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A small molecule pan-inhibitor of Ras-superfamily GTPases with high efficacy towards Rab7

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Received: ; Last Update: March 7, 2013.

Low molecular weight guanine triphosphate hydrolases (GTPases) are GTP-binding enzymes that play pivotal roles in cell biology. Grouped into three subfamilies which are designated by function, Ras, Rho and Rab GTPases are involved in signal transduction, cytoskeleton modulation, and macromolecule cargo transport and degradation, respectively. Mutation and aberrant gene expression levels have been linked to human diseases including cancer, immunodeficiencies, and neurological disorders. A high through-put screen of the Molecular Libraries Small Molecule Repository (MLSMR) identified a compound, ML282, which potently inhibits GTPases from all three subfamilies. Importantly, this represents the discovery of the first reported compound to inhibit Rab GTPases, especially Rab7, the mutation of which is a causal factor in a heritable neuropathy called Charcot-Marie-Tooth Type 2B disease. ML282 inhibits Rab7 with an average EC50 = 53.2 ± 0.35 nM and with high response, as compared to other GTPases in the panel. The subsequent structure activity relationships (SAR) and secondary assays demonstrate its use as a molecular probe for both biochemical and cellular studies.

Assigned Assay Grant #: MH081231-01

Screening Center Name & PI: UNM Center for Molecular Discovery, Larry Sklar

Chemistry Center Name & PI: University of Kansas Specialized Chemistry Center, Jeffrey Aubé

Assay Submitter & Institution: Angela Wandinger-Ness, UNM

PubChem Summary Bioassay Identifier (AID): 1772

Probe Structure & Characteristics

Image ml282fu1

Table 1Probe ML282 Assay Data Summary

CID/ML#Target NameEC50 (nM) [SID]aAnti-target Name(s)EC50 (μM) [SID, AID]aFold SelectiveSecondary Assay(s) Name: EC50 (nM) [SID, AID]a,fSecondary Assay(s) Name: EC50 (nM) [SID, AID]a,gSecondary Assay(s) Name: EC50 (nM) [SID, AID]a
CID 1067700/ML282Rab753.2 ± 0.35 bGlutathione S-transferase>100

AID 602137, AID 602145
>100Cellular viability of U937 cell line: not toxic

EC50 >20000
AID 588369
VLA4 binding in U937 ΔST cell: active

EC50 ~50
AID 602148
EGFR degradation in SCC-12F cell: active
EC50 < 10

AID 602146
AIDs 588394, 588631
CID 1067700/ML282Ras wild Type49.1 ± 1.41b
AID 588388, 588630
CID 1067700/ML282Ras activated51.5 ± 2.83 b
AID 588387, AID 588628
CID 1067700/ML282Cdc42 wild type130.0 ± 23.0 b
AID 588385, AID 588626
CID 1067700/ML282Cdc42 activated91.0 ± 4.0 b
AID 588384, AID 588624
CID 1067700/ML282Rho wild type79.5 ± 3.54 c
AIDs 588479, AID 588622
CID 1067700/ML282Rac1 wild type62.1 ± 32.5 c
AIDs 588479, AID 588622
CID 1067700/ML282Rac1 activated76.5 ± 24.7 c
AIDs 588479, AID 588622
CID 1067700/ML282Rab737.9 ± 10.0 d
AIDs 588479, AID 588622
CID 1067700/ML282Cdc42 wild type64.5 ± 28.3 c
AIDs 588479, AID 588622
CID 1067700/ML282Cdc42 activated129.0 ± 30.0 e
AIDs 588479, AID 588622
a

Reported data is an average of the EC50 values from two separate lots of ML282 (SIDs 57578339 and 85747738) unless otherwise noted

b

Reported dose response data was obtained with 100nM GTP, 1mM EDTA and is an average of the EC50 values from two separate lots of ML282 (SID 57578339 and SID 85747738 reported.

c

Reported dose response data was obtained with 1nM GTP, 1mM Mg2+ and is an average of the EC50 values from two separate lots of ML282 (SID 57578339 and SID 85747738 reported.

d

Reported dose response data was obtained with 1nM GTP, 1mM Mg2+ and represents only SID 85747738

e

Reported dose response data was obtained with 1nM GTP, 1mM Mg2+ and represents only SID 57578339

f

Reported in AID 588369 and AID 588410 for respective SID 57578339 and SID 85747738.

1. Recommendations for Scientific Use of the Probe

Limitations in the current state of the art being addressed by the probe: This is a first-in-class probe that inhibits guanine nucleotide binding. Currently, there no reported compounds that are active against Rab family GTPases, and selective inhibitors of other GTPases are rare. The first reported GTPase inhibitors were discovered using cellular assays in which the anchoring of GTPase to membranes was blocked. Since the localization of GTPase to membranes is mediated through farnesyltransferase and prenyltransferase, the lipid transferase turned out to be the true target. However, since lipid transferases have a broad range of substrates, these inhibitors lack specificity and have been disappointing in clinical trials [1]. Selective and direct GTPase inhibitors, though rare, are possible. Previously, our team identified and reported ML141, a probe that specifically inhibits Cdc42 and its active mutant by blocking nucleotide binding [2]. Brefeldin A, a fungal metabolite, has been determined to promote the formation of an inactive complex of Arf and Arf GEF, thus blocking Arf activation [34]. These compounds have activities in the micromolar range and are specific towards a single GTPase. Through virtual screening, a compound has been discovered to specifically inhibit Rac1 [5]. Compared to the compounds mentioned above, probe ML282 has an improved activity of about 100-fold and shows inhibition towards all of the GTPases tested to date.

Use of the probe and relevant biology to which the probe can be applied: As a non-selective, Ras-family GTPase inhibitor, ML282 will serve several purposes. It will be a useful control compound for identifying specific GTPase inhibitors, analogous to the pan-kinase inhibitor, staurosporine, used in kinase inhibitor screening campaigns being conducted across academia and industry. Secondly, since the functions of subfamily GTPases often overlap, ML282 could block these collective functions, thereby facilitating the discovery of new biological targets other than a GTPase that play a role in a particular biological process. Thirdly, the combined use of a selective and pan-inhibitor will help to define the function of individual GTPases. For instance, if the phenotypes of filopodia formation are the same for cells treated with either a Cdc42-specific inhibitor or the pan-inhibitor, it can be inferred that Cdc42 is exclusively accountable for filopodia formation. Lastly, ML282 is the only reported small molecule compound that shows inhibitory activity for against Rab GTPases, including Rab7. For Rab7-related diseases, ML282 provides a starting point for medicinal chemistry to develop Rab7 specific inhibitors.

2. Materials and Methods

Materials and Instrumentation

GST-tagged GTPases were from Cytoskeleton (Denver, CO) and Rab7 was purified by the assay provider. BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or dipyrromethene boron difluoride) nucleotide analogues (BODIPY FL GTP 2′-(or 3′)-O-[N-(2-aminoethyl)urethane] G-35778 and BODIPY FL GDP 2′-(or-3′)-O-[N-(2-aminoethyl)urethane], G-22360) were from Invitrogen Molecular Probes (Carlsbad, CA). 2-(benzoylcarbamothioylamino)-5, 5-dimethyl-4, 7- dihydrothieno[2,3-c]pyran-3-carboxylic acid (CID 1067700) was from ChemDiv and from University of Kansas Specialized Chemistry Center, which also supplied analogs to CID 1067700. Detergents Nonidet P-40 and Tween 20 were from Sigma (St. Louis, MO) and Bio-Rad (Hercules, CA), respectively. Precision Plus Protein™ Standards were also purchased from Bio-Rad. For cell permeabilization, CelLytic™M Cell Lysis Reagent, Protein Inhibitor Cocktail for Mammalian Cells and phenylmethanesulfonyl fluoride were from Sigma. BCA protein assay kit was from Thermo Fisher Scientific (Waltham, MA). EGF Receptor Rabbit mAb was from Cell Signaling (Danvers, MA). Anti-actin antibody produced in rabbit was from Sigma. Stabilized goat anti-rabbit IgG with peroxidase conjugated and SuperSignal West Dura were from Thermo Fisher Scientific. Recombinant human epidermal growth factor, EGF, was purchased from Invitrogen. Cycloheximide was from Sigma. The sequences of peptide LDV, FITC-LDV, and N-formyl peptide have been described before [6] and are products of Biogen Idec (San Diego, CA).

The CyAnADP flow instrument and Biomek FX are products of Beckman Coulter (Indianapolis, IN). Low volume transfers (100 nL) were done via pintool (V&P Scientific; San Diego, CA). FACScan flow instrument is from Becton Dickinson (Franklin Lakes, NJ). Cyto-Plex™ Microspheres (4.0 μm) are from Thermo Fisher Scientific. Quantum™ FITC-5MESF is from Bangs Laboratories, Inc (Fishers, IN). Countess Cell Counter is from Life Technologies (Carlsbad, CA). The HyperCyt system is from Intellicyt (Albuquerque, NM). ChemiDoc™ XRS+ molecular imager is from Bio-Rad. HyperView software (version 2.5.1, IntelliCyt) was modified to enable specialized analysis functions used in these studies.

Cell culture

U937 ΔST cells (a gift from Dr. Eric Prossnitz, Dept. Cell Biology and Physiology, University of New Mexico, Albuquerque, NM) were grown at 37°C in a humidified incubator with 5% CO2 and 95% air in RPMI 1640 supplemented with 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum. SCC-12F cells (a gift from Dr. Laurie Hudson, Dept. Pharmaceutical Sciences, University of New Mexico, College of Pharmacy, Albuquerque, NM) were grown in a 1:1 mixture of DMEM and nutrient mixture F-12 Ham’s medium supplemented with 10% fetal bovine serum. HeLa cells stably overexpressing Rab7 wild-type (a gift from Dr. Matthew Seaman, Dept. Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge, UK) were grown in MEM with 10% serum or MEM without serum during starvation.

2.1. Assays

Dose-response inhibition

The compound was serially diluted 3–fold and the resulting concentrations in the reactions ranged from 100 μM to 15 nM. The eight GTPases were grouped into two sets: Rab7, H-Ras wild type and its V12 mutant, and Cdc42 and its L61 mutant were assayed in a buffer containing 1 mM EDTA, while Rho, Rac1 and its L61 mutant, and Cdc42 and its L61 mutant were assayed in the same buffer but instead containing 1 mM Mg2+ with Cdc42 and its mutant being tested in both buffers. Besides 1 mM EDTA or 1 mM Mg2+, the buffer contained 30 mM HEPES, pH 7.2, 40 mM KCl, 100 mM NaCl, 0.1% (v/v) NP-40 and 1% BSA. For each multiplex reaction, an extra set of beads was included to serve as scavengers to bind any proteins that dissociate from their assigned bead set and thereby reduce interference due to protein dissociation during the assay. As a control, GST-tagged GFP was bound to a set of glutathione beads in a separate reaction to make sure that the fluorescence drop observed in the assay reaction in the presence of a compound was not due to fluorescence quenching of GFP nor interference of GST-tagged protein binding to beads, but due to the bona fide inhibition of fluorescent nucleotide binding.

The assay protocol has been described previously [2, 7]. Briefly, each individual GST-tagged enzyme was incubated with 4 μm glutathione-coated microsphere beads overnight with gentle rotation. The microsphere beads have different red fluorescence dyes incorporated and therefore could be separated by a flow cytometer. The unbound enzyme was removed by sufficient washing with the buffer. The bead sets were then combined and dispensed to 384-well plates at 5 μL/well. For one plate, 0.5 μg each of the enzymes and 4.0 ×104 of each bead were used. Then 100 nL compound was added using Biomek FX followed by the addition of 5 μL/well of BODIPY-FL-GTP (ribose-linked BODIPY). For assays carried out in EDTA-containing buffer, the final concentration of BODIPY-FL-GTP was 100 nM while in Mg buffer, the concentration of BODIPY-FL-GTP was 1 nM. The plate was incubated at 4 °C for 2 h with gentle rotation. The beads were aspirated and delivered with the HyperCyt® instrument and read on CyAnADP flow cytometer. Forward scatter and side scatter was used to define the whole bead population. FL9 channel (ext/em: 635/750LP nm) was used to separate different bead sets each of which was bound with an individual enzyme. FL1 channel (ext/em: 488/530 nm) measures the FITC fluorescence associated with the beads. The data was analyzed by the HyperView® software developed by Dr. Bruce Edward at the University of New Mexico.

Secondary Assays

Cytotoxicity of compounds

U937 cells were grown to a density of 0.4 × 106 cells/mL. Compounds were added to a final concentration of 20 μM or otherwise indicated. As a control, cells were treated with DMSO of the same volume. After 24h incubation, the density and viability of the cells were measured with Countess Cell Counter according to the protocols from the manufacturer.

LDV binding assay

The procedure followed the established protocols described previously [6,89]. U937 ΔST cells (0.4~0.8 × 106 cells/mL) were constantly stirred with a magnetic stir bar at 500 rpm in a test tube. The fluorescence was recorded on a FACScan flow cytometer. The base line fluorescence of the cells was established for 30s. FITC-LDV at a concentration of 4nM was then added. After the fluorescence stabilized at around 120s, the chemotactic ligand N-formyl peptide was added for stimulation resulting activation of VLA-4 integrin and additional FITC-LDV binding. When a plateau was reached at around 280s, compounds at different concentrations were added to the cell suspension. Fluorescence was continually recorded until no further change was observed. The inhibition percentage was calculated according to Equation 1.

Inhibitionpercentage=(1-remainingfluorescenceaftercompoundtreatmentfluorescenceafterDMSOtreatment)*100%
Equation 1
FITC-LDV dissociation assay

The dissociation kinetics of FITC-LDV was measured by the addition of 100-fold of non-fluorescent LDV (The kinetic constants obtained are the same within experimental errors whether adding 100-fold or 200-fold of non-fluorescent LDV) [6, 9]). The fluorescence decrease was recorded. For the resting state, when a fluorescence plateau was reached after the addition of FITC-LDV, LDV was added to the cell suspension. For the high affinity state, when the fluorescence reached equilibrium after the sequential addition of FITC-LDV and N-formyl peptide, LDV was added. To study the compounds effect, LDV was added at the time point when the fluorescence dropped to the minimum. The fluorescence decrease curve was fitted to either Equation 2 to obtain the dissociation rate constant koff, or Equation 3 to calculate the active receptor percentage according to Equation 4. In Equation 3, kh is the dissociation constant when the integrin is at the high affinity state while kl is the dissociation constant at the resting state. The values are 0.014 s−1 and 0.036 s−1, respectively, as determined in separate experiments using Equation 2.

y = A * exp(−koff * x) + C
Equation 2
y = Ah * exp(−kh* x) + Al * exp (−kl * x) + C
Equation 3
Activereceptorpercentage=/(Ah+Al)Ah*100%
Equation 4
EGFR degradation in SCC-12F cells

SCC-12F cells were seeded in 12-well plates at 0.12 × 106 cells/well and allowed to grow overnight. On the day of the experiment, the cells were starved in serum free medium containing 25 μg/mL cycloheximide for 2 h before compounds at different concentrations were added. The cells were treated for 30 min. Then, ligand EGF was added to 20 nM. At time 0, 15, 30, 60, and 120 min after the EGF addition, the suspension medium was removed and the cell was quickly washed with cold PBS and frozen at −80 °C. For electrophoresis and blotting, CelLytic™M Cell Lysis buffer containing protease inhibitor cocktails was added to the frozen cells to obtain the cell lysates according to the protocols from the manufacture. Protein concentration was quantified using BCA assay kit. For SDS-PAGE, 10 μg protein was loaded to each lane and proteins transferred to nitrocellulose. The nitrocellulose membranes were probed with antibodies directed against EGFR or actin and detected using peroxidase conjugated secondary antibodies and SuperSignal West Dura. Signal intensities were measured and quantified using a ChemiDoc™ XRS+ molecular imager coupled with Image Lab software.

EGFR degradation in Rab7 overexpressing HeLa cells

EGFR degradation was also monitored in HeLa cells with some minor modifications. Briefly, HeLa cells overexpressing GFP-Rab7 wild-type protein were serum starved overnight, incubated with 100 μM ML282 for 3 h, and then treated with cycloheximide and stimulated with 100 ng/ml EGF in serum free medium for 0–180 min. Cell lysates were prepared at various time points and immunoblotted for total EGFR while actin served as a loading control.

2.2. Probe Chemical Characterization

A. Probe Chemical Structure, Physical Parameters and Probe Properties

Figure 1. Probe characteristics for ML282.

Figure 1Probe characteristics for ML282

B. Structure Verification and Purity: 1H NMR, 13C NMR, LCMS, and HRMS Data

Proton and carbon NMR data for ML282 (CID 1067700): Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental proton and carbon data are represented below.

Proton NMR Data for ML282 (CID 1067700):1H NMR (400 MHz, DMSO-d6) δ 14.84 (s, 1H), 13.39 (s, 1H), 11.81 (s, 1H), 7.98 (apparent dd, J = 1.2, 8.4, 2H), 7.74 – 7.62 (m, 1H), 7.55 (apparent t, J = 7.7, 2H), 4.67 (s, 2H), 2.75 (s, 2H), 1.24 (s, 6H).

Carbon NMR Data for ML282 (CID 1067700):13C NMR (101 MHz, DMSO-d6) δ 174.43, 166.86, 165.35, 146.72, 133.08, 132.06, 129.10, 128.75, 128.41, 124.09, 116.50, 70.14, 58.82, 37.27, 26.19.

LCMS and HRMS Data for ML282 (CID 1067700): Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental LCMS and HRMS data are represented below. LCMS retention time: 1.871min. LCMS purity at 214 nm: 92.8%. HRMS: m/z calculated for C18H19N2O4S2 [M++1]: 391.0781, found 391.0777.

C. Solubility

Solubility was measured in phosphate buffered saline (PBS) at room temperature (23 °C). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4. Detection was based on UV absorbance [10]. Probe ML282 (SID 85747738) was found to have an excellent PBS solubility measurement of > 116 μg/mL, or > 297 μM, under these conditions. Solubility was also assessed in the each of the four media used in the individual assays. Probe ML282 (SID 85747738) was determined to have excellent assay media solubility, as depicted in Table 2.

Table 2. Solubility for ML282 in Assay Media.

Table 2

Solubility for ML282 in Assay Media.

D. Stability

Stability was measured under two distinct conditions with ML282 (SID 85747738, Fig. 2). Stability, depicted as closed circles in the graph, was assessed at room temperature (23 °C) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%). Stability data is depicted as a graph showing the loss of compound with time over a 48 hr period with a minimum of 6 time points and providing the percent remaining compound at end of the 48 hr period [10]. Under these conditions, 77% of ML282 remains after 48 hours. It is unknown at this time if or what degradation might be occurring or what this result truly represents.

Figure 2. Graph depicting stability of ML282 after 48 h in PBS (no additives).

Figure 2

Graph depicting stability of ML282 after 48 h in PBS (no additives).

To assess the chemical stability of ML282 and its propensity towards nucleophilic attack, the compound was treated with a range of equivalents of L-glutathione in DMSO for 72 h at 37 °C (11). The three experiments were monitored by LCMS at each of the following time points: 1 h, 2 h, 4 h, 24 h, 48 h, and 72 h. Procedure: To a solution of ML282 (2-(3-benzoylthioureido)-5,5-dimethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxylic acid, SID 85747738), 1.0 mg, 2.56 μmol, 1 eq) in DMSO (1.0 mL) was added:

  1. L-glutathione (1.0 mg, 3.25 μmol, 1.2 eq) and the mixture stirred at 37 °C for 72 h
  2. L-glutathione (1.6 mg, 5.12 μmol, 2.0 eq) and the mixture stirred at 37 °C for 72 h
  3. L-glutathione (2.4 mg, 7.68 μmol, 3.0 eq) and the mixture stirred at 37 °C for 72 h

LCMS analysis of each reaction vial, taken after time (t) = 1 h, 2 h, 4 h, 24 h, 48 h, and 72 h, showed only the presence of 2-(3-benzoylthioureido)-5,5-dimethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxylic acid (SID 85747738). No glutathione conjugate or other peaks were observed. These results suggest that the compound is not generally electrophilic or susceptible to protein-derived nucleophiles

2.3. Probe Preparation

General experimental and analytical details:1H and 13C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500 and 126 MHz respectively) in CDCl3 with 0.03% ™S as an internal standard or DMSO-d6. The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet and m = multiplet. The LCMS analysis was performed on an Agilent 1200 RRL chromatograph with photodiode array UV detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the following parameters: a Waters Acquity BEH C-18 2.1 × 50mm, 1.7 um column; UV detection wavelength = 214 nm; flow rate = 0.4ml/min; gradient = 5 – 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the following parameters: an Agilent multimode source which simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist ionization. Melting points were determined on a Stanford Research Systems OptiMelt apparatus.

2-(3-Benzoylthioureido)-5,5-dimethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxylic acid (SID 85747738, CID 1067700) was purchased from ChemDiv, Inc. (CAS 314042-01-8) and purified by mass-directed reverse-phase chromatography to yield a white solid.

ML282, SID 85747738, CID 1067700: 2-(3-Benzoylthioureido)-5,5-dimethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxylic acid was purchased from ChemDiv, Inc. (CAS 314042-01-8) and purified by mass-directed reverse-phase chromatography to yield a white solid. However, the probe can theoretically be prepared by the route depicted in Figure 3 [12].

Figure 3. Synthetic route for probe and analog generation.

Figure 3

Synthetic route for probe and analog generation.

3. Results

3.1. Dose Response Curves for Probe

The effect of ML282 (CID 1067700) on the binding of BODIPY FL-GTP to eight different GTPases was measured in dose response assays. The GTPases were grouped into two sets and tested in buffers containing either 1 mM EDTA or 1 mM Mg2+. The concentration of the fluorescent guanine nucleotide was held constant near the Kd in each of the buffers. The inclusion of 1 mM EDTA was to chelate any residual Mg2+ which functions to stabilize the guanine nucleotide binding to the GTPases. The buffer containing EDTA could, therefore, facilitate the reversible exchange of the guanine nucleotide and aid in the identification of compounds that inhibit the fluorescent nucleotide binding. However, our previous results showed that Rac1 and Rho enzymes are not stable in the EDTA buffer. Therefore, Rac1 and Rho GTPases were tested in the buffer containing 1 mM Mg2+. For comparison, Cdc42 and its mutant were tested in both buffers and yielded similar EC50 values demonstrating that both buffers can be feasibly used. Based on the dose response curves, ML282 inhibited BODIPY FL-GTP binding by all GTPases tested in both sets of reactions whether the buffer contained EDTA or Mg2+, and the EC50 values were in the nanomolar range (Fig. 4).

Figure 4. Dose-response curves of ML282 (CID 1067700) with different GTPases.

Figure 4

Dose-response curves of ML282 (CID 1067700) with different GTPases. A. The assay buffer contained 1 mM EDTA. B. The assay buffer contained 1 mM Mg2+.

In a control experiment, we coated the beads with GST- tagged GFP. Incubation with compound ML282 did not cause any changes to the beads’ fluorescence. Therefore, the fluorescence decrease we observed in the assay was not due to fluorescence quenching of GFP or to interference of the GST-tagged protein binding to the beads, but due to the bona fide inhibition of the fluorescent nucleotide binding.

According to the Cheng-Prusoff equation [13], the compound IC50 obtained under our assay condition is a close estimate of its Kd. Probe ML282 is the first compound shown to have nanomolar inhibition potency towards multiple GTPases, including Rab7. On the other hand, careful examination of the dose response curves indicates that although the compound inhibited all GTPases tested with similar potency, it appears that the inhibition modalities may be different. The dose response curve for Rab7 fits well with a classical one-site binding model with a hill slope close to one and nearly total inhibition achieved at high compound concentrations (Fig. 4). However, the compound only resulted in partial inhibition of Cdc42 and Ras even at high concentrations. Partial inhibition may suggest the presence of multiple forms of the protein and hint at the involvement of a complex inhibition mechanism. The different behavior of the compound towards various GTPases warrants further investigation and may serve as the basis for identifying a specific inhibitor for Rab7.

3.2. Cellular Activity

ML282 (CID 106770) has no obvious cellular toxicity.

The most useful molecular probes have a generous “therapeutic” window in which they do not demonstrate cellular toxicity. As a model cell line used in biomedical research, U937 cells mature and differentiate in response to a number of soluble stimuli [14]. Also, a mutant form of U937 cells was used in the cellular VLA4 binding assay. Therefore, the cytotoxicity of ML282 and its analogues were evaluated in the U937 cells. The compound was included in the growth medium of U937 cells for 24h. The cell density and viability were subsequently measured. The cells had a growth rate comparable to that treated with the DMSO control and maintained their viability. Thus, ML282 and its analogues demonstrate no obvious cytotoxicity and supports their use in cellular assays.

3.3. Profiling Assays

Broad spectrum target profiling: ML282 was submitted for assessing off-target pharmacology using a Ricerca LeadProfiling® screen made up of 68 assays. The probe was assayed in duplicate at a concentration of 10 μM for all targets.

ML282 did not register any percent inhibition over 50% in this panel. The most significant findings were noted as 40% inhibition of the human serotonin receptor (5-HT2B) at 10 μM. All other findings registered below 22% inhibition at this concentration. Details of the panel results are provided in the Appendix.

4. Discussion

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

GTPases are important for cellular signal transduction, cytoskeleton reorganization, and membrane protein traffic and have been shown to be the causal agents for multiple human diseases by either mutation or expression level changes [15]. For example, the mutation of Ras has been found in one third of all cancers, and Rab and Rho GTPases are also increasingly implicated in cancer as well as other human diseases [16]. In many cases, GTPases are hyper- activated in the disease state. Therefore, the pursuit of GTPase inhibitors has been a universal subject that is carried on by both academic research laboratories and pharmaceutical companies parallel to the search for kinase inhibitors. The latter quest has successfully generated several commercialized drugs. Among others, erlotinib has been used as an EGFR inhibitor to treat non-small cell lung cancer [1718] and pancreas cancer [1920], while imatinib has been used as an ABL inhibitor to treat chronic myelogenous leukemia [21] and gastrointestinal stromal tumors [22]. Compared with the success achieved with kinase inhibitors, the identification of a GTPase inhibitor that can be a useful drug has remained enigmatic. One reason is that GTPases have versatile and overlapping functions so that toxicity has been a challenging issue. In addition, the guanine nucleotide binding affinity is quite high [2324]. There have also been practical hurdles. One of them is the lack of a positive control. The pan-kinase inhibitor staurosporine has been the control compound for both biochemical inhibitor screening and cellular assay assessment [2527]. However, there has not been a versatile GTPase inhibitor that could play an analogous role. Besides the limitation in drug discovery, the lack of a versatile GTPase inhibitor also discourages the function assignment for unknown factors where the intervening GTPase activity needs to be blocked. The current GTPase inhibitors include lipid transferase inhibitors, regulatory protein inhibitors, downstream effector inhibitors, or GTPase inhibitors with specificity for individual GTPases [16. 28]. The first three categories act upon the proteins interacting with the GTPase instead of the GTPase itself, while the specific inhibitors are usually not potent or have limited utility in drug discovery and basic research. The compound ML282 (CID 1067700) that we report here shows potent inhibition towards multiple GTPases in both biochemical and cellular assays and can be an ideal molecular probe for the purpose of drug discovery and fundamental research.

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APPENDIX. Ricerca Profiling Results for ML282

ML282 did not register any percent inhibition over 50% in this panel. The most significant findings were noted as 40% inhibition of the human serotonin receptor (5-HT2B) at 10 μM. All other findings registered below 22% inhibition at this concentration. Details of the panel results are provided below.

Summary

STUDY OBJECTIVE

To evaluate, in Radioligand Binding assays, the activity of compound 125264860 (UK-6, PT# 1156808).

METHODS

Methods employed in this study have been adapted from the scientific literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained. Assays were performed under conditions described in the accompanying “Methods” section of this report.

Where presented, IC50 values were determined by a non-linear, least squares regression analysis using MathIQ™ (ID Business Solutions Ltd., UK). Where inhibition constants (Ki) are presented, the Ki values were calculated using the equation of Cheng and Pursoff (Cheng, Y., Prusoff, W.H., Biochem, Pharmacol. 22:3099–3108, 1973 using the observed IC50 of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the KD of the ligand (obtained experimentally at Ricerca Biosciences, LLC). Where presented the Hill coefficient, (nH), defining the slope of the competitive binding curve, was calculated using MathIQ™. Hill coefficients significantly different than 1.0, may suggest that the binding displacement does not follow the laws of mass action with a single binding site. Where IC50, K­i, and/or nH data are presented without Standard Error of the Mean (SEM), data are insufficient to be quantitative, and the values presented (Ki, IC50, nH) should be interpreted with caution.

RESULTS

A summary of results meeting the significance criteria is presented in the following sections, Complete results are presented under the section labeled “Experimental Results”. Individual responses, if requested are presented under the section labeled “Individual Responses”.

SUMMARY/CONCLUSION

Significant results are displayed in the following table(s) in rank of potency for estimated IC­50 and/or Ki values.

Experimental Results

Cat#Assay NameBatch*Spec.Rep.Conc.%Inh.IC50*KinHR
Compound: 125264860, PT #: 1156808
200510Adenosine A1307264hum210 μM7
200610Adenosine A2A307124hum210 μM12
200720Adenosine A3307115hum210 μM8
203100Adrenergic α1A307444rat210 μM0
203200Adrenergic α1B307445rat210 μM−1
203400Adrenergic α1D307125hum210 μM18
203620Adrenergic α2A307447hum210 μM9
204010Adrenergic β1307126hum210 μM4
204110Adrenergic β2307127hum210 μM6
285010Androgen (Testosterone)AR307551rat210 μM−2
212510Bradykinin B1307276hum210 μM−13
212620Bradykinin B2307235hum210 μM6
214510Calcium Channel L-Type, Benezothiazepine307270rat210 μM−9
214600Calcium Channel L-Type, Dihydropyridine307263rat210 μM10
216000Calcium Channel N-Type307169rat210 μM9
217030Cannabinoid CB1307262hum210 μM−14
219500Dopamine D1307130hum210 μM6
219700Dopamine D2S307454hum210 μM3
219800Dopamine D3307455hum210 μM0
219900Dopamine D4,2307132hum210 μM2
2240410Endothelin ETA307133hum210 μM11
224110Endothelin ETB307415hum210 μM−10
225510Epidermal Growth Factor (EGF)307233hum210 μM−4
226010Estrogen ERα307134hum210 μM0
226600GABAA, Flunitrazepam, Central307261rat210 μM17
226500GABAA, Muscimol, Central307271rat210 μM−1
228610GABAB1A307172hum210 μM−21
232030Glucocorticoid307168hum210 μM7
232700Glutamate, Kainate307279rat210 μM10
232810Glutamate, NMDA, Agonism307135rat210 μM18
232910Glutamate, NMDA, Glycine307136rat210 μM0
233000Glutamate, NMDA, Phencyclidine307272rat210 μM7
239610Histamine H1307137hum210 μM7
239710Histamine H2307138hum210 μM1
239820Histamine H3307158hum210 μM−6
241000Imidazoline I2, Central307281rat210 μM−3
243520Interleukin IL-1307139mouse210 μM22
250460Leukotriene, Cysteinyl CysL T1307554hum210 μM−5
251600Melatonin MT1307162hum210 μM2
252610Muscarinic M1307255hum210 μM−1
252710Muscarinic M2307256hum210 μM2
252810Muscarinic M3307257hum210 μM4
257010Neuropeptide Y Y1307553hum210 μM5
257110Neuropeptide Y Y2307428hum210 μM6
258590Nicotinic Acetylcholine307282hum210 μM−10
258700Nicotinic Acetylcholine α Bungarotoxin307283hum210 μM−3
260130Opiate δ1 (OP1, DOP)307556hum210 μM0
260210Opiate κ(OP2, KOP)307557hum210 μM3
260410Opiate μ(OP3, MOP)307273hum210 μM−1
264500Phorbol Ester307284mouse210 μM6
265010Platelet Activating Factor (PAF)307232hum210 μM8
265600Potassium Channel[KATP]307285ham210 μM1
265900Potassium Channel hERG307269hum210 μM−18
268420Prostanoid EP4307152hum210 μM13
268700Purinergic P2X307432rabbit210 μM8
268810Purinergic P2Y307143rat210 μM9
270000Rolipram307286rat210 μM5
271110Serotonin (5-Hydroxytryptamine) 5-HT1A307163hum210 μM4
271700Serotonin (5-Hydroxytryptamine) 5-HT2B307254hum210 μM40
271910Serotonin (5-Hydroxytryptamine) 5-HT3307154hum210 μM11
278110Sigma σ1307154hum210 μM−1
255520Tachykinin NK1307426hum210 μM5
285900Thyroid Hormone307120rat210 μM9
220320Transporter, Dopamine (DAT)307266hum210 μM−7
226400Transporter, GABA307274rat210 μM2
204410Transporter, Norepinephrine (NET)307265hum210 μM11
274030Transporter, Serotonin (5-Hydroxytryptamine)307280hum210 μM3

Note: Items meeting criteria for significance (≥50% stimulation or inhibition) are highlighted.

*

Batch: Represents compounds tested concurrently in the same assay(s).

R=See Remarks (if any) at end of this section.

ham=Hamster; hum= Human

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