Acetohydroxamic acid salts: mild, simple and effective degradation reagents to counter Novichok nerve agents.

Novichoks is the latest known class of organophosphorus nerve agents to be developed. These highly lethal persistent agents, which exert their toxicity mainly through dermal exposure, pose new major challenges in mitigating their effect, mainly in respect to decontamination and medical countermeasures. Herein we report on the effective degradation of Novichok agents (A-230, A-232 and A-234) by hydroxamic acid salts. This class of α-nucleophiles, with emphasis on the FDA approved drug acetohydroxamic acid, were found to promote rapid hydrolysis of these extremely toxic agents. Using 31P NMR the Novichoks degradation rates were determined to be in time scale of minutes with the following order of reactivity A-230>A-232>A-234. The degradation efficiency was found to be dependent on the nucleophiles, their counter-cations and the specific solvent mixture used. Hence, these scavengers can serve as efficient and mild decontaminants in various scenarios including surfaces, dermal decontamination (as an alternative to active lotions such as the RSDL® kit) and also as a medical countermeasure in the form of "catch-up therapy".

Species-specific lipophilicities of fluorinated diketones in complex equilibria systems and their potential as multifaceted reversible covalent warheads.

Combined molecular, physicochemical and chemical properties of electrophilic warheads can be applied to create covalent drugs with diverse facets. Here we study these properties in fluorinated diketones (FDKs) and their multicomponent equilibrium systems in the presence of protic nucleophiles, revealing the potential of the CF2(CO)2 group to act as a multifaceted warhead for reversible covalent drugs. The equilibria compositions of various FDKs in water/octanol contain up to nine species. A simultaneous direct species-specific 19F-NMR-based log P determination of these complex equilibria systems was achieved and revealed in some cases lipophilic to hydrophilic shifts, indicating possible adaptation to different environments. This was also demonstrated in 19F-MAS-NMR-based water-membrane partitioning measurements. An interpretation of the results is suggested by the aid of a DFT study and 19F-DOSY-NMR spectroscopy. In dilute solutions, a model FDK reacted with protected cysteine to form two hemi-thioketal regioisomers, indicating possible flexible regio-reactivity of CF2(CO)2 warheads toward cysteine residues.

"Catch-up" therapy: combining antidotal treatment with dermal application of AHA following percutaneous VX poisoning in the domestic swine.

Low-volatility organophosphorus chemical warfare agents (OP CWAs) are cholinesterase inhibitors which easily absorb into the skin, leading to the formation of a dermal depot from which they slowly enter the bloodstream. This leads to sustained cholinergic hyperstimulation, which if untreated may lead to death. However, current available countermeasures are not adequate to neutralize the agent residing in the dermal depot. Accordingly, we evaluated the efficacy of the potassium salt of acetohydroxamic acid (880 mg/ml in DMSO/H2O 1:4, AHAK), as a potential "catch-up" therapy lotion intended to neutralize the dermal depot, by penetrating the skin and decomposing it before it reaches the bloodstream. To that end, we compared the clinical outcome following skin surface decontamination combined with antidotal treatment, to that following the same antidotal treatment combined with dermal application of AHAK at the site of VX exposure, against percutaneous poisoning by a lethal neat dose (4 mg/kg) of the low-volatility nerve agent VX, in an unanesthetized swine model. Following skin surface decontamination and antidotal treatment, recurrence of intoxication signs and a prolonged recovery time were observed. In contrast, similar antidotal treatment combined with dermal application of AHAK significantly reduced intoxication signs recurrences and accordingly medical supervision duration needed, paralleled by a significantly faster recovery of whole blood cholinesterase activity. An initial evaluation demonstrated the safety of prolonged whole-body AHAK application. Hence, the AHAK lotion may act as an efficient "catch-up" therapy against percutaneous poisoning by low-volatility OP CWAs, improving the clinical outcome and reducing the burden on medical staff.

Efficient Decontamination of HD by an Electrophilic Iodine/Carboxylate Composite as an Active Sorbent.

The development of new and efficient decontamination methods has become more relevant in recent years, especially with regard to solid-based decontamination and detoxification systems. The majority of powders used today are dealing with the physical adsorption of chemical warfare agents (CWAs) and their removal from sites without actively destroying them. In this work, we have designed and developed an active solid composite matrix combining organic carboxylate salts and N-iodosuccinimide (NIS) for HD decontamination via oxidation. All the reactions and mechanistic studies for the sorption and degradation of CWAs were conducted using direct polarization and cross polarization solid-state magic-angle spinning nuclear magnetic resonance techniques. Performance toward the sorption and detoxification of HD was tested, exhibiting oxidation within minutes in a mild and selective manner to the nontoxic sulfoxide derivative followed by visible formation of iodine. The results indicate that carboxylate moieties in the matrix are important for stabilizing the positively charged sulfonium ion intermediate and for supplying oxygen for hydrolysis in a water-deficient environment. The NaOBz/NIS composite was shown to be the most efficient in sorbing and converting the water-insoluble agent HD to its nontoxic, water-soluble sulfoxide, which could then be removed from the site with mere water, resulting in less environmental damage and quick remediation.

Studying Lipophilicity Trends of Phosphorus Compounds by 31P-NMR Spectroscopy: A Powerful Tool for the Design of P-Containing Drugs.

Systematically studying the lipophilicity of phosphorus compounds is of great importance for many chemical and biological fields and particularly for medicinal chemistry. Here, we report on the study of trends in the lipophilicity of a wide set of phosphorus compounds relevant to drug design including phosphates, thiophosphates, phosphonates, thiophosphonates, bis-phosphonates, and phosphine chalcogenides. This was enabled by the development of a straightforward log P determination method for phosphorus compounds based on 31P-NMR spectroscopy. The log P values measured ranged between -3.2 and 3.6, and the trends observed were interpreted using a DFT study of the dipole moments and by H-bond basicity (pKHB) measurements of selected compounds. Clear signal separation in 31P-NMR spectroscopy grants the method high tolerability to impurities. Moreover, the wide range of chemical shifts for the phosphorus nucleus (250 to -250 ppm) enables a direct simultaneous log P determination of phosphorus compound mixtures in a single shake-flask experiment and 31P-NMR analysis.

Towards catch-up therapy: evaluation of nucleophilic active pharmaceutical ingredients for the treatment of percutaneous VX poisoning, in-vial and in-vitro studies.

Dermal exposure to low volatility organophosphorus chemical warfare agents (OP CWA) poses a great risk to the exposed person. Due to their lipophilic nature, these compounds rapidly absorb into the skin, leading to the formation of a "dermal reservoir" from which they slowly enter the bloodstream causing prolonged intoxication. Traditionally, strategies to counter the toxicity of such substances consist of chemical decontamination/physical removal of the residual agent from the skin surface (preferably as soon as possible following the exposure) and administration of antidotes in the case of intoxication signs. Hence, these strategies are unable to counter a substantial amount of the agent, which accumulates inthe dermal reservoir. More than a decade ago, the concept of a "catch-up therapy" intended to neutralize the dermal reservoir was suggested. Herein, we describe examples of potential "catch-up therapy" lotions - vehicles designed to deliver small nucleophilic molecules into the skin and potentially decompose the remaining CWA before it reaches the blood stream. Eleven nucleophilic compounds, based on approved drugs, were initially screened. They were then tested in various binary solutions, for their detoxification efficacy and degradation ability towards lipophilic OP CWA models such as dibutylphosphofluoridate and o-nitro-phenyl diphenyl phosphate, as well as the nerve agent VX, by means of kinetic 31P NMR and UV-Vis spectroscopy. Of these, the potassium and diethyl ammonium salts of acetohydroxamic acid (AHAK and AHA DEA) in (DMSO/H2O 1:4) were found to be the most active nucleophiles, hydrolyzing VX in practical time scales (t1/2 = 5.28 and 6.78 min, respectively). The vehicle solution DMSO/H2O 1:4 promoted the penetration of substantial amounts of AHA K and AHA DEA through excised pig skin in in-vitro studies, suggesting that such formulations may serve as useful CWA nucleophilic scavengers for both on and within -skin detoxification. These findings may pave the way to a more efficacious treatment against low volatility OP CWA percutaneous poisoning.

A novel approach for the detection and identification of sulfur mustard using liquid chromatography-electrospray ionization-tandem mass spectrometry based on its selective oxidation to sulfur mustard monoxide with N-iodosuccinimide.

A new derivatization strategy for the detection and identification of sulfur mustard (HD) via liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is developed. The method incorporates selective oxidation of the sulfide group by the electrophilic iodine reagent N-iodosuccinimide (NIS) to produce sulfur mustard monoxide (HDSO). The derivatization reaction efficiencies were evaluated with acetonitrile extracts of soil, asphalt, cloth, Formica, and linoleum spiked with HD at concentrations of 50-5000 pg/ml and found to be similar to that with pure acetonitrile. The current derivatization approach is the first to preserve the identity of chloride groups and support HD regulation and evidentiary findings.

Towards Catalytic Antibiotics: Redesign of Aminoglycosides To Catalytically Disable Bacterial Ribosomes.

The emergence of multidrug-resistant pathogens that are resistant to the majority of currently available antibiotics is a significant clinical problem. The development of new antibacterial agents and novel approaches is therefore extremely important. We set out to explore the potential of catalytic antibiotics as a new paradigm in antibiotics research. Herein, we describe our pilot study on the design, synthesis, and biological testing of a series of new derivatives of the natural aminoglycoside antibiotic neomycin B for their potential action as catalytic antibiotics. The new derivatives showed significant antibacterial activity against wild-type bacteria and were especially potent against resistant and pathogenic strains including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Selected compounds displayed RNase activity even though the activity was not as high and specific as we would have expected. On the basis of the observed chemical and biochemical data, along with the comparative molecular dynamics simulations of the prokaryotic rRNA decoding site, we postulate that the rational design of catalytic antibiotics should involve not only their structure but also a comprehensive analysis of the rRNA A-site dynamics.

Oxidative Detoxification of Sulfur-Containing Chemical Warfare Agents by Electrophilic Iodine.

Mild oxidation of sulfur-containing chemical warfare agents was performed in organic medium by electrophilic iodine reagents. Kinetic experiments on sulfur mustard (HD) showed rapid ( t1/2 < 3 min) and selective oxidation to the nonvesicant sulfoxide product (HD-SO) in acetonitrile or propylene carbonate solutions (9% water added) containing excess N-iodosuccinimide (NIS). Molecular iodine solutions in these solvents led to similar results as with NIS but at much slower rates ( t1/2 ∼ 90 min). Higher donor number solvents, such as THF, DMF, or DMSO, showed slower rates with both iodine and NIS. The oxidation of the nerve agent O-ethyl- S-2-( N,N-diisopropylaminoethyl)methylphosphonothioate (VX) selectively to the nontoxic ethyl methylphosphonic acid product exhibited fast rates ( t1/2 = 6 min) using NIS in DMSO solution. In all other solvents tested with VX, rates were slower ( t1/2 ∼ 30-70 min). Oxidation experiments under the same conditions with chloroethyl ethyl sulfide (HD simulant) and O,S-diethyl methylphosphonothioate (VX simulant) led to much faster reaction rates. These transformations are believed to proceed through electrophilic iodine attack on the sulfur moiety and display solvent dependency based on the agents' structural and chemical properties.

Design of Novel Aminoglycoside Derivatives with Enhanced Suppression of Diseases-Causing Nonsense Mutations.

New pseudotrisaccharide derivatives of aminoglycosides that exploit additional interaction on the shallow groove face of the decoding-site rRNA of eukaryotic ribosome were designed, synthesized and biologically evaluated. Novel lead structures (6 and 7 with an additional 7'-OH), exhibiting enhanced specificity to eukaryotic cytoplasmic ribosome, and superior nonsense mutation suppression activity than those of gentamicin, were discovered. The comparative benefit of new leads was demonstrated in four different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Usher syndrome, and Hurler syndrome.

Structural basis for selective targeting of leishmanial ribosomes: aminoglycoside derivatives as promising therapeutics.

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL.

Repairing faulty genes by aminoglycosides: development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations.

New pseudo-di- and pseudo-trisaccharide derivatives of the aminoglycoside drug G418 were designed, synthesized and their ability to readthrough nonsense mutations was examined in both in vitro and ex vivo systems, along with the toxicity tests. Two novel lead structures, NB74 and NB84, exhibiting significantly reduced cell toxicity and superior readthrough efficiency than those of gentamicin, were discovered. The superiority of new leads was demonstrated in six different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Duchenne muscular dystrophy, Usher syndrome and Hurler syndrome.