Drug screening assays for phosphate-generating enzymes


Dr Nick Gee CEO/CSO of Innova Biosciences


1. Methods of phosphate detection

2. Application to assays for phosphate generating enzymes

3. Enzyme activity & calculations

4. Malachite green assays

5. Improved malachite assays with greater stability/linear range

6. PiColorLock reagent – advantages for HTS

7. ATPase/GTPase assays


Do I need to worry about enzyme units? Or calculate units?

For drug screening work – No.

Relative activity +/- drug is main consideration.

When comparing enzyme activity in different samples – Yes.

When purchasing enzymes – not essential, but useful to know about

units and unit definitions so you get the best value if there is more

than one supplier.


The enzyme unit:

The amount of enzyme that converts 1umol of substrate into product

in one minute (under standard assay conditions)

If we have ‘x’ enzyme units in 10ul and ‘x’ units in 1ml we have

the same number of units, but different concentrations (i.e. units/ml)

Specific enzyme activity (purity)

The number of enzyme units per mg of enzyme. In the above example,

the specific activity in each case is the same (i.e. units/ml div. by mg/ml

= units/mg)


Calculating enzyme units:

1. Determine the number of umols of product formed

2. Divide by the assay time (in minutes) (umol per min i.e. enzyme units)

3. Multiply by 1000/assay volume in ul (units per ml of reaction mix)

4. Multiply by total assay volume/vol enzyme added (units per ml enzyme)

5. Multiply by the dilution factor if the enzyme was diluted before use

i.e. if 1/100 diluted, multiply by 100 (units per ml of undiluted enzyme)

Signal

Pi


Terms in the numerator and denominator often cancel out:

Activity = (AxC)/500B where, A = concentration of Pi (mM) determined from the standard curve B = assay time in minutes C = dilution factor of the enzyme

e.g. Innova ATPase assay kit:

© Innova Biosciences ltd. 2013. All rights reserved 8

P O-

O

OH

Substrate O

Substrate P

P O-

O

OH

O H

Pi

Abbreviations:


Substrate P Product OH Pi +

Specific Universal

Product

Enzyme

Three assay approaches:

1. Measure loss of substrate (poor; only ~10% converted)

2. Measure appearance of ‘specific’ product (okay)

3. Measure appearance of universal product (ideal)

Assay of phosphate-generating enzymes:


Substrate 32P Product OH 32Pi +

Assay types (i) radioactive

OH Pi + 14C-Substrate P 14C-Product

e.g. Protein substrates

Pase

e.g. Sugar phosphate

Pase

Pros: high sensitivity, no interference from endogenous Pi

Cons: radioactivity, separation steps


Pi + Dye/Stop Read assay

Assay types (ii) colorimetric, end point assay

e.g. Malachite green assay

Pros: simple, non-radioactive

Cons: interference from Pi in samples, very acidic reagent, risk of

non-enzymatic hydrolysis of phosphorylated substrates

Colour

development Reaction


Pi + Inosine Hypoxanthine + R1P

Assay types (iii) Coupled assays

NP

Uric acid

XDH Tetrazolium

Formazan

Pros: continuous, no acidic reagents

Cons: Complex three-enzyme system, not suitable for identification

of inhibitors in HTS

(Red)


Malachite green assay format – 2 steps:

(i) Generate Pi

(ii) Stop and measure


Very simple assay, but there are things to watch out for…

Pi contamination

Non-enzymatic decay

Precipitation


Substrate P Product OH Pi + Pure enz.

Crude extract > substrate must be hydrolysed only by the enzyme of interest

Or, the enzyme has to be pulled down from the sample before assay

Substrate P Product OH Pi + Crude

(v. specific) Enz 1,2,3,4 ..

Setting up assays. Step 1, Q. Pure enzyme or crude extract?


Setting up assays. Step 2, prepare standard curve

Measure absorbance with known amounts of Pi

Ab

so

rba

nce

Pi

Ideally the standard curve

should be linear

Simple to do, no enzyme, no

substrate.


Setting up assays. Step 3, find linear range for the assay

Choose a reaction time that is convenient; 15 min is typical

Add enzyme, measure amount of product formed

All readings must

fall within the linear

range – if not dilute

enzyme a bit more.

Time (min)

Linear

Non linear

Ab

so

rba

nce


A & B - ‘Standard’ malachite green reagents

C - PiColorLock, modified malachite reagent

Precipitation

within 30-60

minutes is

quite common


PiColorlock Gold/ALS and standard malachite reagents

Standard curves:

range of linearity

& sensitivity vary


PiColorlock malachite dye-Pi complex is stable for many hours

Ideal for drug screening labs or in any situation where each

plate cannot be read immediately after colour development


Inhibitor profile for Li+ on IMPase (IMPA-1)

-7 -6 -5 -4 -3 -2 -1 00

50

100

IC50 187 uM

Log dilution (mM)

% A

cti

vit

y


Interferences in PiColorLock malachite assays

Component Conc. Effect

NaCl 250 mM None

KCl 250 mM None

MgCl2 25 mM None

DTT 0.25 mM Slight signal loss

bME 0.5 mM None

Tris 25 mM None

Hepes 25 mM None

Mes 25 mM None

Mops 25 mM None

BSA 0.1 mg/ml None

BSA 1 mg/ml Precipitation

DMSO 2.5% None

Detergents 0, >0.03% None*


Substrate P Product OH (10%) Pi +

Enzyme

+ Substrate P (90%)

Non-enzymatic hydrolysis – a major problem and its cure.

Many phosphorylated substrates are sensitive to acid e.g. ATP, GTP

Malachite reagents are very acidic > high backgrounds


Background signals with various phosphate detection

reagents using ATP substrate (i.e. no enzyme present)

Background

suppression

system in

PiColorLock


Watch out - Pi may be present before you even start!

Substrate (esp. ATP, GTP) may be contaminated with Pi.

Buffers – do not use phosphate assay buffer!

Tissue/cell extracts contain lots of

inorganic phosphate.

Need to desalt or dialyse your samples,

or use PiBind phosphate-scavenging beads.


Removal of 1 mM P i from buffers

of varying pH with P iBindTM resin

Glycine

pH 2

.3

Ace

tate

pH 5

MES p

H 6

MOPS p

H 7

Hep

es p

H 7

.5

Tris p

H 8

No

resin

Blank

(wat

er)

0

1

2

3

A650


ATP

Applications of malachite assays:

ADP Pi ATPase

ATP AMP + PPi Pi ATPase PPase

PPi 2Pi PPase

-

GTP GDP Pi GTPase

Peptide-P Peptide-OH Pi Phosphatase

X-Pn Pi ?Pase X-Pn-1

Sugar phosphate Sugar Pi SPase


Examples from the literature that use modified malachite reagent:

Type Name Area of interest/Notes

Nuclease/Helicase DNA2 DNA repair

Heat shock protein gp96 Systemic lupus erythromatosis

P-type ATPase PARK9 Parkinsonism

ATPase EG5 Spindle formation

DNA-stimulated ATPase SWSAP1 Recombination repair

GTPase/kinase PARK8 Parkinsonism

GTPase DRP-1 Meiosis

GTPase ARF-6 Regulation of PIP2 synthesis

Nuclear GTPase SLIP-GC Replication

Uridyl transferase NAGUT Tuberculosis, UTP > PPi > Pi

Phosphatase PNPK DNA repair, oligo-P substrate

Na/K ATPase human isoforms Muscle control


Reminder of things to think about...

Is my substrate contaminated with free phosphate?

How quickly can I read my assay plates?

Is my substrate sensitive to acid?

By considering these questions it is easy to determine (i) the best

set up for your assay, (ii) whether you need ‘standard’ or ‘modified’

malachite reagent and (iii) whether sample clean up is required.

Is my enzyme pure or crude?

Does my enzyme contain any free phosphate?

Is my substrate hydrolysed by more than one enzyme?


Summary of Innova products and help…..

• Modified malachite reagent – PiColorlock Gold

with background suppression system for acid labile substrates

• PiBind resin for cleaning up enzyme samples (buffers)

• ATPase/GTPase assay kits with ultra-pure ATP/GTP for low backgrounds

• Guide to enzyme units (free)

• Litre quantities available for HTS


References


Contact

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www.innovabiosciences.com

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Drug screening assays for phosphate-generating enzymes

Science

Transcript of Drug screening assays for phosphate-generating enzymes