Hugo Albrecht

Sulfonylureas and glinides exhibit peroxisome proliferator-activated receptor gamma activity : a combined virtual screening and biological assay approach

Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target the peroxisome proliferator-activated receptor (PPARγ) improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides additionally bind to PPARγ and exhibit PPARγ agonistic activity. This activity was predicted in silico by virtual screening and confirmed in vitro in a binding assay, a transactivation assay, and by measuring the expression of PPARγ target genes. Among the measured compounds, gliquidone and glipizide (two sulfonylureas), as well as nateglinide (a glinide), exhibit PPARγ agonistic activity at concentrations comparable with those reached under pharmacological treatment. The most active of these compounds, gliquidone, is shown to be as potent as pioglitazone at inducing PPARγ target gene expression. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, because it provides evidence that drugs can be designed that target both the sulfonylurea receptor and PPARγ. Targeting both receptors could increase pancreatic insulin secretion and improve insulin resistance. Glinides, sulfonylureas, and other acidified sulfonamides may be promising leads in the development of new PPARγ agonists. In addition, we provide a unified concept of the PPARγ binding ability of seemingly disparate compound classes.

Discovery of 5‐(2‐(Phenylamino)pyrimidin‐4‐yl)thiazol‐2(3H)‐one Derivatives as Potent Mnk2 Inhibitors: Synthesis, SAR Analysis and Biological Evaluation

Phosphorylation of eIF4E by human mitogen‐activated protein kinase (MAPK)‐interacting kinases (Mnks) is crucial for human tumourigenesis and development. Targeting Mnks may provide a novel anticancer therapeutic strategy. However, the lack of selective Mnk inhibitors has so far hampered pharmacological target validation and clinical drug development. Herein, we report, for the first time, the discovery of a series of 5‐(2‐(phenylamino)pyrimidin‐4‐yl)thiazole‐2(3H)‐one derivatives as Mnk inhibitors. Several derivatives demonstrate very potent Mnk2 inhibitory activity. The most active and selective compounds were tested against a panel of cancer cell lines, and the results confirm the cell‐type‐specific effect of these Mnk inhibitors. Detailed cellular mechanistic studies reveal that Mnk inhibitors are capable of reducing the expression level of anti‐apoptotic protein Mcl‐1, and of promoting apoptosis in MV4‐11 acute myeloid leukaemia cells.

Use of FLIPR Membrane Potential Dyes for Validation of High-Throughput Screening with the FLIPR and µARCS Technologies: Identification of Ion Channel Modulators Acting on the GABAA Receptor

Fluorometric imaging plate reader (FLIPR) membrane potential dyes (FMP-Red-Dye and FMP-Blue-Dye) were evaluated for the detection of compounds acting either as positive allosteric modulators or agonists on the GABAA receptor (GABAAR). A stable HEK293 cell line with constitutive expression of the rat GABA AR α1, β2, and γ2 genes was used to establish a functional high-throughput screening (HTS) assay based on measurement of the membrane potential change in living cells. The assay was validated with the FLIPR technology for identification of agonists and positive allosteric modulators using GABA and diazepam as model compounds. The FMP-Red-Dye showed better performance than the FMP-Blue-Dye, and the effects induced by GABA and diazepam were comparable to electrophysiology data. Subsequently, the assay was also validated with an ultra-HTS approach known as microarrayed compound screening (µARCS). The LOPAC library was used in a test screen for an initial assessment of the technology. Finally, the FLIPR and µARCS technologies were tested with a larger screening campaign. A focused library of 3520 putative positive modulators was tested with the FLIPR assay, and a diverse subset of 84,480 compounds was selected for screening with the µARCS technology. All hits were subjected to verification using the FLIPR technology, and confirmed hits were subsequently evaluated by EC50 determination. Finally, selected hits were further confirmed with electrophysiology testing. (Journal of Biomolecular Screening 2008:218-228)

Discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]pyrimidine derivatives as potent Mnk inhibitors: synthesis, structure-activity relationship analysis and biological evaluation.

Phosphorylation of the eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks) is essential for oncogenesis but unnecessary for normal development. Thus, pharmacological inhibition of Mnks may offer an effective and non-toxic anti-cancer therapeutic strategy. Herein, we report the discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]pyrimidine derivatives as potent Mnk inhibitors. Docking study of 7a in Mnk2 suggests that the compound is stabilised in the ATP binding site through multiple hydrogen bonds and hydrophobic interaction. Cellular mechanistic studies on MV-4-11 cells with leads 7a, 8e and 8f reveal that they are able to down-regulate the phosphorylated eIF4E, Mcl-1 and cyclin D1, and induce apoptosis.

Targeting CDK9: a promising therapeutic opportunity in prostate cancer

Cyclin-dependent kinase 9 (CDK9) is a key transcriptional regulator and a lucrative target for cancer treatment. Targeting CDK9 can effectively confine the hyperactivity of androgen receptor and the constitutive expression of anti-apoptotic proteins; both being main causes of prostate cancer (PCa) development and progression. In castrate-resistant PCa, traditional therapies that only target androgen receptor (AR) have become obsolete due to reprograming in AR activity to make the cells independent of androgen. CDK9 inhibitors may provide a new and better therapeutic opportunity over traditional treatment options by targeting both androgen receptor activity and anti-apoptotic proteins, improving the chances of positive outcomes, especially in patients with the advanced disease. This review focuses on biological functions of CDK9, its involvement with AR and the potential for therapeutic opportunities in PCa treatment.

Pharmacologic Inhibition of MNKs in Acute Myeloid Leukemia

The Ras/Raf/MAPK and PI3K/Akt/mTOR pathways are key signaling cascades involved in the regulation of cell proliferation and survival, and have been implicated in the pathogenesis of several types of cancers, including acute myeloid leukemia (AML). The oncogenic activity of eIF4E driven by the Mnk kinases is a convergent determinant of the two cascades, suggesting that targeting the Mnk/eIF4E axis may provide therapeutic opportunity for the treatment of cancer. Herein, a potent and selective Mnk2 inhibitor (MNKI-85) and a dual-specific Mnk1 and Mnk2 inhibitor (MNKI-19), both derived from a thienopyrimidinyl chemotype, were selected to explore their antileukemic properties. MNKI-19 and MNKI-85 are effective in inhibiting the growth of AML cells that possess an M5 subtype with FLT3-internal tandem duplication mutation. Further mechanistic studies show that the downstream effects with respect to the selective Mnk1/2 kinase inhibition in AML cells causes G1 cell cycle arrest followed by induction of apoptosis. MNKI-19 and MNKI-85 demonstrate similar Mnk2 kinase activity and cellular antiproliferative activity but exhibit different time-dependent effects on cell cycle progression and apoptosis. Collectively, this study shows that pharmacologic inhibition of both Mnk1 and Mnk2 can result in a more pronounced cellular response than targeting Mnk2 alone. However, MNKI-85, a first-in-class inhibitor of Mnk2, can be used as a powerful pharmacologic tool in studying the Mnk2/eIF4E-mediated tumorigenic mechanism. In conclusion, this study provides a better understanding of the mechanism underlying the inhibition of AML cell growth by Mnk inhibitors and suggests their potential utility as a therapeutic agent for AML.

An integrated approach for discovery of highly potent and selective Mnk inhibitors: Screening, synthesis and SAR analysis.

Deregulation of protein synthesis is a common event in cancer. As MAPK-interacting kinases (Mnks) play critical roles in regulation of protein synthesis, they have emerged as novel anti-cancer targets. Mnks phosphorylate eukaryotic initiation factor 4E (eIF4E) and promote eIF4E-mediated oncogenic activity. Given that the kinase activity of Mnks is essential for oncogenesis but is dispensable for normal development, the discovery of potent and selective pharmacological Mnk inhibitors provides pharmacological target validation and offers a new strategy for cancer treatment. Herein, comprehensive in silico screening approaches were deployed, and three thieno[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine derivatives were identified as hit compounds. Further chemical modification of thieno[2,3-d]pyrimidine derivative 3 has given rise to a series of highly potent Mnk2 inhibitors that could be potential leads for the treatment of acute myeloid leukemia.

Highly Potent, Selective, and Orally Bioavailable 4-Thiazol-N-(pyridin-2-yl)pyrimidin-2-amine Cyclin-Dependent Kinases 4 and 6 Inhibitors as Anticancer Drug Candidates: Design, Synthesis, and Evaluation

Cyclin D dependent kinases (CDK4 and CDK6) regulate entry into S phase of the cell cycle and are validated targets for anticancer drug discovery. Herein we detail the discovery of a novel series of 4-thiazol-N-(pyridin-2-yl)pyrimidin-2-amine derivatives as highly potent and selective inhibitors of CDK4 and CDK6. Medicinal chemistry optimization resulted in 83, an orally bioavailable inhibitor molecule with remarkable selectivity. Repeated oral administration of 83 caused marked inhibition of tumor growth in MV4-11 acute myeloid leukemia mouse xenografts without having a negative effect on body weight and showing any sign of clinical toxicity. The data merit 83 as a clinical development candidate.

Identification of a Highly Conserved Allosteric Binding Site on Mnk1 and Mnk2.

Elevated levels of phosphorylated eukaryotic initiation factor 4E (eIF4E) have been implicated in many tumor types, and mitogen activated protein kinase-interacting kinases (Mnks) are the only known kinases that phosphorylate eIF4E at Ser209. The phosphorylation of eIF4E is essential for oncogenic transformation but is of no significance to normal growth and development. Pharmacological inhibition of Mnks therefore provides a nontoxic and effective strategy for cancer therapy. However, a lack of specific Mnk inhibitors has confounded pharmacological target validation and clinical development. Herein, we report the identification of a novel series of Mnk inhibitors and their binding modes. A systematic workflow has been established to distinguish between type III and type I/II inhibitors. A selection of 66 compounds was tested for Mnk1 and Mnk2 inhibition, and 9 out of 20 active compounds showed type III interaction with an allosteric site of the proteins. Most of the type III inhibitors exhibited dual Mnk1 and Mnk2 activities and demonstrated potent antiproliferative properties against the MV4-11 acute myeloid leukemia cell line. Interestingly, ATP-/substrate-competitive inhibitors were found to be highly selective for Mnk2, with little or no activity for Mnk1. Our study suggests that Mnk1 and Mnk2 share a common structure of the allosteric inhibitory binding site but possess different structural features of the ATP catalytic domain. The findings will assist in the future design and development of Mnk targeted anticancer therapeutics.

Inhibition of Mnk enhances apoptotic activity of cytarabine in acute myeloid leukemia cells

Cytarabine (Ara-C) is a first line clinical therapeutic agent for treatment of acute myeloid leukemia (AML). However, this therapy is limited due to high rate of resistance and relapse. Recent research has revealed that the poor prognosis and resistance to Ara-C in AML were associated with its abnormally activated MAPK pathways. In this study, we showed a strong synergistic effect of Ara-C with either our Mnk inhibitor (MNKI-8e) or short hairpin RNA (shRNA) mediated knockdown of Mnks in MV4-11 AML cells. We investigated the underlying mechanisms for this synergism. We showed that both MNKI-8e and Mnk shRNAs enhanced the ability of Ara-C to induce apoptosis. We found that Ara-C increased the phosphorylation of Erk1/2, p38 and eIF4E, which correlated with an enhanced level of anti-apoptotic Mcl-1 protein. Inhibition of Mnk activity suppressed the Ara-C-induced MAPK activity, and thus enhanced apoptosis in MV4-11 cells. Taken together, our study suggests that MAPK-Mnk-eIF4E pathway plays a critical role in Ara-C-treated MV4-11 cells and targeting Mnk may be a promising therapeutic strategy for sensitizing leukemic cells to Ara-C therapy.