Discovery of dual inhibitors of the immune cell PI3Ks p110delta and p110gamma: a prototype for new anti-inflammatory drugs

Abstract

PI3Kdelta and PI3Kgamma regulate immune cell signaling, while the related PI3Kalpha and PI3Kbeta regulate cell survival and metabolism. Selective inhibitors of PI3Kdelta/gamma represent a potential class of anti-inflammatory agents lacking the antiproliferative effects associated with PI3Kalpha/beta inhibition. Here we report the discovery of PI3Kdelta/gamma inhibitors that display up to 1000-fold selectivity over PI3Kalpha/beta and evaluate these compounds in a high-content inflammation assay using mixtures of primary human cells. We find selective inhibition of only PI3Kdelta is weakly anti-inflammatory, but PI3Kdelta/gamma inhibitors show superior inflammatory marker suppression through suppression of lipopolysaccharide-induced TNFalpha production and T cell activation. Moreover, PI3Kdelta/gamma inhibition yields an anti-inflammatory signature distinct from pan-PI3K inhibition and known anti-inflammatory drugs, yet bears striking similarities to glucocorticoid receptor agonists. These results highlight the potential of selectively designing drugs that target kinases with shared biological function.

Fig. 1. Basis of selectivity and potency for aryl quinazolinones

A. Crystal structure of PI3Kγ with PIK90, a pan-PI3K inhibitor (Left) and PIK39, a PI3Kδ inhibitor (Right)(Knight et al., 2006). The bi-planar binding mode of PIK39 and alternate positioning of Met804 (red) are illustrated. Chemical compound structures are shown below. B. Compounds in this study introduce affinity elements (yellow) onto selective tolyl quinazolinone (red) scaffold maintaining selectivity for PI3Kγ and PI3Kδ. C. Diversification of SW series affinity elements was achieved through Suzuki-Miyaura couplings with aryl boronic acids and Sonogashira couplings with terminal alkynes, yielding two distinct chemical series.

Fig. 2. Diversification of Tolyl Quinazolinone Series

A. Introduction of affinity elements to PIK293. Series 1 (PI3Kδ/γ) introduces aryl groups and Series 2 (PI3Kδ) introduces alkynyl-linked groups. IC₅₀s of both Series 1 (blue) and Series 2 (red) were graphed. B. Biochemical IC₅₀s and γ/δ ratios for Series 1 and 2 along with benchmark compounds. All compounds were tested at 10μM ATP. C. Crystal structure of SW13(cyan), and 30(pink) as reported (Berndt et al., 2009). Hydrogen bonding elements for SW series are all aligned.

Fig. 3. Effect of selected compounds on THP-1 monocyte signaling

A. THP-1 monocyte signaling pathway leads to phosphorylation of Akt through either RTK-linked Macrophage Colony Stimulating Factor (M-CSF) or GPCR-linked Monocyte chemoattractant protein 1 (MCP-1). B. pAkt was measured by FACS using fluorescent pSer473 Akt antibodies. Quantitated fluorescence was used to determine an EC₅₀ for selected compounds. Biochemical γ/δ ratios are provided for comparison.

Fig. 4. BioMAP Analysis of PI3K Inhibitors

A. BioMAP Systems details. B. BioMAP profiles for PIK90 (pan-PI3K) (top), SW30 (PI3Kδ) (middle), and SW14 (PI3Kδ/γ) (bottom). Red dots represent a 10μM dose, orange corresponds to 3.3μM, yellow represents 1.1μM and green represents 370nM. Levels of proteins were measured by ELISA and presented as log expression ratios [log₁₀(parameter value with inhibitor/parameter value of 0.1% DMSO)]. The gray area represents the 95% prediction interval of the 0.1% DMSO data. Arrows indicate readouts that are expanded in 4C for closer inspection. C. Log Expression Ratios of selected readouts that show divergence between pan-PI3K inhibition and selective PI3K inhibition

Fig. 5. Function Similarity Map of PI3K inhibitors and other compounds

A Function Similarity Map of compounds from the SW series with other anti-inflammatories, PI3K inhibitors, and kinase inhibitors. This map is generated by subjecting the pairwise correlation data of BioMAP profiles to multidimensional scaling. Significant correlations are shown by gray lines. The distance between the compounds is inversely related to the similarity of their profiles. Compounds are color-coded, grouped by target class, and the area of the circle is proportional to the dose.