uHTS Fluorescent assay for identification of activators of hexokinase domain containing I (HKDC1)
The gene HKDC1 (HexoKinase Domain Containing I) was found to encode a fifth mammalian hexokinase. Since hexokinases (HKs) are the first step in glucose metabolism, they play a major role in regulating the metabolic fate of glucose in the tissues they are expressed. Understanding both the proper and pathological metabolism of glucose is obviously of critical importance to deciphering the more ..
BioActive Compounds: 189
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: R21 NS061703-01S1
Assay Provider: Dr. Jeff Johnson, Metabolex, Inc., Hayward, CA.
The gene HKDC1 (HexoKinase Domain Containing I) was found to encode a fifth mammalian hexokinase. Since hexokinases (HKs) are the first step in glucose metabolism, they play a major role in regulating the metabolic fate of glucose in the tissues they are expressed. Understanding both the proper and pathological metabolism of glucose is obviously of critical importance to deciphering the dysregulation of glucose that leads to Type 2 diabetes, a disorder of metabolism that morbidly afflicts 35 million Americans and perhaps close to 200 million worldwide. It is our expectation that HTS to find small molecule activators for HKDC1 will begin the process of identification of probes that can serve to help define the physiological function of this newly described enzyme and also to initiate determination of whether HKDC1 could be a target for diabetes therapeutics.
The expression pattern of HKDC1 is unique among hexokinases, and this may provide some initial clues to its function in biology. Unlike hexokinase I (HK I) which is broadly expressed, HKDC1 has been found selectively in the pancreatic islets of Langerhans, retina, kidney, and the jejunum segment of small intestine. Strikingly, we have found that HKDC1 mRNA and protein are greatly increased in pancreatic islets from diabetic animal models (unpublished data). Increased hexokinase activity in the insulin producing islet cells may participate in the hyperglycemia-induced decline in insulin secretion capacity in Type 2 diabetes, a phenomenon described as glucotoxicity. The retina and kidney also suffer from hyperglycemia-induced complications of diabetes that may be facilitated by the presence of HKDC1 in these tissues. Finally, the jejunum is the major site of glucose absorption from meals, and it is likely that HKDC1 plays a role in this process as well.
One member of HK enzyme family, Hexokinase IV or glucokinase, has been the subject of intense pharmaceutical development of small molecule activators for treatment of Type 2 diabetes. The other members of the family have not been substantially investigated for small molecule activators, but there are good reasons to think that they can be found by HTS and that they would be extremely useful as probes and potentially key components of the effort to target HKs therapeutically. First, most HKs have known allosteric regulatory sites that bind substrates or substrate like molecules and regulate enzyme activity. Allosteric activation of HKDC1 has not yet been demonstrated, but HTS is probably the best approach to obtain such activators. Secondly, the example of glucokinase suggests that there may be additional, as yet undescribed, sites for allosteric activation in the HK family. An activator of HKDC1 would be of great value in determining the physiological role of the enzyme in the tissues where it is expressed.
A. Brief Description of the Assay:
This assay is to look for activators or inhibitors of HKDC1 (Hexokinase domain containing-1, or HK5) enzyme. It will be measured by fluorescence in 1536 well plate format.
Item, Source, Cat#
HK5 Enzyme Stock Solutions, In-house, N/A
Glucose, Sigma-Aldrich, G6152
NADP, Amresco, 0760
Resazurin, Sigma-Aldrich, R7017
ATP, Sigma-Aldrich, A7699
Diaphorase, Sigma-Aldrich, D5540
G6PDH, Sigma-Aldrich, G8404
Tris-HCl pH 8.0, Sigma-Aldrich, T6066
KCl, Sigma-Aldrich, P9541
Tween-20, Sigma-Aldrich, P1379
MgCl2, Sigma-Aldrich, M8266
Bovine serum albumin, Sigma-Aldrich, A7888
Beta-mercaptoethanol, Calbiochem, 444203
Mol. Grade H2O, Mediatech, 46-000-CM
1536-well, black, non-binding plates, Aurora, 00029844
1. Prepare Reagents as described in section D. Recipe.
2. Using LabCyte Echo, transfer 60 nL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48 (final concentration of test compounds is 20 uM, 1% DMSO). 60nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.
3. Spin plates at 1500 rpm for 1 minute on Vspin.
4. Using the Kalypsys with angled tips, add 3 uL/well of Mix 1 (buffer) to col. 1-2 for the positive control.
5. Using the Kalypsys with straight tips, add 3 uL/well of Mix 2 (enzyme) to col. 3-48 for the negative control and test compound wells.
6. Using the Kalypsys with straight tips, add 3 uL/well of Mix 3 (substrate) to col. 1-48.
7. Spin plates at 1500 rpm for 1 minute on Vspin.
9. Incubate plates in the dark at room temperature for 90 minutes.
9. Using a Perkin Elmer Viewlux read the plates utilizing a fluorescent protocol at Ex. 525 nm, Em. 613 nm.
25 mM Tris-HCl pH 8.0, 50 mM KCl, 0.02% Tween-20, 10 mM MgCl2, 0.1 mg/mL BSA, 5 mM BME, mQ H2O
Mix 1 for 2.73 mg/mL stock of HK5 (Prep 2+3):
*663 uM glucose (331.5 uM FAC), 10 ug/mL HK5 (5 ug/mL FAC), 2 mM NADP (1 mM FAC), 200 uM Resazurin (100 uM FAC), in Assay Buffer
Mix 1 for 2.72 mg/mL stock of HK5 (Prep 4+5):
*645 uM glucose (322.5 uM FAC), 15 ug/mL HK5 (5 ug/mL FAC), 2 mM NADP (1 mM FAC), 200 uM Resazurin (100 uM FAC), in Assay Buffer
2 mM ATP (1 mM FAC), 2 U/mL G6PDH (1 U/mL FAC), 0.2 U/mL Diaphorase (0.1 U/mL FAC), in Assay Buffer
700 uM glucose (350 uM FAC), 2 mM NADP (1 mM FAC), 200 uM Resazurin (100 uM FAC), in Assay Buffer
*There are two different stock concentrations of HK5 (2.73 mg/ml prep 2+3, 2.72 mg/ml prep 4+5) that have different FAC in the assay.
E. Special Note:
1. All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
2. Make up assay buffer minus BME in large scale and add fresh BME just before the assay starts.
3. Storage conditions after reagents are made up:
Reagent, Temperature, Notes
Buffer minus BME, 4 oC
HK5, -80 oC
Glucose, -80 oC
NADP, -80 oC
Resazurin, -80 oC, light sensitive
ATP, -80 oC
Diaphorase, -80 oC
G6PDH, -80 oC
Compounds that demonstrated an inhibition of <= -40% at 20 uM concentration are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)