Homology model-assisted elucidation of binding sites in GPCRs.

G protein-coupled receptors (GPCRs) are important mediators of cell signaling and a major family of drug targets. Despite recent breakthroughs, experimental elucidation of GPCR structures remains a formidable challenge. Homology modeling of 3D structures of GPCRs provides a practical tool for elucidating the structural determinants governing the interactions of these important receptors with their ligands.

Evaluation of homology modeling of G-protein-coupled receptors in light of the A(2A) adenosine receptor crystallographic structure.

Homology modeling of the human A(2A) adenosine receptor (AR) based on bovine rhodopsin predicted a protein structure that was very similar to the recently determined crystallographic structure. The discrepancy between the experimentally observed orientation of the antagonist and those obtained by previous antagonist docking is related to the loop structure of rhodopsin being carried over to the model of the A(2A) AR and was rectified when the beta(2)-adrenergic receptor was used as a template for homology modeling. Docking of the triazolotriazine antagonist ligand ZM241385 1 was greatly improved by including water molecules of the X-ray structure or by using a constraint from mutagenesis.

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.

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.

Efficacy and safety of intravenous immunoglobulin in patients with metastatic melanoma.

We have previously reported studies performed both in vitro and in laboratory animals, as well as a case study in humans, suggesting that intravenous immunoglobulin (IVIG) may be beneficial in the treatment of malignancies, including metastatic melanoma. As part of a phase II open label trial, we have administered IVIG to nine patients with metastatic melanoma who had been heavily treated. In two of nine (22%) patients treated every 3 weeks with IVIG (1 g/kg body weight), the disease stabilized.

Function-specific blockage of M(1) and M(3) muscarinic acetylcholine receptors by VX and echothiophate.

Certain organophosphate (OP) cholinesterase inhibitors (ChEIs) are also known to bind to the muscarinic acetylcholine receptor (mAChR). The functional consequences of such binding were investigated here using the following OP compounds: VX, echothiophate, sarin, and soman. VX (charged at physiological pH) and echothiophate (formally charged) inhibited a specific signal transduction pathway in CHO cells expressing either the M(1) or M(3) mAChR.

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.

Engineering of A Adenosine and P2Y Nucleotide Receptors and Their Ligands.

Modification of the ribose moiety of nucleotides and nucleosides has provided new insights into structural and conformational requirements for ligands at P2Y nucleotide receptors and at adenosine receptors (ARs). Methanocarba derivatives (containing a rigid bicyclic ring system in place of ribose) of adenosine, ATP, ADP, UTP, UDP, and other receptor agonist analogs were synthesized. Biological evaluation led to the conclusion that in general the Northern (N)-conformation was favored over the Southern (S)-conformation of the pseudoribose moiety at A and A ARs and at P2Y, P2Y, P2Y, or P2Y receptors, but not P2Y receptors.

Structural determinants of A(3) adenosine receptor activation: nucleoside ligands at the agonist/antagonist boundary.

Mutagenesis of the human A(3) adenosine receptor (AR) suggested that certain amino acid residues contributed differently to ligand binding and activation processes. Here we demonstrated that various adenosine modifications, including adenine substitution and ribose ring constraints, also contributed differentially to these processes. The ligand effects on cyclic AMP production in intact CHO cells expressing the A(3)AR and in receptor binding were compared.

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).

Identification by site-directed mutagenesis of residues involved in ligand recognition and activation of the human A3 adenosine receptor.

Ligand recognition has been extensively explored in G protein-coupled A(1), A(2A), and A(2B) adenosine receptors but not in the A(3) receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A(3) adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His(95), Trp(243), Leu(244), Ser(247), Asn(250), and Lys(152). The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist.