Detection of Cellular Target Engagement for Small-Molecule Modulators of Striatal-Enriched Protein Tyrosine Phosphatase (STEP).

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific enzyme that regulates the signaling molecules that control synaptic plasticity and neuronal function. Dysregulation of STEP is linked to the pathophysiology of Alzheimer's disease and other neuropsychiatric disorders. Experimental results from neurological deficit disease models suggest that the modulation of STEP could be beneficial in a number of these disorders. This prompted our work to identify small-molecule modulators of STEP to provide the foundation of a drug discovery program. As a component of our testing funnel to identify small-molecule STEP inhibitors, we have developed a cellular target engagement assay that can identify compounds that interact with STEP46. We provide a comprehensive protocol to enable the use of this miniaturized assay, and we demonstrate its utility to benchmark the binding of newly discovered compounds.

Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells.

Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain-containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal-regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.

Repeat aortic valve surgery: contemporary outcomes and risk stratification.

OBJECTIVES: Redo aortic valve surgery (rAVS) is performed with increasing frequency, but operative mortality is usually higher compared to that associated with primary aortic valve surgery. We analysed our patients who had rAVS to determine the current outcomes of rAVS as a surgical benchmark in view of the growing interest in transcatheter valve techniques. METHODS: We retrospectively reviewed 148 consecutive patients [median age 67.7 years (interquartile range 54.9-77.6); 68.2% men] who underwent rAVS following aortic valve replacement (81.6%), aortic root replacement (15%) or aortic valve repair (3.4%) between 2000 and 2018. RESULTS: Indications for rAVS were structural valve dysfunction (42.7%), endocarditis (37.8%), non-structural valve dysfunction (17.7%) and aortic aneurysm (2.1%). Valve replacement was performed in 69.7%, and 34 new root procedures were necessary in 23%. Early mortality was 9.5% (n = 14). Female gender [odds ratio (OR) 6.16], coronary disease (OR 4.26) and lower creatinine clearance (OR 0.95) were independent predictors of early mortality. Follow-up was 98.6% complete [median 5.9 (interquartile range 1.7-10.9) years]. Survival was 74.1 ± 3.7%, 57.9 ± 5.1% and 43.8 ± 6.1% at 5, 10 and 14 years, respectively. Cox regression analysis revealed female gender [hazard ratio (HR) 1.73], diabetes (HR 1.73), coronary disease (HR 1.62) and peripheral vascular disease (HR 1.98) as independent determinants of late survival. CONCLUSIONS: Despite many urgent situations and advanced New York Heart Association functional class at presentation, rAVS could be performed with acceptable early and late outcomes. Risk factors for survival were female gender, coronary disease and urgency. In this all-comers patient cohort needing rAVS, only a minority would eventually qualify for transcatheter valve-in-valve procedures.

A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors.

The nonreceptor protein-tyrosine phosphatase (PTP) SHP2 is encoded by the proto-oncogene PTPN11 and is a ubiquitously expressed key regulator of cell signaling, acting on a number of cellular processes and components, including the Ras/Raf/Erk, PI3K/Akt, and JAK/STAT pathways and immune checkpoint receptors. Aberrant SHP2 activity has been implicated in all phases of tumor initiation, progression, and metastasis. Gain-of-function PTPN11 mutations drive oncogenesis in several leukemias and cause developmental disorders with increased risk of malignancy such as Noonan syndrome. Until recently, small molecule-based targeting of SHP2 was hampered by the failure of orthosteric active-site inhibitors to achieve selectivity and potency within a useful therapeutic window. However, new SHP2 allosteric inhibitors with excellent potency and selectivity have sparked renewed interest in the selective targeting of SHP2 and other PTP family members. Crucially, drug discovery campaigns focusing on SHP2 would greatly benefit from the ability to validate the cellular target engagement of candidate inhibitors. Here, we report a cellular thermal shift assay that reliably detects target engagement of SHP2 inhibitors. Using this assay, based on the DiscoverX InCell Pulse enzyme complementation technology, we characterized the binding of several SHP2 allosteric inhibitors in intact cells. Moreover, we demonstrate the robustness and reliability of a 384-well miniaturized version of the assay for the screening of SHP2 inhibitors targeting either WT SHP2 or its oncogenic E76K variant. Finally, we provide an example of the assay's ability to identify and characterize novel compounds with specific cellular potency for either WT or mutant SHP2.