PiColorLockTM detection reagent

Unique properties and applications for measuring phosphate-generating

+44 (0)1223 496170 www.innovabiosciences.com [email protected] Introduction The phosphate detection reagent, PiColorLockTM, changes colour in the presence of inorganic phosphate (Pi) and this property can be exploited to measure any that generates Pi. The reagent is applicable to a large number of enzymes including , , phosphatases, heat- shock proteins and DNA unwinding proteins. The unique formulation of PiColorLock affords enhanced assay linearity, dynamic range and colour stability; it is also possible with PiColorLock to work with unstable substrates (e.g. ATP, GTP) that give high non-enzymatic background signals with other acidic dye-based detection reagents.

Why use colorimetric assays? Assays based on absorbance are simple and attractive in all areas of science. In drug screening environments, absorbance assays can also be miniaturised without loss of performance e.g. when switching from 96-well to 384-well formats, there is no impact on measured values because absorbance is proportional to path length (depth of solution), which can be maintained despite miniaturisation. For virtually all other screening methodologies a reduction in assay volume results in lower assay signals. Colorimetric assays for inorganic phosphate have a number of obvious advantages over assays that involve the use of radioactivity (e.g. 32P/33P) especially in drug screening applications. The principle of the one-step colorimetric assay for phosphate is shown in Figure 1 below.

Figure 1. Universal assay for inorganic phosphate. Phosphorylated substrate (X-P) is acted on by an enzyme releasing X and inorganic phosphate. The reaction is halted by the addition of an acidified dye reagent (orange), which turns green or yellow in the presence or absence of inorganic phosphate.

Methods for measuring inorganic phosphate using dyes (especially Malachite Green) are well known. However, there are two major problems with these reagents (i) the complexes of dye and inorganic phosphate are prone to precipitation and (ii) the reagents are very acidic, which leads to non-enzymatic hydrolysis of many phosphorylated substrates.

+44 (0)1223 496170 www.innovabiosciences.com [email protected] PiColorlock detection reagent has special additives that enhance the stability of dye complexes. Figure 2 shows the effect of adding inorganic phosphate to three commercial dye reagents and incubating for one hour.

Figure 2. Most detection reagents form very unstable dye complexes and samples need to be read promptly. In cuvette assays there can be a total loss of signal if a precipitate forms and settles. In plate assays, the data become extremely erratic when clumps of material form in the light path. PiColorlock complexes are stable for many hours, which allows multiple assay plates or cuvettes to be conveniently set up as a batch and read later using plate stackers or other automated equipment.

Commercial reagents also show widely varying performance with significant differences in linear range and signal-to-background ratios. For example, in Figure 3 below, the maximum signal that can be generated with reagent A is just 0.5 OD units.

Figure 3. For the purposes of comparison, data for PiColorlock ALS reagent (no longer available) is also shown, as it provides similar absorbance values to competitor products, at least at low levels of inorganic phosphate. Competitors’ products are linear over a much narrower range of concentrations. PiColorlock ALS reagent has now been superseded by PiColorlock Gold, which gives higher OD values.

+44 (0)1223 496170 www.innovabiosciences.com [email protected] There are very significant additional benefits of PiColorLock systems if the substrate of the enzyme is unstable in acid. The terminal phosphate in nucleoside triphosphates is particularly prone to non-enzymatic hydrolysis, and further hydrolysis of diphosphates (e.g. ADP, GDP) to their corresponding monophosphates also readily occurs in acidic environments.

Figure 4. shows the effect of incubating ATP (without any added enzyme) with three commercial dye-based detection reagents.

A rising non-enzymatic background signal is seen with all standard dye-based reagents. One of the greatest advantages of PiColorlock detection reagent, especially for ATPase assays or for other assays with acid labile substrates, is complete suppression of non-enzymatic background signals.

Existing phosphate-generating assays can be converted to PiColorlock format very easily because it is not necessary to change the conditions of the normal enzyme reaction. The detection reagent is added at the end of the assay, which stops the reaction and initiates color development. The presence of phosphate (indicated by the formation of a green colour) can be seen within a matter of seconds and full colour development is achieved within 30 minutes.

Phosphate-generating enzymes A large number of enzymes generate inorganic phosphate from phosphorylated peptides and sugars, and from a wide range of nucleotides. Indeed, ATP hydrolysis is one of the most important reactions in biochemistry. While many ATPases are known, many proteins originally named on the basis of other functions also exhibit ATP-hydrolysing activity. Enzymes that can be assayed with PiColorLock include phosphatases, GTPases, helicases, 5’nucleotidases, DNA gyrases, heat shock proteins (HSPs), P-type ATPases, , and ATP-dependent . This is not an exhaustive listing and there are scores of enzyme targets within the various classes. Assays for enzymes that generate phosphorylated molecules can often be configured to generate inorganic phosphate.

+44 (0)1223 496170 www.innovabiosciences.com [email protected] Applications of PiColorLock in drug discovery and basic research

Over 100 publications to date cite the use of PiColorLock, either as a standalone reagent or as a component of NTPase assays kits, also available from Innova Biosciences. Further details and product codes may be found in the Appendix.

(i) Helicases Helicase enzymes unwind DNA strands and are involved in DNA replication and repair processes in viruses, bacteria and human cells. They are important targets for new antiviral, antibiotic, and anti-cancer agents (1,2). Genetic defects in helicase genes in humans are associated with Bloom’s, Werner’s and Rothmund- Thomson’s syndromes. Bloom helicase protein has been expressed and characterised using PiColorock reagent (22). Heng et al. (3) studied the effects of the fluoroquinolone antibiotic fleroxacin on DNA-binding, unwinding and ATPase activities of Bloom (BLM) helicase. They showed that fleroxacin strongly inhibited ATPase and DNA-unwinding activity. PiColorLock has also been used in several studies on the ATPase and RNA/DNA unwinding activities of West Nile virus NS2BNS3 proteinase-helicase protein (4, 5).

(ii) Gyrases/Topoisomerases Gyrases and topoisomerases introduce actual breaks in DNA in order to relax supercoiling and facilitate DNA replication (compare this to helicases, which simply break hydrogen bonds between strands). DNA gyrase is a clinically validated target for novel anti-tuberculosis (TB) agents. Hammeed et al. (6) used PiColorLock reagent in drug screening studies and identified pyrrolamides as a novel class of antimicrobial agents. These compounds were active against drug-sensitive and drug-resistant TB.

(iii) P-type ATPases This sub-family of ATPases catalyses the selective transport of cations across diverse membrane systems at the expense of ATP. The P-type ATPases have a unique catalytic mechanism that distinguishes them from V-ATPases (see below). There are five P-type subfamilies (P1-P5) with further subclassifications (A, B, C, D, E). The best known P-type family member is the Na+/K+ ATPase, and others include the gastric H+/ K+-ATPase, Ca2+=-ATPase from sarcoplasmic reticulum. Several of these are targets for existing drugs or the focus of discovery efforts (for review, see 2).

A number of publications cite the use of PiColorLock to measure Na/K+-ATPases (7, 8, 9). These enzymes can be measured in crude membranes by testing activity in the presence and absence of the specific inhibitor ouabain (e.g. see 7, 8 for example protocols). Hirz et al. used PiColorlock to measure the activity of recombinant human Na,K-ATPase α3β1 isoform expressed in P. pastoris (7).

Cardiac glycosides are well known inhibitors of Na+/K+ ATPase. Katz et al. used PiColorLock to study the effects of glycosides on recombinant detergent-solubilised isoforms and showed that these molecules (but not ouabain) had moderate selectivity for the a2 subunit, supporting the major role of α2 in cardiac contraction and cardiotonic effects of digitalis glycosides (10).

Human ATP13A2 ATPase (PARK9) encodes a member of the P5 subfamily of ATPases. Moore et al. measured the activity of proteins with known mutations in this gene, which have been associated with early onset Parkinsonism (2).

+44 (0)1223 496170 www.innovabiosciences.com [email protected] Spillman et al. studied ATPase activity of membranes prepared from P. falciparum-infected and non- infected human erythrocytes (11). A new class of anti-malarial agents, spiroindolones, which disrupt sodium regulation in the parasite are thought to act at PfATP4 P-type ATPase as mutations in the ATPase are able confer resistance to the drugs. A lead compound KAE609 (cipargamin; formerly NITD609) is currently in phase 2 clinical trials for treatment of malaria.

(iv) Vacuolar ATPases (V-ATPases) V-ATPases are responsible for acidification of several internal cell compartments. There are some useful natural product inhibitors of v-ATPases, including bafolimycin which is extremely selective for v-ATPase over the P- and F-types in all eukaryotic cells (plants, animals and fungi). V-ATPases are implicated in various disease process and are interesting drug targets (12).

Robinson et al. (13) used PiColorLock to study V-ATPases in human macrophages. They showed that helminth parasites secrete defense molecules that target V-ATPases and prevent lysosomal acidification. In the resulting alkalized environment, antigen processing is disrupted and the helminth is better able to survive because the immune response is attenuated.

(v) P-glycoprotein (Pgp) P-glycoprotein is an ATP-dependent pump which is responsible for efflux of toxic metabolites and drugs. It is also implicated in the development of drug resistance by parasites. Kathinathan used PiColorLock to study the properties of the Pgp homologue SMDR2 fromSchistosoma mansoni (14) and determined the IC50 values for several drugs including praziquantal, which is used to treat schisotosomiasis.

(vi) Kinesins Human Eg5 is part of a family of around ten kinesins that play a key role in intracellular transport and cell division. Eg5 is specifically involved in spindle formation and its activity was studied using PiColorLock by Tan et al. (15). They showed that Eg5 associates with Xeroderma pigmentatosum group F excision–repair protein which suggests that the proteins together play a key role in mitosis and DNA repair. The intrinsic ATPase activity of kinesins can be stimulated several hundred fold in the presence of microtubules and this can form the basis of a screen for ATPase inhibitors using colorimetric detection of released phosphate. A number of inhibitors to human Eg5 ATPase have been identified in this way (16).

(vii) Ligases The ATP-dependent MurE from M. tuberculosis is a new target for the development of antimicrobial compounds. The enzyme is critically important in the biosynthetic pathway for peptidoglycan. A number of natural product inhibitors were identified in a PiColorLock screen (17) and the reagent has been cited in other publications on MurE ligase (18, 19).

(viii) Polynucleotide kinase/phosphatase (PNKP) PNKP is involved in DNA repair processes and is a target for the development of small molecule inhibitors to enhance the toxicity of other cancer therapies, including ionising radiation and topoisomerase I inhibitors (US patent 13/375,876; 20).

+44 (0)1223 496170 www.innovabiosciences.com [email protected] (ix) Pyrophosphate generating enzymes (adenylate cyclase & various synthetases/transferases) A subset of enzymes with nucleoside triphosphate as substrate generates inorganic pyrophosphate PPi (i.e. two phosphates joined together) and a nucleoside monophosphate. PPi does not react with dye- based detection reagents, but it can be measured using PiColorLock simply by adding a pyrophosphatase enzyme to the assay system. The pyrophosphatase hydrolyses nascent PPi molecules to generate two molecules of Pi for each molecule of PPi converted. Rossi et al. used PiColorLock to measure the activity of adenylate cyclase AC which converts ATP into cAMP and PPi (21). Singh et al. measured N-acetylglucosamine-1-phosphate uridyltransferase, which produces an important precursor for peptidoglycan biosynthesis. The enzyme is a drug target in tuberculosis.

(x) Other assays involving monophosphate substrates or PNPP The hydrolysis of a wide range of monophosphorylated small molecules can easily be monitored using PiColorLock reagent. This includes substrates acted on by a very specific enzyme (e.g. inositol monophosphatase) or ‘generic’ substrates such as p-nitrophenyl phosphate, which is commonly used to measure alkaline and tyrosine phosphatases. Instead of measuring the yellow p-nitrophenolate anion, the Pi can be quantified with PiColorlock to give around 4-fold greater absorbance readings.

+44 (0)1223 496170 www.innovabiosciences.com [email protected] Appendix

Common pitfalls and technical tips

(i) Contaminating phosphate – often leads to high background Before starting your assay, it is key to ensure that there is no contaminating phosphate in the buffers, reagents or other materials to be used. It is unusual for contamination to come from glassware unless bottles previously contained phosphate. Even then, a normal wash will ordinarily suffice. It almost goes without saying that phosphate buffers should not be used in the assay! A far less obvious source of unwanted phosphate is the enzyme sample itself. For example, homogenates of tissues and cells will contain lots of inorganic phosphate, as it is a natural constituent of cells. Purified enzymes are far less likely to be contaminated, unless of course the purification procedure uses phosphate buffer. Another common source of contamination is the substrate. Use only good quality materials and to store substrates under the correct conditions.

(ii) Detecting and removing contamination Detecting and removing contaminants is simple enough. The individual components (or mixtures lacking either enzyme or substrate) are tested. If there is unwanted phosphate in the enzyme sample, the following clean-up procedures may be used: a. Dialysis of samples (simple, but slow). Phosphate contamination of media is likely to be quite high (mM concentrations) so you will need to dialyse thoroughly (i.e change the dialysis buffer probably once or twice. b. Desalting (fast but limited to small volumes; you also need to be comfortable in running chromatography columns). c. PiBind phosphate-scavenging resin (very simple, fast and ideal for cleaning up small samples of enzyme and also, if required, removing trace phosphate contamination from buffers) http://www. innovabiosciences.com/affinity-resins/pibind-resin.html

(iii) Interfering substances There are very few laboratory chemicals that will interfere with PiColorLock reagent as it contains special colour accelerators. To get the most recent guidance notes on interfering substances and the concentration limits on chemicals that may be used in assays see the latest PiColorLock protocol on www. innovabiosciences.com.

(iv) Non-enzymatic backgrounds There is a very effective background suppression system but care is required to mix in the stabiliser thoroughly, especially with acid labile substrates. Incomplete mixing will give rise to backgrounds with substrates such as ATP.

+44 (0)1223 496170 www.innovabiosciences.com [email protected] Related products

High Throughput Colorimetric ATPase Assays (inc. PiColorLock) This non radioactive colorimetric assay kit contains all the necessary reagents for measuring enzyme activity (everything included in the PiColorlock kit and more) and is ideal for high throughput drug screening. [601-0120, 601-0121, 601-0122]

High Throughput Colorimetric GTPase Assays (inc. PiColorLock) As above, this non radioactive colorimetric assay kit contains all the necessary reagents for measuring enzyme activity (everything included in the PiColorlock kit and more) and is ideal for high throughput drug screening.

[602-0120, 602-0121, 602-0122]

PiColorLock Gold Phosphate Detection System PiColorLock is a phosphate detection reagent for measuring phosphatases, ATPases, GTPases and other enzymes that release inorganic phosphate (Pi). The PiColorLock Gold Phosphate Detection System includes PiColorLock Gold reagent (high sensitivity) as well as Accelerator, Stabiliser and Pi Standard.

[303-0030, 303-0125]

10mM Lyophilized ATP These lyophilized 10mM ATP vials contain ultra high quality ATP to ensure the lowest possible assay background. (Also supplied as part of the ATPase assay kit). [601-9999]

+44 (0)1223 496170 www.innovabiosciences.com [email protected] References

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