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Oxidative stress is considered to be a major contributor to dysfunction in a host of disease states. Reactive oxygen species (ROS) mediate distinct oxidative alterations in biopolymers, including DNA, proteins, lipids, and lipoproteins. Currently, the mechanisms of biochemical reactions underlying oxidative stress are poorly understood because of the instability of ROS. One of the consequences of oxidative stress is one-electron oxidation of tyrosine (Tyr) residues in proteins, which represents a hallmark of this insult and is implicated in the pathogenesis of a number of pathological processes leading to atherosclerosis, inflammatory conditions, multiple system atrophy and several neurodegenerative diseases. Major products of oxidation of Tyr include protein-bound dityrosine and isodityrosine. In this report, the mechanism of tyrosine coupling (including structure and stability of a number of proposed reaction intermediates) is studied by high-level density functional and conventional ab initio methods including B3LYP, MP2, CASSCF, and CASPT2. It is demonstrated that dityrosine and isodityrosine are the most stable structures at all theoretical levels applied. In addition to classical structures of the reaction intermediates, evidence is found for a novel transient structure of Tyr dimer, stacked dityrosyl. This dimer is predicted to exist because of strong electron correlation between two tyrosyl moieties. The counterpoise corrected energy of stacked dityrosyl is below the energy of two tyrosyl radicals by about 95 kJ/mol at the PUMP2/6-31G** level. High proton affinity of tyrosyl radical (about 9.4 eV) suggests that positively charged amino acids in the vicinity of a solvent-exposed Tyr residue may increase the probability of tyrosine coupling.

Right-handed twisting is a fundamental structural feature of β-pleated sheets in globular proteins which is critical for their geometry and function. The origin of this twisting is poorly understood and has represented a challenge for theoretical chemistry for almost 30 years. Density functional theory using the B3LYP exchange-correlation functional and the split-valence 6-31G** basis set has been utilized to investigate the structure and conformational transitions of single and double-stranded antiparallel β-sheet models to determine the driving force for the right-handed twisting. Right-handed twisting is found to be an intrinsic property of a peptide main chain because of the difference in rotational potentials around N(sp²)−Cα(sp³) and C(sp²)−Cα(sp³) bonds. The difference arises from a tendency of the single Cα(sp³)−C(sp²) bonds to eclipse the lone pair of atoms N(sp²), which results in decreasing absolute values of dihedral angles ϕ but not ψ. This tendency is suppressed by hydrogen bonding between adjacent CO and NH groups within single β-strands, and released only when these bonds are disrupted by the interstrand CO···HN hydrogen bonding. The results obtained constitute the following paradigm of the origin of β-sheet twist:  although right-handed twisting of β-sheets in globular proteins is an inherent property of the peptide backbone within single β-strands, it is unleashed by the interstrand hydrogen bonding in multistranded β-sheets. The observed pleating, right-handed twisting, skewed mutual orientation of β-strands, and intrinsic conformational variability of double-stranded antiparallel β-sheet motifs in globular proteins are explained from the first principles.

There is growing evidence that nerve growth factor (NGF) may function as a mediator of persistent pain states. We have identified a novel nonpeptidic molecule, ALE-0540, that inhibits the binding of NGF to tyrosine kinase (Trk) A or both p75 and TrkA (IC50 5.88 +/- 1. 87 microM, 3.72 +/- 1.3 microM, respectively), as well as signal transduction and biological responses mediated by TrkA receptors. ALE-0540 was tested in models of neuropathic pain and thermally-induced inflammatory pain, using two routes of administration, a systemic i.p. and a spinal intrathecal (i.th.) route. Morphine was also tested for comparison in the antiallodynia model using mechanical stimuli. We show that either i.p. or i.th. administration of ALE-0540 in rats produced antiallodynia in the L5/L6 ligation model of neuropathic pain. The calculated A50 values (and 95% confidence intervals) for ALE-0540 administered i.p. and i. th. were 38 (17.5-83) mg/kg and 34.6 (17.3-69.4) microgram, respectively. ALE-0540 given i.th., at doses of 30 and 60 microgram, also blocked tactile allodynia in the thermal sensitization model. Although morphine displayed greater potency [A50 value of 7.1 (5.6-8. 8) mg/kg] than ALE-0540 in anti-allodynic effect when given i.p. to L5/L6-ligated rats, it was not active when administered i.th. These data suggest that a blockade of NGF bioactivity using a NGF receptor antagonist is capable of blocking neuropathic and inflammatory pain and further support the hypothesis that NGF is involved in signaling pathways associated with these pain states. ALE-0540 represents a nonpeptidic small molecule which can be used to examine mechanisms leading to the development of agents for the treatment of pain.