Annika Jenmalm Jensen

Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1

The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.

A non-peptide receptor inhibitor with selectivity for one of the neutrophil formyl peptide receptors, FPR 1.

The neutrophil formyl peptide receptors (FPR1 and FPR2) are members of the G-protein coupled receptor family. The signals generated by occupied FPRs are both pro-inflammatory and anti-inflammatory. Accordingly, these receptors have become a therapeutic target for the development of novel drugs that may be used to reduce injuries in inflammatory diseases including asthma, rheumatoid arthritis, Alzheimers disease and cardiovascular diseases. To support the basis for a future pharmacological characterization, we have identified a small molecular non-peptide inhibitor with selectivity for FPR1. We used the FPR1 and FPR2 specific ligands fMLF and WKYMVM, respectively, and an earlier described ratio technique, to determine inhibitory activity combined with selectivity. We show that the compound 3,5-dichloro-N-(2-chloro-5-methyl-phenyl)-2-hydroxy-benzamide (BVT173187) fulfills the criteria for an FPR1 inhibitor selective for FPR1 over FPR2, and it inhibits the same functional repertoire in neutrophils as earlier described peptide antagonists. Accordingly, the new inhibitor reduced neutrophil activation with FPR1 agonists, leading to mobilization of adhesion molecules (CR3) and the generation of superoxide anion from the neutrophil NADPH-oxidase. The effects of a number of structural analogs were determined but these were either without activity or less active/specific than BVT173187. The potency of the new inhibitor for reduction of FPR1 activity was the same as that of the earlier described FPR1 antagonist cyclosporine H, but signaling through the C5aR and CXCR (recognizing IL8) was also affected by BVT173187.

Small kinase assay panels can provide a measure of selectivity.

In this Letter, a novel strategy for assessment of ligand promiscuity is described. By using a carefully selected small set of kinases together with multivariate statistical methods, a measure of selectivity can be estimated. This will facilitate an appropriate selection of compounds for further development in lead generation and optimization.

Identification Of Akn-032, A Novel 2-Aminopyrazine Tyrosine Kinase Inhibitor, With Significant Preclinical Activity In Acute Myeloid Leukemia

Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput kinase screens. Further characterization showed inhibitory activity on FLT3 enzyme with an IC(50) of 70 nM. Western blot analysis revealed reduced autophosphorylation of the FLT3-receptor in AML cell line MV4-11 cells after exposure to AKN-032. Flow cytometry disclosed cytotoxic activity against MV4-11, but not against non-malignant 3T3-L1 fibroblast cells. Using a fluorometric microculture cytotoxicity assay, AKN-032 was tested against 15 cell lines and displayed a potent cytotoxic activity in AML cell lines MV4-11 (IC(50)=0.4 μM) and Kasumi-1 (IC(50)=2.3 μM). AKN-032 was also highly cytotoxic in tumor cells from AML patients in vitro. Furthermore, AKN-032 demonstrated significant antileukemic effect in a relatively resistant in vivo hollow fiber mouse model. No major toxicity was observed in the animals. In conclusion, AKN-032 is a promising new kinase inhibitor with significant in vivo and in vitro activity in AML. Results from the hollow fiber mouse assay suggest a favorable toxicity profile. Future studies will focus on pharmacokinetic properties, toxicity as well as further clarifying the mechanisms of action of AKN-032 in AML.

Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP Pyrophosphatase 1

The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit-to-lead development.

Membrane-depolarizing channel blockers induce selective glioma cell death by impairing nutrient transport and unfolded protein/amino acid responses.

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741-52. ©2017 AACR.

Chemical Biology Consortium Sweden.

Fredrik Almqvist (a), Per Artursson (b), Hanna Axelsson (c), Marcus Carlsson (a) Inger Cullman (a), Mikael Elofsson (a), Per-Anders Enquist (a), Jonas Eriksson (a), Anders Esberg (a), Anna-Lena Gustavsson (c), Lars Hammarstrom (c), Martin Haraldsson (c), Annika Jenmalm Jensen* (c), Lars Johansson (c), Lucia Lazorova (b), Thomas Lundback (c), Maria Mastej (b), Weixing Qian (a), Kristmundur Sigmundsson (c), Richard Svensson (b), Hanna Uvell (a), Anders Ohman (a) (a)Laboratories for Chemical Biology Umea, Department of Chemistry, Umea University, SE90187 Umea, Sweden. (b) The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Department of Pharmacy, Uppsala University, SE75123 Uppsala, Sweden. (c) Laboratories for Chemical Biology Karolinska Institutet, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE17177 Stockholm, Sweden. E-mail: Annika.Jenmalm-Jensen@ki.se

Diverse heterocyclic scaffolds as dCTP pyrophosphatase 1 inhibitors. Part 2: Pyridone- and pyrimidinone-derived systems

Two screening campaigns using commercial (Chembridge DiverSET) and proprietary (Chemical Biology Consortium Sweden, CBCS) compound libraries, revealed a number of pyridone- and pyrimidinone-derived systems as inhibitors of the human dCTP pyrophosphatase 1 (dCTPase). In this letter, we present their preliminary structure-activity-relationships (SAR) and ligand efficiency scores (LE and LLE).

Abstract 5509: A drug-screening model to identify compounds active in cells under metabolic stress

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background and aim: Promising anticancer compounds often fail in vivo due to lack of efficacy. Drug screenings to identify anticancer agents are usually performed in culture conditions that very poorly represent the complex metabolic tumor environment. Tumor tissues are characterised by low oxygen (hypoxia) and acidic extracellular pH (acidosis). A screen of a drug-library for compounds able to induce cell death in acidic/hypoxic conditions may lead to discover effective drugs targeting quiescent/hypoxic cells, normally considered responsible for tumor relapses after therapy. Methods: The colon carcinoma cell line HCT-116 has been adapted to grow in low pH conditions (pH 6.8). The Prestwick compound library (1200 FDA-approved compounds) was used to validate the assay. Cell viability was measured using the acid phosphatase assay after 48 hours exposure to test compounds. The assay was validated in HCT-116 cells cultured in normoxic and hypoxic conditions. Multicellular spheroids (MCS) were used to evaluate the sensitivity to hit compounds confirmed at least three times in 2D culture conditions. Results: The assay was optimized to perform the screening at pH 6.8 in normoxic and hypoxic conditions. Experiments have demonstrated the ability of cells to survive and grow in hypoxic conditions (1% oxygen) for at least 4 days. The test was optimized in terms of number of plated cells and serum concentration. Hit compounds were identified based on the mean values for quality control plus 3 standard deviations of untreated samples. The screen led to identify 20 hits for both conditions and 11 hits were further confirmed for hypoxic/acidic conditions. Auranofin and Verteporfin showed to better induce cell death in cells adapted or acutely exposed to low pH compared to parental cells and immortalized epithelial cells (RPE1). Both drugs showed cytotoxic effects on cells grown as MCS, suggesting a potential good efficacy of the therapeutic agent in vivo. In addition, the screening of about 10000 compounds of the LCBKI library is ongoing and about 100 hits have been identified. Conclusions: The screening validation on the Prestwick library will hopefully lead to identify potential “off-label” effects of already approved drugs. The screening of larger drug-libraries will hopefully lead to find and characterize more compounds able to target slow proliferating and therapy resistant cells. Citation Format: Paola Pellegrini, Martin Haraldsson, Annika Jenmalm Jensen, Thomas Lundback, Angelo De Milito. A drug-screening model to identify compounds active in cells under metabolic stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5509. doi:10.1158/1538-7445.AM2015-5509