Drug Discovery and Design Lecture 1 PDF

+-----------------------------------------------------------------------+ | Drug discovery and design lecture 1 | | | | - Natural product screening: this involves screening compounds | |...

+-----------------------------------------------------------------------+ | Drug discovery and design lecture 1 | | | | - Natural product screening: this involves screening compounds | | derived from natural sources (like plants, fungi or marine | | organisms) for potential therapeutic effects. | | | | - Drug metabolism studies- these studies assess how a drug is | | metabolised in the body- which is important for understanding its | | efficacy, safety, and optimal dosing. These drug metabolism | | studies can also help predict potential drug interactions and | | adverse effects. | | | | - Clinical observations are observations from clinical trials and | | patient experiences can lead to the discovery of a new drug uses | | or highlights unexpected side effects that might guide further | | research | | | | - Rational approach to drug discovery: This method involves | | figuring out which biological target (like a protein or enzyme) | | is involved in a disease and then designing drugs that | | specifically interact with that target. It usually includes using | | advanced techniques like high-throughput screening and computer | | modelling to find and test potential new drugs | | | | | | | | - Of the 1437, FDA-approved small molecule drugs on the market in | | 2012, 347 (24.1%) were discovered as a result of serendipity | | (luck) | | | | - For anticancer agents, this rises to 31/88 (35.2%) | | | | | | | | - Phases of the cell cycle | | | | | | | | - S-phase: DNA is synthesised and replicated | | | | - M-phase: chromosomes are separated into two new nuclei | | | | - Gap phases (G1 and G2): intervals between the other phases where | | the cell prepares either for DNA synthesis (G1 ) or mitosis (G2) | | | | - Interphase: this is a collective term for the G1, S, and G2 | | phases, which is the period between one mitosis and the next | | | | - ​​G0 phase: A state of quiescence where cells may exit the cell | | cycle from G1 | | | | | | | | - Stages of mitosis: | | | | | | | | - **P (Prophase)** | | | | - **M (Metaphase)** | | | | - **A (Anaphase)** | | | | - **T (Telophase)** | | | | - **C (Cytokinesis** | | | | | | | | - How chemotherapeutic agents interact with the cell cycle | | | | | | | | - Cell-cycle specific agents: These drugs target cells at | | particular phases of the cell cycle, making them effective | | against rapidly dividing cancer cells | | | | - Cell- cycle nonspecific agents: These can act at any phase of the | | cell cycle, including the resting phase (G0), targeting both | | dividing and non-dividing cells. | | | | | | | | - Natural product screening: | | | | | | | | - Lead Compounds Identification: Many drugs have been discovered by | | isolating active ingredients from folklore or traditional | | remedies. This approach has led to the development of effective | | drugs. | | | | - Bioactive Product Isolation: Systematic screening of plant and | | animal extracts has been instrumental in identifying new drugs. | | | | - Global Dependence on Natural Drugs: It notes that 80% of the | | world\'s population uses drugs derived exclusively from natural | | sources. | | | | - Contribution to Modern Medicine: 35% of drugs contain \'active | | principles\' or key elements from natural sources. | | | | - Screening Scope: Despite the vast biodiversity, less than 5% of | | higher plant species have been screened for their potential | | medicinal properties, suggesting a large untapped reservoir for | | future drug discovery. | | | | - Diversity of Plant Constituents: Each plant can contain thousands | | of different constituents, each potentially holding the key to | | new therapeutic agents. | | | | | | | | - Some Anticancer Agents from Natural Origins | | | | | | | | - Paclitaxel (Taxol): Sourced from the Pacific yew tree (Taxus | | brevifolia), this molecule is highly detailed with various | | functional groups, including esters and alcohols, crucial for its | | activity against cancer cells, how? | | | | | | | | - There\'s part of the cell called microtubules, which are | | essential for cells to divide and multiple. Paclitaxel makes | | microtubules very stable and prevents them from breaking apart. | | When this happens, the cancer cells cannot divide and eventually | | die. | | | | | | | | - Vincristine and Vinblastine: These are alkaloid compounds derived | | from the Madagascar periwinkle. They are similar to paclitaxel | | (target microtubules) however instead of stabilising microtubules | | these drugs prevent the microtubules from forming in the first | | place- if the microtubules cant form, the cancer cells cant | | divide which stops them from spreading. Differ slightly in their | | chemical side chains, indicated by R1, R2, and R3. These drugs | | work by binding to tubulin, disrupting microtubule formation, and | | inhibiting cancer cell division. | | | | | | | | - M Phase Specific Chemotherapeutics | | | | | | | | - The M phase is a part of the cell cycle when a cell divides to | | form two new cells. This phase is crucial because it\'s where the | | cell\'s chromosomes (which carry genetic material) are evenly | | split into two new cells. Microtubules, which are like tiny | | threads inside the cell, play a key role here by helping to pull | | the chromosomes apart. | | | | - Basically, the goal of these M phase specific chemotherapeutic is | | stop cancer cells from completing division (which is what happens | | during mitosis)- stopping cell division limits cells ability to | | spread- remember we said that these M phase specific | | chemotherapeutic stop cell division by targeting microtubules | | (which are important structures in cell decision as they help | | distribute chromosomes to new cells. | | | | - There are two classes of M phase specific drugs | | | | | | | | - Vinca alkaloids e.g. Vinblastine: These drugs interfere with the | | formation and function of microtubules by making them unstable. | | By destabilising microtubules, Vinca alkaloids cause the cells to | | stop or \"arrest\" at a critical point in mitosis (the | | metaphase/anaphase transition). This prevents the cancer cells | | from successfully dividing into two new cells | | | | - Taxanes: Unlike Vinca alkaloids, Taxanes stabilise microtubules. | | They promote the polymerization of tubulin (the protein building | | block of microtubules) which leads to the stabilization and | | bundling of microtubules. This also arrests cells in mitosis but | | does so by a different mechanism compared to Vinca alkaloids. | | | | Explanation of taxanes: Taxanes bind to a protein called tubulin, | | which is the building block of microtubules. Unlike other drugs that | | prevent tubulin from forming microtubules, taxanes do the | | opposite---they promote the assembly of tubulin into microtubules and | | then prevent these microtubules from disassembling.Microtubules need | | to be dynamic (constantly assembling and disassembling) for cells to | | divide properly. By stabilizing microtubules, taxanes effectively | | freeze their structure. This abnormal rigidity prevents the | | microtubules from pulling the cell's genetic material apart into the | | two new cells, a process essential for cell division.Because the | | microtubules are frozen, the cell can\'t complete division and | | eventually dies. This stops the multiplication of cancer cells, | | limiting the spread of the cancer. Examples of Taxanes: Paclitaxel | | (Taxol) | | | | - Vinca alkaloids - | | | | | | | | - Different phases of mitosis (cell division that ensures genetic | | material is accurately duplicated and distributed into two | | daughter cells) | | | | ### | | | | ### **Microtubule Function in Cell Division** | | | | - **Microtubules in Mitosis:** Microtubules are essential for | | pulling the chromosomes apart during mitosis, the process of cell | | division. | | | | - **Spindle Fibers:** These are made up of microtubules that | | extend from opposite ends (poles) of the cell and attach to | | chromosomes. They help in aligning and then separating the | | chromosomes so that each daughter cell receives an equal set | | of genetic material. | | | | - **Tubulin Polymerization:** | | | | - Microtubules are composed of a protein called **tubulin**, | | which forms long chains (polymers) of alternating **alpha** | | and **beta** subunits. | | | | - The process of building these long chains is called | | **polymerization**. When microtubules need to shrink, they | | **depolymerize** back into tubulin subunits. | | | | - This constant growth and shrinkage of microtubules is highly | | controlled and necessary for proper cell division. | | | | ### **Microtubule Dynamics** | | | | - **Dynamic Instability:** | | | | - Microtubules exhibit a behavior called **dynamic | | instability**, where one end of the microtubule (the **plus | | end**, primarily made of beta-tubulin) grows while the other | | end (the **minus end**, mainly composed of alpha-tubulin) may | | shrink. This allows microtubules to rapidly reorganize as | | needed during cell division. | | | | - **(treadmilling) Growth at the plus end** and **shortening at | | the minus end** allow microtubules to adjust as they pull | | chromosomes into the correct positions within the dividing | | cell. | | | | ### **Dynamic Instability** | | | | - **What it is**: Dynamic instability is characterised by rapid and | | sudden changes between growth and shrinkage at the ends of a | | microtubule. | | | | - **Key feature**: This process mainly affects the plus end of the | | microtubule (though the minus end can also show dynamic changes | | but at a much slower rate). The plus end alternates between | | phases of rapid growth (polymerization) and rapid shrinkage | | (depolymerization), known as \"catastrophe\" when it shifts from | | growth to shrinkage, and \"rescue\" when it reverts back to | | growth. | | | | - **Purpose**: The primary role of dynamic instability is to allow | | microtubules to quickly explore the cellular environment, such as | | during the formation of the mitotic spindle, which helps in cell | | division. | | | | ### **Treadmilling** | | | | - **What it is**: Treadmilling is a process where the addition of | | subunits at one end and the loss at the other occur | | simultaneously, leading to a net movement of the filament itself. | | | | - **Key feature**: In treadmilling, the plus end and the minus end | | of the microtubule are both active, but in opposite ways: the | | plus end grows as the minus end shrinks. The overall length of | | the microtubule does not change significantly, but there is a | | continuous \"flow\" of tubulin subunits through the microtubule. | | | | - **Purpose**: Treadmilling is important for processes like cell | | migration and maintaining cell polarity, where the position and | | orientation of microtubules are crucial. | | | | ### **Main Differences** | | | | - **Behavior at Microtubule Ends**: Dynamic instability involves | | abrupt switches in growth and shrinkage at one or both ends of | | the microtubule, whereas treadmilling involves a steady state of | | subunit addition and loss at opposite ends. | | | | - **Impact on Microtubule Length**: In dynamic instability, the | | microtubule length can change dramatically and quickly due to | | sudden growth or shrinkage. In treadmilling, the microtubule | | length remains relatively constant, with subunits \"flowing\" | | through the structure. | | | | ### **Drug Interference with Microtubules** | | | | - **Vinca Alkaloids:** | | | | - These are anticancer agents that disrupt microtubule | | formation. | | | | - At high concentrations, these drugs cause microtubules to | | **depolymerize** (break down), preventing the microtubules | | from functioning properly in cell division. | | | | - At lower concentrations, both the growth and shortening of | | microtubules are slowed down, also hindering the cell | | division process. | | | | - **Importance of Microtubule Targeting in Cancer Treatment:** | | | | - Since cancer cells divide rapidly, drugs that interfere with | | microtubule function can halt their division, making these | | drugs effective anticancer therapies. | | | | - The discovery of vinca alkaloids as anticancer agents | | happened by chance; researchers did not originally design | | them to target microtubules. It was only after their | | effectiveness was observed that scientists understood how | | these drugs actually worked | | | | | | | | - Microtubules have two ends, known as the \"plus\" and \"minus\" | | ends. The plus end is typically more dynamic and is where most | | growth and shrinkage occur. | | | | - Vinblastine shows the greatest affinity for binding at this plus | | end. This preference is significant because it allows the drug to | | effectively target the most active part of the microtubule, | | disrupting its normal function. | | | | - At the extreme end of the microtubule, vinblastine binds with the | | highest affinity. This high-affinity binding can prevent the | | addition of new tubulin subunits to the plus end, which is | | critical for microtubule growth. | | | | - By inhibiting this addition, vinblastine can halt microtubule | | elongation, leading to a disruption in microtubule dynamics. This | | disruption is crucial for its anti-cancer effects, as it can halt | | cell division by preventing the proper formation of the mitotic | | spindle, a structure required for segregating chromosomes during | | cell division. | | | | - **Tubulin** is the building block protein that stacks together to | | form microtubules, which are like tiny rods inside cells that | | help give the cell its shape and are crucial for cell division. | | | | - **Vinblastine** is a drug used to treat cancer. It works by | | targeting tubulin, but it can only interact with tubulin when the | | tubulin is free or exposed. | | | | - Now, when tubulin is stacked tightly together to form the | | microtubule (like bricks in a wall), this configuration is called | | the **microtubule lattice**. In this tight arrangement, much of | | the tubulin is hidden inside the structure, much like how the | | inside parts of bricks in a wall aren't exposed to the outside. | | | | - So, when tubulin is incorporated into this tight lattice of the | | microtubule, vinblastine has a harder time reaching it because | | the tubulin is less exposed or accessible. This means that | | vinblastine can\'t bind as easily to the tubulin to exert its | | effects when the tubulin is locked away inside the microtubule | | structure. | | | | - - The binding affinity of vinblastine decreases for tubulin | | that is not at the ends but rather integrated into the existing | | microtubule structure. This difference in binding affinity allows | | vinblastine to specifically target the growing ends of | | microtubules rather than disrupting the stability of already | | formed sections of the microtubule network. | | | | Explanation of above; **Targeting Growing Ends** | | | | - **Growing Ends of Microtubules**: The ends of microtubules are | | where new tubulin units are added as the microtubule grows, | | especially during cell division. These ends are dynamic and have | | exposed tubulin. | | | | - **Action of Vinblastine**: Because vinblastine has a higher | | affinity for the exposed tubulin at these growing ends, it | | primarily targets these areas. By binding to the tubulin here, | | vinblastine disrupts the normal addition of new tubulin units, | | effectively stopping the growth of the microtubule | | | | - **Disrupting Microtubule Growth**: By targeting the growing ends | | and not the stable, already formed parts of the microtubule, | | vinblastine specifically disrupts the formation of new | | microtubule structures necessary for cell division. This action | | prevents cancer cells from dividing and proliferating. | | | | | | | | - Rational drug design = approach to developing medications based | | on the biological target they are meant to act on | | | | - It\'s a three step process: | | | | Vorinostat is a drug used to treat cancer. It works by targeting a | | specific enzyme in the body called histone deacetylase, or HDAC for | | short. This enzyme usually removes certain groups (acetyl groups) | | from proteins called histones, which affects how genes are turned on | | or off in cells. | | | | When Vorinostat stops HDAC from removing these groups, more groups | | stay on the histones. This leads to turning on genes that can slow | | down or stop cancer cells from growing and can even make them die. | | Essentially, Vorinostat helps to control which genes are active in a | | way that fights cancer | | | | - Cancer cells typically grow more rapidly than normal cells. This | | rapid growth increases their demand for nucleotides, which are | | the building blocks for DNA and RNA, essential for cell | | replication and function. (adenine (A), cytosine (C), guanine | | (G), and thymine (T) | | | | - Antimetabolites are a type of chemotherapy drug designed to | | interfere with DNA and RNA synthesis by mimicking the usual | | building blocks of RNA and DNA. When cancer cells try to use | | these mimics, they find themselves unable to produce functioning | | DNA or RNA, which disrupts their growth and survival. | | | | **Types of Antimetabolites and Their Specific Actions**: | | | | - **Pyrimidine Analogues (e.g., 5-fluorouracil)**: These drugs | | resemble pyrimidine bases (C and T) (one of the two types of | | bases in DNA and RNA, the others being purines). When a cell | | attempts to use these analogues instead of the real bases during | | DNA/RNA synthesis, the resulting DNA or RNA is faulty and cannot | | function properly. This disrupts the cell\'s ability to reproduce | | or carry out normal functions, leading to cell death. | | | | - **Purine Analogues (e.g., 6-mercaptopurine)**: These mimic the | | purine bases (A and G) in DNA and RNA. Similar to pyrimidine | | analogues, when incorporated into DNA or RNA during the | | replication process, they lead to errors that can halt cell | | division or cause cell death. | | | | **Dual Mechanisms of Action**: | | | | - **Inhibiting de novo Synthesis of Nucleotides**: These | | antimetabolites can inhibit the enzymes involved in making new | | nucleotides from scratch, which deprives rapidly dividing cancer | | cells of the materials needed for making new DNA and RNA. | | | | - **Incorporation into DNA/RNA**: beyond just resembling nucleic | | acid bases, when these analogues are incorporated into DNA or | | RNA, they disrupt vital processes such as replication (copying | | DNA), transcription (creating RNA from DNA), and translation | | (assembling proteins based on RNA instructions). This disruption | | can lead to the production of malfunctioning proteins or trigger | | cellular mechanisms that lead to cell death. | | | | | | | | - 6-mercaptopurine (Purine Analogues) | | | | 6-MP is designed to trick cancer cells. Here's a simplified | | breakdown: | | | | - Mimicking Natural Molecules: 6-MP looks like the normal building | | blocks of DNA and RNA (called purines). Cancer cells need these | | building blocks to grow and multiply. | | | | - Blocking Building Block Use: When cancer cells try to use 6-MP | | thinking it\'s a normal purine, it messes up their ability to | | make DNA and RNA properly. This stops the cancer cells from | | growing and can lead to their death. | | | | - Stopping an Enzyme: 6-MP also blocks an enzyme called xanthine | | oxidase, which is part of the pathway that creates purines. This | | further starves the cancer cells of what they need to grow | | | | - 6-MP was initially tested in children with acute leukaemia, | | significantly increasing life expectancy from a median of 3-4 | | months to over a year, with more than 50% of cases showing | | remissions. It is primarily used in treating acute lymphocytic | | leukaemia (ALL) and chronic myeloid leukaemia (CML). | | | | - **Immunosuppression**: Apart from its oncological applications, | | 6-MP is also used as an immunosuppressant in the treatment of | | autoimmune diseases. | | | | **Broader Spectrum of Activity** | | | | - **Antimicrobial Effect**: Interestingly, 6-MP inhibits the growth | | of *Lactobacillus casei*, a bacteria found in the human gut and | | dairy products. This suggests that 6-MP can affect bacterial DNA | | synthesis, potentially impacting the gut microbiota during | | treatment. | | | | - **Effect on Solid Tumours**: 6-MP has shown activity against | | mouse sarcoma 180, a type of solid tumour, indicating its | | effectiveness not only against haematological cancers but also in | | treating solid tumours in experimental models. | | | | **Significance** | | | | - The development and application of 6-MP marked a significant | | milestone in pharmacology, illustrating the potential for | | targeted therapies in cancer treatment and beyond. Its ability to | | inhibit both bacterial growth and tumour proliferation underlines | | its broad therapeutic impact, affecting a variety of cellular | | processes. | | | | **1. Production of DNA and RNA:** | | | | - **Nucleotide Requirements:** Both DNA and RNA synthesis require | | purine bases (Adenine, Guanine) and pyrimidine bases (Cytosine, | | Thymine for DNA and Cytosine, Uracil for RNA). | | | | - **Synthesis Pathways:** Cells can synthesise nucleotides via two | | pathways: | | | | - **De novo pathway:** Building nucleotides from scratch. | | | | - **Salvage pathway:** Recycling bases and nucleotides. | | | | - **Cancer Cell Targeting:** Rapidly dividing cancer cells | | preferentially use the de novo pathway, making this process a | | target for chemotherapy drugs to achieve selectivity over normal | | cells. | | | | **2. DNA Replication:** | | | | - **Process Overview:** DNA replication involves unwinding of the | | double helix by DNA helicase, followed by the synthesis of new | | strands by DNA polymerase which adds nucleotides complementary to | | the template strand. | | | | - **Key Enzymes:** | | | | - **DNA Helicase:** Unzips the DNA double helix. | | | | - **DNA Polymerase:** Synthesizes new DNA strands by adding | | nucleotides. | | | | - **DNA Ligase:** Seals gaps between the newly synthesised DNA | | fragments to create a continuous strand. | | | | - **Topoisomerase:** Relieves the twisting and strain ahead of | | the replication fork. | | | | **3. Molecular Events During the S Phase:** | | | | - **Unzipping of DNA:** DNA helicase breaks the hydrogen bonds | | between the base pairs to separate the strands. | | | | - **Base Pairing:** DNA polymerase facilitates the base pairing | | (A-T, G-C) AND catalyses the formation of the sugar-phosphate | | backbone (This involves creating phosphodiester bonds between the | | sugar of one nucleotide and the phosphate group of the next, | | effectively building the new strand.) | | | | - Resulting DNA: Post replication, two identical DNA molecules are | | formed, each consisting of one old and one new strand, known as | | semi-conservative replication | | | | 6-MP Mechanism of Action | | | | ### **How does 6-MP work?** | | | | 1. **Conversion into Active Form**: | | | | - When 6-MP enters the body, it doesn\'t work right away (it\'s | | a prodrug). It first needs to be converted by the body\'s | | enzymes (hypoxanthine-guanine Phosphoribosyltransferase 1 | | (HGPRT1)) into its active forms. The first important form it | | turns into is called TIMP (thioinosine monophosphate). | | | | 2. **Blocking DNA Building Blocks**: | | | | - Normal cells and cancer cells need building blocks to make | | DNA and RNA, which are essential for cell growth and | | survival. One of these building blocks is called IMP (inosine | | monophosphate). | | | | - TIMP, the active form of 6-MP, looks a lot like IMP. Because | | of this similarity, when TIMP is present, it competes with | | IMP for the attention of a specific enzyme (adenylosuccinate | | synthase). | | | | - Normally, this enzyme helps convert IMP into another molecule | | that\'s critical for making DNA and RNA. But when TIMP binds | | to the enzyme, it blocks the conversion. This means the | | cancer cells can't make the DNA and RNA they need to grow and | | multiply. | | | | 3. **Why is Sulphur Important?**: | | | | - 6-MP includes a sulphur atom in its structure, replacing an | | oxygen atom that would normally be in that spot in its | | non-drug counterpart. Sulphur has different chemical | | properties than oxygen---it\'s less electronegative, which | | means it doesn\'t pull electrons towards itself as strongly | | as oxygen does. | | | | - This difference in electronegativity affects how well TIMP | | can mimic IMP. The presence of sulphur means that TIMP can | | bind to the enzyme but doesn\'t allow the enzyme to do its | | job correctly. Thus, the enzyme gets stuck with TIMP and | | can\'t help produce the necessary components for DNA and RNA. | | | | ### **The Outcome** | | | | By blocking this pathway, 6-MP effectively halts the proliferation of | | cancer cells, which are particularly reliant on quickly making new | | DNA and RNA for their rapid growth. Normal cells are affected too, | | but they don\'t rely on this pathway as heavily, which gives 6-MP its | | usefulness in treating cancer selectively. | | | | \* For the above: Inosine monophosphate is a normal nucleoid found in | | cells that play a role in the synthesis of other nucleotides like AMP | | (adenosine monophosphate) and GMP (guanosine monophosphate). IMP has | | a hypoxanthine base (that has a carbon) double bonded to an oxygen. | | | | \*TIMP (thioinosine monophosphate is similar to IMP but it has a | | sulfur atom replacing the oxygen atom that is normally double bonded | | to the carbon in the hypoxanthine base. So, instead of having a | | carbonyl group (C=O), TIMP has a thiocarbonyl group (C=S). | | | | \***Inhibition of Nucleotide Synthesis**: | | | | - **TIMP** is mistaken for **inosine monophosphate (IMP)** by the | | enzyme **adenylosuccinate synthase**, which attempts to use it in | | the synthesis of AMP (adenosine monophosphate). | | | | - However, the substitution of sulfur for oxygen (as in TIMP | | instead of IMP) leads to a reduction in the electropositivity of | | the carbon atom in question, which alters the chemical reactions | | that would normally occur. | | | | ABOVE : When TIMP, a structurally similar molecule to IMP, enters the | | scene (thanks to 6-MP metabolism), adenylosuccinate synthase | | mistakenly tries to use it just like it would use IMP. | | | | - The chemical reaction normally involves a nucleophilic attack on | | the carbon atom at the C6 position of the IMP molecule, | | facilitated by its partial positive charge due to the attached | | oxygen. | | | | - However, in TIMP, the sulfur atom replacing the oxygen reduces | | this partial positive charge. The carbon atom becomes less | | electropositive, decreasing its reactivity towards nucleophilic | | attack. | | | | ### **Consequences of This Inhibition** | | | | 1. **Interruption of AMP Synthesis**: | | | | - Due to the reduced reactivity of the carbon atom in TIMP, the | | enzyme\'s activity is hindered or completely blocked. This | | prevents the normal conversion of IMP to adenylosuccinate and | | subsequently to AMP. | | | | - The inhibition of AMP synthesis disrupts the balance and | | availability of purine nucleotides. | | | | 2. **Effect on Cellular Processes**: | | | | - Nucleotides like AMP are fundamental for DNA and RNA | | synthesis as well as for other cellular functions including | | signaling and repair mechanisms. | | | | - A deficiency in AMP can lead to a broader disruption of | | nucleotide pools within the cell, affecting the synthesis of | | nucleic acids and impairing cell division and growth. | | | | ### **Link to 6-MP\'s MOA in Treating Cancer** | | | | - **Selective Toxicity to Rapidly Dividing Cells**: | | | | - Cancer cells, particularly those in leukemias, proliferate | | rapidly and have a high demand for nucleotides to support | | constant DNA replication and cell division. | | | | - By inhibiting nucleotide synthesis, 6-MP preferentially | | affects these rapidly dividing cells more than normal cells, | | which do not divide as frequently and thus are less dependent | | on rapid nucleotide synthesis. | | | | - **Induction of Cell Death**: | | | | - The shortage of nucleotides leads to DNA replication stress | | and potential replication fork stalling, which can trigger | | cell cycle arrest and apoptosis (programmed cell death). | | | | - This specific targeting makes 6-MP an effective anticancer | | agent, causing the death of leukemia cells while sparing most | | normal cells | | | | so we\'ve talked about chance and natural products. We\'ve talked | | about rational and synthesis. How about if we give a drug if we | | design a drug for a particular application and then we find out that | | drug isn\'t any use, but we then test it in something else (this is | | called clinical observations) | | | | - **MER25 (Ethamoxytriphetol)**, initially developed for | | cardiovascular conditions, was found to have estrogen receptor | | (ER) antagonist properties during clinical trials. This | | serendipitous discovery led to its repurposing for cancer | | therapy. **Tamoxifen**, developed from the chemical foundation | | provided by MER25, incorporated modifications like a double bond | | and an ethyl group to enhance its anti-estrogen efficacy. | | Tamoxifen became a groundbreaking treatment for estrogen | | receptor-positive breast cancer, effectively blocking estrogen | | from stimulating cancer cell growth. This transformation from a | | failed cardiovascular drug to a cornerstone of breast cancer | | treatment exemplifies the potential of drug repurposing in | | pharmaceutical development. | | | | ### **Estrogen Receptor (ER) Antagonists: Historical Background and C | | linical Development** | | | | **Historical Insight into ER and Breast Cancer Treatment**: | | | | - **1896**: George Beatson first demonstrated the beneficial | | effects of estrogen ablative therapy on patients with advanced | | breast cancer. This involved the removal of sources of estrogen, | | such as the ovaries (oophorectomy), which led to improvements in | | breast cancer conditions. | | | | - **1936**: Antoine Lacassagne speculated that antagonizing the | | estrogen receptor might be an effective strategy in preventing | | breast cancer. | | | | **Development of ER Antagonists**: | | | | - Building on early observations and the discovery of MER25 | | (Ethamoxytriphetol), a compound initially developed for | | cardiovascular diseases but found to have ER antagonist | | properties, ICI Pharmaceuticals developed ICI 46,474, known as | | Tamoxifen. | | | | - **Tamoxifen** is categorized as a Selective Estrogen Receptor | | Modulator (SERM), which means it acts differently across various | | tissues: | | | | - **Antiestrogenic**: Inhibits estrogen activity in breast | | tissue, preventing cancer cell growth. | | | | - **Estrogenic**: Mimics estrogen effects in bones, helping | | maintain bone density. | | | | ### **Structure of Estrogen Receptor (ER)** | | | | The ER is a protein with three main functional domains, each with a | | specific role: | | | | 1. **Amino-Terminal Activation Function (AF1)** | | | | - **Role**: Kicks off the process that turns genes on | | (transcriptional activation). | | | | - **Location**: At the start of the protein (N-terminus). | | | | 2. **DNA Binding Domain (DBD)** | | | | - **Role**: Grabs onto specific spots on the DNA called | | estrogen response elements (EREs), allowing the ER to control | | (up or down) the activity of certain genes. | | | | - **Feature**: Contains \"zinc fingers,\" which are structural | | motifs that help the ER grip the DNA properly. | | | | 3. **Carboxyl-Terminus Ligand Binding Domain (LBD)** | | | | - **Role**: Catches and holds onto estrogen, triggering the | | receptor to activate. | | | | - **Feature**: Houses another function called AF2, which helps | | pull in other helper molecules needed to get gene | | transcription going once estrogen is bound. | | | | ### **Function of Estrogen Receptor (ER)** | | | | - **How it Works**: When estrogen (like estradiol) enters a cell, | | it travels to the nucleus and binds to the LBD of the ER. | | | | - **What Happens Next**: Upon binding estrogen, the ER changes | | shape (conformational change), which allows it to interact more | | effectively with the cell\'s gene-transcribing machinery. | | | | ### **Estrogen Binding Mechanism** | | | | This is how estrogen binds to the ER: | | | | 1. **Hydrogen Bonds** | | | | - **Interaction 1**: The phenolic OH (a small, oxygen-hydrogen | | group) on estradiol forms bonds with Glu 353 and Arg 394 in | | the ER. Think of this like a hand (estradiol\'s OH) putting | | on a glove (the ER\'s Glu 353 and Arg 394) --- a perfect fit | | that helps the hormone sit just right. | | | | - **Interaction 2**: The 17β-OH group on estradiol links up | | with His 524. This is another snug interaction, securing the | | hormone firmly in place. | | | | 2. **Hydrophobic Contacts** | | | | - **What They Do**: The rest of the estradiol molecule, which | | is largely \'water-fearing\' or hydrophobic, fits into the ER | | like a puzzle piece among various other amino acids. These | | interactions are crucial because they stabilize the entire | | complex, ensuring that estradiol doesn\'t just fall off once | | it\'s bound. | | | | ### **Easy Way to Remember for Your Exam** | | | | Think of the ER as a highly specialized machine designed to \"read\" | | the presence of estrogen. Here's a simple analogy: | | | | - **ER is like a lock**, and **estrogen is like a key**. | | | | - **AF1 is the power switch**, turning on the machine when the | | right key is inserted. | | | | - **DBD is the part of the lock that reads the key\'s | | pattern**---ensuring only the right key can activate the machine. | | | | - **LBD is where the key fits**. It checks the key (estrogen), and | | if it\'s the right one, it tells the power switch to turn on. | | | | - | | | | ### **Examining the Mechanism at Molecular Level** | | | | **Molecular Interactions**: | | | | - These interactions are crucial for the functionality of ER, | | affecting how it can be targeted by drugs like Tamoxifen to | | provide therapeutic effects against breast cancer. The design of | | ER antagonists like Tamoxifen leverages this detailed | | understanding of molecular interactions to inhibit | | estrogen-induced proliferation in breast cancer cells | | selectively. | | | | **Clinical and Pharmacological Significance**: | | | | - Tamoxifen remains a cornerstone of treatment for ER-positive | | breast cancer due to its dual ability to block or mimic oestrogen | | depending on the tissue context, showcasing an excellent example | | of targeted therapy based on receptor biology. | | | | TAMOXIFEN METABOLISM: (learn in metabolism lectures) | | | | ### **Tamoxifen and Its Metabolism** | | | | ### **Metabolism and Mechanism** | | | | - **Tamoxifen as a Pro-drug: Tamoxifen itself is not very active. | | It is a pro-drug, meaning it needs to be metabolized by the body | | to become active. Its effectiveness largely depends on its | | conversion into more potent compounds.** | | | | - **Conversion by CYP2D6: The enzyme CYP2D6 (Cytochrome P450 2D6) | | in the liver metabolizes Tamoxifen into its active | | forms---endoxifen and 4-hydroxytamoxifen. These metabolites are | | significantly more effective in exerting the drug\'s intended | | effects than Tamoxifen itself.** | | | | - **Impact of Genetic Variations: Not everyone metabolizes | | Tamoxifen efficiently. Some individuals have genetic variations | | in the CYP2D6 gene, known as non-functional alleles (like | | CYP2D6\*4 and \*6), which make them poor metabolizers. This can | | lead to reduced effectiveness of Tamoxifen therapy because less | | of the active metabolites are produced.** | | | | ### **Clinical Considerations** | | | | - **Genotyping and Therapy Efficacy: There is an ongoing debate | | about whether patients should be genotyped for CYP2D6 variations | | before starting Tamoxifen therapy. Although genotyping can | | predict poor metabolizers, it\'s not currently recommended as a | | standard practice before treatment. The concern is that these | | genetic variations can diminish the drug's effectiveness.** | | | | - **Drug Interactions: Patients are advised to avoid drugs that | | inhibit CYP2D6 (like bupropion or fluoxetine), as these can | | further decrease the amount of active metabolites formed, | | undermining the efficacy of Tamoxifen.** | | | | - **Resistance and Mutations: Resistance to Tamoxifen can develop | | due to mutations in cancer cells that affect amino acids capable | | of hydrogen bonding with Aspartate 351 in the estrogen receptor. | | This mutation can change the receptor\'s conformation to one that | | supports cancer cell proliferation even without estrogen, making | | Tamoxifen less effective.** | | | | ### **Discovery and Development** | | | | - **Origins in Cardiovascular Research: Tamoxifen was originally | | developed from a compound named MER25, which was being studied | | for cardiovascular diseases but was found to act as an estrogen | | receptor antagonist.** | | | | - **Effect on Estrogen Receptor: As an estrogen receptor (ER) | | antagonist, Tamoxifen blocks the action of estrogen, which is | | crucial for the growth of some types of breast cancers. By | | competing with estrogen for binding sites on the ER, Tamoxifen | | inhibits the proliferation of ER-positive breast cancer cells.** | | | | - **Tissue-Specific Actions: Tamoxifen has a dual nature depending | | on the tissue type. It acts as an anti-estrogen in breast tissue, | | helping to treat and prevent breast cancer. However, it can | | exhibit estrogen-like effects in other tissues like bones, which | | can be beneficial in reducing bone density loss.** | | | | ### **Clinical Use** | | | | - **Oophorectomy and Breast Cancer: The removal of ovaries, a | | procedure known as oophorectomy, decreases the production of | | estrogen, depriving ER-positive breast cancer cells of their | | growth stimulus. This highlights the role of estrogen in these | | cancers and supports the use of estrogen-blocking therapies like | | Tamoxifen.** | | | | | | | | - **Drug-Food Interactions: Certain drugs and foods can inhibit | | CYP2D6, which is undesirable for patients on tamoxifen. These | | inhibitors prevent tamoxifen from being metabolised into its more | | active form, endoxifen, reducing the drug's effectiveness.** | | | | | | | | - **Grapefruit and grapefruit juice: Can inhibit CYP2D6, | | potentially reducing the effectiveness of tamoxifen by lowering | | its conversion to the active metabolite, endoxifen.** | | | | - **Seville oranges: Similar to grapefruit, contain compounds that | | inhibit CYP2D6.** | | | | - **Certain spices: Such as turmeric, might inhibit CYP2D6.** | | | | - **Green tea: High concentrations of green tea have been reported | | to inhibit CYP2D6.** | +-----------------------------------------------------------------------+