Shi Hua Ang

Spirotetrahydro β-carbolines (spiroindolones): a new class of potent and orally efficacious compounds for the treatment of malaria.

The antiplasmodial activity of a series of spirotetrahydro β-carbolines is described. Racemic spiroazepineindole (1) was identified from a phenotypic screen on wild type Plasmodium falciparum with an in vitro IC50 of 90 nM. Structure−activity relationships for the optimization of 1 to compound 20a (IC50 = 0.2 nM) including the identification of the active 1R,3S enantiomer and elimination of metabolic liabilities is presented. Improvement of the pharmacokinetic profile of the series translated to exceptional oral efficacy in the P. berghei infected malaria mouse model where full cure was achieved in four of five mice with three daily doses of 30 mg/kg.

Discovery of Tetrahydropyrazolopyrimidine Carboxamide Derivatives As Potent and Orally Active Antitubercular Agents

Tetrahydropyrazolo[1,5-a]pyrimidine scaffold was identified as a hit series from a Mycobacterium tuberculosis (Mtb) whole cell high through-put screening (HTS) campaign. A series of derivatives of this class were synthesized to evaluate their structure-activity relationship (SAR) and structure-property relationship (SPR). Compound 9 had a promising in vivo DMPK profile in mouse and exhibited potent in vivo activity in a mouse efficacy model, achieving a reduction of 3.5 log CFU of Mtb after oral administration to infected mice once a day at 100 mg/kg for 28 days. Thus, compound 9 is a potential candidate for inclusion in combination therapies for both drug-sensitive and drug-resistant TB.

Discovery and Optimization of a Porcupine Inhibitor

Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life. Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound O-acyltransferase that palmitoleates the Wnts and hence is essential for their secretion and function. The inhibition of PORCN could serve as a therapeutic approach for the treatment of a number of Wnt-dependent cancers. Herein, we describe the identification of a Wnt secretion inhibitor from cellular high throughput screening. Classical SAR based cellular optimization provided us with a PORCN inhibitor with nanomolar activity and excellent bioavailability that demonstrated efficacy in a Wnt-driven murine tumor model. Finally, we also discovered that enantiomeric PORCN inhibitors show very different activity in our reporter assay, suggesting that such compounds may be useful for mode of action studies on the PORCN O-acyltransferase.

Pharmacophore Model for Wnt/Porcupine Inhibitors and Its Use in Drug Design

Porcupine is a component of the Wnt pathway which regulates cell proliferation, migration, stem cell self-renewal, and differentiation. The Wnt pathway has been shown to be dysregulated in a variety of cancers. Porcupine is a membrane bound O-acyltransferase that palmitoylates Wnt. Inhibiting porcupine blocks the secretion of Wnt and effectively inhibits the Wnt pathway. Using high throughput screening, we have identified a number of novel porcupine inhibitors with diverse scaffolds. The pharmacophore requirements for our porcupine inhibitors were elucidated, and a pharmacophore model is proposed. Our compounds as well as all currently published porcupine inhibitors may be fitted to this model in low energy conformations with good superimposition of the pharmacophore elements. The model also explains the stereochemical requirements of our chiral porcupine inhibitors. The pharmacophore model was successfully used for designing 3 new series of porcupine inhibitors having a tricyclic xantine, a phtalimide, or a piperidine-maleimide scaffold.

Probing the binding mechanism of Mnk inhibitors by docking and molecular dynamics simulations.

Mitogen-activated protein kinases-interacting kinase 1 and 2 (Mnk1/2) activate the oncogene eukaryotic initiation factor 4E (eIF4E) by phosphorylation. High level of phosphorylated eIF4E is associated with various types of cancers. Inhibition of Mnk prevents eIF4E phosphorylation, making them potential therapeutic targets for cancer. Recently, we have designed and synthesized a series of novel imidazopyridine and imidazopyrazine derivatives that inhibit Mnk1/2 kinases with a potency in the nanomolar to micromolar range. In the current work we model the inhibition of Mnk kinase activity by these inhibitors using various computational approaches. Combining homology modeling, docking, molecular dynamics simulations, and free energy calculations, we find that all compounds bind similarly to the active sites of both kinases with their imidazopyridine and imidazopyrazine cores anchored to the hinge regions of the kinases through hydrogen bonds. In addition, hydrogen bond interactions between the inhibitors and the catalytic Lys78 (Mnk1), Lys113 (Mnk2) and Ser131 (Mnk1), Ser166 (Mnk2) appear to be important for the potency and stability of the bound conformations of the inhibitors. The computed binding free energies (ΔGPred) of these inhibitors are in accord with experimental bioactivity data (pIC50) with correlation coefficients (r(2)) of 0.70 and 0.68 for Mnk1 and Mnk2 respectively. van der Waals energies and entropic effects appear to dominate the binding free energy (ΔGPred) for each Mnk-inhibitor complex studied. The models suggest that the activities of these small molecule inhibitors arise from interactions with multiple residues in the active sites, particularly with the hydrophobic residues.

Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors

Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.

Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

Optimization of Selective Mitogen-Activated Protein Kinase Interacting Kinases 1 and 2 Inhibitors for the Treatment of Blast Crisis Leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of leukemic stem cells (LSCs). Inhibition of BCR-ABL1 kinase activity offers long-term relief to CML patients. However, for a proportion of them, BCR-ABL1 inhibition will become ineffective at treating the disease, and CML will progress to blast crisis (BC) CML with poor prognosis. BC-CML is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E), which renders LSCs capable of proliferating via self-renewal, oblivious to BCR-ABL1 inhibition. In vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor of MNK1/2 should reduce the level of phosphorylated eIF4E and re-sensitize LSCs to BCR-ABL1 inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure-activity relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate, ETC-206, which in combination with dasatinib prevents BC-CML LSC self-renewal in vitro and enhances dasatinib antitumor activity in vivo.

Abstract 2134: Rational design of selective MNK 1 and 2 kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia patients

The marketed BCR-ABL tyrosine kinase inhibitor (TKI), imatinib (Gleevec™) is a very successful targeted anti-cancer therapy. It has revolutionized the treatment of early stage or chronic phase (CP) chronic myeloid leukemia (CML). Unfortunately, a proportion of CP patients experience suboptimal responses to BCR-ABL TKIs, and progress to blast crisis (BC) stage of CML with poor survival rate. A potential cause of the resistance to TKI is the elevated level of phosphorylated eukaryotic initiation factor 4E (eIF4E), which has been found to be a consistent feature in patient-derived BC-CML samples. Importantly, both in vivo and in vitro studies have demonstrated that the MAP kinase-interacting serine/threonine-protein kinases 1 and 2 (MNK1/2) phosphorylate eIF4E on Ser209, and that the overexpression of eIF4E drives oncogenesis in a variety of cancers including BC-CML. Furthermore, several reports have indicated that eIF4E phosphorylation at Ser209, as well as eIF4E overexpression, is critical to tumor progression. We found that a BC-CML cell line, K562, that expresses a serine to alanine phospho-mutant at position 209 of eIF4E, shows reduced ability to form tumors in mice compared to wildtype eIF4E. In addition, our recent work has demonstrated the importance of the MNK-eIF4E axis in activating BC leukemia stem cell (LSC) function (Lim et al., PNAS18; 110(25):E2298-307, 2013). These data highlight the critical importance of MNK1/2-dependent eIF4E phosphorylation in cancer progression and maintenance, and suggests that inhibition of MNK1/2 is an attractive therapeutic approach to treat BC-CML. Consequently, we set out to identify selective inhibitors of the MNK1/2 kinases to treat BC-CML patients. Here, we report our hit finding strategy, as well as our hit to lead optimization process. Results describing structure activity relationships, pharmacokinetics properties, and biochemical characteristics of a highly specific MNK1/2 inhibitor, are presented. Our data demonstrate that drug-like molecules can be developed to potently and specifically inhibit the MNK kinases. We also show that simultaneous inhibition of MNK and BCR-ABL is effective at inhibiting BCR-ABL-driven growth and proliferation, as well as inhibiting the MNK-eIF4E-dependent self-renewal function of BC-LSCs. A combination of selective MNK and BCR-ABL inhibitors may provide clinical benefit to BC-CML patients. Citation Format: Kassoum Nacro, Haiyan Yang, Melvyn Wai Tuck Ho, Yoon Sheng Yeap, Lohitha Rao Chennamaneni, Shi Hua Ang, Eldwin Sum Wai Tan, Athisayamani Jeyaraj Duraiswamy, Sharon Lim, Boping Liu, Esther Hongqian Ong, Meng Ling Choong, Shi Jing Tai, Vithya Manoharan, Vishal Pendharkar, Lijun Ding, Yun Shan Chew, Joma Kanikadu Joy, John LW Kuan, Perlyn Z. Kwek, Anders Poulsen, May Ann Lee, Kanda Sangthongpitag, Charles Chuah, Tiong S. Ong, Jeffrey Hill, Thomas H. Keller, Alex Matter. Rational design of selective MNK 1 and 2 kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2134.