KR101285375B1 - Method of Screening Compound Having Antiviral Activity - Google Patents

Abstract

The present invention provides methods for screening novel compounds with antiviral activity using neuraminidase antigens, carbon nanotube semiconductor device sensors, probe stations, and source meter devices. The method according to the invention allows for the simple and rapid screening of novel compounds with antiviral activity.

Description

Screening Compound Having Antiviral Activity {Method of Screening Compound Having Antiviral Activity}

The present invention relates to a method for screening novel compounds having antiviral activity. More specifically, the present invention relates to a method for screening novel compounds with antiviral activity using neuraminidase antigens and carbon nanotube semiconductor device sensors as receptors.

Carbon nanotube field-effect transistors (CNT-FETs) manufactured using semiconducting single-walled carbon nanotubes are being actively researched as nano biosensors that recognize biomaterials. When the target material is bound to the receptor attached to the surface of the carbon nanotube channel of the carbon nanotube semiconductor device sensor, an electric signal of a microcurrent is generated in real time. Research papers have been published that the detection sensitivity can be up to fM as a current analysis instrument. Carbon nanotube semiconductor device sensor can be mass-produced by MEMS process, and low cost and simple operation can be used to observe large quantities of samples by converting the characteristics of reaction between receptor and target material occurring on the surface of carbon nanotube in a short time directly into electric signal. This is a very suitable technique for early drug candidate screening systems of novel drug derivatives.

In general, a probe station and a source meter are semiconductor inspection devices that inspect the electrical characteristics of a semiconductor wafer chip. The probe station and the source meter contact a pad of a semiconductor chip with a fine needle (probe card probe), and then It is a device to check the state (good / bad) of the chip with an electrical signal (Fig. 1). Utility Model Registration No. 286040 uses a probe station for biochip inspection.

Antiviral agents, such as zanamivir and oseltamivir, are hemagglutinin (HA) or enzyme neuraminidase that are present as mushroom-shaped dendritic cells on the surface of influenza A and B viruses. NA). These proteins bind to the membrane surface of infected target cells by cleaving sialic acid residues of sialo glycoproteins and glycolipids. Also, at the end of viral replication, neuraminidase is essential for cleaving sialic acid from the receptor so that the virus is released. WO 92/06691 discloses compounds that bind to neuraminidase and are claimed to exhibit antiviral activity in vivo. US 5,952,375 discloses novel compounds as neuraminidase inhibitors.

Recently, however, there has been an increasing number of reports of persistent mutations in drug-resistant influenza strains and in particular of type A viruses, and such resistant variants or combinations are propagable and completely pathogenic. In this respect, the risk of outbreak of the avian influenza A (H5N1) virus is emerging most recently.

In order to solve such a problem, the development of the method of the simple and rapid screening of the antiviral compound which has a novel structure is desired.

In order to solve the problems desired in the prior art as described above, an object of the present invention is to easily and quickly screen novel compounds having antiviral activity using neuraminidase antigens and carbon nanotube semiconductor device sensors. To provide a way.

In order to achieve the above object of the present invention, the present invention is a method for screening a compound having antiviral activity,

Attaching the neuraminidase antigen to the surface of the carbon nanotube channel of the carbon nanotube semiconductor device sensor,

Attaching a silicon well to a channel of a carbon nanotube semiconductor element sensor to which a neuraminidase antigen is attached, and mounting it to a stage unit of a probe station,

Adding a reaction buffer solution to a silicon well of a carbon nanotube semiconductor sensor mounted on a probe station, and applying a voltage to the gate electrode; and

After placing the sample in a silicon well, a method is provided that includes identifying a change in electrical conductivity using a source meter.

In the present invention, the probe station and the source meter may be used to inspect the electrical characteristics of a conventional semiconductor chip. The probe station includes a stage unit in which a carbon nanotube element sensor is located, a silicon well for loading a sample into a channel of the semiconductor element sensor, a source unit disposed on one side of the stage unit, and the other side of the stage unit to face the supply unit. A drain unit disposed at the gate unit, and a gate unit disposed at the other side of the source and drain stage units (FIG. 2).

Carbon nanotubes are carbons that form a round circle and grow long in the form of a tube. Among them, single-walled carbon nanotubes are hollow, semiconducting single-walled tubes of 1-2 nanometers (millions of millimeters) made of carbon. It is applied to carbon nanotube electronic devices because it has electrical, chemical and physical properties that are superior to existing materials such as better electrical conductivity than copper and better heat transfer ability than diamond. In the method of the present invention, a semiconductor element sensor using a channel made of such carbon nanotubes is used.

The attachment of neuraminidase to carbon nanotube (CNT) channels uses a linker. As the linker, 1-pyrenebutanoic acid succinimidyl ester, biotin linker, and gold nanoparticles are mainly used.

The neuraminidase antigen is a protein located on the surface of influenza A and B viruses and is a functional protein involved in viral host cell infection and proliferation. Protein structure was identified by peptide sequencing and gene sequencing by separating and purifying neuraminidase from influenza virus. Mass production of full sequence protein or oligopeptide antigen was achieved through the development of recombinant and oligopeptide synthesis techniques. This is possible and commercially available.

The carbon nanotube channel to which the neuraminidase antigen is attached is attached to the stage unit of the probe station by attaching a silicon well.

In the state in which the gate voltage is applied to the semiconductor device sensor mounted on the stage unit of the probe station, the sample is placed in a silicon well attached to the carbon nanotube channel region, and then the change in electrical conductivity is observed. It is preferable to use the sample in the concentration of pg / ml ~ ug / ml, the gate voltage is preferably applied in the range of -1 ~ +1 volts.

The change in conductivity is checked by a source meter connected to the probe station's source, drain and gate electrodes. The operating process of the system is a structure in which the driving and data processing software in the source meter collects and displays the electrical signals generated by the driving of the semiconductor device sensor and the sensor. On the display screen of the meter, a graph in which an electric signal flows between the source and the drain of the semiconductor element sensor is displayed.

The principle of the screening method according to the invention is as shown in FIG. 3. When a newly synthesized compound derivative binds to the neuraminidase antigen, a change in electrical conductivity is detected on the graph under the influence of impedance in the current flow between the source and the drain, and the change is confirmed by confirming the change with neuraminidase. It is possible to observe the characteristics of the binding strength between the compound derivatives and the stability pattern of the binding complex and search for a compound that inhibits neuraminidase enzyme activity based on this.

According to the method according to the invention, it is possible to screen novel compounds with antiviral activity simply and quickly. In particular, the method according to the present invention is excellent in reaction specificity by using carbon nanotube channels to which neuraminidase antigen is attached. Due to the characteristics of the semiconductor device, it is possible to detect minute electrical changes, thereby improving detection sensitivity, and the result can be quickly and accurately determined by changing the reaction between the neuraminidase antigen and the target substance directly on the surface of the carbon nanotube. The characteristics of the reaction from the start of the reaction to the end of the reaction, that is, the strength of the initial binding reaction and the stability of the complex after the coupling reaction can be observed in real time. The screening method of the present invention can be used to quickly screen for derivatives of zanamivir or oseltamivir with novel structures.

1 is a photograph showing an example of a probe station and an analyzer.
2 is a schematic diagram showing a schematic structure of a stage unit of a probe station and a carbon nanotube semiconductor element sensor used in the present invention.
3 is a schematic diagram illustrating the principle of binding activity measurement of a receptor (neuramidase) using a carbon nanotube semiconductor element sensor device at a probe station.
4 to 7 show the binding strength and the binding by the electrical conductivity measurement for the sample containing 1 pg / ml, 1 ng / ml of zanamivir (FIGS. 4 and 5) and oseltamivir (FIGS. 6 and 7), respectively. Stability graph.
8 is a graph showing the effect on the electrical conductivity by the non-specific reaction of the phosphate buffer solution (PBS).
9 is a graph showing the effect on the electrical conductivity by non-specific reaction of phenolic compounds.
10 and 11 are graphs of bond strength and bond stability by electrical conductivity measurement for the zanamivir derivatives of Examples 3 and 4, respectively.
12 is a graph showing bond strength and bond stability by measuring electrical conductivity of the zanamivir derivative of Comparative Example 3. FIG.

The present invention can be better understood by the following examples, and the following examples are intended to illustrate the invention and are not intended to limit the scope of protection defined by the appended claims.

Example

A. On the carbon nanotube channel New laminase  Attachment of antigen

A linker (1-pyrenebutanoic acid succinimidyl ester) was washed with a phosphate buffer solution on a sensor attached to a carbon nanotube channel and dried with nitrogen gas. It was added to the nanotube channel and allowed to stand overnight in a 4 ° C. refrigerator to attach the neuraminidase protein to the carbon nanotube surface. Then, washed three times with a phosphate buffer solution, and then reacted with 1 mM ethanolamine at room temperature for 1 hour to remove the unreacted linker activity, and then washed three times with phosphate buffer solution to attach the neuraminidase to the carbon nanotube semiconductor device. The manufacture of the sensor was completed and refrigerated.

B. Sample Preparation

To prepare the Example and Comparative Example samples (Compound + PBS) as shown in Table 1 below:

ingredient density Example 1 Janamivir 1 pg / ml, 1 ng / ml Example 2 Oseltamivir 1 pg / ml, 1 ng / ml Comparative Example 1 PBS (phosphate buffer solution) 1.5mM / ml Comparative Example 2 Phenolic Compounds (Bisphenol A) 1ng / ml

C. Probe  Measurement of electrical conductivity change of the sample in the station device

To the stage unit of the probe station (MST 5000, MSTech Inc.), a carbon nanotube semiconductor device sensor with a neuraminidase antigen prepared in B was attached and silicon wells were attached, and then 10 μl of PBS reaction solution was added to the silicon wells. Input a gate voltage of -0.5 V and stabilize the current flowing between the source and drain of the sensor element in a source meter (Keithley 4200, Keithley Instruments Inc.) connected to the probe station's source, drain, and gate electrode. After confirming that, 1 ul of the samples prepared in B (Examples 1 and 2, Comparative Examples 1 and 2) were added, and a change pattern of the electrical conductivity of each sample was confirmed. The electric conductivity change pattern graphs are shown in FIGS. 4 to 9, respectively.

As can be seen in Figures 4 to 7, the neuraminidase antigen attached to the carbon nanotube semiconductor device sensor used in the screening method of the present invention is very specific with zanamivir (relenza) and oseltamivir (tamiflu) The reaction was carried out, and showed a strong reactivity even in a very small amount of 1 pg / ml (Fig. 4, Fig. 6).

8 and 9, it can be seen that the neuraminidase antigen attached to the carbon nanotube semiconductor device sensor used in the screening method of the present invention does not react with PBS and phenolic compounds.

From the above results, it can be understood that the screening method according to the present invention can specifically screen derivatives of novel structures such as rerenza and Tamiflu, which can inhibit neuraminidase.

D. New Janamivir  Derivative Screening

For the zanamivir derivative having a novel structure as shown in Table 2, the neuraminidase inhibitory activity was searched as in the above A to C, and the electric conductivity change graphs are shown in FIGS. 10 to 12:

Compound structure density Example 3

1ng / ml Example 4

1ng / ml Comparative Example 3

1ng / ml

In the case of the zanamivir derivative of the structure (urea derivative) as in Examples 3 and 4, the binding activity with neuraminidase showed a similar bond strength and pattern of binding stability as zanamivir (Fig. 10 and Fig. 11). It can be seen that it can be applied as a viral agent.

Janamivir derivatives having the same structure as in Comparative Example 3 did not cause a binding reaction with the neuraminidase antigen (FIG. 12).

Therefore, the screening method of the present invention can easily screen derivatives having a neuraminidase enzyme inhibitory activity that can be applied as an antiviral agent among zanamivir derivatives having a novel structure.

Claims (5)

As a method of screening compounds having antiviral activity,
Attaching the neuraminidase antigen to the carbon nanotube semiconductor device sensor,
Attaching a silicon well to a channel region of a carbon nanotube semiconductor element sensor to which a neuraminidase antigen is attached, and mounting the silicon well to a stage unit of a probe station,
Adding a reaction buffer solution to a silicon well of a carbon nanotube semiconductor element sensor mounted on a stage unit of a probe station, and applying a gate voltage; and
Inserting a sample and checking the change in electrical conductivity. The method of claim 1, wherein the attachment of the neuraminidase antigen to the carbon nanotube semiconductor device sensor is via any one linker selected from the group consisting of 1-pyrenbutanoic acid succinimidyl ester, biotin, and gold nanoparticles. How to do. The method of claim 1, wherein the gate voltage ranges from −1 Volt to +1 Volt. The method of claim 1, wherein the neuraminidase antigen is from swine flu virus. The method of claim 1, wherein the sample contains a zanamivir derivative or an oseltamivir derivative.

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