We describe the design and synthesis of highly selective PI3K inhibitors based on a quinazolinone scaffold that engages the methionine switch observed in both PI3Kγ (Knight et al., 2006
) and PI3Kδ (Berndt et al., 2009). Elaboration of this scaffold with aryl and alkyne affinity elements afforded compounds with a wide range of activity against PI3Kδ and PI3Kγ but importantly did not significantly inhibit PI3Kα, PI3Kβ, or any of the 219 protein kinases evaluated. These compounds were used in conjunction with AS605240, a PI3Kγ-directed literature inhibitor, and the pan-PI3K inhibitor PIK90 in order to evaluate the effects of isoform-selective inhibition in single and multi-cellular contexts.
Several PI3K inhibitors with different isoform selectivities were tested in primary human cell models of inflammatory signaling. Pan-PI3K inhibition (PIK90) was highly anti-proliferative to both HUVECs and PMBCs whereas PI3Kδ inhibition (SW30) or PI3Kδ/γ inhibition (SW14) resulted in selective T-cell inhibition. Selective PI3Kδ inhibition (SW30) blocked T-cell cytokine production, resulting in decreased HUVEC E-selectin expression and revealing the potential to elicit a potent anti-inflammatory response without a direct effect on endothelial cells. Interestingly, SW30 and other selective PI3Kδ inhibitors such as IC87114 (Supplementary Material
) did not inhibit E-selectin expression in HUVECs directly stimulated with IFNγ, TNFα, and IL1β (; ) but only did so in the superantigen-stimulated HUVEC/PMBC co-culture conditions (SAg; , Supplementary Material
). This result is consistent with these compounds indirectly
blocking E-selectin expression in HUVECs as a result of lymphocyte TNFα production inhibition (TNFα is known to rapidly upregulate E-selectin expression in HUVECs (Schindler and Baichwal, 1994
)), and this data clearly illustrates the ability of these assays to capture parts of the intercellular communication integral to inflammatory responses.
PI3Kδ/γ inhibition (SW14) was not cytotoxic to HUVECs and displayed more significant anti-inflammatory effects in the HUVEC/PBMC co-cultures. Dual targeting of PI3Kδ/γ resulted in enhanced inhibition of LPS-induced TNFα production () and overall T-cell activation. The inhibitory effects observed with inhibition of PI3Kδ/γ in these assays led us to wonder whether they were due to synergistic inhibition of PI3Kδ/γ or whether PI3Kγ inhibition might be sufficient. The most commonly used PI3Kγ compound in the literature and only PI3Kγ selective molecule we were able to synthesize, AS605240, inhibits all PI3K isoforms at 300nM (Camps et al., 2005
), and primary human cell assays displayed a profile similar to pan-PI3K compounds (Fig. S4; Supplementary Material
Our studies using both new and benchmark compounds show the most effective anti-inflammatory PI3K inhibitor is one that inhibits both PI3Kδ and PI3Kγ. Pan-PI3K inhibitors, on the other hand, demonstrated limited effects on anti-inflammatory markers in T-cell and monocyte-driven environments with concurrent anti-proliferative effects. Nevertheless, it remains possible that selectively inhibiting other combinations of PI3K isoforms, such as PI3Kα/β, PI3Kβ/γ, or PI3Kβ/δ, may also show synergistic anti-inflammatory activity.
Interestingly, additional inhibition of PI3Kα and β (PIK90) did not further suppress inflammatory markers (; LPS, SAg). In fact, when comparing the effects on E-selectin expression (SAg), selective inhibition of PI3Kδ alone was better than pan-PI3K inhibition, but inhibition of PI3Kδ/γ was the most effective (). The most effective isoform combination at inhibition of LPS-induced TNFα production was also PI3Kδ/γ (). Additional PI3Kα/β inhibition (PIK90), in addition to displaying undesirable anti-proliferative properties, did not significantly decrease TNFα expression and was essentially identical to selective PI3Kδ inhibition (). These results together suggest a possible role for PI3Kγ in TNFα production.
Both kinase-dependent and kinase-independent routes exist through which PI3Kγ could regulate TNFα production. PI3Kγ interacts with phosphodiesterase 3B (PDE3B), regulating cyclic AMP (cAMP) levels and activation of protein kinase A (PKA), leading to increased heart contractility (Patrucco et al., 2004
) and was also linked to PDE4 (Kerfant et al., 2007
). PDE4 is the main cAMP hydrolyzing enzyme in immune cells, and has been pursued as a target for generating novel anti-inflammatories (Teixeira et al., 1997
) that suppress LPS-induced TNFα production in monocytes (Souness et al., 1996
). Since PI3Kγ is required for PDE4 activity (Kerfant et al., 2007
), PI3Kγ inhibition may lead to diminished PDE4 activity and explain the TNFα suppression. However, the first link between PDEs and PI3Kγ involved a scaffolding, non-catalytic effect (Patrucco et al., 2004
) and the PDE4-PI3Kγ link was discovered in a PI3Kγ KO mouse (Kerfant et al., 2007
), which has no functional PI3Kγ suggesting that this route may not involve the catalytic activity of PI3Kγ.
TNFα is involved in several autocrine signaling loops (Lisby et al., 2007
) and it is possible that PI3Kγ is involved in amplifying such signals. TNFα activates PI3Kγ in endothelial cells leading to oxidant generation and NF-κB activation (Frey et al., 2006
). NF-κB regulates production of TNFα (Li and Stark, 2002
) so TNFα may activate its own production by NF-κB through PI3Kγ. TNFα can upregulate its own mRNA synthesis in keratinocytes (Lisby et al., 2007
), and if this happens in PBMCs, it provides an attractive model for PI3Kγ involvement in TNFα production and may explain why PI3Kγ inhibition lowers TNFα levels.
Our data suggest that inhibition of PI3Kγ's catalytic activity is sufficient to regulate TNFα levels, but they do not necessarily rule out other mechanisms. It is possible that application of BioMAP analysis to this question may resolve the conflict. If the combination of a PDE4 inhibitor and a PI3Kγ inhibitor did not alter TNFα levels more than either inhibitor alone, this would suggest that PI3Kγ is signaling through PDE4, if however, combined inhibition of PDE4 and PI3Kγ further decreased TNFα levels, this might suggest that they work through independent pathways (as mentioned above) Results obtained by treating with more than one compound may be unreliable because of potential off-target effects, thus a combination of a specific inhibitor with siRNA in a BioMAP system might better resolve the dilemma.
In broad terms, PI3K signaling can be viewed into two divisions, a survival pathway (α/β) and inflammatory PI3K (δ/γ) pathway, yet inhibitors that target both units can lead to functional antagonism. Essentially, inhibition of Class I PI3-Ks is not necessarily additive, and inhibition of PI3-Kα and PI3-Kβ can actually antagonize the effects of more isoform selective inhibition. For example, inhibition of PI3Kδ/γ by SW14 results in suppression of E-selectin or TNFα but additional inhibition of PI3Kα/β using PIK90 actually results in a smaller degree of suppression ().
The actual mechanisms by which PI3Kα/β inhibition may antagonize PI3Kδ/γ inhibition are not perfectly clear. In the case of TNFα suppression, pan-PI3K inhibition with wortmannin and LY294002 has been shown to reverse amlodipine-induced inhibition of TNFα production in LPS-stimulated rat cardiomyocytes (Li et al., 2009
), and of more relevance, treatment of PBMCs and THP-1 monocytes with the same compounds increases LPS induced TNFα expression (Guha and Mackman, 2002
). Both wortmannin and LY294002 have more activity outside of the Class PI3Ks than PIK90, (Knight et al., 2006
; Knight and Shokat, 2005
) which may explain why PIK90 did not increase
TNFα levels, but our results with pan-PI3K inhibition are not necessarily surprising. Studies with pan-PI3K inhibitors cannot identify the actual isoforms responsible for the increase in TNFα levels, but a recent study showed that p110α deficient THP-1 monocytes display increased TNFα production when stimulated by LPS, suggesting that PI3Kα inhibition is responsible for the increase in TNFα production (Lee et al., 2007
). Together this evidence suggests that PI3Kα inhibition increases TNFα production, while PI3Kδ/γ inhibition decreases TNFα production, and when all isoforms are inhibited, both activities are balanced, leaving TNFα levels unchanged, but if PI3Kα is not inhibited (in the case of our PI3Kδ/γ inhibitors) then TNFα levels will decrease.
This work illustrates how targeted chemical inhibition can access information not available from genetic inactivation of one or more PI3K isoforms, which is difficult to obtain because these animals often suffer significant defects during development. Furthermore, results obtained using targeted inhibitors can be different from those obtained in animals with sustained genetic inactivation (Knight and Shokat, 2007
). PI3Kδ/PI3Kγ knockout mice exhibited a more severe immune phenotype than mice lacking either isoform alone, (Swat et al., 2006
; Webb et al., 2005
) but the severity of that phenotype is likely due to sustained absence of both PI3Kδ and PI3Kγ and may not be duplicated with pharmacological treatment.
Many clinical anti-inflammatory agents function through different targets yet all have the property of inhibiting immune cell function while leaving non-immune cells relatively unaffected. We asked if the PI3Kδ/γ inhibitors exhibited this property and which current anti-inflammatory agents they might resemble. The most closely related profile was that of the glucocorticoid receptor (GR) agonist, prednisolone. It is interesting that a particular multi-cellular profile can be achieved through two distinct mechanisms (kinase inhibition vs. nuclear hormone receptor activation). Although prednisolone is an effective anti-inflammatory agent, there have been efforts to identify “dissociating GR agonists” that separate anti-inflammatory from other GR effects (bone loss, cardiovascular disease) that limit their long term use (Schaecke et al., 2007
). The discovery that PI3Kδ/γ inhibition can functionally mimic several anti-inflammatory features of prednisolone opens a new way to improve upon a proven class of anti-inflammatories (GR agonists) while targeting completely different enzymes, and could only have been realized through analysis of this compound series on primary human cells.
Despite the related responses of prednisolone and PI3Kδ/γ inhibitors, the similarities may be limited to the cell types we analyzed. Cell types which have documented GR agonist responses (macrophages, coronary artery cells) are not included in our assays. The exceptionally broad cellular effects of GR agonists would likely be distinguished from PI3Kδ/γ inhibitors if more cell types were analyzed. Despite these caveats, the use of primary human cells provides a powerful early assessment of differential inhibition of important signaling nodes (PI3Ks, nuclear receptors, JNKs, calcineurin, IKK).
With the new availability of small molecules capable of inhibiting the inflammatory PI3Ks (δ/γ) without inhibition of the ubiquitous growth-linked PI3Ks we are poised to begin to resolve the opposing effects of pan-PI3K inhibition and selective inflammatory PI3K inhibition and to begin further validation of PI3Kδ/γ as a target for the treatment of inflammatory disorders.