1843u89 and 2--deoxyuridylic-acid

Thymidylate synthase (TS) catalyzes the synthesis of deoxythymidine monophosphate (dTMP), which is an essential precursor for DNA synthesis. The rationale underlying drug design is to identify compounds that differentially inhibit a viral or parasite enzyme vs. the host homologue. We studied the TS of the white spot syndrome virus (WSSV TS) and the corresponding TS from the host, the marine invertebrate shrimp Litopenaeus vannamei. TS is the only de novo source of dTMP and is essential for host and viral DNA replication. To establish proof of principle, we cloned a full-length TS cDNA from the white shrimp L. vannamei (shrimp TS) that corresponds to a deduced sequence of 289 amino acids and over-expressed it to study inhibition of both shrimp and viral TSs. Steady-state kinetic parameters for both TSs are similar, and dissociation (K(d)) or half maximal inhibitory concentration constants (IC(50)) did not show differential inhibition between the folate analogues. Differences in their amino acid sequence are not reflected in theoretical molecular models of both TSs, since both appear to have identical active sites. These results suggest that the eukaryotic TS active site is very constrained into the functional residues involved in reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP).

Topics: Amino Acid Sequence; Animals; Base Sequence; Catalytic Domain; Cloning, Molecular; Deoxyuracil Nucleotides; Folic Acid; Folic Acid Antagonists; Isoindoles; Kinetics; Models, Molecular; Molecular Sequence Data; Penaeidae; Phylogeny; Quinazolines; Sequence Alignment; Thymidylate Synthase; White spot syndrome virus 1

Using fixed receptor sites derived from high-resolution crystal structures in structure-based drug design does not properly account for ligand-induced enzyme conformational change and imparts a bias into the discovery and design of novel ligands. We sought to facilitate the design of improved drug leads by defining residues most likely to change conformation, and then defining a minimal manifold of possible conformations of a target site for drug design based on a small number of identified flexible residues.. The crystal structure of thymidylate synthase from an important pathogenic target Pneumocystis carinii (PcTS) bound to its substrate and the inhibitor, BW1843U89, is reported here and reveals a new conformation with respect to the structure of PcTS bound to substrate and the more conventional antifolate inhibitor, CB3717. We developed an algorithm for determining which residues provide 'soft spots' in the protein, regions where conformational adaptation suggests possible modifications for a drug lead that may yield higher affinity. Remodeling the active site of thymidylate synthase with new conformations for only three residues that were identified with this algorithm yields scores for ligands that are compatible with experimental kinetic data.. Based on the examination of many protein/ligand complexes, we develop an algorithm (SOFTSPOTS) for identifying regions of a protein target that are more likely to accommodate plastically to regions of a drug molecule. Using these indicators we develop a second algorithm (PLASTIC) that provides a minimal manifold of possible conformations of a protein target for drug design, reducing the bias in structure-based drug design imparted by structures of enzymes co-crystallized with inhibitors.

Topics: Algorithms; Amino Acid Motifs; Binding Sites; Crystallography; Deoxyuracil Nucleotides; Drug Design; Enzyme Inhibitors; Folic Acid Antagonists; Ligands; Membrane Proteins; Pliability; Pneumocystis; Protein Binding; Protein Conformation; Substrate Specificity; Thymidylate Synthase

Thymidylate synthase (TS) is critical to DNA synthesis as it catalyzes the rate limiting step in the only biosynthetic pathway for deoxythymidine monophosphate (dTMP) production. TS is therefore an important target for anti-proliferative and anti-cancer drug design. The TS enzymatic mechanism involves the reductive methylation of the substrate, deoxyuridine monophosphate (dUMP), by transfer of a methylene group from the co-factor, methylenetetrahydrofolate (CH2H4folate), resulting in the production of deoxythymidine monophosphate (dTMP) and dihydrofolate (H2folate). Previous drug design efforts based on co-factor analogues have produced good inhibitors of TS, but poor bioavailability and toxicity have limited their usefulness. BW1843U89, a folate analogue, is a recently developed compound which is an exceptionally strong inhibitor (Ki = 0.09 nM), has good bioavailability and in clinical trials thus far has not demonstrated significant toxicity.. We report the crystal structure of E. coli TS in ternary complex with dUMP and BW1843U89 at 2.0 A resolution. Although the benzoquinazoline ring system of the inhibitor binds to TS in much the same manner as previously determined for H2folate and CB3717, the larger size of the ligand is accommodated by the enzyme through a local distortion of the active site, that is not strictly conserved in both monomers in the asymmetric unit. Several conserved waters that had been previously implicated in mechanistic roles have been displaced.. BW1843U89 forms a ternary complex with dUMP and completes with CH2H4 folate at the active site. Inhibition of TS by BW1843U89 shows four unique aspects in its mechanism of action. BW1843U89 prevents the Michael addition of dUMP to Cys146, in contrast to the mechanisms implicated from crystallography of other quinazoline based inhibitors; displaces a catalytic water from the active site; reorders a peptide loop (Leu72-Trp83) in the active site; and is unique amongst the antifolates in inactivating TS at a stoichiometric ratio of one molecule per TS dimer. Thus, it exploits the principles of negative cooperativity that are increasingly being recognized in the catalytic mechanism of the enzyme per se. The structure suggests that this 'half-the-sites' effect is catalytic and not related to ligand binding. Therefore BW1843U89 is both a competitive inhibitor (at the binding site) and a non-competitive inhibitor at the other site.

Topics: Antineoplastic Agents; Binding Sites; Binding, Competitive; Crystallography, X-Ray; Deoxyuracil Nucleotides; Drug Design; Enzyme Inhibitors; Escherichia coli; Folic Acid Antagonists; Indoles; Isoindoles; Models, Molecular; Molecular Structure; Protein Binding; Protein Conformation; Quinazolines; Thymidylate Synthase

The TS-inhibitory effects induced by a 24-h exposure to the folate-based TS inhibitors CB3717, C2-desamino analogs of CB3717 including D1694, and BW1843U89 were quantitated using the MGH-U1 human bladder carcinoma. The effects of D1694 on the time course of TS inhibition and on intracellular deoxyuridine monophosphate (dUMP) accumulation and deoxyuridine (dUrd) production were evaluated. D1694 and BW1843U89 were the most active TS inhibitors with IC50 values of 2.4 and 0.5 nM, respectively. The C2-desamino C2-methyl dideazafolates were 27-292 times more potent than the parent CB3717 as TS inhibitors. A methyl group at the C2 position of CB3717 had the most dramatic effect, whereas a thiazole substitution for a benzyl added a small benefit and N10 substitution had a limited impact on TS-inhibitory potency and clonogenic survival. There was a significant correlation between the IC50 values for TS inhibition and those for cytotoxic potency obtained for these drugs. LV and thymidine protected cells from these folate-based TS inhibitors. Intracellular dUMP levels following 24 h D1694 (IC50) exposure increased 7-fold. Levels of dUrd effluxing into the media increased up to 4.5 microM following a 24-h exposure to D1694 (IC90). We conclude that (a) C2-desamino C2-methyl dideazafolates are potent TS inhibitors, (b) TS inhibition requires prolonged exposure with these folate TS inhibitors, (c) survival is correlated with inhibition of TS for the folate-based TS inhibitors and (d) the biochemical consequences of TS inhibition include increased dUMP and dUrd levels.

Topics: Cell Survival; Deoxyuracil Nucleotides; Deoxyuridine; Folic Acid; Folic Acid Antagonists; Glutamates; Humans; In Vitro Techniques; Indoles; Isoindoles; Quinazolines; Structure-Activity Relationship; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Human thymidylate synthase is a polymeric protein composed of two subunits with identical primary structures. In this study we determined the binding affinities of 5,10-methylene tetrahydropteroyltetraglutamate (folate substrate) and a group of close structural folate analog inhibitors. Thymidylate synthase bound both mono and polyglutamylated folate substrates and analogs more tightly in the presence of deoxyuridylate. These results and product inhibition studies confirmed that the orders of substrate addition and product release from thymidylate synthase were similar for mono and polyglutamylated substrates. Equilibrium dialysis studies showed that the folate substrate in a ternary complex with deoxyuridylate bound to one of the subunits (site A) with a Kd of 720 nM. The binding of the substrate to the second subunit (site B) was much weaker, and the Kd could not be determined by this method. However, dissociation constants for each subunit could be measured for the folate analog inhibitors, and, depending on the inhibitor, the relative Kd value for each subunit varied substantially. For example, formyl-5,8-dideazafolate and tetraglutamylated 10-propargyl-5,8-dideazafolate bound to both sites with similar Kd values, whereas D1694Glu4 bound to subunit A with a higher affinity (Kd = 1.0 nM) than to subunit B (Kd = 30 nM). In contrast, 1843U89 (mono or diglutamylated form) had a much higher affinity for subunit B (Kd approximately 0.1 nM) compared with subunit A (Kd approximately 400 nM). Enzyme inhibition kinetic analyses showed that the Ki values of 1843U89 were quite low (0.1 nM) and that the inhibition was noncompetitive. In contrast, the other folate analogs inhibited the enzyme via mixed inhibition (i.e. both the Km for the folate substrate and the Vmax were altered). We conclude that the two subunits of thymidylate synthase bind folate substrates and analogs differently and that the asymmetric binding of the ligands is the major factor that determines the inhibition kinetics of each folate analog inhibitor.

Topics: Binding Sites; Carbon Radioisotopes; Deoxyuracil Nucleotides; Folic Acid; Folic Acid Antagonists; Humans; Indoles; Isoindoles; Kinetics; Macromolecular Substances; Mathematics; Protein Binding; Quinazolines; Structure-Activity Relationship; Thymidylate Synthase