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.

Co-and post-translational processing are crucial maturation steps to generate functional proteins. MetAP-2 plays an important role in this process, and inhibition of its proteolytic activity has been shown to be important for angiogenesis and tumor growth, suggesting that small-molecule inhibitors of MetAP-2 may be promising options for the treatment of cancer. This work describes the discovery and structurebased hit optimization of a novel MetAP-2 inhibitory scaffold. Of critical importance, a cyclic tartronic diamide coordinates the MetAP-2 metal ion in the active site while additional side chains of the molecule were designed to occupy the lipophilic methionine side chain recognition pocket as well as the shallow cavity at the opening of the active site. The racemic screening hit from HTS campaign 11a was discovered with an enzymatic IC 50 of 150 nM. The resynthesized eutomer confirmed this activity and inhibited HUVEC proliferation with an IC 50 of 1.9 μM. Its structural analysis revealed a sophisticated interaction pattern of polar and lipophilic contacts that were used to improve cellular potency to an IC 50 of 15 nM. In parallel, the molecular properties were optimized on plasma exposure and antitumor efficacy which led to the identification of advanced lead 21.

The recently disclosed next generation of reversible, selective, and potent MetAP-2 inhibitors introduced a cyclic tartronic diamide scaffold. However, the lead compound 1a suffered from enterohepatic circulation, preventing further development. Nevertheless, 1a served as a starting point for further optimization. Maintaining potent antiproliferation activity, while improving other compound properties, enabled the generation of an attractive array of new MetAP-2 inhibitors. The most promising derivatives were identified by a multiparameter analysis of the compound properties. Essential for the efficient selection of candidates with in vivo activity was the identification of molecules with a long residence time on the target protein, high permeability, and low efflux ratio not only in Caco-2 but also in the MDR-MDCK cell line. Orally bioavailable, potent, and reversible MetAP-2 inhibitors impede the growth of primary endothelial cells and demonstrated antitumoral activity in mouse models. This assessment led to the nomination of the clinical development compound M8891, which is currently in phase I clinical testing in oncology patients.

Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (β1, β2, and β5). LMP7 (β5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.

New phenoxyacetic acid antagonists of CRTH2 are described. Following the discovery of a hit compound by a focused screening, high protein binding was identified as its main weakness. Optimization aimed at reducing serum protein binding led to the identification of several compounds that showed not only excellent affinities for the receptor (41 compounds with K(i) < 10 nM) but also excellent potencies in a human whole blood assay (IC(50) < 100 nM; PGD2-induced eosinophil shape change). Additional optimization of the PK characteristics led to the identification of several compounds suitable for in vivo testing. Of these, 19k and 19s were tested in two different pharmacological models (acute FITC-mediated contact hypersensitivity and ovalbumin-induced eosinophilia models) and found to be active after oral dosing (10 and 30 mg/kg).