uHTS Fluorescent assay for identification of inhibitors of hexokinase domain containing I (HKDC1)
The gene HKDC1 (HexoKinase Domain Containing I) was found to encode a fifth mammalian hexokinase. Since hexokinases 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 more ..
BioActive Compounds: 540
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 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 metabolism that leads to Type 2 diabetes, a disorder of glucose metabolism that morbidly afflicts 35 million Americans and perhaps close to 200 million worldwide. It is our expectation that HTS to find small molecule inhibitors 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we have found that HKDC1 mRNA and protein are greatly increased in pancreatic islets from diabetic animal models (unpublished data).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. A small molecule inhibitor of HKDC1 would be a key component of the effort to define the function of HKDC1 in mammals and could help us begin to understand whether inhibition of HKDC1 activity could be an avenue toward a novel approach to treating Type 2 diabetes and its complications.
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). 60 nL 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 >= 35% 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)