Development and Application of a Sensitive Peptide Reporter to

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Development and Application of a Sensitive Peptide Reporter to Discover 20S Proteasome Stimulators Rachel Coleman, and Darci J. Trader ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00193 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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ACS Combinatorial Science

Development and Application of a Sensitive Peptide Reporter to Discover 20S Proteasome Stimulators Rachel A. Coleman and Darci J. Trader* Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 W. Stadium Ave, West Lafayette, IN 47907 Abstract: To attenuate an over-abundance of cellular protein, it has been hypothesized that the 20S core particle (20S CP) of the proteasome can be chemically stimulated to degrade proteins into non-toxic peptides more quickly. Screening for small molecule 20S CP stimulators is typically performed with a reporter peptide composed of four amino acids and a coumarin group that is released upon proteasome-mediated hydrolysis to generate a fluorescent signal. Screening with this small reporter can lead to false negatives because the reporter peptide is rapidly turned-over without stimulation. To improve the screening for 20S CP stimulators, we have developed a peptide FRET reporter nearly four times more sensitive to stimulation but still amenable for high throughput screening. Through application of our FRET reporter, we have discovered two 20S CP gate-opening stimulators and also a molecule that elicits its mechanism of action through an interaction with a 20S CP active site.

*Corresponding Author [email protected]

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Introduction One of the most basic, essential cellular processes is the degradation of proteins. This is performed through two main pathways. Proteins can be shuttled to the lysosome or degraded by a large enzyme complex known as the proteasome.1 The 26S proteasome is composed of the 19S regulatory particle (19S RP) and the 20S core particle (20S CP), Figure 1.2 For a protein to be degraded by the 26S proteasome, it needs to be tagged with ubiquitin. This tag is then recognized by a subunit of the 19S regulatory particle, and upon this recognition, the 19S RP recruits deubiquitinases to remove the ubiquitin, unwinds the protein to limit its tertiary structure, and then shuttles it into the 20S CP. The 19S Figure 1. The proteasome can degrade proteins through a ubiquitin-dependent process when the 19S RP and 20S CP associate to form the 26S proteasome. The 20S CP can degrade proteins but can only accept small, intrinsically disordered proteins and does so at a slow rate.

RP also interacts directly with the N-termini of the αrings of the 20S CP to open the pore or gate to allow protein substrates to enter.3 It is the responsibility of the 20S CP to hydrolyze the protein with chymotrypsin-, trypsin- and caspase-like activity, into peptides that can then be recycled to make new

proteins or used as antigenic peptides. The 26S proteasome has been previously validated as a therapeutic target. A number of small molecules have been developed that behave as proteasome inhibitors and lead to cytotoxicity in cancers.4 The most well-known is bortezomib (Velcade ®), which is a covalent inhibitor that interacts preferentially with the chymotrypsin-like site on the 20S CP to prevent protein hydrolysis and is prescribed for patients with refractory multiple myeloma. Hydrolysis of proteins is an essential process for all cell types, and disruption of the activity of the proteasome, either the 26S or 20S isoform, can lead to cell death. Unfortunately, as cells age or are affected by protein accumulation diseases such as Parkinson’s, the level of proteins that assemble to form the 19S RP is greatly diminished as compared to younger or healthy cells.5 Without the 19S RP, ubiquitin-dependent protein degradation cannot occur, but the 20S CP can degrade proteins, slowly, in an ubiquitin-independent manner.6 The levels of the proteins that compose the 20S CP remain the same between diseased and healthy cells.7 When the 20S CP is

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not capped by the 19S RP, only small, intrinsically disordered proteins can be degraded, but at a slow rate. There has long been a general hypothesis that if one could increase the rate at which the 20S CP can turn over proteins, this could alleviate the negative effects associated with protein accumulation diseases.8–10 It is believed that this can be done through two mechanisms with small molecules. The first is stimulating the gate of the 20S CP to open, allowing more substrates to enter at a faster rate.11,12 The second mechanism is through an interaction with one of the active sites to increase the turnover rate; we refer to this as an allosteric active site stimulator.13 There have been previous reports of peptides and small molecules that can stimulate the 20S CP. For example, peptides that mimic the C-terminus of select 19S RP ATPases have been described to stimulate the 20S CP.14–16 There have also been natural products reported to behave as 20S CP stimulation agents, including oleuropein, a major component of olive oil, and betulinic acid, originally discovered from the white birch tree.17–19 The aforementioned molecules were discovered by utilizing a small reporter peptide, composed of 3-4 amino acids plus a terminal aminomethyl coumarin group, Figure 2A. Upon interaction with the 20S CP, the amino methyl coumarin group is hydrolyzed from the peptide, and a fluorescent signal accumulates. Because of the small size of the reporter, it can easily diffuse into the 20S CP and be turned over with no stimulation. Therefore, performing a Figure 2. (A) Typically used reporter peptide to monitor the chymotrypsin-like activity of the 20S CP. (B) To more efficiently detect 20S CP stimulation molecules, we developed a FRET reporter to monitor the hydrolysis activity of the 20S CP.

high-throughput

screen

with

this

reporter can result in a significant number of false negatives due to the high basal level of activity of the 20S

CP, making the detection of weak 20S CP stimulators a challenge. To limit the false negative rate and increase the dynamic range of molecules that will be detected during a high-throughput screening campaign, we have developed a fluorescence resonance energy transfer (FRET) reporter to monitor the activity of the 20S CP, Figure 2B. As we report here, our FRET reporter is over three-times more sensitive to stimulation than the traditionally used reporters. We utilized the FRET reporter to screen a library of 800 small

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molecules and discovered three that stimulate the 20S CP. Two molecules were validated as 20S CP-stimulating probes that elicit their effects through opening the gate. These molecules do not affect the 26S isoform, whose gate is already opened by the 19S RP. The third molecule we present here, whose scaffold has been previously unreported to interact with the 20S CP, can stimulate either the gate opened or gate closed proteasome conformation. This result highlights that our FRET reporter can discover both gate opening and allosteric stimulating probes.

Results and Discussion To begin our design of a more sensitive reporter to discover 20S CP stimulating molecules, we hypothesized that having a larger reporter would yield hit molecules with a variety of stimulating capabilities. We also wanted to have a reporter that was more challenging for the 20S CP to degrade without a stimulator present. The small reporter most often used, Figure 2A, to monitor the chymotrypsin-like activity of the 20S CP can easily be turned over with no stimulating molecule present; because of this, it has a very limited dynamic range, meaning that the difference between the basal level of activity and stimulated activity of the 20S CP is very small. There have been other proteasome activity reporters developed, but these too are small peptides of three to four amino acids and are not well suited for stimulation studies.20 Reporters for monitoring the activity of the proteasome in cells have also been developed but cannot differentiate between activity of the 20S CP and the 26S proteasome.21–23 There have also been gel- and NMR-based assays for detecting the activity of the 20S CP, but these are not amenable for high-throughput screening.24,25 To retain the speed and cost effectiveness of a fluorescent assay, we sought to develop a FRET reporter. We gained inspiration from a FRET peptide designed to monitor the activity of HIV protease, which uses the Dabcyl moiety as the FRET acceptor and Edans as the donor.26 The larger size of these FRET moieties compared to the coumarin fluorophore create a more difficult entry for the FRET reporter (Supporting Information Figure S1). In addition, we wanted to synthesize the largest peptide possible to still retain sufficient FRET efficiency (Forster radius = 33 angstroms) and limit any background signal. The amino acids between the FRET pairs were chosen based on cost effectiveness, ease of coupling, and moieties to help with solubility.

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For our initial assays, we needed to determine the amount of time and how often to obtain a fluorescent reading to elucidate the rate of hydrolysis (∆RFU/min). Monitoring the 20S CP activity over a range of time to generate a rate, rather than a single time point, minimizes the error from screen to screen. We next needed to determine the amount of FRET reporter and 20S CP in each well to obtain significant signal. Previous reports have utilized 250 µM of the smaller reporter, Suc-LLVYAMC, and between 1-10 nM of 20S CP to monitor 20S CP activity. We found instead that the FRET reporter had the best response at more than tenfold less (20 µM) than the Suc-LLVY-AMC probe (Supporting Information Figure S2). To determine the increased sensitivity to stimulation that the FRET reporter provides over the smaller reporter, we tested both in the presence of sodium dodecyl sulfate (SDS). SDS has long been considered a 20S CP stimulator.27 It is believed that it denatures the gate opening to allow more substrate to enter, leading to an increase in the amount of fluorescence observed. After determining the EC50 of SDS (Supporting Information Figure S3), we Figure 3. Accumulation of fluorescence upon the cleavage of the amino coumarin group (A) or the FRET (B) reporter by the 20S CP with and without SDS. (C) Direct comparison of the basal level of 20S CP activity to that when SDS is added with the four-amino acid reporter or the FRET. The rate of hydrolysis of the larger FRET reporter increases more in the presence of a stimulator than the four-amino acid reporter.

compared the basal level of activity of the 20S CP to that in the presence of 500 µM of SDS with the FRET reporter or the Suc-LLVY-AMC reporter. For this analysis, the basal level of the 20S CP was set to 100% for both probes, and the percentage of increase in the rate of fluorescent accumulation per minute in the presence of SDS was determined. The average

basal level of the 20S CP with the smaller reporter was determined to be 60 ∆RFU/min, while in the presence of SDS, the rate increased to an average rate of 145 ∆RFU/min, a 2.4-fold increase, Figure 3A. The result with the FRET reporter is shown in Figure 3B. With this reporter, the

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average basal level of the 20S CP was determined to be 2.9 ∆RFU/min, and after treatment with SDS, the rate increased to an average rate of 24 ∆RFU/min, an 8.3-fold increase in the rate. As we had hypothesized, the FRET is hydrolyzed at a much slower rate compared to the SucLLVY-AMC reporter. While this is significant, what is most noteworthy is the dramatic increase in the difference between basal level and stimulated activities of the 20S CP, Figure 3C. The range of stimulating molecules our FRET reporter can detect during a high-throughput screening campaign is larger than that of the Suc-LLVY-AMC probe, as it is 3.5-fold more sensitive to stimulation. We then wanted to determine the efficiency of the FRET reporter to detect inhibition of the 20S CP. We chose to monitor the effect of bortezomib on the hydrolysis of the FRET reporter. Bortezomib, as aforementioned, is a potent inhibitor of the chymotrypsin-like activity of the proteasome. As hydrolysis of the FRET reporter utilizes multiple activities of the 20S CP (Supporting Information Figure S4), we aimed to determine the concentration at which bortezomib prevents the hydrolysis of the FRET reporter. A dose-response assay was performed, revealing that 10 µM bortezomib prevents 9 nM 20S CP from hydrolyzing the FRET reporter (Supporting Information Figure S5) and the percent inhibition decreases

in

a

dose-response

manner

with

decreasing amounts of bortezomib. To confirm that the FRET reporter could detect small molecule stimulators in a dose-dependent manner, we also tested a previously reported small molecule 20S CP stimulator. AM-404, Figure 4A, has been identified as a 20S CP stimulator with the Suc-LLVY-AMC with an EC50≈ 32 µM in cellulo.28 In a similar fashion as the SDS experiment, purified Figure 4. (A) Structure of AM-404. (B) Increase in fluorescence intensity in the presence of AM-404 is dose-dependent. The concentrations of AM-404 had the following increases in the rate of hydrolysis: 50.0 µM, 7-fold increase; 25.0 µM, 5-fold increase; 12.5 µM, 4-fold increase.

20S CP was dosed with decreasing amounts of AM404. At the lowest concentration we tested, AM-404 stimulated the activity of the 20S CP 4-fold, consistent with the results previously published, Figure 4B.28 To verify that the results with SDS

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and AM-404 were not due to an interaction with

the

FRET

reporter,

similar

experiments were performed in which the 20S

CP

was

omitted

(Supporting

Information Tables S1, S2). As a predictor of the effectiveness of the FRET assay for high-throughput Figure 5. Scatter plot of positive (AM-404) and negative (cyclopentanepropanamide) controls used to determine Z-factor for the FRET assay. The mean percent of basal level for the positive control is 504 + 37 and that of the negative control is 111 + 10.

screening, we sought to determine its Zfactor, a statistic in which a value between 0.5 – 1 is indicative of an excellent assay. Using AM-404 as our positive control and

a derivative of AM-404, cyclopentane-propanamide, as a negative control, we determined the Zfactor for this assay to be 0.64 (Figure 5). This data was also used for further calculations to characterize the FRET assay (Supporting Information Tables S5, S6). Collectively, we can conclude that the FRET reporter can effectively detect 20S CP stimulating molecules in a dosedependent manner and is viable for use in a high-throughput screen. We next wanted to utilize the FRET reporter to screen a library of small molecules to find new 20S CP stimulating molecules. We screened the commercially available TimTec NPL-800 library, which is comprised of 800 natural products or natural product derivatives. The library was prescreened at 25 µM in Tris-HCl with no 20S CP to remove compounds that caused fluorescence at the emission wavelength of Edans (85 compounds removed). Next, fresh 96-well plates were prepared that contained 25 µM of the compound, 20 µM of the FRET reporter, and 9 nM of 20S CP in a total volume of 50 µL. The plate was incubated at 37oC and a fluorescence reading was taken every two minutes over a 40-minute period. Wells containing 25 µM of AM-404, wells with no 20S CP, and wells to monitor the basal level of the 20S CP were included in triplicate in every plate. After all compounds were screened in singlet, the rates of 20S CP hydrolysis in the presence of each compound were calculated and compared to the basal level, Figure 6A. We selected hits as those that increased the rate of hydrolysis at least 50%. With this cut off, there were four molecules that required validation. To validate these four compounds as 20S CP stimulating probes, we performed a number of additional assays. The four hits were tested in triplicate at 25 µM in the presence and absence of

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Figure 6. (A) Scatter plot of screening results utilizing the FRET reporter. The black dots represent the triplicates of the 20S CP basal level, which was normalized to zero for each plate of compounds. The red dots are the hit molecules while the blue dots were not considered further. (B) Four primary hit molecules that stimulated the 20S CP at least 50% at 25 µM.

the 20S CP, using new batches of molecules recently purchased (Supporting Information Table S3), to verify the observed increase in fluorescence was due to proteasome-mediated hydrolysis. Molecule 4, Sennoside B, did not perform as it had in the initial screen. After liquid chromatography-mass spectrometry analysis of the compound from the screening library and the new solid obtained, a significant amount of the non-glycosylated product was seen in the new stock (data not shown). At this point, molecule 4 was removed from the hit pool due to the difficulty in obtaining pure product to test and its overall poor drug-like properties. However, molecules 1-3 validated in triplicate at 25 µM. Since we had only ever tested the molecules at 25 µM, we chose to obtain a dose response curve for all three. As shown in Figure 7A-C, all three compounds responded in a concentration-dependent manner. Molecule 3 is the most potent with an EC50 of 7 µM, followed by compound 1 with an EC50 of 10 µM, and compound 2 with an EC50 of 14 µM.

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As previously described, the 20S CP can be capped with the 19S RP to enable ubiquitin-dependent protein degradation. The 19S RP associates directly with the alpharing of the 20S CP, opening the gate to allow substrates to enter. Molecules that behave as gate openers should therefore have no effect on the 26S proteasome because the gate is already open through interaction with the 19S RP. This is indeed the case with molecules 2 and 3, Figure 8A. Neither molecule 2 or 3 affected the rate of the 26S proteasome, providing more evidence these molecules behave by opening the gate of the 20S CP. Molecule 1 does increase 26S stimulation by 45% (Supporting Information Figure S6). From this result, we can hypothesize that molecule 1 induces its stimulation effect through an allosteric interaction with one or more of the hydrolysis sites of the 20S CP. Figure 7. (A-C) Dose response curves for 20S CP stimulation for the three primary hits. The EC50s listed is the concentration of half of the maximum stimulation. Molecule 3 stimulates the 20S CP the most with 150% over basal level followed by molecule 2 at 100% and molecule 3 at 50%. Assays were run in triplicate to generate error bars.

To further evaluate the mechanism of action of molecules 1-3, they were tested for their ability to stimulate the activity of the immunoproteasome (iCP). The iCP and 20S CP contain the same protein subunits that form the gate, but the active site subunits are structurally different. Interferon-gamma stimulates the production of the iCP active sites, which cleave proteins into peptides differently than the 20S CP. The peptides produced by the iCP are more

amenable to be loaded into an MHC-I complex. To support our hypothesis that compounds 2 and 3 are gate openers, they were tested for their ability to stimulate the iCP since the gate subunits are identical to the 20S CP. As shown in Figure 8B compounds 2 and 3 do stimulate the activity of the iCP significantly, while compound 1 shows no appreciable increase in activity (Supporting Information Figure S7). This result implies that molecule 1 has a specific interaction with one of

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the active sites of the 20S CP and less so with the active sites of the iCP whose structures are slightly different.29 The ability of these molecules to enhance the degradation

of

α-synuclein,

an

intrinsically

disordered protein previously reported to be degraded by the 20S CP, was evaluated using a gel assay. The 20S CP (5 nM) was incubated for 2 hr at 37 oC with 200 ng of α-synuclein and dosed with 25 µM of compounds 1-3 and AM-404 as a positive control. Following the 2 hr period, the samples were run utilizing SDS-PAGE. The gel was stained with Coomassie Brilliant Blue and imaged using a LICOR Odyssey. Analysis of the gels was performed using ImageJ and revealed significant Figure 8. (A) Molecules 1-3 were tested in triplicate for their ability to stimulate the 26S proteasome. 1: 145% (p0.05), 3: 115% (p0.05), 2: 191% (p

Development and Application of a Sensitive Peptide Reporter to

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