Figure 2.

K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100

(A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P₃ 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC₅₀ of K306 and MN-100 on tSHIP1 and the EC₅₀ of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P₂ generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P₂ and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC₅₀ was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.