Identification of small molecules that modify the protein folding activity of heat shock protein 70

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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 374 (2008) 371–377 www.elsevier.com/locate/yabio

Identification of small molecules that modify the protein folding activity of heat shock protein 70 Susanne Wise´n, Jason E. Gestwicki * Departments of Pathology and Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA Received 30 September 2007 Available online 14 December 2007

Abstract Molecular chaperones, such as heat shock protein 70 (Hsp70) and its bacterial ortholog DnaK, play numerous important roles in protein folding. In vitro, this activity can be observed by incubating purified chaperones with denatured substrates and measuring the recovery of properly folded protein. In an effort to rapidly identify small molecules that modify this folding activity, we modified an existing method for use in 96-well plates. In this assay, denatured firefly luciferase was treated with a mixture of DnaK and prospective chemical modulators. The luminescence of refolded luciferase was used to follow the reaction progress, and counterscreens excluded compounds that target luciferase; thus, hits from these screens modify protein folding via their effects on the function of the chaperone machine. Using this platform, we screened a pilot chemical library and found five new inhibitors of DnaK and one compound that promoted folding. These chemical probes may be useful in studies aimed at understanding the many varied roles of chaperones in cellular protein folding. Moreover, this assay provides the opportunity to rapidly screen for additional compounds that might regulate the folding activity of Hsp70. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Chaperone; Luciferase; DnaK; High-throughput screen; Dihydropyrimidine

The 70-kDa heat shock proteins, such as heat shock protein 70 (Hsp70)1 and its bacterial ortholog DnaK, are members of a highly conserved and ubiquitously distributed family of molecular chaperones (for reviews, see Refs. [1–4]). One of the major roles of Hsp70 is to assist in the folding of nascent or otherwise unfolded polypeptides. Because this chaperone binds promiscuously to exposed hydrophobic sequences, it is thought to promote the folding of a wide array of proteins [5,6]. In support of this idea, Hsp70 refolds many different substrates in vitro, including firefly luciferase, lactate dehydrogenase, malate dehydrogenase, b-galactosidase, and glucose-6-phosphate dehydrogenase [7–12]. Genetic and biochemical evidence suggests that *

Corresponding author. Fax: +1 734 647 1247. E-mail address: [email protected] (J.E. Gestwicki). 1 Abbreviations used: Hsp70, heat shock protein 70; HTS, highthroughput screen; RRL, rabbit reticulocyte lysate; GuHCl, guanidine hydrochloride; DMSO, dimethyl sulfoxide. 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.12.009

this chaperone may also be important in protein misfolding disorders such as Huntington’s and Parkinson’s diseases [13–19]. Thus, Hsp70 likely is central to a number of cellular processes via its ability to broadly regulate protein folding; however, this complexity makes attempting to understand its specific roles more difficult. Small molecules that modulate the function of Hsp70 could be useful as probes to understand its diverse cellular functions [14,20]. By analogy, chemical inhibitors of Hsp90 have been shown to be useful in studies aimed at understanding that chaperone [21]. Recently, a number of functionalized dihydropyrimidines have been found to have activity against members of the Hsp70 family [15,22–26]. Although the binding site for these molecules is not yet clear, some of them stimulate the ATPase activity of Hsp70, whereas other structurally related molecules inhibit this function. These tools have been useful in preliminary studies of chaperone biology [25], but existing reagents have modest efficacy. One of the major hurdles to the dis-

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covery of new chemical modifiers of Hsp70 is the dearth of high-throughput screens (HTSs) for identifying more potent compounds from large collections of inactive molecules [22]. Recently, the ability of chaperones to restore the enzyme activity of denatured luciferase was developed as an HTS [27]. The refolding of damaged proteins is an important cellular function of chaperones; thus, this platform marks an important addition to the battery of available approaches. The reported HTS method uses rabbit reticulocyte lysate (RRL) as a relatively inexpensive source of chaperones. RRL is a well-known source of cytoplasmic material for in vitro translation experiments and other biochemical assays. This mixture is rich in cellular chaperones such as Hsp90, Hsp60, and Hsp70 [28]; therefore, it is also useful for protein folding studies [29]. For example, this method has been effective at revealing more than 100 potent inhibitors of Hsp90, a major component of the RRL mixture [27,30]. In our studies, we were interested in accelerated discovery of chemical probes that specifically target Hsp70 (and not other components of RRL); thus, we sought to develop a parallel HTS using purified chaperone. Cuvette-based methods for specifically studying Hsp70’s folding functions have been reported [31–33]. Using these sensitive, albeit low-throughput, assays as a guide, we have developed a high-throughput assay in 96-well plates. Using this approach, we uncovered a new activator and five potent inhibitors within a focused collection of dihydropyrimidines. Combined with other emerging methods, this assay should assist in the discovery of new chemical probes for Hsp70.

chaperone concentrations are indicated, but the ratio of DnaK/DnaJ/GrpE was kept constant at 1.0:0.2:0.1 based on previous findings [31–33]. Finally, 1 ll of compound or dimethyl sulfoxide (DMSO) was added. We found that the addition of common screening detergents, such as Triton X-100, has potent profolding activity in this format; thus, these agents were not added. In what might be a related finding, we routinely added compounds to denatured luciferase in the absence of chaperone, and in these cases a few hydrophobic compounds had effects on folding by a chaperone-independent mechanism. These controls were important in hit validation to ensure selective function. Specifically, we were interested only in those compounds that influence folding via their effects on the chaperone, and so those that altered luciferase function independently were removed from consideration as hits. Each reaction was performed in duplicate, and at the indicated times (10, 20, 40, and 60 min) 2 ll of the refolding mixture was removed and added to a white, flat-bottomed, 96-well plate (Corning) that was preloaded with 48 ll of SteadyGlo (Promega). An excess of ATP was included in the SteadyGlo reagent because luciferase itself is also dependent on nucleotide. This feature is particularly important for the experiments in which we studied the ATP dependence of the folding assay. After mixing, the luminescence was measured on a SpectraMax M5 multimode plate reader (Molecular Devices) using a 500-ms integration time. For routine screening, we found that 60 min of refolding was sufficient to give strong signal. We also found that freshly prepared ATP was a critical parameter in this assay, and efforts were made to normalize its concentration using frozen aliquots.

Materials and methods Results Reagents: Proteins and small molecules Refolding of firefly luciferase by purified chaperones The proteins DnaK, DnaJ, and GrpE were expressed and purified as described previously [22]. The chemical library, which is composed of functionalized dihydropyrimidines, was also reported previously [22,26]. Luciferase folding assay Firefly luciferase (0.5 mg/ml, Promega) was denatured in buffer A (25 mM Hepes [pH 7.2], 50 mM potassium acetate, and 5 mM dithiothreitol) containing 6 M guanidine hydrochloride (GuHCl) at room temperature for 60 min. The denatured protein was diluted 1:40 in buffer A and placed on ice for 20 min before refolding. Refolding was initiated by adding 2 ll of the denatured luciferase stock into 48 ll of refolding buffer (28 mM Hepes [pH 7.6], 120 mM potassium acetate, 12 mM magnesium acetate, 2.2 mM dithiothreitol, 0.1 mM ATP, 8.8 mM creatine phosphate, and 35 U/ml creatine kinase). This refolding buffer also contained chaperones that, in the screens, were at final concentrations of 240, 48, and 24 nM for DnaK, DnaJ, and GrpE, respectively. For other experiments, the

Insights into the diverse roles of Hsp70 likely would follow discovery of potent chemical modulators. Toward this goal, we were interested in developing a method for rapidly finding molecules that alter chaperone-mediated protein folding. Using existing methods as a starting point [7], our first goal was to identify the concentrations of purified chaperone that would provide robust signal in 96-well plates. Toward this goal, firefly luciferase was chemically denatured with GuHCl and then diluted into a chaperone-containing mixture (shown schematically in Fig. 1). In these experiments, we employed the bacterial system composed of purified DnaK and the cochaperones DnaJ and GrpE. Without assistance, DnaK is a poor chaperone, but in cells it is assisted by these factors [34,35]. This tricomponent system is the best studied of the chaperone machines; thus, there is interest in developing mechanistic probes for this model. When this mixture was added to denatured luciferase, a time- and concentration-dependent enhancement in signal was observed (Fig. 1). Importantly, luciferase refolded poorly in the absence of chaperones;

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Fig. 1. Investigating the effects of chaperones and nucleotide on the refolding of denatured luciferase. (A) Schematic depiction of the assay. (B) Increasing the levels of the chaperone system improved folding and the luminescence signal by up to 7.5-fold. To find both agonists and antagonists, we chose chaperone concentrations (240, 48, and 24 nM for DnaK, DnaJ, and GrpE, respectively) that produce intermediate activity. (C) The refolding assay is dependent on ATP hydrolysis. In addition, the signal intensity is proportional to the nucleotide levels.

thus, this assay provides specific insight into a chaperonedependent process. Another goal of these experiments was to find conditions that provide intermediate signal intensity. Previous work revealed that dihydropyrimidines can either activate or inhibit ATPase activity [22–24]; thus, we considered it likely that similar positive and negative modifiers of folding function would be found. To facilitate discovery of both activators and inhibitors, we chose our screening conditions (240, 48, and 24 nM for DnaK, DnaJ, and GrpE, respectively) such that both increases and decreases in luminescence intensity could be measured within the dynamic range of the system. Optimizing the ATP concentration For all members of the Hsp70 family, the affinity for hydrophobic substrates is regulated by ATP hydrolysis [36,37]. In addition, in vitro folding of luciferase is known to be ATP dependent [38]. To investigate how nucleotide might affect the signal intensity under HTS conditions, we performed experiments in the presence of either ATP or c-S-ATP. The nonhydrolyzable analog, c-S-ATP, completely blocked folding (Fig. 1); hence, the turnover of ATP is crucial. Next, the effect of ATP concentration on signal intensity was obtained by performing a refolding experiment with varied nucleotide levels. These results showed

a linear increase over a broad range (Fig. 1), and for the purpose of the screen we selected 100 lM ATP for further studies. DnaJ and GrpE are required for refolding activity In cells and in vitro, stimulatory cochaperones are important for chaperone function [34,35,39–41]. To define the roles of these cofactors under our reaction conditions, we examined whether all three components of the trichaperone system (DnaK, DnaJ, and GrpE) are required. Specifically, we performed refolding assays is which each component was systematically omitted. The results showed that the presence of all three proteins was necessary (Fig. 2); refolding in the absence of GrpE was the best tolerated, leading to a 50% decrease, whereas removing DnaK or DnaJ blocked folding almost entirely. Based on these results, we included all three components in the screening experiments. Screening a focused chemical library Using the conditions selected above, we screened a pilot library of dihydropyrimidines [22,26]. The compounds were screened at the relatively low concentration of 1 lM because we found that certain hydrophobic molecules

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decreased refolding activity by more than 20% as inhibitors. Using this definition, we found 10 inhibitors and 8 activators, making up 11% of the compounds in this library. These relatively high hit rates are expected for the screening of structurally focused collections.

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Fig. 2. Folding of denatured luciferase requires all three components of the chaperone machine. The concentration of the chaperones are as follows: DnaK, 240 nM; DnaJ, 48 nM; and GrpE, 24 nM. Each result is the average of two experiments in duplicate, and the error is the standard deviation.

interacted with denatured luciferase by a chaperone-independent mechanism when present at a higher level such as 100 lM (see Supplementary Fig 1). Importantly, we also chose the screening conditions to allow for discovery of compounds that either accelerate or decrease chaperonemediated folding. Consistent with the design, these conditions enabled the discovery of both inhibitors and activators (Fig. 3). As shown in Fig. 3, 159 of the 177 compounds ( 90%) are distributed between 20% below and 20% above the solvent control. Based on this distribution, we define compounds that increased refolding activity by more than 20% as activators and define those that

A total of 13 hits from the screen were chosen for validation by dose dependence experiments. These assays were performed at five concentrations between 1 and 50 lM, and EC50 values were generated. A representative inhibitor, 116-5 c, is shown in Fig. 4 to illustrate the progress curves. In addition, the effect of each compound against luciferase in the absence of chaperones was measured. As mentioned above, we found that certain compounds influenced luciferase activity through a chaperone-independent mechanism (see Supplementary Fig. 1). Thus, it was important to define validated hits as those compounds that specifically modulate chaperone-mediated refolding. Using these criteria, we verified nearly 50% (6/13) of the screening hits. Of these compounds, 116-5c and 115-3b were particularly potent, with EC50 values below 4 lM; interestingly, 1153b was an activator of folding, whereas 116-5c was an inhibitor. Thus, this luciferase-based assay can be used to readily identify potent activators and inhibitors of chaperone-mediated folding. Discussion Hsp70 is involved in a number of essential processes in cells, including the folding of newly synthesized and otherwise unfolded polypeptides [1–4]. Compounds that control the function of this chaperone might be employed to better

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Fig. 3. A dihydropyrimidine library contains both activators and inhibitors. Each result is the average of two experiments in duplicate, and the error is the spread. Of the total collection, 4.5% (8/177) were defined as activators and 5.5% (10/177) were defined as inhibitors.

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Fig. 4. Validation of screening hits. (A) Representative dose–response for an inhibitor, 116-5c. (B) Representative conversion of the dose–response to an EC50 value using the luminescence values at 60 min. These values were calculated in PRISM (GraphPad) using a variable slope sigmoidal fit. (C) Nearly 50% (6/13) of the selected screening hits were validated in the follow-up analysis. Validated hits were defined as those that produced EC50 values below 50 lM with no residual activity against native luciferase. No distinction was made between compounds that activate or inhibit activity, but the intensity and direction of the change are shown in the right column. Each result is based on the average of two experiments in duplicate, and the error is the standard deviation. (D) Chemical structure of the validated hits.

understand its important cellular activities. Unlike Hsp90 [42], Hsp70 inhibitors have become available only recently and more potent compounds likely await discovery. Whereas previous screening studies have focused largely on finding compounds with an effect on the ATPase activity of Hsp70 [22–24], here we have described a complementary method to uncover small molecules that have a direct impact on its folding functions. We envision that this method could be an important addition to a growing battery of methods for finding chemical modulators of Hsp70. Specifically, given the variety of functions ascribed to chaperones and the uncertain relationship between ATP turnover and folding function, a combination of different assays might be required to identify small molecules with the desired profiles. Using this luciferase-based HTS method, we identified a number of compounds that directly adjust the folding activity of the major bacterial chaperone system: DnaK and its cochaperones DnaJ and GrpE. We found that all three components are required for full activity; thus, any screening hits may physically bind to any combination of

these proteins. Despite this necessary complexity, one of the ways in which HTS can be used is to screen collections of related compounds so that structure–activity relationships can be identified. Based on this idea, we focused our initial screens on a collection of dihydropyrimidines because compounds with this scaffold previously were shown to modify Hsp70 [22,24]. Although additional work remains to identify the binding site(s) of these molecules, we found that some of the most potent inhibitors—1165c, 116-10d, and 116-8e—share a similar core structure (Fig. 4). Satisfyingly, these compounds also resemble MAL3-101, the lead candidate that was reported previously [24]. However, it is still unclear why some compounds (e.g., 115-3b) stimulate folding, whereas related molecules (e.g., 116-5c) are inhibitory. A separate group of hits— 116-4d and 115-3b—have lower molecular weight and share a nitrosobenzyl functionality. Thus, these results suggest that there are at least two major types of core structures that alter folding functions. Moreover, these findings do not rule out the possibility that new chemical scaffolds might be uncovered by screening larger libraries

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of diverse molecules. Given the low number of known compounds, the candidates identified in this way may be useful as chemical probes to give further insight into the function(s) of Hsp70.

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Acknowledgments The authors thank S. Warner, P. Wipf, and the University of Pittsburgh’s Center for Chemical Methodologies and Library Development (funded by P50/GM067082) for the generous access to their chemical libraries. B. Barretta provided useful comments on the manuscript. This work was supported by the University of Michigan’s Biomedical Research Council and the McKnight Endowment Fund for Neuroscience.

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