Chemistry 106: Drugs in Society Lecture 19: How do Drugs Elicit an Effect? Interactions between Drugs and Macromolecular Targets 11/02/17 Targets for Therapeutic Intervention: A Comparison of Enzymes to Cell Surface Receptors Learning Objectives 1. Know what blood plasma is, what is meant by plasma concentration (C p ) and how plasma concentration relates to binding of drug targets 2. Know what is meant by volume of distribution (V D ) and half-life (t 1/2 ) and how drug polarity influences each 3. Describe in general terms - the mechanisms by which enzymes and cell surface receptors exert their effects a. Know cell surface receptors communicate an extracellular signal to the interior of the cell b. Know enzymes are biological catalysts that greatly increase the rate at which molecules are made in the body 4. Understand the relationship between populating an enzyme and effect a. Know what is meant by V, V max, E, ES, K D and K m 5. Understand the relationship between populating a receptor and effect a. Know what is meant by E, E max, B, B max, K D and EC 50 6. Understand the concept of spare receptors and how this allows receptors to bind communicating molecules at lower concentrations 7. Describe the similarities and differences between enzyme catalysis and cell surface receptor effects 8. Understand the similarities and differences between enzyme and receptor inhibition 9. Know what is meant by agonist, antagonist, and partial agonist 1
Introduction Recall therapeutic index (TI) is defined as the ratio of MTC/MEC, where MTC = mean toxic concentration in plasma and MEC = mean effective concentration in plasma The question then becomes What is plasma concentration and how a given plasma concentration influence the binding of drugs to drug targets? The figure immediately below is a general scenario for drugs where the site of action is removed from the site of application. However, there are scenarios when we can apply drugs directly for effect and avoid absorption into the systemic circulation. Why would avoiding absorption into the systemic circulation be beneficial? If you inspect the figure closely (and you should) you will see the central compartment is the drug concentration in systemic circulation, which is quantified as plasma concentration, C p. The plasma concentration relates directly to (1) drug concentration at the site of action which in turn is proportional to how much drug interacts with the receptor (2) how much drug is presented to those organs responsible for clearing foreign compounds from the body and thus how long the drug stays in the body 2
Concentration may have a variety of units, but mg per liter is typical Blood plasma is simply blood that has had the cellular component removed o Blood - cells Plasma - clotting factors Serum o Blood =.08L/kg, Plasma =.04L/kg We have learned that the polarity/ionization status of a drug plays a key, since non-polar drugs will have difficulty dissolving in water and tend to hide out in nonpolar tissues and polar drugs will have difficulty crossing cellular membranes A useful way of looking at the influence of polarity is the volume of distribution, V D, which may be defined as the [intravenous] dose/c p 3
Essentially, V D looks at the distribution of a drug as if it were in a very large volume of plasma o (Apparent) Volume of Distribution (V D ) Defined: V D = Dose/Cp Mass/Concentration = Volume (try it!) V D,min =.04L/kg (plasma) or 2.8L/70kg (154lb) individual o It should not be surprising that volume of distribution has a role to play in how long drugs stay in the body, since the blood carries drug to the kidney for excretion and/or to the liver for metabolism In fact, elimination half-life (t 1/2 ) is proportional to V D ` This is a big deal the more a non-polar drug hides out in nonpolar tissues (giving a larger V D ), the longer it takes to get rid of it, since t 1/2 is the time to get rid of half of the dose o A couple of examples should drive the point home 4
Aminoglycoside antibiotic for intraveneous injection V D =.33L/kg 90 % excreted by the kidney unchanged t 1/2 = 2.2 hrs 2 nd line antiemetic for cancer chemotherapy, appetite stimulant for cancer, AIDS. Apparently, has not proven superior to available medications for glaucoma V D 10L/kg Extensively metabolized t 1/2 30 hrs, CNS effects for 4-6 hrs, appetite increase up to 24 hrs Once we have a drug in plasma, it ultimately gets out of the blood stream and into the area where the drug targets are what then? It binds its target: either a receptor or enzyme, both of which are proteins 1 1 There are certain drugs (mainly cancer chemotherapy agents) that bind directly to DNA, but they will not be discussed here 5
The next question becomes how well does a drug bind its particular drug target? Really, why does a drug bind to a given receptor in the first place It is not a bad approximation to view enzymes and cell surface receptors as proteins that bind to specific molecules in a complementary fashion (think hand in a glove here), and recall classes of drugs often have structural similarities which mimic a natural communicating molecule (the endogenous ligand) How changes to a core drug structure 2 relates to the drug s effect ultimately by changing the binding to the target receptor 3 is known as the structure activity relationship (SAR) Since both enzymes and receptors are proteins that may be targets for drugs, we should be able to describe the binding similarly. First, let s briefly look at how enzymes and receptors work Enzymes Enzymes function by the strategic placement of critical amino acid side chains. This allows chemical bonds to be formed as bonds are broken in order to lower the energy of the transition state. As an example, consider the protein chewer (protease) chymotrypsin 2 The core structure which is always retained within a given drug class is known as the pharmacophore 3 Changes to the core structure can also influence the disposition of a drug how well it is absorbed, transported, and ultimately removed 6
Recall acetylcholine esterase, the enzyme that terminates the activation of the acetylcholine receptor by splitting acetylcholine The transition state is that point in a chemical reaction corresponding to the most difficult, highest energy point Since bond making releases energy and bond breaking requires energy, the transition state corresponds to that point where there is maximum bond breaking compared to bond making Enzymes function by allowing bonds to be made while bonds are being broken so the overall energy state of the process does not have to be so high It is not unusual for enzymes to speed up reactions by a factor of many million, essentially turning a chemical reaction on when the enzyme is available and functioning 7
Notice the lower energy state on binding substrate to enzyme. Effective enzyme inhibitors would similarly be of lower energy when bound to the enzyme compared to free enzyme and inhibitor. It then becomes a matter of who binds better and is more abundant, the natural substrate or inhibitor Receptors Receptors recognize a natural communicating molecule (endogenous ligand) and respond by undergoing a shape change which is communicated to the inside of the cell and changes its function. Essentially, by changing shape a receptor communicates a message to the cell, such as The nicotinic acetylcholine receptor (nachr) binding acetylcholine and opening a gate and allowing Na + into muscle cells 8
The muscarinic acetylcholine receptor (machr) binding acetylcholine and changing its shape to allow a G-protein to bind to it inside the cell become activated to carry the signal throughout the cell The cortisol receptor binding cortisol, dimerizing, binding to DNA, and regulating the assembly of the general transcription apparatus (GTA), a theme which holds for all of the steroid hormones. In the figure below, the estrogen receptor bound to estrogen, and the antiestrogen tamoxifen are shown. The portion of the receptor that communicates the signal for growth is shaded green note the dramatic shape change when estradiol vs. tamoxifen bind 9
An interesting variation on the cell surface receptor theme are the Janus kinase receptors: Growth Factors, Janus Kinase Receptors, Ras Genes, and Cancer Here is a cartoon of the Janus (tyrosine) kinase receptor binding endothelial growth factor, dimerizing and activating proteins on the inside of the cell, ultimately leading to changes in the assembly of the GTA, particularly where activating genes necessary for cell proliferation are concerned they are after all binding endothelial growth factor Vascular endothelial growth factor (VEGF) is a subfamily of the greater platlet-derived growth factors (PDGF), responsible for angiogenesis (the generation of new blood vessels from existing ones) Binding of VEGF brings the tyrosine kinase subunits together so they can phosphorylate one another. This leads to recognition by intracellular proteins which carry an SH2 domain, as shown below 10
Chemical principals still apply, as may be seen by the structure of the SH2 domain that surrounds the phosphorylated tyrosine below. This protein structural motif is very commonly found in adapter proteins that aid in activating Ras proteins The name Ras derives from Rat sarcoma. Sarcomas are cancers deriving from mesenchymal cells (which may be very loosely defined as supporting structure cells, and as such relatively rare). They were initially discovered in rat post viral infection 11
Lest you think this isn t particularly important, it is estimated up to 25% of all cancers have mutations leading to spontaneously hyperactive Ras proteins remember, we are talking about the binding of growth factors to the Janus Kinase In the case of both the G-protein coupled receptors and the Janus Kinase receptors, we call the proteins which interact with the receptor inside the cell effector proteins without them the receptor cannot influence the function of the cell Now that we have a better sense of what our targets look like, what are the similarities and differences of binding drug molecules to enzymes and receptors? A Brief Primer on Receptor and Enzyme Kinetics In making comparisons between enzymes and cell surface receptors, note that we are varying the concentration of substrate, ligand, and inhibitor while keeping the amount of enzyme or receptor fixed In the graphs below which relate population of a drug ligand to a cell surface receptor, we see what appears to be an identical curve to that seen with enzymes below, with a gradual flattening of the curve as the concentration of drug is raised; however, notice 2 curves are presented one for drug concentration that produces effect (E) and one for drug concentration that populates the receptor in question (B, analogous to ES below). While related, they are not the same 12
1. EC 50 is the concentration that produces a 50% maximal effect 2. K D is the concentration that populates 50% of the receptors; this is analogous to K m below 3. EC 50 and K D are not equal 13
By considering the relationship between drug concentration and receptor population, as well as drug concentration and effect, the following relationship emerges B B max C C KD This equation is a representation of the concept of spare receptors K D is the intrinsic dissociation constant for a given ligand; consequently, for a fixed concentration (C) of natural signaling molecule (endogenous ligand), increasing the number of receptors on a cell (increased B max ) leads to an increased number of populated receptors, B, as the ratio of B/B max must remain constant to maintain the equality Conversely, an increased number of receptors allows the same number of receptors to be bound at lower concentration (as shown in the following graphic); i.e. in this scenario, the ratio of B/B max decreases as one increases B max, and the only way to maintain the equality is for C to decrease (since K D is a constant indicating how well the receptor and communicating molecule bind to one another) Importantly, consider the effect of a low K D. What is the ratio of receptorligand vs. the maximum receptor-ligand as K D 0? K D? K D values give us a measure of receptor selectivity 14
(Consider that binding to proteins, any proteins, is an equilibrium process that can be driven by raising the concentration of interacting species) The equation does not change whether we are talking about an endogenous ligand or a drug which mimics that natural communicating molecule. An important point to be made is one of homeostasis over time the body will attempt to return to an initial state by reducing the number of receptors. This leads us to the classic model for tolerance and dependence Let s say we had a morphine analog that bound to the opioid mu receptor with a K D of 10 nm and an EC 50 of 10 nm. If the total number of receptors that could be populated (B max ) were reduced from 1000 to 600, what would EC 50 become? B B max C C KD 15
It is interesting to note that at times the cell will temporarily remove the receptor to see if the activating molecule the agonist - will go away. The implications to drug therapy and living well are huge. Mechanisms of Cell Surface Receptor Regulation: Desensitization vs. Down-Regulation Below is a likely mechanism for the desensitization of the -adrenergic receptor 16
Down-regulation of receptors is brought about by removal of the receptor from the surface of the cell The process is mediated by clathrin Notice that at the stage of the early endosome, the receptor can be recycled to the plasma membrane, thus leading to a temporary desensitization process Alternatively, the endosome may fuse with a lysosome and degrade the receptor; when receptor degradation outpaces receptor synthesis down regulation results Both the recycling to the cell surface and lysosomal degradation have been described for the -adrenergic receptor 17
When considering the binding of naturally occurring substrates to enzymes we see a similar relationship between binding and effect; however, the effect is not on a cell but instead on converting the substrate to a product Where enzymes as drug targets are concerned, we can think of blocking the conversion of the naturally occurring substrate as blocking the formation of an endogenous ligand that can subsequently influence the function of a cell The equation governing the rate of conversion of substrate to product for a fixed amount of enzyme is V S velocity max S Km 18
Note the similarity to the equation for the binding of ligand to receptor (K D and K M are essentially the same thing) and as a result the graphs look the same. When there is no substrate, there can be no conversion of substrate to product. When there is no endogenous ligand, there can be no effect of that ligand on the cell this is the x-y intercept. When there is so much substrate all of the available sites on all the enzymes are occupied, it does not matter if you add additional substrate. The same holds true for endogenous ligand and receptor this is where the curve flattens out While enzymes and receptors show similarities, there are important fundamental differences V max for an enzyme occurs when all enzyme molecules E are in the ES form, which is not the case when spare receptors are available A receptor mediated event may continue (say G-protein remaining in the active G-GTP form) after the agonist has left enzymes only function when there is substrate available for them to transform As a practical matter, we generally try to inhibit enzymes, whereas we may try to either block or activate receptors. On these lines, A drug may bind to a receptor and produce either 1. A maximal effect: an agonist (endogenous ligands are also agonists) 2. A sub-maximal effect: partial agonist 3. The complete prevention of receptor activation: an antagonist 19