The nuclease hypersensitivity element III1 (NHE III1) upstream of the P1 and P2 promoters of c-MYC controls 80-90% of the transcriptional activity of this gene. The purine-rich strand in this region can form a G-quadruplex structure that is a critical part of the silencer element for this promoter. We have demonstrated that this G-quadruplex structure can form a mixture of four biologically relevant parallel-loop isomers, which upon interaction with the cationic porphyrin TMPyP4 are converted to mixed parallel/antiparallel G-quadruplex structures.

The human telomeric sequence d[T(2)AG(3)](4) has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na(+)), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K(+)). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K(+) or Na(+). In contrast, in the presence of K(+), Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures.

Cationic porphyrins are known to bind to and stabilize different types of G-quadruplexes. Recent studies have shown the biological relevance of the intramolecular parallel G-quadruplex as a transcriptional silencer in the c-MYC promoter. TMPyP4 also binds to this G-quadruplex and most likely converts it to a mixed parallel/antiparallel G-quadruplex with two external lateral loops and one internal propeller loop, suppressing c-MYC transcriptional activation. To achieve therapeutic selectivity by targeting G-quadruplexes, it is necessary to synthesize drugs that can differentiate among the different types of G-quadruplexes. We have designed and synthesized a core-modified expanded porphyrin analogue, 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28-diselenasapphyrin chloride (Se2SAP). Se2SAP converts the parallel c-MYC G-quadruplex into a mixed parallel/antiparallel G-quadruplex with one external lateral loop and two internal propeller loops, resulting in strong and selective binding to this G-quadruplex. A Taq polymerase stop assay was used to evaluate the binding of TMPyP4 and Se2SAP to G-quadruplex DNA. Compared to TMPyP4, Se2SAP shows a greater selectivity for and a 40-fold increase in stabilization of the single lateral-loop hybrid. Surface plasmon resonance and competition experiments with duplex DNA and other G-quadruplexes further confirmed the selectivity of Se2SAP for the c-MYC G-quadruplex. Significantly, Se2SAP was found to be less photoactive and noncytotoxic in comparison to TMPyP4. From this study, we have identified an expanded porphyrin that selectively binds with the c-MYC G-quadruplex in the presence of duplex DNA and other G-quadruplexes.

Focal adhesion kinase (FAK) is considered as an attractive target for oncology, and small-molecule inhibitors are reported to be in clinical testing. In a surface plasmon resonance (SPR)-mediated fragment screening campaign, we discovered bicyclic scaffolds like 1H-pyrazolo[3,4-d]pyrimidines binding to the hinge region of FAK. By an accelerated knowledge-based fragment growing approach, essential pharmacophores were added. The establishment of highly substituted unprecedented 1H-pyrrolo[2,3-b]pyridine derivatizations provided compounds with submicromolar cellular FAK inhibition potential. The combination of substituents on the bicyclic templates and the nature of the core structure itself have a significant impact on the compounds FAK selectivity. Structural analysis revealed that the appropriately substituted pyrrolo[2,3-b]pyridine induced a rare helical DFG-loop conformation. The discovered synthetic route to introduce three different substituents independently paves the way for versatile applications of the 7-azaindole core.