Dino Marcus

Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity: In vitro and in vivo quantitative characterization

Butyrylcholinesterase purified from human plasma (HuBChE) was evaluated both in vitro and in vivo in mice and rats as a single prophylactic antidote against the lethal effects of highly toxic organophosphates (OP). The variation among the bimolecular rate constants for the inhibition of HuBChE by tabun, VX, sarin, and soman was 10-fold (0.47 to 5.12 x 10(7) M-1 min-1; pH 8.0, 26 degrees). The half-life of HuBChE in blood after its i.v. administration in mice and rats was 21 and 46 hr, respectively. The peak blood-enzyme level was obtained in both species approximately 9-13 hr following i.m. injection of HuBChE, and the fraction of the enzyme activity absorbed into the blood was 0.9 and 0.54 for rats and mice, respectively. The stoichiometry of the in vivo sequestration of the anti-cholinesterase toxicants was consistent with the HuBChE/OP ratio of the molar concentration required to inhibit 100% enzyme activity in vitro. Linear correlation was demonstrated between the blood level of HuBChE and the extent of protection conferred against the toxicity of nerve agents. Pretreatment with HuBChE alone was sufficient not only to increase survivability following exposure to multiple median lethal doses of a wide range of potent OPs, but also to alleviate manifestation of toxic symptoms in mice and rats without the need for additional post-exposure therapy. It appeared that in order to confer protection against lethality nerve agents had to be scavenged to a level below their median lethal dose LD50 within less than one blood circulation time. Since the high rate of sequestration of nerve agents by HuBChE is expected to underlie the activity of the scavenger in other species as well, a reliable extrapolation of its efficacy from experimental animals to humans can be made.

Efficiency of protection of guinea pigs against infection with Bacillus anthracis spores by passive immunization.

ABSTRACT The efficacy of passive immunization as a postexposure prophylactic measure for treatment of guinea pigs intranasally infected with Bacillus anthracis spores was evaluated. Antisera directed either against the lethal toxin components (PA or LF) or against a toxinogenic strain (Sterne) were used for this evaluation. All antisera exhibited high enzyme-linked immunosorbent assay titers against the corresponding antigens, high titers of neutralization of cytotoxicity activity in an in vitro mouse macrophages cell line (J774A.1), as well as in vivo neutralization of toxicity when administered either directly to Fisher rats prior to challenge with the lethal toxin or after incubation with the lethal toxin. In these tests, anti-LF antiserum exhibited the highest neutralization efficiency, followed by anti-Sterne and anti-PA. The time dependence and antibody dose necessary for conferring postexposure protection by the various antibodies of guinea pigs infected with 25 50% lethal doses of Vollum spores was examined. Rabbit anti-PA serum was found to be the most effective. Intraperitoneal injections of anti-PA serum given 24 h postinfection protected 90% of the infected animals, whereas anti-Sterne and anti-LF were less effective. These results further emphasizes the importance of anti-PA antibodies in conferring protection against B. anthracis infection and demonstrated the ability of such antibodies to be effectively applied as an efficient postexposure treatment against anthrax disease.

Large-scale purification and long-term stability of human butyrylcholinesterase: a potential bioscavenger drug

Butyrylcholinesterase from human plasma (HuBChE) is a potential drug candidate for detoxification of certain harmful chemicals that contain carboxylic or phosphoric acid ester bonds. Large quantities of purified HuBChE, displaying a high stability upon long-term storage, are required for the evaluation of its therapeutic capacity and its pharmaceutical properties. Several modifications of a previously reported procedure enabled us to purify the enzyme > 15,000-fold from pools of up to 100 1 of human plasma. The three-step procedure is based on precipitation of plasma proteins by ammonium sulfate (step I) and batch adsorption of HuBChE on procainamide-Sepharose 4B gel (step II). Ammonium sulfate was also employed in the third stage to fractionate the final product from procainamide-containing HuBChE solution. The overall yield (63%) of electrophoretically pure enzyme was significantly higher than that previously reported (34%) for the purification of HuBChE from 12.5 1 of plasma or from 5 kg of Cohn fraction IV-4. Purified HuBChE was stored at 5 degrees C in 10 mM phosphate buffer (pH 7.4) containing 1 mM EDTA and 0.02% NaN3. The specific activity, protein migration on gel electrophoresis, thermostability at 54 degrees C and the mean residence time in the circulation of mice remained essentially constant for at least 46 months. The modifications introduced can provide large quantities of purified enzyme that maintains its activity and bioavailability properties for several years.

Engineering resistance to ‘aging’ of phosphylated human acetylcholinesterase Role of hydrogen bond network in the active center

Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP‐conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30 100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP‐conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.

Production and purification of high molecular weight oligomers of Yersinia pestis F1 capsular antigen released by high cell density culture of recombinant Escherichia coli cells carrying the caf1 operon

Fermentation Expression of F1 by E. coli MC1060 (pBRK-F1) was studied in a 4-liter computer-controlled fermentor using a rich medium devoid of animal products: soy protein extract, trace elements and glycerol as carbon source (SY). This medium enabled efficient bacterial growth to a high cell density (60 OD600, Fig 1A). No loss of the expression plasmid was observed under these conditions. Unexpectedly, high and constant level of F1 expression was observed throughout the fermentation (up to 24 hours) even at 28°C (no temperature shift to 37°C was required, Fig. 1B). Moreover, at a high cell density (>10 OD600), F1 was released from the encapsulated E. coli cells and accumulated up to 0.9 g/L in the culture medium (Fig. 1C).

Molecular Organization of Recombinant Human Acetylcholinesterase

Acetylcholinesterase (abbreviated AChE) occurs in multiple molecular forms in different tissues of vertebrates and invertebrates (reviewed in Massoulie and Bon, 1982; (Silman and Futerman, 1987; Chatonnet and Lockridge, 1989). This heterogeneity is generated through tissue-specific associations of various catalytic and structural subunits. Characterized catalytic subunits are divided into two major types, the T-type and the H-type, both derived from a single gene by alternative splicing Schumacher et al., 1988; Sikarov et al., 1988). Structural subunits include the collagen-like structure (Krejci et al., 1991) that allows attachment to the basal lamina and the 20kD lipid-linked hydrophobic subunit (Inestrosa et al., 1987) which is associated with the mammalian brain enzyme.

Denaturation of Recombinant Human Acetylcholinesterase

The study of the unfolding process of proteins has been a subject of growing interest during recent years. The availability of recombinant cholinesterases in large amounts permits now to use biophysical tools for gaining information about folding and stability of these enzymes.

Large scale production of human lymphoblastoid (Namalva) interferon II. Purification of interferon

SummaryA procedure for large scale purification of human lymphoblastoid (Namalva) interferon (IFN) is presented. The procedure includes concentration by ultrafiltration, gel filtration on Sephadex G-75 and affinity chromatography on anti-INF antibody (polyclonal or monoclonal) columns.The IFN specific activity is increased from 1 × 105 units/mg protein, in the crude solution, to 2 × 107 and 2 × 108 units/mg protein, in the purified preparations obtained after polyclonal or monoclonal antibody columns, respectively. The overall recovery of IFN in the procedure described is 50–55%.

What can be Learned from the use of HuAChE Mutants for Evaluation of Potential Alzheimer’s Drugs

Senile dementia of the Alzheimer’s type (SDAT) is characterised by loss of cholinergic neuronal markers like the enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), in selected brain regions (Bierer et al., 1995). These indications of progressive depletion of cholinergic synapses led to the hypothesis that increasing the central nervous system (CNS) levels of acetylcholine (ACh), through inhibition of AChE, will improve cognition in SDAT (Court & Perry, 1991). This approach is theoretically preferable to other means of cholinergic augmentation since it may amplify the natural temporal pattern of ACh release, rather than globally stimulating the cholinergic system. Yet up to the present most of these agents show only mild to moderate ameliorating effects on memory deficits (Schneider & Tariot, 1994; Kan, 1992). To further optimize the therapeutic efficacy of these agents, a better understanding is needed of the structural features determining their interactions with AChE. The recent progress in the elucidation of structure-function characteristics of AChE, is therefore of considerable importance for these efforts.

AMINO ACIDS DETERMINING SPECIFICITY TO OP-AGENTS AND FACILITATING THE "AGING" PROCESS IN HUMAN ACETYLCHOLINESTERASE

Cholinesterases (ChE’s) are readily phosphylated at the active site serine, by a variety of organophosphorus agents (OP) (Aldrich et al., 1972; Taylor, 1990). The enzyme can be reactivated by various oxime nucleophiles but in certain cases reactivation is thwarted due to a concomitant unimolecular process termed aging (Aldrich et al., 1972). The aged enzyme-OP-conjugate is refractive to reactivation (Gray, 1984) and thus renders treatment, following intoxication with certain OP insecticides or nerve gas agents, extremely difficult (Glickman et al., 1984). Recombinant human acetylcholinesterase (HuAChE) and several selected active site gorge mutants: W86A, E202Q, F295A, F297A, Y337A and E450A of HuAChE were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation by organophosphorus (OP) inhibitors: DFP, paraoxon and soman and subsequent aging of the resulting OP-conjugates. On the basis of these studies we have identified some of the critical elements in the active center that determine specificity to various OP-agents. Trp86, a key element of the active center “anionic subsite” that stabilizes noncovalent complexes of charged ligands by cation - π interactions (Ordentlich et al., 1993a) also contributes to chemical reactivity towards various noncharged OP - inhibitors as manifested by a 5–20 fold decrease in rates of phosphorylation observed for W86A mutant. Replacement of Tyr337 by alanine had a minimal effect on the bimolecular rate constant of inhibition by the OP-molecules. The 20-fold increase in phosphorylation rates of the F295A mutated enzyme by paraoxon is attributed to the better accommodation of the ethoxy moiety of the inhibitor by the less bulky alanine residue. This is consistent with the role of Phe295 in conferring specificity to the acyl pocket (Ordentlich et al., 1993a). The reduction in catalytic efficiency displayed by the E202Q and E450A mutants, and the marked decrease in the rates of both phosphylation and aging is consistent with the proposed role of these residues as key elements of the hydrogen bond network (Ordentlich et al., 1993b). The role of this network is to maintain the functional architecture of the active center and proper positioning of E202, thereby stabilizing the evolving transition states of acylation and phosphylation. The impaired “aging” process displayed by the E202Q and E450A mutants provides a basis for the engineering of novel reactivatable bioscavengers.