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ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases

, , , , , , , , , , , , , , , , , and .

Author Information and Affiliations

Received: ; Last Update: April 5, 2013.

Protein homeostasis, also called proteostasis, is critical for cellular health and its dysregulation is implicated in aging, cancer, metabolic disease, and neurodegenerative disorders. Proteostasis involves compartmentalized cellular responses (e.g. Heat Shock Response in the cytoplasm, Unfolded Protein Response in the mitochondria and endoplasmic reticulum) that limit protein misfolding and aggregation. Diseases of protein conformation are characterized by inefficient induction of these responses. As a result, identification of molecules that activate cellular stress responses and increase proteostasis may be useful for maintaining cell health. Here, we report on high throughput screening efforts that resulted in identification of a novel activator of heat shock protein 70 (Hsp70): ML346. Probe ML346 belongs to the barbituric acid scaffold. ML346 induces HSF-1-dependent chaperone expression and restores protein folding in conformational disease models. These effects are mediated by novel mechanisms involving FOXO, HSF-1, and Nfr-2.

Assigned Assay Grant #: 5 R21 NS056337-02

Screening Center Name & PI: The Scripps Research Institute Molecular Screening Center (SRIMSC), H. Rosen

Chemistry Center Name & PI: SRIMSC, H. Rosen

Assay Submitter & Institution: R. Morimoto, Northwestern University

PubChem Summary Bioassay Identifier (AID): 588815

Resulting Publications

1.
Calamini B, et al. Small-molecule proteostasis regulators for protein conformational diseases. Nat Chem Biol. 2012;8(2):185–96. [PMC free article: PMC3262058] [PubMed: 22198733]

Probe Structure & Characteristics

CID/ML#/SR#StructureTarget NameEC50 (nM) [SID]Anti-target NameEC50 (μM) [SID]Fold SelectiveSecondary Assay(s): [SID]
CID 767276

ML346

SR-01000219514
Image ml346fu1.jpg
Hsp704600
[SID 152186720]		HeLa Cell Toxicity>25 μM
[SID 152186720]		>5-fold

1. Recommendations for Scientific Use of the Probe

As an activator of Hsp70 expression and HSF-1 activity, ML346 is immediately useful in understanding how the heat shock responses in general, and these proteins in particular regulate the processing, folding, and recycling of misfolded proteins. ML346 has good chemical stability, significantly high water solubility, is not reactive with excess glutathione, and is cell permeable. The probe ML346 and its analogs, and any improved derivatives that emerge, will help elucidate roles for activation of Hsp70 and HSF-1 in the prevention and progression of cancers, cellular aging, and metabolic and neurodegenerative disorders.

Introduction

The human heat shock protein 70 (Hsp70) family is evolutionarily conserved among all organisms from archaebacteria to humans, suggesting an essential role in cell survival [1, 2]. Under circumstances of transient cell stress, the heat shock response and activities of molecular chaperones can restore protein homeostasis. In human disease, however, misfolded proteins can accumulate when polyglutamine-expansion proteins are chronically expressed over the life of the cell. Elevated expression of molecular chaperones suppresses protein misfolding/aggregation and toxicity phenotypes in various model systems of disease. Mutations in the respective proteins huntingtin, tau, alpha-synuclein, and superoxide dismutase (SOD1), associated with these diseases, result in the appearance of misfolded species that adopt alternate conformations. These observations led to the proposal that a common feature of diverse diseases of protein conformation is the appearance of alternate folded states that self-associate and form toxic species and protein aggregates.

A role for Hsp70 family proteins in controlling these events has been widely studied. Studies with mammalian tissue culture cells, transgenic mice, Drosophila, and C. elegans have established that the heat shock response can be activated in cells expressing aggregation-prone proteins, suggesting a role for molecular chaperones as an adaptive survival response [3, 4]. Moreover, a direct relationship with polyglutamine diseases is suggested by the co-localization of several heat shock proteins, including Hdj-1, Hdj-2, Hsp70 and ubiquitin with polyglutamine aggregates in the tissues of affected individuals, transgenic mice and tissue culture cells [5]. Finally, overexpression of Hsp70 can suppress the toxicity associated with the accumulation of misfolded proteins [6–8]. High throughput screening initiatives aimed at the identification of compounds that enhance the heat shock response, in particular Hsp70, will provide insights into this conserved cellular process and may lead to novel therapeutics for these devastating disorders.

We developed a high throughput screen that measures activation of the heat shock response in HeLa cells stably transfected with a heat-shock–inducible reporter containing the proximal human Hsp70.1 promoter sequence upstream of a luciferase (luc) reporter gene [9]. The SRIMSC screened this assay against over 196,000 compounds from the Molecular Libraries Probe Production Center Network (MLPCN) library and another 600,000 compounds from the Scripps institutional drug discovery library.

Our strategy was designed to identify cell-permeable small molecule activators. Our HTS effort was complemented by a medicinal chemistry and molecular biology effort intended to optimize properties of the active compounds that were identified, to define their mode of action, to show their relevance, and finally to deliver a potent and selective probe. The resulting probe compound, ML346, belongs to the barbituric acid scaffold. ML346 has been extensively tested by the assay provider in several mechanism-of-action and protein folding assays. Of note, ML346 activates transcription of the Hsp70 promoter and suppresses aggregation of poly-glutamines in a C. elegans model, suggesting the probe has efficacy in modifying protein aggregation and associated toxicity.

The simplicity of the structure of ML346, its relative ease of preparation, its favorable physical characteristics, and its impressive biological properties all contribute to its use as a molecular probe useful for understanding the role(s) of heat shock response in cellular growth and aging.

Much of the data in this probe report has been published in our Nature Chemical Biology manuscript [9] and we refer readers to that publication for details and insights beyond those presented in this probe report.

2. Materials and Methods

Chemistry: All chemical reagents and solvents were acquired from commercial vendors. Reactions were monitored by LC/MS (Thermo/Finnegan LCQ Duo system with MS/MS capability). An Agilent 1200 analytical HPLC was used for quantitative purity assessment. Teledyne-Isco “combiflash” automated silica gel MPLC instruments were used for chromatographic purifications. A 400 Brüker MHz NMR instrument was used for NMR analysis.

Biology: All protocols are reported in the relevant PubChem AIDs and in [9].

Evaluation of compound properties: Solubility, stability, and glutathione reactivity analyses were conducted in accordance with NIH guidelines.

2.1. Assays

Table 1 shows Hsp70-related PubChem AIDs. Descriptions of the assays follow the table.

Table 1. Hsp70 Activators PubChem Summary.

Table 1

Hsp70 Activators PubChem Summary.

Assay Descriptions

Click on the hyperlinked text to see the assay details in PubChem.

Hsp70 Activation Assays (AID 1203, AID 1252)

The purpose of this assay is to determine the ability of compounds from the MLPCN library to act as activators of Hsp70 expression. Induction of the heat shock response by test compound is measured in a HeLa cell line stably expressing a luciferase reporter under control of the human Hsp70 promoter. As designed, a compound that acts as an activator of Hsp70 expression will activate the Hsp70 promoter, which will increase luciferase transcription, and thus increase well luminescence as detected with the appropriate substrate. Compounds were tested in singlicate at a final nominal concentration of 10 micromolar (AID 1203), and in triplicate using a 10-point 1:3 dilution series starting at a nominal test concentration of 100 μM (AID 1252).

The hsp70.1pr-luc HeLa cell line was grown in tissue culture flasks in Dulbecco’s Modified Eagle’s Media supplemented with 10% v/v fetal bovine serum, 1% pen-strep-neomycin antibiotic mixture and 1% Geneticin at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH).

Prior to the start of the assay, cells were resuspended in growth media as above at a concentration of 750,000 cells/mL. Next, 5 μL of well-mixed cell suspension were dispensed into each well of 1536-well plates (3,750 cells per well). After incubation for 4 hours at 37 degrees C, 5% CO2 and 95% (RH), the assay was started by dispensing 50 nL of test compound in DMSO to sample wells, DMSO alone (1% final concentration) to negative control wells, or MG132 (final nominal EC100 concentration of 30 μM, set as 100% activation) to positive control wells. The plates were then incubated for 16 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 μL of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 15 minutes. Luminescence was measured on the ViewLux plate reader. The percent activity was defined using the following mathematical formula:

% Activity = 100*((Test_Compound − Median_Low_Control) / (Median_High_Control −
Median_Low_Control))

Where:

  • Test_Compound is defined as wells containing test compound
  • High_Control is defined as wells containing MG132
  • and Low_Control is define as wells containing DMSO

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.

List of reagents:

  • Dulbecco’s Modified Eagle’s Media (Invitrogen, part 11965-092)
  • Fetal Bovine Serum (Hyclone, part SH 30088.03)
  • Geneticin (Invitrogen, part 10131-027)
  • Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)
  • SteadyLite HTS luciferase substrate (PerkinElmer, part 6016989)
  • 1536-well plates (Greiner, part 789173)
  • T175 HYPERflasks (Corning, part 10010)
  • Reference agonist MG132 (American Peptide, part 81-5-15)
HeLa Cytotoxicity Assays (AID 1259)

The purpose of this assay is to determine the cytotoxicity of test compounds from the MLPCN library identified as active in a previous set of experiments entitled, “Primary cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)” (PubChem AID 1203) and confirmed activity via dose-response assays entitled, “Dose response cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)” (PubChem AID 1252). The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP found in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of ATP. Thus, well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that induce cell death will reduce ATP levels, and therefore reduce well luminescence. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 99 μM. Compounds active in the assays above and inactive in this cytotoxicity counterscreen are considered nontoxic inducers of Hsp70 transcription. Figure 1 indicates that the probe was not toxic.

Figure 1. Probe ML346 is not toxic to HeLa cells.

Figure 1

Probe ML346 is not toxic to HeLa cells. HeLa-luc cells were treated with increasing concentrations of probe ML346 for 24 h. Cell viability is shown.

QPCR Hsp70 Assays (AID 651945)

The purpose of this assay is to determine whether powder samples of a compound identified as transcriptional activators of heat shock protein 70 (Hsp70) modulates the gene expression of Hsp70 involved in the heat shock response and protein homeostasis. In this assay, HeLa cells are incubated with test compound and harvested. RNA is purified and subjected to quantitative reverse transcription (qRT-PCR) of Hsp70 and GAPDH (control). Gene expression is normalized to GAPDH and compared to levels in cells treated with DMSO only. As designed, test compounds that induce an increase in Hsp70 gene expression will result in an increase in amplified RNA product.

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with phenol red buffered with HEPES and supplemented with 10% v/v fetal bovine serum (FBS), 1% L-glutamine, and 100 U/ml penicillin/streptomycin. The cells were treated with test compound, the positive controls celastrol (3 μM), CdCl2 (50 μM) and MG132 (10 μM), or left in vehicle (DMSO). The cells were harvested 4 hours after compound addition for analysis of chaperone expression by quantitative reverse transcription (qRT)-PCR. RNA was purified using the RNeasy Mini kit according to the manufacturer’s instructions. After the reverse transcription reaction, PCR was performed using PCR primers specific for human Hsp70 and GAPDH. PCR products were amplified with Taq polymerase by using standard cycling conditions. Gene expression was normalized to GAPDH and compared to levels in cells treated with DMSO (mRNA levels set as 1.0) using ImageJ. Compounds that induced a minimum of 1.5-fold change in gene expression compared to the DMSO levels (set at 1.0) were active in this assay.

List of Reagents: HeLa cells (provided by Assay Provider); DMEM (Invitrogen); RNeasy Mini kit (Qiagen, part 74106); Taq polymerase (Promega, part M3001); PCR primers were ordered as appropriate. The primer pair sequences for the human Hsp70 and GAPDH gene targets are indicated below:

  • Hsp70 forward: 5′-AGAGCCGAGCCGACAGAG-3′
  • Hsp70 reverse: 5′-CACCTTGCCGTGTTGGAA-3′
  • GAPDH forward: 5′-GTCGGAGTCAACGGATT-3′
  • GAPDH reverse: 5′-AAGCTTCCCGTTCTCAG-3′

The results of this assay reveal that probe ML346 increases the expression of Hsp70 mRNA by 2.4-fold over DMSO control levels (Figure 2), further validating the HTS results. The expression levels are normalized to the GAPDH housekeeping gene.

Figure 2. Probe ML346 (“F1”) induces Hsp70 mRNA expression.

Figure 2

Probe ML346 (“F1”) induces Hsp70 mRNA expression. “D” indicates DMSO control. The other lanes are for other positive controls. See [9] for details.

Chaperone Protein Western blots (AID 651948)

The purpose of this assay is to determine whether powder sample of a test compound identified as transcriptional activators of heat shock protein 70 (Hsp70) induces a change in the protein levels of three chaperones–Hsp70, Hsp40, and Hsp27. This will provide further evidence that the probe can induce the HSR. In this assay, HeLa cells are incubated with test compound, following by harvesting and western blot analysis of the reaction products using standard western blotting techniques. As designed, test compounds that induce a change in protein expression will result in a change in protein signal (compared to DMSO control) detected on the Western blot. Compound was tested at 10 μM.

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with phenol red buffered with HEPES and supplemented with 10% v/v fetal bovine serum (FBS), 1% L-glutamine, and 100 U/ml penicillin/streptomycin. The cells were treated with test compound (10 μM), the positive controls celastrol (3 μM) and MG132 (10 μM), or left in vehicle (DMSO). The cells were harvested 8 hours after compound addition for analysis of chaperone expression by western blot analysis. Cells were lysed in a buffer containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; pH 7.9), 25% v/v glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol and 2 mg/ml of complete protease inhibitor cocktail for 30 minutes on ice. 15 ug of whole cell extracts were run on 7.5% SDS-PAGE gels and transferred to nitrocellulose. Primary antibody incubations were for 12 hours at 4 degrees C in 10% BSA. The following primary antibodies were used: a mouse monoclonal Hsp70 antibody, a mouse monoclonal Hsp40 (alphaHdj-1 clone 25), and a mouse monoclonal Hsp27. All primary antibodies were used at a dilution of 1:10,000, except for the Hsp27 antibody, which was diluted 1:500. The anti-beta-tubulin antibody was diluted 1:5,000 and used to verify equal protein loading. The secondary antibody was an Alexa Fluor 680 goat anti mouse IgG diluted 1:5,000. Western analysis was performed with the Odyssey system. Protein expression levels were normalized to tubulin and compared to levels in cells treated with DMSO (mRNA levels set as 1.0) using ImageJ on an Odyssey LI-COR imaging system. Compounds that induced a minimum of 1.5-fold change in protein expression compared to the DMSO levels (set at 1.0) were active in this assay. As shown in Figure 3, these assays reveal that probe ML346 induces the levels of all three chaperone proteins. There was no effect on tubulin, showing that the probe did not act non-specifically.

Figure 3. Probe ML346 (“F1”) induces chaperone protein expression.

Figure 3

Probe ML346 (“F1”) induces chaperone protein expression. “D” indicates DMSO control. The other lanes are for additional compounds. See [9] for details.

QPCR Hsp70 Downstream Target Assays

The purpose of this assay is to determine whether powder samples of compounds identified as transcriptional activators of heat shock protein 70 (Hsp70) modulate the endogenous gene expression of Hsp70 target genes and other stress-responsive proteostasis network pathways (such as the UPR and the anti-oxidant stress response). This assay confirms the effects of HSR activation by test compounds. We measured the expression of the UPR-inducible gene GRP78/BiP, the antioxidant responsive genes heme oxygenase 1 (HO1) and the regulatory subunit of glutamate-cysteine ligase (GCLM), and the proapoptotic growth arrest- and DNA damage-inducible gene 153 (GADD153, also known as CHOP). This assay employed WT and hsf-1 null mouse embryonic fibroblasts (MEFs). The probe (compound F1) induced multiple responses and strongly induced Hsp70 (Figure 4), the oxidative stress response genes (HO1 and GCLM), and a 2.5-fold upregulation of BiP. This assay also suggests that induction of HO1 by probe ML246 was due to the generation of oxidative stress, as shown by a concerted upregulation of the antioxidant GCLM gene. Further, we observed potent induction of the antioxidant responsive gene HO1 in absence of HSF-1, suggesting that the probe’s action is hsf1-independent, elucidating the probe’s mechanism of action.

Figure 4. a, Wild type (hsf-1+/+) and b, HSF-1 null (hsf-1−/−) (MEFs) were treated for 4 h with probe ML346 (compound F1) at the indicated concentrations.

Figure 4

a, Wild type (hsf-1+/+) and b, HSF-1 null (hsf-1−/−) (MEFs) were treated for 4 h with probe ML346 (compound F1) at the indicated concentrations. Relative levels of multiple cytoprotective genes were measured by real-time PCR (qPCR). See (more...)

CFTR Model (QFRET Assay)

Human cystic fibrosis bronchial epithelial cells (CFBE41o-) stably expressing ΔF508-CFTR as well as H148Q/I152L-YFP (CFBE41o- -YFP) were added to a 96-well black walled plate and grown to confluency in growth media (α-MEM containing 100 U/ml penicillin, 100 U/ml streptomycin, 10% v/v FBS, 2mM L-glutamine, 2 μg/ml puromycin and 0.75 μg/ml G418). Cells were treated with the indicated concentration of compounds in complete growth media and incubated at 37 degrees C, 5% CO2 for 24 hours. Cells were subsequently washed three times with 200 μL of PBS pH 7.4 (137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl2, 1.1 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM Na2HPO4) and equilibrated in 40 μL of PBS pH 7.4 and maintained at 37 degrees C throughout. Cells were stimulated with a final concentration of 10 μM forskolin (fsk) and 50 μM genistein (gen) for 15 minutes prior to addition of PBS + NaI (replacement of NaCl with 137 mM NaI). Fluorescence was monitored every second for a total of 30 seconds (3 seconds prior to addition of NaI and 27 seconds after addition of NaI). Data were normalized to the initial fluorescence to account for variations in the overall starting fluorescence. To ensure that the observed H148Q/I152L-YFP fluorescence quenching was the result of ΔF508-CFTR activation and not the action of additional halide channels, the CFTR specific inhibitor (CFInh-172) was used. This assay shows that probe ML246 is able to restore proteostasis reduce the aggregation and misfolding of ΔF508-CFTR (Figure 5). Probe ML346 also restored CFTR-mediated iodide conductance. Probe ML346 is the first small molecule capable of enhancing the correct folding of proteins expressed in two different cellular compartments.

Figure 5. Probe ML346 restores proteostasis.

Figure 5

Probe ML346 restores proteostasis. CFBE41o- YFP cells were treated with 0.1% DMSO (black), the positive controls 5 μM SAHA (purple), 10 μM Corrector 4a (Corr4a) (grey) and the PRs A3 (dark blue), C1 (royal blue) and Probe ML346 (F1; cyan) (more...)

2.2. Probe Chemical Characterization

The chemical structure of the probe was verified by analysis of its 400 MHz 1H NMR spectra (Figure 6) obtained on a Brüker 400 MHz instrument. The chemical structure was also corroborated by high resolution mass spectroscopy (calc for M+: 273.0875, found: 273.0891). Purity was measured at >98% (LC/MS analysis, confirmed by analytical HLPC analysis. The mass spectrum is shown in Figure 7. HPLC purity data is shown in Figure 8. HPLC data was obtained using an Agilent 1200 analytical HPLC with an Agilent Eclipse XDB-C18 column, 4.6×150mm. The HPLC solvents used were acetonitrile and water with 0.1% formic acid added to each mobile phase as the pH modifier.

Figure 6. 1H NMR spectrum.

Figure 6

1H NMR spectrum.

Figure 7. Mass spectrum, calc for M+1: 273.08, found 273.10.

Figure 7

Mass spectrum, calc for M+1: 273.08, found 273.10.

Figure 8. HPLC spectrum; purity >95%.

Figure 8

HPLC spectrum; purity >95%.

The solubility of the probe ML346 in PBS at pH 7.4 was determined to be 0.64 μM, while in DMES with 10% FBS the solubility was 21.45 μM (see Table 2). Its solubility is fully adequate to provide the high potency seen in cell-based assays and is also adequate for broad use as a biological probe to be used in a variety of aqueous-based media.

Table 2. Solubility at 100 μM; 1% DMSO.

Table 2

Solubility at 100 μM; 1% DMSO.

The probe ML346 has an apparent half-life of 16 hours in 30% PBS-70% DMSO at room temperature when tested at 10 μM protected from light (Figure 9). The apparent half-life was 6 h when this study was performed in 10% PBS-30% DMSO (absence of light) or 2 h under light exposure. Based on these data, exposure of solutions of the probe to light should be minimized (this can be accomplished by wrapping the glassware in foil or by using dark-glass vessels). Concerning stability of ML346, the probe was completely stable in a 7:1 mixture of d6-DMSO:D2O) over 7 days as determined by NMR (Figure 6). Therefore, the apparent instability of the probe compound in PBS (when studied in the dark) is believed to be due to poor solubility under these condition. The potential light-sensitivity of ML346 when in PBS buffer was not an issue in the cell-based and in vivo studies reported in the 2012 Nature Chem. Biol. paper that preceded the submission of this probe report [9].

Figure 9. Stability of Probe ML346.

Figure 9

Stability of Probe ML346.

The probe is stable (>95%) over a 4 hour period in the presence excess glutathione (50 μM) as determined by HPLC analysis. After 6 hours, 79%–89% of the probe remained unchanged. Because glutathione adducts were not detected by LCMS analysis of these experiments, it is believed that the ‘decrease’ in the amount of probe ML346 after 6 hours is due to solubility issues. These data show that the presence of the highly conjugated diene unit does not render the probe highly susceptible to Michael addition.

The probe is stable in DMSO solution at room temperature (no erosion of peak intensity over 48 hours) and is also stable as a free base dry powder. It is also stable under assay conditions, as indicated by potency in various secondary assays that is independent of incubation time.

2.3. Probe Preparation

Probe ML346 was synthesized in a straightforward fashion in a single step using commercially available reagents, as shown in Figure 10. The yield was 87%. Analogs for SAR evaluation were prepared by similar methods.

Figure 10. One Step Synthesis of ML346 (CID 767276).

Figure 10

One Step Synthesis of ML346 (CID 767276).

A solution of barbituric acid (647 mg, 5.05 mmol, 1.01 equiv; purchased from Fluka, #11709) in water (10 mL) was heated at 100 °C until homogeneous. The resulting solution was cooled to 90 °C, and then 4-methoxycinnamaldehyde (811 mg, 5.00 mmol, 1 equiv; purchased from TCI America, #M1012) was added portion-wise. An orange precipitate appeared almost instantly. The mixture was stirred for 12 h at 90 °C, then was allowed to cool to room temperature. The orange precipitate was filtered off, washed three times with water (10 mL) and one time with cold ethanol (10 mL). The solid was dried under vacuum affording the probe (CID 767276) as an orange powder (1.18 g, 87 % yield, >97% purity by NMR and HPLC).

1H NMR (400 MHz, d6-DMSO): δ 10.70 (s, 1H), 10.65 (s, 1H), 7.82 (dd, J = 12.0, 15.4 Hz, 1H), 7.50 (d, J = 12.1 Hz, 1H), 7.19 (d, J = 15.2 Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 6.57 (d, J = 8.8 Hz, 2H), 2.83 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 163.3, 163.1, 162.1, 154.6, 153.4, 150.4, 130.8 (2C), 128.1, 122.1, 114.9 (2C), 114.0, 55.5; HRMS (ESI) calculated for C14H13N2O4 [M+H]+: 273.0875, found: 273.0891; IR (cm−1): 3173, 3059, 2827, 1724, 1657, 1588, 1537, 1505, 1428, 1373, 1317, 1306, 1258, 1232, 1213, 1159, 1047, 1020, 993, 946, 937, 880, 842, 820, 793, 756, 702.

3. Results

3.1. Dose Response Curves for Probe

This HeLa cell-based assay measures the activation of the heat shock response (HSR) in HeLa cells stably transfected with a heat-shock–inducible reporter containing the proximal human Hsp70.1 promoter sequence upstream of a luciferase (luc) reporter gene. The assay was used in the uHTS campaign and in follow-up runs to aid probe development. Using our standard calculation methods, the probe compound yielded an EC50 of 4.6 μM in this assay (SID 152186720).

However, instead of graded, sigmoidal dose-response curves, the dose-response profiles (see Figure 11) of all compounds assayed in the HSE-Luc assay were characterized by an increasing luminescence response with increasing test compound dose, followed by a sharp decrease in response at higher dose concentrations. This type of dose-response profile has been previously reported for this cell line [10]. Therefore the maximum average percent activity measured, and the corresponding concentration at which the maximal activity was observed was used to rank compound potency. As this assay was run in an activation mode, it is not unusual to observe more than 100% activation: it indicates that the compound was more active than the positive control (the proteasome inhibitor MG132 at its EC100, 30 micromolar).

Figure 11. Concentration response curve for probe ML346.

Figure 11

Concentration response curve for probe ML346.

We also profiled the probe compound in a variety of secondary assays, including Cytotoxicity assays, and in vitro and in vivo efficacy assays. These results are discussed in section 3.3 under profiling assays.

3.2. Cellular Activity

The HTS assays and follow-up primary and secondary assays are cell-based, so the probe has confirmed cellular permeability.

3.3. Profiling Assays

We recently published a communication describing the screening effort and the uHTS results [9]. In that communication a great deal of profiling of the screening hit and related leads was disclosed. In particular relevance to this probe report, the assay provider performed an in vivo assay to determine the effect of the probe on protein aggregation. This assay is described in the following sections.

C. elegans Assays for Aggregation and Motility Defects

The purpose of this assay is to determine whether powder samples of the compounds identified as transcriptional activators of heat shock protein 70 (Hsp70) can modulate the characteristics of polyglutamine diseases such as Huntington’s. These assays employed a model organism: C. elegans worms that exhibit age-dependent aggregation of polyQ35-YFP in body wall muscles. Worms with polyQ35 aggregation will exhibit decreased motility. As designed, compounds that reduce the aggregation of polyQ35 without reducing levels of the protein are considered active. This assay also monitored aggregation-associated toxicity.

Worms were maintained according to standard methods, at 20°C on nematode growth media (NGM) with OP50 E. coli (Brenner 1974). The following strains were obtained from the C. elegans Genetic Center (CGC): wild-type (wt) Bristol strain N2, HSF-1 mutant hsf-1(sy441) (PS3551), temperature sensitive strains unc- 52(e669su250) and unc-45(e286) (HE250 and CB286, respectively). The polyglutamine strain expressing 35 CAG-repeats fused with YFP (Q35::YFP) was described elsewhere (AM140 in CGC)[11].

Treatment with compounds was performed in a 96-well plate format (final volume 60 μL), comprising 20 to 25 L2 (larval 2 stage) age-synchronized animals, compound at the appropriate concentration, and OP50 bacteria to a final OD595nm of 0.8 in the microtiter plate. Animals and bacteria were resuspended in S-medium supplemented with streptomycin, penicillin, and nystatin (Sigma, St. Louis, MO). To obtain the age synchronized population of L2 larvae, gravid adults were bleached with a NaOCl solution [250 mM NaOH and 1:4 (v/v) dilution of commercial bleach] and the eggs were allowed to hatch in M9 buffer overnight at 20°C. The first larval stage (L1) animals were transferred to OP50 plates to develop into L2 stage. Animals were washed with M9 buffer, resuspended in S medium, and transferred into 96-well plates. Compounds were dissolved and diluted in 100% DMSO, and animals were incubated at a maximum concentration of 1.5% DMSO to avoid solvent-specific developmental defects and toxicity. The range of final concentrations tested was 0, 1, 5, 10 and 15 μM. OP50-only and DMSO-only controls were used. In addition, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG, Biomol, Plymouth Meeting, PA) (0.5, 1, 5 and 50 μM) was used as positive control for HSR induction. Plates were incubated at 20°C for 4 days. Animals were scored for changes in aggregation (number of fluorescent foci) using the stereomicroscope Leica MZ16FA equipped for epi-fluorescence. Suppression of aggregation was scored positive when ≥50% of worms had a reduction in fluorescent foci, without loss of body-wall fluorescence, compared to DMSO. As shown in the results of Figure 12, probe ML346 is able to suppress the aggregation of polyQ35, as demonstrated by statistically significant reductions in the protein foci. These reductions in protein aggregation correlated with restorations of animal motility (Figure 12c). This result shows the in vivo efficacy of probe ML346 for reducing protein aggregation and associated toxicity.

Figure 12. Probe ML346 (compound F1) reduces aggregation/toxicity in C. elegans models of diseases associated with polyQ expansions.

Figure 12

Probe ML346 (compound F1) reduces aggregation/toxicity in C. elegans models of diseases associated with polyQ expansions. (a) C. elegans expressing YFP-tagged Q35 protein were treated with either DMSO (panel I) or PRs (panels III–V) at different (more...)

PubChem Promiscuity Analyses

Analysis of the PubChem database of assays and small molecules shows that the screening hit (CID 1045135) is active in only a small percentage of all assays in which it has been screened (~1.2%), indicating a lack of non-specific protein binding or polypharmacology, characteristics that likely extend to the probe ML346, a positional isomer of the screening hit. There are no serious issues with regard to the drug-like attributes of the probe [12–14]. There are also no concerns with respect to toxicity structure alerts [15].

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Because of the anti-apoptotic and anti-inflammatory properties of Hsp70, research efforts have attempted to increase Hsp70 expression and activity through the use of small molecules. Several activators of the HSR are available, including natural product derivatives. Radicicol (CID 6323491) and geldanamycin (CID 5288382) that inhibit formation of huntingtin protein aggregates [16]. Celastrol is neuroprotective in animal models of neurodegeneration [17]. Compounds such as bimoclomol (CID 9576891) [18] and arimoclomol (CID 208924), promising candidates for the treatment of ischemia and ALS, respectively, demonstrate the ability as coinducers of the HSR and Hsp70 [19,20]. Although registered, neither of these compounds has been tested in PubChem bioassays.

Protein synthesis inhibitors, amino acid analogues [21,22], proteasome inhibitors such as MG132 (CID 462382; reversible) and lactacystin (CID 6610292; irreversible), and certain NSAIDs increase the HSR, in part by inducing hyperphosphorylation and/or DNA binding activity of HSF-1 [23,24]. However, many of these compounds either do not increase Hsp70 expression or fail to be selective [24]. However, MG132, used as a High Control for Hsp70 activation is not selective for the HSR.

In comparison to the above prior art and control compounds, probe ML346 is the first small molecule shown to restore the correct folding of proteins in both cellular and animal models, without significant cytotoxicity or lack of specificity. The probe is cell permeable and induces specific increases in genes and protein effectors of the HSR, including chaperones such as Hsp70, Hsp40, and Hsp27.

Publications

A communication describing the screening effort and the HTS results has been published in 2012 in the journal Nature Chemical Biology [9].

5. References

1.
Gupta RS, Singh B. Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus. Curr Biol. 1994;4(12):1104–14. [PubMed: 7704574]
2.
Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet. 1988;22:631–77. [PubMed: 2853609]
3.
Satyal SH, et al. Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2000;97(11):5750–5. [PMC free article: PMC18505] [PubMed: 10811890]
4.
Wyttenbach A, et al. Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease. Proc Natl Acad Sci U S A. 2000;97(6):2898–903. [PMC free article: PMC16027] [PubMed: 10717003]
5.
Cummings CJ, et al. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat Genet. 1998;19(2):148–54. [PubMed: 9620770]
6.
Krobitsch S, Lindquist S. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. Proc Natl Acad Sci U S A. 2000;97(4):1589–94. [PMC free article: PMC26479] [PubMed: 10677504]
7.
Kazemi-Esfarjani P, Benzer S. Genetic suppression of polyglutamine toxicity in Drosophila. Science. 2000;287(5459):1837–40. [PubMed: 10710314]
8.
Warrick JM, et al. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat Genet. 1999;23(4):425–8. [PubMed: 10581028]
9.
Calamini B, et al. Small-molecule proteostasis regulators for protein conformational diseases. Nat Chem Biol. 2012;8(2):185–96. [PMC free article: PMC3262058] [PubMed: 22198733]
10.
Westerheide SD, et al. Celastrols as inducers of the heat shock response and cytoprotection. J Biol Chem. 2004 Dec 31;279(53):56053–60. [PubMed: 15509580]
11.
Morley JF, et al. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2002;99(16):10417–22. [PMC free article: PMC124929] [PubMed: 12122205]
12.
Rishton GM. Nonleadlikeness and leadlikeness in biochemical screening. Drug Discov Today. 2003;8(2):86–96. [PubMed: 12565011]
13.
Walters WP. Going further than Lipinski’s rule in drug design. Expert Opin Drug Discov. 2012;7(2):99–107. [PubMed: 22468912]
14.
Lipinski CA, et al. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2001;46(1-3):3–26. [PubMed: 11259830]
15.
Benigni R, Bossa C. Mechanisms of chemical carcinogenicity and mutagenicity: a review with implications for predictive toxicology. Chem Rev. 2011;111(4):2507–36. [PubMed: 21265518]
16.
Hay DG, et al. Progressive decrease in chaperone protein levels in a mouse model of Huntington’s disease and induction of stress proteins as a therapeutic approach. Hum Mol Genet. 2004;13(13):1389–405. [PubMed: 15115766]
17.
Cleren C, et al. Celastrol protects against MPTP- and 3-nitropropionic acid-induced neurotoxicity. J Neurochem. 2005;94(4):995–1004. [PubMed: 16092942]
18.
Jednakovits A, et al. In vivo and in vitro acute cardiovascular effects of bimoclomol. Gen Pharmacol. 2000;34(5):363–9. [PubMed: 11368893]
19.
Nanasi PP, Jednakovits A. Multilateral in vivo and in vitro protective effects of the novel heat shock protein coinducer, bimoclomol: results of preclinical studies. Cardiovasc Drug Rev. 2001;19(2):133–51. [PubMed: 11484067]
20.
Brown IR. Heat shock proteins and protection of the nervous system. Ann N Y Acad Sci. 2007;1113:147–58. [PubMed: 17656567]
21.
Lee YJ, Dewey WC. Effect of cycloheximide or puromycin on induction of thermotolerance by heat in Chinese hamster ovary cells: dose fractionation at 45.5 degrees C1. Cancer Res. 1987;47(22):5960–6. [PubMed: 3664499]
22.
Hightower LE. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides. J Cell Physiol. 1980;102(3):407–27. [PubMed: 6901532]
23.
Kim D, Li GC. Proteasome inhibitors lactacystin and MG132 inhibit the dephosphorylation of HSF1 after heat shock and suppress thermal induction of heat shock gene expression. Biochem Biophys Res Commun. 1999;264(2):352–8. [PubMed: 10529368]
24.
Lee BS, et al. Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage. Proc Natl Acad Sci U S A. 1995;92(16):7207–11. [PMC free article: PMC41308] [PubMed: 7638169]

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