Physicochemical and pharmacotechnical characterization of Prussian blue for future Prussian blue oral dosage forms formulation.

Ferric hexacyanoferrate, Fe4 [Fe(CN)6]3 · xH2O, known as Prussian blue (PB), has proven its effectiveness as an antidote in cases of accidental poisoning or poisoning caused by radioactive materials such as cesium (Cs) and thallium (Tl); which due to their solubility in water, when absorbed by the human body, cause serious damage to vital organs. The local development of a drug with PB as an active ingredient arises as a response to the civil and military needs established within the Ministry's pharmacy request for national defense. This fact contemplates the circumstances related to public health protection in the nuclear, radiological, biological and chemical (NRBQ) of the emergency institutions in health and national security. In this paper and by using various analytical techniques, the characterization of the locally synthesized PB with pharmaceutical quality has been described, as a first step to predict its behavior in the preparation of a drug that contains it as an active ingredient. The research findings demonstrate that locally synthesized PB is suitable for use in oral dosage forms, enabling the local development of drug formulations incorporating PB, thus being able to potentially become a main resource in the treatment of Cs and Tl poisoning in any accidental or intended of the population. This development opens up the possibility of creating drug formulations that incorporate PB at a local level, making it a potentially significant resource in the treatment of Cs and Tl poisoning. The ability to locally produce and utilize PB in oral dosage forms could be crucial in addressing cases of accidental or intentional exposure within the population. This advancement not only contributes to the scientific understanding of PB but also holds promising implications for practical applications in public health and emergency situations.

Probing Factor Xa Protein-Ligand Interactions: Accurate Free Energy Calculations and Experimental Validations of Two Series of High-Affinity Ligands.

The accurate prediction of protein-ligand binding affinity belongs to one of the central goals in computer-based drug design. Molecular dynamics (MD)-based free energy calculations have become increasingly popular in this respect due to their accuracy and solid theoretical basis. Here, we present a combined study which encompasses experimental and computational studies on two series of factor Xa ligands, which enclose a broad chemical space including large modifications of the central scaffold. Using this integrated approach, we identified several new ligands with different heterocyclic scaffolds different from the previously identified indole-2-carboxamides that show superior or similar affinity. Furthermore, the so far underexplored terminal alkyne moiety proved to be a suitable non-classical bioisosteric replacement for the higher halogen-π aryl interactions. With this challenging example, we demonstrated the ability of the MD-based non-equilibrium free energy calculation approach for guiding crucial modifications in the lead optimization process, such as scaffold replacement and single-site modifications at molecular interaction hot spots.

Syntheses of defined sulfated oligohyaluronans reveal structural effects, diversity and thermodynamics of GAG-protein binding.

Binding of sulfated glycosaminoglycans (GAG) to a wide spectrum of extracellular regulatory proteins is crucial for physiological processes such as cell growth, migration, tissue homeostasis and repair. Thus, GAG derivatives exhibit great relevance in the development of innovative biomaterials for tissue regeneration therapies. We present a synthetic strategy for the preparation of libraries of defined sulfated oligohyaluronans as model GAG systematically varied in length, sulfation pattern and anomeric substitution in order to elucidate the effects of these parameters on GAG recognition by regulatory proteins. Through an experimental and computational approach using fluorescence polarization, ITC, docking and molecular dynamics simulations we investigate the binding of these functionalized GAG derivatives to ten representative regulatory proteins including IL-8, IL-10, BMP-2, sclerostin, TIMP-3, CXCL-12, TGF-ß, FGF-1, FGF-2, and AT-III, and we establish structure-activity relationships for GAG recognition. Binding is mainly driven by enthalpy with only minor entropic contributions. In several cases binding is determined by GAG length, and in all cases by the position and number of sulfates. Affinities strongly depend on the anomeric modification of the GAG. Highest binding affinities are effected by anomeric functionalization with large fluorophores and by GAG dimerization. Our experimental and theoretical results suggest that the diversity of GAG binding sites and modes is responsible for the observed high affinities and other binding features. The presented new insights into GAG-protein recognition will be of relevance to guide the design of GAG derivatives with customized functions for the engineering of new biomaterials.

Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization.

Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.

Chemoselective Wittig and Michael ligations of unprotected peptidyl phosphoranes in water furnish potent inhibitors of caspase-3.

Unprotected peptidyl phosphoranes 1 with sequence Ac-L-aspartyl-L-glutamyl-L-valinyl-L-aspartyl are released from polymer support and react with aliphatic and aromatic aldehydes in aqueous medium in a Wittig ligation. Obtained vinyl ketones 6-12 are potent inhibitors of caspase-3. Vinyl ketone 6, derived from formaldehyde, undergoes Michael ligations with thiol nucleophiles furnishing products 14-16, also in aqueous medium. The demonstrated ligation reactions enable the modification of complex functionalized peptides in water providing bioactive protein ligands without side-chain protection.

Alzheimer's disease: identification and development of ß-secretase (BACE-1) binding fragments and inhibitors by dynamic ligation screening (DLS).

The application of dynamic ligation screening (DLS), a methodology for fragment-based drug discovery (FBDD), to the aspartic protease ß-secretase (BACE-1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (Vmax , KM , and kcat ) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3-(3-aminophenyl)-2H-chromen-2-one (1) to be a competitive BACE-1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane-1,2-diamine isostere. Finally, structure-assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment-based BACE-1 inhibitors with up to 30-fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.

Effects of a tacrine-8-hydroxyquinoline hybrid (IQM-622) on Aß accumulation and cell death: involvement in hippocampal neuronal loss in Alzheimer's disease.

Several studies have implicated the enzyme acetylcholinesterase (AChE) as well as several biometals in the pathogenesis of Alzheimer's disease (AD). A multifunctional molecule, the hybrid tacrine-8-hydroxyquinoline (named IQM-622), displays cholinergic, antioxidant, copper-complexing and neuroprotective properties. Using in vitro and in vivo models, we investigated the modulating effects of IQM-622 on amyloid ß-protein (Aß)-induced pathology as well as on chemically induced neurodegeneration by domoic acid. In the first experimental model, we observed a significant decrease in brain Aß deposits in IQM-622-treated APP/Ps1 mice for four weeks. Moreover, IQM-622 promoted the degradation of intracellular Aß in astrocytes, and protected against Aß toxicity in cultured astrocytes and neurons. These findings suggest that the neuroprotective effect of IQM-622 is not only related to AChE inhibition, but also involves other mechanisms, including the modulation of Aß-degradation pathways in AD brain. In this study we also compare the neuronal loss in CA1 hippocampal field of AD patients and of mice treated with domoic acid, giving similar patterns. Thus, we used a second experimental model by killing hippocampal neurons by domoic acid damage, in which IQM-622 increased survival in the CA1 and dentate gyrus regions of the hippocampus. Our observations suggest that administration of IQM-622 may have significant beneficial effects in neurodegenerative diseases, including AD, which course with acute or progressive neuronal death.

New tacrine-4-oxo-4H-chromene hybrids as multifunctional agents for the treatment of Alzheimer's disease, with cholinergic, antioxidant, and ß-amyloid-reducing properties.

By using fragments endowed with interesting and complementary properties for the treatment of Alzheimer's disease (AD), a new family of tacrine-4-oxo-4H-chromene hybrids has been designed, synthesized, and evaluated biologically. The tacrine fragment was selected for its inhibition of cholinesterases, and the flavonoid scaffold derived from 4-oxo-4H -chromene was chosen for its radical capture and ß-secretase 1 (BACE-1) inhibitory activities. At nano- and picomolar concentrations, the new tacrine-4-oxo-4H-chromene hybrids inhibit human acetyl- and butyrylcholinesterase (h-AChE and h-BuChE), being more potent than the parent inhibitor, tacrine. They are also potent inhibitors of human BACE-1, better than the parent flavonoid, apigenin. They show interesting antioxidant properties and could be able to penetrate into the CNS according to the in vitro PAMPA-BBB assay. Among the hybrids investigated, 6-hydroxy-4-oxo- N-{10-[(1,2,3,4-tetrahydroacridin-9-yl)amino]decyl}-4 H-chromene-2-carboxamide (19) shows potent combined inhibition of human BACE-1 and ChEs, as well as good antioxidant and CNS-permeable properties.

Novel tacrine-8-hydroxyquinoline hybrids as multifunctional agents for the treatment of Alzheimer's disease, with neuroprotective, cholinergic, antioxidant, and copper-complexing properties.

Tacrine and PBT2 (an 8-hydroxyquinoline derivative) are well-known drugs that inhibit cholinesterases and decrease beta-amyloid (Abeta) levels by complexation of redox-active metals, respectively. In this work, novel tacrine-8-hydroxyquinoline hybrids have been designed, synthesized, and evaluated as potential multifunctional drugs for the treatment of Alzheimer's disease. At nano- and subnanomolar concentrations they inhibit human acetyl- and butyrylcholinesterase (AChE and BuChE), being more potent than tacrine. They also displace propidium iodide from the peripheral anionic site of AChE and thus could be able to inhibit Abeta aggregation promoted by AChE. They show better antioxidant properties than Trolox, the aromatic portion of vitamin E responsible for radical capture, and display neuroprotective properties against mitochondrial free radicals. In addition, they selectively complex Cu(II), show low cell toxicity, and could be able to penetrate the CNS, according to an in vitro blood-brain barrier model.

Tacrine-melatonin hybrids as multifunctional agents for Alzheimer's disease, with cholinergic, antioxidant, and neuroprotective properties.

Tacrine-melatonin hybrids were designed and synthesized as new multifunctional drug candidates for Alzheimer's disease. These compounds may simultaneously palliate intellectual deficits and protect the brain against both beta-amyloid (A beta) peptide and oxidative stress. They show improved cholinergic and antioxidant properties, and are more potent and selective inhibitors of human acetylcholinesterase (hAChE) than tacrine. They also capture free radicals better than melatonin. Molecular modeling studies show that these hybrids target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. At sub-micromolar concentrations they efficiently displace the binding of propidium iodide from the PAS and could thus inhibit A beta peptide aggregation promoted by AChE. Moreover, they also inhibit A beta self-aggregation and display neuroprotective properties in a human neuroblastoma line against cell death induced by various toxic insults, such as A beta(25-35), H(2)O(2), and rotenone. Finally, they exhibit low toxicity and may be able to penetrate the central nervous system according to an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB).

Novel tacrine-melatonin hybrids as dual-acting drugs for Alzheimer disease, with improved acetylcholinesterase inhibitory and antioxidant properties.

Tacrine and melatonin are well-known drugs with activities as an acetylcholinesterase (AChE) inhibitor and free radical scavenger, respectively. In this work, we report new hybrids of both drugs that display higher in vitro properties than the sum of their parts. As selective inhibitors of human AChE, their IC(50) values range from sub-nanomolar to picomolar. They exhibit a higher oxygen radical absorbance capacity than does melatonin and are predicted to be able to cross the blood-brain barrier to reach their targets in the central nervous system.

Design and synthesis of N-benzylpiperidine-purine derivatives as new dual inhibitors of acetyl- and butyrylcholinesterase.

The synthesis and biological evaluation of N-benzyl-(piperidin or pyrrolidin)-purines are described. Compounds derived from N-benzylpiperidine and N-substituted purines showed moderate acetylcholinesterase inhibition. Preliminary structure-activity relationships and a superimposition of the best compound with the active conformation of donepezil have revealed structural features that have been used in the design of more potent N-benzylpiperidine inhibitors bearing an 8-substituted caffeine fragment and a methoxymethyl linker. These new compounds are interesting dual inhibitors of acetylcholinesterase and butyrylcholinesterase and have been chosen for further optimisation.