PEG-fibrinogen hydrogel microspheres as a scaffold for therapeutic delivery of immune cells.

Recent advances in the field of cell therapy have proposed new solutions for tissue repair and regeneration using various cell delivery approaches. Here we studied a novel topical delivery system of encapsulated cells in hybrid polyethylene glycol-fibrinogen (PEG-Fb) hydrogel microspheres to respiratory tract models. We investigated basic parameters of cell encapsulation, delivery and release in conditions of inflamed and damaged lungs of bacterial-infected mice.

Human Multi-Compartment Platform for Emulating Respiratory Airborne Transmission: From Nose to Pulmonary Acini.

The past decade has witnessed tremendous endeavors to deliver novel preclinical lung models for pulmonary research endpoints, including foremost with the advent of and . With growing interest in aerosol transmission and infection of respiratory viruses within a host, most notably the SARS-CoV-2 virus amidst the global COVID-19 pandemic, the importance of crosstalk between the different lung regions (i.e.

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities.

A novel mechanism for antibody-based anthrax toxin neutralization: inhibition of prepore-to-pore conversion.

Protective antigen (PA), a key component of anthrax toxin, mediates the entry of lethal factor (LF) or edema factor (EF) through a membranal pore into target cells. We have previously reported the isolation and chimerization of cAb29, an anti-PA monoclonal antibody that effectively neutralizes anthrax toxin in an unknown mechanism. The aim of this study was to elucidate the neutralizing mechanism of this antibody in vitro and to test its ability to confer post-exposure protection against anthrax in vivo.

Next generation OP-bioscavengers: a circulatory long-lived 4-PEG hypolysine mutant of F338A-HuAChE with optimal pharmacokinetics and pseudo-catalytic characteristics.

We have shown previously that conjugation of polyethylene glycol (PEG) chains to recombinant human acetylcholinesterase (rHuAChE) results in the extension of its residence time in the circulation of mice and monkeys [1,2]. By profiling the pharmacokinetic behavior of an array of well-defined hypolysine human mutant AChE molecules following PEGylation, we now determine that the duration of these enzyme forms in the circulation of rhesus macaques correlates with their number of appended PEG moieties, and is influenced by the actual location of the PEG chains at the molecule surface, as well. These findings, which concur with those we have previously established in mice, indicate that a common set of rules dictates the circulatory fate of PEGylated HuAChEs in rodents and non-human primates.

Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA.

Effective post-exposure protection against lethal orthopoxviruses infection by vaccinia immune globulin involves induction of adaptive immune response.

The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%).

Accommodation of physostigmine and its analogues by acetylcholinesterase is dominated by hydrophobic interactions.

The role of the functional architecture of the HuAChE (human acetylcholinesterase) in reactivity toward the carbamates pyridostigmine, rivastigmine and several analogues of physostigmine, that are currently used or considered for use as drugs for Alzheimer's disease, was analysed using over 20 mutants of residues that constitute the interaction subsites in the active centre. Both steps of the HuAChE carbamylation reaction, formation of the Michaelis complex as well as the nucleophilic process, are sensitive to accommodation of the ligand by the enzyme. For certain carbamate/HuAChE combinations, the mode of inhibition shifted from a covalent to a noncovalent type, according to the balance between dissociation and covalent reaction rates.

Aging-resistant organophosphate bioscavenger based on polyethylene glycol-conjugated F338A human acetylcholinesterase.

The high reactivity of cholinesterases (ChEs) toward organophosphorus (OP) compounds has led to propose recombinant ChEs as bioscavengers of nerve agents. The bioscavenging potential of recombinant ChEs can be enhanced by conjugation of polyethylene glycol (PEG) moieties, to extend their circulatory residence. However, the ability of exogenously administered ChEs to confer long-term protection against repeated exposures to nerve agents is still limited due to the aging process, whereby organophosphate-ChE adducts undergo irreversible dealkylation, which precludes oxime-mediated reactivation of the enzyme.

Flexibility versus "rigidity" of the functional architecture of AChE active center.

Functional architecture of the AChE active center appears to be characterized by both structural "rigidity", necessary to stabilize the catalytic triad as well as by flexibility in accommodating the different, high affinity AChE ligands. These seemingly conflicting structural properties of the active center are demonstrated through combination of structural methods with kinetic studies of the enzyme and its mutant derivatives with plethora of structurally diverse ligands and in particular with series of stereoselective covalent and noncovalent AChE ligands. Thus, steric perturbation of the acyl pocket precipitates in a pronounced stereoselectivity toward methylphosphonates by disrupting the stabilizing environment of the catalytic histidine rather than through steric exclusion demonstrating the functional importance of the "rigid" environment of the catalytic machinery.

Polyethylene-glycol conjugated recombinant human acetylcholinesterase serves as an efficacious bioscavenger against soman intoxication.

Extensive pharmacokinetic studies in both mice and rhesus macaques, with biochemically well defined forms of native and recombinant AChEs from bovine, rhesus and human origin, allowed us to determine an hierarchical pattern by which post-translation-related factors and specific amino-acid epitopes govern the pharmacokinetic performance of the enzyme molecule. In parallel, we demonstrated that controlled conjugation of polyethylene-glycol (PEG) side-chains to lysine residues of rHuAChE also results in the generation of active enzyme with improved pharmacokinetic performance. Here, we show that equally efficient extension of circulatory residence can be achieved by specific conditions of PEGylation, regardless of the post-translation-modification state of the enzyme.

Comparison of polyethylene glycol-conjugated recombinant human acetylcholinesterase and serum human butyrylcholinesterase as bioscavengers of organophosphate compounds.

Comparative protection studies in mice demonstrate that on a molar basis, recombinant human acetylcholinesterase (rHuAChE) confers higher levels of protection than native human butyrylcholinesterase (HuBChE) against organophosphate (OP) compound intoxication. For example, mice challenged with 2.5 LD50 of O-isopropyl methylphosphonofluoridate (sarin), pinacolylmethyl phosphonofluoridate (soman), and O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothiolate (VX) after treatment with equimolar amounts of the two cholinesterases displayed 80, 100, and 100% survival, respectively, when pre-treatment was carried out with rHuAChE and 0, 20, and 60% survival, respectively, when pretreatment was carried out with HuBChE.

Lessons from functional analysis of AChE covalent and noncovalent inhibitors for design of AD therapeutic agents.

Determination of the 3D-structure of acetylcholinesterase (AChE) of Torpedo californica over a decade ago, and more recently that of human enzyme together with extensive targeted mutagenesis of the mammalian AChEs led to a fine mapping of the multiple functional domains within the active center of the enzyme. Many of the contributions of this active center architecture to accommodation of noncovalent ligands could be deduced from the X-ray structures of the corresponding HuAChE complexes. Yet, Michaelis complexes leading to transient covalent adducts are not amenable to structural analysis.

The role of AChE active site gorge in determining stereoselectivity of charged and noncharged VX enantiomers.

The reactivity of human acetylcholinesterase (HuAChE) toward the chemical warfare agent VX [O-ethyl S-[2-(diisopropylamino)ethyl] methyl-phosphonothioate] and its stereoselectivity toward the P(S)-enantiomer were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere nc-VX [O-ethyl S-(3-isopropyl-4-methyl-pentyl) methylphosphonothioate]. Stereoselectivity of the wild-type HuAChE toward VX(S) is manifested by a 115-fold higher bimolecular rate constant (1.4 x 10(8) min(-1) M(-1)) as compared to that of VX(R).

Functional requirements for the optimal catalytic configuration of the AChE active center.

Functional analysis of the HuAChE active center architecture revealed that accommodation of structurally diverse substrates and other ligands is achieved through interactions with specific subsites such as the acyl pocket, cation binding site, hydrophobic site or the oxyanion hole. Recent studies have begun to unravel the role of this active center architecture in maintaining the optimal catalytic facility of the enzyme through inducing proper alignment of the catalytic triad. The exact positioning of the catalytic glutamate (Glu334) seems to be determined by a hydrogen bond network including several polar residues and water molecules.

Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as of the peripheral anionic site of AChE.

The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.

Is aromaticity essential for trapping the catalytic histidine 447 in human acetylcholinesterase?

Replacement of both the acyl pocket residue Phe295 as well as residue Phe338, adjacent to the catalytic His447 in human acetylcholinesterase (HuAChE), resulted in a 680-fold decline in catalytic activity due to conformational destabilization of the histidine side chain [Barak et al. (2002) Biochemistry 41, 8245]. A possible restriction of this catalytically nonproductive mobility of His447 in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis.

Pressure and heat inactivation of recombinant human acetylcholinesterase. Importance of residue E202 for enzyme stability.

The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild-type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild-type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability.

The aromatic "trapping" of the catalytic histidine is essential for efficient catalysis in acetylcholinesterase.

While substitution of the aromatic residues (Phe295, Phe338), located in the vicinity of the catalytic His447 in human acetylcholinesterase (HuAChE) had little effect on catalytic activity, simultaneous replacement of both residues by aliphatic amino acids resulted in a 680-fold decrease in catalytic activity. Molecular simulations suggested that the activity decline is related to conformational destabilization of His447, similar to that observed for the hexamutant HuAChE which mimics the active center of butyrylcholinesterase. On the basis of model structures of other cholinesterases (ChEs), we predicted that catalytically nonproductive mobility of His447 could be restricted by introduction of aromatic residue in a different location adjacent to this histidine (Val407).

Effect of chemical modification of recombinant human acetylcholinesterase by polyethylene glycol on its circulatory longevity.

Post-translational modifications were recently shown to be responsible for the short circulatory mean residence time (MRT) of recombinant human acetylcholinesterase (rHuAChE) [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959--967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem.

Resolving pathways of interaction of covalent inhibitors with the active site of acetylcholinesterases: MALDI-TOF/MS analysis of various nerve agent phosphyl adducts.

Understanding reaction pathways of phosphylation, reactivation, and "aging" of AChE with toxic organophosphate compounds is both a biochemical and a pharmacological challenge. Here we describe experiments which allowed to resolve some of the less well understood reaction pathways of phosphylation and "aging" of acetylcholinesterase (AChE) involving phosphoroamidates (P-N agents) such as tabun or the widely used pesticide methamidophos. Tryptic digests of phosphylated AChEs (from human and Torpedo californica), ZipTip peptide fractionation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) enabled reproducible signal enrichment of the isotopically resolved peaks of organophosphoroamidate conjugates of the AChE active site Ser peptides.

Does "butyrylization" of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase?

The active center gorge of human acetylcholinesterase (HuAChE) is lined by 14 aromatic residues, whereas in the closely related human butyrylcholinesterase (HuBChE) 3 of the aromatic active center residues (Phe295, Phe297, Tyr337) as well as 3 of the residues at the gorge entrance (Tyr72, Tyr124, Trp286) are replaced by aliphatic amino acids. To investigate whether this structural variability can account for the reactivity differences between the two enzymes, gradual replacement of up to all of the 6 aromatic residues in HuAChE by the corresponding residues in HuBChE was carried out. The affinities of the hexamutant (Y72N/Y124Q/W286A/F295L/F297V/Y337A) toward tacrine, decamethonium, edrophonium, huperzine A, or BW284C51 differed by about 5-, 80-, 170-, 25000-, and 17000-fold, respectively, from those of the wild-type HuAChE.

Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase.

Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed "aging" is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C-O bond, the aging path for adducts with phosphoroamidates (P-N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear.

A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase.

Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking.

Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level.

Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.