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Dive into the research topics where Charles B. Millard is active.

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Featured researches published by Charles B. Millard.


Journal of Neurochemistry | 2002

Anticholinesterases: Medical Applications of Neurochemical Principles†

Charles B. Millard; Clarence A. Broomfield

Abstract: Cholinesterases form a family of serine esterases that arise in animals from at least two distinct genes. Multiple forms of these enzymes can be precisely localized and regulated by alternative mRNA splicing and by co‐ or posttranslational modifications. The high catalytic efficiency of the cholinesterases is quelled by certain very selective reversible and irreversible inhibitors. Owing largely to the important role of acetylcholine hydrolysis in neurotransmission, cholinesterase and its inhibitors have been studied extensively in vivo. In parallel, there has emerged an equally impressive enzyme chemistry literature. Cholinesterase inhibitors are used widely as pesticides; in this regard the compounds are beneficial with concomitant health risk. Poisoning by such compounds can result in an acute but usually manageable medical crisis and may damage the CNS and the PNS, as well as cardiac and skeletal muscle tissue. Some inhibitors have been useful for the treatment of glaucoma and myasthenia gravis, and others are in clinical trials as therapy for Alzheimers dementia. Concurrently, the most potent inhibitors have been developed as highly toxic chemical warfare agents. We review treatments and sequelae of exposure to selected anticholinesterases, especially organophosphorus compounds and carbamates, as they relate to recent progress in enzyme chemistry.


Journal of Molecular Biology | 2002

A Neutral Molecule in a Cation-binding Site: Specific Binding of a PEG-SH to Acetylcholinesterase from Torpedo californica

Gertraud Koellner; Thomas Steiner; Charles B. Millard; Israel Silman; Joel L. Sussman

The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 A resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH(2) groups of the inhibitor, which are engaged in C-H...pi interactions that structurally mimic the cation-pi interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H...O and C-H...pi types.


Protein Science | 2009

Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones

Charles B. Millard; Valery L. Shnyrov; Simon Newstead; Irina Shin; Esther Roth; Israel Silman; Lev Weiner

Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is “native‐like,” and inactivation can be reversed by exposure to reduced glutathione. The allicin‐modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t1/2 ≃ 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine‐N‐oxide, glycerol, and sucrose, and the divalent cations, Ca2+, Mg2+, and Mn2+ can prevent this transition of the native‐like state for >24 h at room temperature. Trimethylamine‐N‐oxide and Mg2+ can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39°C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native‐like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.


Archive | 1998

Crystal Structures of “Aged” Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase

Charles B. Millard; Gitay Kryger; Arie Ordentlich; Michal Harel; Mia L. Raves; Harry M. Greenblatt; Yoffi Segall; Dov Barak; Avigdor Shafferman; Israel Silman; Joel L. Sussman

Organophosphates (OP) are potent transition state (TS) inhibitors which react rapidly with acetylcholinesterase (AChE), and then may undergo an internal dealkylation to produce an irreversibly inhibited, “aged” OP-enzyme conjugate. To understand the structural basis for the stability of aged enzyme, we crystallized and solved the X-ray structures of conjugates obtained by reaction of Torpedo californica (Tc) AChE with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman). After reaction with OP, unbound inhibitor was removed by gel filtration and aging was allowed to proceed to >90% completion. Aged OP-TcAChE was crystallized using PEG-200 and MES buffer at pH 5.8. X-ray data were collected using trigonal crystals, and refined using difference Fourier techniques at 2.2A (DFP), 2.5A (sarin), and 2.2A (soman) resolution. In each structure, the highest positive difference density peak, corresponding to the OP, was observed to be within covalent bonding distance of Ser200. All three structures suggest that the stability of aged AChE derives from interaction of the two resonance oxygen atoms attached to the phosphorus atom with catalytic subsites of the enzyme. Based upon the geometry of the refined structures, we infer that backbone amides of the oxyanion hole (Gly118, Gly119 and Ala201) stabilize one oxygen by hydrogen bonding, while the His440 imidazolium holds the other oxygen in a salt bridge. The conformations of the active sites of aged sarin- and soman-TcAChE are essentially identical and provide structural models for the rate-limiting deacylation TS that occurs during enzyme hydrolysis of the natural substrate, acetylcholine.


Archive | 1998

Activity of Torpedo Californica Acetylcholinesterase in the Crystalline State

Anne Nicolas; Charles B. Millard; Mia L. Raves; Raimond B. G. Ravelli; Jan Kroon; Israel Silman; Joel L. Sussman

Numerous studies have demonstrated that enzymes display catalytic activity in the crystalline state [1]. Factors which might affect activity of a crystalline enzyme include: i. Ability of substrates to reach the active site and of products to exit ii. Chemical composition of the crystallisation mother liquor (ML) iii. Conformational freedom of residues involved in substrate binding and hydrolysis


Archive | 1998

Kinetic and X-Ray Crystallographic Studies of the Binding of ENA-713 to Torpedo Californica Acetylcholinesterase

Pazit Bar-On; Michal Harel; Charles B. Millard; Albert Enz; Joel L. Sussman; Israel Silman

Compared with other clinically useful carbamates, the anti-Alzheimer drug, (+)S-N-ethyl-3-[(l-dimethyl-amino)ethyl]-N-methylphenylcarbamate (ENA-713; Exelon™) has a longer duration of action in vivo and preferentially inhibits acetylcholinesterase (AChE) of the brain cortex and hippocampus. To understand the basic inhibition mechanism of ENA-713, we studied its reactions with Torpedo californica (Tc) AChE in vitro. The apparent bimolecular rate constant for progressive inhibition was low: ki= 6 M-1min-1 (0.067M Na/K phosphate buffer, pH 7.4, 25°C). After carbamylation (10mM ENA-713) and removal of excess inhibitor by gel filtration, spontaneous reactivation of TcAChE was observed with a rate constant of 0.004 min-1 (pH 7.4, 25°C). Reactivation was base-catalyzed at pH 5.5–8.0 with a maximum at pH 7.5, and displayed an activation energy of 16 kcal/mol. The apparent reversible binding constant for TcAChE with ENA-713 was 200μM, whereas the hydrolysis product, 3-[(l-dimethylamino)ethyl] phenol (NAP), had a Ki of 0.5μM at pH 7.4, 25°C. Thus, the product apparently bound more tightly to TcAChE than did the intact carbamate in the reversible complex. Trigonal crystals of TcAChE were soaked with ENA-713, and the structure was solved and refined to 2.2A resolution. The refinement showed that Ser200 of TcAChE was methylethyl-carbamylated, and NAP was bound non-covalently in the active site. Significant contacts of NAP included hydrophobic interactions with Phe330 and Trp84. We conclude that ENA-713 can inhibit AChE by two mechanisms: (1) covalent carbamylation followed by slow decarbamylation; and (2) action of the carbamate as a vector to deliver the leaving group, NAP, which is itself a good reversible anti-AChE.


Biochemistry | 2002

Kinetic and structural studies on the interaction of cholinesterases with the anti-Alzheimer drug rivastigmine.

P. Bar-On; Charles B. Millard; Michal Harel; Hay Dvir; Albert Enz; Joel L. Sussman; Israel Silman


Biochemistry | 1999

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

Charles B. Millard; Gitay Kryger; Arie Ordentlich; Harry M. Greenblatt; Michal Harel; Mia L. Raves; Yoffi Segall; Dov Barak; Avigdor Shafferman; Israel Silman; Joel L. Sussman


Biochemistry | 1997

A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase.

Oksana Lockridge; Renee M. Blong; Patrick Masson; Marie Thérèse Froment; Charles B. Millard; Clarence A. Broomfield


Journal of Molecular Biology | 2000

Active-site Gorge and Buried Water Molecules in Crystal Structures of Acetylcholinesterase from Torpedo californica

Gertraud Koellner; Gitay Kryger; Charles B. Millard; Israel Silman; Joel L. Sussman; Thomas Steiner

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Israel Silman

Weizmann Institute of Science

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Joel L. Sussman

Weizmann Institute of Science

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Michal Harel

Weizmann Institute of Science

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Arie Ordentlich

Israel Institute for Biological Research

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Avigdor Shafferman

Israel Institute for Biological Research

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Dov Barak

Israel Institute for Biological Research

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Gitay Kryger

Weizmann Institute of Science

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Harry M. Greenblatt

Weizmann Institute of Science

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Mia L. Raves

Weizmann Institute of Science

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