Johanna M. Jansen

Design and Synthesis of Orally Bioavailable Benzimidazoles as Raf Kinase Inhibitors

A series of arylaminobenzimidazoles was designed and synthesized as Raf kinase inhibitors. Exploration of the structure-activity relationship resulted in compounds that are potent in vitro and show desirable in vivo properties.

Design and synthesis of 5,6-fused heterocyclic amides as Raf kinase inhibitors.

Two scaffolds based on 5,6-fused heterocyclic backbones were designed and synthesized as Raf kinase inhibitors. The scaffolds were assessed for in vitro pan-Raf inhibition, activity in cell proliferation and target modulation assays, and pharmacokinetic parameters.

Design and Discovery of N-(2-Methyl-5′-morpholino-6′-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3′-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

Inhibition of prenylated KRAS in a lipid environment

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

Teach-Discover-Treat (TDT): collaborative computational drug discovery for neglected diseases.

Teach-Discover-Treat (TDT) is an initiative to promote the development and sharing of computational tools solicited through a competition with the aim to impact education and collaborative drug discovery for neglected diseases. Collaboration, multidisciplinary integration, and innovation are essential for successful drug discovery. This requires a workforce that is trained in state-of-the-art workflows and equipped with the ability to collaborate on platforms that are accessible and free. The TDT competition solicits high quality computational workflows for neglected disease targets, using freely available, open access tools.

Synthesis, Binding Mode, and Antihyperglycemic Activity of Potent and Selective (5-Imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl]amine Inhibitors of Glycogen Synthase Kinase 3.

In an effort to identify new antidiabetic agents, we have discovered a novel family of (5-imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl]amine analogues which are inhibitors of human glycogen synthase kinase 3 (GSK3). We developed efficient synthetic routes to explore a wide variety of substitution patterns and convergently access a diverse array of analogues. Compound 1 (CHIR-911, CT-99021, or CHIR-73911) emerged from an exploration of heterocycles at the C-5 position, phenyl groups at C-4, and a variety of differently substituted linker and aminopyridine moieties attached at the C-2 position. These compounds exhibited GSK3 IC50s in the low nanomolar range and excellent selectivity. They activate glycogen synthase in insulin receptor-expressing CHO-IR cells and primary rat hepatocytes. Evaluation of lead compounds 1 and 2 (CHIR-611 or CT-98014) in rodent models of type 2 diabetes revealed that single oral doses lowered hyperglycemia within 60 min, enhanced insulin-stimulated glucose transport, and improved glucose disposal without increasing insulin levels.

Computational chemistry and drug discovery: a call to action

The practitioners of computational chemistry devoted to drug discovery use computational approaches to unravel complex relationships in chemical and biological datasets. They also use computational tools to design chemical matter using intricate multi-objective optimization methods. In both applications, the discipline formulates hypotheses that inspire a next round of experiments. The impact from these approaches is critically dependent on productive relationships across multiple disciplines for generating and annotating data, as well as for the required follow-up on the hypotheses. Numerous examples of drugs that have been discovered and optimized with contributions from computational chemists exist [1]. Computational methods provide guidance but are by no means able to make perfect predictions. It is, therefore, important that we share details of our work so that as a community we can learn from what works, as well as what fails. To increase the impact of our computational drug-discovery efforts, we present a call to action to our scientific community. This call to action is embodied by the TeachDiscover-Treat (TDT) initiative [2,101]. �ur ultimate goals include strengthening the education of drug-discovery scientists across disciplines and increasing collaboration and scientific rigor within the computational chemistry discipline.

Virtual-screening workflow tutorials and prospective results from the Teach-Discover-Treat competition 2014 against malaria

The first challenge in the 2014 competition launched by the Teach-Discover-Treat (TDT) initiative asked for the development of a tutorial for ligand-based virtual screening, based on data from a primary phenotypic high-throughput screen (HTS) against malaria. The resulting Workflows were applied to select compounds from a commercial database, and a subset of those were purchased and tested experimentally for anti-malaria activity. Here, we present the two most successful Workflows, both using machine-learning approaches, and report the results for the 114 compounds tested in the follow-up screen. Excluding the two known anti-malarials quinidine and amodiaquine and 31 compounds already present in the primary HTS, a high hit rate of 57% was found.

Imidazo[1,2-a]pyridin-6-yl-benzamide Analogs as Potent RAF Inhibitors

A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAFV600E. Medicinal chemistry techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.

Abstract B38: Inhibiting mutated KRAS, a broken switch of effector pathways

Mutated forms of KRAS are no longer able to switch effectors between “on” and “off” states. It is known that the function of KRAS is controlled by key parts in the C-terminus, including six consecutive lysines, a terminal prenyl moiety and a terminal carboxymethyl functional group. We set out to discover compounds which would inhibit the function of mutated KRAS as an activator for effectors. This campaign yielded several compounds that blocked biochemical and cellular functions of KRAS with low micromolar activity while not affecting markers outside of KRAS pathways in cells. In order to understand the mode of binding of these compounds to KRAS, we generated different forms of the protein, including unprenylated truncated and fully processed full-length protein. NMR studies with truncated protein (amino acids 1-169) identified a site at which compound binding stabilized the inactive conformation of KRAS. This site is located adjacent to switch-II and is similar to sites described by others. The Kd determined for this binding event is almost 3 orders of magnitude higher than the IC50 and EC50 values measured in biochemical and cellular assays. In order to understand this difference, we developed a biophysical assay using the Fortebio system which enabled binding studies in a system with full-length prenylated protein in the presence of lipids, to match the context of the biochemical and cellular assays. Micromolar binding to the full-length prenylated KRAS protein was observed in the Fortebio assay and binding was not observed in the absence of prenylation, consistent with the near millimolar Kd observed by NMR for truncated KRAS. Curiously, similar micromolar binding was seen to a peptide derived from the C-terminus of KRAS (amino acids 168-185) with and without prenyl modification while related compounds that do not bind to the full-length prenylated KRAS also do not bind to these peptides. It is still unclear whether binding to the terminal peptide in lipid context is related to the binding site adjacent to switch-II. From a drug discovery perspective, it remains to be confirmed whether current inhibitors can be optimized. Citation Format: Johanna Jansen, Wolfgang Jahnke, Susan Fong, Laura Tandeske, Charles Wartchow, Keith Pfister, Tatiana Zavorotinskaya, Anke Blechschmidt, Dirksen Bussiere, Yumin Dai, Jeff Dove, Eric Fang, David Farley, Jean-Michel Florent, John Fuller, Simona Gokhin, Alvar Gossert, Mohammad Hekmat-Nejad, Chrystele Henry, Julia Klopp, Bill Lenahan, Andreas Lingel, Arndt Meyer, Jamie Narberes, Gwynn Pardee, C Gregory Paris, Savithri Ramurthy, Paul Renhowe, Sebastien Rieffel, Kevin Shoemaker, Sharadha Subramanian, Tiffany Tsang, Stephania Widger, Armin Widmer, Isabel Zaror, Stephen Hardy. Inhibiting mutated KRAS, a broken switch of effector pathways. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B38. doi: 10.1158/1557-3125.RASONC14-B38