Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Summary

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

Abstract

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Introduction

Approximately 25% of the total cellular proteins are membrane proteins and about 60% of them are drug targets^1,2. One of the potential drug targets³, membrane-bound pyrophosphatases (mPPases), are dimeric enzymes that pump proton and/or sodium ion across the membrane by hydrolysis of pyrophosphate into two orthophosphates⁴. mPPases can be found in various organisms⁵ such as bacteria, archaea, plants, and protist parasites, with the exception of humans and animals⁴. In protist parasites, for example Plasmodium fa....

Protocol

1. Protein preparation

NOTE: The expression and purification of TmPPase has been described elsewhere¹³.

1. Prepare 10 mL of the reactivation buffer solution containing 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 6.5, 3.5% (v/v) glycerol, 2 mM dithiothreitol (DTT), and 0.05% dodecyl maltoside (DDM).
2. Prepare 10 mL of the reaction mixture containing 200 mM Tris-Cl pH 8.0, 8.0 mM MgCl₂, 333 mM KCl, and 67 mM NaCl.
   NOTE: Mg²⁺ is required to chelate the pyrophosphate as the substrate of mPPase, K⁺ is required to increase the enzyme activity as TmPPase....

Results

In this protocol, eight compounds (1−8) were tested (Figure 2A) together with IDP, a common inhibitor of pyrophosphatases, as a positive control. Each compound was tested at three different concentrations (1 µM, 5 µM and 20 µM) in triplicate. The workflow of the screening is depicted in Figure 1, starting from sample and reagent preparation until the absorbance measurement at 860 nm.

At the end of this protocol, after the addition of .......

Discussion

Here we report a detailed protocol for simple screening of inhibitors for membrane-bound pyrophosphatase from T. maritima in a 96 well plate format based on Vidilaseris et al.¹⁴. This protocol is inexpensive and based on 12-phosphomolybdic acid, which is formed from orthophosphate and molybdate under acidic conditions and reduced to phosphomolybdenum species with a distinct blue color¹². This method is preferred over other protocols, such as the more sensitive mala.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesive sealing sheet	Thermo Scientific	AB0558
Ammonium heptamolybdate tetrahydrate	Merck	F1412481 636
Ascorbic acid	Sigma-Aldrich	95212-250G
BioLite 96Well Multidish	Thermo Scientific	130188
Dimethyl sulfoxide (DMSO)	Merck	1167431000
8-well PCR Tube Strips 0.2 ml without caps (120)	Nippon genetics	FG-028
Dodecyl maltoside (DDM)	Melford	B2010-100G
Ethanol	Merck	1009901001
Glacial acetic acid	Merck	1000631011
Hydrochloric acid	Sigma-Aldrich	258148-500ML
Imidodiphosphate sodium salt	Sigma-Aldrich	I0631-1G
L-α-Phosphatidyl choline from soybean lecithin	Sigma	429415-100GM
Magnesium chloride	Sigma-Aldrich	8147330500
Multiplate 96-Well PCR Plates	Bio-Rad	MLL9651
MultiSkan Go	Thermo Scientific	10680879
Nepheloskan Ascent (Type 750)	Labsystems
Polystyrene Petri dish (size 150 mm x 15 mm)	Sigma-Aldrich	P5981-100EA
Potassium chloride	Merck	104936
Prism 6 software	GraphPad
QBT2 Heating block	Grant Instruments
Sodium meta-arsenite	Fisher Chemical	12897692
Sodium phosphate dibasic (Pi)	Sigma	S0876-1KG
Sodium pyrophosphate dibasic	Fluka	71501-100G
Trisodium citrate dihydrate	Fluka	71404-1KG