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.

Phenobarbital (PB) alters expression of numerous hepatic genes, including genes involved in xenobiotic metabolism. Phenobarbital-dependent induction of cytochrome P-450 2B1 (CYP2B1) is subject to regulation by cytokines [e.g., by epidermal growth factor (EGF)], hormones [e.g., by growth hormone (GH)], or the cellular redox status. To investigate mechanisms involved in regulation of CYP2B1 transcription, we performed promoter activation studies using primary rat hepatocyte cultures transiently transfected with individual CYP2B1 promoterluciferase reporter gene constructs. The 2679-bp native 5Ј-flanking region of the CYP2B1 gene conferred reporter gene activation by PB and the potent PB-like inducer permethrin (PM). Furthermore, this region mediated EGF-and GH-dependent repression of gene activation by PB-like inducers. A wide promoter mapping strategy with constructs bearing internal CYP2B1 promoter deletions led to identification of a distal responsive CYP2B1 enhancer region at Ϫ2230 to Ϫ2170, encompassing the section equivalent to the 51-bp PB-responsive enhancer module situated in the distal mouse Cyp2b10-5Ј-flanking region. The distal CYP2B1 enhancer region conferred gene activation by PM, repression of PM-dependent activation by EGF, and enhancement of activation by the antioxidant N-acetylcysteine (NAC). Mutational analyses of the region at Ϫ2230 to Ϫ2170 suggested that the mechanisms of PB-dependent induction of CYP2B1 and the modulating effects by EGF or NAC are closely related.

Since their discovery over five decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC Quinolone Resistance Determining Region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gramnegative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex, but does not form significant contacts with residues in the Quinolone Resistance Determining Region.

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Phototoxic properties of systemically applied pharmaceuticals may be the cause of serious adverse drug reactions. Therefore, a reliable preclinical photosafety assessment strategy, combining in vitro and in vivo approaches in a quantitative manner, is important and has not been described so far. Here, we report the establishment of an optimized modified murine local lymph node assay (LLNA), adapted for phototoxicity assessment of systemically applied compounds, as well as the test results for 34 drug candidates in this in vivo photo-LLNA. The drug candidates were selected based on their ability to absorb ultraviolet/visible light and the photo irritation factors (PIFs) determined in the well-established in vitro 3T3 neutral red uptake phototoxicity test. An in vivo phototoxic potential was identified for 13 of these drug candidates. The use of multiple dose levels in the described murine in vivo phototoxicity studies enabled the establishment of no- and/or lowest-observed-adverse-effect levels (NOAELs/LOAELs), also supporting human photosafety assessment. An in vitro-in vivo correlation demonstrated that a drug candidate classified as "phototoxic" in vitro is not necessarily phototoxic in vivo. However, the probability for a drug candidate to cause phototoxicity in vivo clearly correlated with the magnitude of the phototoxicity identified in vitro.