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. The working model of the binding site can then be used for virtual screening of additional ligands that may fit this site, for determining and comparing specificity profiles of related receptors, and for structure-based design of agonists and antagonists. The current review presents the protocol and enumerates the steps for modeling and validating the residues involved in ligand binding. The main stages include (a) modeling the receptor structure using an automated fragment-based approach, (b) predicting potential binding pockets, (c) docking known binders, (d) analyzing predicted interactions and comparing with positions that have been shown to bind ligands in other receptors, (e) validating the structural model by mutagenesis.

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. Automatic agonists docking to both a new homology modeled receptor and the A(2A) AR crystallographic structure produced similar results. Heterocyclic nitrogen atoms closely corresponded when the docked adenine moiety of agonists and 1 were overlaid. The cumulative mutagenesis data, which support the proposed mode of agonist docking, can be reexamined in light of the crystallographic structure. Thus, homology modeling of GPCRs remains a useful technique in probing the structure of the protein and predicting modes of ligand docking.

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. Whereas the charged moieties of pyridostigmine and rivastigmine contribute significantly to the stability of the corresponding HuAChE complexes, no such effect was observed for physostigmine and its analogues, phenserine and cymserine. Moreover, physostigmine-like ligands carrying oxygen instead of nitrogen at position -1 of the tricyclic moiety (physovenine and tetrahydrofurobenzofuran analogues) displayed comparable structure-function characteristics toward the various HuAChE enzymes. The essential role of the HuAChE hydrophobic pocket, comprising mostly residues Trp(86) and Tyr(337), in accommodating (-)-physostigmine and in conferring approximately 300-fold stereoselectivity toward physostigmines, was elucidated through examination of the reactivity of selected HuAChE mutations toward enantiomeric pairs of different physostigmine analogues. The present study demonstrates that certain charged and uncharged ligands, like analogues of physostigmine and physovenine, seem to be accommodated by the enzyme mostly through hydrophobic interactions.

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. The acyl pocket, the cation-binding subsite (Trp86) and the peripheral anionic subsite were also found to be directly involved in HuAChE stereoselectivity toward charged chiral phosphonates, operating through differential positioning of the ligand cationic moiety within the active center. Residue Trp86 is also a part of the "hydrophobic patch" which seems flexible enough to accommodate the structurally diverse ligands like tacrine, galanthamine and the two diastereomers of huperzine A. Also, we have recently discovered further aspects of the role of both the unique structure and the flexibility of the "hydrophobic patch" in determining the reactivity and stereoselectivity of HuAChE toward certain carbamates including analogs of physostigmine. In these cases the ligands are accommodated mostly through hydrophobic interactions and their stereoselectivity delineates precisely the steric limits of the pocket. Hence, the HuAChE stereoselectivity provides a sensitive tool in the in depth exploration of the functional architecture of the active center. These studies suggest that the combination of "rigidity" and flexibility within the HuAChE gorge are an essential element of its molecular design.

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. One patient had stable disease for 8 months; the other for 3 months. No serious adverse events (AEs) attributable to IVIG were observed. We conclude that IVIG therapy may be useful for the treatment of metastatic melanoma. Furthermore, we suggest that the effects of IVIG therapy might be enhanced by its use as an adjuvant in patients without evidence of disease following surgery.

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. Hence, they blocked carbamylcholine (CCh)-induced cyclic adenosine monophosphate (cAMP) synthesis (muM) and had almost no effect on CCh-induced phosphoinositide (PI) hydrolysis. These substances were inactive on forskolin-induced cAMP inhibition signaling in CHO cells expressing M(2) mAChR. In binding studies, using [(3)H]-N-methyl scopolamine ([(3)H]NMS) as the competitor ligand, the ChEIs, VX and echothiophate exhibited binding to rat cortical mAChR with K(i) values in the muM range. The non-charged compounds, sarin and soman, were inert in modulating both cAMP metabolism and PI hydrolysis in CHO cells expressing M(1), M(2), and M(3) mAChRs, and no binding was observed in presence of [(3)H]NMS. These data suggest that VX and echothiophate act as function-specific blockers via a non-classical path of antagonistic activity, implying the involvement of allosteric/ectopic-binding site in M(1) and M(3) mAChRs. The functionally selective antagonistic behavior of echothiophate and VX makes them potential tools for dissecting the interactions of the mAChR with different G proteins.

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. Disruption of this network by replacement of Ser229 by alanine is thought to remove the Glu334 carboxylate from the vicinity of His447 abolishing catalytic activity. The proper orientation of the catalytic histidine side chain is maintained by these polar interactions as well as through "aromatic trapping" by residues lining the HuAChE active center gorge. Thus, replacement of aromatic residues in the vicinity of His447, as in the F295A/F338A or in the Y72N/Y124Q/W286A/F295L/F297V/Y337A (hexamutant which mimicks the aromatic lining of HuBChE) enzymes, resulted in a dramatic decrease in catalytic activity, which was proposed to originate from catalytically nonproductive mobility of His447. Yet, HuBChE is catalytically efficient indicating that "aromatic trapping" is not the only way to conformationally stabilize the His447 side chain. A possible restriction of this mobility in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Both simulations and reactivities of the actual F295X/F338A enzymes, carrying various aliphatic residues at position 295, indicate that of the bulky amino acids, like leucine or isoleucine, only methionine was capable of maintaining the catalytically viable conformation of His447. The F295M/F338A HuAChE was only two-fold less reactive than the F338A enzyme toward acetylthiocholine, and exhibited wild type-like reactivity toward covalent modifiers of the catalytic Ser203. The findings are consistent with the notion that different combinations of steric interference and specific polar interactions serve to maintain the position of His447 and thereby the high efficiency of the catalytic machinery. The two seemingly conflicting demands on the architecture of the active center-flexible accommodation of substrate and optimal juxtaposition of residues of the catalytic triad, demonstrate the truly amazing molecular design of the AChE active center.

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). HuAChE was also 12,500-fold more reactive toward VX(S) than toward nc-VX(S), demonstrating the significance of the polar interactions of the ammonium substituent to their overall affinity toward VX. Indeed, substitution of the cation-binding subsite residue Trp86 by alanine resulted in a decrease of three orders of magnitude in HuAChE reactivity toward both VX enantiomers, with only a marginal effect on the reactivity toward the enantiomers of nc-VX. These results demonstrate that accommodation of the charged moieties of both VX enantiomers depends predominantly on interactions with the aromatic moiety of Trp86. Yet, these interactions seem to limit the stereoselectivity toward the P(S)-enantiomer, which for charged methylphosphonates is much lower than for the noncharged analogs, like sarin or soman. Marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 by asparagine (D74N) practically abolished stereoselectivity toward VX(S) (a 130-fold decrease), while substitution which retained the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers originates predominantly from a different orientation of the charged leaving group with respect to residue Asp74. Such different orientations of the charged leaving group in the HuAChE adducts of the VX enantiomers seem to be a consequence of intramolecular interactions with the bulky phosphorus alkoxy group.

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. Since the rates of formation of the covalent adducts depend predominantly on the stabilities of the corresponding Michaelis complexes, it is essential to characterize the specific interactions contributing to stabilization of these complexes. Functional analysis of interactions with HuAChE enzymes allows for such characterization for carbamates, like pyridostigmine or rivastigmine, much in the same way as that for the noncovalent therapeutic ligands nivalin or aricept. In fact, the observed differences between the affinities toward carbamates and the noncovalent ligands seem to result from specific structural characteristics of the inhibitors rather than from the decomposition path of the particular complex. Replacements at the cation binding site (Trp86), hydrogen bond network (Glu202, Tyr133, Glu450), and hydrophobic pocket result in similar effects for the covalent as well as for the noncovalent inhibitors. Also, while the effects of perturbing the aromatic trapping of the catalytic His447 for pyridostigmine and nivalin were analogous to those for the substrate, the corresponding effects for rivastigmine and aricept were quite different. Thus, elucidation of the functional architecture of the HuAChE active center is bound to be of considerable utility in the current effort to design novel covalent AChE inhibitors as therapeutics for Alzheimer's disease (AD).

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 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.

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. Simulations suggested that of the 12 aliphatic residues substituted at position 295, including hydrophobic and polar amino acids, only methionine was capable of maintaining the catalytically viable conformation of His447. Examination of the reactivities of the actual F295X/F338A HuAChEs showed that indeed the F295M/F338A enzyme was only 2-fold less reactive than the F338A mutant toward acetylthiocholine, while enzymes substituted by the similarly bulky residues leucine and isoleucine were catalytically impaired. Furthermore, only the F295M/F338A enzyme exhibited wild-type-like reactivity toward covalent modifiers of the catalytic Ser203 including the methylphosphonate soman and transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), as well as a facile dealkylation of the F295M/F338A-soman adduct. A different behavior was observed for bulkier ligands which introduce a deformation in the acyl pocket, and therefore their activity seems only marginally affected by the positioning of His447. The findings emphasize the importance of the precise positioning of His447 for catalysis and indicate that, in the absence of aromatic "trapping", restriction of the histidine mobility in F295X/F338A HuAChEs requires a combination of steric interference and a specific polar interaction. The results also underscore the role of the acyl pocket subsite of cholinesterases in maintaining the catalytically viable conformation of the catalytic histidine.

Engineering of A3 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 A1 and A3 ARs and at P2Y1, P2Y2, P2Y4, or P2Y11 receptors, but not P2Y6 receptors. At the hA3 AR a new full agonist, MRS1898, the (N)-methanocarba equivalent of CI-IB-MECA (2-chloro-N 6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine), had a Ki value of 1.9 nM in binding to the hA3 AR expressed in CHO cells. Functional assays confirmed the selectivity of MRS1898, although CI-IB-MECA was even more functionally selective for human A3 vs. hA1 and hA2A ARs. Thirty µM MRS1898 did not induce apoptosis in HL-60 cells, suggesting that some of the proapoptotic effects of CI-IB-MECA may be nonreceptor-mediated. Manipulation of the sequence of A3 ARs through site-directed mutagenesis has led to pharmacologically unique constructs: constitutively active receptors and "neoceptors." Such engineered receptors may later prove to have potential for cardioprotection through gene transfer. Effects of single amino acid replacement were interpreted using a rhodopsin-based model of ligand-A3 receptor interactions, leading to the proposal that a movement of the conserved W243 in TM6 may be involved in AR activation.

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. Notably, the simple 2-fluoro group alone or 2-chloro in combination with N(6)-substitution dramatically diminished the efficacy of adenosine derivatives, even converting agonist into antagonist. Other affinity-increasing substitutions, including N(6)-(3-iodobenzyl) 4 and the (Northern)-methanocarba 15, also reduced efficacy, except in combination with a flexible 5'-uronamide. 2-Cl-N(6)-(3-iodobenzyl) derivatives, both in the (N)-methanocarba (i.e., of the Northern conformation) and riboside series 18 and 5, respectively, were potent antagonists with little residual agonism. Ring-constrained 2',3'-epoxide derivatives in both riboside and (N)-methanocarba series 13 and 21, respectively, and a cyclized (spiral) 4',5'-uronamide derivative 14 were synthesized and found to be human A(3)AR antagonists. 14 bound potently at both human (26 nM) and rat (49 nM) A(3)ARs. A rhodopsin-based A(3)AR model, containing all domains except the C-terminal region, indicated separate structural requirements for receptor binding and activation for these adenosine analogues. Ligand docking, taking into account binding of selected derivatives at mutant A(3)ARs, featured interactions of TM3 (His95) with the adenine moiety and TMs 6 and 7 with the ribose 5'-region. The 5'-OH group of antagonist N(6)-(3-iodobenzyl)-2-chloroadenosine 5 formed a H-bond with N274 but not with S271. The 5'-substituent of nucleoside antagonists moved toward TM7 and away from TM6. The conserved Trp243 (6.48) side chain, involved in recognition of the classical (nonnucleoside) A(3)AR antagonists but not adenosine-derived ligands, displayed a characteristic movement exclusively upon docking of agonists. Thus, A(3)AR activation appeared to require flexibility at the 5'- and 3'-positions, which was diminished in (N)-methanocarba, spiro, and epoxide analogues, and was characteristic of ribose interactions at TM6 and TM7.

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). Indeed, the F295A/F338A/V407F enzyme is 170-fold more reactive than the corresponding double mutant and only 3-fold less reactive than the wild-type HuAChE. However, analogous substitution of Val407 in the hexamutant HuAChE (generating the heptamutant Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F) did not enhance catalytic activity. Reactivity of these double, triple, hexa, and hepta mutant HuAChEs was monitored toward covalent ligands such as organophosphates and the transition state analogue TMFTA, which probe, respectively, the facility of the enzymes to accommodate Michaelis complexes and to undergo the acylation process. The findings suggest that in the F295A/F338A mutant the two His447 conformational states, which are essential for the different stages of the catalytic process, seem to be destabilized. On the other hand, in the F295A/F338A/V407F mutant only the state involved in acylation is impaired. Such differential effects on the His447 conformational properties demonstrate the general role of aromatic residues in cholinesterases, and probably in other serine hydrolases, in "trapping" of the catalytic histidine and thereby in optimization of catalytic activity.

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. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by approximately 3-38-fold, suggesting that these residues were critical for the high affinity of A(3) adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A(3) agonist 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5'-gamma-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp(243) was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A(3) receptor interactions.