O6-methylguanine-DNA methyltransferase (MGMT) （IC50 = 6-9 nM

Lomeguatrib (Compound 10) is an O6-methylguanine methyltransferase (MGMT) inhibitor, with an IC50 of 9 nM in cell-free assay[1] and ∼6 nM in MCF-7 cells. Lomeguatrib (10 μM) considerably improves the growth inhibitory effects of temozolomide in MCF-7 cells (D60=10 μM with Lomeguatrib versus 400 μM without)[2].

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Effect of Lomeguatrib (PaTrin-2) on MGMT activity and temozolomide sensitivity in MCF-7 cells [2]
MCF-7 cells expressed high levels of MGMT (∼1540 fmoles mg−1 total protein). Exposure to Lomeguatrib (PaTrin-2) for 2 h resulted in extensive inactivation of MGMT in MCF-7 cells: the concentration required to inactivate 50% of the MGMT was around 6 nM (Figure 1). The sensitivity of the MCF-7 cells to the growth inhibitory effects of temozolomide was substantially increased by Lomeguatrib (PaTrin-2) . Growth amounting to 60% of control was seen after 400 μM temozolomide alone but following preincubation with 10 μM PaTrin-2, 60% growth occurred at 10 μM temozolomide (Figure 2), indicating a 40-fold increase in sensitivity. PaTrin-2 itself had no growth inhibitory effect.

In MCF-7 xenografts, memeguatrib (20 mg/kg ip) totally inactivates MGMT in less than two hours, but has no discernible effect on tumor growth[2].

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Effect of Lomeguatrib (PaTrin-2) on MGMT activity in host tissues and tumour [2]
Extensive depletion of MGMT activity was seen in all host tissues measured after a single i.p. dose of 20 mg kg−1 PaTrin-2 (Figure 3). Depletion to below the limits of detection occurred in the kidney, while it was to ∼20, ∼35 and ∼40% of pretreatment values in liver, lung and bone marrow, respectively. The nadir was generally between 2 and 8 h and substantial activity (to over 50% of pretreatment levels) had returned by 24 h after dosing. In the MCF-7 xenografts, complete MGMT inactivation was seen between 2 and 8 h, and recovery of levels was only to ∼20% of pretreatment levels by 24 h after dosing. Slower recovery in the xenograft might reflect the relative strength of the human MGMT promoter or that the human protein is more extensively inactivated by PaTrin-2 or its putative metabolites.

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Effect of Lomeguatrib (PaTrin-2) and temozolomide on MCF-7 tumour growth [2]
The median MCF-7 tqt in the vehicle control and PaTrin-2 only groups were ∼21 and ∼17 days, respectively. Neither temozolomide (tqt∼17 days) nor PaTrin-2 alone had any significant effect on xenograft growth. However, the combination of PaTrin-2 and temozolomide resulted in a median tumour quintupling time of ∼43 days representing an increase of ∼22 days). Toxicity, as measured by weight loss, was essentially unaffected by the addition of PaTrin-2 to the temozolomide treatment regimen. At the end of the treatment period, weight loss was ∼5% in both the temozolomide alone and combination groups.

MGMT Assay. [1]
The MGMT assay has been described previously. 45 Briefly, 200 μg of extracted cellular protein from HeLaS3 cells in 200 μL of 70 mM HEPES buffer (with 1 mM dithiothreitol (DTT), 5 mM EDTA, pH 7.8) was incubated at 37 °C with a defined concentration of MGMT inhibitor (added as a DMSO solution). After 30 min an excess of [3H]-methylated DNA (120 000 cpm) was added, and the incubation was continued for an additional 90 min. The reaction was stopped by the addition of 400 μL TCA (13%), and the DNA was hydrolyzed by heating the reaction mixture for 30 min at 98 °C. The precipitated protein was washed three times with 400-μL portions of 5% TCA, solubilized in 0.1 N NaOH, and analyzed by liquid scintillation counting using the cocktail Rotiszint eco plus and a TRI-CARB 2100 TR liquid scintillation analyzer. Enzyme activity was expressed as fmol of [3H]methyl transferred to TCA-insoluble protein material per mg of total cellular protein. Percent inhibition was calculated relative to untreated control samples. Each assay was repeated three times, and IC50 values were determined graphically from plots of percent inhibition vs inhibitor concentration.

MCF-7 cells (a human breast adenocarcinoma cell line) were grown as a monolayer in RPMI medium containing 10% foetal bovine serum, at 37°C in a humidified atmosphere of 5% CO2/95% air. [2]
To determine MGMT inactivation, cells (5 × 10~6) were incubated in the presence of increasing concentrations of Lomeguatrib (PaTrin-2) at 37°C, 5% CO2. After 2 h, cells were pelleted and resuspended in 10 ml PBS. This was repeated three times in order to remove any residual Lomeguatrib (PaTrin-2) . Finally, cells were pelleted and assayed for MGMT activity as previously described. Activity remaining, based on at least three points on the linear part of the protein-dependence curve, was calculated as a percentage of the activity in untreated cells. [2]
To determine toxicity, the MTT [3′ (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] growth inhibition assay, based on the method of Carmichael et al (1987) was employed. Cells (1000 per well) were plated into a 96-well plate and following a 24 h attachment period, Lomeguatrib (PaTrin-2) was added to the cells. After 2 h incubation with Lomeguatrib (PaTrin-2) (10 μM) at 37°C, 5% CO2, increasing doses of temozolomide or vehicle were added and the cells were incubated for a further 4–5 days. At the end of the exposure period, 150 μg MTT was added to each well and plates were incubated for 3 h at 37°C, 5% CO2. The media were removed and the formazan crystals formed in the viable cells were solubilised in 200 μl DMSO. The absorbances at 540 and 690 nm were determined using a Titertek Multiscan ELISA plate reader and growth inhibition calculated as a percentage of the A540–A690 of untreated wells. [2]

Male nude mice (O/Nu: outbred ALPK Nu/Nu) were housed in a sterile environment and allowed free access to food and water. MCF-7 human breast tumour xenograft samples (1–2 mm3) were implanted in the right flank while the mice were under ethrane and halothane anaesthesia and the experiments begun when tumour volumes had reached a suitable size (see below). As MCF-7 tumours are oestrogen receptor positive, they required additional oestrogen for growth. To prepare oestrogen pellets, β-oestradiol (468 mg) was added to 9.7 g silastic and mixed. Curing agent (1.1 g) was added and the mixture spread into three (26 mm × 12 mm × 1 mm) glass formers. These were then incubated at 42°C overnight before being cut into 2 mm × 2 mm × 1 mm cubes (∼2 mg oestradiol per pellet). The pellets were stored at 4°C until insertion subcutaneously at the tail base, simultaneously with the tumour implant and monthly thereafter. Agents were injected i.p. within 15 min of preparation: Temozolomide was injected 1 h after Lomeguatrib (PaTrin-2) or the vehicle control. [2]
To measure MGMT depletion following Lomeguatrib (PaTrin-2), six groups of mice with at least five mice in each group received Lomeguatrib (PaTrin-2) 20 mg kg−1 i.p. as a single dose. At varying times after dosing, animals were terminated by cervical dislocation, and the tissues (xenograft, kidneys, liver and lungs) dissected out and immediately frozen in dry ice. Bone marrow was collected from femora, which were dissected from each mouse. A minimum amount of bone was trimmed from each end and a 21-gauge needle inserted through the epiphyseal cartilage and the bone marrow flushed into an appropriate volume of PBS. Tissue was stored at −70°C until assayed for MGMT activity. [2]
To assess the ability of Lomeguatrib (PaTrin-2) to sensitise human breast tumour xenografts to the tumour growth inhibitory effects of temozolomide, groups of at least six nude mice were treated as follows: the vehicle control group were given corn oil then 20% DMSO in PBS; the temozolomide only group were given corn oil then temozolomide (100 mg kg−1 day−1); the Lomeguatrib (PaTrin-2) only group were given PaTrin-2 (20 mg kg−1 day−1) then DMSO in PBS, and the PaTrin-2 plus temozolomide group were given PaTrin-2 (20 mg kg−1 day−1) then temozolomide (100 mg kg−1 day−1). Drugs or vehicles were administered i.p. once daily for 5 days with a separation of 1 h. Up to 10 and at least six animals were assigned to each group, and mean tumour volume was standardised across the groups at the start of the experiment: thus the control, PaTrin-2, temozolomide and PaTrin-2 plus temozolomide groups had mean tumour volumes of 29.8±7.6 (range 19.0–38.7), 33.2±14.7 (range 16.5–58.7), 35.1±10.9 (range 20.9–52.4) and 30.3±10.0 (range 20.7–44.5) mm3, respectively.

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20 mg/kg; i.p.

Nude mice with human breast tumour xenografts

[1]. Monosaccharide-linked inhibitors of O(6)-methylguanine-DNA methyltransferase (MGMT): synthesis, molecular modeling, and structure-activity relationships. J Med Chem. 2001 Nov 22;44(24):4050-61.

[2]. O6-(4-bromothenyl)guanine reverses temozolomide resistance in human breast tumour MCF-7 cells and xenografts. Br J Cancer. 2005 Nov 14;93(10):1152-6.

A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair. [1]

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Tumour resistance to chemotherapy involving methylating agents such as DTIC (dacarbazine) and temozolomide is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (MGMT). There is considerable interest in improving the efficacy of such O(6)-alkylating chemotherapy by the prior inactivation of MGMT. We have examined the effect of the modified guanine base, O(6)-(4-bromothenyl)guanine (PaTrin-2, Patrin, Lomeguatrib) on MGMT activity and cell or xenograft tumour growth inhibition by temozolomide in the human breast carcinosarcoma cell line, MCF-7. PaTrin-2 effectively inactivated MGMT in MCF-7 cells (IC(50) approximately 6 nM) and in xenografts there was complete inactivation of MGMT within 2 h of dosing (20 mg kg(-1) i.p.) and only slight recovery by 24 h. MGMT inactivation in a range of murine host tissues varied between complete and approximately 60%, with extensive recovery by 24 h. PaTrin-2 (10 microM) substantially increased the growth inhibitory effects of temozolomide in MCF-7 cells (D(60)=10 microM with PaTrin-2 vs 400 microM without). In MCF-7 xenografts, neither temozolomide (100 mg kg(-1) day(-1) for 5 days) nor PaTrin-2 (20 mg kg(-1) day(-1) for 5 days) had any significant effect on tumour growth. In contrast, the PaTrin-2-temozolomide combination produced a substantial tumour growth delay: median tumour quintupling time was increase by 22 days (P<0.005) without any significant increase in toxicity as assessed from animal weight. A PaTrin-2-temozolomide combination may therefore be beneficial in the treatment of human breast cancers. [2]

C10H8BRN5OS

326.17

324.963

C, 36.82; H, 2.47; Br, 24.50; N, 21.47; O, 4.91; S, 9.83

192441-08-0

3025944

Light yellow to yellow solid powder

1.9±0.1 g/cm3

683.8±65.0 °C at 760 mmHg

367.3±34.3 °C

0.0±2.1 mmHg at 25°C

1.797

2.36

2

6

3

18

299

0

BrC1=CSC(COC2=C3N=CNC3=NC(N)=N2)=C1

JUJPKFNFCWJBCX-UHFFFAOYSA-N

InChI=1S/C10H8BrN5OS/c11-5-1-6(18-3-5)2-17-9-7-8(14-4-13-7)15-10(12)16-9/h1,3-4H,2H2,(H3,12,13,14,15,16)

6-[(4-bromothiophen-2-yl)methoxy]-7H-purin-2-amine

PaTrin-2; Lomeguatrib; PaTrin 2; 192441-08-0; PaTrin-2; PaTrin 2; Lomeguatrib [INN]; Lomeguatrib [INN:BAN]; C10H8BrN5OS; 6-[(4-bromothiophen-2-yl)methoxy]-7H-purin-2-amine; PaTrin2; 2-Amino-6-[(4-bromo-2-thienyl)methoxy]-9H-purine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)