Development of Three Orthogonal Assays Suitable for the Identification and Qualification of PIKfyve Inhibitors

Abstract

FYVE-type zinc finger-containing phosphoinositide kinase (PIKfyve) catalyzes the formation of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) from phosphatidylinositol 3-phosphate (PI(3)P). PIKfyve has been implicated in multiple cellular processes, and its role in the regulation of toll-like receptor (TLR) pathways and the production of proinflammatory cytokines has sparked interest in developing small-molecule PIKfyve inhibitors as potential therapeutics to treat autoimmune and inflammatory diseases. We developed three orthogonal assays to identify and qualify small-molecule inhibitors of PIKfyve: (1) a purified component microfluidic enzyme assay that measures the conversion of fluorescently labeled PI(3)P to PI(3,5)P by purified recombinant full-length human 6His-PIKfyve (rPIKfyve); (2) an intracellular protein stabilization assay using the kinase domain of PIKfyve expressed in HEK293 cells; and (3) a cell-based functional assay that measures the production of interleukin (IL)-12p70 in human peripheral blood mononuclear cells stimulated with TLR agonists lipopolysaccharide and R848. We determined apparent K values for both ATP and labeled PI(3)P in the rPIKfyve enzyme assay and evaluated the enzyme's ability to use phosphatidylinositol as a substrate. We also tested four reference compounds in the three assays and showed that together these assays provide a platform that is suitable to select promising inhibitors having appropriate functional activity and confirmed cellular target engagement to advance into preclinical models of inflammation.

Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin A1-reversible aberrant endomembrane dilation

“…Apilimod-dependent inhibition of the PIKfyve-catalyzed PtdIns5P synthesis in vitro has not been tested in the original study characterizing the drug as a PIKfyve inhibitor [ 22 ]. Additionally, a recent report that did examine a perceived PtdIns5P reduction by apilimod using a cell-free microfluidic enzyme assay and a synthetic di-C6 PI substrate yielded a negative result [ 38 ]. To address this paucity, we performed a traditional in vitro lipid kinase activity assay using radiolabeled ATP, a native enzyme substrate and PIKfyve, immunopurified from HEK293 cells.…”

“…[ 22 ] and 5 nM in Ref. [ 38 ]. The discrepancy is likely related to the different or suboptimal conditions of the in vitro assays in those studies [ 22 , 38 ], including non-physiological synthetic substrates with short (C6) acyl chains, the PIKfyve source, incubation times, reaction buffers, methods of detection, etc.…”

“…Whereas we have previously demonstrated that short pretreatment with low doses of BafA1 (15 nM, 40 min) rendered COS7 cells resistant to cytoplasmic vacuolization upon subsequent PIKfyve inhibition by YM201636 [ 30 ], the PI profiles remained to be examined. Moreover, in the light of recent observations for different functional outcomes of YM201636 and apilimod despite that they both inhibited specifically PIKfyve and triggered vacuole formation, such analysis is warranted [ 38 ]. Therefore, to quantify PIs and reveal whether the prevented PtdIns3P rise by BafA1 pretreatment could be reproduced with the YM201636 compound, we performed similar myo -[2- 3 H]inositol labeling experiments in HEK293 cells.…”

“…We report here for the first time that apilimod powerfully inhibits both PtdIns5P and PtdIns(3,5)P 2 synthesis in vitro as well as in intact cells. Given that the two PIKfyve inhibitors apilimod and YM201636 differ in their downstream outcomes [ 38 ], we explored a plausible BafA1-dependent reversal of apilimod-triggered vacuolization with a focus on the underlying cellular mechanism of the rescue effect. We identified attenuated rise in intracellular PtdIns3P and reduced recruitment of the fusogenic EEA1 protein, rather then mitigated PtdIns(3,5)P 2 loss, to be key mechanistic determinants associated with BafA1 prevention of cytoplasmic vacuolization.…”

Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of β-Coronaviruses

“…The solids were collected by filtration and washed with additional saturated NaHCO3 solution, followed by water, and dried in vacuo to give the title compounds, which were used in the next step without further purification. (36). The reaction was carried out according to general procedure A using 33 (8.05 g, 32.1 mmol) in THF (300 mL) and water (35 mL) and DDQ (14.6 g, 64.3 mmol) in THF (130 mL) to afford the title compound (7.45 g, 87% yield).…”