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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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Optimization and characterization of a pan protein arginine deiminase (PAD) inhibitor

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

Author Information and Affiliations

Received: ; Last Update: April 5, 2013.

The protein arginine deiminases (PADs) are a family of Ca2+-dependent enzymes that catalyze the conversion of peptidyl-arginine to peptidyl-citrulline in numerous protein substrates. Disruption of normal PAD activity plays a role in the pathogenesis of multiple inflammatory diseases such as rheumatoid arthritis (RA), chronic obstructive pulmonary disease, ulcerative colitis, multiple sclerosis, psoriasis, Alzheimer’s disease, and in various cancers. PAD inhibitors described in the literature have been useful chemical tools to study the role of PAD enzymes in inflammatory diseases and cancer biology. Most published PAD inhibitors are mechanism-based inactivators belonging to the halogen-amidine chemotype. For emerging targets such as the PADs, it can be difficult to distinguish compound-specific effects from those truly resulting from enzyme inhibition. We therefore initiated a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high throughput screening (HTS) campaign to identify a second PAD inhibitor chemotype.

The PAD4 HTS campaign identified the natural product streptonigrin (SID 11532976) as an irreversible PAD4-specific inhibitor. We describe herein the medicinal chemistry optimization of streptonigrin to the pan PAD probe ML325 (SID 118043677). ML325 inhibits PAD1, 2, 3, and 4 in vitro with IC50 values of 70 nM, 200 nM 170 nM, and 240 nM respectively. In a kinetic assay of inhibition more appropriate for irreversible inhibitors, ML325 has kinact/KI values of 3500, 7300, 1900, and 5300 M−1min−1 for PAD1, 2, 3 and 4 respectively; indicating it has less than 4-fold selectivity among the four family members. Despite its promiscuity within the PAD family, ML325 exhibits high selectivity vs. more than 20 cysteine-reactive proteins as assayed by activity-based protein profiling. ML325 was also demonstrated to inhibit all four PAD isozymes irreversibly and to be non-cytotoxic to NIH-3T3 cells. The complete properties, characterization, and synthesis of ML325 are detailed in this report.

Assigned Assay Grant #: R01 GM079357-01

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

Chemistry Center Name & PI: SRIMSC, H Rosen

Assay Submitter & Institution: Paul R. Thompson, The Scripps Research Institute, Jupiter, FL

PubChem Summary Bioassay Identifier (AID): 463083

Probe Structure & Characteristics

ML#325.

ML#325

CID/ML#Target NameTarget IC50 (nM) [SID, AID]Anti-target Name(s)Anti-target IC50 (μM) [SID, AID]Fold Selective†Secondary Assay(s) Name: IC50 (nM) [SID, AID]
CID 13891339/ML325PAD1,2,3,4PAD1: 70 ± 40 [SID 118043677, AID 588490]
PAD2: 200 ± 50 [SID 118043677, AID 588490]
PAD3: 170 ± 10 [SID 118043677, AID 588490]
PAD4: 240 ± 10 [SID 118043677, AID 588490]		> 20 cysteine-reactive proteins*>50 [SID 118043677, AID 651628]**PAD1: >714
PAD2: >250
PAD3: >70
PAD4: >208		Inhibition Assay: [SID 118043677, AID 588559, AID 588560]
Selectivity Assay: [SID 118043677, AID 651628]
Binding Mode Assay: [SID 118043677, AID 651627; SID 118043677, AID 651861]
Cytox assay: [SID 118043677, AID 651887],
*

As assessed by gel-based competitive ABPP inMCF-7 soluble and membrane proteome fractions with the cysteine-reactive activity-based probe CA-Rhodamine

**

IC50 of the anti-target is defined as greater than the test compound concentration at which less than or equal to 50% inhibition of the anti-target is observed, which is reported in AID 651628. For SID 118043677, no anti-targets were observed for all cysteine-reactive enzymes assayed at 50 μM concentration, so the IC50 is reported as >50 μM.

†

Fold-selectivity was calculated as: >IC50 for anti-target/IC50 for PAD isozyme.

1. Recommendations for Scientific Use of the Probe

Disruption of normal protein arginine deiminase (PAD) activity plays a role in the pathogenesis of multiple inflammatory diseases such as rheumatoid arthritis (RA) (1), chronic obstructive pulmonary disease, ulcerative colitis (2), multiple sclerosis (3), psoriasis, Alzheimer’s disease (4), and in various cancers (5–12). Increased protein citrullination within inflamed tissues from patients with autoimmune diseases such as RA and colitis has been documented (5, 7, 13). Two mechanism-based pan PAD inhibitors, F-amidine and Cl-amidine (14–16) have been important chemical tools in discerning the physiological role of the PADs (15–18). Inflammatory symptoms have been shown to be suppressed in mouse models of colitis and RA by Cl-amidine, the more potent of the two compounds (19). More recently, Cl-amidine, and a related analog were shown to significantly reduce the growth of xenografted tumors (20, 21). ML325, described herein, is a potent pan PAD inhibitor originating from a distinct structural class from the halogen-amidines. As such, it is recommended as a complementary research tool to elucidate the upstream mechanisms that induce PAD expression and the downstream mechanisms by which PADs regulate gene expression in inflammatory diseases and in various cancers.

2. Materials and Methods

All reagents for chemical synthesis were obtained from ThermoFisher or SigmaAldrich. All other protocols are summarized below.

2.1. Assays

Probe Characterization Assays

Solubility

The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Compounds were inverted for 24 hours in test tubes containing 1–2 mg of compound with 1 mL of PBS. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration.

Stability

Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at −80 °C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed at room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.

LC-MS/MS for stability assay

All analytical methods are in MRM mode where the parent ion is selected in Q1 of the mass spectrometer. The parent ion is fragmented and a characteristic fragment ion is monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods are rapidly set up using Automaton® (Applied Biosystems), where the compounds are listed with their name and mass in an Excel datasheet. Compounds are submitted in a 96-well plate to the HPLC autosampler and are slowly injected without a column present. A narrow range centered on the indicated mass is scanned to detect the parent ion. The software then evaluates a few pre-selected parameters to determine conditions that maximize the signal for the parent ion. The molecule is then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass are determined. Three separate collision energies are evaluated to fragment the parent ion and the largest three ions are selected. Each of these three fragment ions is further optimized and the best fragment is chosen. The software then inserts the optimized masses and parameters into a template method and saves it with a unique name that indicates the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern are saved and can be reviewed later.

Determination of glutathione reactivity

One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 50 μM reduced glutathione. Final compound concentration is 10 μM unless limited by solubility. The solution was allowed to incubate at 37 °C for 6 hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized: a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of −129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans (45).

Primary Assays

Primary uHTS assay to identify PAD4 inhibitors in NIH Validation Collection (AID 463073)

Assay Overview: The purpose of this assay is to identify compounds from the NIH Validation Collection that act as inhibitors of human recombinant PAD4. In this assay, human recombinant PAD4 is labeled in an active site-directed manner by the fluorescent ABPP probe rhodamine-conjugated F-amidine (RFA) in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. As designed, test compounds that act as PAD4 inhibitors will prevent PAD4-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization in the well. Compounds were tested in singlicate at a final nominal concentration of 5 μM.

Protocol Summary: Prior to the start of the assay, 10.0 μL of Assay Buffer (50 mM HEPES pH 7.6, 150 mM NaCl, 10 mM CaCl2, 0.01% Pluronic F-127, and 1 mM TCEP) containing 2 μM of human recombinant PAD4 protein was dispensed into 384-well microtiter plates. Next, 50 nL of test compound in DMSO or DMSO alone (0.5% final concentration) was added to the appropriate wells and incubated for 30 minutes at 25 °C. The assay was started by dispensing 1.1 μL of 750 nM RFA probe in Assay Buffer to all wells. Plates were centrifuged and after 5 hours of incubation at 37 °C, fluorescence polarization was read on an Envision microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). Fluorescence polarization was read for 30 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value was obtained via the PerkinElmer Viewlux software. Assay Cutoff: Compounds that inhibited PAD4 >30% were considered active.

Primary uHTS assay to identify PAD4 inhibitors in MLSMR Library (AID 485272)

Assay Overview: The purpose of this assay is to identify compounds that act as inhibitors of human recombinant PAD4. In this biochemical assay, human recombinant PAD4 is labeled in an active site-directed manner by the fluorescent activity-based protein profiling (ABPP) probe rhodamine-conjugated Cl-amidine (RCA) in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. The assay is performed by incubating test compounds with PAD4 for a defined period, followed by addition of the rhodamine-conjugated Cl-amidine probe and measurement of fluorescence polarization at a specific time point. As designed, test compounds that act as PAD4 inhibitors will prevent PAD4-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization in the well. Compounds were tested in singlicate at a final nominal concentration of 6.36 μM.

Protocol Summary: Prior to the start of the assay, 4.0 μL of Assay Buffer (50 mM HEPES pH 7.6, 150 mM NaCl, 10 mM CaCl2, 0.01% Pluronic F-127, and 1 mM TCEP) containing 1.25 μM of human recombinant PAD4 protein was dispensed into 1536-well microtiter plates. Next, 32 nL of test compound in DMSO or DMSO alone (0.64% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 °C. The assay was started by dispensing 1.0 μL of 375 nM RCA probe in Assay Buffer to all wells. Plates were centrifuged and incubated for 20 hours at 37 °C. Prior to reading, plates were equilibrated at room temperature for 5 minutes. Fluorescence polarization was read on Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). Fluorescence polarization was read for 30 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value was obtained via the PerkinElmer Viewlux software. Assay Cutoff: Compounds that inhibited PAD4 ≥14.75% were considered active.

Confirmation uHTS assay to identify PAD4 inhibitors in MLSMR Library (AID 488796)

Assay Overview: The purpose of this assay is to confirm activity of compounds identified as active in the primary uHTS PAD4 screen (AID 485272). In this assay, the RCA probe was used to label PAD4 in the presence of test compounds and analyzed as described above (AID 485272). Compounds were tested in triplicate at a nominal concentration of 6.36 μM.

Protocol Summary: The assay was performed as described above (AID 485272), except that compounds were tested in triplicate. Assay Cutoff: Compounds that inhibited PAD4 ≥14.75% were considered active.

Secondary Assays

Biochemical substrate assay to determine IC50 values of hits from NIH Validation Set (AID 492970)

Assay Overview: The purpose of this assay is to determine the potency of the compounds that were determined to be active in the NIH Validation Collection primary assay (AID 463073). In this assay, human recombinant PAD4 is pre-incubated with test compounds, followed by the addition of the substrate, Na-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE). The percent activity remaining is determined by measuring the amount of citrulline produced using a standard assay that measures changes in citrulline production. Test compounds that act as human recombinant PAD4 inhibitors will prevent the production of citrulline. IC50 values for inhibition of PAD4 were determined from dose-response curves from 2 trials at each inhibitor concentration in an 8-point dilution series from 0 to 100 μM.

Protocol Summary: Human recombinant PAD4 (0.2 μM in Assay Buffer (50 mM Tris-HCl pH 7.6, 50 mM NaCl, 10 mM CaCl2, and 2 mM DTT) was incubated with DMSO or test compound for 15 minutes at 37 °C before the addition of BAEE at a final concentration of 10 mM in 60 μL total reaction volume. The reaction was incubated for 15 minutes at 37 °C, quenched by flash freezing in liquid nitrogen, and 200 μL of a color developing reagent (COLDER), which consists of solution A (80 mM diacetyl monoxime and 2 mM thiosemicarbazide) and solution B (3 M H3PO4, 6 M H2SO4, and 2 mM NH4Fe(SO4)2 in a 1:3 ratio, was added. This mixture was incubated for 30 minutes at 95 °C and the absorbance was measured at 540 nm. The amount of product produced was determined by comparison to a standard curve with known concentrations of citrulline. Assay Cutoff: Compounds with an IC50 ≤10 μM were considered active.

Gel-based ABPP assay for inhibition of PADs1–4 by cherry-picked uHTS hits (AID 588487)

Assay Overview: The purpose of this assay is to determine whether cherry picked HTS hit compounds from AID 488796 can inhibit PADs 1–4 in a gel-based activity-based proteomic profiling (ABPP) assay. In this assay, the target enzyme (human recombinant PAD1, 2, 3 or 4) is incubated with test compound followed by reaction with a rhodamine-conjugated F-amidine (RFA) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as PAD inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.

Protocol Summary: Human recombinant PAD1 (Assay 1), PAD2 (Assay 2), PAD3 (Assay 3), or PAD4 (Assay4) (10 μM) in assay buffer (50 mM NaCl, 10 mM CaCl2, 2 mM DTT, 100 mM HEPES, pH 7.6) was treated with test compound (10 μM) or DMSO for 15 minutes at 37 °C. RFA was added to a final concentration of 1 μM. The reaction was incubated for 30 minutes in the dark at 37 °C, quenched with 2× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the target enzyme (PAD1, 2, 3, or 4) band relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥50% inhibition were considered active.

Biochemical substrate assay for inhibition of PADs1–4 by cherry-picked uHTS hits (AID 588488)

Assay Overview: The purpose of this assay is to determine whether cherry picked HTS hit compounds can inhibit PADs 1–4 using a substrate-based assay. In this assay, the target enzyme (human recombinant PAD1, 2, 3 or 4) is pre-incubated with test compound followed by addition of substrate N-α-benzoyl-L-arginine ethyl ester HCl (BAEE). The percent activity remaining is determined by measuring the amount of citrulline produced using a standard colorimetric absorbance assay. Test compounds that act as PAD inhibitors will prevent the production of citrulline.

Protocol Summary: Human recombinant PAD1 (Assay 1; 0.2 μM), PAD2 (Assay 2; 0.5 μM), PAD3 (Assay 3; 0.5 μM), or PAD4 (Assay 4; 0.2 μM) in assay buffer (50 mM NaCl, 10 mM CaCl2, 2 mM DTT, 100 mM Tris-HCl, pH 7.6) was treated with test compound (10 μM) or DMSO for 15 minutes at 37 °C. Substrate BAEE (10 mM) was added, and the reaction was incubated for 15 minutes at 37 °C in 60 μL total reaction volume. The reaction was quenched by flash freezing in liquid nitrogen, and 200 μL of a color-developing reagent that detects the product citrulline was added; the COLDER solution consists of a solution A (80 mM diacetyl monoxime and 2 mM thiosemicarbazide) and solution B (3 M H3PO4, 6 M H2SO4, and 2 mM NH4Fe(SO4)2) in a 1:3 ratio. This mixture was incubated for 30 minutes at 95 °C, at which point the absorbance was measured at 540 nm. The amount of product produced was determined by comparison to a standard curve with known concentrations of citrulline. Assay Cutoff: Compounds with ≥50% inhibition were considered active.

Biochemical substrate assay for inhibition of PAD4 by synthesized compounds (AID 588559)

Assay Overview: The purpose of this assay is to determine whether synthesized test compounds can inhibit PAD4 using the substrate-based assay described above (AID 588488).

Protocol Summary: The protocol for these assays was as described above (AID 588488). Assay Cutoff: Compounds with ≥50% inhibition were considered active.

Biochemical substrate assay for inhibition of PADs1–4 by synthesized compounds (AID 588560)

Assay Overview: The purpose of this assay is to determine whether synthesized test compounds can inhibit PADs1–4 using the substrate-based assay described above (AID 588488).

Protocol Summary: The protocol for these assays was as described above (AID 588488). Assay Cutoff: Compounds with ≥50% inhibition for PAD1, 2, 3, or 4 were considered active against that enzyme.

Biochemical substrate assay to determine potency of synthesized compounds against PAD4 (AID 588422, AID 588421, AID 588420, AID 588419, AID 588418, AID 588417, AID 588416, AID 588423)

Assay Overview: The purpose of these assays is to determine the potency of synthesized test compounds as inhibitors of human recombinant PAD4 using a biochemical substrate assay as described above (AID 588488). IC50 values for inhibition of PAD4 were determined from dose-response curves from 2 trials at each inhibitor concentration in a:

Protocol Summary: The protocol for these assays was as described above (AID 588488). Assay Cutoff: Compounds with an IC50 ≤1 μM were considered active.

Biochemical substrate assay to determine potency of synthesized compounds against PADs1–4 (AID 588486, AID 588438, AID 588471, AID 588484, AID 588472, AID 588490)

Assay Overview: The purpose of these assays is to determine the potency of synthesized test compounds as inhibitors of human recombinant PADs1–4 using a biochemical substrate assay as described above (AID 588488). IC50 values for inhibition of PADs1–4 were determined from dose-response curves from 2 trials at each inhibitor concentration in a:

  • 7-point dilution series from 0 to 16 μM (PADs1–4) [AID 588484]
  • 7-point dilution series from 0 to 5 μM (PAD1, PAD2, PAD4) or 0 to 2 μM (PAD3) [AID 588486 and AID 588472]
  • 7-point dilution series from 0 to 7.5 μM (PADs1–3) or 0 to 4 μM (PAD4) [AID 588438]
  • 7-point dilution series from 0 to 7.5 μM (PAD1 and PAD4), 0 to 5 μM (PAD2 ), or 0 to 0.5 μM (PAD3) [AID 588471]
  • 7-point dilution series from 0 to 0.75 μM (PAD1), 0 to 1 μM (PAD2 and PAD4 ), or 0 to 0.25 μM (PAD3) [AID 588490]

Protocol Summary: The protocol for these assays was as described above (AID 588488). Assay Cutoff: Compounds with an IC50 ≤1 μM were considered active.

Biochemical substrate assay to determine potency of streptonigrin against PADs1–3 (AID 588462)

Assay Overview: The purpose of these assays is to determine the potency of streptonigrin as an inhibitor of human recombinant PADs1–3 using a biochemical substrate assay as described above (AID 588488). IC50 values for inhibition of PADs1–3 were determined from dose-response curves from 2 trials at each inhibitor concentration in a 7-point dilution series from 0 to 100 μM.

Protocol Summary: The protocol for these assays was as described above (AID 588488). Assay Cutoff: Compounds with an IC50 ≤1 μM were considered active.

Biochemical substrate assay to determine selectivity of synthesized compounds against PADs1–4 (AID 651865, AID 651866, AID 651867, AID 651868)

Assay Overview: The purpose of these assays is to more rigorously determine the potency and selectivity of test compounds as inhibitors of human recombinant PADs 1–4 using a biochemical substrate assay. kinact(app)/KI values for PADs1–4 were determined by measuring the rate of human recombinant PADs 1–4 enzyme inactivation as a function of time at a single inhibitor concentration.

Protocol Summary: Inactivation Reactions [100 mM Tris-HCl, pH: 7.6, 10 mM CaCl2, and 2 mM DTT] proceeded by incubating test compounds (10 μM) with purified PAD isozymes ([PAD1] = 2 μM (Assay 1), [PAD2] = 5 μM (Assay 2), [PAD3] = 5.0 μM (Assay 3), and [PAD4] = 2 μM (Assay 4)) for various time points between 0–60 minutes at 37 °C (60 μL total volume). After the designated time point, an aliquot (6 μL) of the Inactivation Reaction was added to pre-incubated Reaction Buffer [100 mM Tris-HCl, pH: 7.6, 500 mM NaCl, 10 mM CaCl2, and 2 mM DTT] containing N-α-benzoyl-L-arginine ethyl ester-HCl (BAEE, 10 mM final, PADs 2,4) or N-α-benzoyl-L-arginine amide HCl (BAA, 10 mM final, PADs 1,3) to measure the residual activity. The Reaction Mixtures (60 μL total volume) were pre-incubated at 37 °C for 10 minutes before adding aliquots from the Inactivation Mixture. The final reaction proceeded for 15 minutes at which point the reaction was stopped by flash freezing in liquid N2. 200 μL of color-developing reagent, COLDER, that detects the product citrulline was added; the COLDER solution consists of solution A (80 mM diacetyl monoxime and 2 mM thiosemicarbazide) and solution B (3 M H3PO4, 6 M H2SO4, and 2 mM NH4Fe(SO4)2) in a 1:3 ratio. This mixture was incubated for 30 minutes at 95 °C, at which point the absorbance was measured at 540 nm. The amount of product produced was determined by comparison to a standard curve with known concentrations of citrulline. The data obtained at each inhibitor concentration were fit to equation 2 using GraFit version 5.0.11.,

v =v0e−kobst

where v is the velocity, vo is the initial velocity, kobs is the pseudo-first-order rate constant for inactivation, and t is time (46). The kinact(app)/KI were determined by dividing the inactivation rate (kobs) by the concentration of inhibitor. Assay Cutoff: Compounds with a kinact(app)/KI values ≥1500 min−1M−1 were considered active. Compounds with ≥10 fold kinact(app)/KI among PAD isozymes were considered selective.

Selectivity against anti-targets in a complex proteome (AID 651628)

Assay Overview: The purpose of this assay is to assess compound selectivity in a complex proteome using a gel-based activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction with a cysteine-reactive chloroacetamide-rhodamine (CA-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as anti-target inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.

Protocol Summary: Soluble or membrane proteome (50 μL of 1 mg/mL in DPBS) of MCF-7 human cancer cells was treated with 10 μM or 50 μM test compound (1 μL of a 50× stock in DMSO). Test compounds were incubated for 1 hour at 25 °C, followed by the addition of 10 μM CA-Rh (1 μL of 50× stock in DMSO). The reactions were incubated for 1 hour at 25 °C, quenched with 4× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the anti-target bands relative to a DMSO-only (no compound) control. Assay Cutoff: Bands were counted as anti-targets if ≥ 50% inhibition was observed.

Dialysis assay to determine inhibitor binding mode of PAD4 (AID 651627)

Assay Overview: The purpose of this assay is to determine whether powder samples of test compounds inhibit human recombinant PAD4 in a reversible or irreversible manner. In this assay, PAD4 is incubated with test compound or DMSO control followed by dialysis to remove small molecules not tightly/covalently bound to the protein. Substrate N-α-benzoyl-L-arginine ethyl ester HCl (BAEE) is added, and the percent activity remaining is determined by the biochemical substrate assay described above (AID 588488). Test compounds that act as irreversible PAD4 inhibitors will prevent the production of citrulline and prevent enzyme-probe interactions following dialysis relative to the DMSO (no compound) control. In contrast, compounds that act as reversible inhibitors will show high residual enzyme activity following dialysis and can be labeled by active site directed probes.

Protocol Summary: Recombinant PAD4 (0.6 μM in 100 mM Tris-HCl, pH 7.6, 50 mM NaCl, 2 mM DTT, and 10 mM CaCl2, 400 μL total reaction volume) was pre-incubated with test compound (100 μM) or DMSO control, followed by dialysis in 4 L Long-Term Storage Buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 500 mM NaCl, 2 mM DTT, and 10% glycerol) for 20 hours. Residual PAD4 activity was then measured using the biochemical substrate assay described above (AID 588488). Assay Cutoff: Compounds resulting in > 75% reduction in residual PAD4 activity were considered reversibly bound to PAD4; compounds resulting in ≤ 75% reduction in residual PAD4 activity were considered irreversibly bound to PAD4.

Dialysis assay to determine inhibitor binding mode of PADs 1–3 (AID 651861)

Assay Overview: The purpose of this assay is to determine whether powder samples of test compounds inhibit human recombinant PADs 1–3 in a reversible or irreversible manner. In this assay, PADs 1–3 are incubated with test compound or DMSO control followed by dialysis to remove small molecules not tightly/covalently bound to the protein. The substrates N-α-benzoyl-L-arginine ethyl ester HCl (BAEE) or N-α-benzoyl-L-arginine amide HCl (BAA) are added, and the percent activity remaining is determined by the biochemical substrate assay described above (AID 588488). Test compounds that act as irreversible inhibitors of PADs 1–3 will prevent the production of citrulline and prevent enzyme-probe interactions following dialysis relative to the DMSO (no compound) control. In contrast, compounds that act as reversible inhibitors will regain enzyme activity following dialysis and can be labeled by active site directed probes.

Protocol Summary: Recombinant PADs 1–3 (0.6 μM in 100 mM Tris-HCl, pH 7.6, 50 mM NaCl, 2 mM DTT, and 10 mM CaCl2, 400 μL total reaction volume) were pre-incubated with test compound (100 μM) or DMSO control, followed by dialysis in 4 L of Long-Term Storage Buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 500 mM NaCl, 500 μM TCEP, and 10% glycerol) for 20 hours. Residual PAD1, 2, and 3 activity was then measured using the biochemical substrate assay described above (AID 588488). Assay Cutoff: Compounds resulting in > 75% reduction in residual PADs 1–3 activity were considered reversibly bound to PADs 1–3; compounds resulting in ≤ 75% reduction in residual PADs 1–3 activity were considered irreversibly bound to PADs 1–3.

Cell-based absorbance-based assay to assess cytotoxicity of test compounds (AID 651887)

The purpose of this assay is to assess compound cytotoxicity in mouse embryonic fibroblast NIH-3T3 cells using a colormetric MTT assay. In this assay, NIH-3T3 cells are divided into culture plates and are initially treated with test compound, DMSO (vehicle control), or 100% Triton-X (positive control). After incubation of the compounds for 3 days, cell viability is assessed by addition of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the MTT reagent. The relative number of living cells is determined by the reduction of the yellow-colored MTT reagent in living cells, quantified by measuring absorbance at 570 nm. By design, test compounds that are cytotoxic will have a decreased number of living cells to reduce the MTT reagent resulting in concomitant increase in yellow-colored MTT and decrease in purple-colored formazan. Percent inhibition is calculated relative to a DMSO (vehicle control).

Protocol Summary: NIH-3T3 cells were treated with test compound for 72 hours in a 24 well plate. The MTT reagent 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was then added to a final concentration of 0.5 mg/mL and the cells were further incubated for 4 hours. The culture medium was removed and DMSO was added to dissolve the formazan dye. The absorbance of the formazan-containing solution was then measured at 570 nm using a spectrophotometer. EC50 values for cell growth inhibition were determined from dose-response curves, using a:

  • 6-point dilution series from 0 to 10 μM (Compounds 14 and 21)
  • 6-point dilution series from 0 to 100 μM (Compounds 17)

Assay Cutoff: Compounds with a CC50 value of ≤ 5 μM were considered active (cytotoxic) and compounds with an CC50 value of > 5 μM were considered inactive (non-cytotoxic).

2.2. Probe Chemical Characterization

CID 13891339/ SID 118043677 ML325.

CID 13891339/
SID 118043677
ML325

The probe structure was verified by 1H NMR (see Section 2.3) and high resolution MS (m/z calculated for C15H9N3O4 [M+H]+: 296.0666; found: 296.0668). Purity was assessed to be >95% by NMR. Solubility (room temperature) was determined to be > 100 μM in PBS and >100 μM in DMEM medium containing 10% fetal calf serum. Stability in PBS was determined by LC-MS to be >48 hours. ML325 was found to be reactive when incubated for 6 hours at 37 °C in 50 μM GSH (see discussion in Section 3.3).

Table 1Compounds submitted to the SMR collection

DesignationCIDSIDSRIDMLS
Probe13891339118043677SR-02000001200-1MLS004082609
Analog 1265935118043670SR-01000882441-3MLS004082610
Analog 2253693118043673SR-02000001196-1MLS004082611
Analog 3253688118043674SR-02000001197-1MLS004082612
Analog 451049631118043681SR-02000001204-1MLS004082613
Analog 55323583152136272SR-01000721857-5MLS004556149

2.3. Probe Preparation

Figure 1. Synthesis of ML 325.

Figure 1Synthesis of ML 325

Synthesis Overview: Freidlander condensation of 2-amino-3-(benzyloxy)-4-bromobenzyaldehyde (A) with methyl 2-acetylpyridine-6-carboxylate (B) provided 8-(benzyloxy)-7-bromo-2-2′-pyridyl)quinoline-6′-carboxylic acid. Fischer esterification followed by O-debenzylation by TFA afforded methyl 7-bromo-8-hydroxy-2-(2′-pyridyl)quinoline-6′-carboxylate. Nitration of the free phenol gave the nitrophenol. The subsequent, sequential aluminum amalgam reduction and manganese dioxide oxidation provided the 7-bromoquinoline-5,8-dione as a yellow crystalline solid. Direct C-7 sodium azide displacement of the 7-bromoquinoline-5,8-dione afforded the azide. Azide reduction was accomplished by the treatment of the azide with triphenylphosphine followed by treatment of the resulting triphenylphosphine imide with acetic acid. In situ reduction of the quinoline-5,8-dione to the corresponding hydroquinone followed by base-promoted methyl ester hydrolysis provided the desired carboxylic acid probe compound (47) (Probe ML325). 1H NMR (DMSO, 400 MHz) δ 8.88 (d, J = 8.2 Hz, 1H), 8.71 (t, J = 8.0 Hz, 1H), 8.48 (t, J = 8.0 Hz, 1H), 8.24 (t, J = 8.0 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 6.10 (bs, 2H), 5.91 (s, 1H); purity >95% by NMR. ESI-TOF HRMS m/z calculated for C15H9N3O4 [M+H]+: 296.0666, found 296.0668. See also ref (55).

Synthetic Details:N-benzyltrimethylammonium hydroxide (Triton B, 40% solution in MeOH, 13.4 mL, 29.8 mmol, 4.0 equiv) was added to a stirring solution of methyl-2-acetylpyridine-6-carboxylate (B, 1.3345 g, 7.45 mmol) in THF (75 mL) under Argon at 0 °C. A solution of 2-amino-3-(benzyloxy)-4-bromobenzaldehyde (A, 2.51 g, 8.20 mmol, 1.1 equiv) in THF (11 mL) was added to the reaction mixture. The reaction mixture was stirred at 0 °C for 1 hour and at room temperature for 3 hour. After 3 hours, the reaction mixture was diluted with saturated aqueous NH4Cl (50 mL) and H2O (200 mL). The precipitated white solid was collected by filtration, washed with H2O and washed with hexanes to provide the carboxylic acid C (2.91 g, 90%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 8.78 (dd, J = 7.6, 1.4 Hz, 1H), 8.55 (d, J = 8.7 Hz, 1H), 8.33 (d, J = 8.7 Hz, 1H), 8.32 (dd, J = 7.6, 1.4 Hz, 1H), 8.05 (t, J = 7.6 Hz, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.30–7.25 (m, 5H), 5.57 (s, 2H).

The carboxylic acid C (2.91 g, 6.69 mmol) was added to a stirring solution of 10% HCl-MeOH (300 mL) at 0 °C. The reaction mixture was stirred at room temperature for 18 hours. After 18 hours, the reaction mixture was concentrated. The crude reaction product was dissolved in 400 mL CH2Cl2, washed with water (200 mL), washed with saturated aqueous NaCl (200 mL), dried (Na2SO4), and concentrated on a rotary evaporator. Flash chromatography (SiO2, 10% EtOAc/hexanes) provided methyl 8-(benzyloxy)-7-bromo-2-(2′-pyridyl)quinoline-6′-carboxylate D (2.44 g, 81%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 8.77 (d, J = 8.7 Hz, 1H), 8.72 (dd, J = 7.6, 1.3 Hz, 1H), 8.28 (d, J = 8.7 Hz, 1H), 8.18 (dd, J = 7.6, 1.3 Hz, 1H), 7.93 (t, J = 7.6 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.35–7.16 (m, 5H), 5.58 (s, 2H), 4.05 (s, 3H).

Methyl 8-(benzyloxy)-7-bromo-2-(2′-pyridyl)quinoline-6′-carboxylate (D, 2.44 g, 5.43 mmol) was stirred in TFA (60 mL) under Argon at room temperature for 11 hours. After 11 hours, the reaction mixture was concentrated. The resulting residue was washed with hexane to provide methyl 7-bromo-8-hydroxy-2-(2′-pyridyl)quinoline-6′-carboxylate (E, 1.58 g, 81%) as an off-white solid: 1H NMR (CDCl3, 400 MHz) δ 8.77 (d, J = 8.6 Hz, 1H), 8.75 (d, J = 8.0 Hz, 1H), 8.31 (d, J = 8.6 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.06 (t, J = 7.8 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.30 (d, J = 8.8 Hz, 1H), 4.07 (s, 3H).

HNO3 (1 M in CH3NO2, 2.75 mL, 2.75 mmol, 5.0 equiv) was added to a stirring suspension of methyl 7-bromo-8-hydroxy-2-(2′-pyridyl)quinoline-6′-carboxylate (E, 199 mg, 0.55 mmol) in CH3NO2 (10 mL) under Argon at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour. After 1 hour, the reaction mixture was diluted with H2O (100 mL) and extracted with CH2Cl2 (5 × 80 mL). The combined organic layers were dried (Na2SO4) and concentrated. Trituration with hexane provided methyl 7-bromo-8-hydroxy-5-nitro-2-(2′-pyridyl)quinoline-6′-carboxylate (F, 1.68 g, 94%) as a yellow solid: 1H NMR (CDCl3, 400 MHz) δ 9.44 (d, J = 9.2 Hz, 1H), 9.02 (d, J = 9.2 Hz, 1H), 8.78 (s, 1H), 8.72 (dd, J = 7.5, 1.3 Hz, 1H), 8.29 (dd, J = 7.5, 1.3 Hz, 1H), 8.09 (t, J = 7.5 Hz, 1H), 4.08 (s, 3H).

Freshly prepared aluminum amalgam (48) (100 mg, 5 wt equiv) was added to a stirring solution of methyl 7-bromo-8-hydroxy-5-nitro-2-(2′-pyridyl)quinoline-6′-carboxylate (F, 20 mg, 0.050 mmol) in THF-H2O (3.3 mL, 10:1) at 0 °C. The reaction mixture was stirred at 0 °C for 6 minutes. After 6 minutes, the reaction mixture was filtered through Celite. The Celite was washed with EtOAc (25 mL) and concentration of the filtrate afforded the crude aminophenol (G, 16.3 mg, 0.044 mmol) as a brown solid.

Activated manganese dioxide (19.0 mg, 0.22 mmol, 5 equiv) was added to a stirring solution of the crude aminophenol (G, 16.3 mg, 0.044 mmol) in 35% aqueous sulfuric acid (1.5 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 10 minutes. After 10 minutes, the reaction mixture was filtered through Celite. The Celite was washed with H2O (20 mL) and CH2Cl2 (20 mL). The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (2 × 10 mL). The combined organic layers were dried (Na2SO4) and concentrated on a rotary evaporator. Flash chromatography (SiO2, 50% EtOAc/hexanes) provided methyl 7-bromo-5,8-dioxo-2-(2′-pyridyl)quinoline-6′-carboxylate (H, 6.9 mg, 37% over 2 steps) as a yellow solid: 1H NMR (CDCl3, 400 MHz) δ 9.02 (d, J = 8.3 Hz, 1H), 8.91 (dd, J = 7.5, 1.5 Hz, 1H), 8.54 (d, J = 8.3 Hz, 1H), 8.25 (dd, J = 7.5, 1.5 Hz, 1H), 8.05 (t, J = 7.5 Hz, 1H), 7.63 (s, 1H), 4.05 (s, 3H).

A solution of NaN3 (3.0 mg, 0.046 mmol, 1.1 equiv) in H2O (80 mL) was added to a stirring suspension of methyl 7-bromo-5,8-dioxo-2-(2′-pyridyl)quinoline-6′-carboxylate H in THF-H2O (0.4 mL, 4:1) under Argon at room temperature. The reaction mixture was stirred at room temperature for 18 hours. After 18 hours, the reaction mixture was purified by flash chromatography (SiO2, 40%–80% EtOAc/hexanes) to provide methyl 7-azido-5,8-dioxo-2-(2′-pyridyl)quinoline-6′-carboxylate (I, 3.0 mg, 21%) as a brown solid: 1H NMR (CDCl3, 400 MHz) δ 9.00 (d, J = 8.3 Hz, 1H), 8.87 (dd, J = 7.6, 1.5 Hz, 1H), 8.54 (d, J = 8.3 Hz, 1H), 8.24 (dd, J = 7.6, 1.5 Hz, 1H), 8.04 (t, J = 7.6 Hz, 1H), 6.56 (s, 1H), 4.05 (s, 3H) and methyl 7-amino-5,8-dioxo-2-2′-pyridyl)quinoline-6′-carboxylate (J, 7.8 mg, 59%) as a red solid: 1H NMR (CDCl3, 400 MHz) δ 8.93 (d, J = 8.3 Hz, 1H), 8.85 (d, J = 7.8 Hz, 1H), 8.55 (d, J = 8.3 Hz, 1H), 8.21 (d, J = 7.8 Hz, 1H), 8.04 (t, J = 7.8 Hz, 1H), 6.11 (s, 1H), 5.34 (bs, 2H), 4.05 (s, 3H).

To convert remaining azide I to amine J, a solution of PPh3 (2.4 mg, 0.0091 mmol, 1.0 equiv) in anhydrous CH2Cl2 (0.1 mL) was added to a stirring solution of methyl 7-azido-5,8-dioxo-2-(2′-pyridyl)quinoline-6′-carboxylate (I, 3.0 mg, 0.0089 mmol) in dry CH2Cl2 (0.2 mL) under Argon at room temperature. After 10 minutes, the reaction mixture was concentrated and taken up in THF (0.3 mL) and H2O (0.2 mL). HOAc (0.3 mL) was added and the reaction mixture was stirred at room temperature for 12 minutes. Flash chromatography (SiO2, 80% EtOAc/hexanes) and trituration with cold Et2O provided methyl 7-amino-5,8-dioxo-2-2′-pyridyl)quinoline-6′-carboxylate (J, 2.3 mg, 47% over two steps) as a red solid.

A solution of Na2S2O4 (10.8 mg, 0.062 mmol, 1.05 equiv) in H2O (0.5 mL) was added to a stirring suspension of methyl 7-amino-5,8-dioxo-2-2′-pyridyl)quinoline-6′-carboxylate (J, 18.2 mg, 0.059 mmol) in THF (2.5 mL) and H2O (2.0 mL) under Argon at room temperature. After 30 minutes, 1 M aqueous KOH (413 mL, 0.413 mmol, 7.0 equiv) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 1 hour. After 1 hour, the reaction mixture was diluted with H2O (50 mL), acidified with 10% aqueous HCl, and extracted with EtOAc (5 × 50 mL). The combined organic extracts were dried (Na2SO4) and concentrated on a rotary evaporator. The residue was washed with hexane to provide 7-amino-5,8-dioxo-2-(2′-pyridyl)quinoline-6′-carboxylic acid (ML325, 15.1 mg, 87%) as an orange solid: 1H NMR (DMSO, 400 MHz) δ 8.88 (d, J = 8.2 Hz, 1H), 8.71 (t, J = 8.0 Hz, 1H), 8.48 (t, J = 8.0 Hz, 1H), 8.24 (t, J = 8.0 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 6.10 (bs, 2H), 5.91 (s, 1H); purity >95% by NMR. ESI-TOF HRMS m/z 296.0668 (M + H]+, C15H9N3O4 + H+ requires 296.0666).The coupling partners A and B were prepared according to literature precedent as shown in Figure 2 (47, 49).

Figure 2. Preparation of coupling partners A and B.

Figure 2

Preparation of coupling partners A and B.

3. Results

3.1. Dose Response Curves for Probe

IC50 values (see Figure 3) for PAD1 (70 ± 40 nM), PAD2 (200 ± 50 nM), PAD3 (170 ± 10 nM), and PAD4 (240 ± 10 nM) were obtained from in vitro biochemical assays (AID 588486, AID 588438, AID 588471, AID 588484, AID 588472, AID 588490).

Figure 3. IC50 curves for PADs 1–4 with ML325 (SID 118043677) as determined by biochemical substrate assay using the N-α-benzoyl-L-arginine ethyl ester HCl (BAEE) reagent.

Figure 3

IC50 curves for PADs 1–4 with ML325 (SID 118043677) as determined by biochemical substrate assay using the N-α-benzoyl-L-arginine ethyl ester HCl (BAEE) reagent.

3.2. Cellular Activity

The probe ML325 (SID 13891339) and analogs SID 118043674 and SID 118043681 were evaluated for cytotoxicity in NIH-3T3 cells (AID 651887). ML325 had a >15-fold higher CC50 value than either of the other analogs.

3.3. Profiling Assays

To date, ML325 has been tested in 12 bioassays deposited in PubChem (not related to this project), and was not reported to be active in any of them.

Probe ML325 and select analogs were subjected to gel-based competitive ABPP screening using the chloroacetamide-rhodamine (CA-Rh) (51, 52) activity-based probe to assess reactivity against >20 cysteine-reactive proteins in MCF-7 soluble and membrane proteomes visible by one-dimensional SDS-PAGE separation and fluorescent detection. CA-Rh reacts with a variety of enzymes with cysteine nucleophiles, proteins with metal-coordinating cysteine residues, and proteins having reactive cysteines within nucleotide binding domains (51), and has previously been employed for general cysteine-reactive selectivity analysis (52). This medium-throughput proteome-wide screening technique was employed in our medchem optimization of the probe compound, allowing rapid assessment of selectivity, as visualized by disappearance of bands in compound-treated lanes relative to the DMSO-only control. Overall, this analysis revealed no additional off-targets for ML325, even at 50 μM probe concentration, supporting the assertion that it is not a general cysteine-trap and defining a large (>70-fold) selectivity window against all CA-Rh sensitive proteins resolvable by 1D SDS-PAGE.

4. Discussion

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

Pan PAD inhibitors F-amidine and Cl-amidine are mechanism-based inactivators that covalently modify an active-site cysteine in PAD4 that is critical for catalysis (14–16, 35, 38), and they have been important chemical tools in studies of the role of PAD enzymes in inflammatory diseases and cancer biology (10, 15–17, 19, 30, 39, 40, 60–62). Based on the success of these compounds, it would be useful to develop a second structural class of pan PAD probes to prove that the observed effects of F-amidine and Cl-amidine are due to inhibition of PADs, and not another mechanism unique to the halogen-amidine chemotype. Towards this goal, an HTS assay for PAD4 identified streptonigrin as a PAD4-specific inhibitor (42). While this designates streptonigrin as a potential tool for studying PAD4-specific biology, it lacks the pan PAD activity of the halogen-amidines and therefore cannot be used as a direct complementary tool.

In cases where IC50 values are available for comparison, ML325 is the most potent inhibitor of all PADs1–4 in vitro. In a comparison of either IC50 or kinact/KI values for ML325, it was found to be equally potent for all 4 PAD isozymes, with less than 4-fold overall selectivity. This makes ML325 an attractive complementary tool to streptonigrin, F-amidine and Cl-amidine as it is the first pan PAD inhibitor to emerge outside of the halogen-amidine chemotype.

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