ATPase - 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: 2
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 ability to inhibit the ATPase activity of DnaK.
1. Bukau B, Weissman J, Horwich A.
Cell. 2006 May 5;125(3):443-51.
2. Pellecchia M, Montgomery DL, Stevens SY, Vander Kooi CW, Feng HP, Gierasch LM, Zuiderweg ER. Nat Struct Biol. 2000 Apr;7(4):298-303.
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) ATPase activity was monitored using the ADAPTA kit from Invitrogen.
2) The EC80 of our expressed full-length DnaK was determined to be 2 uM.
3) Compounds were assayed in mixtures of 5 at 400 uM each for screening and the antimicrobial peptides at 40 uM. Compounds and peptides were preincubated with DnaK for at least 10 minutes at room temperature.
4) TR-FRET was measured using a Perkin Elmer Victor 2 Multilabel plate reader.
5) Mixtures reducing activity below 90% of DnaK alone were deconvoluted.
Percent inhibition was determined as such: 100% activity was the amount of signal in the presence of 2uM full length DnaK while 0% activity was the signal in the absence of DnaK. The signal of DnaK with compound mixtures was normalized to this span. Mixtures causing <90% activity were deconvoluted. Individual compounds were assayed at four different concentrations and those signals normalized to the same span. IC50s were estimated as the concentrations leading to approximately 50% activity as four data points were not enough to fit to a standard sigmoidal binding model.
Compounds with an estimated IC50 < 20000 uM are defined as actives in the primary screening.
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 linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. 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 for which we do not have a complete titration curve are scored on a scale from 85 to 100 according to the following scale:
Compounds with an estimated 15,000< IC50 <=20,000 uM assigned a Score of 85
Compounds with an estimated 10,000 < IC50 <=15,000 uM assigned a Score of 90
Compounds with an estimated 5,000 < IC50 <=10,000 uM assigned a Score of 95
Compounds with an estimated IC50 <= 5,000 uM assigned a Score of 100
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)