Hi, hopefully in the last section I can vision that synthesis
is a really important part of drug discovery.
So, where does synthesis come in?
Well, it comes in at every aspect of the drug discovery pipeline.
All of those main six milestones that we talked about
earlier in this course are impacted by synthesis.
So the roles of synthesis, there are several for
them.
I'll break it down into four main subtopics.
The first is target identification.
So we want to find some kind of biological target
that we can interact with via a small molecule.
And we can actually find a target through synthetic methods
so we can tag a bioactive drug to help find its biological target.
And that's going to be the main subject of this
video.
The second way that we can involve synthesis in drug discovery is through lead discovery.
So, we can make synthetic libraries of compounds that allow us to find
a lead compound that allows us to start the drug discovery process.
The third way, and probably the way that most people think
that synthetic chemistry can get involved, is through optimization of the lead.
And this is really the domain of medicinal chemistry.
Finally, at the stage of investigational new drugs for the clinical trials and clinical use,
then synthetic chemists are really still vital.
And this type of chemistry is normally referred to as process chemistry.
How do we make the compound on scale? How do we make it efficiently?
How do we make it in a environmentally friendly manner? And so on.
But first, we're going to look at target identification and how synthesis can...
How synthesis can get involved with that part of the drug discovery process.
Okay, so target identification, we can identify regions of a molecule
that can be altered that don't destroy the bioactivity.
So once we've done that, we can then firstly either
immobilize the compound on a solid support and then use that immobilized compound
to capture the biological target, elute it off,
and then identify what it is.
Or b, we can incorporate what's called a photoaffinity tag,
expose the cells or a lysate from the cells to that photoaffinity tag,
irradiate the mixture with UV light, and then identify the tagged biological target.
I'll go through these in a moment in more detail.
We can also label the compound with fluorescent or radioactive tags
to study cellular and whole body distribution.
This is very often done in the drug discovery process.
So the first of those two methods is to immobilize the lead compound onto a solid support.
So we take the small molecule and we can immobilize it
onto the solid substrate directly.
And then we can get the protein extracts or the cell or lysate
and flush them through this column, the solid supported natural product,
and any target that binds to the natural product should be bound to it
and not flush away with the rest of the eluent.
We can then do something to release that biological target
from binding to the natural products such as denature it or put in
some of the free natural product that will compete with the biological target for binding.
And we'll get our biological target enriched out the other side.
We can use more complex, but actually in practice, much more convenient techniques
such as biotin-streptavidin affinity chromatography.
So biotin, I'll talk about this in more detail later on in this video
but, biotin has a really amazing affinity for the biomolecule avidin.
So biotin will stick to avidin, almost like a covalent bond.
If we attach biotin to our natural product, when we get that construct
and flush it through a column that exposes avidin,
the avidin will bind to the biotin and display natural products on the solid surface.
We then get the protein extracts or intact cells or cell lysate,
flush it through the column and once again, our targets will bind to the natural products
and not be flushed to the column.
Then we can do something to release those from the column
such as denature the protein or make something...
make it so that the biotin won't bind to the avidin any longer.
Finally, and we'll go through this a lot more detail in a moment, is photoaffinity labeling.
So we take a more complex construct where we have the natural product or our drug lead,
we have a photoaffinity label and biotin attached to it.
We then pass this through a column with avidin, the biotin binds to the avidin,
and then we flush through the protein extracts or intact cells
but we do an extra step of irradiating that mixture with UV light.
And that leads to a covalent bond being formed between this photolabel
and the target proteins.
So they are now covalently bound and there's going to be no dissociation
between those components.
We can then flush the column to get rid of this association
in between biotin and avidin.
And use all sorts of different techniques to work out what our targets are.
So once we've got our proteins flushed off the bottom here, we can use things like
SDS-PAGE, mass spectrometry, liquid chromatography-mass spec/mass spec
isotopic labeling and all sorts of other techniques to try to identify
what our protein target for our natural product or lead is.
Okay, I mentioned this term photoaffinity labeling. So what does that mean?
Well, here's a little diagram of what it looks like.
So, we take our compound of interest that could be a drug lead or natural product
and we quite often will label it with something like biotin that will allow us to find it easily.
And then we also modify the structure so that it has what we call a photoaffinity label.
So this is a group that becomes really highly reactive when you shine UV light on it.
Now, we take that labeled compound and we expose it to our cellular mixture,
which has a receptor or some kind of target in it that is going to bind
to our lead or our natural product.
We get reversible binding, but hopefully it's strong enough that
a large proportion of our target is bound to this natural product or lead compound.
We then shine a UV light on it and that turns this photoaffinity label
into a highly reactive species that forms a covalent bond
between the receptor or our target and the compound itself.
So now these will not dissociate, this is a irreversible process.
So now we can take the covalently bound target with the natural product
and this handle on it, and subject it to some kind of analytical techniques.
So we could use SDS gel electrophoresis and identify the label target that way.
Or we can break up this target protein through proteolysis so we get peptide sequences
do HPLC purification, identify the labeled peptides.
Because now some of these peptides are actually going to be different molecular weight.
They are going to have this natural product or lead compound
plus some kind of handle attached to them.
So they might be easy to spot through our analytical technique.
We can even break them out further into their individual amino acids
and we may find that particular amino acids are labeled with our particular compound.
And we can identify those easily through techniques like mass spec.
Okay, so that's a little snapshot of how to do photoaffinity labeling.
What kind of groups can we use for this photoaffinity labeling?
So there's a number of different photoactivatable groups that we can use.
One group here are azides.
So if you look over here, azides form these reactive nitrenes through a loss of nitrogen.
Diazo compounds can lose nitrogen to form carbenes.
Whereas, diazonium salts form carbocations as the reactive intermediate.
And diazirines which is these interesting three-member rings over here,
they form carbenes through photoactivation.
And all of these different groups: nitrenes, carbenes, carbocations,
and carbenes are highly reactive.
They don't exist for very long before they react with the target which the compound is bound to.
One of the most common groups that is used in modern drug discovery for photoaffinity labeling
are aryl trifluoromethyl diazirines.
So that's a bit of a mouthful, but it just turns out that the trifluoromethyl group
suppresses side reactions such as isomerization of the diazo group.
So many of these photoactivatable groups can isomerize
or undergo some other types of chemistry.
Whereas, diazirines are relatively stable until you shine the UV light onto them.
So their excellent chemical stability prior to photolysis
allows us to do a lot of synthetic chemistry on them.
And that allows us to incorporate them into really
what can sometimes be quite delicate molecules,
particularly in the case of natural products.
One disadvantage of these types of molecules is that
it can be synthetically challenging to build up these molecules
because now we're looking at fairly complex group that can take some synthetic steps
to introduce into our natural product or our drug lead.
Okay, so a little more about carbenes and nitrenes.
Photoaffinity labeling relies on the photochemical generation
of these highly reactive, short-lived intermediates.
Carbenes and nitrenes are commonly employed
as they are really highly reactive.
They are uncharged, yet they are electron deficient
because they only have six valence electrons.
We know that main group elements want to have the octet, satisfy the octet rule.
So these compounds are seeking out additional bonding partners to form new bonds
and that's what they do with the target to which the compound is bound to.
Typical reactions they undergo are things
like
insertion into a heteroatom-hydrogen bonds, carbon-hydrogen bonds,
and insertion into pi bonds such as carbon-carbon double bond.
So carbene reactivity, for example, in a diazo compound we can get loss of nitrogen.
When you shine a UV light onto the compound, we can generate
two different types of carbenes: singlet or triplet
carbenes.
We're not so interested in the difference between the two for this course.
By all means, look it up in your organic chemistry textbook.
But both of these types of intermediate can insert into these
either heteroatom or carbon-hydrogen bonds, which are abundant within proteins
and other biomolecules to which your lead molecule
or your natural product maybe binding to.
So the photoaffinity label or tag that we can put onto the molecule is good.
We can get a covalent attachment of the molecule onto the protein target.
But once we form that covalent adduct, how do we actually find that molecule
amongst the mixture of compounds or mixture of biological molecules that we might have?
It could be present in very small quantities.
Traditionally, radioactive labels have been used.
So we can use tritium or carbon-14 or other radioactive isotopes
and put them into our substrate that then gets covalently attached.
And we can use radiochemical means to find that covalently attached
compound in the whole biological milieu.
However, more recently biotin labeling has been favored
because you get around a lot of the problems associated with radiochemistry such as
having to use radiochemical techniques and all of the safety issues that go along with that.
The biotin tagging is really useful because of the strong complex
that biotin forms with the biomolecule avidin, which has the dissociation
constant around 10 to the minus 15 moles per liter.
This is incredibly strong binding and it's not a covalent bond but almost as strong as one.
And it's, for all intents and purposes, like having an irreversible covalent bond.
So we can use this in affinity purification where we have an immobilized avidin
displayed on the outside of a insoluble matrix.
We can then use biotin avidin complex via a detection method
called chemiluminescent detection where we generate a chemiluminescent output
through peroxidase activity which has been conjugated to the avidin.
The detection limit for this process is around 10 to the minus 14 moles,
which is incredibly sensitive and is comparable to radioisotopic methods.
The disadvantage of this type of approach as opposed to the radioisotopes
is that biotin is a relatively large and polar group.
And so maybe it could affect the biological activity of the molecule
in which you're attaching it to.
Whereas, if we're just replacing a hydrogen with tritium or a carbon with a carbon-14,
we're really not changing the size or properties of that molecule in a biological context.
So to summarize, chemical probes can be formed from natural products
or lead compounds by linking them to fluorescent or photoaffinity or biotin tags.
We can use these to identify the binding proteins
or the localization of active molecules within cells of interest
such as cancer cells, malaria parasites, and so on.
Hopefully you found this interesting.
And we are going to demonstrate this through a case study
where it's been used to identify new targets in the discovery of new anticancer agents.
Thanks a lot.
