ABSTRACT
Mammalian sialyltransferases play a role in the metastasis of various cancers in humans. Inhibitors of these enzymes will in principle be able to directly inhibit aberrant sialylation in cancer. Inhibitors of ST3Gal-I resembling the donor component of SN1 Transition State structures were previously evaluated as part of a kinetics study. Here, using classical dynamics simulations and free energy perturbation calculations, we rationalize the performance of three of these donor analogue ST3Gal-I enzyme inhibitors. We find to inhibit the mammalian ST3Gal-I enzyme a donor analogue requires configurationally limited functionality. This is mediated by the binding of the inhibitor to the enzyme. The inhibitor's ability to interact with Y194 and T272 through a charged group such as a carboxylate is especially important. Furthermore, a conformational rigid form approximating the donor substrate is central. Here this is achieved by an intramolecular hydrogen bond formed between the carboxylate group and one of the ribose hydroxyl groups of the cytidine monophosphate (CMP) leaving group. This intramolecular interaction results in the donor substrate conformer complimenting the form of the catalytic binding site. Finally the carboxylate charge is essential for electrostatic pairing with the binding site. Substituting this group for an alcohol or amide results in severe weakening of the ligand binding. The carboxylate thus proves an to be an irreplaceable functional group and an essential pharmacophore.
Subject(s)
Carbohydrates/pharmacology , Cytidine Monophosphate/pharmacology , Enzyme Inhibitors/pharmacology , Sialyltransferases/antagonists & inhibitors , Carbohydrates/chemistry , Crystallography, X-Ray , Cytidine Monophosphate/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Models, Molecular , Molecular Conformation , Sialyltransferases/metabolism , Static Electricity , Structure-Activity Relationship , beta-Galactoside alpha-2,3-SialyltransferaseABSTRACT
The herpes simplex virus uracil-DNA glycosylase (hsvUNG) enzyme is responsible for the reactivation of the virus from latency and efficient viral replication in nerve tissue. The lack of uracil-DNA glycosylase enzyme in human neurons and the continuous deamination of cytosine create an environment where the presence of viral uracil-DNA glycosylase is a necessity for the proliferation of the virus. A series of 6-(4-alkylanilino)-uracil inhibitors has been developed that selectively and strongly binds to the hsvUNG enzyme while weakly binding to human uracil-DNA glycosylase (hUNG). Here, by using a combination of sequence and structural comparisons between the two enzymes along with free energy of binding computations and principal component analysis of the ligands, we investigate and rationalize the inhibitory effect of the 6-(4-alkylanilino)-uracil series as a function of alkyl chain length on the hsvUNG. The results of these computations corroborate the experimental finding that the inhibitor with an octyl aliphatic chain selectively binds hsvUNG best. More importantly we find that 6-(4-octylanilino)-uracil's selective inhibition of hsvUNG over hUNG is due to the combination of the solution preconfigured bent conformation of that specific chain length and the position of HIS92 (absent in hUNG) just outside hsvUNG's hydrophobic gorge lying adjacent to its uracil binding pocket. The similarities between the uracil binding pockets in hsvUNG and hUNG obfuscate an understanding of the preferential inhibition of the virus enzyme. However, the differences in the enzymes' shallow hydrophobic grooves adjacent to the binding pockets, such as the gorge we identify here, rationalizes 6-(4-alkylanilino)-uracil with an octyl chain length as an excellent pharmacophore template for hsvUNG inhibitor design.
Subject(s)
Enzyme Inhibitors/pharmacology , Herpesvirus 1, Human/enzymology , Models, Molecular , Uracil-DNA Glycosidase/antagonists & inhibitors , Amino Acid Sequence , Binding Sites , DNA/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Ligands , Molecular Sequence Data , Protein Conformation , Substrate Specificity , Thermodynamics , Uracil/chemistry , Uracil/metabolism , Uracil/pharmacology , Uracil-DNA Glycosidase/chemistry , Uracil-DNA Glycosidase/metabolism , Water/chemistryABSTRACT
We investigated link atom approaches for treating the polar C-O bond with particular reference to the glycosidic bond found in complex carbohydrates. We show that cutting this bond after the oxygen in the QM region and saturating the QM system with a hydrogen link atom leads to greater conformational and configurational accuracy at the boundary compared with cutting the bond before oxygen and saturating the QM system with a halogen link atom to represent the oxygen. Furthermore, we find that balancing the MM atom charges and redistributing the boundary atom charges at the QM/MM boundary minimizes the effect of the link atom, both energetically and structurally. This is illustrated via a series of calculations on a set of carbohydrate and carbohydrate-like model compounds. Finally, we confirm the validity of our model by performing molecular dynamics simulations for a typical disaccharide model compound in water. Our postsimulation conformational and configurational analyses show that the oxygen-to-water hydrogen pair distribution functions and the Φ,Ψ distributions at the glycosidic boundary between the quantum and classical regions compare favorably with results obtained from complete QM and complete MM treatments of the saccharide.
