Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

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 phosphatase. 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 neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. 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 RAS­ERK 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.

Discovery of a ZIP7 inhibitor from a Notch pathway screen.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.

Transgenic mice: fat-1 mice convert n-6 to n-3 fatty acids.

Mammals cannot naturally produce omega-3 (n-3) fatty acids--beneficial nutrients found mainly in fish oil--from the more abundant omega-6 (n-6) fatty acids and so they must rely on a dietary supply. Here we show that mice engineered to carry a fat-1 gene from the roundworm Caenorhabditis elegans can add a double bond into an unsaturated fatty-acid hydrocarbon chain and convert n-6 to n-3 fatty acids. This results in an abundance of n-3 and a reduction in n-6 fatty acids in the organs and tissues of these mice, in the absence of dietary n-3. As well as presenting an opportunity to investigate the roles played by n-3 fatty acids in the body, our discovery indicates that this technology might be adapted to enrich n-3 fatty acids in animal products such as meat, milk and eggs.

Fluopack screening platform for unbiased cellular phenotype profiling.

Gene and compound functions are often interrogated by perturbation. However, we have limited methods to capture associated phenotypes in an unbiased and holistic manner. Here, we describe Fluopack screening as a novel platform enabling the profiling of subcellular phenotypes associated with perturbation. Our approach leverages imaging of a panel of fluorescent chemical probes to survey cellular processes in an unbiased and high throughput fashion. Segmentation-free, whole image analysis applied to Fluopack images identifies probes revealing distinct phenotypes upon perturbation, thereby informing on the function and mechanism of action of perturbagens. This chemical biology approach allows to interrogate phenotypes that tend to be overlooked by other methods, such as lipid trafficking and ion concentration inside the cell. Fluopack screening is a powerful approach to study orphan protein function, as exemplified by the characterization of TMEM41B as novel regulator of lipid mobilization.

An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cells.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.

Identification of TNO155, an Allosteric SHP2 Inhibitor for the Treatment of Cancer.

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.

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor.

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.

Effects of adenoviral gene transfer of C. elegans n-3 fatty acid desaturase on the lipid profile and growth of human breast cancer cells.

BACKGROUND: Current evidence from both experimental and human studies indicates that omega-6 polyunsaturated fatty acids (n-6 PUFAs) promote breast tumor development, whereas long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) exert suppressive effects. The ratio of n-6 to n-3 fatty acids appears to be an important factor in controlling tumor development. Human cells usually have a very high n-6/n-3 fatty acid ratio because they cannot convert n-6 PUFAs to n-3 PUFAs due to lack of an n-3 desaturase found in C. elegans. MATERIALS AND METHODS: Adenoviral strategies were used to introduce the C. elegans fat-1 gene encoding an n-3 fatty acid desaturase into human breast cancer cells followed by examination of the n-6/n-3 fatty acid ratio and growth of the cells. RESULTS: Infection of MCF-7 cells with an adenovirus carrying the fat-1 gene resulted in a high expression of the n-3 fatty acid desaturase. Lipid analysis indicated a remarkable increase in the levels of n-3 PUFAs accompanied with a large decrease in the contents of n-6 PUFAs, leading to a change of the n-6/n-3 ratio from 12.0 to 0.8. Accordingly, production of the eicosanoids derived from n-6 PUFA was reduced significantly in cells expressing the fat-1 gene. Importantly, the gene transfer induced mass cell death and inhibited cell proliferation. CONCLUSION: The gene transfer of the n-3 fatty acid desaturase, as a novel approach, can effectively modify the n-6/n-3 fatty acid ratio of human tumor cells and provide an anticancer effect, without the need of exogenous n-3 PUFA supplementation. These data also increase the understanding of the effects of n-3 fatty acids and the n-6/n-3 ratio on cancer prevention and treatment.

Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays.

Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment.