Deborah E. Shalev

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor

Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids—arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series—and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB1 cannabinoid receptor (Ki = 21.2 ± 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB2 receptor (Ki > 3 μM).

Structural Requirements for Potent Versus Selective Cytotoxicity for Antimicrobial Dermaseptin S4 Derivatives

To better understand the structural requirements for selective cytotoxicity of antimicrobial peptides, seven dermaseptin S4 analogs were produced and investigated with respect to molecular organization in solution, binding properties to model phospholipid membranes, and cytotoxic properties. Native dermaseptin S4 displayed high aggregation in solution and high binding affinity. These properties correlated with high cytotoxicity. Yet, potency was progressively limited when facing cells whose plasma membrane was surrounded by increasingly complex barriers. Increasing the positive charge of the native peptide led to partial depolymerization that correlated with higher binding affinity and with virtually non-discriminative high cytotoxicity against all cell types. The C-terminal hydrophobic domain was found responsible for binding to membranes but not for their disruption. Truncations of the C terminus combined with increased positive charge of the N-terminal domain resulted in short peptides having similar binding affinity as the parent compound but displaying selective activity against microbes with reduced toxicity toward human red blood cells. Nuclear magnetic resonance-derived three-dimensional structures of three active derivatives enabled the delineation of a common amphipathic structure with a clear separation of two lobes of positive and negative electrostatic potential surfaces. Whereas the spatial positive electrostatic potential extended considerably beyond the peptide dimensions and was required for potency, selectivity was affected primarily by hydrophobicity. The usefulness of this approach for the design of potent and/or selective cytolytic peptides is discussed herein.

Cognitive-Performance Recovery of Alzheimer's Disease Model Mice by Modulation of Early Soluble Amyloidal Assemblies†

A rationally designed oligomerization inhibitor interacts with early intermediate assemblies of amyloid-beta polypeptide (Abeta) through the aromatic elements and inhibits their assembly into the toxic oligomers that cause Alzheimers disease by a unique C(alpha)-methylation beta-breakage strategy. The electrostatic potential of the low-energy conformation of the dipeptide inhibitor bound to Abeta is shown.

Complete phenotypic recovery of an Alzheimer's disease model by a quinone-tryptophan hybrid aggregation inhibitor.

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimers disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimers disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimers disease.

Inhibiting α-Synuclein Oligomerization by Stable Cell-Penetrating β-Synuclein Fragments Recovers Phenotype of Parkinson's Disease Model Flies

The intracellular oligomerization of α-synuclein is associated with Parkinsons disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we indentified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinsons disease.

The Butyrylcholinesterase K Variant Confers Structurally Derived Risks for Alzheimer Pathology

The K variant of butyrylcholinesterase (BChE-K, 20% incidence) is a long debated risk factor for Alzheimer disease (AD). The A539T substitution in BChE-K is located at the C terminus, which is essential both for BChE tetramerization and for its capacity to attenuate β-amyloid (Aβ) fibril formation. Here, we report that BChE-K is inherently unstable as compared with the “usual” BChE (BChE-U), resulting in reduced hydrolytic activity and predicting prolonged acetylcholine maintenance and protection from AD. A synthetic peptide derived from the C terminus of BChE-K (BSP-K), which displayed impaired intermolecular interactions, was less potent in suppressing Aβ oligomerization than its BSP-U counterpart. Correspondingly, highly purified recombinant human rBChE-U monomers suppressed β-amyloid fibril formation less effectively than dimers, which also protected cultured neuroblastoma cells from Aβ neurotoxicity. Dual activity structurally derived changes due to the A539T substitution can thus account for both neuroprotective characteristics caused by sustained acetylcholine levels and elevated AD risk due to inefficient interference with amyloidogenic processes.

Backbone cyclic peptidomimetic melanocortin-4 receptor agonist as a novel orally administrated drug lead for treating obesity.

The tetrapeptide sequence His-Phe-Arg-Trp, derived from melanocyte-stimulating hormone (alphaMSH) and its analogs, causes a decrease in food intake and elevates energy utilization upon binding to the melanocortin-4 receptor (MC4R). To utilize this sequence as an effective agent for treating obesity, we improved its metabolic stability and intestinal permeability by synthesizing a library of backbone cyclic peptidomimetic derivatives. One analog, peptide 1 (BL3020-1), was selected according to its selectivity in activating the MC4R, its favorable transcellular penetration through enterocytes and its enhanced intestinal metabolic stability. This peptide was detected in the brain following oral administration to rats. A single oral dose of 0.5 mg/kg in mice led to reduced food consumption (up to 48% vs the control group) that lasted for 5 h. Repetitive once daily oral dosing (0.5 mg/kg/day) for 12 days reduced weight gain. Backbone cyclization was shown to produce a potential drug lead for treating obesity.

Cyclic peptide inhibitors of HIV-1 integrase derived from the LEDGF/p75 protein

Restricting linear peptides to their bioactive conformation is an attractive way of improving their stability and activity. We used a cyclic peptide library with conformational diversity for selecting an active and stable peptide that mimics the structure and activity of the HIV-1 integrase (IN) binding loop from its cellular cofactor LEDGF/p75 (residues 361-370). All peptides in the library had the same primary sequence, and differed only in their conformation. Library screening revealed that the ring size and linker structure had a huge effect on the conformation, binding and activity of the peptides. One of the cyclic peptides, c(MZ 4-1), was a potent and stable inhibitor of IN activity in vitro and in cells even after 8 days. The NMR structure of c(MZ 4-1) showed that it obtains a bioactive conformation that is similar to the parent site in LEDGF/p75.

Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2

We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2Ank-SH3) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2Ank-SH3 binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2Ank-SH3 to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2Ank-SH3 to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2Ank-SH3 with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2Ank-SH3 with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins.

Low viscosity reversed hexagonal mesophases induced by hydrophilic additives.

This study reports on the formation of a low viscosity H(II) mesophase at room temperature upon addition of Transcutol (diethylene glycol mono ethyl ether) or ethanol to the ternary mixture of GMO (glycerol monooleate)/TAG (tricaprylin)/water. The microstructure and bulk properties were characterized in comparison with those of the low viscosity HII mesophase formed in the ternary GMO/TAG/water mixture at elevated temperatures (35-40 degrees C). We characterized the role of Transcutol or ethanol as inducers of disorder and surfactant mobility. The techniques used were rheology, differential scanning calorimetry (DSC), wide- and small-angle X-ray scattering (WAXS and SAXS, respectively), NMR (self-diffusion and (2)H NMR), and Fourier transform infrared (FTIR) spectroscopies. The incorporation of either Transcutol or ethanol induced the formation of less ordered HII mesophases with smaller domain sizes and lattice parameters at room temperature (up to 30 degrees C), similar to those found for the GMO/TAG/water mixture at more elevated temperatures (35-40 degrees C). On the basis of our measurements, we suggest that Transcutol or ethanol causes dehydration of the GMO headgroups and enhances the mobility of the GMO chains. As a result, these two small molecules, which compete for water with the GMO polar headgroups, may increase the curvature of the cylindrical micelles and also perhaps reduce their length. This results in the formation of fluid H(II) structures at room temperature (up to 30 degrees C). It is possible that these phases are a prelude to the H(II)-L(2) transformation, which takes place above 35 degrees C.