Characterization of binding mode of imatinib to human α1-acid glycoprotein.

Imatinib (IMT) is a selective tyrosine kinase inhibitor, used in the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Its strong plasma protein binding was found to belong to the F1*S genetic variant of α(1)-acid glycoprotein (AGP). In this work, comparative AGP binding studies were performed with IMT fragment molecules to reveal which parts of the molecule are important in the high-affinity interaction provoking specific spectral changes. Molecular modeling calculations indicated that IMT docked into the X-ray structure of AGP/F1 adopts a bent, compact conformation. This binding mode is similar to those found in its complexes with some low-affinity kinases and a quinone reductase, being strikingly different from the extended conformation of IMT in its high-affinity kinase targets.

Assessing structure, function and druggability of major inhibitory neurotransmitter gamma-aminobutyrate symporter subtypes.

Ambient level of gamma -aminobutyric acid (GABA), the major inhibitory neurotransmitter of the brain is mediated by neuronal and glial GABA transporters (GATs), members of the sodium and chloride ion-dependent solute carrier family. The neuronal GABA transporter subtype (GAT-1) has already been proven to be the target for the antiepileptic drug Tiagabine. However, druggability of glial GAT-2 and GAT-3 is yet to be established. Recent advances in structure elucidation of a bacterial orthologue leucine transporter in complex with different substrates substantiate homology modeling of human GATs (hGATs). These modeling studies can provide mechanistic clues for structure-based prediction of the potential of medicinal chemistry campaigns. A recently identified characteristic structural feature of the occluded conformation of hGATs is that similar extra- and intracellular gates are formed by middle-broken transmembrane helices TM1 and TM6. Binding crevice formed by unwound segments of broken helices facilitates symport of GABA with Na+ ion via fitting of GABA to TM1-bound Na+ closely inside. Favored accommodation of substrate inhibitors with high docking score predicts efficient inhibition of the neuronal hGAT-1 if the TM1-TM8 binding prerequisite for GABA was used. Docking, molecular dynamics and transport data indicate, that amino acids participating in substrate binding of the neuronal hGAT-1 and the glial hGAT-2 and hGAT-3 subtypes are different. By contrast, substrate binding crevices of hGAT-2 and hGAT-3 cannot be distinguished, avoiding sensible prediction of efficient selective substrate inhibitors. Glial subtypes might be specifically distinguished by interfering Zn2+ binding in the second extracellular loop of hGAT-3. Formation of the unique ring-like Na+-GABA complex in the occluded binding crevices anticipates family member symporters exploring chemiosmotic energy via reversible chemical coupling of Na+ ion.

Molecular dynamics study of active-site interactions with tetracoordinate transients in acetylcholinesterase and its mutants.

The role of active-site residues in the dealkylation reaction in the P(S)C(S) diastereomer of 2-(3,3-dimethylbutyl)methylphosphonofluoridate (soman)-inhibited Torpedo californica acetylcholinesterase (AChE) was investigated by full-scale molecular dynamics simulations using CHARMM: >400 ps equilibration was followed by 150-200 ps production runs with the fully solvated tetracoordinate phosphonate adduct of the wild-type, Trp84Ala and Gly199Gln mutants of AChE. Parallel simulations were carried out with the tetrahedral intermediate formed between serine-200 Ogamma of AChE and acetylcholine. We found that the NepsilonH in histidine H(+)-440 is positioned to protonate the oxygen in choline and thus promote its departure. In contrast, NepsilonH in histidine H(+)-440 is not aligned for a favourable proton transfer to the pinacolyl O to promote dealkylation, but electrostatic stabilization by histidine H(+)-440 of the developing anion on the phosphonate monoester occurs. Destabilizing interactions between residues and the alkyl fragment of the inhibitor enforce methyl migration from Cbeta to Calpha concerted with C-O bond breaking in soman-inhibited AChE. Tryptophan-84, phenyalanine-331 and glutamic acid-199 are within 3.7-3.9 A (1 A=10(-10) m) from a methyl group in Cbeta, 4.5-5.1 A from Cbeta and 4.8-5.8 A from Calpha, and can better stabilize the developing carbenium ion on Cbeta than on Calpha. The Trp84Ala mutation eliminates interactions between the incipient carbenium ion and the indole ring, but also reduces its interactions with phenylalanine-331 and aspartic acid-72. Tyrosine-130 promotes dealkylation by interacting with the indole ring of tryptophan-84. Glutamic acid-443 can influence the orientation of active-site residues through tyrosine-421, tyrosine-442 and histidine-440 in soman-inhibited AChE, and thus facilitate dealkylation.

Origins and diversity of the aging reaction in phosphonate adducts of serine hydrolase enzymes: what characteristics of the active site do they probe?

Molecular mechanics and dynamics combined with semiempirical calculations were carried out for purposes of comparison of the active site characteristics of AChE, trypsin, and chymotrypsin as probed by their diastereomeric adducts with 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman), methylphosphonate monoester anions, and tetravalent carbonyl intermediates of the reactions of the natural substrates in each case. Glu199 is a key residue in the electrostatic catalytic mechanism of AChE, in removal of the leaving group, and possibly by acting as an alternate general base catalyst. "Pushing" of an alkoxy ligand by Glu199 and the numerous small van der Waals interactions promote dealkylation in phosphonate adducts of AChE much more effectively than any other enzyme. A high concentration of negative charge created by the phosphonate ester monoanion and Glu199 adjacent to it fully accounts for the resistance to the attack of even the strongest nucleophile applied for enzyme reactivation. Stabilization of the developing negative charge on the phosphonates in the soman-inhibited PSCS adducts of serine hydrolases is by electrophilic residues in the oxyanion hole (AChE) and the protonated catalytic His. PR diastereomers of soman-inhibited AChE can be accommodated in an orientation in which the oxyanion hole interactions are lost and for which the stabilizing interactions are 17-26 kcal/mol smaller than in the PS diastereomer. The dealkylation reaction is almost equally likely in all diastereomers of soman-inhibited AChE. The stabilizing interaction energies are approximately 4 kcal/mol greater in the PR than in the PS adducts of the soman-inhibited serine proteases. There is 0.60 unit greater partial negative charge on the phosphonyl fragment in the anion of phosphonate monoesters of Ser than at the oxygens of tetravalent carbonyl transients resulting in approximately 12-22 kcal/mol greater stabilization of the former than the latter.

Enantioselective and reversible inhibition of trypsin and alpha-chymotrypsin by phosphonate esters.

Trypsin is inactivated by the levorotatory enantiomers (most likely PS) of 4-nitrophenyl 4-H-, 4-CH3-,4-OCH3-, and 4-Cl-phenacyl methylphosphonates (PMNs) with second-order rate constants between 231 and 884 M-1 s-1. 4-NO2-PMN hydrolyzes before inhibiting the enzyme. The second-order rate constants for the inactivation of alpha-chymotrypsin by the levorotatory enantiomers of the five PMNs are between 37,000 and 770,000 M-1 s-1, and those for the dextrorotatory enantiomers are between 400 and 640 M-1 s-1; the enantioselectivity is 90-1880. Specific rotation [alpha]22D of the faster-reacting enantiomer of 4-CH3-PMN with trypsin and alpha-chymotrypsin is -30 +/- 6 degrees. 31P NMR of the adducts shows a signal at 41.0 ppm, 10 ppm downfield from the parent compound. Results of molecular mechanics and dynamics calculations show that the principal interactions are between the phosphonyl group and constituents of the oxyanion hole and between the aromatic fragment and residues in the binding regions of the enzymes. Trypsin activity returns from its phenacyl methylphosphonyl adducts on the hour time scale and in reversed order to the rates of inactivation within the series. Recovery of alpha-chymotrypsin activity from the adducts formed with the (-) enantiomers is on a slower time scale still, whereas its recovery from the adducts formed with the (+) enantiomers is on the second to minute time scale. The data support a mechanism of reactivation involving rate-determining intramolecular displacement of Ser by the carbonyl hydrate of the phenacyl moiety. The pH-rate profiles for trypsin reactivation from its adducts indicate involvement of an ionizable group with pKa approximately 8.0. The pH dependence and solvent isotope effects are small in most cases. The compounds demonstrate favorable properties for controllable and temporary modulation of enzyme activity.

Efficient product clearance through exit channels in substrate hydrolysis by acetylcholinesterase.

The channels connecting the active site of acetylcholinesterase (AChE) to the protein exterior were mapped by computational techniques in order to find potential exit routes for charged reaction products. 3.9% of the total volume of the AChE monomer is hollow space and over 50% of the void is located in the center; it is partitioned into three chambers, a deep entry channel, below it a wide channel located in a slightly positive region of AChE and ideally suitable for the exit of negatively charged fragments and a small chamber above Trp84 and Met83. The latter serve as gates for the departure of the positively charged choline product of the hydrolysis of acetylcholine into the small cavity. An efficient product clearance is a prerequisite to a very low energy pathway for the irreversible hydrolysis of acetylcholine.