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The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 A resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinson’s disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis. A high throughput screen of our compound collection afforded a number of promising indazole leads which were truncated in order to identify a minimum pharmacophore. Further optimization of these indazoles led to the development of MLi-2 (1): a potent, highly selective, orally available, brain-penetrant inhibitor of LRRK2.

Novel tricyclic derivatives containing an oxazepine, thiazepine, or diazepine ring were studied for their EGFR tyrosine kinase inhibitory activity. While the oxazepines were in general more potent than thiazepines, the diazepines displayed somewhat different structure–activity relationships. Moreover, the diazepines, in contrast to the oxazepines, showed appreciable inhibitory activity against the KDR tyrosine kinase. Furthermore, both oxazepines and diazepines demonstrated significant ability to inhibit autophosphorylation of EGFR in DiFi cells (generally, IC50 values in the single-digit micromolar to submicromolar range).

WW domains are protein modules that bind proline-rich ligands. WW domain-ligand complexes are of importance as they have been implicated in several human diseases such as muscular dystrophy, cancer, hypertension, Alzheimer's, and Huntington's diseases. We report the results of a protein array aimed at mapping all the human WW domain protein-protein interactions. Our biochemical approach integrates parallel synthesis of peptides, protein expression, and high-throughput screening methodology combined with tools of bioinformatics. The results suggest that the majority of the bioinformatically predicted WW peptide ligands and most WW domains are functional, and that only about 10% of the measured domain-ligand interactions are positive. The analysis of the WW domain protein arrays also underscores the importance of the amino acid residues surrounding the WW ligand core motifs for specific binding to WW domains. In addition, the methodology presented here allows for the rapid elucidation of WW domain-ligand interactions with multiple applications including prediction of exact WW ligand binding sites, which can be applied to the mapping of other protein signaling domain families. Such information can be applied to the generation of protein interaction networks and identification of potential drug targets. To our knowledge, this report describes the first protein-protein interaction map of a domain in the human proteome.

All RAS isoforms, including the oncogenic variants, must localize to the plasma membrane (PM) to initiate downstream signaling. H-RAS, N-RAS and the KRAS splice variant KRAS4a all transit to the PM through the Golgi, and rely on cycles of reversible palmitoylation on one or two cysteines in the hyper variable region (HVR) to regulate exchange on and off the PM. In contrast, the KRAS4b HVR contains a series of positively charged lysine residues, which target KRAS4b to regions of the PM rich in negatively charged phospholipids. Though the details have not been fully worked out, evidence suggests that KRAS4b trafficking to and from the PM is mediated by chaperones such as PDE6δ and smgGDS. Since its path to the PM is distinct, and since membrane localization is a requirement for downstream signaling, perturbing the spatial organization of KRAS4b may uncover new molecular targets for KRAS-dependent cancers. To this end, we are developing a high content assay and image analysis pipeline to screen for molecules that disrupt the PM localization of KRAS4b. The assay is based on a doxycycline-inducible GFP-KRASG12V expressing HeLa cell line, and uses confocal microcopy to automatically image cells at sub-micron resolution in multi-well glass bottom plates. To specifically counterstain the PM compartment and nucleus, we use fluorescently labelled Concanavalin A (ConA) and Hoechst (a DNA specific dye) respectively. An image analysis pipeline was developed to segment and quantitate the levels of GFP-KRAS-G12V in the PM compartment. The pipeline includes a supervised machine learning algorithm to convert the grey-scale intensity values of the PM channel (ConA) to the probability that the ConA signal belongs to the PM. These probability values are subsequently used to segment the cell boundary and PM using a graph-cut method and a thresholding technique. We use the nuclear channel to eliminate any segmented boundaries that do not belong to a cell. Calculated Z’ factors using the mean values per well of membrane localized GFP-KRAS-G12V in doxycycline treated and untreated cells show acceptable values (0.7) for high content screening (HCS). Furthermore, as a secondary assay to evaluate hits from the primary HCS, we are developing fluorescence correlation spectroscopy (FCS) techniques to measure the temporal dynamics of GFP-KRAS-G12V membrane localization in live cells. In preliminary FCS experiments, we tested farnesyl thiosalicylic acid (FTS), a molecule reported to dissociate RAS molecules from the PM. We observed increased lateral diffusion (2.38±0.55 μm2/s) in drug treated cells (15-30 mins after treatment) compared to untreated cells (0.85±0.12 μm2/s) consistent with values previously reported using fluorescence recovery after photobleaching measurements (Niv H. et al. J. Biol. Chem. 1999; 274:1606-1613). Taken together, we demonstrate that our screening platform can be used to identify small molecule disruptors of oncogenic KRAS4b spatial and temporal localization in the PM of cells. Citation Format: Alla Brafman, Prabhakar Gudla, Kaustav Nandy, John Columbus, De Chen, Karen Worthy, Stephen Lockett, Thomas Turbyville. Targeting KRAS4b plasma membrane localization in cells. [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 4542. doi:10.1158/1538-7445.AM2015-4542

A novel class of pyrimido[4,5-b]-1,4-benzoxazepines is described as inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. Two compounds display potent EGFR inhibitory activity of less than 1 microM in cellular phosphorylation assays (IC(50) 0.47-0.69 microM) and are highly selective against a small kinase panel. Such compounds demonstrate anti-EGFR activity within a class that is different from any known EGFR inhibitor scaffolds. They also provide a basis for the design of kinase inhibitors with the desired selectivity profile.

Pyrimido-oxazepine based sub-micromolar inhibitors (2–4) for Aurora and FLT-3 were designed and synthesized. These preliminary results supported the potential use of pyrimido-oxazepines as a versatile template for developing specific kinase inhibitors.