Amanda P. Skoumbourdis

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.

Evaluation of thieno[3,2-b]pyrrole[3,2-d]pyridazinones as activators of the tumor cell specific M2 isoform of pyruvate kinase

Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure-activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.

A Virtual Screen for Diverse Ligands : Discovery of Selective G Protein-Coupled Receptor Antagonists

Virtual screening has become a major focus of bioactive small molecule lead identification, and reports of agonists and antagonists discovered via virtual methods are becoming more frequent. G protein-coupled receptors (GPCRs) are the one class of protein targets for which success with this approach has been limited. This is likely due to the paucity of detailed experimental information describing GPCR structure and the intrinsic function-associated structural flexibility of GPCRs which present major challenges in the application of receptor-based virtual screening. Here we describe an in silico methodology that diminishes the effects of structural uncertainty, allowing for more inclusive representation of a potential docking interaction with exogenous ligands. Using this approach, we screened one million compounds from a virtual database, and a diverse subgroup of 100 compounds was selected, leading to experimental identification of five structurally diverse antagonists of the thyrotropin-releasing hormone receptors (TRH-R1 and TRH-R2). The chirality of the most potent chemotype was demonstrated to be important in its binding affinity to TRH receptors; the most potent stereoisomer was noted to have a 13-fold selectivity for TRH-R1 over TRH-R2. A comprehensive mutational analysis of key amino acid residues that form the putative binding pocket of TRH receptors further verified the binding modality of these small molecule antagonists. The described virtual screening approach may prove applicable in the search for novel small molecule agonists and antagonists of other GPCRs.

Synthesis and characterization of a BODIPY conjugate of the BCR-ABL kinase inhibitor Tasigna® (Nilotinib): Evidence for transport of Tasigna® and its fluorescent derivative by ABC drug transporters

Tasigna (Nilotinib) is a BCR-ABL kinase inhibitor recently approved by the Food and Drug Administration, which is indicated for the treatment of drug-resistant chronic myelogenous leukemia (CML). The efflux of tyrosine kinase inhibitors by ATP-binding cassette (ABC) drug transporters, which actively pump these drugs out of cells utilizing ATP as an energy source, has been linked to the development of drug resistance in CML patients. We report here the synthesis and characterization of a fluorescent derivative of Tasigna to study its interaction with two major ABC transporters, P-glycoprotein (Pgp) and ABCG2, in in vitro and ex vivo assays. A fluorescent derivative of Tasigna, BODIPY FL Tasigna, inhibited the BCR-ABL kinase activity in K562 cells and was also effluxed by Pgp- and ABCG2-expressing cells in both cultured cells and rat brain capillaries expressing Pgp and ABCG2. In addition, [(3)H]-Tasigna was found to be transported by Pgp-expressing polarized LLC-PK1 cells in a transepithelial transport assay. Consistent with these results, both Tasigna and BODIPY FL Tasigna were less effective at inhibiting the phosphorylation of Crkl (a substrate of BCR-ABL kinase) in Pgp- and ABCG2-expressing K562 cells due to their reduced intracellular concentration. Taken together, these data provide evidence that BODIPY FL Tasigna is transported by Pgp and ABCG2, and Tasigna is transported by Pgp. Further, we propose that BODIPY FL Tasigna can potentially be used as a probe for functional analysis of Pgp and ABCG2 in cancer cells and in other preclinical studies.

Exploration and optimization of substituted triazolothiadiazines and triazolopyridazines as PDE4 inhibitors.

An expansion of structure-activity studies on a series of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine PDE4 inhibitors and the introduction of a related [1,2,4]triazolo[4,3-b]pyridazine based inhibitor of PDE4 is presented. The development of SAR included strategic incorporation of known substituents on the critical catachol diether moiety of the 6-phenyl appendage on each heterocyclic core. From these studies, (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (10) and (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-[1,2,4]triazolo[4,3-b]pyridazine (18) were identified as highly potent PDE4A inhibitors. Each of these analogues was submitted across a panel of 21 PDE family members and was shown to be highly selective for PDE4 isoforms (PDE4A, PDE4B, PDE4C, PDE4D). Both 10 and 18 were then evaluated in divergent cell-based assays to assess their relevant use as probes of PDE4 activity. Finally, docking studies with selective ligands (including 10 and 18) were undertaken to better understand this chemotypes ability to bind and inhibit PDE4 selectively.

Identification of a Potent New Chemotype for the Selective Inhibition of PDE4

A series of substituted 3,6-diphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were prepared and analyzed as inhibitors of phosphodiesterase 4 (PDE4). Synthesis, structure-activity relationships, and the selectivity of a highly potent analogue against related phosphodiesterase isoforms are presented.

Synthesis and biological evaluation of analogues of the kinase inhibitor nilotinib as Abl and Kit inhibitors.

The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a-c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region.

Elucidation of the structural basis of interaction of the BCR-ABL kinase inhibitor, nilotinib (Tasigna) with the human ABC drug transporter P-glycoprotein

Elucidation of the structural basis of interaction of the BCR-ABL kinase inhibitor, nilotinib (Tasigna) with the human ABC drug transporter P-glycoprotein

Phosphodiesterase 4 inhibitors enhance sexual pleasure-seeking activity in rodents.

Pleasure-seeking deficits, including lack of libido, are a core feature of depression. Animal and preliminary clinical studies both suggest that phosphodiesterase 4 (PDE4) is a target for developing novel antidepressants. This study examined the potential involvement of PDE4 in the pathology of depression in both animal models and human postmortem brains. In humans, PDE4B and PDE4D levels were elevated in cingulate cortical tissue from individuals with major depressive disorder (MDD) compared to controls. Using the female urine smelling test (FUST), a recently refined method for monitoring sexual pleasure-seeking activity in mice, we found that icv infusion of selective potent PDE4 inhibitors enhanced sexual pleasure-seeking activity in male mice that underwent the learned helplessness or serotonin depletion paradigms. The infusion also increased sexual pleasure-seeking activity in naïve male mice. The results suggest that PDE4 may be a plausible contributor to the sexual pleasure-seeking deficits seen in depressed patients; inhibiting PDE4 may restore these deficits.

Abstract LB-224: Identification of a binding site for the tyrosine kinase inhibitor Nilotinib on the human ABC drug transporter, P-glycoprotein as determined by 3D-QSAR, molecular docking and mutational mapping

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Nilotinib (Tasigna®) is a novel tyrosine kinase inhibitor (TKI) that has been used to treat chronic phase chronic myeloid leukemia patients who develop resistance due to the emergence of point mutations within the BCR-ABL kinase domain. It is known to be transported by the ATP binding-cassette (ABC) drug efflux transporters P-glycoprotein (P-gp) and ABCG2, which not only alters the efficacy of nilotinib but might also result in lower bioavailability. Identification of a nilotinib binding conformation in the drug-binding pocket of these transporters is important to understand its interactions and can be used to design the next generation of TKIs that do not interact with ABC drug transporters. A structure-activity relationship (SAR)-based approach for nilotinib was used in this study to identify an initial pharmacophore interacting with P-gp. [125I]-Iodoarylazidoprazosin (IAAP) photolabeling and FACS-based transport of fluorescent substrates were carried out with 26 structural derivatives of nilotinib to evaluate their potency for inhibiting the binding of substrate and the transport function of P-gp. IC50 values from these assays were used for 3D pharmacophore modeling and quantitative structure-activity relationships (QSARs) to propose the spatial arrangement of chemical features that are essential for inhibitory activity of nilotinib towards P-gp. Molecular docking of nilotinib in a homology model of human P-gp was performed to identify interactions with the drug-binding pocket. This docked model was evaluated by mutational mapping studies replacing the Y307, M949 and A985 residues with Cysteine in the primary-binding site, which resulted in the loss of nilotinibs activity to inhibit substrate binding to P-gp. The orientation of nilotinib in this binding pocket was further substantiated by assessing the interaction of nilotinibs structural derivatives with P-gp using photoaffinity labeling, ATP hydrolysis and transport assays. These results demonstrate that the pyridine and pyrimidine rings in nilotinib play a key role in its interaction with the drug-binding pocket, while orientation of the imidazole ring determines the affinity of nilotinib for P-gp. These results for the first time identify the primary binding site of nilotinib in the drug-binding pocket of P-gp and can be exploited to design novel tyrosine kinase inhibitors that would not be recognized by ABC drug transporters. Citation Format: Suneet Shukla, Eduardo E. Chufan, Satyakam Singh, Amanda P. Skoumbourdis, Khyati Kapoor, Matthew B. Boxer, Damien Y. Duveau, Craig J. Thomas, Tanaji T. Talele, Suresh V. Ambudkar. Identification of a binding site for the tyrosine kinase inhibitor Nilotinib on the human ABC drug transporter, P-glycoprotein as determined by 3D-QSAR, molecular docking and mutational mapping. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-224. doi:10.1158/1538-7445.AM2014-LB-224