dyson (4, 5) and Wright (6-8). However, the premise that olfaction involves detection of vibrational frequencies of odorants remains highly speculative because neither the structures of the odorant receptors (ORs) nor the binding sites or the activation mechanisms triggered upon odorant binding to ORs have been established. In 1996-1997, Turin (9-12) elaborated on the undulatory theory of smell, as considered in more detail below, and suggested that a mechanism analogous to inelastic electron tunneling spectroscopy (13) may be involved, where tunneling electrons in the receptor probe the vibrational frequencies of odorants. In 2013, Gane et al. (14) In judging the plausibility...
Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. Understanding the structural organization of amyloid fibrils can be beneficial for the search of drugs to successfully treat diseases associated with protein misfolding. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman (DUVRR) and Nuclear Magnetic Resonance (NMR) spectroscopy combined with hydrogen-deuterium exchange. The compositions of the fibril core and unordered parts were determined at single amino acid residue resolution. All three disulfide bonds of native insulin remained intact during the aggregation process, withstanding scrambling. Three out of four tyrosine residues were packed into the fibril core, and another aromatic amino acid, phenylalanine, was located in the unordered parts of insulin fibrils. In addition, using all-atom MD simulations, the disulfide bonds were confirmed to remain intact in the insulin dimer, which mimics the fibrillar form of insulin.
Photoluminescent molecules are widely used for real-time monitoring of peptide aggregation. In this Article, we detail both experimental and computational modeling to elucidate the interaction between [Ru(bpy)2dppz](2+) and amyloid-β (Aβ(1-40)) aggregates. The transition from monomeric to fibrillar Aβ is of interest in the study of Alzheimer's disease. Concentration-dependent experiments allowed the determination of a dissociation constant of 2.1 μM, while Job plots provided a binding stoichiometry of 2.6 Aβ monomers per [Ru(bpy)2dppz](2+). Our computational approach that combines molecular docking (both rigid and flexible) and all-atom molecular dynamics (MD) simulations predicts that the hydrophobic cleft between Val18 and Phe20 is a plausible binding site, which could also explain the increase in photoluminescence of [Ru(bpy)2dppz](2+) upon binding. This binding site is parallel to the fibril axis, in marked contrast to the binding site of these complexes in DNA (perpendicular to the DNA axis). Other binding sites may exist at the edges of the Aβ fibril, but they are actually of low abundance in an Aβ fibril several micrometers long. The assignment of the binding site was confirmed by binding studies in an Aβ fragment (Aβ(25-35)) that lacked the amino acids necessary to form the binding site. The agreement between the experimental and computational work is remarkable and provides a general model that can be used for studying the interaction of amyloid-binding molecules to Aβ.
Understanding olfaction at the molecular level is challenging due to the lack of crystallographic models of odorant receptors (ORs). To better understand the molecular mechanism of OR activation, we focused on chiral ()-muscone and other musk-smelling odorants due to their great importance and widespread use in perfumery and traditional medicine, as well as environmental concerns associated with bioaccumulation of musks with estrogenic/antiestrogenic properties. We experimentally and computationally examined the activation of human receptors OR5AN1 and OR1A1, recently identified as specifically responding to musk compounds. OR5AN1 responds at nanomolar concentrations to musk ketone and robustly to macrocyclic sulfoxides and fluorine-substituted macrocyclic ketones; OR1A1 responds only to nitromusks. Structural models of OR5AN1 and OR1A1 based on quantum mechanics/molecular mechanics (QM/MM) hybrid methods were validated through direct comparisons with activation profiles from site-directed mutagenesis experiments and analysis of binding energies for 35 musk-related odorants. The experimentally found chiral selectivity of OR5AN1 to ()- over ()-muscone was also computationally confirmed for muscone and fluorinated ()-muscone analogs. Structural models show that OR5AN1, highly responsive to nitromusks over macrocyclic musks, stabilizes odorants by hydrogen bonding to Tyr260 of transmembrane α-helix 6 and hydrophobic interactions with surrounding aromatic residues Phe105, Phe194, and Phe207. The binding of OR1A1 to nitromusks is stabilized by hydrogen bonding to Tyr258 along with hydrophobic interactions with surrounding aromatic residues Tyr251 and Phe206. Hydrophobic/nonpolar and hydrogen bonding interactions contribute, respectively, 77% and 13% to the odorant binding affinities, as shown by an atom-based quantitative structure-activity relationship model.
To elucidate the involvement of individual and inter-related pathological factors [i.e., amyloid-β (Aβ), metals, and oxidative stress] in the pathogenesis of Alzheimer’s disease (AD), chemical tools have been developed. Characteristics required for such tool construction, however, have not been clearly identified; thus, the optimization of available tools or new design has been limited. Here, key structural properties and mechanisms that can determine tools’ regulatory reactivities with multiple pathogenic features found in AD are reported. A series of small molecules was built up through rational structural selection and variations onto the framework of a tool useful for in vitro and in vivo metal–Aβ investigation. Variations include: (i) location and number of an Aβ interacting moiety; (ii) metal binding site; and (iii) denticity and structural flexibility. Detailed biochemical, biophysical, and computational studies were able to provide a foundation of how to originate molecular formulas to devise chemical tools capable of controlling the reactivities of various pathological components through distinct mechanisms. Overall, this multidisciplinary investigation illustrates a structure–mechanism-based strategy of tool invention for such a complicated brain disease.
In this B3LYP study, the catalytic mechanisms for the hydrolysis of the three different peptide bonds (Lys28-Gly29, Phe19-Phe20, and His14-Gln15) of Alzheimer amyloid beta (Abeta) peptide by insulin-degrading enzyme (IDE) have been elucidated. For all these peptides, the nature of the substrate was found to influence the structure of the active enzyme-substrate complex. The catalytic mechanism is proposed to proceed through the following three steps: (1) activation of the metal-bound water molecule, (2) formation of the gem-diol intermediate, and (3) cleavage of the peptide bond. With the computed barrier of 14.3, 18.8, and 22.3 kcal/mol for the Lys28-Gly29, Phe19-Phe20, and His14-Gln15 substrates, respectively, the process of water activation was found to be the rate-determining step for all three substrates. The computed energetics show that IDE is the most efficient in hydrolyzing the Lys28-Gly29 (basic polar-neutral nonpolar) peptide bond followed by the Phe19-Phe20 (neutral nonpolar-neutral nonpolar) and His14-Gln15 (basic polar-neutral polar) bonds of the Abeta substrate.
The mechanism of O(2) reduction by copper amine oxidase from Arthrobacter globiformus (AGAO) is analyzed in relation to the cobalt-substituted protein. The enzyme utilizes a tyrosine-derived topaquinone cofactor to oxidize primary amines and reduce O(2) to H(2)O(2). Steady-state kinetics indicate that amine-reduced CuAGAO is reoxidized by O(2) >10(3) times faster than the CoAGAO analogue. Complementary spectroscopic studies reveal that the difference in the second order rate constant, k(cat)/K(M)(O(2)), arises from the more negative redox potential of Co(III/II) in relation to Cu(II/I). Indistinguishable competitive oxygen-18 kinetic isotope effects are observed for the two enzymes and modeled computationally using a calibrated density functional theory method. The results are consistent with a mechanism where an end-on (η(1))-metal bound superoxide is reduced to an η(1)-hydroperoxide in the rate-limiting step.
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