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{{Template:Sandbox Reserved Lynmarie Thompson}}
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=='''Catechol-O-Methyltransferase'''==
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=='''Fibroblast Growth Factor Receptor/Ponatinib (4uxq) <ref name="one">PMID: 25465127</ref>'''==
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by Julie Boshar, Emily Boyle, Nicole Kirby, Cory Thomas, Connor Walsh
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===Introduction===
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[[Student Projects for UMass Chemistry 423 Spring 2016]]
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<Structure load='3bwm' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
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<StructureSection load='4uxq' size='350' side='right' caption='The Ponatinib-FGFR complex is highly effective for treating CML ([[4uxq]])' scene=''>
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Catechol-O-Methyltransferase (COMT) is an enzyme, which can be either soluble or membrane-bound, that is responsible for the degradation of catecholamine neurotransmitters. This inactivation is accomplished by transferring a methyl group from S-adenosyl methionine (SAM) to the catecholamine, seen <scene name='Sandbox_Reserved_425/Sam-catecholamine_interaction/7'>here</scene>. In this scene, the catecholamine 3,5-dinitrocatechol is represented in blue and SAM is represented in yellow and red, with the red indicating the methyl group being transferred.
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==Introduction==
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One neurotransmitter in this catecholamine family targeted by COMT is dopamine, the neurotransmitter most closely associated with Parkinson's disease. Parkinson's Disease arises out of a lack of dopamine and is characterized by uncontrollable tremors, muscular rigidity, postural instability. At a functional synapse, the action potential prompts release of neurotransmitters like dopamine at the synapse. These neurotransmitters bind to receptors on the postsynaptic membrane, perpetuating the signal. Once the signal has been transmitted, the neurotransmitters are removed from the synapse via reuptake or degradation by enzymes such as COMT. In a person with Parkinson's Disease, dopamine levels are often too low to adequately continue the message to the next neuron. The disease is currently treated with L-DOPA, a dopamine precursor that is converted to dopamine within the brain. However the bioavailability and stability of L-DOPA when used alone is limited. COMT is being investigated as a target for therapeutic agents that would increase the efficacy of L-DOPA. Inhibition of COMT would prevent inactivation of dopamine, leaving higher levels of active dopamine at the synapse and increasing the likelihood of perpetuation of the message to the postsynaptic neuron.
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Potatinib was developed as a treatment option for chronic myeloid leukemia (CML) as other inhibitors in treatment have become ineffective. BCR-ABL is a kinase with a cancerous genetic mutation in chromosome 22 that leaves it always active. Further mutations in BCR-ABL have left earlier drugs that inhibit tyrosine kinases unable to bind in almost 30% of cases after five years of treatment. The newer, mutant BCR-ABL kinase’s ability to develop new resistances has pushed for newer developments in inhibitors, such as Potatinib<ref name="seven">PMID: 21118377 </ref>.
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===Overall Structure===
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Fibroblast growth factor (FGFR) signaling is the factor that normally activates the BCR-ABL kinase. Also, it is the protein behind both tissue development and repair, the disruption of FGFR leads to tumor growth. The activation of BCR-ABL happens through a series of cascading signals that induce proliferation and migration in cells. Mutations in the regulation of the FGFR tyrosine kinases can be diresctly correlated to malignant tumor growth<ref name="one" />. The tyrosine kinase inhibitor Ponatinib has been used to <scene name='48/483882/Activation_loop/1'>bind</scene> to the mutant version of kinase BCR-ABL by the enzyme's specific "DFG-out" conformation (in <font color='turquoise'><b>turquoise</b></font>). This conformation has the phenylalanine group of BCR-ABL flipped out of its hydrophobic binding site. Ponatinib is the first of its kind to be able to inhibit this specific mutation in BCR-ABL of the "DGF-out" conformation<ref name="seven">PMID: 21118377 </ref>.
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<Structure load='2zvj' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
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The Catechol-O-Methyltransferase complex, 4PCM, with a coumarine inhibitor is a monomer that is made up of eight <scene name='Sandbox_Reserved_425/Beta_sheets/1'>Beta Sheets</scene>. These sheets are all parallel except for one. The monomer is also comprised of <scene name='Sandbox_Reserved_425/Alpha_helices/1'>eight</scene> alpha helices. The beta sheets are on the inside of the complex where the alpha helices are on the outside and enclose the beta strands. The 4PCM complex also has <scene name='Sandbox_Reserved_425/Polar-non_polar_residues/1'>polar</scene> and non polar residues on the outside of the complex on the alpha helices. This is because this 4PCM complex can be both trans membrane protein or a peripheral membrane.
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Ponatinib's harmful side effects have caused it to fall under scrutiny from the U.S. Food and Drug Administration (FDA). It has shown to increase chances of deadly blood clotting and restenosis in both arteries and veins with a rate of about 1 in 5 patients. The drug has also shown to increase risk of heart attack and overall worsening of heart disease in patients<ref name="seven" />.
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Catechol-O-Methyl Transferase in complex with an inhibitor, COMT, can normally form a <scene name='Sandbox_Reserved_425/Dimer_2cl5/1'>dimer</scene>. For previous inhibitors that have been used and studied this is true. This dimer can be formed because the ligand that is attached which in this case in a bacterial inhibitor,BIE, allows the complex to be structurally flexible. The dimer that forms creates a <scene name='Sandbox_Reserved_425/Dimer_pocket/1'>Pocket</scene> so that the two monomers fit together nicely and have flexibility. In this pocket the S-Adenosyl Methionine, <scene name='Sandbox_Reserved_425/Dimer_bonds_length/1'>SAM</scene>, donates a methyl group to each of the ligand present that have the magnesium ion attached. The distance between the SAM molecules and the ligand are almost exactly equal distances apart from each other creating an even spacing pocket to form. Our coumarine ligand does not allow for such a convenient <scene name='Sandbox_Reserved_425/Monomer_sam_methyl_transfer/1'>distance</scene> between the SAM molecule and ligand to form when bound together. Even though are complex is more restricted it is the first ligand complex to be studied that does not have a toxicity issue.
 
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==Overall Structure==
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In terms of <scene name='48/483882/Secondary_structure/2'>secondary structure</scene>, FGFR in complex with Ponatinib consists of two domains, which is the characteristic structure exhibited by kinases. The N-terminal domain is the smaller of the two, and it contains a five-stranded beta sheet and an alpha carbon helix. The larger C-terminal domain is primarily alpha helical in structure. The alpha helices are shown in <font color='fuchsia'><b>fuchsia</b></font> and the beta strands are shown in <font color='orange'><b>orange</b></font>. A hinge links the two regions. A network of hydrogen-bonds between three conserved residues – Glu551, Asn535, and Lys627 – exists in the hinge region. This hydrogen-bonding controls the kinase activity of FGFR.
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In its active form, FGFR is dimerized and contains two activated intracellular substrates. The binding of a coreceptor, β-Klotho, stabilizes the activated complex. A DFG moiety is found in BCR-ABL, the conformation of which plays a key role in binding Ponatinib. Another defining feature of active FGFR is its <scene name='48/483882/Hydrophobic_spine/1'>hydrophobic spine</scene>. Four residues in the spine – Leu536, Met524, His610, and Phe631 (in <font color='orange'><b>orange</b></font>) – are highly conserved. A gatekeeper residue is present at the beginning of the hinge, and interactions among the four hydrophobic spine residues link the gatekeeper to Tyr643 in the activation loop. This activation loop is glycine-rich and found in the kinase domain of FGFR<ref name="one" />.
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The structure of Ponatinib is shown as follows:
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[[Image:StructureP.PNG]]
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==Binding Interactions==
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Kinases are the largest drug targets being tested. All kinases possess a biolobal fold that connects the N and C termini by a “hinge” that binds ATP. “Gatekeepers” are other residues that are in the hinge and alter binding capabilities. Mutations to gatekeepers are critical considerations in drug development because they can result in drug resistances<ref name="four">PMID: 25317566</ref>. Ponatinib can bind even in the presence of gatekeeper mutations, like T315I which accounts for 15-20% of all clinically observed mutations and is resistant to all previous generation drugs<ref name="five">PMID: 25219510</ref>. This class of inhibitors can bind deep within the hydrophobic and allosteric pocket that is only accessible in the <scene name='48/483882/Active_sitezoom/1'>DFG-out</scene> conformation (see color chart) which consists of Asp630, Phe631, and Glu632<ref name="four" />.
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===Binding Interactions===
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<center><big>{{Template:ColorKey_N52C3Rainbow}}.</big></center>
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<Structure load='2zvj' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
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Met 40, Leu 198, and Tyr 200 define the <scene name='Sandbox_Reserved_425/Pocket/2'>pocket</scene> for the 4-phenyl-7, 8-dihydroxycoumarine (4PCM) ligand binding site. This also allows us to see how, unlike the ligand described in the Overall Structure section, 4PCM is sterically constrained and unable to form necessary interactions for a dimer configuration. Trp 38 and Pro 174 make <scene name='Sandbox_Reserved_425/Vanderwaals/2'>Van der Waals</scene> interactions with the 4PCM. The magnesium ion interacts with the two <scene name='Sandbox_Reserved_425/Hydroxyl_groups/1'>hydroxyl groups</scene> of the 4PCM. The magnesium ion also aids in the protonation of <scene name='Sandbox_Reserved_425/Lys_144/1'>Lys 144</scene>, causing an electrostatic interaction with a hydroxyl group of 4PCM. The other end of this Lys 144 then acts a hydrogen bond donor for a water molecule in the binding pocket. This water molecule then acts a hydrogen bond donor for the carbonyl group of 4PCM, creating an interesting network of hydrogen bonds. The above interactions stabilize the ligand in the binding pocket. These interactions are somewhat similar to other inhibitors previously used, however some differences do occur that make past inhibitors more stable. In our 4PCM, we can see a bond angle between the sulfur and carbon atoms of <scene name='Sandbox_Reserved_425/Met_40/1'>Met 40</scene> to be significantly less than that of the Met 40 in a COMT complex using <scene name='Sandbox_Reserved_425/3_5-dinitrocatchetol/1'>3,5-dinitrocatchetol</scene> as an inhibitor instead; the change is about 96o. This interaction, as well as the constraining effects of Trp 38 and Pro 174 interactions, actually make 4PCM less stable than nitrocatchetol inhibitors currently being used to treat PD. However, nitrocatchetol inhibitors act as uncouplers, making 4PCM side effects less complex and more attractive.
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Ponatinib binds to the ATP binding pocket between the N and C lobes to shift from the DFG-in to the DFG-out conformation. It spans from the <scene name='48/483882/Hinge/1'>hinge</scene> (<font color='cyan'><b>cyan</b></font>) to the front catalytic pocket. Three sites are engaged in the ATP binding cleft by ponatinib’s aromatic rings. First, imidazo[1,2b]pyridazine occupies the same space as the adenine ring of ATP and forms a <scene name='48/483882/Hingehbond/1'>hydrogen bond</scene> with the amide nitrogen atom of Ala553 (<font color='magenta'><b>magenta</b></font>) in the hinge<ref name="seven" />. Multiple triple bonds help the rest of ponatinib to move further in to the ATP binding pocket. Second, the methylphenyl group displaces the side chain of Lys503 and its aromatic ring binds to the hydrophobic pocket that is formed by Val550 (the <scene name='48/483882/Gatekeeper/1'>gatekeeper</scene> in <font color='yellow'><b>yellow</b></font>), and Met524. This allows Glu520 to hydrogen bond with the amide linkage between the aromatic rings in ponatinib. Third, Phe631 is replaced by ponatinib’s 3-trifluoromethylphenyl group. Asp630 becomes available for hydrogen bonding with the amide linkage between ponatinib’s aromatic rings and lets the piperazine ring hydrogen bond with the catalytic loop which forms the DFG-out conformation<ref name="seven" />.
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==Additional Features==
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Ponatinib is an orally ingested tyrosine kinase inhibitor that has revealed successful avenues of treatment for counteracting the effects of angiogenesis in tumor growth. Besides the inhibition of FGFRs, this agent inhibits tyrosine kinases involved in vascular endothelial growth factor receptors. Ponatinib is considered a third generation TKI that can treat even the most drug-therapy resistant mutations that previous TKIs were incapable of treating<ref name="eight">PMID: 23986642</ref>.
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The brand name for ponatinib is Iclusig. Iclusig received an accelerated approval grant through the Food and Drug Administration. It was mainly prescribed to patients suffering from Chronic Myeloid Leukemia or Acute Lymphoblastic Leukemia who did not make any progress with the first and second generation TKIs. However, the clinical trials data displayed a spike in adverse effects. These consequences include heart failure, stroke, coronary artery disease, loss of blood flow to body parts leading to amputation amongst other narrowing of blood vessels<ref>FDA Drug Safety Communication: FDA investigating leukemia drug Iclusig (ponatinib) after increased reports of serious blood clots in arteries and veins; Drug Safety and Availability; United States Food and Drug Administration (2013). Web. [http://www.fda.gov/Drugs/DrugSafety/ucm370945.htm]</ref>.
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===Additional Features===
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FGFR-4 is abundantly present in human prostate cancer observed in vitro and in mouse model simulations<ref name="nine">PMID: 22573348</ref>. A <scene name='48/483882/Variant/9'>variant</scene> of FGFR-4 with Arg388 replacing Gly388 is implicated with increased human prostate cancer. This variation causes increased receptor stability and activation<ref name="ten">PMID:18670643</ref>. A study revealed that the <scene name='48/483882/Inhibition/1'>inhibition</scene> of FGFR-4 signaling completely curtailed prostate cancer cell lines that were responsible for tumor growth<ref name="nine">PMID: 22573348</ref>. Due to the significant results of diminished cell growth in treated tumors, targeting fibroblast growth factor signaling appears to provide a promising step towards combating aggressive prostate cancer.
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==Quiz Question 1==
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In addition to variation in the gene itself, the COMT gene is also associated with 22q11.2 deletion syndrome, which is characterized by an approximately 3 million base pair deletion on the long arm of chromosome 22 near the q11.2 locus. Deletion of this segment results in the loss of 30 to 40 genes, including the COMT gene, which leads to abnormal regulation of catechol-O-methyltranferase in the brain. Affected persons have only one copy of the gene in each somatic cell and may have symptoms that include heart defects, cleft palate, facial feature differences, recurrent infections, autoimmune disorders, kidney abnormalities, low blood calcium, seizures, thrombocytopenia, feeding difficulties, skeletal differences, hearing loss, learning disabilities, schizophrenia, depression, anxiety, bipolar disorder, ADHD, and autism. Deletion of COMT may explain the increased risk of mental problems, which may result from changes in dopamine concentrations.
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Ponatinib is unique in it's ability to bind to the mutated BCR-ABL because of it's preference to shift to the DFG-out conformation. In theory, if a competitive inhibitor was created by nature to prevent Ponatinib from binding to BCR-ABL to further its drug resistance, what specific structural characteristics would the inhibitor need to possess? Consider the unique binding methods of Ponatinib and the <scene name='48/483882/Active_sitezoom/1'>DFG-out</scene> conformation.
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a. Small, fully conjugated aromatic system with no electronegative substituents, to prevent unwanted hydrogen bonding.
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Variations in the COMT gene such as the Val158Met single-nucleotide polymorphism may increase the risk for mental problems. This mutation is a gain-of-function mutation that allows COMT to catabolize dopamine at up to four times the rate of the normal variant, resulting in lower levels of dopamine and reduced stimulation of post-synaptic neurons. This variant is associated with an increased risk for schizophrenia, difficulties in emotional processing, and has been shown to affect executive functions such as set shifting, response inhibition, abstract thought, and acquisition of rules or task structure.
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b. Multiple ring system, one ring particularly for hydrogen bonding and another capable of binding in a hydrophobic pocket.
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Inhibition of COMT increases the lifetime of dopamine and is therefore an attractive drug target for diseases involving low dopamine levels. Until recently, inhibitors were all of the nitrocatechol structure and included such drugs as entacapone, carbidopa, and benserazide. These nitrocatechol drugs may inhibit oxidative phosphorylation in mitochondria by allowing oxidation to proceed without the production of ATP, thereby uncoupling mitochondria energy production. Newer classes of drugs are hoped to be more effective by being able to inhibit COMT without the risk of uncoupling.
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c. Polymer chain with an ester linkage and a hydroxyl end group .
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===Credits===
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d. Metal center that binds four large, nonpolar hydrocarbon ligands that exhibit significant steric hindrance.
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Introduction - Jessica Royal
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==See Also==
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*[[Fibroblast growth factor receptor]]
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*[[4uxq]]
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*[[4v01]]
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*[[4v04]]
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*[[4v05]]
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Overall Structure - Stephanie Bristol
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==Credits==
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Drug Binding Site - Emily Brackett
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Introduction - Emily & Cory*
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Additional Features - Anh Huynh
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Overall Structure - Nicole* & Connor
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===References===
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Drug Binding Site - Julie* & Cory
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Additional Features - Connor* & Nicole
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Quiz Question 1 - Julie & Emily*
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==References==
<references/>
<references/>
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Tsuji, E., Okazaki, K., & Takeda, K. (2009). Crystal structures of rat catechol-O-methyltransferase complexed with coumarine-based inhibitor. Biochemical and Biophysical Research Communications, 378(3), 494-497.
 

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This Sandbox is Reserved from January 19, 2016, through August 31, 2016 for use for Proteopedia Team Projects by the class Chemistry 423 Biochemistry for Chemists taught by Lynmarie K Thompson at University of Massachusetts Amherst, USA. This reservation includes Sandbox Reserved 425 through Sandbox Reserved 439.


Fibroblast Growth Factor Receptor/Ponatinib (4uxq) [1]

by Julie Boshar, Emily Boyle, Nicole Kirby, Cory Thomas, Connor Walsh

Student Projects for UMass Chemistry 423 Spring 2016

The Ponatinib-FGFR complex is highly effective for treating CML (4uxq)

Drag the structure with the mouse to rotate
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