P2X7 antagonists for CNS indications: recent patent disclosures.

P2X7, a ligand-gated purinergic ion channel, has been at the center of intense efforts in the pharmaceutical industry in the last 15 years due to the growing appreciation of its role in inflammation. Since 2008-2009, increased focus on CNS available compounds has led to the publication of various patents on behalf of several pharmaceutical companies. This patent review aims at analyzing the recent patent literature (2008-2016) with a particular emphasis on those patents that are thought to deal with CNS penetrant compounds on the basis of their physicochemical features, the assays described in the patents and the uses these compounds are claimed for.

Piecing Together Cell-like Systems.

Several laboratories are pursuing the synthesis of cellular systems from different directions, including those that begin with simple chemicals to those that exploit existing cells. The methods that begin with nonliving components tend to focus on mimicking specific features of life, such as genomic replication, protein synthesis, sensory systems, and compartment formation, growth, and division. Conversely, the more prevalent synthetic biology approaches begin with something that is already alive and seek to impart new behavior on existing cells. Here we discuss advances in building cell-like systems that mimic key features of life with defined components.

Fluorescent proteins and in vitro genetic organization for cell-free synthetic biology.

To facilitate the construction of cell-free genetic devices, we evaluated the ability of 17 different fluorescent proteins to give easily detectable fluorescence signals in real-time from in vitro transcription-translation reactions with a minimal system consisting of T7 RNA polymerase and E. coli translation machinery, i.e., the PUREsystem. The data were used to construct a ratiometric fluorescence assay to quantify the effect of genetic organization on in vitro expression levels. Synthetic operons with varied spacing and sequence composition between two genes that coded for fluorescent proteins were then assembled. The resulting data indicated which restriction sites and where the restriction sites should be placed in order to build genetic devices in a manner that does not interfere with protein expression. Other simple design rules were identified, such as the spacing and sequence composition influences of regions upstream and downstream of ribosome binding sites and the ability of non-AUG start codons to function in vitro.

Intravesicle isothermal DNA replication.

BACKGROUND: Bacterial and viral DNA replication was previously reconstituted in vitro from component parts 1234. Significant advances in building minimal cell-like structures also have been made recently 567. Combining the two approaches would further attempts to build a minimal cell-like structure capable of undergoing evolution by combining membrane encapsulation and genome replication. Towards this end, we attempted to use purified genomic replication protein components from thermophilic bacterial sources to copy strands of DNA isothermally within lipid vesicles. FINDINGS: Bacterial replication components (such as helicases and DNA polymerases) are compatible with methods for the generation of lipid vesicles. Encapsulation inside phospholipid vesicles does not inhibit the activity of bacterial DNA genome replication machinery. Further the described system is efficient at isothermally amplifying short segments of DNA within phospholipid vesicles. CONCLUSIONS: Herein we show that bacterial isothermal DNA replication machinery is functional inside of phospholipid vesicles, suggesting that replicating cellular mimics can be built from purified bacterial components.

Synthesis and evaluation of new endomorphin-2 analogues containing (Z)-alpha,beta-didehydrophenylalanine (Delta(Z)Phe) residues.

New endomorphin-2 (EM-2) analogues incorporating (Z)-alpha,beta-didehydrophenylalanine (Delta(Z)Phe) in place of the native phenylalanine in EM-2 are reported. Tyr-Pro-Delta(Z)Phe-Phe-NH(2) {[Delta(Z)Phe(3)]EM-2} (1), Tyr-Pro-Phe-Delta(Z)Phe-NH(2) {[Delta(Z)Phe(4)]EM-2} (2), and Tyr-Pro-Delta(Z)Phe-Delta(Z)Phe-NH(2) {[Delta(Z)Phe(3,4)]EM-2}(3) have been synthesized, their opioid receptor binding affinities and tissue bioassay activities were determined, and their conformational properties were examined. Compound 2 shows high mu opioid receptor selectivity and mu agonist activity comparable to those of the native peptide. The conformation adopted in solution and in the crystal by N-Boc-Tyr-Pro-Delta(Z)Phe-Phe-NH(2) (8) is reported.

N-(tert)-butyloxycarbonyl)-beta,beta-cyclopentyl-cysteine (acetamidomethyl)-methyl ester for synthesis of novel peptidomimetic derivatives.

It has been recently reported that thiol groups could play an important role in the protection of neuronal cells in Alzheimer's disease (AD), prion disease (CJD) and Parkinson's disease (PD). Also bucillamine, that is a pseudo dipeptide possessing a thiol group capable to form an internal disulfide bridge, has relevant scavenger properties used in therapy for the treatment of arthritis. Furthermore, many sulphur containing compounds show strong chelating properties to heavy metals. Due to the crucial role of thiol groups in a variety of detoxicant biological systems, we report the synthesis of a racemic beta,beta-dialkyl-substituted, fully protected, cysteine derivative as a suitable intermediate in the synthesis of novel biological active peptides.

Synthesis and evaluation of new endomorphin analogues modified at the Pro(2) residue.

Six new endomorphin analogues, incorporating constrained amino acids in place of native proline have been synthesized. Residues of (S)-azetidine-2-carboxylic acid (Aze), 3,4-dehydro-(S)-proline (Delta(3)Pro), azetidine-3-carboxylic acid (3Aze) and dehydro-alanine (DeltaAla) have been used to prepare [Delta(3)Pro(2)]EM-2 (1), [Aze(2)]EM-1 (2), [Aze(2)]EM-2 (3), [3Aze(2)]EM-1 (4), [3Aze(2)]EM-2 (5) and [DeltaAla(2)]EM-2 (6). Binding assays and functional bioactivities for mu- and delta-receptors are reported. The highest affinity, bioactivity and selectivity are shown by peptides 2 and 3 containing the Aze residue.

Synthesis and bioactivity of chemotactic tetrapeptides: fMLF-OMe analogues incorporating spacer aminoacids at the lateral positions.

A small library of N-For and N-Boc tetrapeptidic analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe), obtained by incorporating three different spacer aminoacids (Gly, betaAla and Pro) between the native residues of Met and Leu (N-For- and N-Boc-Met-Xaa-Leu-Phe-OMe; Xaa2 series) and Leu and Phe (N-For- and N-Boc-Met-Leu-Xaa-Phe-OMe; Xaa3 series), have been synthesized and examined for their biological activity as agonists and antagonists. Chemotaxis, lysozyme release and superoxide anion production have been measured. All the N-For analogues maintain good to moderate chemotactic activity with the betaAla3 15 model reaching the maximum value. All the N-Boc tetrapeptides are efficient chemotactic antagonists. Conversely, with the exception of the moderate antagonistic activity exhibited by the N-Boc Xaa2 models against lysozyme release, all the other N-Boc analogues do not show significant activity against both superoxide anion and lysozyme release.

Novel chemotactic For-Met-Leu-Phe-OMe (fMLF-OMe) analogues based on met residue replacement by 4-amino-proline scaffold: synthesis and bioactivity.

cis-(2S,4S) 4-amino-proline (cAmp) and trans-(2S,4R) 4-amino-proline (tAmp) residues, bearing N-For or N-Boc substituents at the two amino groups, have been incorporated into the potent chemotactic agent fMLF-OMe in place of the N-terminal native (S)-methionine to give the analogues 17a-19a and 17b-19b. The new ligands have been examined for their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils as agonists and antagonists. Compounds 19a and 19b, bearing two N-For groups at the proline scaffold, are active and selective chemoattractants. The ligand 18b, containing N-For at the 4-amino group of the N-Boc-tAmp residue, exhibits significant chemotactic antagonism. The influence of the different substitution at the N-terminal position of the new analogues is discussed.