LOD ~7.5 nM LOD ~3.2 nM

Fast and Direct Measurement of Glycolysis Reactants by SPE-MS/MS: Conversion of Fructose-6-phosphate to Fructose-1,6-bisphosphate as a Case Study Peter T. Rye and William A. LaMarr SLAS 2014, Poster #197

Abstract/Introduction

Conclusions

Measuring Glycolysis Reactants Using SPE-MS/MS

Allosteric activation of PPi-PFK by F16bP and F26bP

• The RapidFire High-throughput Mass Spectrometry System is a novel platform for the analysis of glycolysis reactions that leverages the fast (8.5 seconds per sample) and direct measurement of substrates and products by SPE-MS/MS.

• The conversion of fructose-6-phosphate to fructose-1,6-bisphosphate by PPi-dependent Phosphofructokinase (PPi-PFK) was used as a model system to demonstrate the utility of SPE-MS/MS for measuring in vitro glycolysis reactions.

• Concentration-response curves were conducted for ten different reactants within the glycolysis pathway, and the results demonstrate that the detection strategy has good sensitivity (average limit of detection = 5.4 nM), dynamic range (nM to µM = three orders of magnitude), and linear response (average R^2 = 0.9982).

• The ability of SPE-MS/MS to measure multiple reactants within the glycolysis pathway, utilizing a single set of parameters, demonstrates the potential of this platform for pathway based analyses in drug discovery.

Direct Measurement of PPi-PFK Substrate and Product Inhibition of PPi-PFK by Phosphate

Glycolysis is the ten-step metabolic pathway that converts glucose into pyruvate, and produces cellular energy in the form of ATP. Some tumors exhibit glycolytic rates that are two hundred times higher than normal tissues, and, as a result, the enzymes that participate in this pathway are focal points of cancer research. Biochemical assays to identify glycolysis inhibitors have been developed; however, many assays suffer from indirect readouts of coupled reactions, which can increase the risk of data artifacts. Here, a novel assay platform for analyzing glycolysis reactions is presented that leverages the fast and direct measurement of substrates and products by SPE-MS/MS.

Using the RapidFire High-throughput Mass Spectrometry System by Agilent, a single analytical method was developed that enables multiple glycolysis reactants to be measured at the same time. Eleven-point concentration-response curves, including a zero, were conducted for ten different reactants within the glycolysis pathway. The results demonstrate that the detection strategy has good sensitivity (average limit of detection = 5.4 nM), dynamic range (nM to µM = three orders of magnitude), and linear response (average R^2 = 0.9982). Direct analyses from 96- or 384-well plates were sustained at 8.5 seconds per sample, permitting the measurement of 10,000 wells a day.

To test the detection method on reactions, the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate by PPi-dependent Phosphofructokinase (PPi-PFK) was used as a model system. Data that characterize the allosteric activation of PPi-PFK are presented. Additionally, competitive and noncompetitive inhibitors of PPi-PFK are validated to highlight how direct detection of reactants can simplify the design and interpretation of mechanistic experiments. In summary, a novel SPE-MS/MS method for measuring glycolysis reactants is presented that demonstrates unprecedented accuracy, robustness, and versatility.

Inhibition of Glycolysis for Anticancer Treatment

IC50 ~2.4 mM

MDPA

An eight-point inhibition curve (50 mM Tris 7.5, 5 mM MgCl2, 1 mM DTT, 18 µM NaPPi, 100 µM F6P, and 10 µM 13C6-F16bP) was conducted using MDPA, a pyrophosphate analog, and the IC50 was calculated to be ~2.4 mM (top left). Substrate titrations were carried out in the presence of inhibitor, illustrating that MDPA inhibits PPi-PFK noncompetitively to F6P (top right) and competitively to PPi (bottom right).

Noncompetitive with F6P

MDPA does not affect Vmax

Competitive with PPi

Inhibition of PPi-PFK by a Pyrophosphate Analog (MDPA)

IC50 ~750 µM

An eight-point inhibition curve (50 mM Tris 7.5, 5 mM MgCl2, 1 mM DTT, 18 µM NaPPi, 100 µM F6P, and 10 µM 13C6-F16bP) was conducted using phosphate, and the IC50 was calculated to be 750 µM (top left). Substrate titrations were carried out in the presence of inhibitor, illustrating that phosphate inhibits PPi-PFK noncompetitively to both F6P (top right) and PPi (bottom right).

Noncompetitive with F6P

Noncompetitive with Pi

Kact = 3.4 µM Kact = 3.0 µM

• To circumvent non-linear enzyme kinetics, F26bP can be added to the assay

• For our studies, assays were supplemented with saturating concentrations of 13C6-F16bP

• No detriment to assay sensitivity

• provided an internal standard for an additional option for data normalization

Measurement of Multiple Glycolysis Reactants

O OH

OHHO

H2O4P OH

O OH

OHHO

H2O4P PO4H2

Fructose-6-phosphate (F6P)

Fructose-1,6-bisphosphate (F16bP)

PPi-PFK PPi Pi

activation inhibition

F26bP, F16bP Pi

R More active

T Less

active

PPi-PFK has a tetrameric structure, composed of two α and two β subunits, that shifts into a more active conformation upon the binding of allosteric effectors.

Kact of F16bP and F26bP for PPi-PFK

Substrate and Enzyme Titrations on PPi-PFK Reactions

2DG (Phase I/II for prostate cancer) Lonidamine (Phase II/III for benign hyperplasia)

3PO (Preclinical)

TLN-232/CAP-232 (Phase II for MRCC)

• The “Warburg effect” is the observation that most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation.

• Targeting the glycolysis pathway may preferentially kill malignant cells.

Porporato PE, Dhup S, Dadhich RK, Copetti T, Sonveaux P. Front Pharmacol. (2011) Aug 25;2:49.

Hill slope of 2.8 ½ Vmax at 2 mM F6P KM = 18 µM

R^2 of fit to 2.5 µM LOD (nM)

G 6P glucose-6-phosphate 0.9939 6.5

F 6P fructose-6-phosphate 0.9998 7.5

F 1,6bP F ructose-1,6-bisphosphate 0.9999 3.2

G 3P glyceraldehyde-3-phosphate 0.9954 5.8

2,3bP G 2,3-bisphosphoglycerate (isomer of 1,3-bP G ) 0.9983 18.5

3P G 3-phosphoglycerate 0.9995 1.0

2P G 2-phosphoglycerate 0.9991 4.0

P E P phosphoenolpyruvate 0.9988 1.0

AT P adenosine triphosphate 0.9983 1.7

ADP adenosine diphosphate 0.9990 4.7

MS/MS Transitions Q1 (unit)

Q3 (unit)

CE CAV

glucose-6-phosphate 259.0 79.0 65 2 fructose-6-phosphate 259.0 79.0 65 2 Fructose-1,6-bisphosphate 338.9 79.0 65 2 13C6-fructose-1,6-bisphosphate 345.0 79.0 65 2 glyceraldehyde-3-phosphate 168.9 79.0 35 5 2,3-bisphosphoglycerate (isomer of 1,3-bPG)

264.9 79.0 45 5

3-phosphoglycerate 184.9 79.0 80 7 2-phosphoglycerate 184.9 79.0 40 7 phosphoenolpyruvate 167.0 79.0 65 7 adenosine triphosphate 506.0 79.0 35 5 adenosine diphosphate 426.0 79.0 35 5

Rapid Fire Conditions

Solvent A H2O + 6 mM octylammonium acetate

Solvent B 80% acetonitrile in water

SPE Cartridge C4 RF State 1: Aspirate 600 ms (sip sensor)

RF State 2: Load/Wash 3000 ms RF State 3: Elute 4000 ms RF State 4: Re-equilibrate 500 ms

Pump 1 flow rate 1.5 mL/min

Pump 2 flow rate 1.25 mL/min

Pump 3 flow rate 1.25 mL/min

MS Source Parameters ESI (-) Agilent 6490 Drying gas temp. 225 °C Drying gas flow 15 L/min Sheath gas temp. 300 °C Sheath gas flow 12 L/min Nebulizer pressure 20 psi Nozzle voltage 1000 V Capillary voltage 2000 V

Eleven-point concentration-response curves, plus a zero, were conducted for ten different reactants within the glycolysis pathway. The results demonstrate that the detection strategy has good sensitivity (average limit of detection = 5.4 nM), dynamic range (nM to µM = three orders of magnitude), and linear response (average R^2 = 0.9982).

The conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F16bP), by PPi-PFK, was chosen as a model system to illustrate the simplicity of measuring glycolysis reactants by SPE-MS/MS. Eleven-point concentration response curves, plus a zero, were conducted for the reaction substrate (bottom left) and product (bottom right), demonstrating good sensitivity, dynamic range, and linear response.

PPi-PFK is activated by F16bP, the reaction product, causing the progress curve for the reaction (top right) to have a sigmoidal shape. Comparison of product formation in reactions that contain 10 µM F26bP, to those that do not, shows F26bP also has a significant effect on enzyme activity (bottom right).

The saturation curve of PPi-PFK for F6P (top left) was sigmoidal, indicating that F6P binding by one enzyme subunit positively influences the F6P binding by the other enzyme subunit. The Hill coefficient was calculated to be 2.8 and half-maximal velocity was reached at 2 mM F6P. The saturation curve of PPi-PFK for PPi (top right) was hyperbolic with a KM in the range of 18 µM. From these enzymatic determinations, an enzyme titration time course was conducted with conditions adequate for screening (bottom left). Plotting the initial velocities against the concentration of enzyme used, provided a linear response (bottom right).

Phosphate

Saturation curves for F16bP and F26bP were both hyperbolic with Kact values of 3.0 and 3.4 µM, respectively.

no activator 10 µM F26bP

µM MDPA µM MDPA µM MDPA µM MDPA

µM Pi µM Pi µM Pi µM Pi

µM MDPA µM MDPA µM MDPA µM MDPA

µM Pi µM Pi

µM Pi µM Pi