2.1. Assays

The detailed description and protocols for the primary HTS and additional assays can be found in the “Assay Description” section in the PubChem BioAssay (http://pubchem.ncbi.nlm.nih.gov/) view under the AIDs as listed in Table 2, and to Leung, C. K., et al. High-throughput Screening and Biosensing with Fluorescent C. elegans Strains. J. Vis. Exp. (51), e2745, DOI: 10.3791/2745 (2011) and Leung, C. K., et al. An ultra high-throughput, whole-animal screen for small molecule modulators of a specific genetic pathway in Caenorhabditis elegans. PLoS One (in Press) 2013 for detailed worm culture and dispensing protocols.

Table 2

Summary of Assays and AIDs.

2.2. Probe Chemical Characterization

Chemical name of probe compound. The IUPAC name of the probe is N-(3-chloro-4-((4-chlorobenzyl)-oxy)-5-ethoxybenzyl)-cyclopentanamine. The actual batch prepared, tested and submitted to the MLSMR is archived as SID 161004951 corresponding to CID 70789776. ML358 does not have any chiral centers.

Figure 1Chemical Structure of ML358

Synthesis and Structural Verification Information of probe SID 161004951 corresponding to CID 70789776 (See Scheme 1)

Scheme 1Synthesis of ML358, conditions: i) 4-chlorobenzyl bromide, K₂CO₃, CH₃CN, 80 °C, 8 h, 70%; ii) a. cyclopentylamine, sodium triacetoxyborohydride, THF-Acetic acid (4:1), 23 °C, 24 h; b. 4M HCl (dioxane), dioxane, 23 °C, 59%

Figure 2¹H NMR and LC-MS spectra of ML358

2A¹H NMR Spectrum of ML358 (500 MHz, Methanol-d₄)

2B¹³C NMR Spectrum of ML358 (125 MHz, Methanol-d₄)

2CLC/MS for ML358

2DMS spectrum for ML358

Solubility and Stability of ML358 in PBS at room temperature. The solubility of ML358 was investigated (Figure 3) in aqueous buffers at room temperature by monitoring the amount of starting ML358 apparently remaining after incubation at room temperature in either PBS (pH 7.4) or 1:1 PBS:acetonitrile (v/v). ML358 was stable in PBS:acetonitrile with 100% remaining after 48 hrs. The apparently lower stability (25%) in neat PBS is a reflection of pH dependent solubility. Thus, the stability value is artificially low due to compound precipitation vs. degradation. As noted in the Summary of in vitro ADME/T properties (Section 3.4, Table 5 below), ML358 has good solubility (<103 μM) at pH 5.0, 6.2 and 7.4 in pION buffer but lower solubility in PBS (<54 μM) at pH 7.4.

Figure 3

Stability of ML358 in 1× PBS at RT.

Table 5

Summary of in vitro ADME Properties of SKN-1 inhibitor probe ML358.

MLS# verifying submission of probe molecule and five related samples submitted to the SMR collection. This probe is not commercially available. A 27 mg sample of ML358 synthesized at SBCCG has been deposited in the MLSMR (Evotec). Table 3 summarizes the deposition of the Probe and 5 analogs.

Table 3

Probe and Analog Submissions to MLSMR (Evotec) for nematode SKN-1 pathway inhibitors.

2.3. Probe Preparation

Synthesis of ML358: 3-chloro-4-((4-chlorobenzyl)oxy)-5-ethoxybenzaldehyde (A): The phenol derivative (CAS 70842-33-0) purchased from Enamine (100 mg, 0.50 mmol), 4-chlorobenzylbromide (123 mg, 0.60 mmol), and potassium carbonate (207 mg, 1.50 mmol) were combined and charged with 5 mL of acetonitrile. The vial was capped and heated in an 80 °C block for 8 hours. After TLC (10% ethyl acetate in hexanes) indicated completion, the mixture was diluted with 10 mL of water and chilled in the refrigerator. The white precipitate obtained after decanting the solution was triturated twice with ca. 3 mL of hexanes, each time drawing away the supernatant by pipet. The white solid was dried to give 113.7 mg (70%) of A. ¹H NMR (500 MHz, Chloroform-d) δ 9.87 (s, 1H), 7.59 – 7.42 (m, 3H), 7.43 – 7.34 (m, 3H), 5.18 (s, 2H), 4.19 (q, J = 7.0 Hz, 2H), 1.52 (t, J = 7.0 Hz, 3H). TLC: R_f = 0.25, 10% ethyl acetate in hexanes; MS: weak response, no data.

N-(3-chloro-4-((4-chlorobenzyl)oxy)-5-ethoxybenzyl)cyclopentanamine hydrochloride (ML358): The aldehyde A (112 mg, 0.345 mmol) was dissolved in 4 mL of tetrahydrofuran. The solution was charged with 1 mL of glacial acetic acid and 59 mg (0.689 mmol) of cyclopentylamine. After stirring for ca. 10 minutes at room temperature, sodium triacetoxyborohydride (219 mg, 1.03 mmol) was added as solid in portions over ca. 1 minute. The whitish turbid mixture was stirred for ca. 24 hours and then was transferred into 15 mL of 4 N NaOH. The pH was verified to be ca. 14 by pH paper, and the mixture was extracted with two 15 mL portions of chloroform. The organics were dried over magnesium sulfate and concentrated to give 144.8 mg of a colorless oil. Flash chromatography (25 mL of silica gel, eluting with 50% ethyl acetate in hexanes followed by 5% methanol in dichloromethane) returned the purified free base, which was dissolved in 2 mL of 1,4-dioxane and charged with 0.5 mL of 4 N HCl in dioxane. Concentration gave a white solid salt, which was dissolved in ca. 3 mL of 1:1 water:acetonitrile and lyophilized to afford 88.1 mg (59%) of the salt ML358 as a low density white solid. ¹H NMR (500 MHz, Methanol-d₄) δ 7.59 – 7.43 (m, 2H), 7.43 – 7.29 (m, 2H), 7.25 – 6.98 (m, 2H), 5.11 (s, 2H), 4.18 (q, J = 7.0 Hz, 2H), 4.14 (m, 2H), 3.59 (p, J = 7.3 Hz, 1H), 2.17 (m, J = 9.7, 6.9, 3.3 Hz, 2H), 1.91 – 1.78 (m, 2H), 1.70 (m, J = 14.5, 5.5 Hz, 4H), 1.50 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, Methanol-d₄) δ 154.99, 145.93, 137.18, 135.11, 131.31, 129.93, 129.59, 129.43, 124.14, 114.91, 74.87, 66.14, 60.58, 50.82, 49.53, 30.78, 25.06, 15.14; HRMS (ESI+ve): Calculated for C₂₁H₂₆Cl₂NO₂, [M+H] = 394.1335, observed [M+H] = 394.1333.

3.1. Dose Response Curves for Probe

Figure 4Selectivity of ML358 inhibition for SKN-1 pathway vs. heat-shock induced pathway and the non-cytotoxic effect of ML358 on worm health

3.2. Cellular Activity

ML358 is active in cells because the primary, confirmatory and secondary assays were all conducted in an whole organism screen in a nematode, so by inference it is cell active. The probe ML358 was profiled further in a set of assays in both nematodes and human cell lines to characterize its activity and assess its potential for broader utility (Table 4). The SKN-1 target reporter strain (VP596 Pgst-4::GFP; Pdop-3::RFP) was used to test primary activity of the SKN-1 inhibitor using acrylamide (500 mg/L) or juglone (25 μM). We used another reporter strain (QV87 Pgst-30:: GFP; Pdop-3::RFP) to test the inhibitors on a second SKN-1 target promoter using acrylamide (500 mg/L) and juglone (25 μM). The strain (QV65 Phsp-16.2GFP; Pdop-3RFP) is induced by heat independent of SKN-1 and was used to test selectivity of the inhibitor to the SKN-1 pathway. In addition, Pgst-4::GFP is constitutively expressed in a SKN-1 gain-of-function mutant strain harboring the same (strain name: QV130 skn-1(k1023); Pgst-4::GFP). Activity using this genetic mutant suggests the inhibitor can specifically act through the SKN-1 pathway to regulate Pgst-4::GFP expression. No significant inhibition of Nrf2, the human homolog to SKN-1, in both LnCap and IMR-32 cell lines was observed up to 10 μM.

Table 4

Additional Profiling in Cellular Assays.

We also note that the scaffold structure represented by ML358 has no substantial chemical liabilities. However, there is a potential for metabolism to ortho-quinone or quinone-methide intermediates.

Cell Viability with MTT. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a cytotoxicity assay that relies on the cellular conversion of a tetrazolium salt to a formazan compound. The formation of the formazan product can be measured using a 96-well plate reader at an absorbance of 570nm. Cell viability was measured according to the manufacturer's instructions. (Celltiter 96 protocol from Promega.)

Nrf2 Reporter Assay. The luciferase reporter assay relies on activation of the Nrf2-responsive antioxidant response element (ARE) found upstream of many phase II detoxification enzymes. An ARE-luciferase reporter construct was transfected into LNCaP and IMR32 cells. 24 h after transfection, cells were treated with 10 uM of ARE-activators, SF or tBHQ, respectively, for 1.5 h before addition of compounds. After an additional 24 h of treatment, the ability of compounds to inhibit SF/tBHQ-driven activation of the ARE-luc was measured based on luminescence output. SF/tBHQ activation with DMSO solvent control was set at 100% ARE-luc activation.

3.3. Profiling Assays

As a pro forma activity, the SBCCG is committed to profiling all final probe compounds and in certain cases key informative analogs in the PanLabs full panel as negotiated by the MLPCN network. Additional commercial profiling services will be considered for funding by SBCCG as deemed appropriate and informative. ML358 was evaluated in a detailed in vitro pharmacology screen as shown in Table 5, below:

ML358 achieved concentrations >400 × IC₅₀ in aqueous buffer between a pH range of 5.0-7.4 with increasing solubility as pH is lowered as expected for a basic amine (-NH) containing compound. In PBS, ML358 attained a solubility >54 μM, which is >225 times its IC₅₀.

The PAMPA (Parallel Artificial Membrane Permeability Assay) assay is used as an in vitro model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. Consistent with its solubility data, ML358 exhibited very good permeability with increasing pH of the donor compartment.

Plasma protein binding is a measure of a drug's efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. ML358 was highly plasma protein bound.

Plasma stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which can strongly influence the in vivo efficacy of a test compound. Drug candidates are exposed to enzymatic processes (proteinases, esterases) in plasma, and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. ML358 showed good stability in both human plasma and mouse plasma.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. ML358 showed moderate stability and was ∼60% metabolized in human and mouse liver microsomes within 1 hour.

ML358 showed no toxicity (>50 μM) towards immortalized Fa2-N4 human hepatocytes and >30-fold index in MTT cytotoxicity (CellTiter96, Promega) in LnCap and IMR-32 cells versus potency against SKN-1 inhibition in nematodes.

Cell Viability with ATP-lite. Hepatic toxicity of compounds was determined with Fa2N-4 immortalized human hepatocytes using the ATP-lite 1-step assay (Perkin Elmer)assay according to the manufacturer's instructions. Fa2N-4 cells (XenoTech, Kansas City, KS) were seeded at 50,000 cells/well, and incubated with a range of concentrations of the test compound (0.01 μM-50 μM) in MFE support media for 24 hrs at 37°C, 5% CO₂. At the end of the experiment, cell viability was determined by cellular ATP levels using the ATP-lite kit according to the manufacturer's instructions. Luminescence was measured on the Infinite M200 plate reader (Tecan US). The concentration of each compound that killed 50% of the cells (LC50) was calculated by non-linear regression analysis using a log(inhibitior) vs response equation with a variable slope, using the statistic software package Prism4 (GraphPad, San Diego, CA).

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

A SciFinder and Integrity Prous search was performed on March 9, 2012 to assess the state of prior art as it relates to SKN-1. The following terms were searched: SKN-1, SKN-1 inhibitors. The assay providers are not aware of any SKN-1 inhibitors from the published literature and none were found upon conducting this prior literature search. However, two references for a diallyl trisulfide (a key garlic constituent) [6] and curcumin (active ingredient in turmeric) [7] both appear to increase C. elegans lifespan, and this life extension in part mapped to SKN-1 activation. On October 2, 2012 and March 28, 2013, chemists at Sanford-Burnham repeated the SciFinder search, using the terms “SKN-1” and SKN-1 inhibitor”. The search was limited to 2012-2013. The first query identified 15 sources. No new art was identified which disclosed small molecule inhibitors that would potentially impact the goals of this program. Of general interest, the search revealed biological art disclosing, “HCF-1 prevents the nuclear accumulation of SKN-1 and represses the transcriptional activation of SKN-1 specifically at target genes involved in cellular detoxification pathways” [8]; TOR Signaling affects longevity in part by regulating SKN-1/Nrf [9]; and life span in nematode acts via insulin/IGF-1-like signaling [10].

Currently available drugs target only a specific enzyme or enzyme class and/or affect homologous human targets. By selective targeting of the SKN-1 pathway present in diverse nematode clades, ML358 can potentially lead to drug candidates that could be used as adjuvants to increase the efficacy of current anthelmintics. ML358 is the first reported small molecule that inhibits activation of the SKN-1 pathway. It is also the first probe developed against a nematode gene regulatory pathway and is expected to drive a new series of research into the regulation and function of this core detoxification and longevity pathway in C. elegans and parasitic nematodes. ML358 is a potent (IC₅₀ = 0.24 μM, E_max = 100%) and selective inhibitor of the SKN-1 pathway in C. elegans. ML358 is non-toxic to C. elegans (LC₅₀ > 64 μM) and thus may be less susceptible to resistance. We have shown that ML358 inhibits transcriptional activity of gst-4 in a SKN-1 gain-of-function mutant strain. This result demonstrates that the compound blocks the SKN-1 pathway. No significant inhibition of Nrf2, the human homolog to SKN-1, was observed up to 10 μM indicating a potential application to human use. ML358 is non-toxic in Fa2N-4 immortalized human hepatocytes (LC₅₀ > 50 μM) and has >30-fold difference in cytotoxicity in LnCap and IMR-32 cell lines compared to its potency in C. elegans. In addition, ML358, has good solubility, permeability, and chemical and metabolic stability in human and mouse liver microsomes. Optimizing the potency and plasma protein binding and attenuating any reactive metabolites will improve the probe's utility.

While, the molecular target of the SKN1 pathway inhibition is not known, ML358 strongly inhibits induction of two core SKN-1 target gene promoters (gst-4 and gst-30) during exposure to two different electrophiles/reactive oxygen species generators (Table 4). Results with the SKN-1 gain-of-function mutant (Table 4) indicate that the target of ML358 is either SKN-1, or an essential component of the SKN-1 pathway. WDR-23 interacts directly with SKN-1 and functions with the DDB1/CUL4 E3 ligase to repress SKN-1 [18]. However, the SKN-1 gain-of-function mutant protein does not interact with WDR-23 (Leung and Choe, unpublished data) suggesting that ML358 works independently of WDR-23 and the E3 ligase. Glycogen synthase kinase 3 and p38 MAPK regulate SKN-1, but both require WDR-23 [18]. Other components of the SKN-1 pathway are poorly defined. If SKN-1 is not the direct target, then ML358 will likely lead to identification of a new critical pathway component, which would be an important discovery for the fields of drug detoxification, stress biology, and longevity.

Nematodes infect over a billion people world-wide and current control programs rely on mass administration of drugs that were developed for veterinary use over three decades ago. Heavy use of these drugs in veterinary medicine rapidly resulted in widespread resistance, a major concern for their use in humans. Genetic and molecular research tools for parasitic nematodes are extremely limited and therefore pharmacological probes are the most promising approach for studying the role of specific pathways such as SKN-1.

The SKN-1 protein is present in at least four out of five nematode clades [1]. ML358 potency will likely be highest in species closely related to C. elegans. C. elegans is in Clade V, together with human hookworms, which infect 0.5-1.0 billion people world-wide, cause extensive disability, and are currently the target of global mass drug administration programs [11].

We plan to evaluate ML358 against homologous proteins from parasitic strains to extend the utility of this probe. Furthermore, our long-term plan includes: 1) characterization of probe mode-of-action in C. elegans, 2) characterization of in vivo bioactivity in C. elegans, 3) identification of protein target, 4) testing activity on homologous proteins from parasitic species, and 5) test the probes in parasitic nematodes.