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| Type | HazMat fee for 500 gram (Estimated) |
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Structure of Methotrexate
CAS No.: 59-05-2
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The BI-3802 was designed by Boehringer Ingelheim and could be obtained free of charge through the Boehringer Ingelheim open innovation portal opnMe.com, associated with its negative control.
Methotrexate, an antifolate compound, is a nonspecific inhibitor of the dihydrofolate reductase(DHFR) of bacteria and cancerous cells as well as normal cells. It forms an inactive ternary complex with DHFR and NADPH and can interfere with the growth of certain cells of the body, especially cells that reproduce quickly, such as cancer cells, bone marrow cells, and skin cells.
Synonyms: Amethopterin; CL14377; NCI-C04671
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Search for reports by entering the product batch number.
Batch number can be found on the product's label following the word 'Batch'.
Search for reports by entering the product batch number.
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Search for reports by entering the product batch number.
Batch number can be found on the product's label following the word 'Batch'.
Search for reports by entering the product batch number.
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Identification of efflux substrates using a riboswitch-based reporter in Pseudomonas aeruginosa
Verónica Urdaneta-Páez ; Randy Hamchand ; Karen Anthony ; Jason Crawford ; Alan G. Sutherland ; Barbara I. Kazmierczak
Abstract: Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention, and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide-sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis. In the first step, a collection of structurally diverse compounds enriched in antifolate drugs was screened for ZTP (5-aminoimidazole-4-carboxamide riboside 5′-triphosphate) riboswitch reporter activity in efflux-deficient P. aeruginosa, allowing us to identify compounds that entered the cell and disrupted the folate pathway. These initial hits were then rescreened using isogenic efflux-proficient bacteria, allowing us to separate efflux substrates from efflux avoiders. We confirmed this categorization by measuring intracellular levels of select compounds in the efflux-deficient and -proficient strain using high-resolution liquid chromatography-mass spectrometry (LC-MS). This simple yet powerful method, optimized for high-throughput screening, enables the discovery of numerous permeable compounds that avoid efflux and paves the way for further refinement of the physicochemical and structural rules governing efflux in this multidrug-resistant Gram-negative pathogen. IMPORTANCE: Treatment of Pseudomonas aeruginosa infections has become increasingly challenging. The development of novel antibiotics against this multidrug-resistant bacterium is a priority, but many drug candidates never achieve effective concentrations in the bacterial cell due to its highly restrictive outer membrane and the action of multiple efflux pumps. Here, we develop a robust and simple reporter system in P. aeruginosa to screen chemical libraries and identify compounds that either enter the cell and remain inside or enter the cell and are exported by efflux systems. This approach enables the development of rules of compound uptake and retention in P. aeruginosa that will lead to more rational design of novel antibiotics.
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Keywords: Pseudomonas aeruginosa ; antimicrobial resistance ; drug efflux
Show More >
| CAS No. : | 59-05-2 |
| Formula : | C20H22N8O5 |
| M.W : | 454.44 |
| SMILES Code : | O=C(O)[C@@H](NC(C1=CC=C(N(CC2=NC3=C(N)N=C(N)N=C3N=C2)C)C=C1)=O)CCC(O)=O |
| Synonyms : |
Amethopterin; CL14377; NCI-C04671
|
| MDL No. : | MFCD00064370 |
| InChI Key : | FBOZXECLQNJBKD-ZDUSSCGKSA-N |
| Pubchem ID : | 126941 |
| GHS Pictogram: |
|
| Signal Word: | Danger |
| Hazard Statements: | H301-H341-H360-H372 |
| Precautionary Statements: | P202-P260-P264-P280-P301+P310-P405 |
| Class: | 6.1 |
| UN#: | 1544 |
| Packing Group: | Ⅲ |
| Target |
|
In Vitro:
| Concentration | Treated Time | Description | References |
| Ramos cells | 20 μM | 72 hours | To investigate the effect of FTCD depletion on methotrexate sensitivity, results showed that FTCD depletion significantly reduced the sensitivity of Ramos cells to methotrexate | Nature. 2018 Jul;559(7715):632-636 |
| HEL cells | 5 μM | 3 days | To investigate the effect of FTCD depletion on methotrexate sensitivity, results showed that FTCD depletion significantly reduced the sensitivity of HEL cells to methotrexate | Nature. 2018 Jul;559(7715):632-636 |
| BHK-CD16a | 10 μM | Used for culturing BHK-CD16a cells to study ADCC activity of CD19 antibodies | Front Immunol. 2022 Aug 31;13:957874. | |
| BT-474 cells | 0.1 µM | 72 h | MTX treatment increases the intracellular concentration of the metabolite AICAR, resulting in AMPK activation. Methotrexate-induced AMPK activation leads to decreased one-carbon metabolism gene expression and cellular proliferation as well as increased global bioenergetic capacity. | Sci Rep. 2020 May 12;10(1):7838. |
| mouse embryonic fibroblasts (MEFs) | 0.1 µM | 72 h | MTX caused a strong increase (~30 fold) in endogenous AICAR levels in breast cancer cells and mouse embryonic fibroblasts (MEFs) | Sci Rep. 2020 May 12;10(1):7838. |
| A549 cells | 1 μM | To investigate the effects of MTX on Th17 cell differentiation, results showed that MTX-treated A549 cells co-cultured with PBMCs significantly increased the frequency of Th17 cells. | EBioMedicine. 2024 Oct;108:105339. | |
| Molt4 cells | 90 nM | To evaluate the synergy between methotrexate and SHIN2, results showed that methotrexate increased sensitivity to SHIN2 | Leukemia. 2021 Feb;35(2):377-388. |
In Vivo:
| Administration | Dosage | Frequency | Description | References |
| C57BL/6 nude mice | Acute lymphoblastic leukemia mouse model | Intraperitoneal injection | 80 mg/kg ginger extract and 5 mg/kg MTX | Five times a week for ginger extract and once a week for MTX, lasting two months | To evaluate the anti-leukemic activity of ginger extract alone or in combination with MTX on ALL mouse models, results showed that combined treatment significantly increased survival rate and reduced damages in brain and liver tissues. | J Cell Mol Med. 2021 May 3;25(13):6148–60 |
| DBA/1J mice | Collagen-induced arthritis (CIA) mouse model | Intraperitoneal injection | 23 mg/kg | Thrice a week for 38 days | To evaluate the effect of MTX on arthritis in CIA mice, the results showed that MTX significantly alleviated arthritis symptoms in CIA mice. | Clin Transl Med. 2022 Dec;12(12):e1148. |
| DBA/1J female mice | Bovine collagen-induced arthritis (CIA) model | Intragastric administration | 0.92 mg/kg | Twice a week for 30 days | To evaluate the therapeutic effect of Methotrexate on arthritis in CIA mice, the results showed that Methotrexate was effective in controlling inflammation but had no obvious benefits in regulating weight loss and mechanical allodynia | Front Pharmacol. 2021 May 28;12:693777 |
| DBA/1 mice | Collagen-induced arthritis (CIA) model | Oral gavage | 2 mg/kg | Every 3 days for 20 days | To evaluate the therapeutic effect of MTX on CIA model mice, the results showed that MTX significantly alleviated arthritis symptoms and footpad swelling. | Front Immunol. 2018 Nov 27;9:2762 |
| SKG mice | RA-ILD model | Intraperitoneal injection | 7.5 mg/kg | Twice weekly for 12 weeks | To investigate the effects of MTX on pulmonary inflammation in the RA-ILD mouse model, results showed that MTX exacerbated pulmonary inflammation, increased immune cell infiltration, and fibrosis. | EBioMedicine. 2024 Oct;108:105339. |
| Mice | NOTCH1-driven mouse primary T-ALL model | Intraperitoneal injection | 10 mg/kg | Administered every 5 days for 4 weeks | To evaluate the synergy between methotrexate and SHIN2, results showed that the combination significantly extended mouse survival | Leukemia. 2021 Feb;35(2):377-388. |
Clinical Trial:
| NCT Number | Conditions | Phases | Recruitment | Completion Date | Locations |
| NCT01404429 | Rheumatoid Arthritis | PHASE4 | COMPLETED | 2025-07-12 | New OPD Block, Rheumatology Cl... More >>inic, Level 3, Chandigarh, 160012, India Less << |
| NCT01314417 | Non-infectious Intermediate an... More >>d Posterior Uveitis Less << | PHASE1|PHASE2 | TERMINATED | 2025-01-13 | National Institutes of Health ... More >>Clinical Center, 9000 Rockville Pike, Bethesda, Maryland, 20892, United States Less << |
| NCT02777736 | Diffuse Large B-cell Lymphoma | PHASE3 | UNKNOWN | 2025-12-19 | University Hospital Brno-Bohun... More >>ice, Brno, 62500, Czech Republic|University Hospital Hradec Králové, Hradec Králové, 50005, Czech Republic|University Hospital Ostrava, Ostrava, 708 52, Czech Republic|University Hospital Pilsen, Pilsen, 304 60, Czech Republic|University Hospital Kralovske Vinohrady, Prague, 100 34, Czech Republic|General University Hospital Prague, Prague, 12808, Czech Republic Less << |
| NCT02076997 | Cancer | COMPLETED | 2019-05-10 | Texas Children's Hospital, Hou... More >>ston, Texas, 77030, United States Less << | |
| NCT01187264 | Psoriasis | PHASE4 | COMPLETED | 2025-09-09 | Postgraduate Institute of Medi... More >>cal Education and Research, Chandigarh, U.T, 160012, India Less << |
| NCT03757364 | Nail Psoriasis | COMPLETED | 2018-09-30 | University of Warmia and Mazur... More >>y, Olsztyn, Warminsko-mazurskie, 10-900, Poland Less << | |
| NCT00006168 | Liver Cirrhosis, Biliary | PHASE3 | COMPLETED | 2025-03-04 | Keck School of Medicine at U.S... More >>.C., Los Angeles, California, 90033, United States|U California Medical Center, San Francisco, California, 94143, United States|Yale University School of Medicine, New Haven, Connecticut, 06520-8019, United States|Emory University School of Medicine, Atlanta, Georgia, 30322, United States|Saint Louis University, St. Louis, Missouri, 63104, United States|University of Nebraska Medical Center, Omaha, Nebraska, 68198-3285, United States|Cleveland Clinic, Cleveland, Ohio, 44195, United States|Oregon Health Sciences University, Portland, Oregon, 97201, United States|Albert Einstein Medical Center, Philadelphia, Pennsylvania, 19141, United States|UT Southwestern Medical Center at Dallas, Dallas, Texas, 75235-9151, United States|Medical College of Virginia, Richmond, Virginia, 23298-0711, United States|University of Washington Medical Center, Seattle, Washington, 98195, United States Less << |
| NCT06289205 | Proliferative Vitreoretinopath... More >>y Less << | PHASE1|PHASE2 | RECRUITING | 2025-05-25 | Sahiwal Medical College & Teac... More >>hing Hospital, Sahiwal, Pakistan, Sahiwal, Punjab, 57000, Pakistan Less << |
| NCT06300333 | Ectopic Pregnancy | NOT_YET_RECRUITING | 2025-03-25 | - | |
| NCT04677868 | Acute GVH Disease | PHASE2 | UNKNOWN | 2025-06-22 | Peking University Institute of... More >> Hematology,Beijing, Beijing, Beijing, 100044, China Less << |
| NCT05315128 | Non-melanoma Skin Cancers | PHASE2 | COMPLETED | 2021-12-06 | Zagazig university, Zagazig, S... More >>harkia, 44519, Egypt Less << |
| NCT05353829 | Rheumatoid Arthritis | PHASE4 | ACTIVE_NOT_RECRUITING | 2026-12-31 | Region S?rmland, Eskilstuna, S... More >>weden|Region Dalarna, Falun, Sweden|Region G?vleborg, G?vle, Sweden|Region Norrbotten, Lule?, Sweden|V?stra G?talandsregionen, Sk?vde, Sweden|Akademiskt specialistcentrum, Stockholm, Sweden|Danderyds sjukhus, Stockholm, Sweden|Karolinska sjukhuset, Stockholm, Sweden|Region V?sternorrland, Sundsvall, Sweden|Region V?sterbotten, Ume?, Sweden|Reumatologisk klinik, V?ster?s, V?ster?s, Sweden|Reumatologsektionen ?rebro universitetssjukhus, ?re, Sweden|Region J?mtland H?rjedalen, ?stersund, Sweden Less << |
| NCT02553018 | Arthritis, Rheumatoid | PHASE3 | COMPLETED | 2025-08-17 | SARAUX, Brest, France|HUDRY, P... More >>aris, France Less << |
| NCT04379219 | Cesarean Scar Pregnancy | PHASE4 | UNKNOWN | 2025-12-20 | Mansoura University, Mansoura,... More >> Dakahliya, Egypt Less << |
| NCT03535519 | Rheumatoid Arthritis | UNKNOWN | 2020-08-31 | Hospital General Universitario... More >> Gregorio Mara?ón, Madrid, 28007, Spain Less << | |
| NCT02248792 | Severe Plaque Type Psoriasis | PHASE4 | UNKNOWN | - | Narayana Med College and Hospi... More >>tal, Nellore, AP, 524003, India Less << |
| NCT05208034 | Ectopic Pregnancy | COMPLETED | 2021-07-30 | Nishtar Medical University Hos... More >>pital, Multān, Pu, 66000, Pakistan Less << | |
| NCT00952016 | Advanced Colorectal Cancer | PHASE2 | TERMINATED | 2025-09-14 | The Royal Marsden NHS Foundati... More >>on Trust, Sutton, Surrey, SM2 5PT, United Kingdom Less << |
| NCT02897011 | Rheumatoid Arthritis|Influenza... More >>|Methotrexate Less << | UNKNOWN | 2025-08-17 | Seoul National Univ. Bundang H... More >>ospital, Bundang, Gyeonggi-do, 463-870, Korea, Republic of|Seoul National University Hospital, Seoul, 110-744, Korea, Republic of|SMG-SNU Boramae Medical Center, Seoul, 156-707, Korea, Republic of Less << | |
| NCT02074319 | Schizophrenia|Schizoaffective ... More >>Disorder|Schizophreniform Disorder Less << | PHASE1 | COMPLETED | 2025-08-15 | Abasi Shaheed Hospital, Karach... More >>i, Sindh, 72000, Pakistan|Civil hospital Karachi, Karachi, Sindh, 72000, Pakistan|Institute of Behavioural Sciences, Karachi, Sindh, 72000, Pakistan|Karwn e Hayat, Karachi, Sindh, 72000, Pakistan Less << |
| NCT00791531 | Malaria | PHASE1 | COMPLETED | 2025-08-09 | Kenya Medical Research Institu... More >>te, Center for Clinical Research, Nairobi, 00200, Kenya Less << |
| NCT06001125 | Arthritis|Arthralgia | PHASE2 | RECRUITING | 2025-11-29 | Cross Cancer Institute, Edmont... More >>on, Canada Less << |
| NCT06649136 | Rheumatoid Arthritis | PHASE4 | RECRUITING | 2026-09-30 | Medical University of Vienna, ... More >>Vienna, 1090, Austria Less << |
| NCT04830878 | Proliferative Vitreoretinopath... More >>y Less << | PHASE1 | WITHDRAWN | 2025-04-24 | Byers Eye Institute at Stanfor... More >>d University and Lucille Packard Children Hospital, Palo Alto, California, 94303, United States Less << |
| NCT00298012 | Spondylarthropathies|Spondylit... More >>is, Ankylosing Less << | PHASE4 | WITHDRAWN | - | Rheumatism Foundation Hospital... More >>, Heinola, Finland|Helsinki University Central Hospital, Helsinki, Finland|Central Finland Central Hospital, Jyv?skyl?, Finland|Kuopio University Hospital, Kuopio, Finland|Tampere University Hospital, Tampere, Finland Less << |
| NCT04482543 | Proliferative Vitreoretinopath... More >>y Less << | PHASE2|PHASE3 | UNKNOWN | 2021-09-01 | Ophthalmic Research Center, Te... More >>hran, Iran, Islamic Republic of Less << |
| NCT02097173 | Psoriasis | PHASE1 | COMPLETED | - | Clinical Trials of Texas, Inc.... More >>, San Antonio, Texas, 78229, United States Less << |
| NCT01860690 | Ectopic Pregnancy | PHASE4 | UNKNOWN | 2025-02-14 | Dr. Zvi Klein, Kefar-saba, Isr... More >>ael Less << |
| NCT04958538 | Acute Graft Versus Host Diseas... More >>e Less << | PHASE2 | UNKNOWN | 2023-01-30 | Peking University Institute of... More >> Hematology,, Beijing, Beijing, 100044, China Less << |
| NCT00243750 | Ankylosing Spondylitis | PHASE2 | UNKNOWN | 2025-11-05 | Charité Campus Benjamin-Frankl... More >>in, Rheumatology, Berlin, 12200, Germany Less << |
| NCT04360785 | Spondyloarthritis | COMPLETED | 2023-02-27 | Hopital Pellegrin, Bordeaux, F... More >>rance|Hopital Ambroise Paré, Boulogne-Billancourt, France|Hopital La Cavale Blanche, Brest, France|CHU Trousseau, Chambray-lès-Tours, France|CH Sud Francilien, Corbeil-Essonnes, France|Hopital Mondor, Créteil, France|Kremlin Bicetre hospital, Le Kremlin-Bicêtre, France|CH Le Mans, Le Mans, France|Hopital Saint Philibert, Lille, France|CHU Nantes, Nantes, France|CHR Orléans, Orléans, France|Hopital Bichat, Paris, France|Hopital Cochin, Paris, France|Hopital la Pitié Salpétrière, Paris, France|Hopital Saint Antoine, Paris, France|CH Pontoise, Pontoise, France|CHU Rouen, Rouen, France Less << | |
| NCT00035074 | Inflammatory Bowel Disease | COMPLETED | - | Childrens Hospital of Philadel... More >>phia, Philadelphia, Pennsylvania, 19104, United States Less << | |
| NCT02715947 | Psoriasis Vulgaris | PHASE2 | UNKNOWN | 2025-12-17 | Xiangya Hospital Central South... More >> University, Changsha, Hunan, 410008, China Less << |
| NCT00194272 | Ectopic Pregnancy | PHASE3 | COMPLETED | 2025-06-07 | University of Southern Califor... More >>nia Women's and Children's Hospital, Los Angeles, California, 90033, United States|University of Miami, Miami, Florida, 33101, United States|University of Pennsylvania Reproductive Research Unit, Philadelphia, Pennsylvania, 19104, United States Less << |
| NCT01393405 | Ulcerative Colitis | PHASE2 | COMPLETED | 2025-04-17 | University of Southern Califor... More >>nia, Los Angeles, California, 90033, United States|University of Colorado, Denver, Colorado, 80045, United States|Shafran Gastroenterology, Winter Park, Florida, 32789, United States|Atlanta Gastroenterology, Atlanta, Georgia, 30342, United States|Northwestern University, Chicago, Illinois, 60611, United States|The University of Chicago, Chicago, Illinois, 60637, United States|Indiana University IU Health, Indianapolis, Indiana, 46202, United States|University of Iowa, Iowa City, Iowa, 52242, United States|University of Kentucky, Lexington, Kentucky, 40536, United States|University of Louisville, Louisville, Kentucky, 40202, United States|University of Maryland, Baltimore, Maryland, 21201, United States|Metropolitan Gastroenterology Group, PC, Chevy Chase Clinical Research, Chevy Chase, Maryland, 20815, United States|Massachusetts General Hospital, Boston, Massachusetts, 02114, United States|Henry Ford Health System, Novi, Michigan, 48377, United States|Minnesota Gastroenterology, Plymouth, Minnesota, 55446, United States|Mayo Clinic, Rochester, Minnesota, 55905, United States|Dartmouth College, Lebanon, New Hampshire, 03756, United States|Mt Sinai School of Medicine, New York, New York, 10029, United States|Asheville Gastroenterology Associates, Asheville, North Carolina, 28801, United States|University of North Carolina, Chapel Hill, North Carolina, 27599, United States|Charlotte Gastroenterology & Hepatology, Charlotte, North Carolina, 28207, United States|Case Western Reserve University, Cleveland, Ohio, 44106, United States|Great Lakes Gastroenterology, Mentor, Ohio, 44060, United States|University of Pennsylvania, Philadelphia, Pennsylvania, 19104, United States|Penn State University, State College, Pennsylvania, 17033, United States|Medical University of South Carolina, Charleston, South Carolina, 29425, United States|Baylor University, Houston, Texas, 77030, United States|University of Utah, Salt Lake City, Utah, 84132, United States|Harborview Medical Center, Seattle, Washington, 98104, United States|University of Washington, Seattle, Washington, 98195, United States|University of Wisconsin, Madison, Wisconsin, 53792, United States Less << |
| NCT03327116 | Atopic Dermatitis | COMPLETED | 2018-12-01 | Hospital das Clínicas of the U... More >>niversity of S?o Paulo Medical School, Department of Dermatology, S?o Paulo, SP, 05403900, Brazil Less << | |
| NCT05054426 | Diffuse Large B Cell Lymphoma|... More >>Central Nervous System Lymphoma Less << | PHASE3 | RECRUITING | 2025-10-08 | Peking union medical college h... More >>ospital, Beijing, Beijing, 100005, China Less << |
| NCT02383901 | Osteosarcoma | COMPLETED | 2025-05-16 | Semmelweis Egyetem II. Sz. Gye... More >>rmekklinika, Budapest, 1094, Hungary|Oslo University Hospital, Oslo, N-0424, Norway|Intytut Matki Dziecka Klinika Chirurgii Onkologicznej Dzieci i M?odzie?y, Warszawa, 01 211, Poland|Sk?ne University Hospital, Lund, 221 85, Sweden|Karolinska University Hospital, Sweden, Stockholm, SE-17176, Sweden Less << | |
| NCT00656591 | Child Mortality | UNKNOWN | 2025-12-08 | International Child Health, IC... More >>H, London, WC1N 1EH, United Kingdom Less << | |
| NCT03531073 | Psoriatic Arthritis | COMPLETED | 2024-12-31 | Oxford University Hospitals NH... More >>S Trust, Oxford, Oxfordshire, OX3 7LD, United Kingdom Less << |
| Bio Calculators | ||||
| Preparing Stock Solutions | ![]() |
1mg | 5mg | 10mg |
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1 mM 5 mM 10 mM |
2.20mL 0.44mL 0.22mL |
11.00mL 2.20mL 1.10mL |
22.01mL 4.40mL 2.20mL |
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