Novel N-Acylethanolamide Derivatives Affect Body Weight and Energy Balance.

Introduction - The obesity pandemic is multifactorial. Nutritional, pharmacologic and surgical interventions are limited in reach and efficacy, raising need for new therapeutics. Aims - Characterization of anorexigenic and cognitive effect and central mechanism of action of novel N-acylethanolamide derivatives. Methods - Sabra mice divided to similar experimental groups, injected IP with: oleyl-L-leucinolamide (1 A), linoleyl-L-leucinolamide (4 A), linoleyl-L-valinolamide (5 A), oleyl-oxycarbonyl-L-valinolamide (1 B), oleyl-oxycarbonyl-D-valinolamide (2 B), oleylamine-carbonyl-L-valinolamide (3 B), oleylamine-carbonyl-D-valinolamide (4 B), and oleyl-L-hydroxyvalineamide (5 B). Control group with vehicle. Body weight and food consumption followed for 39 days. Motor activity and cognitive function by open field test and eight-arm maze. Mice sacrificed and mechanism of action investigated by qPCR. The genes analyzed involved in energy balance and regulation of appetite. Catecholamines and serotonin evaluated. Results - Compounds 1 A, 5 A, 1 B-4 B, caused significant weight loss of 4.2-5.6 % and 5 A, 1 B-4 B, improved cognitive function following 8 i. p. injections of 1 mg/kg during 39 days, by different mechanisms. 5 A, 3 B and 4 B decreased food consumption, whereas 1 A, 5 A and 2 B increased motor activity. 1 A, 4 A, 1 B and 3 B elevated SIRT-1, associated with survival. POMC upregulated by 1 B and 2 B, CART by 1 B, 2 B and 1 A. NPY and CAMKK2 downregulated by 5 A. 4 B enhanced 5-HT levels. 4 A, 5 A, 1 B, 4 B, 5 B decreased FAAH, showing long lasting effect. Conclusions - These new compounds might be developed for the treatment of obesity and for improved cognitive function.

Peptide Bond Formation in the Protonated Serine Dimer Following Vacuum UV Photon-Induced Excitation.

Possible routes for intra-cluster bond formation (ICBF) in protonated serine dimers have been studied. We found no evidence of ICBF following low energy collision-induced dissociation (in correspondence with previous works), however, we do observe clear evidence for ICBF following photon absorption in the 4.6-14 eV range. Moreover, the comparison of photon-induced dissociation measurements of the protonated serine dimer to those of a protonated serine dipeptide provides evidence that ICBF, in this case, involves peptide bond formation (PBF). The experimental results are supported by ab initio molecular dynamics and exploration of several excited state potential energy surfaces, unraveling a pathway for PBF following photon absorption. The combination of experiments and theory provides insight into the PBF mechanisms in clusters of amino acids, and reveals the importance of electronic excited states reached upon UV/VUV light excitation.

Sourcing Herod the Great's calcite-alabaster bathtubs by a multi-analytic approach.

Herod "the Great", king of Judea in the second half of the first century BC, was known for his building projects, wealth, and political power. Two of his personal calcite-alabaster bathtubs, found in the Kypros fortress and the palace of Herodium, are among the very limited archaeological evidence of his private life. It seemed plausible that they were imported from Egypt, the main source of calcite-alabaster in ancient periods. Yet, the recent identification of a calcite quarry in the Te'omim cave, Israel, challenges this hypothesis. Here, we developed an approach for identification of the source of calcite-alabaster, by combination of four analytical methods: ICP, FTIR, ssNMR and isotope ratio. These methods were then applied to Herod's bathtubs demonstrating that they were indeed quarried in Israel rather than in Egypt.

Beta-Carotene derivatives as novel therapy for the prevention and treatment of autistic symptoms.

Autistic Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by impaired social interaction & communication as well as restricted and repetitive behavior. The currently reported incidence of ASD is 1-2%, and it increases dramatically to 10-20% in families predisposed to ASD. To date, there is no effective way to treat or prevent ASD, and only symptomatic treatment with limited efficacy is available. Oxytocin (Oxt) enhances affiliative behavior and improves social cognition. Social deficits characteristic of autism may be related to dysfunctional Oxt neurotransmission. Thus, administration of Oxt may relieve ASD, however it has a short plasma half-life and poor Blood Brain Barrier (BBB) permeability. CD38, a multifunctional ecto-enzyme expressed in brain and immune cells, was found to be critical for social behavior via regulation of Oxt secretion. All-trans retinoic acid (ATRA) is a potent inducer of CD38 and improves social behavior, but it is toxic and teratogenic. We have shown that beta-carotene has a similar therapeutic effect. The present study aimed to investigate the activity of novel beta-carotene derivatives in rescuing low sociability found in BTBR mice, providing an in vivo "proof of principle" that beta-carotene derivatives are potential agents to prevent/ameliorate the reappearance of ASD in high-risk populations for ASD. Beta-carotene and its synthetic analogs were administered orally to newborn BTBR mice with ASD associated like behavior. After 2 months, they were tested (at dosages of 0.1 and 1.0 mg/kg) by cognitive (T-maze spontaneous alteration and neurological score) and behavioral tests (reciprocal social interaction, repetitive grooming / bedding behavior), previously shown as indicators for autistic behavior. The following biochemical and molecular biology parameters were also examined: serum Oxt; gene expression in hippocampus and hypothalamus of CD 38, Oxt, Oxt receptor, BDNF, and retinoic acid receptor. The new compounds were significantly more effective than control. The most effective compounds, both in the behavioral tests and in their biochemical effects, were (3R,3'R)-astaxanthin bis(N-Cbz-l-alanine ester) (3B(and (3S,3'S)-astaxanthin bis(N,N-dimethylglycine ester (5). They did not exert any neurological symptoms. Thus, beta-carotene derivatives may have the potential to prevent and/or ameliorate autistic symptoms when administered orally after birth to newborns of families predisposed to autism.

Targeting Necrosis: Elastase-like Protease Inhibitors Curtail Necrotic Cell Death Both In Vitro and in Three In Vivo Disease Models.

Necrosis is the main mode of cell death, which leads to multiple clinical conditions affecting hundreds of millions of people worldwide. Its molecular mechanisms are poorly understood, hampering therapeutics development. Here, we identify key proteolytic activities essential for necrosis using various biochemical approaches, enzymatic assays, medicinal chemistry, and siRNA library screening. These findings provide strategies to treat and prevent necrosis, including known medicines used for other indications, siRNAs, and establish a platform for the design of new inhibitory molecules. Indeed, inhibitors of these pathways demonstrated protective activity in vitro and in vivo in animal models of traumatic brain injury, acute myocardial infarction, and drug-induced liver toxicity. Consequently, this study may pave the way for the development of novel therapies for the treatment, inhibition, or prevention of a large number of hitherto untreatable diseases.

Development and characterisation of SMURF2-targeting modifiers.

The C2-WW-HECT-domain E3 ubiquitin ligase SMURF2 emerges as an important regulator of diverse cellular processes. To date, SMURF2-specific modulators were not developed. Here, we generated and investigated a set of SMURF2-targeting synthetic peptides and peptidomimetics designed to stimulate SMURF2's autoubiquitination and turnover via a disruption of the inhibitory intramolecular interaction between its C2 and HECT domains. The results revealed the effects of these molecules both in vitro and in cellulo at the nanomolar concentration range. Moreover, the data showed that targeting of SMURF2 with either these modifiers or SMURF2-specific shRNAs could accelerate cell growth in a cell-context-dependent manner. Intriguingly, a concomitant cell treatment with a selected SMURF2-targeting compound and the DNA-damaging drug etoposide markedly increased the cytotoxicity produced by this drug in growing cells. Altogether, these findings demonstrate that SMURF2 can be druggable through its self-destructive autoubiquitination, and inactivation of SMURF2 might be used to affect cell sensitivity to certain anticancer drugs.

Gas Phase Bond Formation in Dipeptide Clusters.

Protein bonds between amino acids are one of the most important biological linkages that create life. The detection of amino acids in the interstellar environments and in meteorites may lead to the suggestion that amino acids came from outer space and that peptides bonds may have been created in the gas phase. Here we show experimentally the creation of covalent bonds, most likely peptide bonds, between serine dipeptides in the gas phase. More specifically, we show that spraying a solution of Ser-Ser dipeptides results, in addition to dipeptide clusters, in a peak with the same mass as the serine tetrapeptide, which also has the same fragmentation pattern. Moreover, we show that this mass is formed upon collision induced dissociation of clusters containing four serine dipeptides. Thence, if the dipeptide can be generated abiotically the polymerization process may occur spontaneously.

Beta-carotene as a novel therapy for the treatment of "Autistic like behavior" in animal models of Autism.

Autism-affected individuals are characterized by lower plasma oxytocin and its ectoenzyme regulator CD38. Oxytocin, a hypothalamic hormone secreted upon the release of CD38, plays a role in social behavior and bonding. All-trans retinoic acid is a potent inducer of CD38 and can be used as a novel therapeutic strategy in autism. We investigated the role of beta-carotene in rescuing autistic-like behavior in BALB/c and BTBR mice. Beta-carotene derivatives are preferred as they are neither toxic nor teratogenic. Beta-carotene at 0.1-5.0 mg/kg was administered orally to BALB/c and BTBR newborn mice on days 1-7. They were tested at age 2-3 months for five behavioral tests for "autism"; in addition, brain CD38, oxytocin, oxytocin receptor, Brain Derived Neurotrophic Factor (BDNF) and retinoic acid receptor gene expression, serum oxytocin levels, and neurological score were evaluated. Beta-carotene administered at birth significantly increased T-maze alternations and led to longer time spent with an unfamiliar mouse in the "three-chamber test" and less time spent in the empty chamber. Furthermore, enhanced activity in the open field test; increased time spent in the reciprocal social interaction test; decreased grooming and bedding behaviors; and enhanced brain CD38, oxytocin, oxytocin receptor, BDNF, retinoic acid gene expression, and serum oxytocin levels. No changes in neurological score were observed. Beta-carotene oral supplementation to BALB/c and BTBR mice at birth significantly reduced restricted and stereotyped behaviors and interests, increased social interactions and communication, CD38, and oxytocin, probably by enhancing brain neuroplasticity without toxicity. Thus, beta-carotene administered after birth to newborns of families predisposed to "autism" has the potential to prevent/ameliorate" autistic like behavior". These results support further clinical studies.

How does the exosite of rhomboid protease affect substrate processing and inhibition?

Rhomboid proteases constitute a family of intramembrane serine proteases ubiquitous in all forms of life. They differ in many aspects from their soluble counterparts. We applied molecular dynamics (MD) computational approach to address several challenging issues regarding their catalytic mechanism: How does the exosite of GlpG rhomboid protease control the kinetics efficiency of substrate hydrolysis? What is the mechanism of inhibition by the non-competitive peptidyl aldehyde inhibitors bound to the GlpG rhomboid active site (AS)? What is the underlying mechanism that explains the hypothesis that GlpG rhomboid protease is not adopted for the hydrolysis of short peptides that do not contain a transmembrane domain (TMD)? Two fundamental features of rhomboid catalysis, the enzyme recognition and discrimination of substrates by TMD interactions in the exosite, and the concerted mechanism of non-covalent pre-catalytic complex to covalent tetrahedral complex (TC) conversion, provide answers to these mechanistic questions.

Ligand-Substitution Reactions of the Tellurium Compound AS-101 in Physiological Aqueous and Alcoholic Solutions.

Since its first crystallization, the aqueous structure of the tellurium-containing experimental drug AS-101 has never been studied. We show that, under the aqueous conditions in which it is administered, AS-101 is subjected to an immediate ligand-substitution reaction with water, yielding a stable hydrolyzed oxide anion product that is identified, for the first time, to be TeOCl3-. Studying the structure of AS-101 in propylene glycol (PG), an alcoholic solvent often used for the topical and oral administration of AS-101, revealed the same phenomenon of ligand-substitution reaction between the alcoholic ligands. Upon exposure to water, the PG-substituted product is also hydrolyzed to the same tellurium(IV) oxide form, TeOCl3-.

The Anticancer Activity of Organotelluranes: Potential Role in Integrin Inactivation.

Organic Te(IV) compounds (organotelluranes) differing in their labile ligands exhibited anti-integrin activities in vitro and anti-metastatic properties in vivo. They underwent ligand substitution with l-cysteine, as a thiol model compound. Unlike inorganic Te(IV) compounds, the organotelluranes did not form a stable complex with cysteine, but rather immediately oxidized it. The organotelluranes inhibited integrin functions, such as adhesion, migration, and metalloproteinase secretion mediation in B16F10 murine melanoma cells. In comparison, a reduced derivative with no labile ligand inhibited adhesion of B16F10 cells to a significantly lower extent, thus pointing to the importance of the labile ligands of the Te(IV) atom. One of the organotelluranes inhibited circulating cancer cells in vivo, possibly by integrin inhibition. Our results extend the current knowledge on the reactivity and mechanism of organotelluranes with different labile ligands and highlight their clinical potential.

Biolabile peptidyl delivery systems toward sequential drug release.

Compact carriers for peptidyl delivery systems (PDSs) loaded with various drugs were synthesized using a simple and convenient solid phase organic synthesis strategy, including semi-orthogonal functional group protection schemes. Each attachment point of the compact carrier can thus be bound to an anticancer agent through a biodegradable covalent link. Chemo- and biostability experiments of a model peptidyl platform loaded with three different drugs revealed pH and liver homogenate (metabolic) dependent sequential release behavior. The versatility of this approach will serve to expedite the preparation of PDS libraries. This approach may prove useful for applications suitable for personalized medicine where multiple drug delivery is required in a sequential and controlled fashion.

Stepwise Versus Concerted Mechanisms in General-Base Catalysis by Serine Proteases.

General-base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp-His-Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser-His dyad) inhibition by an isocoumarin derivative. The sampled MD trajectories of averaged pKa  values of catalytic residues were QM calculated by the MD-QM/SCRF(VS) method on molecular clusters simulating the active site. Differences between concerted and stepwise mechanisms are controlled by the dynamically changing pKa  values of the catalytic residues as a function of their progressively reduced water exposure, caused by the incoming ligand.

α-Aminoisobutyric Acid Leads a Fluorescent syn-bimane LASER Probe Across the Blood-brain Barrier.

Penetration of the blood brain barrier (BBB) by appropriate fluorescent probes remains a challenge in optical imaging and diagnostics. We designed, synthesized and observed the in vivo BBB penetration of a LASER syn-bimane probe. Results demonstrate that the Aib transporter unit in our probe may lead a fluorescent bimanyl moiety across the BBB.

Antimicrobial benzodiazepine-based short cationic peptidomimetics.

Antimicrobial peptides (AMPs) appear to be good candidates for the development of new antibiotic drugs. We describe here the synthesis of peptidomimetic compounds that are based on a benzodiazepine scaffold flanked with positively charged and hydrophobic amino acids. These compounds mimic the essential properties of cationic AMPs. The new design possesses the benzodiazepine scaffold that is comprised of two glycine amino acids and which confers flexibility and aromatic hydrophobic 'back', and two arms used for further synthesis on solid phase for incorporation of charged and hydrophobic amino acids. This approach allowed us a better understanding of the influence of these features on the antimicrobial activity and selectivity. A novel compound was discovered which has MICs of 12.5 µg/ml against Staphylococcus aureus and 25 µg/ml against Escherichia coli, similar to the well-known antimicrobial peptide MSI-78. In contrast to MSI-78, the above mentioned compound has lower lytic effect against mammalian red blood cells. These peptidomimetic compounds will pave the way for future design of potent synthetic mimics of AMPs for therapeutic and biomedical applications.

"Switch off/switch on" regulation of drug cytotoxicity by conjugation to a cell targeting peptide.

Bi-nuclear amino acid platforms loaded with various drugs for conjugation to a peptide carrier were synthesized using simple and convenient orthogonally protective solid-phase organic synthesis (SPOS). Each arm of the platform carries a different anticancer agent linked through the same or different functional group, providing discrete chemo- and bio-release profiles for each drug, and also enabling "switch off/switch on" regulation of drug cytotoxicity by conjugation to the platform and to a cell targeting peptide. The versatility of this approach enables efficient production of drug-loaded platforms and determination of favorable drug combinations/modes of linkage for subsequent conjugation to a carrier moiety for targeted cancer cell therapy. The results presented here potentiate the application of amino acid platforms for targeted drug delivery (TDD).

A new method for filtering of reactive "warheads" of transition-state analog protease inhibitors.

In light of the major contribution of the reactive warhead to the binding energy trend in reversible covalent transition-state analog inhibitors of serine and cysteine hydrolases, would it be possible to rationally design and quickly filter such warheads, especially for large-scale screening? The previously defined W1 and W2 covalent descriptors quantitatively account for the energetic effect of the covalent bonds reorganization, accompanying enzyme-inhibitor covalent binding. The quantum mechanically calculated W1 and W2 reflect the warhead binding energy by modeling of the enzyme-inhibitor reaction core. Here, we demonstrate the use of these descriptors for warhead filtering, and examine its scope and limitations. The W1 and W2 descriptors provide a tool for rational design of various warheads as universal building blocks of real inhibitors without the requirement of 3D structural information about the target enzyme or QSAR studies. These warheads could then be used as hit structural templates in the subsequent optimization of inhibitors recognition sites.

Anti-neoplastic activity of 1,3-diaza-2-functionalized-adamantan-6-one compounds against melanoma cells.

Four series of 1,3-diaza-2-functionalized-adamantan-6-one derivatives, bearing at the 2 position SO, SO2, POCl and PO2H functional groups, were synthesized via a key quadruple Mannich reaction, followed by transformation of an aminal functionality into the final 2-thia- and 2-phospha compounds. The compounds were tested for cytotoxic activity against the mouse B16-F10 melanoma cell line. Malignant melanoma is notorious for its high resistance to chemotherapy, and new anti-melanoma drugs are urgently needed. The 2-thia compounds exhibited poor proliferation inhibition activity, but the 2-phospha derivatives showed significant activity, with IC50 values of 10-60 µM. The compounds induced cell death by G2/M cell cycle arrest, which led to apoptosis, as determined by Annexin V-FITC/PI staining, mitochondrial membrane potential changes assessed by the JC-1 reagent, caspases 3 and 7 activation, and morphological changes.

Application of EMBM to Structure-Based Design of Warheads for Protease Inhibitors.

Most CADD tools handle non-covalent enzyme inhibitors, despite the growing interest of the pharma industry in covalent inhibitors. We have recently introduced an enzyme mechanism-based method, EMBM, as a computational tool for binding trend analysis and prediction of chemical sites (CS) of reversible covalent enzyme inhibitors. In the current study we demonstrate the utility of EMBM to structure-based applications. In this mode, the energy of the enzyme-inhibitor covalent bond is accounted for by the W1 and W2 covalent descriptors we have developed, whereas the non-covalent interactions between the inhibitor CS and the enzyme active site can be estimated directly on the 3D structure of the enzyme-inhibitor complex.