Konstantinos Beis - Antibodies + Drug Design Flashcards

Question

What are some examples of groups that can be used to re-create tetrahedral intermediates of transition states?

Answer 1

Once we have identified T.S. analog we have our Hapten which can bind an antibody BUT haptens are too small to illicit an immune response Thus, in order to overcome this problem we design a linker to join our hapten to a carrier protein - big enough to illicit an immune response

Answer 2

Once we have our hapten conjugated to a carrier protein it is time to **immunize** **Conventional Antibody production** Inject/immunise mice with compound --\> illicits an immune response --\> kill the mice and remove spleen (B-cells located) --\> isolate antibodies (using antiserum?) that can catalyze the reaction Problem – Killed source - if you are successful in obtaining an antibody you can not make more **Monoclonal Antibody Production** Immunize mice --\> kill mice --\> remove spleen --\> mix spleen cells and myeloma cells (M.C. LACK HGPRT – important for nucleic acid synthesis) in HAT medium --\> unless fusion has taken place the cells will not survive --\> isolate different clones --\> test and identify clones that have catalytic activity Useful technique as we retain a source of the antibodies --\> immortal cell line

Answer 3

Determine kinetic parameters of the antibodies - assess which antibodiy is most catalytically active Examine parameters such as... **Km** – Antibodies binding Hapten – affinity **Kcat** – reaction per unit time **Kcat/ Km** – catalytic efficiency at low concentration From the parameters we can identify 15A10 as having significant activity

Answer 4

Toxicity assays - Cocaine Toxicity in the Rat Question we are trying to answer - Do these antibodies help with survival rates? Examine rats’ survival after infusion of an LD90 (lethal dose to kill 90% of mice) (16 mg/kg) cocaine with abzyme The effect of mAb 15A10 on survival was significant at - Graph shows that at higher concentrations of mAb 15A10 there was a higher survival rate

Answer 5

Both cocaine and TS analog were crystallized in presence of eitehr cocaine and TS analog --\> both cases they bound in the hypervariable regions - binding to CDR loops Interestingly, they also allowed the antibody to react with cocaine for a few minutes before capturing X-ray diffraction data and they were able to show... The crystal structure with the product – Ecgonine methyl ester and benzoate in the active site - breakdown products of cocaine

Answer 6

Examining active site of antibody there was a lack of normal residues associated with acid-base catalysis – instead replaced by tyrosine residues that form hydrogen bonds **Mechanism** 1. Cocaine enters active site - stabilized by hydrogen bonds 2. Activated Water molecule performs nucleophilic attack on carbonyl carbon 3. Tetrahedral intermediate formed – -ive charge is stabilized by tyrosine 4. Followed by its collapse --\> release of two products Simpler mechanism than normal acid-base

Answer 7

Prodrug – Precusor molecule is introduced into the body which only forms the active drug once it is metabolised - Useful as drug itself can be highly toxic itself - limit side effects - Able to deliver high concentrations of active drug - protects against rapid metabolism and clearance - Improve targetting of drug to specific regions

Answer 8

**Basic idea** --\> is that you introduce prodrug and catalytic antibody into patient --\> the catalytic antibody is targeted towards a specific tissue, hence when the prodrug enters it can become metabolized by the catalytic antibody into the active form **Benefits?** 1. Minimizes toxicity 2. Allows for drug targeting 3. More complex chemistry to be performed (harder to do with normal chemistry) - more verstaile tool In this case we would have to design a abzyme against the prodrug TS --\> to favour formation of active drug

Answer 9

1. Haptens fail to generate antibodies that catalyse the desired reaction 2. Haptens may closely resemble the final product (stabilize it) - slow product release or inhibition – preventing the antibody for catalyzing more reactions 3. It might be difficult to synthesize the desired hapten (chemistry) 4. The catalytic efficiency depends on the solvent exposed binding site? 5. Introduction of foreign antibody might elicit immune response

Answer 10

Use chimeric or humanized antibodies 1. Chimeric consist of variable regions (VL and VH) derived from a mouse and constant regions derived from human. ~65% human 2. Humanized therapeutic mAbs are predominantly derived from a human source except for the CDRs, which are murine. \>90% human

Answer 11

1. Catalytic Abs can be raised against specific haptens 2. Catalyse desired reactions 3. Consider different analogues, linkers and carrier proteins 4. Catalytic Abs have the potential to treat addictions

Answer 12

Antibody–Drug Conjugates (ADC) --\> Antibodies used to deliver a drug to a specific site

Answer 13

Cancers (many different types!) can be treated by: 1. Surgery 2. Chemotherapy – several side effects and resistance to anticancer drugs 3. Radiotherapy 4. Hormone therapy 5. Combination of therapies is usually required **Recurring problem** - target/impact healthy cells – leads to side effects

Answer 14

**Antibody–Drug Conjugates** (ADCs) are monoclonal antibodies or antibody fragments attached to biologically active molecules through chemical linkers with labile bonds **Advantage** Deliver highly cytotoxic agents directly to tumour cells without affecting other dividing cells in the body – targeting antigens on Tumour cells When the drug is bound to the antibody, the chemotherapeutic ‘‘payload’’ no longer circulates systemically and is therefore **doesn’t negatively impact healthy cells**

Answer 15

Regular antibody conjugated to a drug with a linker Antibody binds to cell surface receptor --\> internalization --\> release of payload --\> block cellular process in nucleus or cytosol leading to apoptosis of cancer cells

Answer 16

**Mechanism** An ADC binds to an antigen on the surface of a cancer cell and then internalises, after which the highly cytotoxic payload molecule is released, typically by lysosomal cleavage **Requirements** ADC needs to retain the selectivity of the original monoclonal antibody (bind to the specific antigen) while being able to release the attached cytotoxic payload in concentrations high enough to kill the targeted tumour cells. **Componenets** Three key components to an ADC: the **antibody** (which antigen will it bind), the **linker** (Cleaved/non-cleaved) and the **payload** (Pathway you desire to inhibit?)

Answer 17

1. ADCs are designed to **directly target and kill cancer cells**, and so the antibody has to be able to recognise and bind to its corresponding **antigen localized on the tumour cell.** 2. Once bound to the antigen, the entire antigen–ADC complex is then internalized through **receptor-mediated endocytosis.** 3. The internalization process proceeds with the formation of a clathrin-coated early endosome, containing the ADC–antigen complex --\> release of the ADC from the receptor which is recycled 4. Once inside the lysosome, the ADC is degraded and free cytotoxic payload released into the cell, leading to cell death. 5. The mechanism of cell death will depend on the type of cytotoxic payload.

Answer 18

Another crucial requirement is to ensure that a sufficient concentration of payload reaches the interior of the cancer cells to guarantee their death It is estimated that, even if the overall mechanism of action of an ADC works at an efficiency of 50%, **only 1–2% of the administered payload will reach the tumour cells** - a lot of it will be degraded Therefore, it is important that the chosen payload is sufficiently cytotoxic to exert an effect at very low concentrations

Answer 19

- Wider therapeutic windown --\> less likley to be toxic as there is a higher maximum tolerated dose and lower minimum effective dose

Answer 20

1. Antigen Selection 2. Cleavable linker 3. Non-cleavable linker 4. Site of conjugation

Answer 21

A/B) Shows relationship between the DAR and rate of degradation --\> the more drugs we conjugate to the antibody – the quicker the rate of clearance (more susceptible to protease) but the more drug we attach the greater the payload --\> trade-off Hence, there is an optimal mid-point which yields that highest efficacy (DAR 3.5-4)

Answer 22

**Cleavable linkers** Cleavable linkers utilise the differences in conditions between the bloodstream and the cytoplasm within tumour cells Take advantage of the **change in environment** once the ADC–antigen complex has internalized it triggers cleavage of the linker and release of the active payload Cleavable linkers are divided into three main sub-categories: (1) Acid-labile (e.g., hydrazones) (2) Reducible (e.g., disulphides) (3) Enzyme-cleavable (e.g., peptides).

Answer 23

**Non-cleavable linkers – Why are they used?** 1. Drug (usually) remains attached to mAB - not effected by pH, reducing environment or enzymes 2. In the lysosome – mAB is degraded by compound + linker is not! 3. Increased plasma stability 4. Lower risk of systemic toxicity due to premature release of the payload 5. Better therapeutic window, with improved stability and tolerability

Answer 24

1. Prevent modifying antibody to the point where they induce an immune response 2. DAR --\> typical average DAR of 3.5 or 4 - Species that have a DAR of more than 4 have been shown to lead to lower tolerability, higher plasma clearance rates and decreased efficacy in vivo 3. Retaining antibody selectivity (antigen binding) Conjugation Example:

Answer 25

1. Thiosuccinimide linkage, which is formed through the reaction of **thiols** and alkyl maleimides --\> thiosuccinimide formation is slowly reversible under physiological conditions 2. Conjugation to the *terminal amines of lysine residues* using an amide ester

Answer 26

1. Engineered Cys – Thiomab 2. Insertion of unnatural amino acids – tight regulation of our payload with a specific amino acid that is not found naturally 3. Enzyme-assisted ligation 4. Glycan remodelling and glycoconjugation - remember that antibodies are glycosylated 5. Amino‑terminal engineered serine --\> problem may change overall properties of antibody 6. Native cysteine rebridging 7. Highly loaded ADCs at specific sites

Answer 27

Market Name - Kadcyla T-DM1 - DM1 conjugated with trastuzumab (antibody against HER2) DM1 --\> Binds tubulin to cause mitotic arrest and cell death **Research findings?** Conjugates were evaluated for in vivo pharmacokinetics and antitumor activity In vivo studies revealed that the higher the linker stability the higher the antitumor activity T-DM1 was approved in 2013 for HER2-positive breast cancer treatment

Answer 28

1. ADCs are potent drug delivery tools 2. Low toxicity – relative to other cancer treatments 3. Care should be taken with linker design, payload choice 4. Linker can be cleavable or not 5. Bystander effect desirable or not

Answer 29

Remember that there is a large number of small molecules (e.g., vitamins, hormones) circulating through the body --\> counterproductive for immune system to illicit an autoimmune response

Answer 30

**Cannot no diffuse out through the membrane** forcing the drug to remain in the cell + similarly it **can’t diffuse into the nucleus** (through the nuclear membrane) remains in the cytoplasm instead of moving out of the cell or into the nucleus – helps with the concentrating the active drug Why? Linker is fairly hydrophilic + increases size by 500-800 Da - repels from the hydrophobic membrane + the size inhibits free diffusion

Answer 31

**Trial and error of changing DAR** - how many drugs you load onto the antibody. If we overload with hydrophobic molecules (ADC - drugs) – the antibodies will precipitate together

Answer 32

Cancer cells have efflux protein in the membrane that can efflux the drugs --\> problematic if our drug enters and does not induce apoptosis it allows our cancer cell to develop mutations (mutations that benefit cell survival) in the transporter which will allow it to be exported --\> like antibiotic resistance idea This is exacerbated by the fact that the cancer cells divide quickly facilitating this rapid evolutionary process Thus.... making it favourable to use multiple therapeutics to maximize the effectiveness

Answer 33

You want to choose your conjugate so that it will be most effective at targeting the specific process that you are interested in inhibiting For Example... Target soluble enzyme --\> you will want your conjugate drug to be hydrophilic Target a membrane protein --\> you will want your conjugate drug to be hydrophobic Note - normally we are most interested in soluble enzymes within cancer cells.

Answer 34

The drug will enter the metabolism --\> The human body can detoxify these compounds – making them inactive

Answer 35

Profiling of antigens that are expressed differentially in cancer cells relative to healthy cells --\> Mass spec profiling Also, worth looking at glycosylation – at the antigens tend to be heavily glycosylated - possible target

Answer 36

1. High Throughput Screening (HTS) – wet lab setup - most commonly used by Pharma But... - Expensive - False positives - Assay variability or errors in data 2. Virtual Screening (VS) --\> main focus of lectures 3. Combination of HTS and VS

Answer 37

1. Expensive robotics are required to screen for candidates – large scale 10⁷ 2. Very robust assay required – Biological activity meter 3. Interaction analysis of successful hits - between drug and target 4. Complement HTS with structural data and using in-silico methods

Answer 38

CADD methods are classified into ligand-based methods (LBDD) and structure-based (SBDD)… **Structure-based methods** require the 3D information of the target to be known - 3D conformation of target protein of interest **Ligand-based methods** are used when the 3D structure of the target is not known. Rather... They use information about the molecules that bind to the target of interest. Hits are identified, filtered and optimized to obtain potential drug candidates that will be experimentally tested in vitro - information about the substrate or inhibitor

Answer 39

1. Start with library of compounds 2. Filter the library depending on Mw, physical properties, etc. 3. Virtual HTS - LBDD or SBDD **LBDD - focus of lecture** 1. Searching for similarity in library towards ligand (known) of interest – identify hits 2. Synthesize or purchase hits 3. Evaluate in the lab a) Biological activity - refine b) No biological activity - Start again

Answer 40

Takes into account different kinds of molecular information of the known ligand such as 1. 2D molecular fingerprint 2. 3D molecular shape 3. Molecular and electronic properties 4. 3D pharmacophore Image shows the two different LBDD strategies

Answer 41

1. Similarity searching 2. Pharmacophore mapping 3. Machine learning

Answer 42

Reasoning for using bits --\> facilitates processing for computer

Answer 43

Pharmacophore is the **substructure** of a molecule that is **responsible for its pharmacological activity** --\> A set of geometrical constraints between specific functional groups that enable the molecule to have biological activity Basically, similar functional groups in similar organisation but the scaffold of the protein changes --\> this is how drug companies overcome patents In this search both valence angles and torsion angles can be included

Answer 44

When searching we may have reference compounds of known activity, which we search against a database that contains other actives and decoy compounds **using our filters** **A good similarity measure** will cluster the known actives at the top of the ranking --\> appropriate filters Known **active compounds not appearing** in ranked list --\> suggestive that we have to adjust our search parameters

Answer 45

Nope! We assume that all (or the majority) of the known actives bind to the same location of the protein 1. **Generate our pharmacophores** --\> identify **pharmacophoric features** (hydrogen bond donors and acceptors, lipophilic groups, charges) + their **geometrical arrangement** of all actives that match with a low-energy conformation 2. **Databse search** --\> find all molecules in a database that can match it in a low-energy conformation (energetically possible) Scaffold-hopping possible --\> needs to match the pharmacophore

Answer 46

- Most methods assume similar binding modes - One or more rigid molecules are preferred - The ligands should be diverse (otherwise too many common features that are not involved in binding) Procedure 1. **Prepare molecules** (e.g. tautomeric form, protonation state), generate 3D structure and conformations (if required) 2. Use pharmacophore software/tool to generate pharmacophores (depending on input it may be biased or unbiased) 3. Select preferred pharmacophore model(s) and validate them experimentally --\> are they really the core functional groups in binding our protein?

Answer 47

1. Pharmacophoric features in each ligand are identified (Donors, acceptors, hydrophobic groups, etc) 2. Ligands aligned so the corresponding features are overlaid 3. Conformational space explored 4. Scoring system: number of features, goodness of fit to features, conformational energy, volume of the overlay, etc

Answer 48

Basically, the way in which we generate our model should resemble the way in which the database was created - same feature definitions (functional elements) + protocol used to define pharmacophores --\> **ensures accuracy** **For example,** What tolerance should we use? What distance between two pharmacophores is acceptable?

Answer 49

**Quantitative-Structure Activity Relationships** (QSAR) tries to establish quantitative relationships between descriptors and the target property capable of predicting activities of novel compounds Basically using information about the chemical behaviour of each functional element to help predict activities of the new compounds Require descriptors that accurately convey chemically-relevant information to the **machine learning models**

Answer 50

**Takeaway message** --\> from assays and previous knowledge (chemically behaviour), the machine learning model will predict the activity of the novel compound After the prediction you have to test it out experimentally

Answer 51

Key difference between LBDD and SBDD is that the structure of the protein is known --\> Either **structure-based methods** (X-ray etc.) or **homology modelling** can be used to determine structure Note - More commonly used relative to LBDD 1. Start with Library 2. Filter compounds 3. Virtual HTS – Use different types of docking 4. Score Hits 5. Evaluate using experimental techniques 6. Process can be repeated for optimization

Answer 52

- Solve the structure of the target at high resolution – X-ray or Cryo-EM (resolution is improving) or alternatively use homology modelling (known homologous proteins) - Solve the structure in the presence of the substrate if possible --\> determining binding interaction – Alternatively, use inhibitor - Use this structure as a template for screening of small molecules - Redesign the molecules to fit and/or bind better

Answer 53

virtual HTS - Perform docking in-silico After In-silico screening... - Test activity (enzymatic and pharmacokinetics) - Solve crystal structures to verify binding mode

Answer 54

- The energetics of protein-ligand interactions are complicated - Both proteins and ligands can be quite **flexible** - dynamics adds extra complexity - Many target-binding ligands in-silico are not good drug candidates - The structures of many important drug targets are difficult to determine – Lack of structural information e.g. Many GPCRs - Plus structures need to be **high resolution** - in order to determine all the Ligand-protein interactions

Answer 55

Correlations that are constructed between the **features of chemical structure** in a set of candidate compounds and **biological activity**, such as potency, selectivity and toxicity. Basically... Linking chemical structurs/groups to biological activity - kinda like pharmacophores **How can these biologically relevant groups be identified?** 1. X-ray crystallography can be used to identify important interactions between drug and protein 2. Test for biological activity with modified compounds and comparing them with the original compound – modify groups and check impact on activity a) Modification shows a significant lower activity, then the group that has been modified must be important b) If the activity remains similar, then the group is not essential

Answer 56

**SBDD follows the Lipinski’s rule of Fives** - Rule(s) of thumb that were created to predict the likeliness that a drug would be orally active in humans 1. MW less than 500 – easier diffusion across membrane + easier to modify 2. Fewer than 5 H-bond donating groups 3. Fewer than 10 H-bond accepting groups 4. LogP between -1 and +5 – experimentally determined using the partition of molecule in octanol and H2O – indicates hydrophobicity

Answer 57

1. In silico to **identify potential lead compounds from a database** - From which we score, rank and filter a set of chemical structures 2. **Pose prediction** – What will the algorithm – predict the interaction at the active site - Requires to have a high resolution structure - Identify key residues in the binding site - Dock compound by selecting proper stereochemistry

Answer 58

Ligand is treated as a **rigid structure** and **only** the **translational** and **rotational** degrees of freedom are considered Means that... Different ligand conformations are docked separately plus... Protein is considered rigid **Goal** - satisfy as many of the **positional** and **chemical descriptors** of the active site as possible **Process** 1. Determine **plausible conformations** of ligand (prior to docking) - match binding site shape --\> geometrical descriptors are combined with chemical and electrostatic descriptors - needs to match shape and chemical nature of active site 2. Orient ligand in the active site using simple geometry descriptors, like spheres or triangles --\> Satisfy stereochemistry, no clashes 3. Rank/score the different poses based on receptor affinity

Answer 59

**First principle scoring** – Parameters - Molecular Mechanics force field. Force fields typically contain **intra-molecular terms** from the ligand: Bond lengths, Bond angles, Dihedral terms And **inter-molecular terms**: Van der Waals contacts (non-polar) Electrostatic interactions (polar) All together - Ebind = EIntra + Enonpolar + Epolar **Empirical scoring** – similar to first principal score - looks at DG Score’s ligands very rapidly ΔGbind= ΔG0 + ΔGpolar · Σf(Complex) + ΔGnon-polar · Σf(Complex) + ΔGrot · Nrotatable-bonds - ΔG0, ΔGpolar, ΔGnon-polar, and ΔGrot are empirically parameterised weights - f(Complex) is a penalty function aimed at penalising any unfavourable interactions

Answer 60

Fast as it is less computationally demanding – screen many ligands quickly

Answer 61

**Flexible docking** **Ligand flexibility along torsion angles** is considered in the docking process --\> increased accuracy ligand accommodation in binding site Resulting in different binding models - **Protein is considered “rigid”**- small flexibility is allowed a) Large protein conformational changes challenging for docking b) Small conformational changes can be accommodated

Answer 62

A) Structure are required to be **high resolution structure** since otherwise... 1. Water molecules not resolved 2. Electron density for flexible side chains might be weak 3. Hydrogen bonds are absent - 1Å or better resolution is optimal but can be artificially added at lower resolutions (less accurate) 4. pKa of the active site side chains - crystallisation buffer 5. Hard to distinguish protonation state and tautomeric form of the ligand B) Torsion angles provide flexibility to the ligand but **too many rotatable bonds can be a problem** E.g., Taxol – very computationally demanding

Answer 63

Structure based fragment screening (de novo) De novo synthesis - examine which regions in the active site are involved in binding from which we place fragments which can be linked to create biologically active molecules Build into the active site and link fragments to create bioactive compounds **How can we identify the fragments?** 1. Examine possible poses & conformations (search algorithm) --\> Orientation of molecule in binding site - More flexible as we are dealing with smaller molecules/fragments 2. **Predicting energetics** of protein-ligand binding (scoring function) - Binding affinity & Thermodynamics

Answer 64

Lead identification by Fragment evolution Screen for fragments - Fragment is know to bind to the active site. The fragment acts as a building block for the construction of the lead molecule - The lead molecule is **evolved by building away**

Answer 65

Lead identification by Fragment linking - identify two fragments that bind and link them together Fragment 1 binds to one site Fragment 2 binds to an adjacent site Fragments joined together by a linking group that allows the lead molecule to span both sites – increasing bioactivity

Answer 66

**Lead identification by fragment self-assembly** This involves... Identifying fragments that bind to the active site and are able to self-assemble into a single molecule - two fragments bind followed by a catalytic reaction performed by the enzyme, linking the two fragments together Note - either one (e.g. turning it into a good nucleophile) or both can be modified for the reaction to occur The protein is used to **self-select or to catalyze the synthesis of its own inhibitor** without covalent attachment of the protein to the inhibitor --\> linking two fragments increased binding affinity (nM/µM --\> fm) Can only be done with proteins that possess enzymatic activity

Answer 67

**Lead progression by fragment optimization** - Optimize or modify properties of the lead compound - Re-engineered to address optimization of a particular property (e.g. selectivity, cell-based activity, oral activity or efficacy)

Answer 68

**Thymidylate synthase** This function mThymidilate synthase maintains the dTMP pool, critical for DNA replication and repair. Cancer cells express elevated levels The enzyme has been of interest as a target for cancer chemotherapeutic agents.

Answer 69

1. Researchers attempted to find molecules that resembled the **co-factor tetrahydrofolate** --\> X-ray structure of the TS available allowing for identification of key interactions 2. Identified potentual lead from docking 3. They proceeded to modify the ligand in order to attempt to increase binding affinity and water solubility --\> but in-silico binding did not match X-ray - lower affinity than expected 4. Further redesigning - introduction of amide group --\> Wet-lab results and in-silico predictions matched up

Answer 70

Lead is validated by re-testing checking for... 1. **X-ray Structure assesment** 2. **Potency**: the amount of drug required for its specific effect to occur; inverse of the EC50 3. **Efficacy**: the maximum strength of the effect itself, at saturating drug concentrations. 4. **Pharmacokinetics**: determining the fate of xenobiotics. Extend and rate of adsorption, distribution, metabolism, and excretion (ADME). 5. **Pharmacodynamics**: determining the biochemical and physiological effects of drugs, the mechanism of drug action, and the relationship between drug concentration and effect. 6. **Chemical optimization** 7. **Patentability**

Answer 71

**Drug metabolism** is the metabolic breakdown of drugs by a living organisms --\> metabolic products produced are less pharmacologically active in turn reducing the toxicity of the drug - Liver is the main site of drug metabolism but specific drugs may undergo biotransformation in other tissues - Drug metabolism uses pathways that are normally used for the biosynthesis of endogenous substrates - has to be a degree of resemblance to natural compound to enter the pathway - Drug metabolism has to be taken into account when designing novel drugs

Answer 72

Drug metabolism is required to convert lipophilic compounds into more hydrophilic compounds --\> excretion Why? Lipid soluble non-polar compounds remain in the blood and tissues and maintain their pharmacological effects for much longer - result in undesirable side-effects

Answer 73

Phatmacokinetics - Drug Absorbtion, Metabolism, Distribution, and Elimination (ADME) e.g. Orally administered drug, dissolve in the GI tract and absorbed through the gut --\> enter the liver and into the blood stream/circulation

Answer 74

The physical and chemical properties of a drug determine its success to reach its target Chemical stability – e.g. stable in the stomach? Metabolic stability – e.g. no interaction with other metabolites Successful Absorption – e.g. cross membranes 1. Volume of distribution (reach target) and clearance influence Half-Life 2. Clearance and Absorption will influence oral bioavailability - how much reaches the blood stream

Answer 75

Phase I and Phase II Transformations - Role of detoxifying **Phase I transformations** – introduce or unmask a functional group (make inactive), e.g., by oxygenation or hydrolysis (OH, -SH, -NH2, -COOH, etc.) These metabolites are often inactive + Can be excreted readily **Phase II transformations** – generate highly polar derivatives (conjugates) for excretion; glucoronide, sulfate, acetate, amino acids (large functional added so must be excreted through the feces)

Answer 76

**1^o type of modification - Oxidation** - Addition of oxygen or removal of hydrogen --\> The first and most common step involved in the drug metabolism - Liver is the main organ where oxidation takes place by cytochrome P450 - Increased polarity of the oxidized products; increased water solubility and excretion in urine **Oxidation, can occur on…** - Aliphatic or aromatic hydroxylation - N-, or S-oxidation - N-, O-, S-dealkylation – e.g. removal of methyl

Answer 77

**Reduction** - Removal of oxygen or addition of hydrogen - Less common than oxidation - Cytochrome P450 system is involved in some of these reactions. Other reactions are catalyzed by reductases present in different sites within the body. **Reduction can be classified into…** 1. Nitro reduction to hydroxylamine/ amine 2. Carbonyl reduction to alcohol

Answer 78

Hydrolysis - reaction between a compound and water Results in the addition of water – improves metabolites polarity Different enzymes catalyze the hydrolysis of drugs: a) Esterase b) Amidase Ester or amide to acid, alcohol or amine

Answer 79

Oxidation enzymes – all these enzymes found in the liver 1. Cytochrome P450 monooxygenase system 2. Alcohol dehydrogenase 3. Aldehyde dehydrogenase 4. Flavin-containing monooxygenase system 5. Monoamine oxidase

Answer 80

Reduction enzymes 1. NADPH-cytochrome P450 reductase 2. Reduced (ferrous) cytochrome P450

Answer 81

Hydrolysis enzymes 1. Esterases and amidases 2. Epoxide hydrolase

Answer 82

Phase I relies on microsomal mixed-function oxidase system (cytochrome P450 dependent) The mixed-function oxidase is found in microsomes (endoplasmic reticulum) of many cells (liver, kidney, lung, and intestine) P450 requires **oxygen and a reducing system (NADPH)**—one atom of oxygen is transferred to the substrate, and the other undergoes a two-electron reduction and is converted to water

Answer 83

Cytochrome P450 (CYP) 3A4 - Catalyzes **hydroxylation or epoxidation** of various substrates CYP requires another enzyme **NADPH-cytochrome P450 reductase** which is a flavoenzyme containing one molecule of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) --\> used for electron transfer to CYP Both Cytochrome P450 and NADPH-cytochrome P450 reductase are associated with the membrane

Answer 84

Cytochrome P450 can accommodate a large variety of functional group as the cavity adjacent to the Heme is large, as shown below.

Answer 85

When phase I products are **not sufficiently hydrophilic or inactive** to be eliminated, the drugs or metabolites formed from phase I reaction undergo phase II reactions Phase I reactions provide a functional group in the molecule to undergo phase II reactions --\> this group is modified in phase II via **conjugation reactions** forming **more polar and water-soluble** products. Require coenzyme + transferases for conjugation (liver) Note - most conjugates are inactive and not toxic --\> highly polar and unable to pass through membrane

Answer 86

1. **Glucuronidation** by UDP-Glucuronosyltransferase (-OH, -COOH, -NH2, -SH) 2. **Sulfation** by Sulfotransferase: (-NH2, -SO2NH2, -OH) 3. **Acetylation** by acetyltransferase (-NH2, -SO2NH2, -OH) 4. **Amino acid conjugation** (-COOH) 5. **Glutathione** **conjugation** by Glutathione-S-transferase 6. **Fatty acid conjugation** 7. **Condensation reactions**

Answer 87

**Phase 1** - Prodrug converted to active compound Salicylic acid **Phase 2** - Conjugate to allow for excretion – different routes

Answer 88

**Glucuronidation** - transferring glucuronate sugar moiety (a-D-glucoronic acid) Most important phase II pathway for drugs and endogenous compounds Products are often excreted in the bile Enterohepatic recycling may occur due to gut glucuronidases - recycling of glucuronate Requires enzyme UDP-glucuronosyltransferase (UGT) to transfer sugar moiety on to target

Answer 89

N- or O-glucuronidation **N-glucuronidation** can be added to amines, amides and sulphonamides which are added in phase 1 **O-glucuronidation** - creation of an ester or ether linkage using carboxylic acids, phenols and alcohols

Answer 90

**Sulfation** Major pathway for **phenols** (also alcohols, amines and thiols). Requires the enzyme PAPS (3’-Phosphoadenosine-5’-phosphosulfate) Phenols can be modified by both Sulfation or Glucuronidation but usually… a) Sulfation at low substrate concentrations b) Glucuronidation at higher substrate concentrations

Answer 91

**Acetylation** - Targets aromatic amines and sulfonamides - Requires N-acetyltransferase and the co-factor acetyl-CoA Important in sulfonamide metabolism because acetyl-sulfonamides are less soluble than the parent compound and may cause renal toxicity due to precipitation in the kidney - **can be more harmful so must be considered**

Answer 92

Stearic and palmitic acids are conjugated to drug by esterification reaction

Answer 93

Active CoA-amino acid conjugates that react with drugs via N-Acetylation --\> Glycine, Glutamine, Arginine

Answer 94

**Glutathione Conjugation** Tripeptide Gly-Cys-Glu; conjugated by glutathione-S-transferase (GST) Conjugated compounds can subsequently be attacked by g-glutamyltranspeptidase and a peptidase product can be further acetylated  metabolite acts as a peptide than can be further digested and acetylated

Answer 95

Remove functional groups susceptible to enzymes E.g. Tolbutamide can be excreted directly whereas Chlorpropamide needs to enter phase I metabolism

Answer 96

Making drugs less resistant to metabolism: If a drug is too resistant to metabolism, it can pose problems as well (toxicity, long-lasting side effects). Add functional groups that are susceptible to metabolic enzymes – allow it to easily enter phase I metabolism

Answer 97

**Efflux transporters** detoxify cells from ‘toxic compounds’; eg P-glycoprotein One of the primary proteins involved in multidrug resistance in the treatment of cancers --\> cancer cell use the efflux transporters to drive out export of drugs ATP-binding cassette (ABC) –superfamily- requires ATP hydrolysis to drive the export

Answer 98

Rate and pathway of drug metabolism are affected by **species, strain, sex, age, hormones, pregnancy, and liver diseases** **Drug metabolism is stereospecific** – drugs may contain different enantiomers – specific enantiomers may be more toxic (pre/post-metabolism) For example... **Enantiomers act as two different xenobiotics** – different metabolites and pharmacokinetics Sometimes the inactive enantiomer produces toxic metabolites or may inhibit metabolism of active isomer

Answer 99

**Drugs can bind to plasma proteins** – determines bioavailability as in the bound state the drug is not bioactive Only unbound compound is available for distribution into tissues **Acidic drugs** tend to bind to **albumin**. **Basic drugs** bind to **alpha-1 acid glycoprotein**. a) 0-50% bound = negligible b) 50-90% = moderate c) 90-99% = high d) \>99% = very high % of drug reaching bloodstream = bioavailability

Answer 100

**Drug metabolism of Prodrugs** – rely on **Phase I metabolism** to active the drug Prodrugs are compounds that are inactive, but are converted in the body to an active drug by metabolic enzymes

Answer 101

**1. Improve membrane permebility using the addition of esters** Note - If a carboxylic acid is important for drug binding to its target, but it prevents the drug from crossing a membrane, **temporarily “hide” it as an ester**. Once in the blood, it is hydrolyzed to the active form by esterase’s But.... There is a balance that must be reached as if it’s too hydrophobic it will prefer the membrane environment and not diffuse into the cell 2. **N-methylation - amines can be methylated to increase hydrophobicity allowing movement across membranes** **-** N-demethylation is a common liver metabolic reaction --\> can be removed by liver

Answer 102

**Membrane transporter** - mimic substrates that have transporters to cross membrane

Answer 103

Create a pro-drug that is slowly converted to active drug --\> allows for slow and prolonged release of active drug into the body Example - 6-mercaptopurine Immune suppressant (organ transplants), but it is eliminated from the body quickly. Hence, a prodrug is used as it is slowly converted to the drug allowing longer activity.

Answer 104

Salicylic acid is a painkiller, but phenolic -OH causes gastric bleeding. Aspirin has an ester to mask this toxic group until it is hydrolyzed in the blood to form the active pro-drug salicylic acid

Answer 105

Phase I and II metabolism make drugs less toxic and more hydrophilic

Answer 106

More H-bonds means that the ligand is likely to bind very tightly - making it hard to reverse the reaction (undseriable) Hence, having 5H bonds allows for tight binding but not too tight to the point where the binding is hard to reverse If you desire a tight binder than you would use more than 5H bonds

Answer 107

Monoclonal - single type of antibody targeting a single epitope Polyclonal - Several types of antibody for the same target but different epitope

Answer 108

In 2D fingerprinting we are attempting to describe what groups/elements are present and how they are linked Bit collision arises when you have **similar chemical groups which can’t be distinguished** unless you indicate the position of the group in the molecule More bit collision - more false positive results --\> reality to be worse hit than predicted

Answer 109

The structures present in database often come from crystal structure elucidation, in which the molecule adopts its lowest energy state But this might not be ideal for docking in our protein --\> removing it as a possible candidate

Answer 110

2D fingerprints and hashed fingerprints are similar but hashed fingerprints includes structural information --\> but you can still get bit collisions in both

Answer 111

Sometimes the linked ligand is unable to bind directly e.g. due to steric clashes

Answer 112

Glutathione resembles a tripeptide --\> Peptidase recognize this Gly-Cys-Gln motif resulting in cleavage This leaves an amine which can be acetylated - things to consider when thinking about drug metabolism

Answer 113

**Not common but It is possible** if there are multiple functional groups that can be modified. But we need to remember that the molecule needs to re-enter the P450 enzyme for example and this might not be possible after the initial modification

Konstantinos Beis - Antibodies + Drug Design Flashcards

(185 cards)