Dual 5-HT3 and 5-HT6 Receptor Antagonist FPPQ Normalizes Phencyclidine-Induced Disruption of Brain Oscillatory Activity in Rats.

Schizophrenia is a severe mental disorder featuring psychotic, depressive, and cognitive alterations. Current antipsychotic drugs preferentially target dopamine D2-R and/or serotonergic 5-HT2A/1A-R. They partly alleviate psychotic symptoms but fail to treat negative symptoms and cognitive deficits. Here we report on the putative antipsychotic activity of (1-[(3-fluorophenyl)sulfonyl]-4-(piperazin-1-yl)-1H-pyrrolo[3,2-c]quinoline dihydrochloride) (FPPQ), a dual serotonin 5-HT3-R/5-HT6-R antagonist endowed with pro-cognitive properties. FPPQ fully reversed phencyclidine-induced decrease of low-frequency oscillations in the medial prefrontal cortex of anaesthetized rats, a fingerprint of antipsychotic activity. This effect was mimicked by the combined administration of the 5-HT3-R and 5-HT6-R antagonists ondansetron and SB-399 885, respectively, but not by either drug alone. In freely moving rats, FPPQ countered phencyclidine-induced hyperlocomotion and augmentation of gamma and high-frequency oscillations in medial prefrontal cortex, dorsal hippocampus, and nucleus accumbens. Overall, this supports that simultaneous blockade of 5-HT3R and 5-HT6-R-like that induced by FPPQ-can be a new target in antipsychotic drug development.

An In Silico Methodology That Facilitates Decision Making in the Engineering of Nanoscale Protein Materials.

Under the need for new functional and biocompatible materials for biomedical applications, protein engineering allows the design of assemblable polypeptides, which, as convenient building blocks of supramolecular complexes, can be produced in recombinant cells by simple and scalable methodologies. However, the stability of such materials is often overlooked or disregarded, becoming a potential bottleneck in the development and viability of novel products. In this context, we propose a design strategy based on in silico tools to detect instability areas in protein materials and to facilitate the decision making in the rational mutagenesis aimed to increase their stability and solubility. As a case study, we demonstrate the potential of this methodology to improve the stability of a humanized scaffold protein (a domain of the human nidogen), with the ability to oligomerize into regular nanoparticles usable to deliver payload drugs to tumor cells. Several nidogen mutants suggested by the method showed important and measurable improvements in their structural stability while retaining the functionalities and production yields of the original protein. Then, we propose the procedure developed here as a cost-effective routine tool in the design and optimization of multimeric protein materials prior to any experimental testing.

Rational engineering of a human GFP-like protein scaffold for humanized targeted nanomedicines.

Green fluorescent protein (GFP) is a widely used scaffold for protein-based targeted nanomedicines because of its high biocompatibility, biological neutrality and outstanding structural stability. However, being immunogenicity a major concern in the development of drug carriers, the use of exogenous proteins such as GFP in clinics might be inadequate. Here we report a human nidogen-derived protein (HSNBT), rationally designed to mimic the structural and functional properties of GFP as a scaffold for nanomedicine. For that, a GFP-like ß-barrel, containing the G2 domain of the human nidogen, has been rationally engineered to obtain a biologically neutral protein that self-assembles as 10nm-nanoparticles. This scaffold is the basis of a humanized nanoconjugate, where GFP, from the well-characterized protein T22-GFP-H6, has been substituted by the nidogen-derived GFP-like HSNBT protein. The resulting construct T22-HSNBT-H6, is a humanized CXCR4-targeted nanoparticle that selectively delivers conjugated genotoxic Floxuridine into cancer CXCR4+ cells. Indeed, the administration of T22-HSNBT-H6-FdU in a CXCR4-overexpressing colorectal cancer mouse model results in an even more efficient selective antitumoral effect than that shown by its GFP-counterpart, in absence of systemic toxicity. Therefore, the newly developed GFP-like protein scaffold appears as an ideal candidate for the development of humanized protein nanomaterials and successfully supports the tumor-targeted nanoscale drug T22-HSNBT-H6-FdU. STATEMENT OF SIGNIFICANCE: Targeted nanomedicine seeks for humanized and biologically neutral protein carriers as alternative of widely used but immunogenic exogenous protein scaffolds such as green fluorescent protein (GFP). This work reports for the first time the rational engineering of a human homolog of the GFP based in the human nidogen (named HSNBT) that shows full potential to be used in humanized protein-based targeted nanomedicines. This has been demonstrated in T22-HSNBT-H6-FdU, a humanized CXCR4-targeted protein nanoconjugate able to selectively deliver its genotoxic load into cancer cells.

Lead mobilization during calcium disodium ethylenediaminetetraacetate chelation therapy in treatment of chronic lead poisoning.

It has long been established that chronic lead (Pb) poisoning is a cause of renal insufficiency. However, although easily diagnosed, there is still no treatment available that will revert this type of poisoning. We report a study performed on 56 male Wistar rats administered Pb in drinking water (500 ppm Pb acetate) over a 90-day period. Twenty-one non-Pb-exposed animals served as the control group. Seven animals from each group were killed days 60 and 90. At the end of the 90-day period, 21 of the Pb-exposed animals were treated with disodium monocalcium EDTA (50 mg/kg/d for 5 days) intraperitoneally and 21 animals were administered serum saline by the same route. Three treatment courses were administered, separated by 9 days free of treatment. Seven animals from each subgroup were killed at the end of each treatment course. Pb levels were determined in blood, urine, liver, brain, kidney, and bone. Treatment with EDTA led to a greater and more rapid reduction in Pb contents in the brain and kidney. The decrease in hepatic Pb levels in the treated group of animals was similar to that in the group administered placebo. Bone Pb levels also failed to show a response to the chelating agent. Use of EDTA appears to result in a reduction in Pb deposits in such critical organs as the kidney and brain. However, the chelating agent does not seem to have access to bone Pb deposits, such that the skeleton becomes a permanent source of poisoning for other tissues.

Experimental lead nephropathy: treatment with calcium disodium ethylenediaminetetraacetate.

Chronic lead poisoning may cause hypertension, gout, and renal insufficiency. Most experimental poisoning studies have involved the use of high doses over short periods (ie, acute poisoning). Although chelating treatment leads to remission of acute lead nephropathy, its effects in the treatment of chronic poisoning are unclear. The aims of this study were to evaluate renal alterations produced during chronic lead poisoning and their progression when poisoning was over and to determine the efficiency of chelating treatment with calcium disodium ethylenediaminetetraacetate (EDTA). In this study, 56 male Wistar rats were administered lead in drinking water (500 ppm lead acetate) over 90 days. The control group consisted of 21 nonexposed rats. Seven rats from each group were killed on days 60 and 90. At the end of the 90-day period, 21 of the lead-exposed rats were treated with disodium monocalcium EDTA (50 mg/kg/d x 5 days) intraperitoneally, and 21 were administered serum saline by the same route. Three treatment courses were given separated by 9 days free of treatment. Seven rats from each subgroup were sacrificed at the end of each treatment course. Main findings related to poisoning were hypertrophy and vacuolization of medium and small arteries; mucoid edema and muscular hypertrophy in arterioles; loss of cell brush borders, cell loss, and intranuclear inclusion bodies in the proximal tubule; and fibrosis and the presence of infiltrates in the interstitial component. Treatment with EDTA slowed the progression of most alterations. No damage associated with the use of the chelating agent was observed. Longer term studies of the effects of this drug are required to establish whether the damage caused by lead poisoning may be reversed.

Trapping of Cyclopropenyl Radicals by 5,5-Dimethyl-1-pyrroline-N-oxide.

The possible generation of cyclopropenyl radicals by ultraviolet irradiation of different cyclopropenyl derivatives in fluid solution and in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin trap has been detected by electron paramagnetic resonance. The spectra consist of doublets of triplets in which the ß-hydrogen splitting is larger than that of the nitrogen, in good agreement with data reported in the literature for other DMPO adducts.This methodology is unprecedented in the study of these transient radical species, and these results suggest the participation of cyclopropenyl radicals in the photosensitized decarboxylation of N-(2-cyclopropenylcarbonyloxy)phthalimides.

Practical synthesis of a dithiane-protected 3',5'-dialkoxybenzoin photolabile safety-catch linker for solid-phase organic synthesis.

The solution-phase preparation of the 3',5'-dialkoxybenzoin photolabile safety-catch linker 16 is described. Pivotal to this convenient synthesis is the selection of appropriate orthogonal protecting groups for the alkoxy functionalities present. The new linker can be readily loaded onto any standard amine-terminating resin under peptide-coupling conditions, without the need to protect the secondary alcohol functionality, and subsequently loaded with substrate. Alternatively, the loading efficiency of sterically hindered substrates can be enhanced by preloading the semiprotected linker variant 10 in solution prior to immobilization onto the resin. This second generation of benzoin photolabile safety-catch linkers provides greater control of both linker loading and resin attachment and should prove to be a more versatile and convenient form of the linker.

Studies on the chemical stability and functional group compatibility of the benzoin photolabile safety-catch linker using an analytical construct.

A chemical stability study of the benzoin photolabile safety-catch linker (BPSC) has been carried out using a dual-linker analytical construct to establish its compatibility with a range of commonly employed solid-phase reaction conditions. As a result of this study, the dithiane-protected benzoin linker was shown to be reactive only toward strong acids and fluoride nucleophile. Furthermore, a scan of diverse functional groups thought to be unstable toward the safety-catch removal conditions has also been carried out. These data should provide assistance in future utilization of the BPSC for syntheses.

Studies on the chemical stability and synthetic utility of an oxazolidine linker for solid-phase chemistry.

A chemical stability study on the oxazolidine linker system has been carried out using a dual-linker analytical construct within a parallel reaction scan. The study established the compatibility of the oxazolidine platform with a wide range of commonly employed synthetic reaction conditions including nucleophilic, oxidizing, and reducing conditions. The scan was further used to probe and optimize acidic conditions under which the oxazolidine could release the substrate from the solid-support and to identify reagents that could cleave while retaining other acid-labile groups. The solid-phase synthesis of a small molecular array established the utility of oxazolidine aldehyde 1 as a building block for asymmetric chemistry while exploiting the data generated by the reaction scan.