Major applications

FBDD (Fragment-based Drug Discovery) is a new drug discovery method to find fragment compounds which bind to target proteins, and to optimize them to make lead compounds. Fragment compounds have simple structures, small molecular weight, and low affinity.

AP-3000 has the following unique properties and a best system suited for fragment screening.
- High sensitivity that makes it possible to detect compounds with molecular weight of 100Da.
- Throughput (3,840 assays/day) equivalent to the size of fragment libraries(103~104)
- Small protein consumption.
- Automatic calculation of KD values (Data mining software, Spotfire DecisionSite for AP-3000 is installed.)

Fragment screening is carried out as follows.
(1)Setting screening conditions (optimization of protein immobilization, etc.)
(2)Prescreening of a library (at one high concentration level) to eliminate nonspecific binding compounds.
(3)Main screening of the library (at one high concentration level) to identify hit compounds
(4)Dose response measurement to confirm reproducibility of hit compounds and to calculate affinity constant.
Because of its high throughput and automatic analysis functions, AP-3000 can find hit compounds quickly in a short time frame and calculate the affinity constant automatically by measuring the dose response.

Day 1

(1)Optimization of protein immobilization conditions (6 conditions):

Day 2

(2)3,840 compounds prescreening*1:

3,840 dp*2

Day 3

(2)3,840 compounds prescreening:

3,840 dp

Day 4

(2)3,840 compounds prescreening:

3,840 dp

Day 5

(3)3,840 compounds Main screening*1:

3,840 dp

Day 6

(3)3,840 compounds Main screening:

3,840 dp

Day 7

(3)3,840 compounds Main screening:

3,840 dp

Day 8

Review of main screening results/ preparation of analyte plates for dose response.

Day 9

(4)120 Compounds Dose response:

1,920 dp

Day 10

(4)120 Compounds Dose response:

1,920 dp

Day 11

(4)120 Compounds Dose response:

1,920 dp

Day 12

(4)120 Compounds Dose response:

1,920 dp

Day 13

(4) 96 Compounds Dose response:

1,536 dp

Day 14

Review of dose response measurement results.

Total 32,256 dp

*1

Pre-screening: screening to see how a compound library behaves
Main screening: screening of the library from which some nonspecific binding compounds are eliminated.

*2

dp:data points

*3

The hit rate is 5%, and the dose response of hit compounds is done at 8 points x n=2 per one compound.

Day 1

(1)Optimization of protein immobilization conditions(6 conditions):

Day 2

(2)2,304 compounds Prescreening:

2,304 dp

Day 3

(3)2,304 compounds Main screening:

2,304 dp

Day 4

Review of main screening results/ preparation of analyte plates for dose response.

Day 5

(4)115 Compounds dose response:

1,840 dp

Day 6

Review of dose response measurement results.

Total 6,448 dp

It is required to measure multiple compounds repeatedly at the same area of protein immobilization in order to minimize protein consumption. However, if there are strong nonspecific binding compounds, ordinary washing does not do thorough dissociation, and measurements of the compounds that follow those nonspecific binders get affected, causing incorrect binding signals for them. Therefore it is recommended to do prescreening to eliminate nonspecific binding compounds before the main screening.

AP-3000 has the capability to skip injection of specific compounds on analyte plates into sensors. Using this function, the compounds which the prescreening identifies as those not to be further measured do not get injected into the flow paths in sensors and therefore will not be measured. Accordingly, analyte plates for the prescreening and those for the main screening are exactly same, and therefore reconstructing of plates is not necessary. This makes preparation of analyte plates simple and easy.

SPR methods have not been used for the random library screening because of their low throughput. AP-3000 changes this, and makes SPR well suited for the random library screening, because of the following reasons:

- High throughput ( Maximum of 3,840 compounds/day)
- Label free binding assays
- Fully automated, running for 24 hours unattended.
- Small protein consumption
- Automatic calculation of KD values (Data mining software, Spotfire DecisionSite for AP-3000 is installed.)
- Function to recover the substrate solution incubated in the flow paths where enzymes are immobilized. Enzymatic activities can be confirmed by measuring the recovered substrate solution.
- Function to customize measurement protocols according to experiment goals (AP-3000 Planner Software is installed.)

Label free binding assays by AP-3000 do not require building of complicated assay protocols. Therefore, this widely increases a range of target proteins to select from. Even when measurements cannot be completed within one day because of the library size being large, it is possible to plan ahead using AP-3000 Planner Software. With this software, the number of days, and the amount of consumables, reagents, and buffers, which are required for a full process of the screening can be planned, prior to the screening,
Once the needed target proteins, compounds, reagents and buffers are set, and the program is started, the screening for 24 hours is completed automatically without any attendance.

In recent years, screening with a library focused on each target protein has become popular. AP-3000 is well suited for the focused library screening because of the following reasons.
- Throughput capacity matching the number of compounds in a focused library (Maximum of 3,840 compounds/day).
- Label free binding assays.
- Fully automated, running for 24 hours unattended.
- Small protein consumption
Automatic calculation of KD values (Data mining software, Spotfire DecisionSite for AP-3000 is installed.)
- Function to recover the substrate solution incubated in the flow paths where enzymes are immobilized. Enzymatic activities can be confirmed by measuring the recovered substrate solution.
- Function to customize measurement protocols according to experiment goals (AP-3000 Planner Software is installed.)

For instance, when screening is done on a focused library targeting proteins such as kinase, the target protein sometimes loses its activity at the time of immobilization. It is possible to confirm if or not there is a loss of activity by measuring the binding between the immobilized protein and positive control compounds. But still there is a possibility of nonspecific binding taking place between positive control compounds and the protein which has lost activity or been denatured.
The above problem can be solved by the following function unique to AP-3000. AP-3000 can recover the substrate solution incubated in the flow paths where target proteins are immobilized. Enzymatic activities can be confirmed by measuring the recovered substrate solution. This enables satisfactory screening.

At the same time, using the data mining software, Spotfire DecisionSite for AP-3000, the following can be automatically calculated:
- Dissociation rate constant (kd )
- Affinity constant (KD )
- Binding activity (Stoichiometric ratio between immobilized proteins and binding compounds)
It is easy to get verification and feedback of the measurement results promptly, because analysis can be carried out by looking at dose response curves.

For the secondary screening, the hit rate is extremely high because the compounds have been already identified as hit compounds by the random screening, and therefore there is a large possibility for these compounds to include tight binders.
AP-3000 is good for the secondary screening because of the following reasons.
- High throughput ( Maximum of 3,840 compounds/day)
- Automatic calculation of KD values (Data mining software, Spotfire DecisionSite for AP-3000 is installed.)
Easy measurement without tight binders (Capability to skip injection of specific compounds on analyte plates into sensors.)
- Label free binding assays
- Fully automated, running for 24 hours unattended.
- Small protein consumption
- Function to recover the substrate solution incubated in the flow paths where enzymes are immobilized. Enzymatic activities can be confirmed by measuring the recovered substrate solution
- Function to customize measurement protocols according to experiment goals (AP-3000 Planner Software is installed.)

It is required to measure multiple compounds repeatedly at the same area of protein immobilization in order to minimize protein consumption. However, if there are tight binders, ordinary washing does not do thorough dissociation, and measurements of the compounds that follow those tight binders get affected, causing incorrect binding signals for them. Therefore it is recommended to do prescreening to eliminate tight binders before the secondary screening.
AP-3000 has the capability to skip measurements of compounds at any specific locations on a plate injection of specific compounds on analyte plates into sensors. Using this function, the compounds identified as those not to be further measured do not get injected into the flow paths in the sensors and therefore will not be measured. Therefore reconstructing of plates is not necessary. This makes preparation of analyte plates simple and easy.
It is also possible to customize measurement protocols to dissociate tight binders by using the functions such as extending the dissociation time, using regeneration solutions, and increasing the number of washes.

AP-3000's sensor is made of the flow-paths and prisms all combined together. AP-3000 has following properties and because of them, it is good for screening of antibodies.
- High throughput (Maximum of 3,840assays/day)
- Ease of maintenance. Because of wide and short flow paths, even highly concentrated samples which are easily precipitated do not clog up the flow paths. (Disposable sensor/pipette tips)
- Fully automated, running for 24 hours unattended.
- Automated calculation of physical constant (Data mining software, Spotfire DecisionSite for AP-3000 is installed)

Especially since the flow-path is wide and short, crude samples injected do not clog up the path, and even when the path gets clogged up, it is easy for a user to replace it with a new one by simply changing the disposable sensor. Thus measurement can be restarted right away, because the vender's services are not required.

As computing technology advances, there is an increasing trend to do In-Silico screening of a virtual library based on the idea of SBDD(Structure Based Drug Design). Because of the following properties, AP-3000 is ideally suited for screening of In-Silico derived compounds.
- High throughput ( Maximum of 3,840 compounds/day)
- Easy measurements without non specific binding compounds (Capability to skip injection of specific compounds on analyte plates into sensors.)
- Ease of maintenance. Because of wide and short flow paths, even compounds which are easily precipitated do not clog up the flow paths. (Disposable sensor/pipette tips)
- Label free binding assays
- Fully automated, running for 24 hours unattended.
- Small protein consumption
Automatic calculation of KD values (Data mining software, Spotfire DecisionSite for AP-3000 is installed.)
- Function to customize measurement protocols according to experiment goals (AP-3000 Planner Software is installed.)

By combining In Silico screening and direct binding assays on AP-3000, real binders for target proteins, which have not been previously available, can be found efficiently.

Because of the following properties, AP-3000 is applicable to screening plasma samples from living bodies.
- High throughput (3,840 compounds/day)
- Ease of maintenance. Because of wide and short flow paths, even crude samples which are easily precipitated do not clog up the flow paths.(Disposable sensor/pipette tips)
- Fully automated, running for 24 hours unattended.
- Function to customize measurement protocols according to experiment goals (AP-3000 Planner Software is installed.)

Measurements are fully automated, which allows expansion of experiments capacity.

AP-3000 is well suited for binding assays of agricultural compound library.
Solubility of low molecular compounds for agricultural chemicals is low, which often causes precipitation during preparation of analyte plates. AP-3000 has a function to skip injection of specific compounds on analyte plates into sensors, and thus avoid the precipitated compounds to be injected and measured in the sensors. At the same time, AP-3000's own unique sensors that have flow-paths and prisms all in one offer the following features:
- Flow-paths are wide and short, and do not get clogged up easily.
- Sensors with the flow-paths can be replaced by users.

Because of these features, even if precipitated compounds go through, they do not clog up the flow-paths, and should the flow paths get clogged up, users can solve the problem simply by replacing the sensors without the vender services. Thus flexibility for experiments is increased.