Ting Xie

Therapeutic Targeting of Oncogenic K‐Ras by a Covalent Catalytic Site Inhibitor

We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling.

EGFR Mutations and Resistance to Irreversible Pyrimidine-Based EGFR Inhibitors.

Purpose: Mutant selective irreversible pyrimidine-based EGFR kinase inhibitors, including WZ4002, CO-1686, and AZD9291, are effective in preclinical models and in lung cancer patients harboring the EGFR T790M gefitinib/erlotinib resistance mutation. However, little is known about how cancers develop acquired resistance to this class of EGFR inhibitors. We sought to identify and study EGFR mutations that confer resistance to this class of agents. Experimental Design: We performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in EGFR-mutant (sensitizing alone or with concurrent EGFR T790M) Ba/F3 cells and selected drug-resistant clones. We evaluated the sensitivity of EGFR inhibitors in models harboring drug-resistant EGFR mutations. Results: We identified 3 major drug resistance mutations. EGFR L718Q, L844V, and C797S cause resistance to both WZ4002 and CO-1686 while, in contrast, only EGFR C797S leads to AZD9291 resistance. Cells containing an EGFR-sensitizing mutation, Del 19 or L858R, in conjunction with L718Q, L844V, or C797S retain sensitivity to quinazoline-based EGFR inhibitors, gefitinib and afatinib. The C797S mutation, in the presence of Del 19 or L858R and T790M, causes resistance to all current EGFR inhibitors, but L858R/T790M/C797S remains partially sensitive to cetuximab which leads to disruption of EGFR dimerization. Conclusions: Our findings provide insights into resistance mechanisms to irreversible pyrimidine-based EGFR inhibitors and identify specific genomic contexts in which sensitivity is retained to existing clinical EGFR inhibitors. These findings will guide the development of new strategies to inhibit EGFR. Clin Cancer Res; 21(17); 3913–23. ©2015 AACR. See related commentary by Ayeni et al., p. 3818

In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C.

Significance SML-8-73-1 (SML) is the first example, to our knowledge, of a GTP-competitive inhibitor of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras). A high-resolution structure of K-Ras G12C bound to SML shows K-Ras in an inactive conformation. In situ proteomic-based chemical profiling of SML demonstrates that SML is highly selective for K-Ras G12C over other small GTPases. A novel chemosensor-based assay allows measurement of covalent reaction rates between K-Ras G12C and SML and enables characterization of this reaction in the context of millimolar concentrations of GTP and GDP, well in exccss of what is found in living cells. These results demonstrate that even in the presence of high concentrations of GTP and GDP, SML is able to exchange into the GN site. Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.

Pharmacological targeting of the pseudokinase Her3

Her3 (ErbB3) belongs to the epidermal growth factor receptor tyrosine kinases and is well credentialed as an anti-cancer target but is thought to be “undruggable” using ATP-competitive small molecules because it lacks significant kinase activity. Here we report the first selective Her3 ligand, TX1-85-1, that forms a covalent bond with Cys721 located in the ATP-binding site of Her3. We demonstrate that covalent modification of Her3 inhibits Her3 signaling but not proliferation in some Her3 dependent cancer cell lines. Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3 dependent signaling. Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met. These results suggest that small molecules will be capable of perturbing the biological function of Her3 and the approximately 60 other pseudokinases found in human cells.

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.

Development of small molecules targeting the pseudokinase Her3

Her3 is a member of the human epidermal growth factor receptor (EGFR) tyrosine kinase family, and it is often either overexpressed or deregulated in many types of human cancer. Her3 has not been the subject of small-molecule inhibitor development because it is a pseudokinase and does not possess appreciable kinase activity. We recently reported on the development of the first selective irreversible Her3 ligand (TX1-85-1) that forms a covalent bond with cysteine 721 which is unique to Her3 among all kinases. We also developed a bi-functional compound (TX2-121-1) containing a hydrophobic adamantane moiety and the same warhead of TX1-85-1 that is capable of inhibiting Her3-dependent signaling and growth. Here we report on the structure-based medicinal chemistry effort that resulted in the discovery of these two compounds.

Abstract 4832: Novel EGFR mutations that cause drug resistance to irreversible pyrimidine but not quinazoline based EGFR inhibitors

Background: Oncogenic EGFR T790M causes drug resistance to quinazoline based EGFR kinase inhibitors by increasing ATP affinity (Yun PNAS 2008). Mutant selective irreversible pyrimidine EGFR kinase inhibitor, WZ4002, is effective in non-small cell lung cancer (NSCLC) models harboring EGFR T790M (Zhou Nature 2009). We aimed to determine potential mechanisms of resistance to WZ4002 and explore alternative strategies to overcome acquired resistance to pyrimidine based EGFR inhibitors. Methods and Results: We performed an ENU mutagenesis screen in Ba/F3 cells expressing EGFR L858R, L858R/T790M, Del E746_A750 and Del E746_A750/T790M followed by culture in the presence of WZ4002 (100 nM or 1 μM). Using RT-PCR, we sequenced resistant clones for secondary EGFR mutations. No EGFR T790M mutations were identified. We detected novel secondary EGFR L718Q (9/27; 33%) or L844V (1/27; 3%) mutations in the drug resistant cells. We also recovered the EGFR C797S (1/27; 3%) mutation previously known to prevent covalent binding and decrease potency of WZ4002. Unlike EGFR T790M, EGFR L718Q and EGFR L844V did not lead to constitutive EGFR phosphorylation, were not transforming in Ba/F3 cells and required EGF for proliferation and survival. The EGFR L858R/L844V Ba/F3 cells were resistant to WZ4002 (IC50 0.7 μM) but sensitive to irreversible quinazoline EGFR inhibitors CL-387,785, HKI-272 (neratinib) and BIBW2992 (afatinib) (IC50 values all Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4832. doi:1538-7445.AM2012-4832

Abstract A178: Structure guided development of irreversible inhibitors for TAK1

Background: Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is serine/threonine kinase belonging to mitogen-activated protein kinase kinase family that can promote tumor cell survival by modulation of the tumor microenvironment, mediation of stress responses and suppression of pro-apoptotic signaling. Moreover, TAK1 has been identified as essential for the survival of certain KRAS-dependent cancer cells and has therefore been studied as a therapeutic target. Several TAK1 inhibitors including LYTAK1, NG25 and fungal isolate 5Z-7-oxozeanol (5Z-7) have been reported, but these compounds are relatively non-selective as illustrated by 5Z-7 which inhibits numerous other kinases including PKD2, IKKα, Mnk2, Flt3, Flt4, KDR, Trk, PDGFRα, MKK4, NLK, MEK at modest concentrations. Furthermore, 5Z-7 is a resorcyclic lactone with a complex cyclic structure, making it difficult to synthesize derivatives that might have with better selectivity and potency. In an effort to develop new TAK1 inhibitors with excellent selectivity and synthetic accessibility, we studied a series of pyrimidine-based covalent inhibitors which target CYS-174, a cysteine adjacent to the ATP binding site of TAK1. Methods: Structure-guided compound evolution was used to guide design of compounds. Candidate covalent compounds were evaluated for relative binding affinity to TAK1, and the ability to covalently label recombinant TAK1 protein. Co-crystal x-ray structures of selected compounds were additionally solved to guide iterations of compound design. Compounds were evaluated for anti-proliferative activity in TAK1-dependent cell lines from 3 distinct cancer types. Results: Co-crystal structures of TAK1 in complex with the pyrrolopyrimidine-based compound CPT1691 showed an unexpected binding mode leading to the hypothesis that substitution of the pyrrolopyrimidine for a simpler pyrimidine would provide synthetically simpler routes to compounds which covalently bind to Cys-174. This substitution was tolerated, yielding potent TAK1-binding compounds which were further evolved to optimize differential selectivity between TAK1 and other highly related kinases such as MEK1, ERK2 and FLT3. Additional co-crystal structures of these compounds were solved. These inhibitors showed anti-proliferative activity in various TAK1-dependent colon cancer cells (LoVo, SW620, SK-CO-1), pancreatic cancer cells (PANC-1, AsPc-1 and Colo357FG cell lines) and renal cancer cells. Conclusions: Covalent pyrimidine-based TAK1 probes provide an effective means of TAK1 inhibition that may have value as therapeutic agents and scientific tools. Citation Format: Deepak Gurbani, Li Tan, Scott Ficarro, John C. Hunter, William Singer, Faviola B. Vazquez, Ting Xie, Sang Min Lim, Jarrod Marto, Nathanael S. Gray, Kenneth D. Westover. Structure guided development of irreversible inhibitors for TAK1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A178.

Abstract PR07: Crystal structure of K-Ras G12C bound to an active site inhibitor

Activating K-Ras mutations are common oncogenic events in many cancers but effective direct-acting inhibitors have not been developed. Targeting the nucleotide binding site of GTPases like K-Ras has long been dismissed as a possibility because of the high affinity of these proteins for their natural ligands GTP and GDP. We previously reported the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggested that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. Here we report a crystal structure of K-Ras G12C labeled with SML-8-73-1. The structure reveals that when bound to SML-8-73-1, K-Ras presents in an inactive conformation. This first-in-class covalent K-Ras inhibitor suggests that irreversible targeting of the K-Ras guanine-nucleotide binding site is a viable therapeutic strategy for inhibition of Ras signaling. This abstract is also presented as Poster B54. Citation Format: John Hunter, Sang Min Lim, Scott B. Ficcarro, Hwan Geun Choi, Deepak Gurbani, Ting Xie, Jarrod A. Marto, Nathanael S. Gray, Ken Westover. Crystal structure of K-Ras G12C bound to an active site inhibitor. [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 PR07. doi: 10.1158/1557-3125.RASONC14-PR07