BackgroundPreviously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions.ResultsHere we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i) enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii) enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii) substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine.ConclusionThe experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand), although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria.

Potentiometric equilibrium measurements were made for some metal ions (M(II) = Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Ca(II), Sr(II), and Ba(II)) with guanine (A) in a 1:1 (M(II):A) ratio and with cytosine, cytidine, 5-bromocytosine, 5-azacytosine, and 5-fluorocytosine as primary ligands (L) and guanine as secondary ligand in a 1:1:1 (M(II):L:A) ratio at (25.0, 35.0, and 45.0) °C and I = 0.1 mol·dm-3 NaNO3 in aqueous solution. The experimental pH-titration data were analyzed by using a BEST computer program in order to evaluate the formation constants of various intermediate species and their relative distribution. The experimental conditions were selected in such a way that the self-association of the nucleobases and their complexes due to stacking interaction was negligibly small, so that only the neutral monomeric and hydroxo ternary complexes were studied. The enthalpy (Δf H°) and entropy (Δf S°) changes for the formation of binary and ternary complexes were calculated from temperature coefficient data. The δΔf S° values are positive for all the metal ligand systems. The negative δΔf H° values indicate the extra stabilization of most of the ternary complexes by the exothermic enthalpy change (δΔf S° = ΔT S° − ΔB S° and δΔf H° = ΔT H° − ΔB S° where ΔT S°, ΔT H° and ΔB S°, ΔB H° are the entropy and enthalpy values associated with the ternary and binary complexes, respectively). On the basis of IR data for metal complexes with the 5FC−G mispair, it has been proposed that the guanine is bonded to metal ions through N1/C6O and N7, whereas cytosine and its derivatives are bonded through N3 atoms in ternary complexes.

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Solution studies were performed pH-metrically to study the interaction of Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) metal ions with 5-fluorouracil (5FU) and histamine (Hm) separately (binary) and in the presence of each other (ternary) at 25±0.1 °C temperature and a constant ionic strength of 0.1 M NaNO3 in aqueous solution. The ternary complexes have been found to be more stable than the corresponding binary complexes as shown by the positive value of ΔlogK. The species distribution curves have been obtained using the computer programme BEST. On the basis of species distribution results, efforts were also made to prepare some mixed complexes of Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) ions by performing the reaction of their metal nitrates, 5FU and Hm in aqueous ethanol medium at suitable pH. The isolated solid complexes were characterized by different physico-chemical method in order to suggest the possible binding site of the ligands and the structure of the resultant complexes. All these complexes were checked for their antitumour activity by injecting in Dalton's lymphoma (DL) and Sarcoma-180 (S-180) bearing C3H/He mice. The results indicate that some complexes have good antitumour activity both in vivo and in vitro.