hello hydroxychloroquine hydroxychloroquine hydroxychloroquine oh okay so what target is
the drug aiming for in the body and what sort of human trials have been done to confirm that the
drug indeed actually works as you think I feel good about it oh that's great that you trust
it that's really good you know... that's where it all starts, but for the rest of the community the
rest of the world to trust it and to accept that it's actually going to do what you say it does,
to want to spend money on it you know not waste their money buying the drug or worse it could be
dangerous to their bodies you're going to have to show that you've done some tests you've measured
the safety of it and make sure that it's doing what you think it is doing yeah yeah there're
some studies that you should have done hi today we're going to be talking about the
process of discovering drugs for the treatment of disease so let's do that we're gonna look at
three main components of the process which is identifying a target, target identification,
then high-throughput screening to identify a small molecule that can be progressed on to make a drug
and then the final step in the process is looking at making the molecule more drug-like and you'll
understand what that means as we're going through the process so let's get started. A background:
Who gets involved in drug discovery? who are the key players that may be interested in looking at
discovering new drugs? So as you all may guess pharmaceutical companies and industries were for
the longest time the main players but more and more research and academic institutions are very
much involved in this space too. So you start off then in this process with target... what's
called a target identification this is where you identify a target in the disease phenotype.
Phenotype just means what it looks like. To do this you check hundreds of thousands of what
what are called small molecules or compounds - small molecule compounds - for their ability
to alter what the disease looks like. The modern approach/the current approach is to more focus on
a target. We will be clarifying the drug discovery process we will learn that a compound does not
become a drug until it's gone through several steps and it passes through those processes
that's then and only then would a compound become a drug it needs to be made drug- like the compound
that you find in the drug discovery process needs to be made drug-like and we'll cover what that
means. It needs to be tested in preclinical trials so usually animal models well definitely animal
models and then it needs to be ultimately tested in humans in human volunteers in clinical trials
before it can be released and you know if you have a look at the process, a lot of compounds,
the majority don't get to this stage. so let's have a big picture view of the drug discovery
process, so the best thing to do that to use an example malaria for if I want to intervene with
malaria as a disease what are the things that I need to consider? I first need to understand
the symptoms that identifies malaria. I need to understand the unique symptoms that identifies
the disease. I need to understand what causes the symptoms..maybe... what is called the etiological
agent so the actual... you need to understand what you know the organism that causes it whether it be
viral bacterial or whatever component is causing the disease. it's good to get a grasp of that we
know that in the case of malaria it's a parasite that's inside the mosquito it's not actually the
mosquito so you know the system needs to be studied to actually identify the real reason
the real cause and then you have to look at how how does this purse are actually interact with
the body to cause the problems to cause the disease that it does what does the parasite
once it's inside the body what does it need in order to be able to survive inside the body and
once we know that then we can attack what it needs and that's essentially what you're doing
in treating it by a drug the drug will target in this case what the parasite needs to survive if
we can stop that then obviously we can intervene in the disease. so if we can look at... we can
look for structural entities or compounds that interact with that parasite and stop
the parasite's ability to cause disease inside the body. I've called it 'structural entities'
because at this point in a drug discovery process it's not a drug it's a chemical structure that
interacts. well the thing is, the chances of finding a molecule that interacts with the
disease - in this case a parasite - that gives it an outcome that we desire is very very low so
you can't just look at a handful of compounds and and come out with something successful.
Typically hundreds of thousands - not hundreds to thousands but hundreds of thousands of compounds
of structural motifs are checked and this is what's called screening and so the main part
of drug discovery a crucial component of it is the high throughput screening the more readily
recognisable.... the high throughput screening component which will be looking at. so you check
these compounds/structures/ chemical entities against the... your target. In drug discovery
usually... the target identification is a crucial part but in the high throughput screening lab,
that's not their job. Their job is to screen the small molecules with your target so you usually
have to have identified your target, validated the target - as we will discuss - and then you
go into the screening to find compounds that are interacting with your target. It's called
high-throughput because you know it's not just a handful of compounds which you could have just
done on your lab bench it's hundreds of thousands of compounds so the system is usually automated
you're using robotics to handle to be able to process that many compounds. so if any of the
compounds or small molecule structures are noticed of flagged they... as you see this
is called 'hits' in that they are interacting with the target of the disease in a way that is
promising then it is studied further with the hope and aim of making it into a drug also how
do chemists even know where to start? like how do they know what structures or compounds? well
the best way to to do something is to just copy an existing one right so then you know that in nature
there're some compounds or chemicals or structures that we know kill bacteria or we know kill fungi
so you can sort of study those and get an idea of what their structures are like, and then that
can give you a starting point to creating new structures and the word novel comes up again a
novel structure that doesn't exist. it can be sort of... using that scaffold create more and more and
more and more hundreds and thousands of these that in a new disease setting can be checked to
see if it's interacting. then you need an assay an assay simply means an experimental setup that
allows you to check if any of the compounds can interact with your target. You need to miniaturize
the experiments: you need to go from the standard experimental volume such as an experiment that you
is done in an Eppendorf tube or in a test tube or a larger format, you need to miniaturize it what
do we mean by miniaturise it? we're going to be checking hundreds and thousands of compounds this
is going to be expensive reagents are expensive your reporter the this experimental setup all the
components going to be expensive and since we're checking hundreds of thousands of compacts we
need to look you know check it...lower.. reduce you know reduce it to the smallest volume that
still allows us to detect differences it still allows us to have a solid experiment what is
called a robust experiment. there are statistics and measurements you can use to check that you
do have a robust experiment we'll get into that. we have an assay, we have a target,
we have an assay an experiment that's going to let us probe the system to check that you know
our target is being interacted with in a way to be able to detect it that's your assay so now let's
go to the heart of the matter well at least what's readily associated with the process so that comes
to the high throughput side so the high throughput side the first thing you do is to what's called an
assay transfer you transfer the assay into a highly reduced volume as I already alluded to
so that keeping all the components you're not changing any components you start with what
works and you're just reducing the volume so what you aim for is that you have a good signal at the
low volume for detecting the target and how it is altered. we need to use a few parameters to check
that it's suitable to go into this high throughput which means miniaturized format so that one of
the first things one of their parameters you can check... you can check the signal-to-noise ratio.
the signal-to-noise ratio compares your ability to detect a signal to when there is no signal - so
just the noise - and it actually factors in the standard deviation. standard deviation,
if this is foreign to you no worries I have a video on here that introduces statistical concept
in a really accessible way so you can have a look at that to see what standard deviation means. then
you have a signal to background the signal to background basically compares the ability to
detect a real signal with the background generated by the components used in the assay it divides
average signal by the average background you know the the noise the.... the buffers the reagents
give their own signal so you factor that in versus when you've got a real event a real signal and so
that's what your signal to background adjust for. oh there's also another parameter a statistic
that is used here and it's the Z prime and the Z factor like we said you start off with an assay
that works in a test tube or in your large volume format, you've miniaturized it to 384 or 1536
-well format and then you are going to look at a pilot screen what's a pilot screen? a pilot
screen is just an initial screen where... you know we mentioned it's going to be like hundreds
of thousands of compounds you can just check a few of those compounds you can check five thousand...
between five thousand to ten thousand diverse range of compounds to gauge its robustness and hit
rate estimate before going into an expensive proper screen. At this point you only screen
at one concentration just one concentration but at the pilot stage you actually do it in
duplicates just to see how reproducible it is and then at that point you can actually calculate the
statistic that we didn't talk about earlier which is your *Z factor* to really look at your ability
to pick a real signal from a negative signal you've done the pilot screen the pilots when
you do that one concentration the concentration that's usually used is 10 micromolar but you know
that all depends on your target and the kind of assay you're doing in some systems you may
use... start off at 20 micromolar or maybe more. So you've done the pilot with that concentration
then we come to what is called the primary screen this is the screen this is only done when you
know everything's going well and you're at a good place so this screen you select one concentration
again 10 micromolar usually could be more could be less and depend on what you understand about your
system and then you only do one when well simpler kit nor duplicate so you really have to add this
before you get here make sure that everything is working well but you don't just assume it's
working well you have some parameters that you can check so you can do positive controls on
the plate you can put negative controls. not you can, you will, put positive controls put negative
controls and you can use that to calculate your your z factor/score. after that you have
to select what you consider us potential hits. you factor in the z factor if it's a good read
out a robust read out then you can trust what's on the plate that you see as 'hits' flagged because
they're like three standard deviations away and you have to confirm them these are the compounds
that have potential or as we say in this field putative potential activity while you're doing
the primary screen you would have also by now you know you should have an idea of another
assay - this is very crucial- another assay that will allow you to weed out false positives. false
ones that come up as positive but they're really not positives you know they were interacting with
the things you were using... maybe your d...your readout...you know.... because usually you're
using a reporter/ something to report to you that signal... something is interacting with your drug,
so there could be components and drugs in there that interact with that reporter and
not really with your target and gives you a false positive or it interacts... or some
of the drugs actually stop your reporter from reporting and you think that means something,
so you also need a...f... another crucial assay in addition to your main experimental assay to
see that.... when compounds are interacting: so that is called a counter screen so you have the
main screen then a counter screen and also of course you need what is called an orthogonal
assay and that means you know like if I used light detection... light emission... as my reporter to
tell me when a compound is interacting in the way that I want, then I can use a different
way of detecting that interaction so if I used bioluminescent reporter to see the target is
interacting the molecule then I could use maybe fluorescence as a reporter and if I still see
it in that setting then I know yes it's really it just confirms it you know I just use a different
different technology to confirm that interaction so that's an orthogonal assay. let's say you've
done all that you've confirmed your hits then the next step in a high-throughput screening process
is to determine the potency of the compounds using both the main assay and the counter screen
you're going to determine the potency of the of the compounds that you've identified and you do
this with a ten-point dose response curve and this allows you to really know if there's a real
interaction then as you diluted out or increase it out, starting at a high concentration as you
dilute it out, the signal will go down, down, down you know you do it for ten-points and that
means you do a serial dilution of the compound. if you're not familiar with serial dilutions you're
in luck I did an earlier video all building up to this video and it also gives you an idea of how
potent the molecule is and you need that data to be able to draw a curve and determine how potent
the drug is and here the measurement is called an effective concentration 50 (EC50) you may also be
interested not in the effective concentration but the inhibitory concentration 50 so your
ec50 or ic50 can be covered... can be measured once you do your dose... 10-point dose response.
there's still one step in the high throughput side, yes it's a long process, you've heard of
drugs taking 30 years to get to the market or 30 years or more. Give the compound to be assessed
to make sure that the compound that is flagged as potential activity it's actually what you think
it is. so you do this by sending it to a mass spectrometry lab they do liquid chromatography
mass spectrometry to identify the compound because sometimes it could be a contaminant of
the compound when it was synthesized that's giving you the activity, in which case giving that you
know progressing with that compound and making a drug will not result in the interaction with
the target that you had seen you so you have to do check via mass spectrometry that the molecule, the
compound is really what it is. you're still not done you actually have to modify the compound to
make it more drug-like. it is highly unlikely that what you find at that point would be ready for use
as a drug. why is that? well you know that they'll be if you know you're taking this drug if you're
taking it orally there are few obstacles the drug has to go... the molecule has to go through before
it can be available to the target as the stomach the pH of the stomach for instance, highly acidic
it's very very low pH it's a pH of 2 so the molecule needs to be soluble at that pH and not
just soluble, but stable at that pH. You'll have to look at getting rid of side effects you know if
there're some off-target activity... it interacts with... uh yeah it interacts with your target
that's great, but maybe it also interacts with some other targets in the body you can't have that
or you can't have it if it's particularly bad, you know you can reduce that you can look at reducing
its interaction with other targets in the body toxicity is an issue you're gonna have to make
sure you assess if it's toxic and you can do that with cell lines but ultimately these are going to
have to be tested it in whole organism settings so first off with any animal models, you can't
avoid that and then you go into human volunteer trials and at this point you're really working
with the chemists, the medicinal chemists to make incremental changes that will allow the molecule
to behave in a drug-like way that you can then test in whole organisms beginning with animals
and then human volunteers. Wow so you can see that there are a lot of processes that goes through. so
we might look at the Z prime... calculating the Z prime in another video but for now we can leave
it here where we've looked at the process you know target identification, target validation, finding
an essay to let you see how the target is behaving and then going on to finding a small molecule that
interact with it, confirming that that molecule really interacts with a checking the potency of
it and then assessing the purity of the molecules first and formost and then you know working with
medicinal chemists to ensure that you can make the molecule more drug-like before you can go into
whole organisms like animal models and then human volunteer trials. thank you once again for tuning
in if you have questions happy to hear them and yeah if if it was helpful do give it a thumbs up,
subscribe if you want to hear more of this content because I definitely think we look... do
a separate video on z prime calculation and yeah I'll catch in the next video. God bless you. Bye