A Synthetic Agonist to Vasoactive Intestinal Peptide Receptor-2 Induces Regulatory T Cell Neuroprotective Activities in Models of Parkinson's Disease.

A paradigm shift has emerged in Parkinson's disease (PD) highlighting the prominent role of CD4+ Tregs in pathogenesis and treatment. Bench to bedside research, conducted by others and our own laboratories, advanced a neuroprotective role for Tregs making pharmacologic transformation of immediate need. Herein, a vasoactive intestinal peptide receptor-2 (VIPR2) peptide agonist, LBT-3627, was developed as a neuroprotectant for PD-associated dopaminergic neurodegeneration. Employing both 6-hydroxydopamine (6-OHDA) and α-synuclein (α-Syn) overexpression models in rats, the sequential administration of LBT-3627 increased Treg activity without altering cell numbers both in naïve animals and during progressive nigrostriatal degeneration. LBT-3627 administration was linked to reductions of inflammatory microglia, increased survival of dopaminergic neurons, and improved striatal densities. While α-Syn overexpression resulted in reduced Treg activity, LBT-3627 rescued these functional deficits. This occurred in a dose-dependent manner closely mimicking neuroprotection. Taken together, these data provide the basis for the use of VIPR2 agonists as potent therapeutic immune modulating agents to restore Treg activity, attenuate neuroinflammation, and interdict dopaminergic neurodegeneration in PD. The data underscore an important role of immunity in PD pathogenesis.

Manganese-Enhanced Magnetic Resonance Imaging for Detection of Vasoactive Intestinal Peptide Receptor 2 Agonist Therapy in a Model of Parkinson's Disease.

Neuroprotective immunity is defined by transformation of T-cell polarity for therapeutic gain. For neurodegenerative disorders and specifically for Parkinson's disease (PD), granulocyte-macrophage colony stimulating factor or vasoactive intestinal peptide receptor 2 (VIPR2) agonists elicit robust anti-inflammatory microglial responses leading to neuronal sparing in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. While neurotherapeutic potential was demonstrated for PD, there remain inherent limitations in translating these inventions from the laboratory to patients. One obstacle in translating such novel neurotherapeutics centers on the availability of suitable noninvasive methods to track disease progression and therapeutic efficacy. To this end, we developed manganese-enhanced magnetic resonance imaging (MEMRI) assays as a way to track a linkage between glial activation and VIPR2 agonist (LBT-3627)-induced neuroprotective immunity for MPTP-induced nigrostriatal degeneration. Notably, LBT-3627-treated, MPTP-intoxicated mice show reduced MEMRI brain signal intensities. These changes paralleled reduced astrogliosis and resulted in sparing of nigral tyrosine hydroxylase neurons. Most importantly, the data suggest that MEMRI can be developed as a biomarker tool to monitor neurotherapeutic responses that are relevant to common neurodegenerative disorders used to improve disease outcomes.

Selective VIP Receptor Agonists Facilitate Immune Transformation for Dopaminergic Neuroprotection in MPTP-Intoxicated Mice.

Vasoactive intestinal peptide (VIP) mediates a broad range of biological responses by activating two related receptors, VIP receptor 1 and 2 (VIPR1 and VIPR2). Although the use of native VIP facilitates neuroprotection, clinical application of the hormone is limited due to VIP's rapid metabolism and inability to distinguish between VIPR1 and VIPR2 receptors. In addition, activation of both receptors by therapeutics may increase adverse secondary toxicities. Therefore, we developed metabolically stable and receptor-selective agonists for VIPR1 and VIPR2 to improve pharmacokinetic and pharmacodynamic therapeutic end points. Selective agonists were investigated for their abilities to protect mice against MPTP-induced neurodegeneration used to model Parkinson's disease (PD). Survival of tyrosine hydroxylase neurons in the substantia nigra was determined by stereological tests after MPTP intoxication in mice pretreated with either VIPR1 or VIPR2 agonist or after adoptive transfer of splenic cell populations from agonist-treated mice administered to MPTP-intoxicated animals. Treatment with VIPR2 agonist or splenocytes from agonist-treated mice resulted in increased neuronal sparing. Immunohistochemical tests showed that agonist-treated mice displayed reductions in microglial responses, with the most pronounced effects in VIPR2 agonist-treated, MPTP-intoxicated mice. In parallel studies, we observed reductions in proinflammatory cytokine release that included IL-17A, IL-6, and IFN-γ and increases in GM-CSF transcripts in CD4(+) T cells recovered from VIPR2 agonist-treated animals. Moreover, a phenotypic shift of effector to regulatory T cells was observed. These results support the use of VIPR2-selective agonists as neuroprotective agents for PD treatment. SIGNIFICANCE STATEMENT: Vasoactive intestinal peptide receptor 2 can elicit immune transformation in a model of Parkinson's disease (PD). Such immunomodulatory capabilities can lead to neuroprotection by attenuating microglial activation and by slowing degradation of neuronal cell bodies and termini in MPTP-intoxicated mice. The protective mechanism arises from altering a Th1/Th2 immune cytokine response into an anti-inflammatory and neuronal sparing profile. These results are directly applicable for the development of novel PD therapies.

Computational design of a ß-peptide that targets transmembrane helices.

The design of ß-peptide foldamers targeting the transmembrane (TM) domains of complex natural membrane proteins has been a formidable challenge. A series of ß-peptides was designed to stably insert in TM orientations in phospholipid bilayers. Their secondary structures and orientation in the phospholipid bilayer was characterized using biophysical methods. Computational methods were then devised to design a ß-peptide that targeted a TM helix of the integrin α(IIb)ß(3). The designed peptide (ß-CHAMP) interacts with the isolated target TM domain of the protein and activates the intact integrin in vitro.

Environment- and sequence-dependence of helical type in membrane-spanning peptides composed of ß3-amino acids.

Transmembrane (TM) ß-peptides comprised of acyclic ß(3)-amino acids demonstrate equilibrium between 12- and 14-helical structures in an environment- and sequence-dependent manner. Circular dichroism (CD) spectra of TM ß(3)-peptides may be described as linear combinations of the 12- and 14-helical CD spectra. The apparent malleability of ß(3)-substituted acyclic ß-peptides has practical implications for foldamer design, as it suggests that both the 14-helix and 12-helix might be reasonable platforms for molecular recognition.

Computational design of a self-assembling ß-peptide oligomer.

The first computationally designed self-assembling oligomer consisting of exclusively ß-amino acids (ßAAs) is presented. The packing of a ß-3(14) helix into coiled-coils of varying stoichiometries as a function of amino acid sequence is examined. ß-Peptides with hVal repeating every third residue in the sequence appeared to have a strong propensity to pack into hexameric bundles. The designed sequence was synthesized and characterized with CD spectroscopy, NMR, and analytical ultracentrifugation, suggesting that the peptide adopts a well-folded hexameric structure.

Development of a rotamer library for use in beta-peptide foldamer computational design.

Foldamers present a particularly difficult challenge for accurate computational design compared to the case for conventional peptide and protein design due to the lack of a large body of structural data to allow parametrization of rotamer libraries and energies. We therefore explored the use of molecular mechanics for constructing rotamer libraries for non-natural foldamer backbones. We first evaluated the accuracy of molecular mechanics (MM) for the prediction of rotamer probability distributions in the crystal structures of proteins is explored. The van der Waals radius, dielectric constant and effective Boltzmann temperature were systematically varied to maximize agreement with experimental data. Boltzmann-weighted probabilities from these molecular mechanics energies compare well with database-derived probabilities for both an idealized alpha-helix (R = 0.95) as well as beta-strand conformations (R = 0.92). Based on these parameters, de novo rotamer probabilities for secondary structures of peptides built from beta-amino acids were determined. To limit computational complexity, it is useful to establish a residue-specific criterion for excluding rare, high-energy rotamers from the library. This is accomplished by including only those rotamers with probability greater than a given threshold (e.g., 10%) of the random value, defined as 1/n where n is the number of potential rotamers for each residue type.