Discovery and Optimization of 2H-1λ2-Pyridin-2-one Inhibitors of Mutant Isocitrate Dehydrogenase 1 for the Treatment of Cancer.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are oncogenic for a number of malignancies, primarily low-grade gliomas and acute myeloid leukemia. We report a medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-2H-1λ2-quinoline-2,5(6H)-dione screening hit against the R132H and R132C mutant forms of isocitrate dehydrogenase (IDH1). Systematic SAR efforts produced a series of potent pyrid-2-one mIDH1 inhibitors, including the atropisomer (+)-119 (NCATS-SM5637, NSC 791985). In an engineered mIDH1-U87-xenograft mouse model, after a single oral dose of 30 mg/kg, 16 h post dose, between 16 and 48 h, (+)-119 showed higher tumoral concentrations that corresponded to lower 2-HG concentrations, when compared with the approved drug AG-120 (ivosidenib).

Assessing inhibitors of mutant isocitrate dehydrogenase using a suite of pre-clinical discovery assays.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2). We present a comprehensive suite of in vitro preclinical drug development assays that can be used as a tool-box to identify lead compounds for mIDH drug discovery programs, as well as what we believe is the most comprehensive publically available dataset on the top mIDH inhibitors. This involved biochemical, cell-based, and tier-one ADME techniques.

Glycoconjugated Site-Selective DNA-Methylating Agent Targeting Glucose Transporters on Glioma Cells.

DNA-alkylating drugs continue to remain an important weapon in the arsenal against cancers. However, they typically suffer from several shortcomings because of the indiscriminate DNA damage that they cause and their inability to specifically target cancer cells. We have developed a strategy for overcoming the deficiencies in current DNA-alkylating chemotherapy drugs by designing a site-specific DNA-methylating agent that can target cancer cells because of its selective uptake via glucose transporters, which are overexpressed in most cancers. The design features of the molecule, its synthesis, its reactivity with DNA, and its toxicity in human glioblastoma cells are reported here. In this molecule, a glucosamine unit, which can facilitate uptake via glucose transporters, is conjugated to one end of a bispyrrole triamide unit, which is known to bind to the minor groove of DNA at A/T-rich regions. A methyl sulfonate moiety is tethered to the other end of the bispyrrole unit to serve as a DNA-methylating agent. This molecule produces exclusively N3-methyladenine adducts upon reaction with DNA and is an order of magnitude more toxic to treatment resistant human glioblastoma cells than streptozotocin is, a Food and Drug Administration-approved, glycoconjugated DNA-methylating drug. Cellular uptake studies using a fluorescent analogue of our molecule provide evidence of uptake via glucose transporters and localization within the nucleus of cells. These results demonstrate the feasibility of our strategy for developing more potent anticancer chemotherapeutics, while minimizing common side effects resulting from off-target damage.

Discovery of Macrocyclic Pyrimidines as MerTK-Specific Inhibitors.

Macrocycles have attracted significant attention in drug discovery recently. In fact, a few de novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure-based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MerTK)-specific inhibitors. An enzyme-linked immunosorbent assay (ELISA) in 384-well format was employed to evaluate the inhibitory activity of macrocycles in a cell-based assay assessing tyrosine phosphorylation of MerTK. Through structure-activity relationship (SAR) studies, analogue 11 [UNC2541; (S)-7-amino-N-(4-fluorobenzyl)-8-oxo-2,9,16-triaza-1(2,4)-pyrimidinacyclohexadecaphane-1-carboxamide] was identified as a potent and MerTK-specific inhibitor that exhibits sub-micromolar inhibitory activity in the cell-based ELISA. In addition, an X-ray structure of MerTK protein in complex with 11 was resolved to show that these macrocycles bind in the MerTK ATP pocket.

Discovery of Mer specific tyrosine kinase inhibitors for the treatment and prevention of thrombosis.

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.

Hydrogen peroxide induced activation of gene expression in mammalian cells using boronate estrone derivatives.

Keeping the boron out of the ER: A genetic switch was engineered that activates gene expression in the presence of H(2)O(2). The use of a boronate group on an estrone molecule allows for activation of gene expression through binding of the estrogen receptor only when the boron group is oxidized by H(2)O(2). This sensor is highly sensitive and specific for H(2)O(2).

Stabilization and photochemical regulation of antisense agents through PEGylation.

Oligonucleotides are effective tools for the regulation of gene expression in cell culture and model organisms, most importantly through antisense mechanisms. Due to the inherent instability of DNA antisense agents, various modifications have been introduced to increase the efficacy of oligonucleotides, including phosphorothioate DNA, locked nucleic acids, peptide nucleic acids, and others. Here, we present antisense agent stabilization through conjugation of a poly(ethylene glycol) (PEG) group to a DNA oligonucleotide. By employing a photocleavable linker between the PEG group and the antisense agent, we were able to achieve light-induced deactivation of antisense activity. The bioconjugated PEG group provides stability to the DNA antisense agent without affecting its native function of silencing gene expression via RNase H-catalyzed mRNA degradation. Once irradiated with UV light of 365 nm, the PEG group is cleaved from the antisense agent leaving the DNA unprotected and open for degradation by endogenous nucleases, thereby restoring gene expression. By using a photocleavable PEG group (PhotoPEG), antisense activity can be regulated with high spatial and temporal resolution, paving the way for precise regulation of gene expression in biological systems.

Heterotaxin: a TGF-ß signaling inhibitor identified in a multi-phenotype profiling screen in Xenopus embryos.

Disruptions of anatomical left-right asymmetry result in life-threatening heterotaxic birth defects in vital organs. We performed a small molecule screen for left-right asymmetry phenotypes in Xenopus embryos and discovered a pyridine analog, heterotaxin, which disrupts both cardiovascular and digestive organ laterality and inhibits TGF-ß-dependent left-right asymmetric gene expression. Heterotaxin analogs also perturb vascular development, melanogenesis, cell migration, and adhesion, and indirectly inhibit the phosphorylation of an intracellular mediator of TGF-ß signaling. This combined phenotypic profile identifies these compounds as a class of TGF-ß signaling inhibitors. Notably, heterotaxin analogs also possess highly desirable antitumor properties, inhibiting epithelial-mesenchymal transition, angiogenesis, and tumor cell proliferation in mammalian systems. Our results suggest that assessing multiple organ, tissue, cellular, and molecular parameters in a whole organism context is a valuable strategy for identifying the mechanism of action of bioactive compounds.

Photocleavable polyethylene glycol for the light-regulation of protein function.

PEGylation is commonly employed to enhance the pharmacokinetic properties of proteins, but it can interfere with natural protein function. Protein activity can thus be abrogated through PEGylation, and a controllable means to remove the polyethylene glycol (PEG) group from the protein is desirable. As such, light affords a unique control over biomolecules through the application of photosensitive groups. Herein, we report the synthesis of a photocleavable PEG reagent (PhotoPEG) and its application to the light-regulation of enzyme activity.

Tricyclic alkaloid core structures assembled by a cyclotrimerization-coupled intramolecular nucleophilic substitution reaction.

A facile approach to tricyclic alkaloid core structures was developed by sequencing a pyridine-forming [2 + 2 + 2] cyclotrimerization reaction with an intramolecular nucleophilic substitution. This methodology enabled the facile assembly of the spiroindolinone framework of citrinadins A and B, and cyclopiamine B.

A general approach to triphenylenes and azatriphenylenes: total synthesis of dehydrotylophorine and tylophorine.

A convergent and flexible synthesis of substituted triphenylenes, azatriphenylenes, and the cytotoxic alkaloids dehydrotylophorine and tylophorine has been developed.