Konstantinos Beis - Antibodies + Drug Design Flashcards
What are antibodies?
An immunoglobulin protein produced by the immune system as an defense against foreign agents (known as antigens) any foreign molecule, virus, bacteria etc.
Each antibody has a region that binds specifically to a particular antigen which it neutralizes – can have a variety of different targets
It is typically made up of large heavy chains and small light chains
What is a catalytic antibody?
Catalytic Antibodies (abzyme) - Antibodies that behave like enzymes
Generally speaking how do humans produce antibodies? What are the two main systems at play?
- Humoral (antibody-mediated) immune system - antibodies produced in response to freely circulating pathogens - direct recognition of antigen by B cells which produces plasma cells (produce antibody) and memory cells
- Cellular (cell-mediated) immune system – antibodies produced in response to intracellular pathogens –> cell exposes antigen on surface (MHC – membrane protein complexed with antigen), which is recognized by T-Cell which activates/helps transform B cells into antibody providing plasma cells
What are the different immunoglobulin/antibody classifications?
5 different groups – IgG, IgA, IgM, IgD, IgE
Fakts:
- IgM - is the first antibody to appear in response to initial exposure to antigen
- IgE associated with allergic response
- They exist in different states – monomers, dimer, pentamer
Outline the general structure of the IgG protein
IgG structure – Best characterized
Characteristic Y-shape – usually homodimeric - the two monomers are connected by disulphide bridges which connect the heavy chains together
Note - Disulphide bridges also exist between the Heavy and Light chain
Each monomer consists of a Heavy and Light chain
a) Heavy Chain – High Mw
b) Light Chain – Low Mw
Antibody is heavily glycosylated – meaning that the Fc region can itself initiate a immune response but this is not common
What are the different regions within the immunoglobulins e.g. IgG?
Constant region (denoted by C) – high degree of A.A. conservation
Hyper/Variable region (Denoted by V & HV) - when antigens bind + lower A.A. Conservation
The antibody can be further divided into the Fab and FC region
FC - easily crystallized
Fab - Antigen binding site
Breakdown the different parts of the IgG light chain
Breakdown the different parts of the IgG Heavy chain
Why is IgG normally used in the formation of catalytic antibodies?
Easily modified and the most common immunoglobulin, which is distributed between the blood and extravascular fluid.
What is the structure of the Immunoglobulin fold?
Two Beta sheets (built from anti-parallel Beta-strands) that are sandwiched together
Image - Loop region holds together the Variable and constant chain
In the variable domain you can see blue loop regions corresponds to the amino acid sequence that varies, providing specificity –> These loops are known as the complementary-determining regions (CDR) or Hypervariable Loops
How many CDRs/hyper-variable loops do the light and heavy chains have?
Each Light and Heavy chain variable domain will have 3 CDRs or hypervariable regions – CDR L1-3 or H1-3
All these CDR loops are all used to recognize an epitope/antigen
Across antibody species there is no conserved conformation within these regions –> Insertions, deletions and Amino acid sequence differ.
What does the following crystal structure show?
Example Crystal Structure of a Fab
Shows CDR H3 from the Heavy chain which extends significantly out – stabilized by a range of hydrogen bonds
Note - for high specificity we normally get the contribution of both Heavy and light chains CDRs
Why do we want to use catalytic antibodies and why do we not just use enzymes?
On a chemical level whats the benefit of using catalytic antibodies/abzymes?
Abzymes – Use antibodies to catalyze reactions
- Already present in body – doesn’t produce an immune response
- High affinity & specificity –> favours desired reaction coupled with less side reactions/effects
How do they act?
- They have structural complementarity for the transition state –> reduce the activation energy to reach TS
- Consequence? - strong binding of TS with high association constant, enhances the rate of reaction
- Abzymes also reduce rotational entropy
What is a problem associated with using an catalytic antibody that has a high specificity for the subtrate?
High affinity for substrate, hence it will help stabilize the Enzyme substrate complex (ES) (lower in energy) – increasing the energetic barrier to reach the TS.
Solution - We select antibodies for our transition state - Helps to stabilizes that rather than ES
Definition of antigen and hapten?
What is Cocaine?
Alkaloid from the coca plant
Cocaine acts as a serotonin–norepinephrine–dopamine reuptake inhibitor –> leads to increased extracellular concentrations of these neurotransmitters – keeps the neurons firing
How does cocaine acts as a serotonin–norepinephrine–dopamine reuptake inhibitor in the brain?
Normal Processing
- Dopamine released by vesicle into the synaptic cleft
- Binds to the dopamine receptor on the post-synaptic membrane – inducing a signal
- Dopamine is released
- Re-uptake transporter transports dopamine back into the pre-synaptic neuron where it is degraded by MAO to form monoamines which are no longer active
Cocaine action
- Cocaine Binds to the dopamine re-uptake transporter –> preventing dopamine reuptake. Hence, it remains in the synaptic cleft where it can continuously stimulates the action of dopamine receptor
Outline how cocaine is normally degraded in the body
Cocaine Degradation –> Slow process performed by enzymes in the body (can be degraded prior to crossing BBB) - involves hydrolysis the benzoyl ester and the methyl ester
Different methods of degradation normally found in the body
- Enzyme found in the N.S. (Butyrylcholinesterase) – forms ecgonine methyl ester + Benzoic acid –> benzoyl ester hydrolysis
- Enzyme found in liver (liver carboxylesterase) –> methyl ester hydrolysis forming forming benzoylecgonine + methanol
- Enzyme found in the N.S –> Demethylation by N-demethylase followed by Butyrylcholinesterase action (benzoyl ester hydrolysis) to form Norecgonine methyl ester + benzoic acid
All of the products from Cocaine breakdown are harmless which can be secreted out of the body
What is the role of Butyrylcholinesterase?
Butyrylcholinesterase (BChE) – Main enzyme involved in degradation
BChE hydrolyses butyrylcholine BUT It can also hydrolyse toxic compounds that contain an ester – Cocaine is one as well
Human esterases are slow to degrade cocaine
- > 11 isoforms found in liver, brain, heart
Outline Butyrylcholinestarase mechanism of action on butyrylcholine.
Acid-based catalysis with a glutamate, Histidine and serine
- Glutamate hydrogen bonds the histidine, allowing histidine to act as base to remove serine -OH hydrogen - driving nucleophilic attack to the ester bond (Carbonyl carbon)
- Formation of Transition state
- Collapse of the negative charges - histidine is now acting as a acid (donating it’s hydrogen)
- Incoming water displace choline
- Histidine acts a base removing a proton from water, allowing for the -OH to nucleophilically attack the carbonyl
- 2nd Tetrahedral intermediate formed followed by it’s collapse (collapse of negative charge)
- Release of the final product & active site returned to starting state
Outline mechanism of Butyrylcholinestarase degradation of cocaine - general sequence of events
- Activation of serine to perform initial nucleophilic attack on carbonyl carbon
- Tetrahedral intermediate
- Collapse of negative charges
- Release of Ecgonine
- Incoming water molecule – activated by histidine
- -OH nucleophilically attack modified serine
- Tetrahedral intermediate
- Collapse of negative charge
- Release of benzoic acid a regeneration of active site
When designing the catalytic Abs against cocaine, what are some things to think about/take into consideration? What are we doing in practise?
Objective –> Ab recognizes, binds, and catalyzes cocaine. The products must be released so the Ab is recycled and free (unlike naturally occurring antibodies)
What do we have to do in practise?
- Predict transition states, from which we create TS analogs that match TS
- Produce a Hapten that is stable and mimic structural and electronic properties
- Epitope must be large enough to bind well with Abs
- Design and choice of Linker and carrier protein
- Hapten + carrier elicits immune response producing desried Abs which stabilize the TS of the reaction
What is the transition state produced in cocaine hydrolysis which we can use for Abzyme production?
Transition state – Tetrahedral intermediate
In order to create an antibody that targets cocaine we need an antibody that binds and stabilizes a T.S. analogue
Basically need to re-create the T.S. so that an antibody against it can be created (we can’t capture the exact molecule – educational guess)
Example of an analog for Tetrahedral intermediate is replacing the carbon in the tetrahedral intermediate to a phosphate - cannot be attack nucleophilically by H2O – stable
What are some examples of groups that can be used to re-create tetrahedral intermediates of transition states?
Once you have identified your TS analog, what do we do next?
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
Things to consider when designing a linker?
Apart from the linker design and carrier type, what other thing must you consider before conjugation?
What are the two methods we use to produce our desired catalytic antibodies?
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
After isolating catalytic antibodies/abzymes, what is done next?
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
After identifying succesful abzymes, how are toxicity assays performed?
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
What did the crystal structure of reveal show about the binding of cocaine and TS analog with the abzyme?
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
What was the proposed mechanism by which the abzyme degraded cocaine?
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
- Cocaine enters active site - stabilized by hydrogen bonds
- Activated Water molecule performs nucleophilic attack on carbonyl carbon
- Tetrahedral intermediate formed – -ive charge is stabilized by tyrosine
- Followed by its collapse –> release of two products
Simpler mechanism than normal acid-base
What is a prodrug and why is it beneficial to use?
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
How can we combine both pro-drugs and catalytic antibodies?
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?
- Minimizes toxicity
- Allows for drug targeting
- 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
What are the limitations of catalytic Abs?
- Haptens fail to generate antibodies that catalyse the desired reaction
- Haptens may closely resemble the final product (stabilize it) - slow product release or inhibition – preventing the antibody for catalyzing more reactions
- It might be difficult to synthesize the desired hapten (chemistry)
- The catalytic efficiency depends on the solvent exposed binding site?
- Introduction of foreign antibody might elicit immune response
How can we create therapeutic/catalytic Abs that don’t elicit an immune response in the host?
Use chimeric or humanized antibodies
- Chimeric consist of variable regions (VL and VH) derived from a mouse and constant regions derived from human. ~65% human
- Humanized therapeutic mAbs are predominantly derived from a human source except for the CDRs, which are murine. >90% human
Apart from acting as a catalytic antibody, what other useful functions can antibodies perform?
Summary of Catalytic antibody lecture content?
- Catalytic Abs can be raised against specific haptens
- Catalyse desired reactions
- Consider different analogues, linkers and carrier proteins
- Catalytic Abs have the potential to treat addictions
What are antibody-drug conjugates?
Antibody–Drug Conjugates (ADC) –> Antibodies used to deliver a drug to a specific site
What are the current treatments of cancer and is the recurring problem?
Cancers (many different types!) can be treated by:
- Surgery
- Chemotherapy – several side effects and resistance to anticancer drugs
- Radiotherapy
- Hormone therapy
- Combination of therapies is usually required
Recurring problem - target/impact healthy cells – leads to side effects
Why do antibody-drug conjugates provide a useful solution to treating cancer?
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
What is the general concept behind using antibody-drug conjugates?
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
What are some examples of ADCs that have already been approved?
General mechanism, requirements and componenets of a ADC?
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?)
Outline the steps by which ADCs target cells and become internalized
- 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.
- Once bound to the antigen, the entire antigen–ADC complex is then internalized through receptor-mediated endocytosis.
- 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
- Once inside the lysosome, the ADC is degraded and free cytotoxic payload released into the cell, leading to cell death.
- The mechanism of cell death will depend on the type of cytotoxic payload.
Why is it important to choose a payload that is sufficiently cytotoxic?
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
Benefits of using ADCs relative to chemotherapy?
- Wider therapeutic windown –> less likley to be toxic as there is a higher maximum tolerated dose and lower minimum effective dose
What are the three main things to consider when designing an ADC?
What are the major challenges that face researchers when designing ADCs?
When designing a ADC linker what do we have to consider?
- Antigen Selection
- Cleavable linker
- Non-cleavable linker
- Site of conjugation
Why does the selection of target antigen potentially pose problems?
What is the relationship between DAR and the rate of degradation? What can we learn from this?
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)
What are cleavable linkers? What are the different types?
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).
How do acid-labile linkers work? What problems are associated with it?
How do reducible linkers work? What problems are associated with it?
How do enzyme-cleavable linkers work? What problems are associated with it?
Apart from rely on Cathepsin, what other enzyme is used for Enzyme-cleavable linkers?
Outline the general mode of action of ADC’s with cleavable linkers, starting from antigen binding to apoptosis
Why are non-cleavable linker used for ADCs?
Non-cleavable linkers – Why are they used?
- Drug (usually) remains attached to mAB - not effected by pH, reducing environment or enzymes
- In the lysosome – mAB is degraded by compound + linker is not!
- Increased plasma stability
- Lower risk of systemic toxicity due to premature release of the payload
- Better therapeutic window, with improved stability and tolerability
Outline the mode of action of ADCs with non-cleavable linkers
Example of a Non-cleavable Linker
When conjugating our drug to the antibody what do we have to consider?
- Prevent modifying antibody to the point where they induce an immune response
- 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 - Retaining antibody selectivity (antigen binding)
Conjugation Example:
What are two examples of functional groups on our antibody which we tend to use for conjugation?
- Thiosuccinimide linkage, which is formed through the reaction of thiols and alkyl maleimides –> thiosuccinimide formation is slowly reversible under physiological conditions
- Conjugation to the terminal amines of lysine residues using an amide ester
How is conjugation with thiol groups performed?
What is one possible solution to thiol conjugations lack of DAR control?
How is site-specific conjugation performed with THIOMAB technology?
How is conjugation with lysine residues performed? What problems are associated with this form of conjugation?
What are some further potential strategies that could improve conjugation?
- Engineered Cys – Thiomab
- Insertion of unnatural amino acids – tight regulation of our payload with a specific amino acid that is not found naturally
- Enzyme-assisted ligation
- Glycan remodelling and glycoconjugation - remember that antibodies are glycosylated
- Amino‑terminal engineered serine –> problem may change overall properties of antibody
- Native cysteine rebridging
- Highly loaded ADCs at specific sites
What is the toxic payload/warhead? What properties are we looking for? What cellular structures/organelles are we targetting?
What is the largest group of ADCs currently being trialed?
What are some other examples of drugs that are being used for ADCs?
What is the Bystander effect?
What are the 4 different forms of ADC toxicity (think good & bad)?
What do we have to remember when using antibodies (Think immune response)?
Summary of things to consider with ADCs
What is an example of ADC that is approved for targetting HER2-positive breast cancer treatment?
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
Summary of ADCs
- ADCs are potent drug delivery tools
- Low toxicity – relative to other cancer treatments
- Care should be taken with linker design, payload choice
- Linker can be cleavable or not
- Bystander effect desirable or not
Is there any particular reason why our cocaine T.S. analog is too small to elicit an immune response?
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
When using a Non-cleavable linker, why is more active drug compound delivered to the target?
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
How do you prevent the aggregation of antibodies caused by payloads hydrophobicity?
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
What is one reason why cancer cells are often immune/resistant to chemotherapy/drugs?
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
How to select appropriate conjugate?
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.
What happens to the drug once the cell undergoes apoptosis?
The drug will enter the metabolism –> The human body can detoxify these compounds – making them inactive
How to identify target antigens on cancer cells?
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
Outline the different steps in the Drug Discovery proces/program?
What are different way to identify new hits/drugs for a target molecule?
- High Throughput Screening (HTS) – wet lab setup - most commonly used by Pharma But…
- Expensive
- False positives
- Assay variability or errors in data - Virtual Screening (VS) –> main focus of lectures
- Combination of HTS and VS
What are the limitations associated with HTS?
What are some essential requirements when running a HTS screen?
- Expensive robotics are required to screen for candidates – large scale 107
- Very robust assay required – Biological activity meter
- Interaction analysis of successful hits - between drug and target
- Complement HTS with structural data and using in-silico methods
What are the two methods that you can divide Computer-aided drug discovery (CADD) into?
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
Difference between SBDD and LBDD?
Outline the overall process of virtual HTS?
- Start with library of compounds
- Filter the library depending on Mw, physical properties, etc.
- Virtual HTS - LBDD or SBDD
LBDD - focus of lecture
- Searching for similarity in library towards ligand (known) of interest – identify hits
- Synthesize or purchase hits
- Evaluate in the lab
a) Biological activity - refine
b) No biological activity - Start again
What type of information is used in LBDD to filter/search library?
Takes into account different kinds of molecular information of the known ligand such as
- 2D molecular fingerprint
- 3D molecular shape
- Molecular and electronic properties
- 3D pharmacophore
Image shows the two different LBDD strategies
What are the three methods that LBDD searching can be divided into?
- Similarity searching
- Pharmacophore mapping
- Machine learning
Outline what is meant by the LBDD similarity search method
What filters are commonly used for a similarity search?
Outline how 2D fingerprints of molecules are created?
Reasoning for using bits –> facilitates processing for computer
Outline what is meant by Hashed fingerprinting (2D property filter)? What is meant by bit collision?
What is the similarity coefficient used for ranking 2D fingerprints?
What is scaffold hoping?
What is meant by 3D fingerprinting?
What is a pharmacophore?
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
Explain what is going on in the attached image (hint - 3D fingerprinting)
After performing 3D fingerprinting, what do we do?
When performing a search in a database, what should we look out for?
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
Does 3D shape search require us to know the protein structure?
Nope!
We assume that all (or the majority) of the known actives bind to the same location of the protein
- 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
- 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
What is the most common pharmacophore model?
When selecting a representative set of actives what should we consider?
- 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
- Prepare molecules (e.g. tautomeric form, protonation state), generate 3D structure and conformations (if required)
- Use pharmacophore software/tool to generate pharmacophores (depending on input it may be biased or unbiased)
- Select preferred pharmacophore model(s) and validate them experimentally –> are they really the core functional groups in binding our protein?
Things to consider for database matches - i.e. how do we know if we have a good match?
- Pharmacophoric features in each ligand are identified (Donors, acceptors, hydrophobic groups, etc)
- Ligands aligned so the corresponding features are overlaid
- Conformational space explored
- Scoring system: number of features, goodness of fit to features, conformational energy, volume of the overlay, etc
When searching a database, what should we keep in mind about the database itself?
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?
Case study (LBDD) - Inhibition of Human Thymidylate Synthase and Dihydrofolate Reductase Enzymes
Why do we care about these two enzymes? What known substrate was used for LBDD?
What is the LBDD QSAR Model?
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
What is the idea behind QSAR?
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
Outline the general process of SBDD
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
- Start with Library
- Filter compounds
- Virtual HTS – Use different types of docking
- Score Hits
- Evaluate using experimental techniques
- Process can be repeated for optimization
What are the three main SBDD methods?
Basic principles of SBDD?
- 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
What does virtual HTS entail?
virtual HTS - Perform docking in-silico
After In-silico screening…
- Test activity (enzymatic and pharmacokinetics)
- Solve crystal structures to verify binding mode
Examples of drugs designed using SBDD
Main challenges when performing SBDD?
- 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
What are the criteria/parameters/descriptors used for SBDD?
What are Structure activity relationsips (SAR) and how can we identify them?
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?
- X-ray crystallography can be used to identify important interactions between drug and protein
- 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
What is Lipinski’s rule of Fives?
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
- MW less than 500 – easier diffusion across membrane + easier to modify
- Fewer than 5 H-bond donating groups
- Fewer than 10 H-bond accepting groups
- LogP between -1 and +5 – experimentally determined using the partition of molecule in octanol and H2O – indicates hydrophobicity
What are the two main parts of SBDD virtual screening (docking)?
- In silico to identify potential lead compounds from a database
- From which we score, rank and filter a set of chemical structures -
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
What does the Rigid docking search algorithm do?
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
- 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
- Orient ligand in the active site using simple geometry descriptors, like spheres or triangles –> Satisfy stereochemistry, no clashes
- Rank/score the different poses based on receptor affinity
What are the two different scoring methods for different ligand docking poses (SBDD)?
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
Benefit of Rigid docking?
Fast as it is less computationally demanding – screen many ligands quickly
What does the flexible docking search algorithm entail?
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
Before performing flexible docking (SBDD), what do we need to consider?
A) Structure are required to be high resolution structure since otherwise…
- Water molecules not resolved
- Electron density for flexible side chains might be weak
- Hydrogen bonds are absent - 1Å or better resolution is optimal but can be artificially added at lower resolutions (less accurate)
- pKa of the active site side chains - crystallisation buffer
- 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
What does De Novo screening (SBDD) entail?
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?
- Examine possible poses & conformations (search algorithm) –> Orientation of molecule in binding site - More flexible as we are dealing with smaller molecules/fragments
- Predicting energetics of protein-ligand binding (scoring function) - Binding affinity & Thermodynamics
What is Lead identification by Fragment evolution (De-novo synthesis technique?
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
What is Lead identification by Fragment linking (De-novo synthesis technique)?
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
What is Lead identification by Fragment self-assembly (De-novo synthesis technique)?
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
What is the last step for any De novo synthesis technique?
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)
Difference between fragment based approahces (building into) and High throughput screening?
Why is Thymidylate synthase an enzyme of interest for drug researchers?
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.
Case-study - What in-silico docking steps were performed for screening for drug canditates against Thymidylate synthase?
- Researchers attempted to find molecules that resembled the co-factor tetrahydrofolate –> X-ray structure of the TS available allowing for identification of key interactions
- Identified potentual lead from docking
- 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
- Further redesigning - introduction of amide group –> Wet-lab results and in-silico predictions matched up
Once a lead has been identified from screening, what criteria does the potential drug need to meet?
Lead is validated by re-testing checking for…
- X-ray Structure assesment
- Potency: the amount of drug required for its specific effect to occur; inverse of the EC50
- Efficacy: the maximum strength of the effect itself, at saturating drug concentrations.
- Pharmacokinetics: determining the fate of xenobiotics. Extend and 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
- Patentability
Examples of two HIV-protease inhibitors designed using computer modelling?
What is drug metabolism?
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
Why is it important to convert lipophilic compounds hydrophilic during drug metabolism?
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
What does Pharmacokinetics refer to?
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
Why are pharmacokinetics important to conisder when thinking about the drug dosage?
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
- Volume of distribution (reach target) and clearance influence Half-Life
- Clearance and Absorption will influence oral bioavailability - how much reaches the blood stream
What are the different phases in drug metabolism?
Role of Phase I and II transformations in drug metabolism?
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)
In phase I metabolism, what is the primary form of modification?
1o 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
Outline the reduction reactions that take place in phase I metabolism
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…
- Nitro reduction to hydroxylamine/ amine
- Carbonyl reduction to alcohol
Outline the hydrolysis reactions that take place in phase I metabolism
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
What are the main oxidation enzymes in drug metabolism?
Oxidation enzymes – all these enzymes found in the liver
- Cytochrome P450 monooxygenase system
- Alcohol dehydrogenase
- Aldehyde dehydrogenase
- Flavin-containing monooxygenase system
- Monoamine oxidase
What are the main reduction enzymes in drug metabolism?
Reduction enzymes
- NADPH-cytochrome P450 reductase
- Reduced (ferrous) cytochrome P450
What are the main hydrolysis enzymes in drug metabolism?
Hydrolysis enzymes
- Esterases and amidases
- Epoxide hydrolase
What system does phase I metabolism normally rely on?
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
What is Cytochrome P450 (CYP) 3A4? Where is it located? What other enzyme does it require?
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
What is the role of the heme group in Cytochrome P450?
Can Cytochrome P450 accomodate a wide range of substrates?
Cytochrome P450 can accommodate a large variety of functional group as the cavity adjacent to the Heme is large, as shown below.
Outline the reaction cycle of the heme-group in cytochrome P450
What is the role of Phase II transformation?
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
What are the different chemical reactions that take place in Phase II metabolism?
- Glucuronidation by UDP-Glucuronosyltransferase (-OH, -COOH, -NH2, -SH)
- Sulfation by Sulfotransferase: (-NH2, -SO2NH2, -OH)
- Acetylation by acetyltransferase (-NH2, -SO2NH2, -OH)
- Amino acid conjugation (-COOH)
- Glutathione conjugation by Glutathione-S-transferase
- Fatty acid conjugation
- Condensation reactions
What are the phase I and II steps performed by the body when processing aspirin?
Phase 1 - Prodrug converted to active compound Salicylic acid
Phase 2 - Conjugate to allow for excretion – different routes
What is the most important phase II pathway for drugs and endogenous compounds?
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
What are the two different types of glucoronidation?
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
What should you know about Phase II metabolism sulfation?
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
What should you know about Phase II metabolism acetylation?
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
What should you know about Phase II metabolism Fatty Acid Conjugation?
Stearic and palmitic acids are conjugated to drug by esterification reaction
What should you know about Phase II metabolism Amino Acid Conjugation?
Active CoA-amino acid conjugates that react with drugs via N-Acetylation –> Glycine, Glutamine, Arginine
What should you know about Phase II metabolism Glutathione Conjugation?
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
How can we make drugs more resistant to metabolism?
Remove functional groups susceptible to enzymes
E.g. Tolbutamide can be excreted directly whereas Chlorpropamide needs to enter phase I metabolism
How can we make drugs less resistant to metabolism?
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
How are metabolites normally exported from the cell?
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
What are factors that can influence the metabolism of a single drug?
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
Why is it important to consider the amount of drug that binds to plasma proteins?
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
What stage of metabolism do pro-drugs rely on?
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
How can we improve membrane permeability of drugs?
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
- 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
Apart from increase membrane solubility to improve membrane permeability, can we also hijack transporters?
Membrane transporter - mimic substrates that have transporters to cross membrane
How can pro-drug concept be used to extend the life of a drug?
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.
How was aspirin made less toxic?
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
General role of Phase I and II metabolism?
Phase I and II metabolism make drugs less toxic and more hydrophilic
For Lipinski’s rule of five, is there any particular reason why fewer than 5 h bond donating groups and 10 h acceptor groups in the compound is preferred?
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
What is the difference between monoclonal and polyclonal antibody?
Monoclonal - single type of antibody targeting a single epitope
Polyclonal - Several types of antibody for the same target but different epitope
Can u explain in detail about bit collision? Why different structures can be represented by the same bit?
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
For rigid docking, can you explain why some good candidates will be excluded due to stereochemistry? Why would they be good candidates if there is steric clash?
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
Can you get bit collisions in both 2D and hashed fingerprinting?
2D fingerprints and hashed fingerprints are similar but hashed fingerprints includes structural information –> but you can still get bit collisions in both
Why lead identification by fragment self-assembly sometimes useful?
Sometimes the linked ligand is unable to bind directly e.g. due to steric clashes
In glutathione conjugation, how does digestion of glutathione by peptidases relate to acetylation of the products?
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
In drug metabolism, can multiple modifications happen to a single molecule in phase 1?
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
