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High-Throughput Screening for Small Molecules Modulating DnaK's ATPase Activity, Papers of Health sciences

A study conducted by chang et al. To identify small molecules that modulate the atpase activity of dnak, a molecular chaperone. The researchers used a high-throughput screening method and discovered several compounds that inhibit or enhance the atpase activity of dnak. The study also explores the potential mechanisms of action of these compounds and their implications for understanding the function of dnak and its role in protein folding.

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High-throughput screen for small molecules that modulate
the ATPase activity of the molecular chaperone DnaK
Lyra Chang
a
, Eric B. Bertelsen
b
, Susanne Wise
´n
a
, Erik M. Larsen
a
,
Erik R.P. Zuiderweg
b
, Jason E. Gestwicki
a,c,*
a
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA
b
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
c
Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
Received 1 July 2007
Available online 22 August 2007
Abstract
DnaK is a molecular chaperone of Escherichia coli that belongs to a family of conserved 70-kDa heat shock proteins. The Hsp70
chaperones are well known for their crucial roles in regulating protein homeostasis, preventing protein aggregation, and directing sub-
cellular traffic. Given the complexity of functions, a chemical method for controlling the activities of these chaperones might provide a
useful experimental tool. However, there are only a handful of Hsp70-binding molecules known. To build this area, we developed a
robust, colorimetric, high-throughput screening (HTS) method in 96-well plates that reports on the ATPase activity of DnaK. Using this
approach, we screened a 204-member focused library of molecules that share a dihydropyrimidine core common to known Hsp70-bind-
ing leads and uncovered seven new inhibitors. Intriguingly, the candidates do not appear to bind the hydrophobic groove that normally
interacts with peptide substrates. In sum, we have developed a reliable HTS method that will likely accelerate discovery of small mol-
ecules that modulate DnaK/Hsp70 function. Moreover, because this family of chaperones has been linked to numerous diseases, this
platform might be used to generate new therapeutic leads.
2007 Elsevier Inc. All rights reserved.
Keywords: Dihydropyrimidine; Heat shock protein; Hsp70; Malachite green; Stress response
DnaK is an extensively studied member of the family of
70-kDa heat shock proteins. These proteins are evolution-
arily conserved at the amino acid level; for example, Esch-
erichia coli DnaK and human Hsp70 are 46% identical and
65% similar [1]. Moreover, they are functionally conserved;
like other Hsp70s, DnaK acts as a molecular chaperone
that assists in protein folding, aids in trafficking of newly
synthesized polypeptides, and helps dissolve protein aggre-
gates [2–6]. In humans, Hsp70s have also been implicated
in numerous diseases, including cancer, neurodegenerative
disorders, and viral infections [7–12]. However, the com-
plexity of their functions has made studying the specific
roles of Hsp70 challenging. Even for the relatively well-
studied DnaK, numerous questions with regard to how this
chaperone carries out its myriad cellular tasks remain
unanswered.
DnaK is able to take part in a variety of pathways
because nearly all proteins contain hydrophobic sequences
that are suitable as substrates for chaperone binding.
Despite the diversity of potential clients, binding affinity
is tightly regulated. Specifically, DnaK (like all Hsp70s)
consists of three domains: a 25-kDa substrate-binding
domain (SBD),
1
a 45-kDa N-terminal nucleotide binding
domain that harbors ATPase activity, and a 10-kDa helical
0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2007.08.020
*
Corresponding author. Fax: +1 734 764 1247.
E-mail address: gestwick@umich.edu (J.E. Gestwicki).
1
Abbreviations used: SBD, substrate-binding domain; DSG, 15-deoxy-
spergualin; SAR, structure-activity relationships; HTS, high-throughtput
screening; MG, malachite green, PMSF, phenylmethylsulfonyl fluoride;
DTT, dithiothreitol; DMSO, dimethyl sulfoxide.
www.elsevier.com/locate/yabio
ANALYTICAL
BIOCHEMISTRY
Available online at www.sciencedirect.com
Analytical Biochemistry 372 (2008) 167–176
pf3
pf4
pf5
pf8
pf9
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High-throughput screen for small molecules that modulate

the ATPase activity of the molecular chaperone DnaK

Lyra Chang

a

, Eric B. Bertelsen

b

, Susanne Wise´n

a

, Erik M. Larsen

a

Erik R.P. Zuiderweg

b

, Jason E. Gestwicki

a,c,*

a (^) Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA b (^) Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA c (^) Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA

Received 1 July 2007 Available online 22 August 2007

Abstract

DnaK is a molecular chaperone of Escherichia coli that belongs to a family of conserved 70-kDa heat shock proteins. The Hsp chaperones are well known for their crucial roles in regulating protein homeostasis, preventing protein aggregation, and directing sub- cellular traffic. Given the complexity of functions, a chemical method for controlling the activities of these chaperones might provide a useful experimental tool. However, there are only a handful of Hsp70-binding molecules known. To build this area, we developed a robust, colorimetric, high-throughput screening (HTS) method in 96-well plates that reports on the ATPase activity of DnaK. Using this approach, we screened a 204-member focused library of molecules that share a dihydropyrimidine core common to known Hsp70-bind- ing leads and uncovered seven new inhibitors. Intriguingly, the candidates do not appear to bind the hydrophobic groove that normally interacts with peptide substrates. In sum, we have developed a reliable HTS method that will likely accelerate discovery of small mol- ecules that modulate DnaK/Hsp70 function. Moreover, because this family of chaperones has been linked to numerous diseases, this platform might be used to generate new therapeutic leads. Ó 2007 Elsevier Inc. All rights reserved.

Keywords: Dihydropyrimidine; Heat shock protein; Hsp70; Malachite green; Stress response

DnaK is an extensively studied member of the family of 70-kDa heat shock proteins. These proteins are evolution- arily conserved at the amino acid level; for example, Esch- erichia coli DnaK and human Hsp70 are 46% identical and 65% similar [1]. Moreover, they are functionally conserved; like other Hsp70s, DnaK acts as a molecular chaperone that assists in protein folding, aids in trafficking of newly synthesized polypeptides, and helps dissolve protein aggre- gates [2–6]. In humans, Hsp70s have also been implicated in numerous diseases, including cancer, neurodegenerative disorders, and viral infections [7–12]. However, the com- plexity of their functions has made studying the specific roles of Hsp70 challenging. Even for the relatively well-

studied DnaK, numerous questions with regard to how this chaperone carries out its myriad cellular tasks remain unanswered. DnaK is able to take part in a variety of pathways because nearly all proteins contain hydrophobic sequences that are suitable as substrates for chaperone binding. Despite the diversity of potential clients, binding affinity is tightly regulated. Specifically, DnaK (like all Hsp70s) consists of three domains: a 25-kDa substrate-binding domain (SBD), 1 a 45-kDa N-terminal nucleotide binding domain that harbors ATPase activity, and a 10-kDa helical

0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.08.

  • (^) Corresponding author. Fax: +1 734 764 1247. E-mail address: gestwick@umich.edu (J.E. Gestwicki).

(^1) Abbreviations used: SBD, substrate-binding domain; DSG, 15-deoxy- spergualin; SAR, structure-activity relationships; HTS, high-throughtput screening; MG, malachite green, PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; DMSO, dimethyl sulfoxide.

www.elsevier.com/locate/yabio

ANALYTICAL

BIOCHEMISTRY

Available online at www.sciencedirect.com

Analytical Biochemistry 372 (2008) 167–

‘‘lid’’ region [2,13]. Allosteric communication between these modules provides regulatory control. The ATP- bound form of DnaK has a ‘‘loose’’ configuration and poor affinity for peptide substrates [13,14]. Hydrolysis of the nucleotide causes conformational changes in the adjacent SBD that enhance the affinity for the peptide substrate [2,15]. Thus, ATP hydrolysis is one driving force behind chaperone structure and function. Importantly, DnaK’s intrinsic rate of nucleotide hydrolysis is slow (0.14 lmol ATP/lmol DnaK/min vide infra) compared to other typi- cal ATPases, such as porcine Na,K-ATPase ( 75 lmol ATP/lmol enzyme/min) [16]. This modest intrinsic rate permits tight regulation of the cycle by cochaperones. For example, the binding of the cochaperone DnaJ stimu- lates ATP hydrolysis (and, thus, enhances affinity for sub- strate), while the nucleotide exchange factor GrpE promotes ADP release to complete the catalytic cycle [17]. In addition to its role in ATP hydrolysis, DnaJ is thought to recruit DnaK into its various cellular functions and, consequently, there are typically more DnaJ-like pro- teins in a cell than core DnaK/Hsp70 chaperones [4–22]. For example, there are 6 DnaJ homologs in E. coli, 22 in Saccharomyces cerevisiae, and 41 putative family members in humans. In this way, cochaperones steer DnaK/Hsp into various combinatorial partnerships and likely coordi- nate different responses to cellular stress, signaling, or pro- tein aggregation. In contrast to the questions surrounding Hsp70 function, the roles of the related heat shock protein Hsp90 are becom- ing increasingly well characterized. This is especially true in relation to Hsp90’s prominent roles in cancer. In part, knowledge about Hsp90 has been accelerated by the avail- ability of potent and selective chemical inhibitors, such as geldanamycin and radicicol [23]. In addition, these reagents have facilitated the development of anticancer drugs that are currently undergoing clinical trials [24–27]. This success sug- gests that a parallel ‘‘chemical genetic’’ approach might be used to dissect the complex Hsp70 network and, moreover, that this process might also lead to the discovery of new Hsp70-based therapeutics. Of particular interest are com- pounds that can selectively recognize specific DnaK–DnaJ combinations because these complexes are likely to be involved only in subsets of the total chaperone functions. To date, only a handful of small molecules that bind DnaK or Hsp70 have been reported. Among the first part- ners identified was the polyamine 15-deoxyspergualin (DSG), which binds Hsp70 in pull-down assays and, later, was found to enhance its steady state ATPase activity by 20–40% [23–31]. Based on structural similarity to DSG, a small-scale (40 compounds) search for new Hsp70 modu- lators led to the discovery of NSC 630668-R/1 (R/1), which inhibits ATPase activity and blocks Hsp70-mediated traf- ficking of polypeptides [32]. More recently, another collec- tion of 30 dihydropyrimidines related to R/1 was studied using single-turnover ATP hydrolysis reactions [33]. In that work, unique classes of chaperone modulators were uncov- ered: some directly inhibited ATPase activity whereas oth-

ers, such as MAL3-101, selectively blocked the ATPase- enhancing ability of specific J domain proteins [33]. Inter- estingly, despite their diverse activities, many known Hsp70-binding compounds share a central dihydropyrimi- dine core and vary only in their pendant functionality. However, this is not the only chemical scaffold that has affinity for Hsp70-class proteins. For example, a family of acylated benzamido derivatives was reported to bind DnaK’s SBD, inhibit its chaperone function, and thereby display antibacterial activity [34]. Thus, multiple chemical classes have been reported to modify Hsp70’s functions. Despite the potential uses of these reagents, structure– activity relationships (SAR) that govern potent effects on ATPase activity are not yet clear. One factor contributing to this lack of information is that only a small number of compounds have been screened in low-throughput formats. We hypothesized that high-throughput screening (HTS) would be a useful platform for identifying new, potent chemical modulators and discerning SAR between similar compounds. Surprisingly, Hsp70 has not been subjected to extensive HTS. In contrast, several assays have been developed to uncover new inhibitors of Hsp90 [35–39]. One of these plat- forms [38] employes the inorganic phosphate chelator mala- chite green (MG) to monitor the ATPase activity of Hsp90. The MG assay is colorimetric and has the advantages of being robust, cost effective, and suitable for automated screening [40–42]. These advantages led us to explore whether this method could be employed to screen for com- pounds that alter the ATPase activity of DnaK. Because of DnaK’s relatively slow ATP turnover rate, this assay required modification to improve the signal and reduce the noise from spontaneous nucleotide hydrolysis. Importantly, we found that, using optimized conditions, the signal was lin- ear for up to 4 h; therefore, an end-point measurement was sufficient to represent the steady state rate. Using this assay, we discovered seven new inhibitors of DnaK from a focused collection of 204 dihydropyrimidines. This result suggests that the MG assay is a useful platform for finding small-mol- ecule modulators. These selected compounds, in turn, might be applied as chemical genetic tools to elucidate DnaK’s biology and might be used in future development of medi- cines that target Hsp70-related diseases.

Materials and methods

Protein expression and purification

DnaK, DnaJ, and GrpE proteins were expressed in E. coli BL21(DE3) using T7-based vectors. DnaK and GrpE were expressed at 37 °C, whereas DnaJ was expressed at 25 °C to increase the fraction of soluble pro- tein. All purification steps were carried out at 4 °C. Protein concentration was estimated by Bradford assay, using bovine serum albumin as the standard. Following purifica- tion, proteins were frozen on liquid nitrogen and stored at  80 °C until use.

nal increased with DnaK levels (Fig. 1A). At constant DnaK (0.6 lM), ATP concentration was varied from 0. to 8.0 mM, and we found that, above 4 mM, the OD (^620) was independent of nucleotide (Fig. 1B). Based on these studies, we chose 0.6 lM DnaK and 1 mM ATP for screen- ing conditions because these parameters provided good sig- nal while minimizing reagent costs.

Influence of DnaJ and GrpE concentrations

During its normal physiological function, DnaK is assisted by the action of DnaJ and GrpE [2,17]. More- over, certain small molecules that are known to modulate Hsp70’s ATPase activity are active only in the presence of cochaperones [33]. Therefore, we sought to develop a screen protocol that includes these components. Our approach was to vary the levels of recombinant DnaJ or GrpE and monitor effects on ATP hydrolysis by DnaK. In these studies, we were interested in both the rate enhancement and the cochaperone concentration that yielded half-maximal stimulation (K0.5 ). Rate enhance- ment is calculated by comparing the turnover rate of the cochaperone-stimulated system against the rate due to DnaK alone and the K0.5 can be used to approximate the affinity for DnaK. Using the MG assay, we found that the K0.5 of DnaJ was around 0.8 lM and that it pro- vided up to 11-fold stimulation of ATPase activity (Fig. 2A). On the other hand, GrpE had better apparent affinity for DnaK (K0.5 = 0.19 lM) but stimulated ATP- ase activity only approximately 7-fold (Fig. 2B). These results are generally consistent with the findings of McCarty et al. [49] who used single-turnover ATP assays to determine that when DnaK was fixed at 0.7 lM, 1.4 lM DnaJ and 0.7 lM GrpE stimulate DnaK ATPase by 13-fold and 1.3-fold, respectively.

0.4 0.5 0.6 0.7 0.8 0.9 (^0 2 4 6 )

0

DnaK (μM)

OD

620

Increasing [DnaK] enhances MG signal

OD

620

ATP (mM)

A B Increasing [ATP] enhances MG signal

Fig. 1. Dependence of MG signal on the concentration of DnaK and ATP. (A) The concentration dependence of DnaK was observed at 1 mM ATP. (B) DnaK was fixed at 0.6 lM while changing the concentration of ATP. The OD 620 value of the ATP control (OD 620 = 0.25) was subtracted in all figures shown. These results are the average of triplicates and the error is standard deviation.

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pmole ATP/ μg DnaK /min

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620

OD

620

Stimulation by DnaJ

Stimulation by GrpE

GrpE (μM)

pmole ATP/ μg DnaK /min

GrpE (μM)

A

B

Fig. 2. Stimulatory effect of cochaperones DnaJ and GrpE. The ATPase activity of DnaK at different concentrations of (A) DnaJ and (B) GrpE was measured at 1 mM ATP and 0.6 lM DnaK. The OD 620 value was determined after 3 h incubation at 37 °C. In the insets, the OD 620 values were converted into ATP hydrolysis rates based on a standard curve. These results are the average of triplicates and the error is standard deviation.

We were interested also in finding a ternary combination that would provide the most robust signal and the broadest dynamic range because this information could be used to select optimal screening conditions. To explore this idea, we fixed the concentration of DnaK (0.6 lM) and either DnaJ (1.0 lM) or GrpE (0.9 lM) and changed the concen- tration of GrpE or DnaJ, respectively. We discovered that, at a saturating level of GrpE, the K0.5 of DnaJ increased to around 3.1 lM and ATPase activity was stimulated an additional 15.5-fold compared to the DnaK:GrpE complex in the absence of DnaJ. This ternary combination led to a 130-fold stimulation over DnaK alone (Fig. 3A). Alterna- tively, when DnaJ and DnaK were held constant, the K0.5 of GrpE did not change. Under these conditions, we observed a 3.5-fold stimulation over DnaJ alone and only a 30-fold total increase in ATP hydrolysis (Fig. 3B). Next, the rate of DnaK’s ATPase activity over time was mea- sured at different combinations of DnaK, DnaJ, and GrpE. A linear increase in all conditions up to 4 h was observed

(Fig. 4). Finally, we sought to ensure that this platform faithfully reproduces known kinetic parameters of the enzyme. The Vmax and kcat of each reaction condition were calculated and, as shown in Table 1, we found that these parameters were in good agreement with previous data [49,50]. Thus, we hypothesized that, despite the slow turn- over rate of DnaK, these cochaperone-stimulated condi- tions appear to be sufficient for screening.

Screening a focused chemical library

Using the optimized reaction conditions, we screened a small chemical library for compounds that modulate DnaK activity (Fig. 5). This compound collection is composed of 204 dihydropyrimidines that are structurally similar to the lead candidate MAL3-101 [33]. These compounds were assembled from three sources: ‘‘cherry-picked’’ molecules from commercially available collections, those synthesized by the UPCMLD, and those generated by our group. Together, these compounds form a focused library from which we sought to identify potent modulators of DnaK. Using the MG assay, we screened at 200 lM in 96-well plates. During our first screening attempt, we found that most (90%) of the compounds displayed weak ATPase- stimulating ability (Supplemental Fig. 1). We suspected that this might result from nonspecific binding of the largely hydrophobic compounds to the SBD. It has been reported that detergents can remove promiscuous hits and improve the reliability of HTS methods [51]. Therefore, we repeated the screen in the presence of 0.01% Triton X-100. Intrigu- ingly, the mild stimulation effect disappeared, whereas the activity of the inhibitors remained largely unchanged or became more pronounced. We defined compounds that decreased activity >20% as inhibitors and, by this definition, seven inhibitors were identified (3.5% of the library; Table 2). These results demonstrated that the MG assay can be used to readily screen a chemical collection.

Confirming ‘‘hits’’ from the screen

From the seven hits, we picked four inhibitors, 0116-2F, 0116-4G, 0116-7G, and 0116-9E, for further study (Fig. 6). To confirm these hits and generate IC 50 values, we studied their effects (between 50 and 400 lM) on DnaK’s ATPase activity. Importantly, we found that the inhibitory effect was reproducible and the IC 50 values were between 120 and 200 lM (Fig. 6). At saturating concentrations, 0116-4G, 0116-7G, and 0116-9E provide 50% inhibition of DnaK ATPase activity, whereas the potency of 0116-2F is modest (80% original activity). The effects of the validated inhibitors were independent of detergent (Supplemental Fig. 4).

Characterization of the binding of candidate compounds to DnaK

The substrate-binding domain of DnaK has affinity for exposed hydrophobic regions on unfolded polypeptides.

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A

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Fig. 3. Optimization of the concentrations of DnaJ and GrpE for screening. (A) DnaJ stimulates ATP hydrolysis at fixed DnaK (0.6 lM) and GrpE (0.9 lM). (B) GrpE promotes the ATPase activity of 0.6 lM DnaK and 1.0 lM DnaJ. In the insets, the OD 620 values were converted into ATP hydrolysis rates based on a standard curve. The OD 620 value was determined after 1.5 h incubation at 37 °C. These results are the average of triplicates and the error is standard deviation.

we described a MG-based assay and the application of this method to uncover small molecules that inhibit the bacte- rial Hsp70, DnaK. This method is reliable (Z @ 0.7; S/N

  1. [54], low cost, and potentially amenable to the 384- well plate format [55]. By carefully varying the levels of chaperone and stimulatory cochaperones, we were able to arrive at a ratio that provided good dynamic range and sig- nal-to-noise. Moreover, because this system contains both chaperone and cochaperone, it provides the opportunity for uncovering compounds that modify either protein. Of course, this feature requires subsequent follow-up studies to isolate the binding site and mode of action. Finally, we observed a linear increase in OD 620 signal for greater than 4 h, which is a feature that permits usage of simple endpoint measurements. As a preliminary test of this method, we screened 204 compounds that share a dihydropyrimidine core similar to the previously reported lead, MAL3-101 [33]. In this rel- atively small collection, we successfully identified seven inhibitors (>20% inhibition). All selected inhibitors (4 com- pounds) were reproducible and their IC 50 values ranged from approximately 120 to 200 lM. While these potencies are modest, the library size was not large and we would predict that activity might be improved following addi- tional lead optimization and structural studies.

We designed this assay to report on compounds that modulate the function of the DnaK:DnaJ:GrpE chaperone machine. Thus, any ‘‘hits’’ from these screens might be expected to inhibit the complex by a number of distinct mechanisms, including direct binding to DnaK’s SBD or disrupting the interactions between DnaJ and DnaK. To illustrate the follow-up studies that we envision will be needed to deconvolute the binding site of hits, we per- formed exploratory experiments to identify the binding site for dihydropyrimidines on the chaperones. We considered it likely that these compounds bind to the SBD of DnaK and, therefore, modulate ATPase function. This mecha- nism would be similar to that thought to be invoked by other Hsp70-binding ligands, such as acidic glycolipids, phospholipids, and fatty-acylated benzamido derivatives. These compounds, with structures similar to geranylgeran- iol, are reported to bind at the SBD [34,56], and the forma- tion of an Hsp70-acidic lipid complex was hypothesized to play a role in chaperoning membrane proteins [56]. Sur- prisingly, we determined that, unlike geranylgeraniol, the activity of three selected dihydropyrimidines was indepen- dent of peptide competition or addition of detergent (Fig. 8). This finding suggests that dihydropyrimidines do not interact with the hydrophobic pocket of the SBD. However, we observed that adding peptide alters the com- pound’s IC 50. For example, the IC 50 of 0116-7G drops two- fold (from 130 to 65 lM) in the presence of peptide substrate. This observation suggests that the compounds might have different affinity for the apo- and substrate- bound conformations of DnaK. Further structural studies will be required to elucidate the binding site on the chaper- one, but these studies suggest that binding occurs outside the SBD.

0 70 140 210 Compound Number

120%

100%

80%

60%

40%

20%

Inhibitors

% of Control

Fig. 5. Screening of a 204-compound focused library. Each spot represents the average of triplicate wells for a single compound and the error is standard deviation. Triplicates were performed to demonstrate that screening results are reproducible. All compounds were screened at 200 lM with 0.6 lM DnaK, 1.0 lM DnaJ, 0.9 lM GrpE, 0.01% Triton X-100, and 1 mM ATP. Compounds resulting in <80% original DnaK ATPase activity were defined as inhibitors. As a control, 200 lM each compound was incubated with 1 mM ATP and 0.01% Triton X-100 and the resultant OD 620 value was subtracted from the measured signal at the presence of DnaK, DnaJ, and GrpE for the specific compound before calculating the percentage of control activity.

Table 2 Distribution of the screening results

Class % Control Hits % Total compounds

Inhibitor < 50 1 0. 50–80 6 3 Inactive 80–90 46 23 90–100 149 73

In conclusion, we have developed a HTS for the Dna- KÆDnaJÆGrpE system. This platform has potential for both uncovering new modifiers of DnaK and studying its steady state kinetics. It is worth noting that the identity of the chaperone can be varied to favor discovery of selective modulators. For example, we performed screens against rabbit Hsp70 and found a number of unique hits (L.C. and J.E.G., unpublished results). Likewise, the combina- tion of chaperone and cochaperone might be varied to identify selective inhibitors of distinct pairs. Therefore, we expect that this assay could be used to discover com- pounds that block specific chaperone pathways. These chemical modulators might be used to study chaperone biology and may provide leads for developing Hsp70-tar- geting therapeutics and antibiotics.

Acknowledgments

The authors thank Stephan Warner, Peter Wipf, and the University of Pittsburgh’s Center for Chemical Methodol-

NH

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Fig. 6. Dose-dependence curves and IC 50 values for selected inhibitors. The curves were fitted in GraphPad Prism using the equation: y = Min + (MaxMin)/(1 + 10 ^ ((LogIC 50 ·) * HillSlope)); (x = log[compound]).

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(^620) peptide (μM)

Fig. 7. Peptide substrate stimulates ATPase activity. The experiment was carried out at 0.6 lM DnaK, 1 mM ATP, and the OD 620 signal was measured after 3 h incubation at 37 °C. In the inset, the OD 620 values were converted into ATP hydrolysis rates based on a phosphate standard curve. These results are the average of triplicates and the error is standard deviation.

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