We report here the 1.62 Å crystal structure of an intramolecular quadruplex DNA formed from a sequence in the promoter region of the c-kit gene. This is the first reported crystal structure of a promoter quadruplex and the first observation of localized magnesium ions in a quadruplex structure. The structure reveals that potassium and magnesium ions have an unexpected yet significant structural role in stabilizing particular quadruplex loops and grooves that is distinct from but in addition to the role of potassium ions in the ion channel at the centre of all quadruplex structures. The analysis also shows how ions cluster together with structured water molecules to stabilize the quadruplex arrangement. This particular quadruplex has been previously studied by NMR methods, and the present X-ray structure is in accord with the earlier topology assignment. However, as well as the observations of potassium and magnesium ions, the crystal structure has revealed a highly significant difference in the dimensions of the large cleft in the structure, which is a plausible target for small molecules. This difference can be understood by the stabilizing role of structured water networks.

Multitarget drugs have been to be found effective in controlling complex diseases. However, how to design multitarget drugs presents a great challenge. We have developed a computer-assisted strategy to screen for multitarget inhibitors using a combination of molecular docking and common pharmacophore matching. This strategy was successfully applied to screen for dual-target inhibitors against both the human leukotriene A(4) hydrolase (LTA4H-h) and the human nonpancreatic secretory phospholipase A2 (hnps-PLA2). Three compounds screened from the chemical database MDL Available Chemical Directory were found to inhibit these two enzymes at the 10 microM level. Moreover, one synthetic compound matching the common pharmacophores inhibits LTA4H-h and hnps-PLA2 with IC(50) values of 35 nM and 7.3 microM, respectively. The common pharmacophore model can also be used to search small molecule databases or collections of existing inhibitors, as well as to guide combinatorial library design to search for ideal multitarget inhibitors.

High-resolution crystal structures of the DNA duplex sequence d(CGCGAATTCGCG)2 complexed with three minor-groove ligands, are reported. A highly-conserved cluster, of eleven linked water molecules, has been found in the native and all three ligand-bound structures, positioned at the boundary of the A/T and G/C regions where the minor groove widens. This cluster appears to play a key structural role in stabilizing non-covalently-binding small molecules in the AT region of the B-DNA minor groove. The cluster extends from the backbone phosphate groups along the mouth of the groove and links to DNA and ligands by a network of hydrogen bonds that help to maintain the ligands in position. This arrangement of water molecules is distinct from, but linked by hydrogen-bonding to the well-established spine of hydration, which is displaced by bound ligands. Features of the water cluster and observed differences in binding modes help to explain the measured binding affinities and thermodynamic characteristics of these ligands on binding to AT sites in DNA.

A quadruplex sequence from the promoter region of the c-KIT gene forms a stable quadruplex, as characterized by crystallographic and NMR methods. Two new crystal structures are reported here, together with molecular dynamics simulation studies on these quadruplex crystal structures and an NMR structure. The new crystal structures, each in a distinct space group and lattice packing arrangement, together with the existing structures, demonstrate that the c-KIT quadruplex fold does not change with differing environments, suggesting that quadruplex topological dynamism is not a general phenomenon. The single and dinucleotide loops in these structures show a high degree of conformational flexibility within the three crystal forms and the NMR ensemble, with no evidence of clustering to particular conformers. This is in accord with the findings of high loop flexibility from the molecular dynamics studies. It is suggested that intramolecular quadruplexes can be grouped into two broad classes (i) those with at least one single-nucleotide loop, often showing singular topologies even though loops are highly flexible, and (ii) with all loops comprising at least two nucleotides, leading to topological dynamism. The loops can have more stable and less dynamic base-stacked secondary structures.