Discovery and Characterization of a Biologically Active Non–ATP-Competitive p38 MAP Kinase Inhibitor

Abstract

Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with none receiving regulatory approval. One characteristic shared by all of the compounds that failed clinical trials is that they are all adenosine triphosphate (ATP)-competitive p38 inhibitors. Seeing this lack of mechanistic diversity as an opportunity, we screened ~32,000 substances in search of novel p38 inhibitors. Among the inhibitors discovered is a compound that is both non-ATP competitive and biologically active in cell-based models for p38 activity. This is the first reported discovery of a non-ATP-competitive p38 inhibitor that is active in cells and, as such, may enable new pharmacophore designs for both therapeutic and basic research to better understand and exploit non-ATPcompetitive inhibitors of p38 activity.

NCI Program for Natural Product Discovery: A Publicly-Accessible Library of Natural Product Fractions for High-Throughput Screening

“…The model library of SPE fractions, comprising more than 2,000 fractions and their respective parent extracts, was screened in the NCI-60 Human Tumor Cell Lines Screen − and in several biochemical assays including those for inhibition of the protease MALT1 (unpublished results), the kinase p38, and the phosphodiesterase TDP1 with the aim of identifying prefractionation methods that showed (1) fractions with enhanced biological activity relative to the parent extract, (2) concentration of active components across fractions, and (3) a higher confidence in observed hits. For the plants included in this study, each of the parent organic extracts were inactive in the screening assays, and only one extract, a Thai collection of Fluegga virosa (Euphorbiaceae), exhibited activity in the fractions.…”

“…37 The high throughput target-based in vitro assays including MALT1, p38, and TDP1 were conducted by the Molecular Targets Program within the Center for Cancer Research, National Cancer Institute (Frederick, MD, USA) as detailed elsewhere. 38,39 ■ ASSOCIATED CONTENT * S Supporting Information…”

The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting

“…It should also be stressed that the drug targeting of a viral kinase such as pUL97 may not exclusively be limited to classical ATP-competitive types of kinase inhibitors including MBV. This strategy entails also untypical, thus far therapeutically untapped possibilities of kinase targeting, i.e., non-ATP-competitive modes of targeting [193][194][195]. It is quite conceivable that additional research work may reveal prototypes of non-ATP-competitive substrate inhibitors of pUL97 that could be directed to blocking the phosphorylation of individual pUL97 substrates, without inactivating the functionality of the pUL97 kinase domain.…”

In Vitro Ubiquitination Platform Identifies Methyl Ellipticiniums as Ubiquitin Ligase Inhibitors

“…To further ensure that these compounds were CBLB-specific inhibitors, these confirmed screening leads were also screened in tandem against the serine–threonine kinase p38 in a previously published HTS format (data not shown). 17 This tandem screening and availability of resupply further reduced our confirmed primary-screening actives to a pool of eight distinct chemical scaffolds capable of reducing the CBLB-dependent screening signal by at least 50% at 40 µM while not reducing the p38 signal by more than 10% ( Fig. 3 ).…”

“…p38 kinase dose-response testing was carried out as previously published. 17 Primary lead (and derivatives) compound IC 50 determination was carried out manually in a similar manner to that of primary screening. Dry resupplied compounds were resuspended in DMSO at a concentration of 100 mM.…”