ITC - based assay for small molecule DnaK Modulators targeting the beta-domain.
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 ..
BioActive Compounds: 9
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 Proposal Number: XO1 MH078942
Assay Provider: Dr. Maurizio Pellecchia, Sanford-Burnham Medical Research Institute, San Diego, CA
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.
In an earlier assay we screened libraries of small molecules for their ability to interact with the substrate binding domain of DnaK(AID 1033). In this assay, using a construct of DnaK corresponding to only the b-domain (residues 393-507), the most promising hit and its analogs were tested to determine their binding affinity to DnaK using Isothermal Titration Calorimetry.
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1. The gene coding for the E. coli DnaK substrate binding (beta) domain (393-507) was amplified with PCR and subcloned into pET21a using the NdeI and BamHI cloning sites. The resulting protein contains 17 extra amino acid residues (MGSSHHHHHHGLVPRGS) at the N-terminus.
2. The protein was expressed in the Escherichia coli strain BL21(DE3) pLysS and purified using Ni2+ affinity chromatography.
3. DnaK, human Hsp70, DnaJ, and GrpE were purchased from Stressgen (Ann Arbor, MI).
4. The model substrate NRLLLTG was synthesized by the Medical College of Wisconsin (Milwaukee, WI).
1) Titrations were done using a VP-ITC from MicroCal (Northampton, MA).
2) Full-length DnaK or SBD were used at 100 uM in 20 mM sodium phosphate buffer (pH 7.4) and 0.5-5% DMSO.
3) Compounds were used at 1-1.5 mM (10-15x protein concentration) in the same buffer.
4) Compounds were also tested for their ability to affect the binding of the model substrate NRLLLTG. Full-length DnaK was first titrated with a compound to saturation based on evolved heats.
5) The compound/DnaK(FL) complex was then titrated with model substrate NRLLLTG.
6) Data were analyzed using MicroCal Origin software provided by the ITC manufacturer (MicroCal, Northampton, MA).
Compounds with Kd for either the SBD or FL protein < 10 uM are defined as actives in this assay.
Titrations to saturated flDnaK:
Unsaturated model substrate binds with a Kd of 33.4 +/- 10.8 uM, dH ~-10000 cal/mol and dS of ~ -10 cal/(mol*T). Unsaturated ATP binds with a Kd of 0.01 uM, dH ~-100000 cal/mol and dS of ~ >40 cal/(mol*T). In all cases where flDnaK saturated with compound was titrated with peptide, affinity for the peptide increased by an order of magnitude and the binding mode of the peptide went from being entirely enthalpically driven to being both enthalpically and entropically. One compound was tested for its ability to affect ATP binding. After being saturated with compound SID-56427267, the affinity of DnaK(FL) for ATP decreased by 2 orders of magnitude.
All entropy and enthalpy measurements are approximations. All values for dH and dS are approximate.
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. Its utilization for the assay is described below.
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 and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response data and is not applicable in this assay.
3)) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues.
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. 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 are scored on a scale from 85 to 100 according to the following scale. If both the substrate binding domain(SBD) and full length(FL) were tested the lower of the two was used for the score:
Compounds with an estimated Kd >7.5 uM assigned a Score of 85
Compounds with an estimated Kd 5 - <=7.5 uM assigned a Score of 90
Compounds with an estimated Kd 2.5 - <= 5 uM assigned a Score of 95
Compounds with an estimated Kd <= 2.5 uM assigned a Score of 100
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)