Diisocyanates used in polyurethanes are commonly prepared by phosgenation of petroleum-sourced diamines. This involves highly toxic phosgene and produces corrosive HCl, limiting synthetic applications. In our search for a renewable source for diisocyanates, we have developed a practical methodology for the production of isocyanates from algae-biomass-derived fatty acids or other renewable sources. This technique utilizes flow chemistry to prepare and convert high-energy intermediates, thus mitigating safety concerns. By the use of continuous flow, acyl azides are prepared from hydrazides and subsequently heated to undergo Curtius rearrangement, affording isocyanates in one scalable process. The method is efficient, safe, and sustainable, offers an opportunity to prepare isocyanates and diisocyanates from renewable feedstocks, and is amenable to distributed manufacturing processes.

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.

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.