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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 1

, , , , , , , , and .

Author Information

,1 ,1 ,1 ,2 ,2 ,2 ,1 ,1 and 1,3.*

1 The Broad Institute Probe Development Center, Cambridge, MA
2 New York University, New York, NY
3 Howard Hughes Medical Institute, Chemistry and Chemical Biology, Harvard University, Cambridge, MA
*Corresponding author email: gro.etutitsnidaorb@ydomracl

Received: ; Last Update: March 11, 2011.

Chagas disease is a tropical disease caused by the parasite Trypanosoma cruzi (T. cruzi), which is endemic to Central and South America. Approximately 13 million people are infected with the parasite, and 25–30% of infected patients suffer from irreversible damage to the heart and digestive tract resulting in disability and death within 20 years of infection. Few treatments are available with limited effectiveness only against the early (acute) stages of disease, significant toxicity, and widespread drug resistance. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, small-molecule inhibitors of T. cruzi, which will aid the development of more potent and selective therapies for both the acute and chronic stages of Chagas disease. Of the 303,224-screened compounds, 35 compounds were chosen based on their selectivity, potency, and chemical tractability. Of those, 27 dry powder-validated compounds were retested in the primary screen, secondary assays, and an orthogonal screen. Three scaffolds were prioritized to identify potential probes. One of these compounds (CID 6875690/ML164) displayed greater than 100-fold selective inhibition of T. cruzi confirmed in immunofluorescent imaging assays and is inactive against host cells at the highest tested dose. This new probe should be very useful in future cell-based investigations and in vivo studies of T. cruzi inhibition.

Assigned Assay Grant No.: 1R03MH085673-01

Screening Center Name and PI: Broad Institute Probe Development Center, Stuart Schreiber

Chemistry Center Name and PI: Broad Institute Probe Development Center, Stuart Schreiber

Assay Submitter and Institution: Ana Rodriguez, New York University

PubChem Summary Bioassay Identifier (AID): 1885

Probe Structure and Characteristics

Compound Summary in PubChem

IUPAC Chemical NameN-(3,4-dimethylphenyl)-N′-[(E)-(5-nitrofuran-2-yl)methylideneamino]butanediamide
PubChem CID6875690
Molecular Weight358.34862 g/mol
Molecular FormulaC17H18N4O5
XLogP3-AA2.2
H-Bond Donor2
H-Bond Acceptor6
Rotatable Bond Count6
Exact Mass358.12772
Topological Polar Surface Area130
Solubility (PBS, μM)< 1
CID/ML No.Target NameIC50/EC50 (nM) [SID, AID]Anti-targetIC50/EC50 (μM) [SID, AID]Fold Selective*Secondary Assay(s) IC50/EC50 (nM) [SID, AID]
6875690/ ML164Trypansoma cruzi replication34.3 [SID 87219036, AID 2630]NIH/3T3 Cell ToxicityInactive [SID 87219036, AID 2586]34.3 nM vs inactiveImmunofluorescence Static [SID 87219036, AID 2630]
*

Selectivity = anti-target IC50/target IC50.

Recommendations for scientific use of the probe

The goal of this project is to identify a small-molecule inhibitor of the protist Trypanosoma cruzi (T. cruzi), the causative parasite of Chagas disease. Although a few treatments for Chagas disease are available, these treatments are limited because they are only effective against the early (acute) stages of disease and have significant toxicity to the patient (1, 2, 3). Novel antitrypanosomal agents will aid in developing a drug treatment for both the acute and chronic stages of Chagas disease. This probe (CID 6875690/ML164) is expected to inhibit the replication of T. cruzi without having effects on the viability of the mammalian host cell.

The probe (CID 6875690/ML164) described in this report inhibits the replication of T. cruzi with an IC50 of 34.3 nM and is inactive toward mammalian cells (NIH/3T3) up to concentrations of 19,500 nM. In immunofluorescent imaging assays, the compound inhibited T. cruzi amastigote replication within host cells. Since amastigote morphology was maintained, this probe (CID 6875690/ML164) is categorized as a trypanostatic (static) compound.

This probe (CID 6875690/ML164) provides a valuable tool that will allow microbiologists investigating Chagas disease to identify new targets in T. cruzi, which may potentially lead to better drugs. Furthermore, the Assay Provider, Dr. Ana Rodriguez and her colleagues, will continue their own studies with this probe (CID 6875690/ML164) in a mouse model of Chagas disease.

1 Introduction

Scientific Rationale

Chagas disease is a tropical parasitic disease caused by the parasite Trypanosoma cruzi (T. cruzi). Approximately 13 million people are infected with the parasite, which is endemic to Central and South America and results in 14,000 deaths per year (2, 3). Twenty-five to thirty percent of infected patients suffer from irreversible damage to the heart and digestive tract resulting in a high incidence of disability and death within 20 years of infection (1, 2). Chagas disease is the leading cause of heart failure in the region.

In Figure 1, the drugs currently used to treat Chagas disease are: benznidazole A (Rochagan®, Radanil®, Hoffman-La Roche) and nifurtimox B (Lampit®, Bayer), whose anti-T. cruzi activities were discovered more than 30 years ago. Both of these drugs are effective only in the acute phase of infection, rather than the key chronic stage. They often do not completely eliminate the parasite despite long-term administration, while exhibiting unacceptable side effects (such as nausea, vomiting, weight loss, neurological effects, and signs of testicular and ovarian injury). Both treatments are actually nonspecific for T cruzi; in addition, resistance to these drugs is becoming more widespread. Thus, there is a clear need for more potent and selective therapies.

Figure 1. Current Treatments for Chagas Disease.

Figure 1

Current Treatments for Chagas Disease. Benznidazole (Rochagen®, Radanil®, Hoffman-La Roche) (A); Nifurtimox (Lampit®, Bayer) (B)

Several recent reports have revealed promising new lead compounds that target T. cruzi, including inhibitors of the cysteine protease cruzain and inhibitors of sterol demethylase (4). However, while the protease inhibitors (see Figures 2A, 2B, and 2C) exhibit excellent activity in biochemical assays (0.079 μM to 2 μM), they are inactive or demonstrate high micromolar activity in parasite inhibition in cell culture (1 μM to inactive) (4,5,6). Thus, there is a continued unmet need to identify scaffolds that are potent in cell culture against T. cruzi and nontoxic to the mammalian host cell. Our approach overcomes this problem of lack of cellular activity by employing a phenotypic screen measuring parasite replication in a host cell. Counterscreening in the mammalian host filters out toxic compounds.

Figure 2. Recent Inhibitors of T. cruzi Cysteine Proteases.

Figure 2

Recent Inhibitors of T. cruzi Cysteine Proteases.

There are currently four compounds in clinical trials for Chagas disease (see Figure 3). Benznidazole (see Figure 1A) initially developed for treatment of acute Chagas disease is in Phase III trials to expand its use to include the treatment of chronic Chagas cardiomyopathy. Three antifungal drugs from Merck (see Figure 3A), Takeda (see Figure 3B), and Eisai (structure not disclosed) are in Phase I or II trials for expanding their use to include treatment of Chagas disease. Two heart arrhythmia treatments (see Figure 3C and Figure 3D) are the most frequent treatments for the chronic stage heart issues associated with Chagas disease and have shown anti-parasitic activity in some patients. The only novel compound currently in the pipeline is a peptide mimic from the Sandler Center for Drug Discovery (see Figure 3E) which is in preparation for a Phase I safety trial. Since all of these compounds are nonspecific for T. cruzi, the side effects may be problematic as is the case for benzidazole in its current use. Therefore, a selective, nontoxic inhibitor of T. cruzi replication could be of significant use to the scientific community.

Figure 3. Compounds in Clinical Trials for Chagas Disease.

Figure 3

Compounds in Clinical Trials for Chagas Disease.

This project aims to identify novel antitrypanosomal agents for the treatment of Chagas disease. One key design of the project is to target the parasites co-cultured with host cells and to demonstrate little to no toxicity to the host cell. This screening campaign has identified a probe (CID 6875690/ML164),N-(3,4-dimethylphenyl)-N′-[(E)-(5-nitrofuran-2-yl)methylideneamino]butanediamide, which has an IC50 of 34.3 nM and has no toxicity towards NIH/3T3 cells at the highest concentration tested (i.e., 19.5 μM).

2 Materials and Methods

The methods in this section were either performed as described in Bettiol et al. (i.e., immunofluorescence) (7) or modified for high throughput screening (co-culture and host cell toxicity).

Materials and Reagents

T175 culture flasks with vented caps were obtained from BD Falcon, and hyperflasks were obtained from Corning (Corning, NY; Catalog no.10024). Disposable sterile filter units (500 ml or 1 L; pore size, 0.20 μm were obtained from Nalgene (Catalog no. 566-0020). Dulbecco’s modified Eagle’s medium (DMEM) with Phenol Red, high glucose, with L-glutamine and sodium pyruvate was obtained from Cellgro (Mediatech Inc, Manassas, VA; Catalog no. 10-013-CM).

Penicillin-streptomycin-L-glutamine (PSG, Catalog no. 10378-016), FBS-heat inactivated fetal bovine serum (FBS, Catalog no. 16140-089), and 0.25% Trypsin-EDTA 1X (Catalog no. 25200-072) were purchased from Gibco-Invitrogen. Sterile horse serum, from donor herd (if appearance of epimastigotes) was obtained from Sigma (Catalog no. H1270). Sterile, Ca++/ Mg++-free Phosphate Buffered Saline (PBS) 1X was prepared in house.

Nonidet P-40 (NP40, now called Igepal CA 360) was obtained from Fluka (Sigma-Aldrich, St. Louis, MO; Catalog no. 56741) and Gal-Screen® Buffer B was obtained from ABBiosciences (Allston, MA; Catalog no.T1031).

Cell Lines

  • The following cell lines were used in this study: LLC-MK2 cells (rhesus monkey kidney epithelial cell line) and NIH/3T3 cells (mouse embryonic fibroblastic cell line) were initially obtained from the Assay Provider, then from ATCC. T. cruzi expressing β-galactosidase (T. cruzi -β-gal: Tulahuen strain, clone C4) was obtained from the Assay Provider with derivation as described in Buckner et al. (8).
  • Alexa Fluor 488 goat anti-rabbit IgG secondary antibody was from Molecular Probes®, Invitrogen (Carlsbad, CA).
  • Polyclonal rabbit anti-T. cruzi was a gift from Dr. B. Burleigh, Harvard School of Public Health, Boston, MA).

2.1 Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided in Appendix A (Table A1). Refer to Appendix B for the detailed assay protocols.

2.1.1 T cruzi Inhibition Assay (AID Nos.1885, 2044, 2294, 2630, 2396, 493197)

For cell propagation: 90% DMEM, Phenol Red, 10% FBS, and 1% PSG were mixed and filtered through a 0.2-microns membrane. The cells were kept at 4°C, and then warmed up to 37°C in a water bath before use.

For T. cruzi culture and assays: 98% DMEM, Phenol Red, 2% FBS, and 1% PSG were mixed and filtered through a 0.2-microns membrane. The cells were kept at 4°C, and then warmed up to 37°C in a water bath before use.

Solutions: Gal-Screen + 0.05% NP40. Using a Gal-Screen base kit, Buffer B (Catalog no. T2361) was mixed with 1:25 substrate (Catalog no. T2359) with a 1:400 dilution of 20% NP40.

NIH/3T3 Cell Culture. NIH/3T3 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 in 50 ml total of medium.

LLC-MK2 Cell Culture. LLC-MK2 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 flasks in 50 ml total of medium. Cells were usually passaged twice a week at 1:4 to 1:8 ratios.

Parasite Culture: Tcruzi β-gal (Tc). T. cruzi -β-gal were cultivated in DMEM supplemented with 2% FBS and 1% PSG in T175 flasks with vented caps (important to avoid spills!) in 50 ml total of medium.

2.1.2 Growth Inhibition Assay for HTS (384-well plates)

The medium was warmed up with 2% FBS/DMEM. The parasites were harvested in 50-ml tubes, and spun for 10 minutes at 2200 rpm. Approximately 15 ml of media was aspirated, and the samples were incubated for 3 to 5 hours. The NIH/3T3 cells were trypsinized (refer to cell culture protocol). When the NIH/3T3 cells were detached, the cells were harvested in DMEM, 2% FBS, and 1% PSG, then counted using the Nexcelom Cellometer. The cells were diluted to 166,667 cells/ml, and then added to a flask and plated 5,000 cells/30 μL per well using a standard cassette multiwell drop Combi. The cells were incubated for 3 hours, then T. cruzi cells were counted, diluted to 0.250 million cells/ml, and transferred to a 2-liter flask. Then, 50 nL compounds/DMSO were pinned to each well with NIH/3T3 cells. Next, 20 μL/well of parasites (5000 T. cruzi) were added with a standard cassette multiwell drop Combi on slow speed, and incubated for 4 days (or a minimum of 90 hours). Gal-Screen was prepared with 0.05% NP40, 30 μL per well were dispensed in a 384-well plate, incubated for 60 minutes, and the luminescence was read using Envision (Perkin-Elmer) at 0.1 sec/well.

2.1.3 Cell Toxicity Assay: NIH/3T3 Cells (AID Nos. 2010, 2586, 493247)

For the cell toxicity assay with NIH/3T3 cells, the same materials as for T. cruzi co-culture assay were used. NIH/3T3 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 in 50 ml total of medium.

2.1.4 Intracellular T. cruzi Immunofluorescence Assay (AID No. 2632)

Fifty thousand NIH/3T3 cells were seeded on sterile glass coverslips in 12-well plates and allowed to adhere overnight. Five million T. cruzi parasites were added (mechanism of inhibition 100:1) and allowed to infect for 2 hours in DMEM plus 2% FBS and PSG. Parasites were rinsed out 3X with PBS, and compounds were added at 10X their IC50 (as determined in AID 2044 and AID 2294). Infected cells were further incubated for 4 days and fixed for 15 minutes with 4% paraformaldehyde.

Fixed cells on coverslips were rinsed with PBS and permeabilized for 15 minutes in PBS with 0.1% Triton X-100. After blocking for 20 minutes in PBS with 10% goat serum, 1% bovine serum albumin (BSA), 100 mM glycine, and 0.05% sodium azide, the cells were incubated for 1 hour at room temperature with a polyclonal rabbit anti-T. cruzi at 1:2000 dilution. After rinsing, an Alexa Fluor 488 goat anti-rabbit IgG secondary antibody was added for 1 hour at a 1:800 dilution. DNA was stained with DAPI, and coverslips were mounted with anti-fade mounting media. Images were taken using an inverted Olympus IX70 microscope with a 60X oil objective.

2.2 Probe Chemical Characterization

The probe (CID 6875690/ML164) 1 was purchased from Enamine (ID T0512-1877); however it could be synthesized starting from 5-nitro-2-furancarboxaldehdye 2 in one step as shown in Scheme 1, based on literature precedent (9). Treatment of 2 with N-(3,4-dimethylphenyl)-3-hydrazinylpropanamide 3 would provide the probe 1 (CID 6875690/ML164, SID 87219052), N-(3,4-dimethylphenyl)-N′-[(E)-(5-nitrofuran-2-yl)methylideneamino]butanediamide. The probe was determined to have a solubility of <1 μM in PBS at room temperature.

Scheme 1. Synthesis of the Probe.

Scheme 1

Synthesis of the Probe.

The probe (CID 6875690/ML164) and five analogs were submitted to the SMR collection (MLS002725693, MLS002725690, MLS002725681, MLS002725680, MLS002729069).

The 1H NMR (300 MHz, DMSO-d6) spectra and LC-MS chromatograms of the probe (CID 6875690/ML164) and analogs (CID 6875684, CID 6884367, CID 6872741, CID 9689369, CID 9563094, CID 5344250, CID 6875617, CID 6322955, CID 9557463, CID 6875630, CID 5332109, CID 21210609, CID 9558304, CID 5334382, CID 5349954, CID 9640748) are provided in Appendix C.

2.3 Probe Preparation

Not applicable. The probe (CID 6875690/ML164) was purchased from Enamine (ID T5012-1877).

3 Results

Probe Attributes

  • Inhibits extracellular parasite invasion into host cell or inhibits intracellular parasitic replication.
  • Non-toxic to host cells (100-fold selectivity towards parasite vs. host cells)

3.1 Summary of Screening Results

Figure 5 displays the critical path for probe development.

Figure 5. Critical Path for Probe Development.

Figure 5

Critical Path for Probe Development. In the primary screen, 303,224 compounds were screened. Of these, 4,063 compounds were available for evaluation in the primary screen retest at dose and secondary assay. Of the 3,005 that passed this filter, 27 dry (more...)

A high-throughput screen of 303,224 compounds (AID 1885) was performed in duplicate utilizing the recombinant Tulahuen strain of T. cruzi stably expressing beta-galactosidase reporter co-cultured with host cell, mouse fibroblast NIH/3T3 (8). Signal was normalized to neutral (DMSO) controls, and a 55% inhibition cutoff at a screening concentration of 3.75 μM average was used to define a hit. Next, 4,394 hits were identified as inhibitors of T. cruzi replication when co-cultured with host cells and, of these, 4,063 were available as cherry picks. The cherry-picked compounds were retested in dose in the primary assay using co-cultured T. cruzi strain with host cells to confirm their inhibitory activity (AID 2044, 2294). From the 4,063 compounds retested at dose concentrations, 4,016 compounds (99%) re-confirmed in this assay. In parallel, these 4,063 compounds were included in toxicity assays against host cell NIH/3T3 (AID 2010) to determine if these compounds were cytotoxic to mammalian cells and thus, false positives. This secondary screen identified 1,011 compounds that reduced viability of NIH/3T3 cells; therefore, these were excluded as viable hits.

After completing retests and a secondary assay at dose from DMSO stocks, 3,005 compounds were clustered into groups with 70% similarity. The 25 clusters with at least five members were given to a team of chemists; 35 representative compounds, at least one from each cluster, were prioritized based on molecular weight, presumed solubility, and lack of toxic or reactive functionality. Of the 35 compounds selected, 27 authentic dry powders were obtained. From this set of dry powder compounds, a piperidine series was prioritized.

Additional analogs were ordered and tested in the primary and secondary toxicity assay. The results are reported in Table 1, Table 2, and Table 3. Also, these compounds were tested in an additional immunofluorescence assay to determine the mode of action of the compounds (AID 2632).

Intracellular T. cruzi was treated with the probe compound (CID 6875690/ML164). The compound inhibited replication of T. cruzi within the mammalian host cell but did not lyse T. cruzi. Therefore, the compound was classified as a ‘static’ inhibitor. Related analogs were also tested (AIDs 2630, 2586, 2632).

3.2 Dose Response Curves for Probe

Figure 6 displays dose response curves for the probe.

Figure 6. Dose-dependent Activities of the Probe (CID 6875690/ML164) in Target and Counterscreen Assays.

Figure 6

Dose-dependent Activities of the Probe (CID 6875690/ML164) in Target and Counterscreen Assays. Primary screen from dry powders (IC50 34.3 nM) (AID 2630) (A); NIH/3T3 (inactive) Toxicity (AID 2586) (B).

The compound (CID 6875690/ML164) met the defined probe criteria by displaying greater than 100-fold selective inhibition of T. cruzi vs. NIH/3T3 (34.3 nM vs. inactive). Furthermore, the inhibition of T. cruzi replication was confirmed in immunofluorescent imaging assays (AID 2632). The compound inhibited replication as compared to DMSO control condition and the compound did not lyse the cells. The probe (CID 6875690/ML164) has acceptable solubility and stability in aqueous conditions.

3.3 Scaffold/Moiety Chemical Liabilities

A search of PubChem for the probe compound (CID 6875690/ML164) revealed that the probe has been tested in 255 BioAssays and was confirmed as active only in our assays. Therefore, the compound is not promiscuous. The compound has a drug-like structure with no obvious chemical liabilities that would be a concern.

The PBS stability of the probe compound (CID 6875690/ML164) was monitored over 48 hours, and the data is presented in Figure 7. After 24 hours, 87.8% of the compound was remaining; however, the amount of compound decreased to 51.9% between 24 and 48 hours. Recovery with acetonitrile was done at each time point to account for any precipitation and showed that no precipitation occurred.

Figure 7. Stability of the Probe (CID 6875690/ML1164) in PBS.

Figure 7

Stability of the Probe (CID 6875690/ML1164) in PBS.

3.4 SAR Tables

The tables above reveal interesting SAR involving structural perturbations of the probe scaffold. For example, replacing the nitro group (entry 1, Table 1) with hydrogen (entry 2) or methyl (entry 3) leads to complete loss of activity (Table 1).

While the eastern portion of the scaffold is more tolerant to modification, anilines and amides derived from anilines seem to be favored over other substituents as can be seen by comparing Table 2 and Table 3. In general, addition of substituents to the aromatic amine or amides results in greater potency (entries 1–4, Table 2).

Table 1SAR Showing the Nitrofuran Subunit is Essential for T.Cruzi Inhibition

SAR Analysis for T. cruzi
Image ml164fu2.jpg
Potency (nM) mean ± S.E.M. (n=replicates)Target to Antitarget Fold Selectivity
EntryCIDSIDBroad No.*RnT. cruzi inhibitionnToxicity
1687569087219052BRD-K64970062P
Image ml164fu3.jpg
334.3±3.13inactive>500
Solubility: < 1 μMPurity: >95%
2534995487556790BRD-K91047982P
Image ml164fu4.jpg
3inactive-NDNA
Solubility: 105.4 μMPurity: >95%
3964074887556791BRD-K17628612P
Image ml164fu5.jpg
3inactive-NDNA
Solubility: 17.3 μMPurity: >95%

NA=Not applicable; ND = Not determined

*

P = Purchased

Table 2SAR Showing the Influence of Aniline and Amide Substituents on T.Cruzi Inhibition

SAR Analysis for T. cruzi
Image ml164fu6.jpg
Potency (nM) mean ± S.E.M. (n=replicates)Target to Antitarget Fold Selectivity
EntryCIDSIDBroad No.*RnT. cruzi inhibitionnToxicity
1687569087219052BRD-K64970062P
Image ml164fu7.jpg
334.3±3.13inactive>500
Solubility: < 1 μMPurity: >95%
2687568499351071BRD-K30246397P
Image ml164fu8.jpg
33.00311400±0.847>500
Solubility: 5.9 μMPurity: 91%
3688436787219044BRD-K85660637P
Image ml164fu9.jpg
330.8±9.7317550>500
Solubility: NDPurity: >95%
4687274187556788BRD-K73819439P
Image ml164fu10.jpg
361.3±14.8335600±14000>500
Solubility: NDPurity: 94%
5968936999351075BRD-K60134467P
Image ml164fu11.jpg
3124±59.835350±79943
Solubility: NDPurity: 87%
6956309499351068BRD-K56690068P
Image ml164fu12.jpg
3130±31.3323100±7230178
Solubility: NDPurity: 79%

ND = Not determined

*

P = Purchased

Table 3SAR Showing the Influence of Other Substituents on T.cruzi Inhibition

SAR Analysis for T. cruzi
Image ml164fu13.jpg
Potency (nM) mean ± S.E.M. (n=replicates)Target to Antitarget Fold Selectivity
EntryCIDSIDBroad No.*RnT. cruzi inhibitionnToxicity
1534425087556789BRD-K67470788P
Image ml164fu14.jpg
3145±263inactive>500
Solubility: >500 μMPurity: >95%
2687561787219027BRD-K02558072P
Image ml164fu15.jpg
3193±63319500101
Solubility: >500 μMPurity: >95%
3632295599351074BRD-K32049792P
Image ml164fu16.jpg
3317±103321200±61167
Solubility: NDPurity: 96%
49557463104170274BRD-K38455523P
Image ml164fu17.jpg
3335±121323600±570070
Solubility: 12.4 μMPurity: >95%
5687563099351076BRD-K52487866P
Image ml164fu18.jpg
3501±234321500±118043
Solubility: 37.9 μMPurity: 87%
6533210999351064BRD-K75855222P
Image ml164fu19.jpg
3680±491326700±927039
Solubility: NDPurity: 92%
721210609104170282BRD-K79949483P
Image ml164fu20.jpg
3752±1113inactive>500
Solubility: NDPurity: >95%
89558304104170276BRD-K41404496P
Image ml164fu21.jpg
3838±513322300±511027
Solubility: NDPurity: 81%
95334382104170280BRD-K81091498P
Image ml164fu22.jpg
31470±800328000±408019
Solubility: NDPurity: 94%

ND = Not determined

*

P = Purchased

It is interesting that switching the xylene in (entry 1, Table 2) to naphthalene in (entry 3, Table 2) or 2,4-dichlorophenyl in (entry 2, Table 2) results in an increase in potency, but increases toxicity. Replacement of the aromatic amine with morpholine (entry 1, Table 3) results in a 4-fold decrease in potency; however, it increases solubility to >500 μM.

We next investigated other types of substituents on the eastern side of the molecule. Replacement of the hydrazone amide with a carbamate (entry 2, Table 3) also resulted in an increase of solubility to >500 μM; however, it increases toxicity. Alkyl substituents result in a slight increase in solubility, but a greater than 10-fold decrease in potency (Table 3). Compound CID 6875690 should serve as a useful probe for cell biology research because of its low molecular weight and good physical properties. Furthermore, the aromatic ring could be further substituted if an analog were sought with optimized drug metabolism pharmacokinetic (DMPK) properties.

The activity of the piperidine series of analogs in the T. cruzi inhibition assays are shown in Figure 7.

Figure 7. Activity of Piperidine Series Analogs in T cruzi Inhibition Assays (AID 2630).

Figure 7

Activity of Piperidine Series Analogs in T cruzi Inhibition Assays (AID 2630). Representative curves of sixteen analogs tested in the primary screen at dose. CID 6875684 (A), CID 6884367 (B), CID 6872741 (C), CID 9689369 (D), CID 9563094 (E), CID 5344250 (more...)

3.5 Cellular Activity

As the primary assay is a phenotypic, cell-based assay, the low nanomolar (nM) activity of the probe (CID 6875690/ML164) as described above by definition indicates excellent permeability and good physical properties.

3.6 Profiling Assays

Not applicable.

4 Discussion

4.1 Comparison to Existing Art and Feature/Benefits of the New Probe

The current state of the art for probe molecules in cell culture assays against T. cruzi is a potency of about 1 μM (6), whereas our new probe (CID 6875690/ML164) is much more potent at 34.3 nM, a greater than 30-fold improvement. Also, the current treatments for Chagas disease are toxic to host cells. Probe (CID 6875690/ML164) is inactive against host cells at the highest tested dose (19.5 μM), a difference of greater than 500-fold compared with its’ activity against T. cruzi. These features should make this probe (CID 6875690/ML164) very useful as a probe molecule in cell assays and potentially facilitate future in vivo studies.

Investigation into relevant prior art entailed searching the following databases: SciFinder, Patent Lens, PubChem, and PubMed. The search terms applied and hit statistics are provided in Table 4. Abstracts were obtained for all references returned and were analyzed for relevance to the current project. The searches were performed on and are current as of February 16, 2011.

Table 4. Search Strings and Databases Employed in the Prior Art Search.

Table 4

Search Strings and Databases Employed in the Prior Art Search.

4.2 Mechanism of Action Studies

In order to elucidate how this probe (CID 6875690/ML164) works, an immunofluorescent assay was employed to determine its mode of action and the extent of inhibition of T. cruzi replication in the host cell. To quantitate relative inhibition of parasite replication, 50 infected cells were selected for analysis for the probe (CID 6875690/ML164) and each analog. The number of infected cells with more than four amastigotes (where the parasite has invaded and is actively replicating) was divided by the total number of infected cells. As observed by immunofluorescence, all compounds in this series inhibited T. cruzi replication compared with control, but the parasite maintained defined amastigote bodies. Therefore, the mode of action was determined as static. No diffuse staining in the cytosol of host NIH/3T3 cells was observed in any case, suggesting no lysis of the amastigotes as described in Bettiol et al. (7). Therefore, none of the compounds were categorized as lytic.

Additional experiments could be developed to identify the actual molecular target of this probe (CID 6875690/ML164). A phenotypic cell-based screening approach is extremely powerful for identifying novel compounds of interest when the exact mechanisms of parasite growth are not fully understood. At the Broad Institute, we have developed a robust, scalable method for confident identification of the protein targets of small molecules in their cellular context called stable isotope labeling by amino acids in cell culture (SILAC). SILAC-based target identification technology overcomes prior difficulties with affinity-based target identification methods. This technology is routinely applied at the Broad Institute to identify targets of a variety of small molecules with drug-like properties, including kinase inhibitors, immunophilin modulators, and others.

Another method for target identification would be to develop a T. cruzi strain resistant to the probe (CID 6875690/ML164). In brief, T. cruzi could be co-cultured with the probe and compound-resistant parasites would be identified after culturing through many passages. A genomic approach would then be undertaken to identify the gene(s) that differed in the resistant strain versus the wild type. The identification of common mutated genes across multiple experiments could narrow the list of possible targets. Biochemical and biophysical approaches could then be applied for further characterize and optimize the probe (CID 6875690/ML164) for in vivo testing.

4.3 Planned Future Studies

The probe (CID 6875690/ML164) will be tested for its inhibition of related parasites, Leishmani major and Leishmania amazonensis, with the aim of developing a treatment for Leishmania infections.

The probe (CID 6875690/ML164) and certain analogs are also being tested in a mouse model of Chagas disease using fluorescently tagged T. cruzi. Compounds are being dosed intravenously and the extent of T. cruzi replication is assessed by relative fluorescence in the infected area. These studies are ongoing at the University of Georgia by Drs. A. Rodriguez and R. Tarleton.

5 References

1.
De Souza W. Basic cell biology of Trypanosoma cruzi. Curr Pharm Des. 2002;8:269–285. PubMed. [PubMed: 11860366]
2.
Tarleton RL, Reithinger R, Urbina JA, Kitron U, Gürtler RE. The challenges of Chagas Disease: grim outlook or glimmer of hope. PLoS Med. 2007 Dec 27;4(12):e332. [PMC free article: PMC2222930] [PubMed: 18162039] [Cross Ref]
3.
WHO Special Program for Research and Training in Tropical Diseases. Report of the Scientific Working Group on Chagas Disease. World Health Organization; 2005.
4.
Konkle ME, Hargrove TY, Kleshchenko YY, von Kries JP, Ridenour W, Uddin MJ, Caprioli RM, Marnett LJ, Nes WD, Villalta F, et al. Indomethacin amides as a novel molecular scaffold for targeting Trypanosoma cruzi sterol 14α-demethylase. J Med Chem. 2009;52(9):2846–2853. PubMed. [PMC free article: PMC2744100] [PubMed: 19354253]
5.
Mott BT, Ferreira RS, Simeonov A, Jadhav A, Ang KK, Leister W, Shen M, Silveira JT, Doyle PS, Arkin MR, et al. Identification and optimization of inhibitors of trypanosomal cysteine proteases: cruzain, rhodesain, and TbCatB. J Med Chem. 2010;53:52–60. PubMed. [PMC free article: PMC2804034] [PubMed: 19908842]
6.
Brak K, Kerr ID, Barrett KT, Fuchi N, Debrath M, Ang K, et al. Nonpeptidic tetrafluorophenoxymehtyl ketone cruzain inhibitors as promising new leads for Chagas disease chemotherapy. J Med Chem. 2010;53:1763–1773. PubMed. [PMC free article: PMC2838180] [PubMed: 20088534]
7.
Bettiol E, Samanovic M, Murkin AS, Raper J, Buckner F, Rodriguez A. Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening. PLoS Negl Trop Dis. 2009;3(2):e384. Epub 2009 Feb 24. PubMed. [PMC free article: PMC2639639] [PubMed: 19238193] [Cross Ref]
8.
Buckner FS, Verlinde CL, La Flamme AC, Van Voorhis WC. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrob Agents Chemother. 1996;40(11):2592–2597. PubMed. [PMC free article: PMC163582] [PubMed: 8913471]
9.
Aguirre G, Cabrera E, Cerecetto H, Di Maio R, González M, Seoane G, Duffaut A, Denicola A, Gil MJ, Martínez-Merino V. Design, synthesis and biological evaluation of new potent 5-nitrofuryl derivatives as anti-Trypanosoma cruzi agents. Studies of trypanothione binding site of trypanothione reductase as target for rational design. Eur J Med Chem. 2004 May;39(5):421–431. PubMed. [PubMed: 15110968]

Appendix A Assay Summary Table

Table A1Summary of Completed Assays and AIDs

PubChem AIDTypeTargetConcentration Range (μM)Samples Tested
1885PrimaryT. cruzi co-cultureAverage 3.75 μM303,224
2044PrimaryT. cruzi co-culture100 nM-6.0 μM4063
2294PrimaryT. cruzi co-culture4.0 nM-1.0 μM319
2630PrimaryT. cruzi co-culture0.11 nM-19.5 μM42
2396PrimaryT. cruzi co-culture0.11 nM-6.8 μM27
493197PrimaryT. cruzi co-culture3 nM-19.5 μM46
2010SecondaryNIH3T3 Toxicity100 nM-6.0 μM4063
2586SecondaryNIH3T3 Toxicity0.11 nM-19.5 μM27
493247SecondaryNIH3T3 Toxicity3 nM-19.5 μM46
2632OrthogonalInhibition of T. cruzi replicationSingle dose; 10x IC50 as defined in AID 2204 and 229427
1968SummaryNANANA

NA= not applicable

Appendix B Detailed Assay Protocols

T cruzi Inhibition Assay Protocol (AID Nos.1885, 2044, 2294, 2630, 2396, 493197)

Cell Propagation

  1. 90% DMEM+Phenol red, 10% FBS, 1% PSG.
  2. Mix and filter through 0.2 microns membrane.
  3. Keep at 4°C. Warm up to 37°C in water bath before use.

T. cruzi Culture and Assays

  1. 98% DMEM+Phenol red, 2% FBS, 1% PSG.
  2. Mix and filter through 0.2 microns membrane.
  3. Keep at 4°C. Warm up to 37°C in water bath before use.

Cell Culture Protocols

NIH/3T3 Cell Culture

  1. Aspirate medium.
  2. Rinse cells with 10 ml PBS/plate.
  3. Aspirate PBS.
  4. Add 5 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells.
  5. Place the dishes back in the incubator for 5 minutes.
  6. Check that the cells are detaching.
  7. Add 20 ml of medium, pipette up and down to detach all the cells.
  8. Dilute into 2% FBS/DMEM for assay or 10% FBS/DMEM for propagation.

LLC-MK2 Cell Culture

  1. Aspirate medium.
  2. Rinse cells with 10 ml PBS/plate.
  3. Aspirate PBS.
  4. Add 5 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells.
  5. Place the dishes back in the incubator for 5 minutes.
  6. Check that the cells are detaching.
  7. Add 20 ml of medium, pipette up and down to detach all the cells.
  8. Dilute into 2% FBS/DMEM for assay or 10% FBS/DMEM for propagation.

T. cruzi β-gal (Tc) Parasite Culture

  1. The day before infection (or at least 2–3 hours before), plate 3 million LLC-MK2 cells/T175 in DMEM+10% FBS.
  2. Either thaw T. cruzi in from liquid nitrogen stock into 50 ml 2% FBS/DMEM or remove the media from a propagating LLC-MK2 co-culture; in other words, harvesting the medium containing the free T. cruzi in 50-ml Falcon tubes.
  3. Spin 10 minutes at 2200 rpm.
  4. Aspirate the supernatant until 15 ml is left.
  5. Place back the tubes in the incubator delicately (so as not to disturb the pellet).
  6. Incubate minimum 3 hours at 37°C.
  7. Take supernatant to fresh tube.
  8. To count: Mix 75 ml T. cruzi with 25 ml 16% PFA, mix, put 75 ml on Nexcelom cellometer cassette.
  9. Wait for 2–5 minutes to let the T. cruzi settle; use the T. cruzi method saved on the M10 machine, press display image, focus, and count.
  10. Aspirate LLC-MK2 media (used 10% for plating) and replace with 2% FBS/DMEM.
  11. Plate 17–35 million on T. cruzi on fresh LLC-MK2.
  12. Change medium after 2 days (Use 2% FBS/DMEM).
  13. Harvest T. cruzi trypomastigotes on Day 6 and Day 7.

Growth Inhibition Assay Protocol for HTS (384-well plates)

  1. Warm up medium 2% FBS/DMEM.
  2. Harvest parasites in 50-ml tubes (1 flask per tube).
  3. Spin 10 minutes at 2200 rpm.
  4. Aspirate media approximately 15 ml and place them on a rack in the incubator to let trypomastigotes swim out of the pellet for 3–5 hours.
  5. In the meantime, trypsinize NIH/3T3 cells as described in cell culture protocol.
  6. When NIH/3T3 are detached, harvest them in DMEM- 2% FBS and 1% PSG and count using the NIH3T3 program using the Nexcelom Cellometer.
  7. Dilute cells to 166,667 cells /ml and add to flask.
  8. Plate 5,000 cells/30 μL per well using standard cassette multiwell drop Combi, adding at a fast speed.
  9. Put back in incubator for 3 hours to allow cells to attach.
  10. Count T. cruzi as described in the parasite culture protocol.
  11. Dilute to 0.250 million /ml and transfer to a 2-liter flask.
  12. Add to a 2- liter flask, stirring.
  13. Pin 50 nL compounds/DMSO to each well with NIH/3T3s.
  14. Add shortly after 20 μL per well of parasites (5000 T. cruzi) with standard cassette multiwell drop Combi on slow speed.
  15. Incubate for 4 days (or minimum 90 hours).
  16. Prepare Gal-Screen with 0.05% NP40.
  17. Add 30 μL per well of 384-well plate.
  18. Incubate for 60 minutes.
  19. Read using the ultrasensitive luminescence program on the Envision (Perkin-Elmer, 0.1 sec/well).

Cell Toxicity Assay Protocol: NIH/3T3 Cells (AID Nos. 2010, 2586, 493247)

  1. Warm up medium 2% FBS/DMEM.
  2. Trypsinize NIH/3T3 cells as described in cell culture protocol.
  3. When NIH3T3s are detached, harvest them in DMEM- 2% FBS and 1% PSG and count using the NIH3T3 program using Cellometer Auto T4 (Nexcelom Biosciences).
  4. Dilute cells to 166,667 cells /ml and add to flask.
  5. Plate 5,000 cells/50 μL per well using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed in a tissue culture hood.
  6. Put back in incubator for 3 hours to allow cells to attach.
  7. Pin 50 nL compounds/DMSO to each well.
  8. Incubate for 4 days (or minimum 90 hours).
  9. On day of substrate addition, prepare CellTiter-Glo.

CellTiter-Glo® Protocol

  1. Thaw the CellTiter-Glo Buffer, and equilibrate to room temperature prior to use. For convenience the CellTiter-Glo Buffer may be thawed and stored at room temperature for up to 48 hours prior to use.
  2. Transfer the appropriate volume of CellTiter-Glo Buffer into the amber bottle containing CellTiter-Glo Substrate to reconstitute the lyophilized enzyme/substrate mixture.
  3. Mix by gently vortexing, swirling, or by inverting the contents to obtain a homogeneous solution.
  4. Dilute solution 1:3 with PBS.
  5. Take out plates from incubator and incubate at room temperature for 30 minutes.
  6. Add 30 μL/well using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed.
  7. Allow the plate to incubate at room temperature for 10 minutes.
  8. Read using the ultrasensitive luminescence program on the Envision (Perkin Elmer, 0.1 sec/well).

Intracellular T cruzi Immunofluorescence Assay Protocol (AID No. 2632)

  1. Seed NIH/3T3 cells on sterile glass coverslips in 12-well plates and allow cells to adhere overnight.
  2. Add T. cruzi parasites (MOI 100:1) and allow to infect for 2 hours in DMEM+2% FBS and PSG.
  3. Rinse parasites out 3 times with PBS, and add compounds at 10X their IC50 (as determined in AID 2044 and AID 2294).
  4. Incubate infected cells for 4 days and fix for 15 minutes with 4% paraformaldehyde.
  5. Rinse fixed cells on coverslips with PBS and permeabilize for 15 minutes in PBS with 0.1% Triton X-100.
  6. Block for 20 minutes in PBS with 10% goat serum, 1% bovine serum albumin (BSA), 100 mM glycine, and 0.05% sodium azide.
  7. Incubate cells for 1 hour at room temperature with a polyclonal rabbit anti-T cruzi at 1:2000 dilution.
  8. Rinse, then add an Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (Molecular Probes, Invitrogen) for 1 hour at a 1:800 dilution.
  9. Stain DNA with DAPI, and mount coverslips with anti-fade mounting media.
  10. Take images using an inverted Olympus IX70 microscope with a 60X oil objective.

Appendix C NMR and LC Data of Probe and Analogs

1H NMR (300 MHz, DMSO-d6) Spectra of Probe (CID 6875690/ML164).

1H NMR (300 MHz, DMSO-d6) Spectra of Probe (CID 6875690/ML164)

LC-MS Chromatogram of Probe (CID 6875690/ML164).

LC-MS Chromatogram of Probe (CID 6875690/ML164)

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875684.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875684

LC-MS Chromatogram of Analog CID 6875684.

LC-MS Chromatogram of Analog CID 6875684

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6884367.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6884367

LC-MS Chromatogram of Analog CID 6884367.

LC-MS Chromatogram of Analog CID 6884367

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6872741.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6872741

LC-MS Chromatogram of Analog CID 6872741.

LC-MS Chromatogram of Analog CID 6872741

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9689369.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9689369

LC-MS Chromatogram of Analog CID 9689369.

LC-MS Chromatogram of Analog CID 9689369

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9563094.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9563094

LC-MS Chromatogram of Analog CID 9563094.

LC-MS Chromatogram of Analog CID 9563094

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5344250.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5344250

LC-MS Chromatogram of Analog CID 5344250.

LC-MS Chromatogram of Analog CID 5344250

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875617.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875617

LC-MS Chromatogram of Analog CID 6875617.

LC-MS Chromatogram of Analog CID 6875617

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6322955.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6322955

LC-MS Chromatogram of Analog CID 6322955.

LC-MS Chromatogram of Analog CID 6322955

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9557463.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9557463

LC-MS Chromatogram of Analog CID 9557463.

LC-MS Chromatogram of Analog CID 9557463

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875630.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875630

LC-MS Chromatogram of Analog CID 6875630.

LC-MS Chromatogram of Analog CID 6875630

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5332109.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5332109

LC-MS Chromatogram of Analog CID 5332109.

LC-MS Chromatogram of Analog CID 5332109

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 21210609.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 21210609

LC-MS Chromatogram of Analog CID 21210609.

LC-MS Chromatogram of Analog CID 21210609

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9558304.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9558304

LC-MS Chromatogram of Analog CID 9558304.

LC-MS Chromatogram of Analog CID 9558304

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5334382.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5334382

LC-MS Chromatogram of Analog CID 5334382.

LC-MS Chromatogram of Analog CID 5334382

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5349954.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5349954

LC-MS Chromatogram of Analog CID 5349954.

LC-MS Chromatogram of Analog CID 5349954

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9640748.

1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9640748

LC-MS Chromatogram of Analog CID 9640748.

LC-MS Chromatogram of Analog CID 9640748

Appendix D Compounds Submitted to BioFocus

Table A2Probe and Analog Information

BRDSIDCIDP/AMLSIDML
BRD-K64970062-001-01-5872190526875690PMLS002725687ML164
BRD-K85660637-001-01-387219044884367AMLS002725693NA
BRD-K67470788-001-01-6875567895344250AMLS002725680NA
BRD-K02558072-001-01-172190276875617AMLS002725690NA
BRD-K91047982-001-01-675567905349954AMLS002725681NA
BRD-K17628612-001-01-2875567919640748AMLS002729069NA

NA= Not applicable; A = analog; P = probe

Selectivity = anti-target IC50/target IC50.

P = Purchased

P = Purchased

P = Purchased

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