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Growing malignant tumors must evade destruction by the immune system, a hurdle some malignancies overcome by attracting immune-suppressive regulatory T-cells (Tregs)1. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Tregs, and IKZF2 deficiency enhances immune responses to tumors in mice2, suggesting IKZF2 may be an attractive target for cancer immunotherapy. Here we describe the discovery and characterization of DKY709, the first molecular glue degrader of IKZF2/4 which spares IKZF1/3. DKY709 was identified through a recruitment-guided medicinal chemistry campaign that redirected the degradation selectivity of CRBN binders towards IKZF2. The IKZF transcription factor selectivity of DKY709 was rationalized by the X-ray structure of the CRBN-DKY709-IKZF2(ZF2) ternary complex. Upon exposure to DKY709, human Tregs showed reduced suppressive activity and exhausted T-effector cells recovered IFNγ production. In vivo, oral treatment with DKY709 drove a rapid and sustained degradation of IKZF2 including in humans and led to delayed tumor growth in mice with humanized immune systems and enhanced immunization responses in monkeys. DKY709 is a first-in-class, potent and selective oral IKZF2/4 degrader currently being investigated in a phase 1 clinical trial as an immune-enhancing agent for cancer immunotherapy.

Amine containing aryl ethers are common pharmacophore motifs that continue to emerge from drug discovery efforts. As amino alcohols are readily available building blocks, practical methodologies for incorporating them into more complex structures are highly desireable. We report our efforts to explore the application of Pd-catalyzed C-O coupling methods to the arylation of 1,2- and 1,3-amino alcohols. We established reliable conditions, under which we explored the scope and limitations of the transformation. The insights gained have been valuable in employing this methodology within a fast-moving drug discovery environment, which we anticipate will be of general interest to the synthesis and catalysis communities.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

SHP099, a selective inhibitor of signalling meditator SHP2 with drug-like properties, has an allosteric mechanism of action whereby it stabilizes SHP2 in an auto-inhibited conformation, and suppresses RAS–ERK signalling and proliferation in receptor-tyrosine-kinase-driven cancer cell lines and mouse tumour xenograft models. The tyrosine phosphatase SHP2 is a key mediator of receptor tyrosine kinase (RTK) signalling, as well as being important in immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth, and SHP2 is a potential, but so far elusive, therapeutic target in cancer. Pascal Fortin and colleagues report the development of a selective SHP2 inhibitor with drug-like properties. The inhibitor, SHP099, has an allosteric mechanism of action whereby it stabilizes SHP2 in an auto-inhibited conformation. It also suppresses RAS–ERK signalling to inhibit RTK-driven proliferation in human cancer cell lines and mouse tumour xenograft models. The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1,2,3,4,5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2,3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6,7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8,9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

A fast method was developed to determine the amount of anions and cations present in discovery samples using a reagent free ion chromatography system. The system separates and quantifies counterions present in novel discovery compounds from an accurately weighed sample of 1 mg. The advantage of using these methods in the discovery phase and early development to accurately characterize salt identity and stoichiometry has been demonstrated. Likewise, a further advantage is the ability to accurately quantify undesirable counterions that may be present due to process related manipulation of material and to quantify salt equivalents for “mixed” salt samples. The instrumentation described also has the ability to utilize a reagent-free automated eluent generation approach which in addition to saving time, eliminates the variability that can occur with manually prepared eluents. Several examples have been collated to highlight the usefulness of this approach within discovery.

Two routes aimed at the manufacture of chiral exo-2-norbornyl thiourea (1) on large scale are described. The first approach involves five chemical steps and hinges on a classical resolution via diastereomeric salt formation. The synthesis utilizes amine 2 as the resolution handle. The second approach includes two chemical steps and a chiral chromatography of (±)-1. Despite the larger initial investment necessary to acquire the chiral stationary phase used in the chromatographic approach, the shorter reaction sequence and efficiency of the chromatographic separation make the second route a more attractive option for long-term applications.

Tumorigenesis is a multistep process in which oncogenes play a key role in tumor formation, growth, and maintenance. MET was discovered as an oncogene that is activated by its ligand, hepatocyte growth factor. Deregulated signaling in the c-Met pathway has been observed in multiple tumor types. Herein we report the discovery of potent and selective triazolopyridazine small molecules that inhibit c-Met activity.

Angiogenesis is vital for solid tumor growth, and its prevention is a proven strategy for the treatment of disease states such as cancer. The vascular endothelial growth factor (VEGF) pathway provides several opportunities by which small molecules can act as inhibitors of endothelial proliferation and migration. Critical to these processes is signaling through VEGFR-2 or the kinase insert domain receptor (KDR) upon stimulation by its ligand VEGF. Herein, we report the discovery of 2,3-dihydro-1,4-benzoxazines as inhibitors of intrinsic KDR activity (IC 50 < 0.1 microM) and human umbilical vein endothelial cell (HUVEC) proliferation with IC 50 < 0.1 microM. More specifically, compound 16 was identified as a potent (KDR: < 1 nM and HUVEC: 4 nM) and selective inhibitor that exhibited efficacy in angiogenic in vivo models. In addition, this series of molecules is typically well-absorbed orally, further demonstrating the 2,3-dihydro-1,4-benzoxazine moiety as a promising platform for generating kinase-based antiangiogenic therapeutic agents.

The non-receptor protein tyrosine phosphatase (PTP) SHP2 is an important component of RTK signaling in response to growth factor stimulus and sits just upstream of the RAS-MAPK signaling cascade. The first oncogenic phosphatase to be identified, SHP2 is dysregulated in multiple human diseases including the developmental disorders Noonan and Leopard syndromes, as well as leukemia, lung cancer and neuroblastoma where aberrant activity of SHP2 leads to uncontrolled MAPK signaling. Cancer-associated activating mutations in SHP2 impart an “auto-on” state of the enzyme, boosting basal activity by shifting the equilibrium away from the auto-inhibited state. Reduction of SHP2 activity through genetic knockdown suppresses tumor growth, validating SHP2 as a target for cancer therapy. SHP099, a recently reported potent and selective allosteric inhibitor of SHP2, stabilizes the auto-inhibited form of SHP2 through interactions with the N-terminal SH2 and C-terminal PTP domains of the protein. SHP099 suppresses MAPK signaling in RTK amplified cancers resulting in suppressed proliferation in vitro and inhibition of tumor growth in mouse tumor xenograft models. Together, these data demonstrate the therapeutic potential of SHP2 inhibition in the treatment of cancer and other RAS/MAPK-linked diseases. Citation Format: Michael G. Acker, Ying-Nan P. Chen, Matthew J. LaMarche, Ho Man Chan, Peter Fekkes, Jorge Garcia-Fortanet, Jonathan R. LaRochelle, Brandon Antonakos, Christine Hiu-Tung Chen, Zhuoliang Chen, Vesselina G. Cooke, Jason R. Dobson, Zhan Deng, Fei Feng, Brant Firestone, Michelle Fodor, Cary Fridrich, Hui Gao, Huai-Xiang Hao, Jaison Jacob, Samuel Ho, Kathy Hsiao, Zhao B. Kang, Rajesh Karki, Mitsunori Kato, Jay Larrow, Laura R. La Bonte, Gang Liu, Shumei Liu, Dyuti Majumdar, Matthew J. Meyer, Mark Palermo, Minying Pu, Edmund Price, Subarna Shakya, Michael D. Shultz, Kavitha Venkatesan, Ping Wang, Markus Warmuth, Sarah Williams, Guizhi Yang, Jing Yuan, Ji-Hu Zhang, Ping Zhu, Stephen C. Blacklow, Timothy Ramsey, Nicholas J. Keen, William R. Sellers, Travis Stams, Pascal D. Fortin. Conformational activation and allosteric inhibition of SHP2 in RTK-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2017-2084