Molecular Medicine: Gleevec and Chronic Myelogenous Leukemia. Dec 14 & 19, 2006 Prof. Erin O Shea Prof. Dan Kahne

Transcription

1 Molecular Medicine: Gleevec and Chronic Myelogenous Leukemia Dec 14 & 19, 2006 Prof. Erin Shea Prof. Dan Kahne 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 h. Dasatinib Alberts p McMurry p

3 Kinase-Mediated Phosphate Transfer - R H R + P P P H H H Bring substrates together H 2 R H R H H - - P P P Mg 2+ H 3 Aspartic Acid Residue from Kinase H H H rient substrates H 2 Lysine Residue from Kinase eutralize charge in TS Increase nucleophilicity of - R H R P P H H P - - H 2 As you heard from Dan, kinases accelerate the rate of phosphate transfer in the following ways: (1) by bringing substrates together; (2) by positioning substrates in the correct orientation for phosphotransfer; (3) by neutralizing unfavorable negative charge in the transition state; and (4) by increasing the nucleophilicity of the oxygen in the hydroxyl phosphoacceptor. n the next few slides we will talk about how the conserved structure and sequence of protein kinases allows these enzymes to do the things listed above that are crucial for catalysis. 3

4 Proximity Effect: A Kinase Brings the Two Substrates Together ATP protein substrate ne way in which kinases accelerate phosphotransfer reactions is by binding the two reacting species so that they are in close proximity and properly oriented for reaction. As you heard from Dan, the rate of a chemical reaction involving two molecules in solution depends on three factors: (1) the frequency with which the molecules collide; (2) the probability that molecules collide in the right orientation; and (3) the probability that the molecules that collide have enough energy to react. When kinases bind their substrates, the binding energy goes a long way towards paying the entropic penalty of immobilizing ATP and the protein substrate in the correct orientation for phosphotransfer. 4

5 The Mg-ATP binding pocket Glu 91 Glycinerich loop (P-loop) Lys 72 Mg 2+ Mg 2+ ATP Ser 53 Mg 2+ Many of the invariant residues found in the protein kinases are found in the Mg-ATP binding pocket. Three conserved sequence motifs located in the small lobe are associated with Mg-ATP binding. ne such motif is a glycine rich loop (also called the P-loop), containing Ser 53 that lies between the first and second beta-strands. Ser 53 makes a particularly important contact between its backbone amide (blue) and the gamma-phosphate of ATP (orange/red). In addition to the glycine rich loop, there are two conserved charged residues in the -terminal lobe: Lys 72 and Glu 91. The carboxylate of Glu 91 forms an ion pair with the amino group of the lysine side chain. This interaction helps position the protonated amino group of Lys 72 for interaction with the alpha and beta phosphates of ATP. Thus, the small lobe anchors the ATP and positions a positively charged lysine side chain near two of the negative charged phosphate groups. This lysine plays a key role in helping to stabilize the additional negative charge that builds up in the transition state. 5

6 The Catalytic Center ATP Mg 2+ Asp 184 Mg 2+ Asn 171 Tyrosine substrate Asp 166 Lys 168 The large lobe contains residues that are responsible for catalysis. These residues position the essential magnesium ions and the gamma phosphate of ATP, and provide a general base to deprotonate the peptide hydroxyl nucleophile. The large lobe contains two important elements we will discuss: the catalytic loop and the activation loop. The catalytic loop contains Asp 166, Lys 168, Asn 171, and Asp 184. Asp 166 positions the hydroxyl nucleophile of the tyrosine substrate and functions as a general base to deprotonate the hydroxyl nucleophile. Lys 168 is involved in interacting with the gamma phosphate of ATP. Asn 171 and Asp 184 are both involved in orienting the magnesium ions in the active site. Magnesium is required to both position the tri-phosphate and to neutralize unfavorable charge-charge interactions that would build up in the transition state during nucleophilic attack on the phosphate. 6

7 A Minimalist View of Kinase-Mediated Phosphate Transfer - R H R + P P P H H H H 2 R H R H H - - P P P Mg 2+ H 3 Aspartic Acid Residue from Kinase H Asp 166 H H H 2 Lys 72 Lysine Residue from Kinase - R H R P P H H P - - H 2 This slide highlights key interactions provided by kinases that accelerate the phosphotransfer reaction. Asp 166 deprotonates the tyrosine hydroxyl, increasing nucleophilicity of the oxygen. Lys 72 and the essential magnesium ion help stabilize the negative charges on the triphosphate. Charge neutralization is central to catalysis because the slow kinetics of phosphorylation reactions derive in large part from the highly unfavorable formation of an additional negative charge on a ATP molecule that already contains several negative charges in close proximity. 7

8 Kinases are Regulated by Phosphorylation of an Activation Loop Inactivated form Active site not accessible Activated form Active site accessible P Unphosphorylated; inactive Phosphorylated; active We have talked about kinase structure and how kinases accelerate phosphotransfer. Kinases also contain other features that allow for regulation of activity. For example, the large lobe contains an important region called the activation loop that can exist in two different conformations. In one conformation, the enzyme is inactive because the activation loop causes a change in the kinase structure which blocks access to the active site, hindering binding of peptide substrate. In the active conformation, the activation loop is oriented away from the active site, allowing the peptide substrate to bind to the enzyme. The active conformation of the loop is stabilized by phosphorylation of a residue within the loop. This reaction can either be autophosphorylation - catalyzed by the kinase itself - or it can be catalyzed by another kinase in the cell. We will take a closer look at the details of the activation loop on the next slide. 8

9 The Activation Loop Modulates Active Site Conformation Inactivated form Active site not accessible Mg-ATP not oriented Activated form Active site accessible Mg-ATP oriented Salt Bridge Unphosphorylated Threonine (Thr 197) Phosphorylated Threonine When the activation loop of the kinase is not phosphorylated, the loop is in a conformation that changes the kinase structure to block access to the active site. Additionally, in this conformation the kinase is unable to bind Mg-ATP in an orientation that supports phosphorylation. In contrast, when the activation loop is phosphorylated, the phosphate on Thr 197 participates in a network of interactions that alter the active site, enabling the proper positioning of the magnesium ion which is required for stabilization of the transition state. The rearranged active site in the activated form of the kinase is accessible to the peptide whereas it is not in the inactivated form (the black arrow indicates the cleft that is accessible to the peptide substrate in the phosphorylated, active form of the kinase, but not in the inactive form). ote the dramatic conformational changes of the protein in the vicinity of Thr 197 upon phosphorylation! We said that single amino acid changes and post-translational modifications can have dramatic effects on protein structure (and, therefore, activity). This slide provides a beautiful example. 9

10 Src Family of Protein Kinases We have just described the general structure of protein kinases, the mechanism of ATP binding and catalysis, and one aspect of their regulation (the activation loop). Earlier we told you about CML, which is the result of a protein fusion between a region of the bcr gene and the abl gene. Although the catalytic domain of Abl is completely unchanged in the Bcr-Abl fusion protein, the fusion somehow causes the Abl kinase to be constitutively active. To understand how this misregulation occurs, we need to talk about how Abl is normally regulated. Abl is a member of a large class of kinases called the Src family. Src and Abl have homology in the kinase domain, and also in two domains called Src homology 2 (SH2) and Src homology 3 (SH3). Both Src and Abl also are modified at the amino terminus with a fatty acid called myristate. The regulation of Src is complex and well understood. 10

11 The Architecture of Src: The Closed Conformation Src activity is tightly controlled within cells. To achieve this regulation, Src has an elaborate built-in regulatory apparatus. Src can exist in inactive and active forms; the regulatory apparatus controls the switch between these two states. The Src regulatory apparatus consists of the following parts: a latch consisting of a phosphorylatable tyrosine (Tyr 527); a clamp consisting of 2 peptide binding modules called Src Homology 3 domain (SH3 domain) and a Src Homology 2 domain (SH2 domain); and a switch region which is the activation loop containing a phosphorylatable residue we discussed previously (Tyr 416). The inactive form of the kinase is shown on this slide. As you can see, Tyr 527 on the C- terminal peptide tail attached to the large kinase lobe (the C-lobe) is phosphorylated. The SH2 domain recognizes this phosphorylated peptide containing Tyr 527; this structure is referred to as the latch. When the SH2 domain binds to the phosphotyrosine on the tail of the C-lobe, the linker domain between the -terminal lobe of the kinase (the -lobe) and the SH2 domain is extended into a conformation that is recognized by the SH3 domain. The SH2 domain and SH3 domain together act as a clamp that maintains the kinase in an inactive, closed conformation when the C-terminal tail is phosphorylated. In the inactive, closed conformation seen on this slide, the catalytic cleft is made rigid (through interaction of the C-lobe of the kinase with the SH2 domain), hindering access of ATP and substrate. Additionally, in the closed conformation, Tyr 416 in the activating loop (the switch ) is less accessible, and is therefore more difficult to phosphorylate. Both of these effects work together to keep the kinase inactive. 11

12 The Latch, the Clamp, and the Switch : The Regulatory Apparatus of Src Src Inactive closed Inactive open Active open The Src kinase can exist in three forms a fully active open form, an inactive open form and inactive closed form. In order for the Src kinase to proceed from the inactive, closed conformation to the open, active conformation it must be unlatched by dephosphorylation of Tyr 527, unclamped by the release of the SH3 and SH2 domains from the linker joining them to the kinase domain (facilitated by binding of another protein (ligand) to the SH2 and SH3 domains), and finally switched on via phosphorylation of Tyr 416 in the activation loop. 12

13 Abl Uses a Different Latch Mechanism from Src Src Abl If you look at the sequence alignments of Src and c-abl, you will see that they are closely related. They both contain SH2 and SH3 domains as well as the two-lobed kinase domain that is responsible for kinase activity. They both employ a mechanism of activation requiring an unlatching event, an unclamping event and a switching event. The important difference between these two kinases involves the way in which the enzyme is unlatched. In Src family members, we just saw that dissociation of the C-terminal tail, mediated by dephosphorylation of Tyr 527, unlatches the closed complex. c-abl does not contain a residue homologous to Tyr 527. Instead, its latch involves an interaction between the -terminal domain of the peptide with a hydrophobic pocket in the C-lobe of the kinase domain. This interaction is mediated by a long lipid chain (a myristate), that is attached to the -terminus of the protein. Unlatching is accomplished by the release of myristate. How ature accomplishes this is still unknown. However, one possible explanation is that a myristate binding protein associates with the myristate chain and pulls it out of the way. 13

14 ormal Regulation of Abl Abl Inactive closed Inactive open Active open Following unlatching, or dissociation of the myristate chain from the C-terminal lobe of the kinase, unclamping occurs when the SH2/SH3 domains bind to another phosphorylated protein during a signaling cascade. Finally, switching to the active form of the enzyme occurs when the tyrosine in the activation loop of the kinase domain is autophosphorylated. 14

15 Bcr-Abl Lacks a Latch Missing in fusion! Bcr-Abl, the fusion protein encoded on the Philadelphia chromosome, is a chimeric protein in which the cap portion of Abl has been removed and replaced with amino acids from Bcr. (Chimera is a Greek word describing a being that is half-man and half-monster. When we say chimeric protein, we simply mean that the protein is derived from two different protein parents.) This change does not affect the sequence of the kinase domain, but it does affect the latching mechanism of Abl. We learned that the latching mechanism is important for inhibition of the kinase. In Bcr-Abl, the cap is missing, the -terminus does not get myristoylated, and therefore the protein is constitutively active. 15

16 The Missing Latch Has Disastrous Consequences kinase Destabilized closed conformation (missing latch!) Inactive open Active open Because the latch is missing, the inactive closed conformation is less stable. The equilibrium shifts towards the the inactive open form of the protein, which can be activated by a single autophosphorylation event on the activation loop. The protein spends far more time in the active open state, where it phosphorylates other proteins that are ultimately involved in transcribing all kinds of genes involved in cell proliferation. Thus, removing the latching mechanism from this one protein has disastrous consequences. 16

17 Gleevec (STI-571; imatinib) CH 3 H 3 C H H Gleevec (also called STI-571 or imatinib) is a small molecule that was discovered by researchers at ovartis in a screen for kinase inhibitors. It is a derivative of 2- phenylaminopyrimidine. 17

18 Gleevec binds to and stabilizes this conformation Gleevec Binds the Inactive Form of Bcr-Abl kinase Inactive closed Inactive open Active open In investigating the mechanism of Gleevec, researchers found that the molecule does not bind to the active form of Bcr-Abl. Instead, Gleevec selectively recognizes the structure of the inactive form of Bcr-Abl, stabilizing the closed conformation. Gleevec binds partly overlapping with where the adenine ring of ATP normally binds to the kinase. In order for Gleevec to access the ATP binding site, the active site cleft must be open. Therefore, the function of Gleevec depends on the equilibrium between the inactive, closed and inactive open forms of the kinase - if the kinase was always in the inactive, closed state, Gleevec could not gain access to its binding site. 18

19 Gleevec Binds to the Closed, Inactive Conformation of Abl Steric clash! Gleevec + inactive conformation Gleevec + active conformation o clash w/ closed activation loop Clashes w/ open activation loop!!! Crystallographic studies have revealed the molecular basis for Gleevec s selectivity for the inactive, closed conformation of Bcr-Abl. In the active conformation of Abl, there are steric clashes between the activation loop and the drug that destabilize binding. In the inactive conformation, there are no such clashes; Gleevec occupies the space normally taken by ATP and makes a large number of contacts to the protein that contribute to high affinity binding. 19

20 Le Chatelier s Principle and Gleevec Gleevec binds to and stabilizes this conformation kinase Inactive closed Inactive open Active open If Gleevec binds the inactive, closed conformation of Bcr-Abl, how does it inactivate the kinase? Le Chatelier s principle, which you heard about from Dan, is at work. When Gleevec binds to the broken but closed conformation of Bcr-Abl (broken because the latch doesn t work), inactive, closed kinase bound to Gleevec is removed from the equilibrium above. As a consequence of removing kinase bound to Gleevec, the remaining kinase equilibrates between the inactive, closed and inactive, open forms - this re-establishes the equilibrium that exists between these 2 forms and decreases the amount of inactive, open form available to be activated by phosphorylation on the activation loop. The net result is that the amount of kinase that is in the active, open form is reduced, and the amount in the inactive, closed form is increased (some will be bound to Gleevec and some will not). ote that this is a completely different mechanism of inhibition than what you learned about for HIV protease and the protease inhibitors. Those inhibitors bind tightly to the active enzyme because they resemble the transition state of the reaction. In contrast, Gleevec exploits the complex regulation of Abl and binds to the inactive state. Gleevec does not resemble the transition state at all. If Src family kinases share this conserved mode of regulation, does Gleevec inhibit these kinases as well? The answer is no - this is because the inactive conformations of the other Src kinases differ subtly from that of Abl. 20

21 Gleevec is ot a Cure for All CML Patients 90% of patients in early stages of CML respond to treatment with Gleevec 96% of responding patients still exhibit Bcr- Abl mra expression - stem cells? 16% of patients relapse within 42 months of treatment Main cause of relapse are Bcr-Abl mutations Although >90% of patients treated with Gleevec respond by re-establishing normal blood counts (normal numbers of white blood cells), most of these patients still exhibit detectable expression of Bcr-Abl. This suggests that there is a subpopulation of cells that are not killed by Gleevec. It is not well understood why this subpopulation of cells remains, but recent research suggests that it may be a stem cell population that can divide and renew itself (generate more stem cells indefinitely). ne idea is that the stem cell population is less dependent on Bcr-Abl for proliferation, or that these cells are better able to eliminate the drug by pumping it out of the cell. Unfortunately, 16% of the patients that respond to Gleevec relapse with 3-4 years of beginning treatment. The main cause of relapse is mutation of Bcr-Abl, which renders cells resistant to the effects of Gleevec. These mutations could be pre-existing in the stem cell population, or they could develop in the stem cell population as the disease progresses and stem cells go through more and more divisions of self-renewal (there is a probability of mutation in each round of cell division). 21

22 Modeling CML Treatment and Gleevec Resistance S = stem cell P = progenitor D = differentiated TD = terminally differentiated S P D TD A model of CML treatment and resistance has been developed that has provided insights into the origins of resistance and strategies to treat the disease. This model considers 4 stages in the development of normal blood cells: stem cells (S), which are the most immature and which can continuously renew their own population; progenitor cells (P), which have lost the ability to self-renew; differentiated cells (D), which have begun to develop into specialized cell types; and terminally differentiated cells (TD), which are fully mature and specialized. Bcr-Abl leads to a slow expansion of stem cells and accelerates the rate with which these cells produce progenitors and differentiated cells. The model suggests that treatment with imatinib (Gleevec) affects the rate with which progenitors, differentiated, and terminally differentiated cells are produced, but that it does T affect the rate of stem cell division/renewal. Therefore, the rapid reduction in Bcl-Abl mra when patients are treated with Gleevec results from a decrease in the rate of production of differentiated (D) and terminally differentiated (TD) cells and subsequent loss of these populations (they have finite lifetimes, so if production is stopped, the population decreases). With continued treatment, there is a more gradual decline in Bcr-Abl corresponding to a reduction in the the progenitor population (P), but the stem cell population (S) is not affected. When resistant mutations develop in the stem cell population there is a rise in Bcr-Abl mra corresponding to an increase in the progenitors, produced by the stem cells. This is followed by a rapid rise in Bcr-Abl mra arising from development of the progenitors into differentiated and terminally differentiated populations - this corresponds to relapse. ote that this is another example of steady-state, similar to the one described by Andrew for the treatment of AIDS patients with combination therapy. 22

23 Gleevec Resistant Mutations Analyze 32 patients who have relapsed - find Abl kinase domain mutations in 29/32! Resistant mutations must preserve kinase activity Mutations found in 13 different residues in the Abl kinase domain Contact with Gleevec P-loop (glycine-rich loop) Activation loop How do mutations in P-loop and activation loop cause resistance? How do mutations in Bcr-Abl allow Abl to escape inactivation by Gleevec? When 32 patients who had relapsed were studied, 29/32 of them were found to have mutations in the Abl kinase domain that preserve kinase activity (and therefore preserve the ability of Abl to drive cell proliferation in the absence of growth factors), but render Abl insensitive to Gleevec treatment. Mutations were found in 13 different residues in the Abl kinase domain. The mutations fall into three groups: (1) contact with Gleevec; (2) P-loop (glycine rich loop); and (3) activation loop. The first category of mutations are easy to understand, as they are in amino acids that make direct contacts with the drug to stabilize the bound state. It is more difficult to understand how the latter two groups affect the ability of Gleevec to inhibit Abl. 23

24 P-loop * * Gleevec Resistant Mutants * activation loop * contact with Gleevec Insight into the mechanism of resistance comes from mapping these residues onto the structure of Abl. This slide shows the 13 mutations (pink balls) observed in patients mapped onto the structure of the Abl kinase domain (green) bound to Gleevec (red). The three mutations identified in patients that are involved in residues that make direct contacts to Gleevec are indicated with an asterisk (*). How do mutations in the P-loop (glycine-rich loop) and the activation loop confer Gleevec resistance if these residues do not make contacts to Gleevec? These residues are all involved in the conformational change that the kinase domain undergoes to convert to the inactive form that binds Gleevec. For example, the phosphate binding loop (P-loop or glycine-rich loop) is distorted in the Abl-Gleevec structure to help form a hydrophobic pocket for part of the drug. Additionally, the activation loop of Abl has to be in a closed conformation, participating in a network of interactions that help stabilize the drug binding site. Mutations in the P-loop and activation loop destabilize the closed conformation of Abl, reducing binding of Gleevec. 24

25 Dasatinib Dasatinib Gleevec (imatinib) Fortunately for patients who have relapsed, there is now a new drug available. Dasatinib, shown on this slide, was approved by the FDA in June 2006 for the treatment of Gleevecresistant CML. As you can see, the structures of Dasatinib and Gleevec are quite different. It turns out that these two drugs work by very different mechanisms. As you have heard, Gleevec works by binding to and stabilizing the inactive conformation of Abl. We saw that Gleevec resistant mutants destabilize the inactive conformation of Abl to which Gleevec binds, thereby reducing binding of Gleevec. Gleevec-resistant versions of Abl are still inhibited by Dasatinib because this drug works by binding the active conformation of Abl. 25

26 Structure of Abl-Dasatinib Compared to Active Kinase This slide shows the conformation of the active site of Abl bound to dasatinib (green) compared to the structure of an active kinase (gray). Residues important for catalysis are highlighted in red and blue. These two structures are superimposable, demonstrating that Abl bound to dasatinib adopts an active kinase structure. This contrasts with Gleevec, which binds to the inactive conformation of Abl. 26

27 Abl Bound to Imatinib (Gleevec) and Dasatinib If we compare the structures of the active site regions of Abl bound to imatinib (Gleevec) versus dasatinib, we see dramatic differences in the conformation of the kinase. Dasatinib (green) sits in the ATP-binding pocket and protrudes into solution, whereas imatinib (pink) fits partly in the ATP-binding pocket but also extends towards the activation loop. When Abl is bound to Gleevec (pink), the activation loop is folded back toward the ATP-binding site, forming interactions with the P-loop and Gleevec and stabilizing the inactive Abl conformation. F382 (shown as a van der waals surface representation of pink dots) interacts with the pyrimidine ring of Gleevec and also with residues in the P-loop. In contrast, in the structure with dasatinib bound, F382 is in a hydrophobic pocket away from the P-loop. Gleevec is unable to bind this conformation because of steric clashes with F382 and also the activation loop. Current thinking is that combination therapy with Gleevec and Dasatinib may be the most effective way to treat CML. The rationale for this is similar to combination therapy for the treatment of HIV in which the probability of obtaining resistant mutants that can escape both drugs is extremely low - it is the product of the probabilities of obtaining resistance to each drug alone. 27

28 Summary Protein kinases contain a domain that folds into a conserved structure Protein kinases catalyze phosphorylation through proximity, orientation, and electronic effects Multiple mechanisms regulate the Src family of kinases Bcr-Abl is missing a critical mode of control, causing it to be constitutively active Gleevec binds to the inactive, closed form of Abl, shifting the equilibrium towards this form Gleevec resistant mutants destabilize the closed conformation, destabilizing Gleevec binding Dasatinib binds the active conformation of Abl, explaining its efficacy against Gleevec-resistant mutants Combination therapy may be the best treatment for CML 28