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BioAssay: AID 1033

NMR Based Screening Assay for the substrate binding domain of the chaperone DnaK

The misregulation of protein folding often results in a variety of deleterious consequences on cellular function that range from the accumulation of protein aggregates leading to neurological disorders, to the inhibition of apoptosis in cancer cells. Several essential components of the protein folding machinery have been identified. For example, molecular chaperones interact with misfolded more ..
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 Tested Compounds
 Tested Compounds
All(3650)
 
 
Active(8)
 
 
Inactive(3642)
 
 
 Tested Substances
 Tested Substances
All(3684)
 
 
Active(9)
 
 
Inactive(3675)
 
 
AID: 1033
Data Source: Burnham Center for Chemical Genomics (SDCCG-A044-DnaK-NMR)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-02-09
Modify Date: 2010-10-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 8
Related Experiments
AIDNameTypeProbeComment
1494ATPase - based assay for small molecule DnaK Modulators targeting the beta-domainConfirmatory depositor-specified cross reference
1495ITC - based assay for small molecule DnaK Modulators targeting the beta-domain.Confirmatory depositor-specified cross reference
1501Small molecule DnaK Modulators targeting the beta domain.Summary1 depositor-specified cross reference
1503Minimal Inhibitory Concentration assay in E. Coli for small molecule DnaK Modulators targeting the b-domain.Confirmatory depositor-specified cross reference
1505Minimal Inhibitory Concentration assay in Y. pseudotuberculosis for small molecule DnaK Modulators targeting the beta-domainConfirmatory depositor-specified cross reference
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: XO1 MH078942
Assay Provider: Dr. Maurizio Pellecchia, Sanford-Burnham Medical Research Institute

The misregulation of protein folding often results in a variety of deleterious consequences on cellular function that range from the accumulation of protein aggregates leading to neurological disorders, to the inhibition of apoptosis in cancer cells. Several essential components of the protein folding machinery have been identified. For example, molecular chaperones interact with misfolded proteins and facilitate their refolding into native states. In E. coli, the chaperone DnaK is part of a multi-subunit complex that efficiently refolds proteins. Small molecules that inhibit DnaK could lead to a better understanding of the mechanism of chaperones and their importance in other diseases. Inhibitors of DnaK might eventually be developed into novel antibiotics.

DnaK consists of three domains: a 44 kDa nucleotide binding domain (residues 1−392), a 13kDa substrate binding domain (residues 393-507) and a 10 kDa alpha helical domain (residues 508-638). It has been proved that the substrate binding domain play a central role in the functions of chaperones. In addition, a deep hydrophobic pock of the substrate binding domain makes it a good target for the small organic molecules. Here, we propose to use unbiased NMR-based screening to identify some small molecules that bind to the DnaK substrate binding domain, which will be further developed into potentially chemical tools against DnaK.
Protocol
1.Protein expression and purification
The gene coding for the E. coli Dnak substrate binding domain (393-507) was amplified with PCR and subcloned into pET21a using the NdeI and BamHI cloning sites. The resulting protein contains proteins with 17 extra amino acid residues (mgsshhhhhhglvprgs) at the N-termini. The protein was expressed in the Escherichia coli strain BL21(DE3) plys S and purified using Ni2+ affinity chromatography. The uniformly 15N-lableled DnaK was produced by growing the bacteria in M9 minimal media containing 15NHCl as the sole nitrogen source. The NMR samples were dissolved in 20 uM sodium phosphate buffer (pH 7.5) containing 90%/10% (H2O/D2O) or 99.5% D2O.
2.SBCCG NMR Screening Compound Library
We have assembled a scaffold library composed of ~ 4,000 compounds. The compounds have been selected based on their anticipated use as building blocks or scaffolds components of further optimized molecules. The scaffold library has been acquired from three different sources and the chemical structures of the library have been deposited into PubChem. In line with the general NIH Molecular Libraries Screening Centers Network (MLSCN) library, we have also included a collection of 602 Natural Products (MicroSource) that could be screened by NMR
3. NMR Based Screening
A. Ligand binding was monitored by comparing the aliphatic region of 1D 1H NMR spectra of a 20 uM DnaK (393-507) solution (20 uM sodium phosphate buffer at pH 7.5 containing 90%/10% H2O/D2O or 99.5% D2O; T= 300 K) in the presence and in absence of compounds tested at a final concentration of 125 uM. Compounds were initially tested at mixtures of 10, and then individual compounds for those mixtures that caused significant perturbations in the spectrum (>0.08 ppm) were subsequently tested for an estimated Kd with a single titration point (3:1 ligand:protein molar ratio, protein at 30 uM concentration). Compounds with an estimated Kd values of < 500 uM are subsequently measured by a complete NMR titration.
B. Hit compounds with estimated Kd values of 200 uM or less are selected for further SAR studies including Kd determination by ITC. Analogues are selected and purchased directly from commercial sources and tested for binding to the target. SAR data are further analyzed to design additional analogues to be chemically synthesized.
4.Binding Constant Determination by NMR
Dissociation equilibrium constants (Kd) of ligands were determined by monitoring the protein chemical shift changes as a function of ligand concentration. Kd of ligand is initially estimated with a single titration point according to the following equation:
Kd=[To](1-p)([Lo]-[To]*p)/([To]*p)
To and Lo are total concentration of target protein and ligand, respectively. The parameter p represents the fractional population of bound versus free species at equilibrium, which for fast exchanging ligands is measured as:
p = (^obs - ^f)/(^sat - ^free)
^obs is the observed receptor chemical shift during the titration, and ^free and ^sat are the chemical shifts for the receptor in the unbound and fully bound (saturated) states, respectively.
Hit compounds (those with the highest estimated binding affinity:in this case those with Kd values of 500 uM or better) are subsequently determined more accurately by a more complete NMR titration. At least six data points were collected for different concentration of ligand and Kd can then be determined by a non-linear least squares fit of p versus [Lo].
p = {([To]+[Lo]+Kd)- Sqr(([To]+[Lo] + Kd)**2 -4[Lo][To])}/2[To]

Comment
A. The score is an estimate that reflects the binding affinity of the compound for the target and the accuracy of the method used to measure the dissociation constants. Compounds with an estimated Kd > 4000 uM are assigned a Kd of 4001 uM. Compounds for which we do not have a complete titration curve are scored on a scale from 0 to 40 according to the following scale:
Kd > 4000 uM Assigned Score 0
1000 500 < Kd < 1000 uM Assigned Score 20
100 < Kd < 500 uM Assigned Score 30
Kd < 100 uM Assigned Score 40
Kd values for compounds that scored 30 or better are treated as active and are subsequently measured more accurately by NMR titration. These molecules are scored on a scale from 50 to 100 as follows:
100 50< Kd < 100 uM Assigned Score 60
10< Kd < 50 uM Assigned Score 70
1 < Kd < 10 uM Assigned Score 80
0.1< Kd < 1 uM Assigned Score 90
Kd < 0.1 uM Assigned Score 100
B. Compounds that reduce protein solubility as detected by an overall reduction of target NMR signal intensities upon addition of a 1:5 protein-ligand ratio (protein concentration 10-50 uM) are considered potential aggregators. This phenomenon is accounted in the score as -1 and the observation is directly stated as a comment.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Kd_QualifierThis qualifier is to be used with the next TID, Kd. If the qualifier is ">", the Kd result is greater than that value. If the qualifier is "<", the Kd result is less than that value.String
2KdDissociation ConstantFloatμM
Additional Information
Grant Number: XO1 MH078942

Data Table (Concise)
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