Visual representation of how breast cancer affects people around the world.Stress Canada and US.
Cell cycle regulation:Results in abnormal growth of cells, and can be caused by several different biochemical abnormalities.Environmental factors: mainly to factors that results in mutation to the genes.For 4-stagesjust mention:Stages 0-3 is characterized based on size and localization and it is benign.Stage 4 is malignant
Inflammatory = cancer cells blocking of the lymph channels in the breast resulting in inflammation.Paget’s = Cancer cell collects in or aroundthe nipple.
A: DuctsB: LobuleC: Dilated section of ducts to hold milk (Pagets)D: NippleE: Fats
From “Understanding Breast Cancer” – Joy OgdenChemotherapy is usually used in conjunction with surgery.Stresstamoxifen = estrogen analog so it inhibits growth signals by preventing estrogen from binding the site.And herceptin = monoclonal antibody that binds to HER2 (Human Epidermal Receptors) receptors, prevents dimerization. Prevents growth but does not kill cancer cells. IV delivery. Used to treat metastatic breast cancer.Note that a combination of several treatment options may be used.At the end of going through this say why we need to do further research into trying to solve it Because all of this shit isn’t clean. And they have to be used in conjunction with each otherIMMUNOTHERAPY disadvantage: Distribution of malignant cells is heterogeneous.High interstitial pressure within tumourEND WITH:“By looking at successful drugs and their target, we can postulate certain characteristics that are indicative of a good drug target for treating breast cancer”
Talk about how current drugs target only some of them. For example, taxols target microtubules which are highly expressed and involved in cell division. Our drug, however, would target all of themHighly expressed specifically in cancer cells (only) – suggesting that this target is an important factor for cancer progression and thus MAY increase specificity to its inhibition.Cell division – Identify novel inhibitory mechanism in the hopes to design a more specific drug to the target and reduce the side-effects observed in current drug treatments.
“Which has led us to find PAK1 as a target because of its unique mechanism of action that our drug will target”
PAK1 activates cd1 that is over-expressed in 70% of breast tumoursNote that these are only examples of each type of function pak1 can be involved withMolecular hub – refer to how many functions from pak1 (apoptosis, cell proliferation, cell motility) IN CONTEXT WITH CANCER!!!!!!!!!Cell motility involved for metastasisApoptosis, PAK1 turns off this pathway promoting cell survivialCell proliferation which leads to uncontrolled cell growth
Overexpressed – drug will work on many breast tumorsNo drugs – potential to be a novel pathway -> more research/drugs. Preliminary research in to targeting Pak1 for cancer Novel ways – many different mechanisms that can be inhibitedSTARTS WITH:“Further evidence supporting why PAK1 is a good targeting is because….”END WITH:“It can be targeted through different mechanisms which takes advantage of the activation of PAK1”
“PAK1 is initially found in an inactive dimerized form through a trans inhibition between the two monomers”Pak1 in dimerized state, inactive by trans inhibition - P21 GTPases binds allowing conformation change which leads to monomer formationHowever at this stage, Pak 1 not fully activasted, still exists a flexible that sterically blocks kinase domain to provent activityTO be fully activated, PAK1 first undergoes autophosphorylation at T423 which is found in loopFollowing this phosphorylation , subsequent autophosphorylations occur to fully activate this enzyme
We have this target and hypothesized two ways to inhibit its function. We have think if this is feasible and a proof of principle for each approach.
First idea was to prevent monomerization of PAK1.
Autoinhibition targeting in Bcr-Abl which also has an activation loop. Imatinib locks the enzyme in its inactive form by preventing phosphorylation at Asp381. Chronic myelogenous leukemia Bcr-Abl is a human lukemia oncogeneImatinib was one of the first type II kinase inhibitors to be found.
Cytotoxicity: Determine which compounds in our library can kill breast cancers.Kinase Activity: Filter out compounds that do not inhibit PAK1 activity.Dimerization: Going to more specifics to our mechanistic target. “From here we will perform this assay in order to specifically narrow down our hits to Dimerization“After we have our hits from our activity assay, we will figure out the compounds that inhibit PAK1 in our proposed model. Through the dimerization assay, we will select for compounds that fit our proposed models of inhibition.”“At the same time, this assay will eliminate common kinase inhibitors such as ATP analogues which we are uninterested in.” ELISA“however, we would still like to select for compounds that inhibit the phosphorylation at t423. In order to do this, we will be performing an ELISA-based assay.” Cross reactivity:Determine how specific our leads are to PAK1 by testing against other PAK family members as well as other kinasesADME“Following identification of such leads, we will look in to ADME”
MCF-7 – standard breast cancer cell lineMCB MB-134 – carcinoma cell line that overexpressesPAK1Culture cell lines approximately 4-7 days according to standard protocols in Eagle Minimum Essential Media.Partition the cell culture into several 96-well plates with OD of 10,000 cells per well.~ 40 plates a day => 80 compounds per plate => 10 days in total to partition all necessary wells for the 32000 compounds.16 controls per plate.Positive Controls: 20 nM of Taxol need 0.06 mg 1mg = $40 somethingNegative Controls: DMSO Total volume in each well of 180 µL.Add compounds (10 µM) and compare ODs with control to decide which ones go through.Run this experiment in triplicates for the two cells lines.10uM is upper range of drug concentration that can go in to a body (class)taxol 4-7 nm
PAK1 monomers with different fusion proteins ( cerulean/Venus) along with CDC42 in wellsand add in compoundsCompounds that don’t promote dimerization will result in no fluorescence at 528nmCompounds that do promote dimerization will result in larger Venus emission wavelength * if questions are asked…. Cerulean emission peak overlaps Venus excitation wavelength during dimerization… increased energy increases Venus emission signal
FRET based to determine protein-protein interactionNEGATIVE CONTROL: no compounds = monomers of PAK1 = NO SIGNALLLActivity controls : ensure compounds are not interfering with emission spectra
Mobilized PAK1 to well plate through PAK1 specific antibody. Compounds and p21GTPase is added If compound binds to allosteric site and prevents T423 phosphorylation… primary antibody to UNPHOSPHORYLATED T423 will bind.. Secondary antibiody with Horseradish Peroxidase binds to primary and HRP substrate is added leading to positive signalIf compound doesn’t bind, primary antibody cannot bind thus no signal
Cytotoxicity: Determine which compounds in our library can kill breast cancers.Kinase Activity: Filter out compounds that do not inhibit PAK1 activity.Dimerization: Going to more specifics to our mechanistic target. “From here we will perform this assay in order to specifically narrow down our hits to Dimerization“After we have our hits from our activity assay, we will figure out the compounds that inhibit PAK1 in our proposed model. Through the dimerization assay, we will select for compounds that fit our proposed models of inhibition.”“At the same time, this assay will eliminate common kinase inhibitors such as ATP analogues which we are uninterested in.” ELISA“however, we would still like to select for compounds that inhibit the phosphorylation at t423. In order to do this, we will be performing an ELISA-based assay.” Cross reactivity: determining specificity to PAK1 by testing against other PAKS and other kinasesADME“Following identification of such leads, we will look into cross reactivity with other PAK groups and other kinases to determine that our compound is specific to PAK1, and to ADME”
Mouse which is injected with breast cancer cells (MCF-7), allow tumourgenesis to occurr.. Then administer drug (IV or orally)Sacrifice mouse and examine tissue to determine decrease in cancer cells
Herceptin: 20-30% of breast cancers overexpress HER2 receptor $100,000 for treatment per year. PAK1: 50% overexpressed in breast cancerThis is compelling evidence that PAK1 will be as good if not better than targeting HER2By targeting novelMechanism of inhibition of PAK1 this will leads us to a viable potent therapeutic with great potential.
Breast cancer statsHerceptin is one of the most popular treatment of breast cancer, and have a revenue of 2 billion.
Bring you back to thisworldmap to show the global potential that phortune pharmaceuticals can offer in the treatment of breast cancer.
Adapted from Joy Ogden: Understanding Breast Cancer (2004)Only 1 possible factor in each stage is mentioned
Aurora Kinase relation some sort of a mechanism that regulates or gets regulated by aurora kinaseLocalization on the spindle apparatusPresent in almost all stages of the cell cycleCo-localizes with H3 to phosphorylate serine 10 on aurora kinase? - LOOK UP TO MAKE SURE
Fix this slide
Solubility:The shake flask method. Place solute in a flask with octabol and water. Shake the flask and separate out the two solvents and measure the solute in each by SpectroscopypKa
Breast Cancer<br />pHortunepHarmaceuticals<br />Biochem 4H03<br />November 19th, 2010<br />
Breast cancer is the top cancer in women worldwide…<br />
and is increasing particularly in developing countries where the majority of cases are diagnosed in late stages<br />Breast cancer is the top cancer in women worldwide <br />- World Health Organization, 2010<br />
Cancer<br />Loss of cell cycle regulation<br />Caused by environmental or genetic factors<br />4-stage tumor classification<br />Benign or Malignant <br />Ogden, Joy. Understanding Breast Cancer. UK: John Wiley & Sons Ltd, 2004.<br />
Classification of Breast Cancer<br />Non-Invasive<br />Lobular (LCIS)<br />Ductal (DCIS)<br />Invasive<br />Lobular<br />Ductal<br />Inflammatory<br />Paget’s<br />
Breast Cancer Kills<br />Metastatic cells redirect resources from non-cancerous cells<br />Metastasis to vital organs is lethal<br />Ogden, Joy. Understanding Breast Cancer. UK: John Wiley & Sons Ltd, 2004.<br />
Treatment Options<br />Ogden, Joy. Understanding Breast Cancer. UK: John Wiley & Sons Ltd, 2004.<br />
What is our goal<br />Target should be:<br />Highly expressed<br />Involved in cell proliferation<br />Involved in cell motility<br />Anti-apoptotic<br />Identify novel inhibitory mechanism<br />
PAK1 as a Target<br />57% of breast tumoursoverexpressPAK1<br />55% protein overexpression in breast cancer cells<br />Constitutively expressed PAK1 stimulates carcinogenesis in mice.<br />No current drugs on the market<br />Can be targeted through different mechanisms<br />Dummler, B, et al. (2009) Cancer Metastasis Rev.28: 51-63.<br />Kumar, R ., et al. (2006) Nature Reviews Cancer6: 459-471<br />
PAK1<br />PAK1<br />p21<br />P<br />PAK1<br />PAK1<br />P<br />P<br />p21<br />P<br />Activation of PAK1<br />Lei, M., et al. (2000) Cell. 102: 387-397<br />
PAK1<br /> Inhibitors of Monomerization<br />p21<br />T423 Phosphorylation Inhibitors<br />PAK1<br />p21<br />P<br />Our Drug Target<br />
Small Molecules Promote Dimerization<br />Nitric Oxide Synthase (NOS) is an active homodimer<br />Imidazole and 1-phenol imidazole promotes dimerization <br />Given this finding, the promotion of dimerization by small molecules is feasible<br />= imidazole or 1-phenol imidazole<br />NOS<br />NOS<br />Sennequier, N. (1999) J.Biol. Chem. 274, 930-938.<br />
Promoting Autoinhibition<br />im<br />Bcr-Abl<br />Imatinib<br />An inhibitor that binds to the autoregulatory loop of the kinase, Bcr-Abl<br />Imatinibstabilizes the autoinhibitedstate of the protein by interacting with Asp381<br />Found to be highly selective and reasonably effective (Ki = 37 nM)<br />Bcr-Abl<br />active<br />Bcr-Abl<br />Imatinib stabilized autoinhibited inactive confirmation<br />Cheetham, G.M. (2004).Curr. Opin. Struct. Biol. 14, 700–705.<br />
Current Methods of Administration<br />Intravenous<br />Herceptin<br />Oral<br />Tamoxifen<br />Imatinib<br />Kinase inhibitor specific for cancer cells<br />
Conclusion<br />PAK1 is overexpressed more than receptor required for Herceptin<br />Proposed robust strategy and novel mechanism of inhibition of PAK1<br />
$8.7 billion in sales worldwide (2009)<br />Projected $16.5 billion in 2016<br />Herceptin revenue: $4.8 billion (2009)<br />GlobalData. Breast Cancer – Drug Pipeline Analysis & Market Forecasts to 2016<br />Maggon, K. et al. (2008). Global Cancer Market Review: World Best Selling Top 10 anti-Cancer Drugs. Knol publishing. <br />
PAK1 biology - briefly<br />Controls adhesion-induced Rac1 activation and cell spreading by regulating Rac1-b-Pix interaction.<br />Modulates cytoskeleton dynamics and cell mobility at the leading edge through phosphorylation of multiple substrates.<br />During mitosis, PAK1 is recruited to the centrosomes where it becomes activated and phosphorylates Aurora-A and Plk1, both important regulators of mitotic events.<br />Promotes cell proliferation through phosphorylation of c-Raf and MEK<br />PAK protects cells from apoptosis via multiple mechanisms.<br />
PAKs as therapeutic targets<br />Expression of a constitutively active PAK1 mutant increases cell motility, anchorage-independent growth, and invasiveness in MCF-7 breast cancer cells and leads to development of metastatic mammary tumors and other types of breast lesions in a transgenic mouse model.<br />NF1 and NF2 are both pathologically and molecularly distinct diseases, but they both involve mitogenic signaling pathways that are downstream of Ras and impinge upon the regulation of PAK activities.<br />In NF1, PAKs are upstream effectors that result in the full activation of the Ras-MAPK pathway, which is one of many oncogenic signaling cascades.<br />In NF2 patients, loss of Merlin is associated with elevated levels of Rac-GTP accompanied by abnormal PAK1 activation<br />
MCF-7 Culturing Protocol<br />Use Eagle's MEM, supplemented with 10% FBS, 1% penicillin/streptomycin. Can also add non essential amino acids (0.1 mM), Insulin (10ug/mL) and Sodium pyruvate (1mM). Add 10nM estrogen to media for a 3-4x increase in cell numbers. Maintain temperature at 37Â°C in humidified, concentrated CO2 (5%) atmosphere.<br />Once MCF-7 cells reach approximately 90% confluence on plates, remove media and passage cells by rinsing with 1xPBS twice.<br />Add 2-3 mL of warm (37°C) 0.25% Trypsin- 0.53 mM EDTA solution to cells to disperse cell layer. Observe under an inverted microscope. Dispersal should happen between 5 and 15 minutes. If cells are not detaching properly, place flask back in 37°C incubation chamber. Do not incubate for more than 3 minutes or so. Note: Do not agitate the chills during dispersal, either by hitting or shaking the flask. This may cause clumping as the cells detach.<br />Once MCF-7 cell layer is dispersed (3min at 37°C) deactivate Trypsin by adding 5 ml cells/Trypsin-EDTA to 10mL of complete growth medium (see step 1) in sterile tube. Aspirate cells by gently pipetting<br />Centrifuge cells in growth medium for 5 minutes at 125x g-force.<br />Remove trypsin/growth medium suspension from tube.<br />Resuspend pellet (MCF-7 cells) in 10 mL fresh growth medium (see step 1)<br />Plate 1 mL of suspension to each new plate containing 9 mL original growth medium (see step 1), and incubate at 37Â°C in humidified 5% CO2 atmosphere.<br />
Cytotoxicity Assay<br />Culture cell lines approximately 4-7 days according to standard protocols in Eagle Minimum Essential Media.<br />Partition the cell culture into several 96-well plates with OD of 10,000 cells per well.<br />~ 40 plates a day 80 compounds per plate 10 days in total to partition all necessary wells for the 32000 compounds.<br />16 controls per plate.<br />Positive Controls: 20 nM of Taxol need 0.06 mg 1mg = $40 something<br />Negative Controls: DMSO <br />Total volume in each well of 180 µL.<br />Add compounds (10 µM) and compare ODs with control to decide which ones go through.<br />Run this experiment in triplicates for the two cells lines. <br />
Luciferase Assay<br />Add ATP, PAK1, MBP, and cdc42-GTPys + previous hits to a 384-well plates + luciferase + luciferin.<br />Positive Control: 1) ATP + Luciferase + Luciferin. <br />Activity Control: 1) ATP + Luciferase + Luciferin + PAK1 + sphingosine + MBP + drug.<br />Negative Control: 1) Luciferase + Luciferin + no ATP.<br />2 “active controls” per compound. 24 compounds per plate on a 96 well plate. (Assuming 1% hit…14 plates*3 (for triplicates) = 42 plates)<br />
FRET Assay<br />PCR and clone human PAK1 with cerulean and venus into expression vectors (2 vectors each with a tag) and transform E. coli cells with the vectors. <br />Express and purify using Ni column and then cutting the HIS-tag out.<br />With cerulean, add Cdc42-GTP + phosphatase to get monomers. In parallel, do the same with venus-tagged PAK1. <br />Add venus-tagged PAK1 to Cerulean-tagged PAK1 + drug.<br />Excite at 423nm (cerulean will absorb and emit at 475nm) and venus (YFP) absorb at 515nm and will emit at 528nm. <br />Cerulean and Venus work in 5 nm radius; our protein is approximately 4.5 nm radius from N termini from one monomer to C termini of another. <br />Controls<br />1) Two proteins that dimerize together with the cerulean and the venus tag. <br />2) No drug. <br />3) Cerulean fusion protein with drug. Excite at 423 and observe at 475 to make sure that the drug does not affect the abs/ems spectra. <br />4) venus fusion protein with drug. Excite at 515 and observe at 528 to make sure that the drug does not affect the abs/ems spectra.<br />
T423 Phosphorylation Assay<br />Attach Ab specific for PAK1 to a high binding plate, and incubate it with PAK1.<br />Incubate plate with drugs + cdc42-GTPys + ATP.<br />Incubate primary Ab specific to T423, then add secondary Ab for detection.<br />Negative Control: Everything except for ATP. <br />Positive Control: Everything except for drugs.<br />
Drug Localization<br />Low-resolution whole-body autoradiography<br />Routine in preclinical ADME studies, but need high levels of drug accumulation<br />Informs about high-capacity low-affinity binding sites<br />High-resolution autoradiography<br />Using this method, the focus of study can emphasize routes of delivery, adsorption, distribution, metabolism, and excretion<br />Can create a drug homunculus<br />Autoradiography combined with immunocytochemistry (co-localization) can further contribute to the characterization of target cells through simultaneous demonstration of radiolabelled drug and a cancer marker<br />