Inhibition of Human Class I vs Class III Phosphatidylinositol 3

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Inhibition of Human Class I vs Class III Phosphatidylinositol 3'-Kinases Matthew Hassett, Anna Sternberg, and Paul David Roepe Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00413 • Publication Date (Web): 18 Jul 2017 Downloaded from http://pubs.acs.org on July 22, 2017

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Biochemistry

Inhibition of Human Class I vs Class III Phosphatidylinositol 3'-Kinases Matthew R. Hassett, Anna R. Sternberg and Paul D. Roepe* Dept. of Chemistry and Dept. of Biochemistry & Cellular & Molecular Biology Georgetown University 37th and O Streets NW Washington DC 20057

*

Address correspondence to PDR; email: [email protected], tel: 202 687 7300

ACS Paragon Plus Environment

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Abstract Most investigations of phosphatidylinositol 3'-kinase (PI3K) drug inhibition have been via assays based on ADP appearance or ATP consumption (e.g. Liu, Q. et al. (2011) J. Med. Chem. 54, 14731480). However, at least some PI3K isoforms show basal ATPase activity in the absence of PI lipid substrate(s), which may complicate quantification of drug potency, isoform specificity of some drugs, and synergy for drug combinations. In this study, we probe the class I vs class III isoform specificity of a selected set of phosphatidylinositol 3'-kinase (PI3K) inhibitors using a simple,

inexpensive,

semi

high-throughput

assay

that

quantifies

production

of

phosphatidylinositol 3'-phosphate (PI3P) from phosphatidylinositol (PI). Results are compared to previous data largely generated using ATPase activity assays. Good agreement between EC50 values computed via ATPase assays vs the reported PI3P formation assay is found for most drugs, but with a few exceptions. Also, for the first time, drug inhibition of class I vs class III enzymes is compared side-by-side with the same assay for the important class I specific inhibitors GSK2126458 ("Omipalisib") and NVP-BGT226 ("BGT226") currently in clinical development for advanced solid tumors.


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Biochemistry

Introduction

Phosphorylated phosphatidylinositol (PI) derivatives are involved in many different cell signaling pathways, including vesicle trafficking, DNA synthesis, autophagy, and apoptosis1-4. Defects in the metabolism of PI 3'-phosphorylated derivatives have been implicated in human cancers and other diseases, and not coincidentally there are a variety of chemotherapy strategies that envision the use of phosphatidylinositol 3’-kinase (PI3K) inhibitors5-7. PI3K enzymes are classified as class I, II, or III based largely on enzyme kinetic parameters, substrate preferences, and the nature of the phosphorylated PI products produced, and they can also be distinguished based upon domain structure and cofactor dependencies2-3,8-12. The three PI3K classes also differ with respect to their role in cellular physiology8. For example, dysfunctional class I PI3K mediated AKT signaling has been observed in a variety of human cancers7 whereas the yeast class III PI3K, Vps34, plays a vital role in vesicle trafficking and autophagy10,13-15. Volinia et al. first identified the yeast Vps34 human homolog and Petiot et al. confirmed this enzyme's role in activating autophagy, as well as a contrasting autophagy inhibitory effect for the class I PI3Ks9,16. While class I and II PI3K enzymes can both produce phosphatidylinositol 3'-phosphate (PI3P), formation of the majority of cellular PI3P is attributed to Vps34 activity17. Vps34 is the only known class III PI3K and is potentially a specific and unique drug target2,8. Biochemical studies have revealed additional important distinctions between human PI3K enzymes. Beeton et al. compared lipid kinase activities of two purified recombinant class I PI3K enzymes which can utilize multiple substrates18. At 500 µM substrate, the p110β (PI3Kβ) isoform showed lower lipid kinase activity, however, upon titration of substrate, the authors


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found that PI3Kβ is more than twice as active as the p110α (PI3Kα) class I isoform at low substrate concentrations18. For human Vps34, which utilizes only PI as substrate, Volinia et al. found enhanced activity in the presence of a Mn2+ cofactor relative to Mg2+, which is not observed for class I or class II PI3K isoforms9. Based on these differences, and because of their considerable potential medical value, numerous small molecule PI3K inhibitors have been developed in the hopes of perfecting PI3K isoform - specific drugs. However, despite considerable previous work identifying and developing PI3K inhibitors, few studies have directly compared potency vs multiple enzyme isoforms under the same assay conditions. In particular, no studies to our knowledge have directly compared drug inhibition of human class I vs class III PI3K enzyme activities by multiple PI3K inhibitors using the same quantitative assay and PI as substrate. Rusten and Stenmark have reviewed the various assays that have been used to measure PI kinase activity1. While several approaches for assaying PI kinase activity have been developed, not all can be used quantitatively. The most commonly used assays for PI3K activity utilize either radiolabelled-ATP or various ADP / ATP dependent spectrophotometric reporters to quantify consumption of ATP or release of ADP.

They

essentially measure depletion of ATP, which acts as phosphate donor. Although useful, these approaches do not directly measure PI3P production and in some cases interpretation can become complicated since some PI3Ks possess basal ATPase activity19. A few studies have characterized PI3K mediated PI3P production using radiotracer methods, but these are time-consuming, expensive, and complex1. Echelon Biosciences (Salt Lake City, UT) offers convenient PI3K assay kits that measure PI3P production in 96-well ELISA format using a PI3P specific antibody20-21, however, the data that are generated are qualitative


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Biochemistry

with regard to definition of enzyme parameters and molar quantification of drug inhibition. Another approach for analyzing class I or II enzymes relies on fluorescence polarization or FRET measurements and competition between product and a fluorescent reporter for binding to (for example) a pleckstrin domain - containing protein22,23. These are limited to specific PI3K isoforms and are not specific for production of PI3P. Using available Echelon anti-PI3P antibody, we have developed a modified PI3K activity ELISA that directly quantifies enzyme mediated PI3P production through competitive antibody binding. Characterization of the assay followed by direct, side-by-side comparison of the human class I (p110β/p85α) and class III (Vps34) PI3K enzymes further defines the class specificity of important PI3K inhibitors. We find some discrepancy in reported class specificities of some PI3K drugs, and are also able to characterize, for the first time, inhibition of class I (p110β/p85α) vs class III (Vps34) PI3K enzymes by important new PI3K drugs for which no published data were previously available. In the following paper this issue we use this assay to characterize the unique class III PI3K found in the malarial parasite P. falciparum.

Materials and Methods Materials Purified human Vps34 class III PI3K, purified human class I PI3K (p110β/p85α), antimouse IgG-HRP, and 3,3’,5,5’-tetramethylbenzidine were purchased from Sigma (St. Louis, MO). Biotinylated PI3P, PI3P (diC8), PI (diC8), and purified anti-PI3P antibody were purchased from Echelon Biosciences (Salt Lake City, UT). Streptavidin coated plates were purchased from Thermo Fisher (Waltham, MA). PI3K inhibitors were a kind gift of Dr. Craig Thomas, National


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Center for Advancing Translational Science (NCATS), (Rockville, MD). All other reagents and chemicals were reagent grade or better, purchased from Sigma, and used without further purification.

Quantitative PI3P ELISA Assay Streptavidin coated plates were washed three times with Tris buffered saline / 0.1% tween-20 pH 7.4 (TBS-T). 10 pmoles of biotinylated PI3P dissolved in H2O were added to each well and incubated for two hours with light shaking. Plates were covered with plateseal (Sigma) to prevent potential solvent loss due to evaporation. In general enzyme reactions were carried out in Eppendorf tubes, with HsVps34 dissolved in 10 mM Tris / 150 mM NaCl / 10 mM MnCl2 , pH 7.4 (TrisNaCl + Mn), and p110β (HsPI3Kβ) dissolved in TrisNaCl + 10 mM MgCl2 (TrisNaCl + Mg) Substrate concentrations were varied (see results) but were typically 50 μM ATP and 16 μM PI. Before initiation of enzyme mediated reactions via addition of PI and ATP, PI3K enzyme (20 nM) was incubated for 30 minutes at 37oC with drug or buffer alone. When examining drug inhibition reaction time was varied (see results) but was typically 5 min for HsVps34 or 20 min for Hsp110β (see results). Reactions were quenched via addition of EDTA to a final concentration of 17 mM. Reactions were then diluted with detection buffer (10 mM Tris / 150 mM NaCl / 7.5 mM EDTA / 1 mM DTT, pH 7.4). No evaporation of sample was observed visually. Each reaction was then split into three equal volumes and used for triplicate quantification of PI3P. Data shown are typically the results of three independent assays each done in triplicate (9 determinations in total).


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Biochemistry

Plates coated with PI3P as above were washed three times with TBS-T, and quenched reaction solutions were added to the wells along with anti-PI3P antibody at a final concentration of 0.5 μg/mL. The plate was incubated for 1 hr with shaking. Biotinylated PI3P attached to the plate and exogenous PI3P (either from the enzyme reaction or PI3P manually added to construct standard curves; see Results) then compete for binding of anti-PI3P antibody. Plates were then washed three times more with TBS-T. After washing, only antibody bound to biotinylated PI3P attached to the well remains, as any antibody bound to exogenous PI3P is washed away. Anti-mouse IgG-HRP was then added at a final concentration of 1.25 μg/mL and the plate incubated for 30 min with light shaking. The plate was again washed with TBS-T, and 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well and the plate incubated 30 min at RT to develop HRP signal. Development was quenched via addition of 1N sulfuric acid, and absorbance at 450 nm (6 nm bandwidth) quantified using a Victor3V 1420 multilabel counter microtitre plate reader (PerkinElmer Waltham, MA). As shown in Results, the acquired signal is inversely proportional to moles PI3P produced by the PI3K enzyme. Any addition of exogenous PI3P (either from purified PI3P added to generate a calibration curve or produced via PI3K mediated conversion of PI) decreases the absorbance signal that is recorded. The maximum signal decreases upon the addition of increasing PI3P, and continues to decrease to a minimum seen near the highest amounts of PI3P in the standard curve (see below). Standard curves were generated for every experiment, with each point analyzed in triplicate.

Results


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Development of ELISA - based methods for detecting PI3P has been a significant advance20 and these assays are quite useful for empirical quantification of relative amounts of PI3P. In order to directly quantify and compare the specificity and potencies of PI3K inhibitors vs multiple PI3K isoforms, we wished to further optimize an ELISA that could quantify moles PI3P produced per mg enzyme per unit time, which is not immediately possible with commercially available kits. As confirmed below, we suspected that commercial anti-PI3P antibody might show lower affinity binding to PI substrate, potentially complicating interpretation without additional calibration and control experiments. Figure 1 is a schematic representation of the PI3P competition ELISA. As described in Methods, biotinylated PI3P (grey triangles) is first bound to avidin (black circles) coated 96 well polystyrene plates. Variable PI3P (white triangles) is added exogenously (either as pure PI3P to calibrate the assay, or as PI3P produced via a PI3K catalyzed reaction, see below) (Fig. 1, panel A,B). Anti-PI3P antibody (black "Y") is then added, and both bound vs free PI3P then compete for antibody, such that greater PI3P in solution outcompetes antibody binding to PI3P that is bound to the plate (compare panel A vs B). Solution PI3P / antibody complex is then washed away, and antibody bound to the plate is detected as described in Methods. Higher signal corresponds to less PI3P produced, since there is less soluble PI3P to compete for antiPI3P antibody binding to the PI3P that is attached to the plate (that is, the signal generated is inversely proportional to the amount of PI3P formed by the enzyme, compare panel A’ vs B’). However, precise quantification of enzyme activity via this method relies on careful quantification of [PI], [biotinylated PI3P] coated to the wells, and other variables as described below.


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Biochemistry

Figure 1: Optimization of ELISA adapted from the Echelon class III PI3K kinase kit to measure mol product formed per unit time. In A, biotinylated PI3P (grey triangles) is attached to a streptavidin coated well (black circles). Differing amounts of exogenous PI3P (white triangles) produced by activity of the enzyme, or in pure form to generate standard curve data, are then added. Panel B shows more exogenous PI3P than panel A. Depending on the amount of exogenous PI3P, an anti-PI3P antibody (black “Y”) then competes for binding to exogenous PI3P vs biotinylated PI3P bound to the plate. Panel B shows higher levels of exogenous PI3P meaning less antibody binds to biotinylated PI3P bound to the plate relative to the situation depicted in panel A.

After washing, a secondary antibody (asterisk) is added for

chemiluminescent detection of bound PI3P. Wells with more antibody bound to biotinylated PI3P (A’ vs B’) yield higher signal which is inversely proportional to PI3P produced by the enzyme (see Methods).

We titrated known [PI3P] added exogenously vs known [PI] added exogenously, to test specificity of the anti-PI3P antibody. As shown in Fig. 2A (left), when plates are coated with 10


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pmoles of biotinylated PI3P per well (see Methods), antibody signal is progressively lost as added [PI3P] is increased (solid black circles), and as expected is lost completely when exogenous PI3P is increased from 10 to 100 pmoles (solid black circles, Fig. 2A). When PI alone is added exogenously, there is no decrease in signal for added [PI] ≤ 100 pmoles, but antibody binding to PI is clearly apparent at > 100 pmoles of PI (open circles, Fig. 2A). At [PI] ≥ 200 pmoles, very significant signal is lost, demonstrating that at these higher concentrations PI competes effectively vs bound PI3P for available anti-PI3P antibody, and would therefore affect quantification of exogenous (unbound) PI3P produced by PI3K from PI. This is shown more clearly in Fig. 2B (right) where bound PI3P is kept fixed at 10 pmole/well and exogenous PI3P is titrated (x axis) in the absence (black circles) vs presence of variable concentrations of PI. In the presence of 50 pmoles PI (open circles / dotted line, Fig. 2B) or 100 pmoles PI (black triangles, dashed line), titration curves vs added exogenous PI3P are nearly identical to the curve obtained for exogenous PI3P titration in the complete absence of PI (black circles / solid line Fig. 2B). However, as [PI] is increased further (open triangles, black squares, open squares, see caption), signal - to - noise is progressively lost due to progressively increased competition between PI and PI3P for available antibody. Based on these data, we standardized assay conditions such that the concentration of PI substrate was fixed at 100 pmoles/well (corresponding to 16 μM in the assay tube, see Methods) and bound (biotinylated) PI3P was fixed at 10 pmole/well. At this ratio, we find that the balance between assay signal - to - noise and the rate of enzymatic PI3P production (see below) is optimized. This is fortuitous since 16 µM is near estimates of PI substrate Km for human PI3K isoforms17,24 permitting analysis under initial rate conditions and clear quantification of drug inhibition. We find that increasing


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amounts of bound PI3P further at fixed PI lowers assay signal to noise, whereas lowering PI at fixed bound PI3P begins to (not surprisingly) significantly decrease enzyme activity as [PI] moves farther from Km (not shown).

Figure 2: Titration of PI substrate to determine specificity of anti-PI3P antibody. A) Titration of PI3P (black circles) vs PI (open circles). Reactivity vs PI3P appears approximately 10x higher in affinity (see text). (B) PI competes with PI3P for antibody binding. Increasing amounts of PI added in the presence of PI3P titration generates curves that illustrate dampening of PI3P specific chemiluminescence. Constant values of PI that were added to each PI3P standard curve, from top (black circles) to bottom (white squares) are 0, 50, 100, 150, 200, and 400 pmoles. We find that 100 pmol of PI / well yields the largest dynamic range without sacrificing measurement of enzyme activity (see text).

With antibody signal at ratios of substrate PI vs bound PI3P calibrated, we standardized assay conditions and investigated the kinetics of PI3P production from PI via purified Hs class I (p110β/p85α) and Hs class III (Vps34) PI3K enzymes (Fig. 3). As shown (Fig 3A left panel vs Fig. 3B left panel), with 100 pmoles/well (16 μM) PI and 50 µM ATP (near Km, see below), the rate of


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HsVps34 (class III PI3K) mediated production of PI3P is at least 3 - fold faster relative to that of class I p110β/p85α (approximately 104 vs 33 nM / min / mg, respectively, c.f. Table 1).

A

B

Figure 3: Enzyme characterization. Kinetic characterization of human phosphatidylinositol 3’kinases (PI3Ks) HsPI3Kβ (A) and HsVps34 (B). PI3K enzyme (20 nM) was reacted with PI (16 µM) and ATP (50 µM) as described in the methods. Representative kinetic data for each enzyme are shown. (A: HsPI3Kβ PI3P kinetics (initial rate 19.2 nmol/min/mg), ATP titration (Km,app (ATP) 196 µM), pH titration (optimal pH 6.0), B: HsVps34 PI3P kinetics (initial rate 109 nmol/min/mg), ATP titration (Km,app (ATP) 34.4 µM), pH titration (optimal pH 8.0); summarized in Table 1.)


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Biochemistry

HsPI3Kβ This Study

SEM

Previously Published

32.6 nmol/min/mg

4.85

13-19 nmol/min/mg [25]

Optimal pH

pH 6.79

0.61

pH 6.5-6.7 [26]

Km,app (ATP)

209 μM

18.5

26-166 μM [26-28]

This Study

SEM

Previously Published

104 nmol/min/mg

12.0

78-216 nmol/min/mg [29]

Optimal pH

7.89

0.11

pH 7-8 [9,20,27-29]

Km,app (ATP)

41.7 μM

5.75

9.3-120 μM [17,27-28]

Specific Activity

HsVps34

Specific Activity

Table 1: ELISA validation using human PI3Ks. The modified PI3K activity ELISA optimized for in vitro PI3K analysis was validated with class I and class III human PI3Ks (PI3Kβ and Vps34) respectively. Averaged data from multiple independent trials with pure PI as substrate (each trial in triplicate) are shown vs previously published values (with citation; note some earlier measurements may use a mixture of substrates and quantify activity via ATP consumption, not production of PIP3). pH and Km, app (ATP) values are extracted from studies or manufacturer protocols analyzing immuno-purified or recombinant human PI3Ks in vitro.

ATP was then titrated for each enzyme at these initial rate conditions and data revealed apparent Km for ATP near 210 μM and 40 μM for class I vs class III human enzymes, respectively (compare Fig 3A middle panel vs Fig. 3B middle panel; see also Table 1 for a summary). Our calculation of ATP Km for HsVps34 is similar to Km previously reported using commercial "ADP Glo" kit optimization27 whereas we find Km for the class I enzyme that is slightly higher than the range reported previously26-28 via assays that quantify enzyme activity by ADP production


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monitored from ATP luminescence, loss of FRET signal, or radio tracer methods26-28 (see Table 1). We also varied pH under these initial rate conditions and observed pH optima near 6.5 and 7.8 for class I vs class III Hs PI3K enzymes, respectively (Fig 3A right panel vs Fig. 3B right panel, and Table 1), again consistent with previous reports. Table 2 summarizes drug inhibition data culled from the literature for key inhibitors vs class I, II, and III PI3Ks, as well as Hs PI4K and Hs PIPK (see caption). In a number of these reports, drug inhibition is measured via a decrease in ATP consumption or decreased production of ADP. Others measure inhibition via drug competition for substrate binding33, loss of reporter fluorescence due to PIP3 production35-36, or radiolabelled PI3P detection via TLC, scintillation counting, or membrane capture assays19,30,39-42. However, perhaps due to the lack of convenient inexpensive assays that quantify production of non-radioactive PI3P in terms of mol PI3P / time / mg enzyme, the majority of available reports have measured PI3K enzyme inhibition via ATP consumption/ADP production or radiolabelled PI3P production. Only one study to our knowledge39 has assayed both class I and III PI3K enzymes side-by-side using the same quantitative assay (a scintillation proximity assay using radiolabelled ATP), and four other studies19,30,40,42 have examined both classes side-by-side using radiolabelled ATP (with product then spotted on either TLC plates or nitrocellulose).


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Biochemistry

Inhibitor

Class I PI3K

Class II PI3K

Class III PI3K

PI4K

Torin1 [30-33]

0.25-1.8 µM (α) 0.2-4.9 µM (β) 171 nM (γ)

176 nM (α) 549 nM (β)

0.533-3 µM

6.68 µM (β)

Torin2 [33-34]

4.68 nM (α) 180 nM (β) 5.67 nM (γ)

28.1 nM (α) 24.5 nM (β)

8.58-14 nM

18.3 nM (β)

INK128 [35]

219 nM (α) 5.29 µM (β) 221 nM (γ)

> 100 nM (α) > 1 µM (β)

> 1 µM

> 1 µM

GSK2126458 [36]

0.04 nM (α) 0.019 nM (α app Ki) 0.13 nM (β app Ki) 0.06 nM (γ app Ki)

NVP-BGT226 [37]

4 nM (α) 63 nM (β) 38 nM (γ)

SAR405 [38]

> 10 µM

> 10 µM

1.2 nM

PIK-III [39]

3.96 µM (α) > 9.1 µM (β) 3.04 µM (γ)

PIK93 [19,40-42]

39-48 nM (α) 590 nM (β) 4-17 nM (γ)

16 µM (α) 140 nM (β)

18 nM

4.43 µM (β)

307-617 nM

1.1 µM (α) 17-19 nM (β)

PIPK

0.99-2 µM

> 100 µM

Table 2: Previously published EC50 values for PI3K inhibitors against known human PI kinases. Previously published EC50 values for a variety of PI3K inhibitors vs human PIK isoforms. Cited studies from which the values originated reported inhibitor activities against immuno-purified or recombinant enzyme in vitro.

Two of these studies39,42 report drug inhibition data for only one drug, while the other three19,30,40 report inhibition by more than one drug, but these comparisons do not quantify


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mol PI3P produced / unit time / mg enzyme. Two other studies31,34 have examined both enzymes side-by-side using the same assay, but by measuring ADP production, and without examining inhibition by class I vs class III - specific PI3K inhibitors. Finally, only one study to our knowledge reports inhibition by multiple drugs vs both class I and class III enzymes side-by-side using the same assay, in this case via measurement of ADP38. In sum, these experiments have revealed important concepts but they were not designed to quantify mol PI3P product / unit time / mg enzyme as in the present work, and in general did not quantitatively compare inhibition of product formation by a range of isoform specific drugs. We therefore measured drug inhibition of purified class I vs class III PI3K enzymes sideby-side by quantifying moles PI3P produced from PI at varied [drug] for 8 well known PI3K inhibitors (Fig. 4).


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Biochemistry

PIK-III

SAR405

GSK2126458

NVP-BGT226

INK128

PIK93

Figure

4:

PI3K

chemical Shown are the

inhibitor structures.

Torin1

Torin2

structures of

the 8 PI3K inhibitors used in this work vs class I and class III human PI3K isoforms.


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This panel of 8 drugs was chosen specifically due to their chemical diversity, range of activity and range of previously reported PI3K isoform specificities. Clear class specificity was observed for some drugs. For example, Fig. 5A,C shows raw data for (no) inhibition of class I vs (potent) inhibition of class III enzyme by PIK-III. In other cases, clear inhibition of PI3P production was noted for both class I and class III enzymes. For example, Fig. 5B,D shows raw data for inhibition of class I and class III enzymes by GSK2126458, which has been suggested to be specific to class I PI3K enzyme36. However, direct side-by-side comparison of inhibition of class I vs class III enzyme production of PI3P has not previously been reported for GSK2126458. We indeed find effective GSK2126458 EC50 of approximately 1.34 nM vs 364 nM for the two enzymes, respectively (Table 3), consistent with the previously reported isoform specificity of this drug. Relatedly, we measure class III specificity for inhibition by SAR405 and other PI3K drugs similar to that which can be estimated or surmised from previous studies (summarized in Table 3). However, we find that potency and isoform specificity for some PI3K inhibitors differs compared to previous reports that were based primarily on ATPase activity assays.

For

example, we find PIK93 to be a somewhat more potent inhibitor of the class III PI3K when inhibition is measured via PI3P production, and we find lower potency vs class I PI3K for Torin1 and Torin2 when potency is measured via PI3P production from PI. We also note impressive nanomolar level potency of the drug NVP-BGT226 vs the human class III PI3K that to our knowledge

has

not

previously


been

reported.

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Biochemistry

HsPI3Kβ Drug

EC50

SEM

HsVps34

Published EC50

EC50

SEM

Published EC50

Measured Fold Specificity vs HsVps34 Measured

Published

SAR405

> 10 µM

--

> 10 µM [38]

1.11 nM

0.14

1.2 nM [38]

> 9000

> 8000

PIK-III

> 10 µM

--

> 9.1 µM [39]

9.4 nM

1.31

18 nM [39]

> 746

> 500

Torin2

400 nM

30.2

180 nM [33]

11.5 nM

0.70

8.58-14 nM [3334]

35

21

Torin1

> 10 µM

--

0.2-4.9 µM [30,32-33]

784 nM

29.8

0.533-3 µM [3033]

> 12.8

0.375

NVP-BGT226

55.8 nM

15.4

63 nM [37]

7.03 nM

1.96

--

7.9

--

PIK93

539 nM

55.8

590 nM [19,40]

220 nM

25.8

307-617 nM [19,40,42]

2.5

1.9

INK128

6.84 µM

0.37

5.29 µM [35]

5.62 µM

0.63

> 1 µM [35]

1.2

5.3

GSK2126458

1.34 nM

0.14

0.13 nM (app Ki) [36]

364 nM

39.8

--

0.004

--

Table 3. Evaluation of PI3K inhibitor class specificity. Effective concentrations for half-maximal inhibition (EC50) of PI3P activity were measured for a panel of PI3K inhibitors vs purified HsPI3Kβ and HsVps34. Average data from at least 3 independent trials performed in triplicate for each drug against each PI3K enzyme are shown and compared vs previously published values (with citation). Values shown in italics contrast with those previously observed for the inhibitor, and values in bold italics have not been previously measured.


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Biochemistry

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A

B

C

D

Page 20 of 33

Figure 5: Enzymatic inhibition using PI3K inhibitors. Representative data vs HsPI3Kβ (A,B) and HsVps34 (C,D) enzymes is shown. PI3K enzyme was pre-incubated with varying concentrations of drug (x axis) before enzymatic reactions were initiated as described in methods. Representative inhibition curves for each enzyme are shown, the midpoint of exponential curve fits to these data (using Sigmaplot) defines EC50. A: HsPI3Kβ + PIK-III (no significant inhibition below 10 µM), B: HsPI3Kβ + GSK2126458 (EC50 = 1.3 nM; see Table 3), C: HsVps34 + PIK-III (EC50 = 9.4 nM), D: HsVps34 + GSK2126458 (EC50 = 364 nM).


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Biochemistry

Discussion In this paper, we have further optimized a simple ELISA - based approach so that it can be used to quantify PI3K enzyme activity in terms of moles PI3P produced from moles PI substrate / unit time / mg enzyme. Earlier studies using ELISA methods typically report enzyme activity as "percent control" or another empirical measure, however, direct quantification of drug potency or the nature of drug combination inhibition (synergistic, additive, or antagonistic) of enzymes requires the ability to quantify activity in terms of moles product produced per unit time from known moles substrate. We find that such quantification is indeed possible via the ELISA approach, but requires careful titration of antibody specificity for PI substrate vs PI3P product. Since class I and class II PI3Ks can also utilize additional substrates (PI4P and PI4,5P22,3) to produce small amounts of other 3'-phosphorylated products, careful systematic titration of any antibody used to detect these other products vs relevant substrate, would also need to be done if quantification of these other enzymatic activities is desired. We have validated the optimized assay by comparing two different purified human PI3K enzyme isoforms side-by-side, and have then quantified inhibition of both isoforms by a panel of PI3K drugs. All of these drugs have previously been tested vs at least one of the two isoforms, but with one exception39 in all published examples to our knowledge, any previous drug inhibition comparisons vs multiple PI3K isoforms could only be done qualitatively relative to control. Since some PI3K enzymes are known to have basal ATPase activity in the absence of substrate, and since some consume multiple substrates, enzyme activity measurements via ATP consumption can formally have multiple interpretations. Our aim in this study was to assist ongoing consolidation of the wide range of reported EC50 values for these drugs vs different


21

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Page 22 of 33

PI3K isoforms. Quantitative improvements in the ELISA assay format reported here allow for convenient calibration of activity in terms of mole product per mole substrate per unit time. Directly quantifying product formation from known amounts of substrate is a particularly important way to quantify the potency of drug inhibition of an enzyme. The optimized PI3P assay presented here is a relatively fast and inexpensive semi high-throughput method that reduces the costs associated with other plate - based PI3K enzyme assays that rely on radiolabelled substrates or other expensive reagents. Our initial studies of PI3K inhibitors using this quantitative PI3P assay verifies a number of previous observations made with ATP consumption assays but also highlights new conclusions. Although much progress has been made recently, relatively few PI3K drugs have been found to be potent and class specific, and few have been quantified for their direct effects on PI3P production. No previous studies have examined inhibition via a panel of PI3K drugs vs both class I and class III enzymes, side - by - side, using an assay that quantifies PI3P production. The first generation Torin drug (Torin1) is known to be class specific vs mTOR kinase activity30-32. The second generation Torin (Torin2) shows increased potency against mTOR, however, also shows decreased class specificity33-34. Class I PI3K specific drugs GSK2126458 and NVP-BGT226 have previously been measured to be potent vs multiple class I PI3Ks. In all these cases however, basal ATPase activity of PI3K enzymes might complicate quantification of potency and specificity to some extent, whereas direct measurement of PI3P production eliminates those complexities. We find that for Torin1 and Torin2, quantification of PI3P production reveals that both inhibitors have sub-micromolar activity vs class III PI3K enzyme, with Torin2 being quite potent


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Biochemistry

(EC50 = 11.5 nM). Via PI3P production, we are able to quantify for the first time approximately 250 - fold specificity vs the class I PI3K enzyme, relative to class III, for the important drug GSK2126458. For the first time, we also quantify activity for NVP-BGT226 vs class I and class III enzymes side-by-side and note impressive (EC50 = 7 nM) activity vs class III enzyme. Although NVP-BGT226 is thought of as class I PI3K specific, we can find no previously published data vs other PI3K classes, and our side-by-side quantification of PI3P production suggests the drug is mildly class III PI3K specific relative to class I.

This conclusion perhaps merits further

investigation in cell based assays and other experiments. A co - crystal structure for GSK2126458 bound to a human class I PI3K has been reported36 and reveals key enzyme - drug interactions involving enzyme residues K833, Y867 and V882. Via alignment of the human class III PI3K sequence, these residues are found to be K, Y and I, respectively, in the Hs Vps34 (class III PI3K) enzyme (data not shown). Thus, it is possible that subtle changes in structure near position 882 alter GSK2126458 potency for some PI3K enzymes. With respect to NVP-BGT226, again, we find that the compound is quite potent (EC50 = 7 nM) vs class III enzyme, even though it is typically characterized as a class I inhibitor37. When quantified via production of PI3P, we find that BGT226 potency vs Hs Vps34 (class III PI3K) lies between that of SAR405 and PIK-III, which have both recently been discovered to be potent class III specific inhibitors38-39. Unlike SAR405 and PIK-III however, BGT226 retains significant potency vs class I PI3K enzyme. Apparently, the structural differences in PIK-III relative to BGT226 "tune" potency away from class I enzyme without necessarily improving potency vs class III enzyme.


23

Biochemistry

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Page 24 of 33

In sum, we have shown that it is possible to use ELISA - based methods to quantify moles product / unit time / unit enzyme for PI 3'-kinases and to use such methods to quantitatively compare class I vs class III PI3K drug inhibition.

Despite some conflicting

classification of these inhibitors via earlier ATP consumption assays and PIP3 production assays, our results are largely in agreement with previous data in the literature. For example, our data are consistent with previous work showing that the inhibitors SAR405 and PIK-III are highly Vps34 (class III) specific

38-39

. In addition, although Torin1, Torin2, and INK128 have been

reported to be selective mTOR inhibitors30-35, previous studies have reported Torin1 and Torin2 potency against PI3Ks as well, which we find to be similar to what is measured here via PI3P production. However, although NVP-BGT226 and GSK2126458 have been reported to be selective for class I PI3K36-37, we find that these PI3K inhibitors also possess activity vs HsVps34 (class III PI3K), with BGT226 being mildly class III specific relative to class I. Additional work perhaps focusing on additional substrates for class I PI3K isoforms should further aid quantification of class specificity for these important PI3K inhibitors.


24

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Biochemistry

Acknowledgements We thank Dr. Craig Thomas (National Center for Advancing Translational Science) for PI3K inhibitors and helpful discussions.

We also thank our laboratory colleagues Dr. Amila

Siriwardana and Mr. Bryce Riegel for technical assistance. Supported in part by NIH RO1 AI111962.


25

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For Table of Contents use only.


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Inhibition of Human Class I vs Class III Phosphatidylinositol 3

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