Molecular Medicine: Gleevec and Chronic Myelogenous Leukemia

Transcription

1 Molecular Medicine: Gleevec and Chronic Myelogenous Leukemia Dec 14 & 19, 2006 Prof. Erin Shea Today w e are going to talk about the dev elopment of a remarkable drug called G leev ec w hich is used to treat a dev astating form of cancer called chronic my elogenous leukemia, or C ML. A s y ou w ill see, this drug represents a new and improv ed w ay of treating cancer. A dditionally, the mechanism by w hich the drug w orks is fundamentally different from the w ay in w hich y ou learned that most drugs, like IV protease inhibitor, function. 1

2 Cancer, Kinases and Gleevec: 1. What is CML? a. Blood cell maturation b. Philadelphia Chromosome c. Bcr and Abl 2. Protein kinases a. Structure b. Mechanism of catalysis c. Regulation 3. Kinases important in CML a. Src Family b. Regulation of Src family kinases c. Abl and its regulation d. Bcr-Abl and misregulation e. Gleevec structure, selectivity, and mechanism of action f. Le Chatelier s Principle g. Gleevec resistance Lecture Readings Alberts p McMurry p

3 Maturation of Blood Cells Red blood cells carry oxygen White blood cells fight infection Stem cells (Leukemia is characterized by aberrant white blood cell proliferation) Platelets are involved in blood clotting There are many different ty pes of blood cells that form in the bone marrow, w hich contains special cells called stem cells. These stem cells giv e rise to red blood cells, w hich carry oxy gen, a v ariety of w hite blood cell ty pes, w hich are inv olv ed in fighting infection, and platelets, w hich are inv olv ed in blood clotting. Leukemia is a disorder inv olv ing excessiv e production of w hite blood cells. We w ill focus on a ty pe of leukemia, C hronic My elogenous Leukemia (C ML), inv olv ing uncontrolled proliferation of w hite blood cells. C ML is a disease that affects about 8,000 people a y ear in the United States. Patients initially present w ith sy mptoms that include fatigue and a v ery high w hite blood cell count w ith no ev idence of v iral or bacterial infection (common causes of high w hite blood cell lev els). The definitiv e diagnosis is based on the identification of a genetic abnormality, bcr-abl, w hich w e w ill talk about later. A s the disease progresses patients become anemic and prone to infections because their blood v olume is completely filled w ith immature w hite blood cells. There are not enough red cells to carry oxy gen and there are not enough macrophages to fight off common microbes. 3

4 Gleevec Until the dev elopment of Gleev ec, show n on this slide, the prognosis for patients w ith C ML w as poor. In order to understand the importance of Gleev ec in the history of drug discov ery, it is useful to consider w hat options w ere av ailable before Gleev ec. 4

5 Chemotherapeutic Agents 2 2 Methotrexate (blocks DA replication) Taxol (blocks mitosis) Camptosar (blocks DA replication) 5-FU (blocks DA replication) F Gleevec Before G leev ec, traditional treatments for cancer relied on the concept that tumor cells grow more rapidly than normal cells. The strategy for treating cancer w as, therefore, to try to block proliferation of cells. With the exception of Gleev ec, all the compounds show n on this slide block key steps in the cell div ision cy cle. F or example, dihy drofolate reductase inhibitors such as methotrexate block thy midine biosy nthesis, thus prev enting DA replication (so cells cannot proceed through S phase). 5-fluorouracil (5-F U) also inhibits DA replication, both by blocking the formation of thy midine nucleotides (5-F U inhibits a key enzy me inv olv ed in thy midine bisosy nthesis) and by interfering w ith correct DA sy nthesis (5-F U nucleotides are incorporated into the grow ing DA molecule). 5-F U can also interfere w ith RA sy nthesis. Like 5-F U and methotrexate, C amptosar prev ents cells from proceeding through S phase. C amptosar prev ents progression through S phase by inhibiting topoisomerase II (Topo II), an enzy me that facilitates DA unw inding to enable replication. F inally, taxol stabilizes microtubules, w hich causes mitotic arrest. Blocks in the cell cy cle lead to apoptosis, or programmed cell death. A lthough many more cancer cells die than normal cells because the cancer cells div ide more rapidly, all these chemotherapeutic agents w ill kill any normal cells that happen to be div iding. Therefore, they are v ery toxic, and people on cancer chemotherapy get quite sick from the treatment itself. A s y ou w ill learn, Gleev ec is different. Gleev ec is one of the first mechanism-based anticancer drugs. It inhibits the enzy me - a protein kinase - that causes unchecked proliferation in the first place. Most cell ty pes in the body don t need this protein kinase and so the drug is relativ ely non-toxic. 5

6 Growth Factors Regulate Growth and Proliferation of ormal Cells Growth factor ormal cells CML white blood cells Growth and proliferation Growth and proliferation Growth factor-dependent proliferation Growth factor-independent proliferation We hav e talked about signaling cascades that control grow th and proliferation of normal cells. Grow th factors circulating in the blood at low lev els are detected by cells v ia membrane receptors; binding of grow th factors initiates a signaling cascade, inv olv ing the activ ation of protein kinases w ithin the cell, ultimately resulting in changes in gene expression and other cell properties that tell cells to grow and div ide. The w hite blood cells in C ML are not responsiv e to extracellular cues that w ould normally regulate their grow th and div ision. Instead, they are stuck in an activ ated state because a particular kinase that should be regulated by extracellular signals is alw ay s on (activ e). The kinase that is permanently activ e in C ML leads to unregulated cell grow th and proliferation. The unregulated state of this kinase results from a genetic abnormality called the Philadelphia chromosome. 6

7 A Chromosomal Translocation Causes CML c-bcr Ph 1 The Philadelphia chromosome encodes a fusion protein, Bcr-Abl bcr-abl c-abl q+ 22q- The Philadelphia chromosome is a truncated form of chromosome 22 in w hich a gene rearrangement has occurred. The bottom part of chromosome 9 exchanges w ith the bottom part of chromosome 22, resulting in a longer than normal chromosome 9 containing genes from chromosome 22 (9q+) and a shorter than normal chromosome 22 containing genes from chromosome 9 (22q-). (Using a microscope, it is easy to see the difference betw een normal chromosomes and the defectiv e chromosomes in a C ML patient based on length.) The translocation ev ent joins parts of tw o genes, abl and bcr, to produce a new gene called bcr-abl. This new gene encodes a fusion protein called Bcr-A bl that has unregulated A bl activ ity. This translocation does not occur in the germ cells - rather, it occurs in a single bone marrow stem cell and is passed on to all of the descendents of that stem cell. n the next slides, w e w ill learn more about how the bcr-abl translocation causes cells to become cancerous. 7

8 What do Bcr and Abl do? Ph 1 22q- bcr-abl Bcr (breakpoint cluster region) has a serine/threonine kinase, GEF, and GTPaseactivating domains Abl - tyrosine kinase important in control of cell growth Bcr-Abl - constitutively active tyrosine kinase (misregulated Abl activity) The abl gene found normally on chromosome 9 encodes a ty rosine kinase that is important in cell grow th. The activ ity of this ty rosine kinase is tightly controlled in normal cells. In contrast, the Bcr-A bl fusion protein has unregulated (constitutiv e) high activ ity that causes cells to proliferate ev en in the absence of extracellular cues. 8

9 Protein Kinases Share a Conserved Catalytic Domain Src ErbB EGF receptor Conserved regions glycine-rich loop catalytic lysine aspartic acid There are ov er 500 protein kinases encoded by the human genome. We know this because w e can recognize protein kinases based on common sequence features. Protein kinases in eukary otic cells share a cataly tic domain of ~250 amino acids (show n in green) containing sev eral highly conserv ed sequences (gly cine-rich loop, cataly tic ly sine, aspartic acid). The sequences of protein kinase family members frequently div erge outside the cataly tic domain. Some of these div ergent sequences enable each kinase to recognize the specific set of proteins it phosphory lates, or to bind to structures that localize it in specific regions of the cell. The cataly tic domain of protein kinases folds into a conserv ed structure. This structure positions the conserv ed sequences to form the substrate binding and activ e sites of the enzy me w hich are crucial for phosphate transfer. To understand more about the function of protein kinases and how they become misregulated, in the next few slides w e w ill look at the conserv ed sequences and structure of protein kinases. 9

10 The Catalytic Domains of Protein Kinases ave Similar Structures Small lobe () (phosphate binding loop) Large lobe (C) (catalytic loop - phosphate transfer) Cleft (peptide and ATP binding) The conserv ed cataly tic domain of protein kinases consists of tw o lobes: a smaller - terminal lobe comprised primarily of beta-sheet structure and a larger C -terminal lobe that is primarily alpha-helical. The -terminal lobe contains a conserv ed phosphate binding loop, w hich positions the terminal phosphate of A TP for transfer. The C -terminal domain, also contains a conserv ed loop that is important for cataly zing phosphate transfer to the hy droxy l of a peptide side chain. Both substrates of the kinase, the A TP and the peptide, are bound in the cleft betw een the tw o lobes. The function of the kinase depends on its ability to alter its structure subtly - to open the cleft betw een the large and small lobes so that the substrates can gain access. 10

11 Conserved Residues in Protein Kinases ATP Binding Anchors small lobe to large lobe Catalytic Base & Mg binding Structural Stability Gly 50 Gly 52 Gly 55 Lys 72 Glu 91 Phe 100 Tyr 101 Asp 166 Asn 171 Asp 184 Glu 208 Asp 220 Arg 280 The boundaries of the cataly tic core w ere defined initially on the basis of the sequence similarities shared by all members of the protein kinase family. Throughout this cataly tic core are scattered more than a dozen residues that are nearly inv ariant in ev ery protein kinase. These residues serv e as fixed points that must be structurally aligned in a similar manner in all of the protein kinases. A lthough they are far apart in primary sequence, many are in close proximity to one another in the folded structure. A n analogy is seen in the serine proteases in w hich the residues of the cataly tic triad, is-a sp-ser, are distant in primary sequence but are in close proximity in the folded structure and form part of the activ e site. If one considers the common functions shared by all protein kinases, it is reasonable to assume that most of the conserv ed residues are associated w ith those shared functions. The basic shared functions common to all protein kinases are M g-a TP binding and the ability to cataly ze transfer of the gamma phosphate of A TP to a suitably positioned nucleophile. In contrast, peptide substrate recognition differs for each kinase. 11

12 Kinase-Mediated Phosphate Transfer - R R + P P P Bring substrates together 2 R R - - P P P Mg 2+ 3 Aspartic Acid Residue from Kinase rient substrates 2 Lysine Residue from Kinase eutralize charge in TS Increase nucleophilicity of - R R P P P A s y ou heard from Dan, kinases accelerate the rate of phosphate transfer in the follow ing w ay s: (1) by bringing substrates together; (2) by positioning substrates in the correct orientation for phosphotransfer; (3) by neutralizing unfav orable negativ e charge in the transition state; and (4) by increasing the nucleophilicity of the oxy gen in the hy droxy l phosphoacceptor. n the next few slides w e w ill talk about how the conserv ed structure and sequence of protein kinases allow s these enzy mes to do the things listed abov e that are crucial for cataly sis. 12