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| Recent research has showed that there are possible conserved features to the this pore within other pores in other types of gram-negative bacteria. Proteins include; AidaI of E. coli, BrkA of B. pertussis, Hap of Hemophilus influenzae and IgA protease and App of N. meningitidis. Much of the these proteins show low conservation within their alpha helix's yet they all have a long transversing alpha helix that leads into the 12 sheeted beta barrel. Due to much of the research that is being done within Neisseria meningitidis' NalPβ protein, its crystal structure is being used in order to compare against other autotransporter secreting proteins. | | Recent research has showed that there are possible conserved features to the this pore within other pores in other types of gram-negative bacteria. Proteins include; AidaI of E. coli, BrkA of B. pertussis, Hap of Hemophilus influenzae and IgA protease and App of N. meningitidis. Much of the these proteins show low conservation within their alpha helix's yet they all have a long transversing alpha helix that leads into the 12 sheeted beta barrel. Due to much of the research that is being done within Neisseria meningitidis' NalPβ protein, its crystal structure is being used in order to compare against other autotransporter secreting proteins. |
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| == Experimental Mutation ==
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| <Structure load='3eig' size='500' frame='true' align='right' caption='DHFR methotrexate' scene='Insert optional scene name here' />
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| The features of DHFR ligand binding, specifically to methotrexate can be observed and analyzed through various molecular docking and mutation experiments. The a structurally engineered variant of the <scene name='Sandbox_58/Arg_31-35_norm/1'>native human DHFR</scene> altered the F 31 residue of the protein to R, and the Q 35 residue of the protein to E in an attempt to explore the specifics of the methotrexate affinity for DHFR active site residues, resulting in varied active site residues from phenylalanine and glutamine to <scene name='Sandbox_58/Arg_31_glutamine_35/1'>arginine and glutamate</scene>. This mutated enzyme featured a 650x decrease in affinity for the ligand, methotrexate, but retained an amount of methotrexate interaction similar to the enzyme in its native state with native substrates. Crystal structure analysis revealed that the lack of cooperative action and presence of residue disorder lead to the significant decrease in methotrexate activity with the resulting <scene name='Sandbox_58/3eig_active_site/1'>active site</scene>. The arginine residue at place 31, was specifically observed in numerous conformations, a characteristic unique to the mutated enzyme, and the probable cause of the loss of polar contacts and binding affinity between methotrexate and DHFR. A loss of van der Waal forces due to the conformations of the side chains along with an unfavorable placement of Glu-35 causing an “unfavorable electrostatic contact” with methotrexate’s “glutamate portion.” Interestingly this variant was found to display a greater decrease in methotrexate affinity than the decrease in affinity of Dihydrofolate, found to be 9x, evident of catalytic efficiency retention which hold many drug binding resistance implications<ref>Volpato, J., Yachnin, B., & Blanchet, J. (2009). Multiple conformers in active site of human dihydrofolate reductase F31R/Q35E double mutant suggest structural basis for methotrexate resistance.. Journal Biol. Chem., 284, 20079-20089. </ref>.
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| [[Image:2011-03-10 2241.png|500|left|thumb| Methotrexate Variant Residue Interaction ]]<ref>DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE. (n.d.). RCSB Protein Database. Retrieved March 10, 2011, from www.rcsb.org/pdb/results </ref>
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| == Pharmaceutical Implications ==
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| Methotrexate’s inhibition of cellular replication causes it to have an increased toxic response on cells performing DNA replication especially rapidly proliferating cells. These cells display decreased growth and division due to a lack of nucleoside biosynthesis metabolites, resulting in decreased dTMP. Methotrexate is able to interfere with rapid cell growth in this manner, specifically infecting cells including skin cells, bone marrow cells, and often cancer cells, making methotrexate an effective cancer treatment drug. Other DHF analogs exist which can be useful as anticancer agents or antibacterial agents, through inhibition of DHFR
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| <ref>Methotrexate Information from Drugs.com. (n.d.). Drugs.com | Prescription Drugs - Information, Interactions & Side Effects. Retrieved March 10, 2011, from http://www.drugs.com/methotrexate.html</ref>.
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| == Drug Treatment ==
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| Methotrexate is often used in a regimental approach to the chemotherapeutic treatment of cancers and other diseases involving replicating tissue. A variety of specific cancer types have been treated with methotrexate including head, lung, skin, or breast cancer. Methotrexate has also been used in the treatment of various autoimmune diseases. Rheumatoid arthritis and psoriasis have also utilized methotrexate as a treatment method, presumably to diminish immune function. The mechanism of methotrexate in these instances varies from the inhibition of DHFR, but involves inhibition of enzymes involved with purine metabolism resulting in various types of immune suppression including inhibition of T cell activation. Because of this altered mechanism, treated patients are often administered folate to offset the antifolate characteristics of methotrexate. The targeting of rapidly replicating cells allows methotrexate to function as an abortifacient as well. These uses of methotrexate need to be carefully monitored with proper dosage because methotrexate is embryotoxic, carcinogenic, and teratogenic<ref>Marks, J. W. (2008, January 8). Methotrexate. Medicine Net. Retrieved March 10, 2011, from www.medicinenet.com/methotrexate/article.htm </ref>.
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| Trexall is a drug, methotrexate tablet, used as an antimetabolite for treatment of neoplastic diseases, severe rheumatoid arthritis and psoriasis<ref>Trexall. (2007, November 20). The RX List. Retrieved March 10, 2011, from www.rxlist.com/trexall-drug.htm </ref>.
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| == Treatment ==
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| Methotrexate can be administered orally as well as intravenous, intramuscular, subcutaneous, or intrathecal injection. Dosage amount is a crucial aspect of any methyltrexate treatment because of the serious side affects, and often results in dosages being taken rarely more than once or twice a week. The immune system, blood cells, and other rapidly replicating cells including liver, lungs, kidneys are often succeptable to damage which requires regular tests. Side effects from this drug can be common and sever including neutropenia, hair loss, nausea, dermatitis, and anemia, often representative of the antimetabolite function of methotrexate. Stomatitis is not commonly seen with weekly doses, but daily doses for 5 consecutive days often results in these symptoms including renal impairment, toxicity, and possible failure. Myelosuppression may develop with increased dosages, enhancing tissue damage resulting most commonly from radiation of cancer patients. Additional drugs including antibiotics can often result in adverse side effects, and increased methotrexate retention due to additional drugs can often lead to a dangerous increase in concentration of methotrexate in the blood
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| <ref>Schwartza, S., & Borner, K. (2007). Glucarpidase (Carboxypeptidase G2) Intervention in Adult and Elderly Cancer Patients with Renal Dysfunction and Delayed Methotrexate Elimination After High-Dose Methotrexate Therapy. The Oncologist, 12(11), 1299-1308.</ref>.
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| The nature of this treatment type can requires the “rescue” of a patient, through withdrawal of the inhibitor and possible administering of thymidine or folic acid based drugs to prevent the toxicity sometimes seen in beneficial rapidly replicating cells. Leucovorin is often administered for this rescuing effect. Leucovorin or folinic acid is a derivative of THF, and can be readily converted to tetrahydrofolate overcoming the effect of methotrexate because it bypasses the dihydrofolate reductase mechanism to produce THF<ref>Sirotnak, F., Dorick, D., & Moccio, D. (1978). Murine Tumor ModelsRescue Therapy in the L1210 Leukemia and Sarcoma 180 Optimization of High-Dose Methotrexate with Leucovorin . CANCER RESEARCH, 38, 345-353. Retrieved March 10, 2011, from cancerres.aacrjournals.org/content/38/2/345.full.pdf </ref>.
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| == Pharmacokinetics ==
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| Dosage size of methotrexate is extremely important because of the antimetabolic function of the drug, therefore many pharmacokinetic properties must be considered prior to treatment. Methotrexate is a dicarboxylic acid, although with a pKa of 4.8 and 5.5 is weak and often ionized in physiological conditions. Bioavailability following oral absorption is dose dependent, with 60 percent at doses lower than 30 mg/m2, and at concentrations above 80 mg/m2, there is only 20 percent bioavailability, percentages that can be increased with intramuscular administering of the drug. Only about 5 percent of the total loss of the oral dose is due to bacterial degradation. The kidney, spleen, liver, gallbladder, as well as the skin display the highest levels of methotrexate upon treatment. This drug does not cross the blood brain barrier efficiently, but the distribution to the kidney and liver may be prolonged with higher doses extending drug clearance time. Methotrexate can be metabolized through the liver and intracellular mechanisms, and the kidneys are capable of excreting from 80 to 90 percent of the drug without metabolizing methotrexate<ref>Methotrexate. (2010, September 1). CCO Formulary. Retrieved March 10, 2011, from www.cancercare.on.ca/pdfdrugs/methotre.pdf </ref>.
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| ==Additional Information==
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| For additional information see: [[Pharmaceutical Drugs]]
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| == References ==
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| <references/>
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