1
Q
explain the drug discovery process
A
(takes 10-15 years)
1. identify POI which is related to a medical need i.e. emergence of certain disease, antibiotic resistance etc.
2. understand POI’s relevant mechanism (deduce from structure, information of active site by X-ray/ NMR/ cryoEM)
3. The information will be used in the selected screening method to identify “hits” or compounds that result in inhibition of the POI activity
4. Select a lead compound from the “hits” (most effective) which can be used in the drug development stage (optimization using medicinal chemistry, analog synthesis, animal testing)
- 2 possible outcomes:
- If the lead compounds end up ineffective/ toxic to animals, it will be reassessed (repeat optimization/ select for another lead compound from “hits”/ repeat screening for new “hits”)
- If it is successful at animal testing stage, drug candidates can then be established for further clinical testing in humans
2
Q
what are the 3 possible screening methods
A
- High Throughput Screening (HTS)
- Virtual Screening (VS)
- Combination of HTS and VS
3
Q
explain High Throughput Screening (HTS)
A
- Uses a big library of compounds, prepared in an arrayed format, to perform assays w/ POI
- The POI activity w/ the compounds (eg inhibition of POI) can be measured by
fluorescent or luminescent detection, colourimetry, or light scatter - Mostly used by big pharmaceutical companies
4
Q
what are the disadvantages of HTS
A
- Expensive (high quality assays & specialized equipment)
- False positives
- Assay variability or errors in data
- Small amount of compound screened (largest = 10^7) relative to possible chemical diversity space (10^60)
5
Q
explain Virtual Screening/ In silico drug screening
A
- automatic evaluation of large libraries of compounds using computer programs (selects and scores compounds based on input parameters)
- similar to HTS = relies on assessing a large, diverse library of compounds
-difference= compounds contained in databases rather than physically assembled into arrays. - Used in Computer-Aided Drug Discovery (CADD)
6
Q
what is CADD
A
- Entails many different computational methods like virtual screening, virtual library design, lead optimization, de novo design
- classified into ligand-based methods (LBDD) and structure-based (SBDD)
7
Q
how is the method of CADD chosen
A
- based on availability of structural data of protein and ligands
8
Q
what methods are used for both unknown POI & ligand information
A
- Library design: (when no info about both POI & ligand)
- ligand structure = identify a collection of compounds that can inhibit/ bind the POI by HTS, VS,
- POI structure - use Alphafold to predict 3D structure
- De Novo design: (when have info about POI, not ligand)
-Develop ligands based on small fragments that will occupy active site & inhibit protein
9
Q
outline LBDD
A
- 3D structure of POI = not known
- Use information about the molecules that bind to the target of interest (S/Inhibitor)
- Hits are identified, filtered, and optimized to obtain potential drug candidates that will be experimentally tested in vitro.
10
Q
outline SBDD
A
- know 3D information of POI
-can classify positives from false positives
11
Q
describe the prominent steps in SBDD and LBDD approaches
A
- For SBDD (to obtain known POI structure) = Homology modelling & validation
* The target-template alignment leads to the modelling of 3D structure of POI - model is validated by Ramachandran plot (using PROCHECK) - narrow down database of ligands by virtual screening to obtain set of “candidate ligands” eg. several methods
* SBDD: molecular docking, pharmacophore design
* LBDD: pharmacophore design, QSAR - Docking process by virtual screening: ligands and POI are allowed to interact w/ each other (using docking software)
* SBDD = many ligands are screened against the known POI
* LBDD = the chemical properties of single ligand is used to screen against many POI targets
12
Q
what are the libraries used in virtual screening
A
Combinatorial library design = library of ligands constructed based on known structure of POI and compounds that mimic its substrate/ inhibitor = higher success rate because scaffolds can already bind POI
Non-combinatorial library = bought database (files) of compounds
13
Q
describe LBDD (when its used, why is it important)
A
- no POI structural information.
- Exploit knowledge of ligand structure which binds POI
- Key to understand pharmacophores of the ligand structure.
- Pharmacophores have to be kept the same, but all else is modifiable = unlikely
to negatively affect binding - LBDD methods can be divided into:
-Similarity searching = based on 2D descriptors/ 3D descriptors
-Pharmacophore mapping
-Machine learning
14
Q
describe similarity searching (LBDD)
A
- looks for molecules with similar properties/structures to the reference molecules.
- involves screening the compound of interest against a large database, then ranking the matches in order of high to low similarity to the reference.
-The top-ranking compounds are selected for biological testing based on several quantitative measuring methods
15
Q
what is the measure of similarity based on (LBDD)
A
- Physicochemical properties: eg. MW, MR, logP (partition coefficient: shows solubility of substrate in an aq environment or membrane)
- 2D properties: eg. fingerprints, topological indices, maximum common substructures
* need quantitative basis to measure and rank compounds according to similar structures to known ligand
- Similarity coefficient: A quantitative measure of similarity between 2 molecules (parent compound and a similar compound in the library) - 3D properties: eg fingerprints, molecular fields
16
Q
explain 2D fingerprinting (2D properties) (LBDD)
A
- use a binary vector model (bit-string)
- Each bit in the bit string represents 1 molecular fragment
– can design the bit string according to preference by selecting certain functional groups as a bit - The bit string for a molecule records the presence 1 or absence 0 of each fragment in the molecule
- each molecule in library will be assigned a unique bit string
- Similarity is based on determining the number of bits that are common between structures of query and compounds in the library
- More (1) bits in common = greater similarity
- Will only get info. on what functional groups are present
17
Q
explain hashed fingerprinting (2D properties) (LBDD)
A
- uses bit string, but also contains information on the structure (topology) – how the atoms are connected to each other in 2D space, but still no info. about stereochemistry (3D structure)
- Each fragment is processed using many different hashing functions, each of which sets a single bit in the fingerprint
- When assigning the bit string, each bit also considers the order of functional groups – only when the functional groups are in same order, will consider as present (1)
18
Q
what is the problem with hashed fingerprinting
A
Bit collision = the same bit can equate to multiple patterns (generate false positive)
* A few bit collisions in the fingerprint are ok, but too many may result in losing information in the fingerprint
19
Q
what happens once the 2D/hashed fingerprints are defined (LBDD)
A
generate: Similarity (Tanimoto/ Jaccard) coefficient
* define % similarity according to the bit strings for each compound in the library
T(a, b) = Nc / (Na + Nb - Nc)
a = bits in reference structure
b= bits in database structure
c = bits in common in ref and database
- can run search against the library to filter for certain compounds that meet a certain % similarity criteria: If 2D = similar, mostly just assume that will have similar 3D structure
20
Q
explain scaffold hopping (3D properties) (LBDD)
A
- purpose is to find a molecule with the desired pharmacophore (same activity/ function) as the query compound but w/ different core structure
- Considers if the molecules occupy same 3D volume, space and binding site as the substrate
21
Q
what is the principle of 3D fingerprinting (3D properties) (LBDD)
A
- to break down different functional groups in a compound of interest to reconstruct a model for potential drugs
*like in 2D, once 3D library search is done, must score & rank obtained hits to narrow down the compounds selected for further experiments in the wet lab
22
Q
explain the 3D fingerprinting process (LBDD)
A
- Define point of functionalities in the molecule (defined by same function does not have to be the exact same functional group)
- Connect them into triplets and obtain associated distance - can now deduce how they connect in 3D space (more point of functionalities = more triplets detected = more accuracy)
- Develop bit string for diff. triplets
- Use the defined bit string to search for compound in database that satisfy the
following properties:
- pairs of atoms at given distance range
- triplets of atoms and associated distance
- pharmacophore pairs and triplets (donors, acceptors, aromatic centers etc.)
- valence angles
- torsion angles
23
Q
explain the flexibility of parameters in 3D fingerprinting
A
- can be strict or flexible w/ parameters
- hits can have completely diff. sequence/ structure but must satisfy functional properties
24
Q
explain 3D shape search (LBDD)
A
- Molecules are aligned in 3D
- Similarity score is based on common volume occupied
- Method = generate spherical volume of known ligand, then find molecules with volumes that are sufficiently similar to the spherical volume
-Molecules can be dissimilar in 2D but similar in 3D or vice-versa
-Due to diff. rotations around bonds & etc., must decide how the screening is conducted
25
what is the principle of pharmacophore mapping (LBDD)
Pharmacophore generation & searching
* to determine pharmacophores of a molecule & search for them in library
(molecular features that are vital for the biological activity of a compound)
* Protein structure is not required
* Assumes that all (majority) of the known actives bind to the same location
26
explain pharmacophore generation (LBDD)
* Identify pharmacophoric features and how they are organized in 3D space
27
explain pharmacophore searching (LBDD)
* Given a pharmacophore, find all molecules in a database that can match it in a low- energy conformation
* Scaffold-hopping possible
- Doesn’t require structural similarity
- Just needs to match the pharmacophore
* Can also do 4-point model = better feeling for dimensionality of compound but too computationally demanding
28
in a database, you must: (LBDD)
1. Ensure diversity of ligands (tautomers/protonations/ conformational space
should be accounted for)
2. Must define molecules in the database using same protocol and parameters used to generate pharmacophores
3. Generate cutoffs/ allowed tolerance (what Angstrom range to give such that a match is
indicated?) - more or less strict
4. have actives and decoys
-but not ideal for scaffold hopping because the system looks for dissimilarity in core structure
29
what are actives
a compound known to bind the POI
- A good similarity measure will cluster the known actives at the top of the ranking
- if you pick up actives = ensure accuracy of searching criteria
- if you don’t pick up actives = defined parameters/ criteria= not optimal
30
what are decoys
compounds known to not bind or inhibit POI
- if decoys are shown as similar to the reference molecule = design algorithm is erroneous, not optimal
31
explain quantitative-structure activity relationship (QSAR) (LBDD)
machine learning
* tries to establish quantitative relationships between descriptors and the target property – so can predict activities of compounds from 2D properties
* eg. inhibition property, toxicity – can eliminate certain compounds known that will not pass animal testing, etc. or eliminate false positives
* QSAR models require descriptors that accurately convey chemically-relevant information to the machine learning models
32
what are the 3 main methods of SBDD
1. Structure and known inhibitor design
* Known inhibitor or co-factor is modified to improve binding affinity or selectivity
2. Virtual High Throughput Screening (vHTS)
* Docking of small molecules into the crystal structure which are scored and ranked
3. De novo design
* A molecule is designed from scratch to bind in the active site by docking and connecting fragments to create full molecules. These molecules are then scored and ranked.
33
explain structure and known inhibitor design (SBDD)
* Need high-resolution X-ray structure of target protein.
* Solved structure should be in the presence of the substrate -residue orientations in active site are known
* Use this structure as a template for screening of small molecules
* Can redesign the molecules:
-if modification leads to increased activity = groups modified allow molecules to fit and/or bind better
-if modification leads to decreased activity = groups modified are biologically important for binding.
-if modification leads to no change = group modified are not important, & can thus be variable
34
explain the principle of Virtual High Throughput Screening (vHTS) (SBDD)
* dock small molecules with the crystal structure to identify leads- then scored and ranked
* lead = toxic = can modify to avoid toxic side effects
* Must test lead activity using assay (enzymatic and pharmacokinetics)
* Ideally should also solve crystal structure of ligand to verify binding mode
35
what are the challenges of vHTS
The energetics of protein-ligand interactions are complicated:
* Must also consider involvement of solvation of binding site (water molecules mediating H bond between ligand and substrate)
* Both proteins and ligands can be quite flexible
* Many target-binding ligands are not good drug candidates
* The structures of many important drug targets are difficult to determine = helped by Alpha fold
36
what is the method of vHTS
1. Perform docking to identify leads – which are scored and ranked
* need high resolution Xray of POI to know residues involved in binding -so when docking, can fit the ligand by selecting proper stereochemistry
2. Once identified leads, can perform Pose prediction: to identify which chemical groups of the ligands are important for binding and specificity
Once identify database of leads (Substrate/inhibitor), describe by molecular descriptors:
* 1D-: chemical composition & physicochemical properties (eg. MW,hydrophobicity)
* 2D-: chemical topology: how functional groups are associated with each other (eg. Connectivity indices, degree of branching, degree of flexibility)
* 3D-: the spatial arrangement of chemical groups (eg. shape, volume, functionality, surface area)
3. Can also identify activities associated with certain structures of leads using Structure Activity Relationships (SAR)
* Correlations that are constructed between the features of chemical structure in a set of candidate compounds and parameters of biological activity, such as potency, selectivity and toxicity
* Used to: Identify groups of the lead compound that are important to biological activity
* X-ray crystallography can also be used to identify important interactions between drug and protein
37
what is the Lipinski’s rule of 5s for good drug candidiates (SBDD)
1. MW < 500
2. Fewer than 5 H-bond donating functions
3. Fewer than 10 H-bond accepting functions
4. Calculated logP between –1 and +5
* Calculated from partition of compound in artificial membrane (made by mixing n-
octanol = mimicking lipid bilayer; with water = mimic aq environment)
* Describes solubility
* High log P = hydrophobic; negative = very water soluble
* P = [D] lipid / [D] water = log P
38
Explain rigid docking (SBDD)
* The ligand is treated as a rigid structure and only the translational and rotational degrees of freedom (rotation of whole molecules in 3D) are considered – no rotation between bonds of functional groups etc.
* If have diff ligand conformations (known to have rotatable bonds), each conformation is docked separately (pre-rotate bonds before docking)
39
what are the pros/cons of rigid docking
* Advantage: fast & not computationally demanding
* Dis adv: can’t rotate any bonds although rotations are known to prevent steric clash –
lose many possible hits
40
what is the process of rigid docking
1. Define enzyme active site in geometrical shapes (triangles/ spheres)
2. Match & rank ligand that can satisfy the defined shape
* Can also define functionalities within the active site – if active site = known to have H bond donor, set one of the ligand ranking criteria to be having a H bond acceptor at a certain position
41
explain flexible docking
* Allows some degree of ligand flexibility along torsion angles during docking process + small conf. changes at binding site can be accommodated
42
what are the pros/cons of flexible docking
* Dis Adv: more comp demanding
* Adv: allow for molecules to adopt optimal binding pose
43
Why is scoring necessary (for ranking) (SBDD)
* Many different poses of the same ligand need to be ranked based on their affinity with receptor to identify positive hits from other poses
44
explain first principle scoring (SBDD)
-Rank ligand based on force fields generated with molecular mechanic information, can be:
* Intra molecular = bond lengths, angles, dihedrals,
* Inter molecular VDW contacts (non-polar), Electrostatic interactions (polar)
E bind = E intra + E nonpolar + E polar
45
explain empirical scoring (SBDD)
* Based on Gibbs free energy of binding - better since also account for entropy/enthalpy in binding
* Also include a penalty function to account for unfavorable interactions
* Values are empirically determined from experiments
ΔGbind = ΔG0 + (ΔG polar x Σ f-complex + (ΔG nonpolar x Σ f-complex) + ΔG rot x non-rotatable bonds
46
what are some considerations before docking (SBDD)
* Water in structure
* Tautomeric forms (eg. keto-enol forms can appear to have the same electron densities
but are diff. in terms of being H bond acceptor/donors, so may interact differently)
* Weak electron density of sidechain of aa = can result in wrong assignment
* pKa and Protonation state (if protons are not resolved in the structure, will affect H
bond interpretation required in docking programs) – a problem with charged residue since easily lose H, but if know crystallisation buffer & pH of environment, can deduce protonation state
* Complications from rotatable bonds due to flexible torsion angles
* Ring conformations may not be distinguishable (chair or boat)
47
explain de novo design (SBDD)
* When No info. known about ligand
* Active site is treated as an empty pocket = molecules are designed from scratch by searching small fragments that form favourable interactions with the active site
48
what is the process in de novo
1. define binding pocket & interaction sites (aa residues involved in binding)
2. search for fragments that can satisfy the active site 3D space and volume OR can use lattice strategy, in which binding pocket is defined as a 2D lattice & search fragments that satisfy defined lattice
49
what are some parameters involved in de novo screening
1. Search of possible poses & conformations (search algorithm) = Orientation of molecule in binding site
2. Predicting energetics of protein-ligand binding (scoring function) = Binding affinity, and thermodynamics
50
what are the 3 main methods to form a complete lead molecule in De Novo design
Docking:
* Dock larger molecular entities that you already know from experiments, have favourable
interactions in the active site
Building (lead identification by fragment evolution)
* start with 1 fragment known to make favorable contacts with an interaction site
* then build from that fragment to form a complete molecule
Linking:
-lead identification by fragment linking
-lead identification by fragment self-assembly (using enzymes)
51
explain Linking (lead identification by fragment linking)
* separately place fragments (small functional groups) identified to make favorable
contacts with each interaction site and join them to form a complete molecule
* fragments are joined by a linking group/ core template
52
explain linking (Lead identification by fragment self-assembly)
* Only works with enzymes – the protein target is an enzyme, which is used to perform the linking resulting in its own inhibition
* Advantageous as both components may be too large to accommodate active site, but are able to bind effectively individually
53
optimization (SBDD)
* Once complete molecule has been formed:
* Optimize or modify properties of the lead compound
* Re-engineered to address optimization of a particular property (eg. selectivity, cell- based activity, oral activity or efficacy)
54
what must you ensure throughout the whole method (SBDD)
For each method & after every step, must assess the compounds’ properties after growing/ linking the fragment if more/ less desirable
Once hits have been identified from the screening, they are validated by re-testing them and checking the purity and structure of the compounds.
55
what criteria must the leads fulfill (SBDD)
* Potency = the amount of drug required for its specific effect
* Efficacy= the maximum strength of the effect itself
* Pharmacokinetics = rate of adsorption, distribution, metabolism, and excretion (ADME).
* Pharmacodynamics= determining the biochemical and physiological effects of drugs, the mechanism of drug action, and the relationship between drug concentration and effect.
* Chemical optimization= binding affinity and favorable accommodation in active site
* Patentability
56
describe a case study of SBDD
Thymidylate synthase
* generates dTMP from dUMP using 5,10 Methylene tetrahydrofolate
* dTMP = critical for DNA replication and repair, so the enzyme has been of interest as a
target for cancer chemotherapeutic agents.
* A lead was identified by docking
* favorable binding is verified by Xray crystallography
* Original lead identified = hydrophobic, so performed in silico screening to obtain compound with more solubility & in silico synthesis by adding more H bonding group
-but results in diff. interaction than expected, thus decreased binding affinity
* further analysis and redesign: add amide group & result in favorable binding + increased binding affinity
* must verify in silico screening by structural analysis eg. Xray crystallography, NMR, CryoEM or functional assay
57
describe HIV-1 protease inhibitor example
* Squanivir (Roche) & Indinavir (Merck) designed by modeling chemical structures on the computer to fit inside of the active site of HIV-1 protease using the X-ray crystal structure
58
describe the neuramidase drugs
* Relenza and Tamiflu designed from known structure of neuramidase pocket and its substrate – sialic acid
59
what is drug metabolism defined as
metabolic breakdown of drugs by living organisms, usually through specialized enzymatic systems
60
what happens to a drug after ingestion
* drug compounds can enter the body’s biosynthetic pathways of endogenous substrates (eg. hormones, cholesterol, bile acids) to be metabolized. This is possible because drugs resemble the natural compounds.
* These chemical alterations are known as “biotransformation”, which occur primarily in the liver and sometimes, in other tissues (depending on the type of drug compound)
61
what are the results of drug metabolism
-Most metabolic products are inactive because they have been broken down into non-toxic compounds which are then excreted from the body. However, there are some exceptions:
* Inactive drug compound is metabolized and become active eg. prodrugs
* Drug compound is metabolized and become more toxic/carcinogenic
62
describe drug conversion to lipophilic compounds
Drug metabolism is required to convert lipophilic compounds into more hydrophilic compounds to be excreted
* If the lipid soluble non-polar compounds are not metabolized, they will remain in the blood and tissues and maintain their pharmacological effects for much longer
63
describe the metabolic pathway of hydrophilic drugs
-enters the stomach
- enters the circulation system
-enters liver where it is metabolized.
-metabolites secreted out of the body by kidney in urine
(problem: can be secreted too quickly – can have techniques to prolong life of hydrophilic drugs)
64
describe the metabolic pathway of lipophilics drugs
If no metabolism: stomach - circulation system- not metabolized by liver -remain hydrophobic - can’t be secreted by kidney – remain in blood & tissue - can result in side effects from its prolonged activity
If some are metabolized: stomach - circulation system - some metabolized to hydrophilic compounds in the liver -hydrophilic metabolites secreted by kidney in the urine - some that are not metabolized will still be hydrophobic and remain in the bloodstream
- This partial metabolism results in attenuation of the prolonged effect of the drug
If all are metabolized: stomach - circulation system & travel to target tissues -completely metabolized by the liver - all are converted to hydrophilic molecules - secreted by the kidney via urine.
65
what is pharmacokinetics (PK)
the study of how an organism affects a drug or the study of the absorption, distribution, metabolism, and excretion (ADME) processes of a drug
66
explain ADME at different organs
* Absorption: orally administered drug dissolves in GI tract and is absorbed by the
stomach and small intestine
* Metabolism: liver
* Distribution: circulatory system (bloodstream)
* Elimination: kidney (urine), colon (feces)
67
why is pharmacokinetics important
- provide understanding about the physical and chemical properties of a drug which is important in drug development because they will determine the drug’s success to reach its target.
* affect':
-Chemical stability eg. is it stable in the stomach?
-Metabolic stability ex. half-life: how well/ how fast a drug is metabolized?
-Successful Absorption eg. ability to cross membranes
* helps establish optimal dosage
68
explain optimal dosage
Optimal dosage = drug concentration in plasma in therapeutic window to elicit desired effects of the drug
* below = no effect on target – because metabolized too quickly/ doesn’t absorb efficiently
* exceed = toxic overdose – because metabolic system can’t cope with conc. of the ingested drug, resulting in prolonged effect & will already generate damage to the body by the time it is detoxified
69
explain how drug metabolism occurs in 3 phases
Phase 0: where absorption of the drug compound occurs eg. via passive diffusion through the membrane or through a transporter.
1. The drug will then enter Phase I, where the drug gets metabolized/detoxified. Usually phase I = sufficient to make the compounds inactive, so it can be secreted out via urine.
2. if phase I could not completely inactivate the drug, it will enter Phase II, where inactive compounds will be secreted as faeces.
3. Some compounds would enter Phase III when there is a resistance against the drug. This is when different transporters pump the drug out of the target cell without undergoing metabolism.
70
explain phase I transformations of drug metabolism
involve introduction or unmasking of a functional group ex. (OH, -SH, -NH2, -COOH, etc.)
* These metabolites are often inactive & can be excreted readily via urine
1. oxidation
2. reduction
3. hydrolysis
71
explain phase I metabolism -oxidation
Addition of oxygen or removal of hydrogen
* The first and most common step in the drug metabolism
* Liver - cytochrome P450
* Increased polarity of oxidized products = increased water solubility = readily excreted in urine
72
what are some groups likely to be oxidized in phase 1 metabolism
* Aliphatic or aromatic hydroxylation
* N-, or S-oxidation
* N-, O-, S-dealkylation
73
what are some common oxidation enzymes
* Cytochrome P450 monooxygenase system
* Alcohol dehydrogenase
* Aldehyde dehydrogenase
* Flavin-containing monooxygenase system
* Monoamine oxidase
74
phase 1 metabolism usually occurs by:
cytochrome P450 enzyme in the microsomal mixed-function oxidase system
* Oxygen and a reducing system (NADPH) is required
* An atom of oxygen is transferred to the substrate
* Another oxygen atom undergoes 2 e- reduction to form a water molecule
75
where is the microsomal mixed-function oxidase found
in microsomes (ER) of many cells (liver, kidney, lung, and intestine)
76
describe the structure of cytochrome p450 (CYP) 3a4
* Associated with the membrane by being bound or anchored to the membrane, but it is not a TM protein – mostly accept hydrophobic drugs
* Contains a heme group, or protoporphyrin IX, an iron(III) porphyrin cofactor.
-Molecular oxygen binds to the heme after reduction of ferric (Fe3+) to ferrous (Fe2+) iron and is converted to a reactive form which is used in many oxygenation reactions
* The required reducing system involves another enzyme: NADPH-cytochrome P450 reductase, a flavoenzyme that contains one molecule of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)
-Role = to transfer e- to the CYP450 enzyme for its function
77
what is unique about cyp450
able to metabolize many diff. compounds/ functional groups
* Binding site has a large cavity where the drug binds & a heme is coordinated by Cys residue
Lack selectivity because the site = very promiscuous - as long as a compound can fit in the BS and can orient itself correctly to the heme group, it can be metabolized and inactivated
* SO: although we don’t have a lot of types of CYP450 – the ones we do have = capable of detoxifying a huge number of compounds
78
describe the mechanism of oxidation by cyp450 enzymes
1. drug into active site of enzyme that is in ferric (Fe3+) heme bound state - drug is bound but has no coordination with heme.
2. Reduction of heme center by NADPH-cytochrome P450 reductase, transferring e- to reduce ferric (3+) to ferrous heme (2+)
3. Reduction of heme allows molecular oxygen to bind to the iron = ferrous oxy species
4. 2nd reduction by second e- = ferric peroxy species (overall charge = 2-, and iron = 3+) - TS
5. Oxidation occurs as a proton is transferred onto one of the bound oxygens = compound 0 (overall charge = 1-) = ferric hydroperoxo species
6. The addition of a 2nd proton results in release of a water molecule = compound 1 = ferryl oxo species
7. The free radicals formed in compound 1 attack the bound substrate, forming a covalent adduct
8. Incoming water molecule displaces hydroxylated substrate and regenerates the binding site
9. Another substrate can come in to replace the bound water and repeat the cycle
79
why is heme important in oxidation of drugs
the ability of heme group to change oxidation state allows it to catalyze the oxidation of drugs.
80
what is the quality control mechanism of cyp450
If in ferric hydroperoxo species, there’s no good coordination w/P450 NADPH reductase for correct e- transfer, or if the bound substrate can’t be metabolized, the mechanism will fall apart and the enzyme will revert back to the ferric heme species.
This is a good quality control mechanism of the system to ensure that compounds that aren’t supposed to be metabolized/ compounds with incorrect orientation in the BS will not be able to be metabolized.
81
describe reduction in phase 1 metabolism
Removal of oxygen or addition of hydrogen
* Less common than oxidation
* Cytochrome P450 system is involved in some reactions, while other reactions are
catalyzed by reductases present in different sites within the body
82
what are some groups likely to be reduced
* Nitro - hydroxylamine/ amine
* Carbonyl - alcohol
83
explain hydrolysis in phase 1 metabolism
addition of water to hydrolyse a bond
* gives more polar metabolites
* Different enzymes catalyse the hydrolysis of drugs ex. esterase, amidase
84
what are some groups likely to be hydrolysed
* ester to acid + alcohol
* amide to amine
85
what are some common hydrolysis enzymes
* Esterases and amidases
* Epoxide hydrolase
86
explain phase 2 transformations
generate highly polar derivatives - conjugate large molecules to the compounds which does not permit secretion via urine, so secrete via faeces (ex. glucoronide, sulfate, acetate, amino acids)
* Phase I reactions provide a functional group in the molecule to undergo phase II reactions.
-Phase II reactions modify functional groups by a conjugation reaction - capable of converting these metabolites to more polar and water-soluble products.
* Require coenzyme (which is transferred to the drugs)
* Conjugation catalyzed by transferases (liver)
* Most conjugates are biologically inactive and nontoxic because they are highly polar and unable to cross cell membrane
87
phase II metabolism (example) - glucoronidation
1. Glucuronidation by UDP-Glucuronosyltransferase (occur on: -OH, -COOH, -NH2, -SH)
* Conjugation to D-glucuronic acid, a sugar moiety, which increases hydrophilicity of the compound
* Products are often excreted in the bile
* Enterohepatic recycling may occur due to gut glucuronidases – enzyme in the liver which can cleave the added D-glucuronic acid = modified/ metabolized drug becoming active again
* requires the formation of UDP glucoronic acid
88
explain the formation of UDP glucoronic acid
1. A phosphorylase transfers UTP to α-D-
glucose 1 phosphate = release of PPi & formation of α-D- glucose1-UDP
2. UDPG dehydrogenase oxidizes the α-D- glucose1 UDP = release of 2NADH & formation of the active compound called α-D-glucose 1UDP- glucoronic acid UDP (a cofactor participating in the glucuronidation by transferring the D- glucuronic acid to the drug)
89
explain the 2 types of glucoronidation
* N-glucuronidation
-Occurs with amines (mainly aromatic)
-Occurs with amides and sulfonamides
* O-glucuronidation
-by ester linkages with carboxylic acids
-by ether linkages with phenols and alcohols
90
phase II metabolism (example) - sulfation
by Sulfotransferase: (on: -NH2, -SO2NH2, -OH)
* Conjugation to sulfate
* Major pathway of detoxifying drugs that contain phenols (also alcohols, amines and
thiols)
* Require cofactor: PAPS (3’-Phosphoadenosine-5’-phosphosulfate)
* Like glucuronidation, reacts with aromatic amines:
- At low substrate conc., sulfation is preferred over glucuronidation
-At high substrate conc., glucuronidation is preferred
91
describe the formation of PAPS
* ATP phosphorylase adds sulfate group to ATP, releasing PPi, forming APS
* Phosphokinase phosphorylates APS, forming PAPS
92
phase II metabolism (example) -acetylation
by acetyltransferase (on: -NH2, -SO2NH2, -OH)
* Occurs on aromatic amines and sulfonamides
* Requires N-acetyltransferase and the co-factor acetyl-CoA
* Undesirable effect because acetyl-sulfonamides are less soluble than the parent compound and may cause renal toxicity due to precipitation in the kidney – if the drug goes through this pathway, must redesign to remove site of acetylation
93
considering the importance of which metabolic pathway drug will enter
- for phenols, have to consider which metabolic pathway the drug will enter (sulfated or glucuronidated or acetylated) because will impact if the drug becomes more/ less toxic & will determine other pathways that the drug will react with
-knowing the metabolic pathway that the drug will enter = key in drug design – a lot of drugs fail in clinic due to metabolism not being understood ex. acetylation might occur in real life although not predicted during the development process
94
phase II metabolism (example) - amino acid conjugation
on: -COOH
* Active CoA-amino acid conjugates that react with drugs by N-acetylation eg. Gly, Glu, Arg
* makes the drug compound more prone to solubility
95
phase II metabolism (example) - glutathione conjugation
by Glutathione-S-transferase (GST)
* Occurs at tripeptide Gly-Cys-Glu
* Conjugated compounds can subsequently be attacked by g-glutamyltranspeptidase, cleaving off the glutathione moiety – products with free amine can then undergo another phase II metabolic pathway ex. acetylation (must consider as well during drug development)
96
phase II metabolism (example) - fatty acid conjugation
(addition of a big lipid to the compound)
* Stearic and palmitic acids are conjugated to drug by esterification reaction
97
final example of phase II metabolism
condensation reaction
98
same compound can undergo different types of phase II metabolism to be deactivated (example)
Aspirin (a pro-drug)
* Aspirin = inactive.
Phase I metabolism via hydrolysis by CYP450 forms the active component of the drug – salicylic acid.
- phase II metabolism to be detoxified – can undergo many types ex: sulfation of OH group, attachment of glucuronic acid, or diff. modifications by gentisic acid, salicyluric acid eg. addition of glycine moiety
99
what are some considerations during drug development
1. Making drugs more resistant to metabolism – to control the rate of metabolism as sometimes, if too fast may result in insufficient/ineffective drug activity
* By removal of functional group that are susceptible to phase I metabolism
* Eg. removal of methyl group from tolbutamide (an antidiabetic) to form chlorpropamide – contains Cl, a halogen, instead which is metabolized by CYP450 less easily/ less fast
2. Making drugs less resistant to metabolism: If too resistant, constant circulation can pose problems (eg. toxicity, long-lasting side effects)
* Add functional groups that are susceptible to metabolic enzymes
* Eg. addition of methyl group to facilitate binding to CYP450
100
explain phase III of drug metabolism
involve efflux transporters detoxifying cells eg. P-glycoprotein by exporting the drug outside the cell
* Usually undesirable effects because there is no control of how a transporter will excrete drug out of the cell & can lower the effective concentration of the drug inside the cell
* Driven by eg. ATP-binding cassette (ABC) superfamily - requires ATP hydrolysis to drive the export
101
what are some factors affecting metabolism
-Rate and pathway of drug metabolism are affected by species, strain, sex, age,
hormones, pregnancy, and liver diseases (affects functionality of CYP450 – due to mutations or down regulation of its expression) – affects type of drug given/ dosage of drugs that can be taken
* Stereoisomers: Drug metabolism is stereospecific
-Enantiomers act as two different xenobiotics – have different metabolites and PK
-Only 1 enantiomer will be active against the POI
-Sometimes metabolism of the inactive enantiomer can produce toxic metabolites
or may inhibit metabolism of active isomer
-Must consider if drug will be given as a racemic mixture or a pure compound
* Bioavailability - affecting drug dosage:
-A lot of drugs will bind to plasma proteins - only unbound compound will reach the bloodstream and is available for distribution into tissues (usually availability to reach target tissues = very low)
- Acidic drugs tend to bind to albumin
- Basic drugs bind to ⍺-1 acid glycoprotein
- Eg. 90% of Aspirin will bind to plasma proteins = 10% available
- Eg. 99% of Ibuprofen binds = 1% available
- Eg. 20-35% Morphine binds = 65-80% available
102
what are prodrugs
compounds that are inactive, but are converted in the body to an active drug by metabolic enzymes.
Non-enzymatic process such as hydrolysis can also occur but it’s usually unstable and undesirable because there is no control over where the prodrug will be converted into its active form.
103
what are the 3 purposes of prodrugs
1. improving membrane permeability
2. extending the life of a drug
3. reducing toxicity/side effects
104
explain the purpose of prodrug (1-improving membrane permeability)
* Esterification = carboxylic acid can be important for drug binding to its target, but it also prevents the drug from crossing a membrane. The carboxylic acid temporarily be hidden as an ester. Once the drug is in the blood, it is hydrolyzed to the active form by esterases.
* N-methylation = amines may be methylated to increase hydrophobicity. N-methyl groups will be removed in the liver.
* Membrane transporter = drugs can be designed/ modified to mimic substrates of membrane transporters, so they’ll be able to cross the membrane. Once crossed, those modified group can be removed
-eg. modification of dopamine into levodopa, an amino acid that is transported across the membrane by a membrane transporter. Once crossed, carboxy end is removed to generate dopamine
105
explain the purpose of prodrug (2-extending the life of a drug)
A prodrug that is slowly converted to the active drug to allow longer activity
- Eg. 6-mercaptopurine is an immune suppressant for (organ transplants) but is eliminated from the body quickly. By being given in the form of azathioprine, slow conversion via cleaving of the functional group allows longer effect of the drug in the bloodstream
106
explain the purpose of a prodrug (3 - reducing toxicity/side effects)
eg. Salicylic acid is a painkiller, but its phenolic -OH causes gastric bleeding (can develop stomach ulcers).
- given in the form of Aspirin which has an ester to mask this toxic group until it is hydrolyzed & converted into active moiety of salicylic acid in the liver.
107
how prodrugs work
* simple chemical derivatives that require only 1-2 chemical or enzymatic transformation steps to get the active parent drug compound
* modification usually involves masking of the drug by additional functional groups. Once the FG is removed by specific enzymes, prodrug becomes active.
* Prodrug-to-drug conversion can occur before/during/after absorption or at specific site in the body
can:
* Encourage patient acceptance – reduced pain at site of injection, improved taste and odour
108
explain the characterization of drugs
Biopharmaceutical Classification System (BCS)
* characterizes drugs based on solubility and permeability measures
I - high solubility, high permeability
II - low sol, high perm (although drug is not water-soluble, can travel to tissue v easily)
III - high sol, low perm (drugs may carry some functional groups that does not allow them to cross the membrane)
IV - low sol, low perm (hydrophobic drug attacking a membrane protein)
Class II & III drugs can be modified into prodrugs to increase solubility/permeability
109
types of prodrugs
1. Carrier-linked prodrugs = involve attaching a secondary functional group to the drug in order to be carried or to mask its activity/property. It can be further categorized into bipartie, tripartite, and mutual prodrugs
2. Bioprecursors = a prodrug that when metabolized, will transform in a new active compound, different from the parent drug. Unlike carrier-linked prodrugs that already contains a masked active drug, bioprecursors requires the transformation process to form the active drug.
110
what is the ideal drug carrier
1. can mask the FG until it reaches the site of action i.e., drugs taken orally are modified to withstand stomach acidic environment
2. localize the drug at the site of action
3. release the drug chemically/ enzymatically, eg. cause change in pH
4. minimize host toxicity
5. added FG should be biodegradable, biochemically inert & nonimmunogenic. Once that FG is cleaved, it should not interfere with other metabolic pathways or the immune system – must be secreted readily
6. Easily prepared/inexpensive
7. Chemically and biochemically stable in dosage form (so can deliver as a pill/ liquid)
111
explain carrier linked prodrugs
* Esters, amides = major groups of carrier-linked prodrugs. Drugs w/ alcohols and carboxylic acids can be esterified to give ester prodrugs
* Esters are useful in modifying the lipophilicity of drugs.
-Aliphatic esters = improve lipid solubility (more hydrophobic)
-phosphate esters = improve water solubility
* Esterase (a hydrolase enzyme) can cleave off the ester group from the prodrug = active drug compound
* The bond connecting the carrier group to the drug compound must be easy to remove, nontoxic, and biologically inactive.
112
what are some considerations of prodrug design
* Parent drug: should have amenable functional groups. Common ammenable FGs: hydroxyl, carboxylic acid, amine, phosphate, carbonyl groups . Produce diff. types of prodrugs: esters -most common, carbonates, carbamates, amides, phosphates, and oximes.
* Promoiety: added functional groups should be safe and rapidly excreted from the body
* The absorption, distribution, metabolism, excretion (ADME) and PK properties of the prodrug and the parent drug need to be understood eg. where it will be metabolized
* Formation of degradation by-products: must not react with other chemical entities/ processes in the body
113
explain the general mechanism of esterases (acid-base catalysis)
1. Activation of Ser: His acts as a base to obstruct a H from Ser, so it can make a nucleophilic attack on the ester bond
2. results in a –ve charged tetrahedral intermediate, stabilized by backbone of Gly & Asn
3. His now acts as an acid, donating H to the intermediate, resulting hydrolysis of the ester bond (cleavage of functional group) and a covalently modified Ser
4. Activation of water molecule: His acts as a base to obstruct a H from a water molecule, so it can nucleophilically attack the covalently modified Ser, forming another tetrahedral intermediate
5. His acts as an acid to donate H back to the Ser residue in the intermediate, resulting in the release of the active drug compound
114
explain modifications for alcohol-containing drugs
* Acylation w/ aliphatic or carboxylic acids = decrease water solubility
* Acylation w/ carboxylic acids containing amino or additional carboxylates = increase water solubility
* Addition of phosphate or sulphate esters = increase water solubility ( esters may not be good substrates for esterases/sulfatases/phosphatases -not accommodated in binding site – esp. hydrophobic ones, but can be readily hydrolyzed by changing the pH of the environment) - P is good for drugs for parenteral administration (eg. injection)
* Addition of aromatic or aliphatic functional groups = increase hydrophobicity
* Addition of phosphate functional group at hydroxyl and amine groups = increase water solubility -very stable and easily converted to parent drug by sulfatase and phosphatase so it is common
* Addition of carbonates and carbamates groups at carboxyl, hydroxyl or amine groups
-more stable than the corresponding esters but are more susceptible to hydrolysis than amides
115
explain carrier-mediated absorption
some prodrugs are designed to have structural features that would allow them to be taken up by specific membrane transporters in the intestinal epithelium.
o Mimic substrates of a specific transporter to cross the membrane without improving hydrophobicity (hijack transporter)
o they target specific transporters for polar or charged drugs that have negligible passive absorption.
o Peptide transporters are attractive targets; widely distributed throughout the small intestine with high transport capacity and broad substrate specificity – substrates = usually degraded proteins – so can conjugate the drug with a peptide
116
how can prodrugs improve site-selective drug delivery
* passive drug enrichment in the organ (ex. conjugation to tissue-targeting ligands)
* through transporter-mediated delivery (ex. conjugated to transporter-associated ligands)
* by selective metabolic activation through enzymes (ex. prodrugs = activated by enzymes that are specifically expressed at a higher level in certain tissues)
* by antigen targeting (ex. conjugated to antibodies)
117
what are the most common targeted sites for site-selective drug delivery
* CNS (drug should be highly site-selective, and the parent drug should exhibit prolonged retention within the brain tissue)
* tumours (target an inactive prodrug selectively to tumour cells, where the active drug may then be released without causing toxicity to normal, healthy tissue)
* liver = prodrugs are usually rapidly bioconverted
118
explain the principle of prolonged duration of drug action by prodrugs
Highly lipophilic prodrugs of several steroids and neuroleptics are slowly released in the circulation from the site of intramuscular injection and result in a prolonged duration of action.
Once released from the injection site, prodrugs are usually rapidly bioconverted.
* increase half-life in circulation so not metabolized and secreted easily = prolonged effect
* Eg. modification of the nonsteroidal anti-inflammatory (antiarthritis) drug tolmetin sodium to the glycine conjugate increases potency and extends the peak concentration from 1 to 9 hrs because of the slow hydrolysis of the amide linkage
119
what is a bipartite drug + example
drug compound directly linked to 1 carrier
* The carrier alters the compound’s lipophilicity, making it less/more soluble.
* Hydrolytic cleavage is required to release the carrier from the drug at target site
Ex. Latanoprost (Xalatan) is used to treat glaucoma.
* The lipophilic isopropyl ester prodrug can be hydrolysed by corneal esterase to give biologically active acid. Without this modification, this acidic molecule will be excreted very quickly. The lipophilic moiety allows the drug to have a longer lasting effect.
120
what is a tripartite drug
-carrier connected to a drug through a linker
* Alter lipophilicity depending on carrier (if hydrophilic/ hydrophobic)
* Requires enzyme for hydrolytic cleavage at the linker region to release carrier + drug, AND spontaneous release of any remaining parts of linker that is associated with the drug (that will interfere with drug functionality)
* Advantageous over bipartite because can modify property of linker region to make it more stable/ easier to cleave
121
describe the mechanism of activation of a tripartite prodrug
1. Ester hydrolysis by esterase to release carrier, but still left with remains of linker region (eg. aldehyde functional group) & drug is still inactive
2. Spontaneous removal of linker as a secondary by-product (eg. by aldehyde accepting a H from a water molecule = released as formaldehyde)
- By-product must be inactive & shouldn’t interfere with other enzymes/ pathways
3. Release of active drug
122
describe ampicillin as an example
has poor oral absorption and rapid onset resistance by bacteria
* Pivampicillin = a pivaloyloxymethyl ester tripartite prodrug of the β-lactam antibiotic, ampicillin.
- The prodrug uses a –CH2– linker to link ampicillin and the pivalic acid carrier.
- Pivampicillin is thought to have greater biovailability because the ester group provides greater lipophilicity
123
what is a mutual prodrug + example
* Consists of two synergistic drugs attached to each other (bi -directly- or tripartite -via linker)
* One drug is the carrier for the other - mask each other’s function/property
* Once metabolized, will release 2 active drug compounds
Sultamicillin: a mutual tripartite prodrug of the antibiotic ampicillin and the β-lactamase inhibitor, sulbactam.
-The modification improved PK properties i.e. Sultamicillin is more readily absorbed compared to Sulbactam.
-Sultamicillin is broken down into ampicillin and sulbactam in the body. β-lactamase producing strains of bacteria are affected by the combined effects of the antibiotic, ampicillin and the β-lactamase inhibitor, sulbactam.
* Can also reduce the effect of antibiotic resistance cus attacking bacteria w/ 2 antibiotics
124
what are bioprecursors
prodrug that when metabolised results in a new compound which is active or it can be metabolised further to the active drug (requires 2nd metabolic step to be activated)
* Unlike carrier-linked prodrugs that are active drugs linked to a carrier and released by hydrolysis, bioprecursors cannot be converted to the active drug by simple cleavage of a group of the prodrug
* Activated in the body by oxidation or reduction – not functional until goes through Phase I metabolism
* Eg. goes through removal of ester AND phase 1 metabolism in order to become active
125
what are some examples of bioprecursors
Sulfasalazine: is approved for the treatment of inflammatory bowel disease and rheumatoid arthritis
* After an oral dose, only a limited amount of the prodrug (10–30%) is absorbed from the small intestine, so most of the dose reaches the colon
* Activated by reduction of its azo bond by anaerobic bacteria in the lower bowel to an anti-inflammatory agent: mesalazine and the antibacterial agent: sulfapyridine
126
explain Macromolecular drug carrier systems
* Prodrug that involves conjugation of compound to a carrier protein ex:
-Glycoproteins, lipoproteins
-Hormones
-Antibody drug-conjugates
-Peptides
-Synthetic polymers
* Inactive until reach target site where it is converted into active drug – requires esterase and spontaneous cleavage of linker region
127
what are the pros and cons of macromolecular drug carrier systems
* Advantages: specific site targeting, low toxicity, reduced premature drug metabolism
* Disadvantages: may not be well absorbed, maybe immunogenic (induce immune response)
128
what are the challenges designing prodrugs
* synthesis can be complex: adding ester is relatively simple, however, conjugation of groups such as proteins and antibodies is more complex
* Controlling the site and rate of bioconversion and metabolism: cannot assess the exact site of reactions and the exact metabolic pathway of the administered prodrugs
* Interpretation of the preclinical results is complicated by species differences in prodrug bioconversion
* Complex analytical profiling is needed and requires analysis of the prodrug, the parent drug and each of their respective metabolites
- The toxicity of not only the prodrug and drug but also the released promoieties or byproducts needs to be considered
* Stability must be adequate to allow drug synthesis or isolation at scale as well as formulation
129
how can cancers be treated
-surgery
-chemotherapy: several side effects because untargeted (attacks every tissue in the human body in the hope of killing cancer cells) & resistance to anticancer drugs
-radiotherapy
-hormone therapy
combination of therapies usually required
-another approach: antibody-drug conjugates
130
what are antibody drug conjugates (ADCs)
-monoclonal antibodies or fragments attached to biologically active molecules through chemical linkers with labile bonds
131
explain ADCs
* antibodies are tumour-specific; will target the antigens on tumour cells
* while bound to the antibody, the chemotherapeutic payload (drug) no longer circualtes systemically so is tolerated by healthy cells
* delivers highly cytotoxic agents directly to tumour cells w/o affecting other dividing cells in the body (no side-effects)
132
what is the mechanism of action of ADC
ADCs are designed to directly target & kill cancer cells so Ab has to be able to recognise & bind to corresponding Ag localized on tumour cell
1. the Ab's variable region binds the epitope on the targeted tumour-ideally the Ag is tumour cell specific but in many cases it also presents on healthy cells
2. once bound to the Ag, the entire Ag-ADC complex is endocytosed through receptor-mediated endocytosis
-internalization process proceeds w/the formation of a clathrin-coated early endosome containing the ADC-Ag complex
3. once inside the lysosome, ADC is cleaved and cytotoxic payload (CP) is released into the cell, leading to cell death. mechanism of cell death depends on the type of cytotoxic payload
-receptor is then recycled and Ab is degraded in the lysosome
133
what are the 3 key components to an ADC
Ab, linker and CP = each of which requires careful optimisation
134
what are considerations of the antibody
-ADC needs to retain the selectivity of the original Ab while being able to release the CP in concentrations high enough to kill the targeted tumour cell
-upon modification, Ab should not change properties; should bind to same Ag, shoudn't modify key parts of Ab that will render it inactive
135
what are considerations of the linker
-must consider if you want the linker still conjugated to the drug after lysosomal cleavage or removed
-will affect cytotoxicity of neighbouring cells
136
what are considerations of the payload type
-need to ensure that sufficient concentration of payload reaches the interior of cancer cell to guarantee death
-it is estimated that even if overall mechanism of action of ADC works at 50% efficiency, only 1-2% of administered CP will reach the tumour cell
-also important to chose CP that is sufficiently cytotoxic to exert an effect at v.low concentrations
-ADC allows much larger therapeutic window, so delivery at higher dosage is posible
137
what are challenges in designing ADCs (associated w/different steps of ADC mechanism)
1. CIRCULATION: ADC released into the bloodstream
-stable linker required to minimize premature release of payload and off-target toxicity
-pH of the bloodstream could affect cleavage of linker
2. BINDING: mAb component of ADC binds to tumour antigen
-mAB must retain high affinity
3. INTERNALISATION: ADC-Ag complex undergoes receptor-mediated endocytosis
-inefficient internalisation due to limited target antigen level may prevent cytotoxin from reaching its threshold concentration within the cell
4. RECYCLING: fraction of ADCs bind to FcRn receptors in early endosomes and are transported back out of the cell
-excessive binding to FcRns can reduce the amount of CP released in cell
5. RELEASE: lysosomes fuse w/late endosomes and release active CP
-ADC has to efficiently release the CP in active form
-must choose mechanism of release
6. ACTION: cytotoxin interferes w/critical cellular machinery resulting in cell apoptosis
-potency of release payload must be sufficient to kill cell even at low concentration
138
what are antibodies
-an immunoglobin protein produced as an immune defense against foreign agents
-each Ab has a region that binds w/high specificity to particular antigen which it neutralizes
-typically made of large heavy chains and small light chains
139
describe the antibody/immunoglobulin classification
IgG = v.well established in labs, know how to manipulate so preferred for ADCs
IgA (dimer) = found in epithelial cells in lungs
IgM (pentamer) = first antibody to appear in response to initial exposure to antigen
IgE = associated w/allergic responses
140
what is the IgG structure
-heterotetramer of 2LC + 2HC
-each LC linked to HC via diS bond; 2HC also linked via diS
-both LC & HC have a constant (same conserved aa sequence across all Ab's) & variable region (diff aa sequence between diff IgG)
-heavily glycosylated
141
describe immunoglobulin fold
-each domain is structured as an Ig fold; consists of ß-strand arrangements
-apart from linking all subunits together, diS also keep each domain together
142
describe the variable region of Abs
-sequences of the variable domain varies in the loop region called complementary determining region (CDR) -where it binds the Ag allowing for Ag specificity
-L & H chains variable region fold to yield 3 CDR in each chain to form walls of Ab binding groove
- 6 CDR of an Ab is involved in Ag binding (3 from HC/LC)
-all contacts of Ab with Ag comes from CDR loop; aa sequence and length of each CDR varies to allow binding to specific antigens
-the rest of the components provide mechanical stability
143
describe the ADC linker region
-Ab's for ADC are not made by the immune system; synthesised in the lab by generating an immune response in mice and subsequently chemically modifying the Ab
-must consider: Ag selection, linker: cleavable or non-cleavable, site of conjugation
144
antigen selection
* ideally want an Ag specific to tumour cells, but extremely rare because tumour cells are healthy cells w/defective programming
* instead look ate differential expression (upregulation) of specific proteins by tumour cells
-requires substantial expression by tumour cells but limited expression by healthy cells so ADC has more probability to bind to tumour
* also consider the amount of drug molecules conjugated to the antibody: drug-to-antibody ratio (DAR)
145
antigen selection
* ideally want an Ag specific to tumour cells, but extremely rare because tumour cells are healthy cells w/defective programming
* instead look ate differential expression (upregulation) of specific proteins by tumour cells
-requires substantial expression by tumour cells but limited expression by healthy cells so ADC has more probability to bind to tumour
* also consider the amount of drug molecules conjugated to the antibody: drug-to-antibody ratio (DAR) -on average, current clinical-stage ADCs is limited to 3.5-4
-there are also limited # of antigens on the tumour cell surface so amount of drug delivered by ADCs into tumour cells is low
-once identified Ag of interest & generated Ab, must purify and modify the Ab
146
antibody-drug linkers
* when Ab is modified with linker & drug, will lead to change in solubility and how the drug is endocytosed, clearance time, circulation efficiency
-must consider the stability of the Ab w/drug, Ab retention of the drug, & drug stability upon release
-eg. upon conjugation w/drugs = faster clearance, so max efficiency is 3.5-4 drugs/Ab
147
cleavable linkers
-cleaved upon internalization of antibody -by using the differences in conditions between the bloodstream & the cytoplasm within tumour cells (eg, pH, reducing env)
-the change in environment once the ADC-antigen complex has internalized, triggers cleavage of the linker and release of the active payload
* divided into 3 main sub-categories:
-acid-labile (eg. hydrazones) -cleavable by change in pH
-reducible (eg. diS)
-enzyme-cleavable (eg. peptides) -eg. glucuronide moiety cleavead by glucuronidases in cell metabolism
148
acid-labile cleavable linkers
-remain stable in neutral pH
-use the low pH in endosome/lysosome of the tumour cell to cleave the conjugation and release the drug from the ADC
-clinical studies indicate that acid-labile cleavable linkers are associated with non-specific release of the payload (released in any acidic environment) which can lead to systemic toxicities
149
reducible cleavable linkers
* rely on the difference in reduction potential between the intracellular compartment of a tumour cell vs the blood based on glutathione concentration gradients
-tumour cells have high-conc of glutathione so higher reducing environment
* diS are stable at neutral pH but susceptible to Nu attack from thiols
* abundance of thiol molecules within tumours, especially generated during stress, like hypoxia, because thiols are involved in survival and growth of tumour cells
150
enzyme-cleavable linkers
* take advantage of hydrolytic enzymes capable of recognising and cleaving particular peptide sequences contained within linkers, ensuring that ADC only undergoes cleavage in the lysosomal environment and not in the plasma
-usually peptidases cleave the peptide bond
* can also rely on metabolism -eg. ß-glucoronidase, which can release payloads from ß-glucoronide-containing linkers
-present in lysosomes & over-expressed in some tumour cell types
-mechanism: ADC w/ß-glucoronide linker enters metabolic pathway and is cleaved by ß-glucoronidase and CP is released
-ß-glucoronide linker is hydrophilic, can potentially reduce aggregation during conjugation compared w/constructs containing dipeptide-based or other linker types
151
what is the mode of action of cleavable linkers
1. bind to specific Ag, internalization of ADC-Ag complex according to clathrin-dependent mechanism
2. transfer into lysosomes
3. lysosomal cleavage of the linker between the trigger and the spacer which degrades to release the drug
4. transfer of drug to tubulin (MMAE, MMAF, DM1/4), nucleus (calicheamicin), or cytosol
5. CP then leads to cell death
152
advantage of cleavable linkers
able to enter and affect pathways in the nucleus so can target DNA
153
disadvantage of cleavable linker
* diffusion of the drug to neighbouring cancer cells can result in bystander killing effect (all drugs except MMAF)
-MMAF because of charged character cannot cross the membrane so does not exhibit bystander killing effect (good and bad)
-if the neighbour is also a cancer cell (eg. solid tumour w/huge cell mass in prostate & breast cancer), Ab only binds to Ag on surface of outer cell mass -leading to increase in success rate of killing tumour cells
-if neighbour is a healthy cell it will lead to systemic toxicity
154
non-cleavable linkers
-drug usually remains attached to mAB
-engaged in lysosomal degradation of Ab only
155
mode of action of non-cleavable linkers
1. ADC recognises specific Ag, internalization of complex ADC-Ag through clathrin-dependent mechanism
2. transfer into lysosome (linker is not cleaved but Ab is degraded)
3. complete digestion of mAB to release the active corresponding metabolite amino-acid-NCL-drug
4. transfer to tubulin or cytoplasm
5. payload leads to cell death of the Ag-positive cancer cell
156
advantage of non-cleavable linkers
* increased plasma stability -not dependent on pH/reducing env
-lower risk of systemic toxicity due to less premature release of CP & drug = unable to be exported to neighbouring cell (no bystander effect)
-good for blood tumour circulating around the body
-not good for solid tumours because will only kill the cell that Ab binds to -if there is no space for Ab to diffuse to the centre of the cell mass, it will not be able to kill
-better control of inhibiting pathway, better therapeutic window w/improved stability & tolerability
157
disadvantage of non-cleavable linkers
-limited effect because unable to target pathways in the nucleus, only in cytosol
-left with linker/part of Ab attached to drug after Ab degradation
158
Site of conjugation
* Ab is relatively large molecule of ~90kDa w/ ~600-1200 residues, so many sites of conjugation (both on LC & HC); can modify in any region except ABS where CDR loops are (or will lose Ag specificity)
* typical average DAR = 3.5-4 drugs conjugated per Ab
* must consider type of chemistry involed in modification:
-thiol chemistry (uses Cys)
-amine chemistry (uses Lys on cell surface)
-disadvantage of both = no control of which specific residues will be modified
159
conjugation via thiol groups
-monoclonal IgG Ab's contain many diS bonds
-the inter-chain thiol groups are the most desired sites of attachment for CP
-but they are not free/accessible because they are involved in formation of diS linking LC & HC
160
conjugation via thiol groups process
1. DiS bonds are reduced with agents eg. tris, phosphine, DTT, or 2-mercaptoethylamine prior to conjugation
2. react w/ a linker-payload complex containing suitable electrophilic moiety (will make Nu attack on free thiols) eg; maleimide, N-hydroxysuccinimide
3. must oxidise again to reform some interchain diS
161
disadvantage of conjugation via thiol groups
-unable to control which diS will be reduced; once the reducing agent is added, all diS will be reduced
-cannot control which Cys will be modified despite being able to control the number of molecules added in the modification
-most importantly, Ab may lose integrity (if reduce both LC-HC and HC-HC diS)
162
overcoming limitations of unspecific conjugation (thiol groups)
partial reduction
-add red.agent at a conc that will not fully reduce diS with the anticipation that some non-important diS will be reduced to prepare for conjugation w/linker, while the important ones remain intact
site-specific conjugation by the THIOMAB technology:
-THIOMABs are Ab's engineered w/reactive Cys residues which have a THIOMAB cap at specific sites
-can perform full reduction reduce all diS, followed by oxidation to reform all important diS except the Cys residues w/THIOMAB caps (too far apart from each other to form diS)
-end up with specific Cys residues available for modifications
advantage: has defined stoichiometry and sites for payloads w/o disruption of interchain diS bond
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conjugation via lysine residues
-lysine amines are v.effective for payload conjugation because they are well-exposed and their amino groups are good nucleophiles
-lys conjugation reaction involves formation of stable amide bonds using activated ester of CP, typically an O-succinimide reagent such as NHS or maleimide
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what are the adv/disadv of conjugation via lysine residues
adv: don’t need to reduce key Cys residues
disadv: lys is a v.common aa & can have many Lys residues on Ab, unable to control the site/number of conjugating molecule & high likelihood of modifying ABS/CDR loop
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strategies to improve conjugation via lysine residues
* Engineered Cys
* Insertion of unnatural amino acids (not found in genetic code, can control specific site/ number)
* Enzyme-assisted ligation
* Glycan remodelling and glycoconjugation (modify sugars found on surface – not popular because Ab = heavily glycosylated)
* Amino terminal engineered serine
* Native cysteine rebridging
* (Highly loaded ADCs at specific sites)
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type of drug that is conjugated to the linker
* high toxicity
-cytotoxicity is required since delivery is limited by Ag copy number
-payloads need to be active in low nM or picoM regions
-need to posses favourable physicochemical properties eg. hydrophilic/hydrophobic balance, and good stability
* drug compound must be able to maintain its chemical properties upon conjugation w/ Ab or linker
* must consider the site on the drug that will be conjugated. esp important for non-cl linker
*bystander effect: may or may not need depending on type of target
-blood tumour: can lead to toxicity so not needed
-solid tumour: need it because drug compound can cross the membrane and kill neighbouring cells
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auristatins
* largest groups of ADCs in clinical trials are those based on monomethyl auristatin E (MMAE) and MMAF
* synthetic analogues of dolastatin 10 (natural antimitotic drug extracted from the sea hare Dolabella auricularia)
* too toxic to be used in its unconjugated form
-high potency, water-solubility, stability under physiological conditions and suitably for attachment of stable linkers
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other examples:
Tubulysins: inhibit microtubule polymerization during mitosis to induce cell death
Calichaemicins: binds to minor groove of DNA and cleaves dsDNA in site-specific manner
Duocarmycins: DNA minor-groove alkylating agent
Benzodiasepines: PBDs bind to DNA minor groove in a sequence-specific manner
Doxorubicin: an actinomycete-derived antimitotic anticancer agent that is routinely used in clinic
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bystander effect
* solid tumours often express target antigen in a hetergenous manner. ADCs that selectively kill only Ag+ve cells and spare neighbouring Ag-ve cancer cells may be ineffective in eradicating such tumpours
* ADCs can be designed to kill not only Ag+ve cells but also other cells in vicinity, w/o expression of the target Af on their surface (bystander effect)
* need to be able to cross biomembranes and kill neighbouring cells
* bystander effect is not always desirable; may choose a non-cl linker
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ADC toxicity
* via unsufficient uptake of ADC so results in premature release of CP, which can freely circulate and cause systemic toxicity
* independent uptake of ADC resulting in CP affecting untargeted cells
* bystander effect
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ADC administration to humans
*Ab used to make ADC are usually produced from animals (usually mice), because can engineer Ab at specific sites
* mice Ab has different structure to humans (<70% similarity) so upon injection of mice Ab into human body will active immune response against it.
To fix this:
chimeric and humanized antibodies
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chimeric Abs
-consists of variable regions derived from a mouse and constant regions derived from human w/total similarity ~65% to human Abs
-can reduce the intensity of immune response, but not completely
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humanized Abs
-therapeutic mABs predominantly derived from a human source (same aa sequence as human Ab) except fro CDR loops which are generated from mice
->90% similarity to human Ab so can significantly reduce immune response
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summary when designing ADC, must consider:
* cleavable vs NC linker
* which pathway to target; which effect to generate?
* bystander effect Y/N
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example of ADC development: Ab-drug conjugate for breast cancer
Trastuzumab: anti-HER2 antibody
-approved for use in 1998, often administered in combination w/ paclitaxel or docetaxel, 2 microtubule stabilizers
-effective in eliminating cancer cells by an Ab-dependent cytotoxicity mechanism; but some tumours do not respond satisfactorily to the treatment
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T-DM1
trastuzumab conjugated w/cytotoxic agent DM1
-developed w/aim of improving cytotoxicity of trastuzumab while taking advantage of its tumour selectivity
-conjugates were evaluated for in vivo pharmacokinetics and antitumour activity
-in vivo studies revealed that the higher the linker stability, the higher the anti-tumour activity
-based on this infor, drug Kadcycla was developed
-T-DM1 approved in 2013 for HER2-positive breast cancer treatment
PS
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