Old exam october 2022 Flashcards
A. The development of modern pharmaceuticals that are effective in treating disease is a rather long, complex, and costly process that consists of different phases.
- Describe the overview of the main phases and processes of drug discovery and drug development that are necessary to go from a therapeutic concept to a final product
After deciding the therapeutic concept, e.g. what you want to treat you start by identifying the target. The target may be a protein, enzyme, receptor, or other molecule that is involved in a biological pathway or process that contributes to the disease. It can be selected through biological knowledge, screening of compound libraries etc.
After identifying a potential target, the next step is to validate its relevance to the disease. This can be done through in vitro assays, animal studies or patient samples.
For lead finding this involves screening large libraries of compounds to identify those that show activity against the target. Lead finding may involve a variety of approaches, such as high-throughput screening for example. High-throughput screening is screening large libraries of compounds to test sometimes thousands of compounds at the same time. The goal of HTS is to identify a small number of lead compounds that shows activity against target of interest to then be optimized.
Lead optimization includes improving their efficacy, safety, and pharmacokinetic properties. This involves modifying the structure of the lead compounds through iterative rounds of synthesis and testing to identify compounds with improved activity, selectivity, and pharmacological properties. This will then lead to a candidate drug.
Preclinical development includes testing the drug candidate in laboratory and animal studies to examine the efficacy, safety, toxicity and pharmacokinetics of the drug.
Clinical development includes testing it on humans in a series of clinical trials. Phase 1 on a small group of healthy individuals to determine the drugs safety and side effects, phase 2 on a large number of patients with the disease to determine the efficacy and further evaluate safety. Phase 3 is a even larger trial that is meant to determine efficacy, safety and optimal dosage.
Regulatory approval is submitting the data from the previous stages for approval.
B. Today, there are mainly two basic strategies for drug discovery.
- Activity centered discovery and (2p)
- Target centered discovery. (2p)
Explain these two strategies and give an example of one drug that have originated from each of these two strategies.
Activity based discovery means identifying a small molecule that has an effect either on cultured cells or in animal models and then optimize the properties of the molecule and solve the mechanism of action. Salicylic acid from the bark of vitpil was discovered by activity based discovery.
Target based discovery you first begin to understand the disease mechanism, identify a “druggable” target for example an enzyme, a receptor, ion channel or nuclear receptor, and show that the target is coupled to disease mechanism in for example animal models. Then one identifies a lead series and optimizes the properties of these lead molecules. Imatinib is an anticancer drug that was discovered by target based discovery.
A. Historically, conventional small molecule drugs have been discovered from synthetic chemistry or from natural products.
- Which of the following drugs has been identified from synthetic chemistry or from natural products?
o Heparin (0.5p)
o Antiepileptic drugs (0.5p)
o Sulfonamides (0.5p)
o Ciclosporin (0.5p)
Natural compounds:
o Heparin
o Sulfonamides
o Ciclosporin
Antiepileptic drugs some are natural and some are synthetic
B. After discovering a potential drug candidate, the mechanism of action of the drug, as well as the target for the drug, needs to be confirmed.
- How can the mechanism of action and the potential target be validated? (2p)
In vitro assays: In vitro assays involve testing the drug candidate’s activity against isolated target proteins or cell cultures. By exposing the drug candidate to specific protein targets or cells, researchers can observe its effects and determine if it interacts with the intended target. These assays can help identify the mechanism of action and provide preliminary evidence for target validation.
Animal models and in vivo studies: Animal models are used to evaluate the efficacy of the drug candidate in a whole organism and assess its impact on disease-related processes. By administering the drug candidate to animal models with the targeted disease or condition, researchers can observe its effects on physiological parameters, biomarkers, or disease progression. These studies can provide further evidence for the mechanism of action and target validation.
C. Drug companies often have access to large libraries of molecules.
- What is the process used to evaluate these compounds as potential drugs? (1p)
- Briefly describe this process. (2p)
High throughput screening.
Assay Development: The first step in high-throughput screening is to develop an assay that can measure the desired activity or interaction related to the target of interest. This could involve designing a biochemical or cellular assay that reflects the function or activity of the target.
Compound Library: A diverse collection of chemical compounds, often referred to as a compound library, is assembled for screening. These libraries can contain thousands of different compounds.
Screening Process: The compound library is screened using an automated system that can rapidly and accurately dispense small amounts of each compound into the assay wells. Robotic systems are commonly used to handle the high volume and perform the screening process. The compounds are typically tested at multiple concentrations to assess their potency.
Data Acquisition and Analysis: As the compounds are tested, the screening system collects data on the response of the assay to each compound. This could be measured as a change in fluorescence, absorption etc.
Hit Confirmation and Validation: Compounds that exhibit activity or interaction with the target in the primary screening are referred to as “hits.” These hits are further confirmed and validated through secondary screenings, which involve repeating the assay with the hit compounds to ensure reproducibility and eliminate false positives. Additional assays may be employed to evaluate the specificity, potency, and other properties of the hit compounds.
Hit-to-Lead Optimization: Once confirmed, the hit compounds undergo further optimization through medicinal chemistry and structure-activity relationship studies. This involves modifying the chemical structure of the hit compounds to improve their potency, selectivity, pharmacokinetic properties, and other drug-like characteristics. The goal is to identify lead compounds that have the potential to become drug candidates.
D. Clinical trials can be divided into 4 different phases. Explain the objective and purpose of these four phases, in more detail as compared to question 1. (4p)
Clinical trials are research studies that are conducted to evaluate the safety and effectiveness of medical interventions, such as drugs, vaccines, or medical devices, in humans. Clinical trials are typically divided into four different phases, each with its own objective and purpose.
Phase 1:
The primary objective of Phase 1 clinical trials is to evaluate the safety and tolerability of the medical intervention in a small group of healthy volunteers. This phase also aims to determine the appropriate dosage and identify any potential side effects of the intervention. Phase 1 trials typically involve 20 to 100 participants and can last several months.
Phase 2:
The primary objective of Phase 2 clinical trials is to evaluate the effectiveness and safety of the medical intervention in a larger group of patients with the condition or disease targeted by the intervention. This phase also aims to determine the optimal dosage and identify any potential side effects of the intervention. Phase 2 trials typically involve several hundred participants and can last up to two years.
Phase 3:
The primary objective of Phase 3 clinical trials is to confirm the effectiveness and safety of the medical intervention in a much larger group of patients with the targeted condition or disease. This phase also aims to identify any rare or long-term side effects of the intervention. Phase 3 trials typically involve several thousand participants and can last several years.
Phase 4:
The primary objective of Phase 4 clinical trials is to monitor the long-term safety and effectiveness of the medical intervention after it has been approved and made available to the general population. This phase also aims to identify any rare or long-term side effects of the intervention that may have been missed during the earlier phases. Phase 4 trials involve a large number of participants and can last for many years.
How would you categorize the following drugs and their toxic side-effects? Where in the matrix above would you place them?
- CNS depression from Ethanol (0.5p)
- Liver cirrhosis from Ethanol (0.5p)
- Gastrointestinal damage from Arsenic (0.5p)
- Skin cancer from Arsenic
Local or systemic chronic or acute
CNS depression from Ethanol: Acute and systemic.
Liver cirrhosis from Ethanol: Chronic and systemic.
Gastrointestinal damage from Arsenic: Acute and local
Skin cancer from Arsenic: Chronic and local
B. Many different models for studying toxicity are used during drug discovery and drug development. Briefly, describe the three general models, in silico, in vitro and in vivo, (including why/how/when they are used for studying toxicity) and give at least one specific example of an assay that is used for each general model.
- in silico, max 250 words (2p)
- in vitro, max 250 words (2p)
- in vivo, max 250 words (2p)
In Silico:
In silico methods use computational modeling and simulations to predict the potential toxicity of compounds before they are synthesized or tested in animals. These methods are cost-effective, fast, and provide an efficient way to screen a large number of compounds in a relatively short amount of time. In silico models can also provide insights into the mechanism of toxicity and help in the rational design of safer drugs.
In Vitro:
In vitro methods involve testing the toxicity of a compound in a controlled environment outside of a living organism. These methods use cell cultures or isolated tissues to determine the potential toxicity of a compound. In vitro assays provide a reliable and cost-effective way to screen compounds for toxicity, and they can also provide mechanistic insights into the toxic effects of compounds.
In Vivo:
In vivo methods involve testing the toxicity of a compound in living organisms such as mice, rats, or non-human primates. In vivo methods provide the most relevant information on the potential toxicity of a compound as they take into account the complexity of biological systems and the potential interactions between the compound and various organs and tissues.
C. Investigating and understanding drug specificity, both during and after drug discovery/development, is an important task. Especially if drugs are targeting specific receptors such as the ERBB2 receptor and not the ERBB1 receptor.
- Visualize using dose-response curves one drug that is specific for the ERBB2 receptor and not the ERBB1 and a full agonist. Also visualize using dose-response curves one drug that is not specific for neither ERBB1 nor ERBB2, but rather targets both receptors as full agonists.
There are multiple classes of NPS drugs
- Describe three of these phases that are often lacking and describe the danger of skipping these phases when releasing a new drug. Denoted the three different areas that you describe as 1, 2 and 3.
Preclinical Phase:
The preclinical phase is the initial stage of drug development that involves extensive laboratory testing and animal studies before human trials can begin. This phase aims to assess the drug’s safety, pharmacokinetics, and potential efficacy. Skipping or neglecting this phase can have severe consequences. Without preclinical testing, there is a higher risk of exposing human subjects to potentially harmful or ineffective drugs. Animal studies provide valuable insights into a drug’s toxicity, potential side effects, and overall safety profile. By skipping this phase, unknown risks and unforeseen adverse reactions may arise during clinical trials, putting human lives at risk.
So both medical chemisists optimizing the drug to decrease side effects, increase safety etc and also animal testing prior to testing on humans.
Phase I Clinical Trials:
Phase I clinical trials are conducted on a small number of healthy volunteers to evaluate the drug’s safety, dosage range, and potential side effects. The primary goal of this phase is to determine the drug’s tolerability and establish a safe dosage range for subsequent trials. Neglecting Phase I trials can lead to serious safety concerns during later phases. Phase I trials are designed to uncover any adverse reactions or unexpected toxicities in a controlled environment. If this phase is skipped, there is a higher probability of administering an unsafe or poorly tolerated dose to patients in later stages, potentially causing harm or even fatalities.
B. There are multiple classes of NPS drugs, either they are divided into groups based on their pharmacological structure or their mechanism of action. Please, indicate which group (1-4) has either of the mechanisms of action described below: (4p)
Groups of compounds:
1. Synthetic opioids
2. Synthetic cannabinoids
3. Cathinones
4. Phenethylamines
Main mechanism of action:
A. Inhibition of the re-uptake transporters for serotonin, dopamine and/or Noradrenaline.
B. Activation of the μ-receptor
C. Binds to traceamine-associatedreceptor 1 (TAAR1) and inhibits vesicular monoaminetransporter 2 (VMAT2) in monoamineneurons.
D. Activation of the CB1 receptor
Cathinones:
Cathinones are a class of synthetic stimulant drugs that are structurally similar to cathinone, a natural stimulant found in the khat plant. The mechanism of action of cathinones involves the release of neurotransmitters such as dopamine, serotonin, and norepinephrine from presynaptic nerve terminals. Cathinones act as reuptake inhibitors of these neurotransmitters, leading to an increase in their concentration in the synaptic cleft and enhancing their stimulatory effects.
In particular, cathinones have a high affinity for the dopamine transporter (DAT) and the serotonin transporter (SERT), blocking the reuptake of these neurotransmitters and increasing their extracellular concentration. This results in a feeling of euphoria and increased energy levels. Cathinones also have some activity at the norepinephrine transporter (NET), contributing to their stimulant effects.
Synthetic Cannabinoids:
Synthetic cannabinoids are a class of drugs that act on the same receptors as delta-9-tetrahydrocannabinol (THC), the active ingredient in cannabis. These drugs are structurally diverse, and their mechanism of action varies depending on the specific compound. However, in general, synthetic cannabinoids act as full agonists at the cannabinoid type 1 receptor (CB1) and the cannabinoid type 2 receptor (CB2).
Activation of CB1 receptors in the brain leads to a range of psychoactive effects, including altered perception, impaired memory, and euphoria. CB2 receptors are mainly found in immune cells and may play a role in inflammation and pain regulation.
Synthetic Opioids:
Synthetic opioids are a class of drugs that act on opioid receptors in the brain, spinal cord, and other organs. These drugs mimic the effects of endogenous opioids such as endorphins, enkephalins, and dynorphins. Synthetic opioids can be classified into three main categories: full agonists, partial agonists, and antagonists.
Full agonists, such as fentanyl and morphine, activate opioid receptors fully and produce strong analgesic and euphoric effects. Partial agonists, such as buprenorphine, activate opioid receptors but have a lower efficacy than full agonists and produce less euphoria and respiratory depression. Antagonists, such as naloxone, block opioid receptors and can reverse the effects of opioid overdose.
Activation of opioid receptors leads to a range of effects, including pain relief, sedation, and euphoria. Opioid receptors are mainly found in the central nervous system, where they modulate the transmission of pain signals and affect mood and behavior. The mu-opioid receptor is the primary target of opioids, and activation of this receptor leads to the majority of opioid effects.
Biopharmaceuticals or Biologics are drugs made from oligonucleotides, proteins, peptides, or polysaccharides. Major blockbuster drugs are today Biologics.
A. Oligonucleotides for gene expression interference is considered viable and promising for a wide range of diseases. However, the use of oligonucleotides has major challenges.
- Explain two problems that are associated with gene therapy.
Delivery Challenges: One significant challenge in gene therapy is efficiently delivering oligonucleotides to target cells or tissues. Oligonucleotides, including small interfering RNA (siRNA) or antisense oligonucleotides (ASOs), are large molecules that need to overcome various barriers to reach their intended site of action. These barriers include cellular membranes, extracellular matrices, and clearance mechanisms in the body. Additionally, specific tissues or organs may be difficult to target due to their location or physiological properties. Developing effective delivery systems that can protect the oligonucleotides, facilitate their cellular uptake, and ensure their release at the desired site remains a significant hurdle in gene therapy.
Off-Target Effects: Another challenge associated with gene therapy using oligonucleotides is the potential for off-target effects. Oligonucleotides are designed to target specific genes or gene sequences to interfere with their expression. However, unintended interactions or binding to other genes or non-targeted regions of the genome can occur. This can lead to off-target effects, such as the suppression or alteration of unintended genes, potentially causing unwanted side effects or toxicity. Achieving high specificity and minimizing off-target effects is crucial to ensure the safety and efficacy of oligonucleotide-based gene therapy approaches.
D. Proteins and peptides have been used as drugs for a rather long time and were previously isolated from natural sources, but today most protein drugs are derived from recombinant DNA-technology.
- Plasmids are very popular genetic vectors for protein expression. Draw a general schematic of a plasmid and explain its key elements.
Promoter: This is a DNA sequence that is recognized by RNA polymerase and initiates transcription of the gene of interest. The promoter can be specific to certain types of cells or tissues, or it can be a strong, constitutive promoter that is active in many cell types.
Start codon: This is the DNA sequence that signals the start of translation of the gene of interest. The most common start codon is ATG.
Gene of interest: This is the DNA sequence that encodes the protein of interest that is to be expressed in the host cell.
Stop codon: This is the DNA sequence that signals the end of translation of the gene of interest. There are three possible stop codons: TAA, TAG, and TGA.
Terminator: This is a DNA sequence that signals the end of transcription of the gene of interest. It is usually located downstream of the stop codon.
Selection marker: This is a DNA sequence that confers resistance to a particular antibiotic or other drug. This allows for selection and isolation of cells that have taken up the plasmid and are expressing the gene of interest.
Origin of replication: This is a DNA sequence that allows the plasmid to replicate independently of the host cell’s chromosomal DNA. This ensures that the plasmid is maintained in the host cell and that the gene of interest is continuously expressed.
Explain what a selection marker could be and why it is crucial for recombinant DNA-technology using plasmids.
A selection marker is a gene that confers a selectable phenotype, allowing researchers to distinguish cells that have taken up a recombinant plasmid from those that have not. In recombinant DNA technology, selection markers are used to ensure that cells that have taken up the desired recombinant plasmid are selected for and propagated, while cells that have not taken up the plasmid are eliminated.
Selection markers can confer a variety of selectable phenotypes, such as antibiotic resistance or resistance to toxic chemicals. For example, a plasmid may contain a gene that confers resistance to the antibiotic ampicillin. When the plasmid is introduced into bacteria, only those bacteria that have taken up the plasmid will be able to grow on a culture medium containing ampicillin, while bacteria that have not taken up the plasmid will be unable to grow. This allows researchers to selectively propagate cells that contain the desired recombinant plasmid and eliminate those that do not.
In recombinant DNA technology, plasmids are often used as vectors to introduce foreign DNA into cells. By including a selection marker in the plasmid, researchers can ensure that only cells that have successfully taken up the plasmid are selected for and propagated, increasing the efficiency of the cloning process. Selection markers are therefore crucial for recombinant DNA technology, as they allow researchers to identify and propagate cells that contain the desired recombinant plasmid, while eliminating those that do not.
- Explain why many plasmids include two selection markers.
Plasmids used in genetic engineering often include two selection markers, one for selection and one for counter-selection. This is done to increase the efficiency and accuracy of the selection process and to reduce the occurrence of false positives.
The selection marker is the gene that confers a selectable phenotype, allowing cells that have taken up the plasmid to be identified and selected for. For example, a selection marker might be a gene that confers antibiotic resistance or resistance to a toxic chemical.
The counter-selection marker is a gene that allows cells that have lost the plasmid to be identified and eliminated. For example, the counter-selection marker may be a gene that confers sensitivity to an antibiotic or toxin that the cells would normally be resistant to due to the presence of the plasmid. This means that cells that have lost the plasmid will not survive when grown on a medium containing the counter-selection agent.
By including both a selection marker and a counter-selection marker, researchers can increase the accuracy of the selection process. Cells that have taken up the plasmid and have the desired phenotype (e.g., antibiotic resistance) will grow on the selection medium, while cells that have lost the plasmid and have the undesired phenotype (e.g., antibiotic sensitivity) will be eliminated on the counter-selection medium. This reduces the occurrence of false positives, which can result from cells that have taken up the plasmid but do not have the desired phenotype, and increases the accuracy and efficiency of the genetic engineering process.
