Week 6 Dr. Greig: Where Do Drugs Come From (Parts 4-6)

Question 1

what do we use when we need a new drug but have no starting point?

Answer 1

use high-throughput screening

Question 2

once we find a protein of interest via target validation, how do we find which chemicals might bind to that protein and change the proteins function? Manual way and implications

Answer 2

• obvious answer: look in our own storeroom of chemicals (library of compounds in the lab)
• test all of these chemicals against the protein to see which will bind to the target
• it is shown that around 10^60 compounds will bind to the protein and follow through the impossible challenge in the body as discussed last lecture
• but we cannot test all of these compounds bc we don’t have enough matter to create it or space to store these compounds

Question 3

how are chemical space and biological space analogized? and how does this analogy show the problem in drug creation?

Answer 3

Drug-like chemical space can be regarded as a vast ocean (most of it unexplored), and biological space can be regarded as tiny islands scattered throughout this ocean.

only the chemicals around the biological island will react with it

problem:
how are we supposed to find the chemicals that react with the drug target (island) when its a huge ocean of compounds?

Question 4

how has nature narrowed down our search in compounds that react with our desired drug target?

Answer 4

we have done past research which has shown what types of chemicals react with what type of targtes. nature has grouped these chemicals and targets together so that if have a target that for instance looks like histamine, we can look at these chemical libraries associated with histamine and guided by nature to see what types of chemicals may react with the histamine-like target.

Question 5

however, what is the problem with natures guidance?

review

Answer 5

Problem is that we are now sifting through chemicals against targets for which we have nothing to guide us …targets labelled undruggable

Question 6

what does high-throughput screening of chemical libraries do?

Answer 6

when we have all these chemicals, some of which may be undruggable, we just randomly test their effects on the target to see what it does.

for instance we may test the chemicals for their activity on
- an enzyme (does the chemical do anything to block the enzyme?)

• cells expressing receptor/pathway (does the chemical do anything to block the ligand from binding to its recptor and activating it?/prevent the activation of a pathway)
• or on model organisms (for ex. when testing antibiotics, does this antibiotic kill this model bacterium)

the rare chemicals that give a positive response are called “hits”

Question 7

what are the two divisions of screens?

Answer 7

Target-based screening:
Known target, looking for chemicals that modulate target or pathway

Phenotypic screening: - only looking for desired effect – doesn’t care how it works just cares if it works:
* Can I kill my osteoclast?
* Can I kill the tuberculosis bacterium?
* Find active compound, then consider how it works

Question 8

what is the difference in way of thinking from back in the golden age and now? particularly when it comes to screening

Answer 8

Back in the golden age of drug discovery, all we had was phenotypic screening as we didn’t have the required biology techniques or knowledge to do target-based screening

Some will argue that this is why we are now struggling and should just get back to finding drugs that work, instead of trying to understand the biology and mechanisms

Question 9

what is the main goal of high-throughput screening?

Answer 9

Chemical libraries will be screened for their ability to modulate the target protein, or for their ability to cause a favourable cellular change. Narrow down the search for a druggable chemical

Question 10

what does the setup and results look like for high- throughput screenings

Answer 10

• Protein or cells are added to each well.
• A different compound from your library is added to each well
• Complex assay components are added…. …a simple result is obtained
• test readout could be a simple color change
• we will know that the chemical has modulated or changed that target because that particular well will fluoresce

Question 11

what is our assay targeting when it changes color in the well?

Answer 11

when we have a chemical that blocks a receptor, we can test if the downstream reactions simply blocked the target or if it actually stopped the activation (we want the latter)

For instance, if we had a calcium-sensing dye, that reacted with calcium to give a fluorescent compound, then that is an example of a simple assay which will tell us if the downstream reaction occured

If the downstream reactions are halted = no increase in fluorescence = the compound effectively interfered with the activation process rather than merely blocking the receptor.

Question 12

are the assays complex

Answer 12

yes the assays are complex and make use of complex biology, but the test yields a simple result – mere color change

Question 13

what is a good model for phenotypic screening

Answer 13

zebrafish embryos are tiny transparent and easily grown to use

they are larvae, so not animals thus ethical for drug discovery

they are also transparent so we can actually see the effects of the chemicals on embryo development and whether they effect the pathway of interest

Question 14

what is in silico libraries and screening? what is a problem with screening though?

Answer 14

computational screenings that can test an enormous amt of products. we can just ask the computer to tell us what compound is the best for the target

the libraries are stored on a digital database

problem:
there are some false positives - not useful if we get so many products that may work instead of 10 that definitely work

Question 15

what is fragment screening

Answer 15

rather than finding the perfect drug in a library or a compound that is similar enough, we instead try to find a small bit of the desired compound.

then with this we can put together all the different discovered bits to find the ideal match of a compound

Question 16

what does the future of screening look like

Answer 16

will transition to in silico screening so we dont have to manually look through a library and conduct trials

instead we will find accurate compounds via computational power to find a starting point.

Question 17

what does potency mean

Answer 17

how little a drug is required to have the desired effect

• how strongly a drug interacts with its target

Question 17

how do we improve potency as we develop a drug

Answer 17

we have a starting point bioactive compound

• we insert it in the drug target and try to improve the potency by growing the drug to match exactly with the drug target
• for instance if a target has 4 pockets of space and the starting point fills one of them up, we would develop the starting point to fill all 4 pockets tightly for best interaction.
• with only 1 or 2 filled = poor potency
• with 3 or 4 filled = good potency
• essentially more/better binding interactions between evolving drug and target = improving potency
• the drug is trying to get a better “grip” on the receptor

Question 18

the more potent a drug is the…?

Answer 18

• less likely it hits other targets (less toxic)
• less needed to dissolve (less for solubility)
• less you need it to be absorbed (permeability)
• less problems you have in the body – more effective

Question 19

why can greater potency sometimes lead to better selectivity and avoid toxicity. use an example to explain

Answer 19

for example let’s say we have four drug targets, all very similar in shape but all have different properties.

inhibition to target A will cure epilepsy but inhibition to target B C or D will cause really bad side effects

if we have a drug that has low potency, it will have fewer binding properties, and will be able to fit in all of the targets. This means it will not be good enough to cure epilepsy, but at least the side effects wont occur because its not fully gripped on.

If we have a higher potency, we will have more binding interactions. we can grow the drug so it binds properly to target A to cure epilepsy, but not bind properly to the other targets, thus preventing side effects.

thus more potency led to selectivity and avoidance to toxicity!

Question 20

what is on target toxicity? expand on epilepsy example

Answer 20

if we inhibit target A stopping epilepsy, but later we find target A is needed for normal brain function, we will decrease epilepsy but increase side effects

this trade off is known as on-target toxicity

this means improved potency will simply mean that the side-effects are also caused at a lower drug concentration

ex. cancer drugs are potent and have therapeutic and toxic effects at very low concentrations

Question 21

what is pharmacaphore? what are electronic properties? how are they associated with another?

Answer 21

the pharmacophore is the parts of a structure on the hit compound (as identified via screening) that bind to the target on their 3D orientation.

ex. cocaine and procaine are very different structures but have the same pharmacophore

it is based on the electronic properties - target doesn’t care about the exact nature of the chemical group – only what it does

so if a substance has the same pharmacophore or the same electronic properties, they can both bind to the same associated target

Question 22

how do we improve a compound?
1. simplification via modification

Answer 22

we take the starting compound and remove or modify each growth then check if it made a difference to the activity

so if we modify a compound and there is a…

loss of activity = essential to the chemical

no change in activity = not necessary to the structure thus can remove for simplification

Question 23

how do we improve a compound?
2. structural extension

(increase potency abd selectivity)

Answer 23

structural extension of the compound to increase potency and find additional binding interactions

makes the molecule much better

structural extension can also increase selectivity.
for instance if we have two very similar receptors, but one is larger, the same non-selective drug may fit into both receptors, but if the drug is structurally extended, it may only now fit into the bigger receptor (selective)

Question 24

how do we improve a compound?
3. rigidification

Answer 24

sometimes drugs can be flexible when its binded with its drug target causing parts of the drug to unbind with the target

by rigidifying the drug we can ensure that the drug stays in contact with the drug target for longer thus improving the compounds potency and selectivity

Question 25

Case study: how do we improve the potency of a compound from the first step of identifying the hit compound to clinical candidate.
ex. osteoporosis

questions:
what is the condition?

what are the two cell types?

how do bones change through the ages?

what do bones look like normally vs in osteoporosis ?

what is the starting point drug here?

describe phenotypic screening for osteoclasts and osteoblasts?

Answer 25

osteoporosis: when bones are every week and can snap

two bone cell types:
* osteoclasts = remove bone
* osteoblasts = build bone

• two cells respond to each other to keep bones fresh and healthy

bones through the ages:
when we are young:
our osteoblasts are more active = build bone

older:
osteoclasts work more to deteriorate bone

bones normally and osteopathically:
normally = honeycomb structure

Osteoporosis = wider honeycomb gaps and less bone material

starting point:
* found a drug that kills osteoclasts (cells that remove bone), but did not promote osteoblasts (cells that build bone)
* thus we want to take this starting point and manipulate it to make a drug which is more selective and potent into killing the same number of osteoclasts at a lower concentration

phenotypic screening for osteoclasts:
* we split the starting point molecule into two parts: part A and part B
* we then make an analog = different A connected to different B
* add the different analogs to the wells with some osteoclasts to see which combination kills the osteoclasts
* with the results (lets say 4 AB combinations worked well), synthesize new patterns using the succeeding A and B parts.
* test and retest to see which A and B parts are the best

phenotypic screening for osteoblasts:
* repeat the same screening but add osteoblasts as well to ensure that the combinations that were potent for the osteoclasts don’t kill the osteoblasts
* those that do are toxic
* we need a selective drug to osteoclast killing but doesn’t affect any other cell type, like osteoblasts

Question 26

what are IC50s for potency

Answer 26

The concentration at which a drug achieves 50% its maximal effect
Lower IC50 = more potent = “better”

Question 27

what is the median potency for approved drugs? what does this mean?

Answer 27

20 nm

this means drugs which have very low IC50s like in the micromolar range, still have a long way of modification to go

Question 28

what do we modify the structure of the compound with to improve the potency and investigate the structure- activity relationship

Answer 28

we can use any functional groups which fit desirably in the target spaces.

The target has spaces which can be filled, these are:
* Large or small
* Polar or hydrophobic
* Electron rich or electron poor

Question 29

what is the acronym for pharmacokinetics

Answer 29

ADME(T)

Absorption
Distribution
Metabolism
Excretion
(Toxicology)

Question 30

what are we trying to achieve when designing a drug

Answer 30

• we want to take orally
• take it less than 3 times in 24 hrs
• enough of the drug needs to reach the target

for ex. if the IC50 is 5 nm, that wont be enough to cure a headache or stop cancer

but, for 90% effect we may need 50 nm of the drug. that may be enough to cure the headache and maybe enough for the cancer.

Question 31

explain how drugs concentration changes in your body and what implications this has

Answer 31

when you take a drug it is at high concentrations in your body, but then falls to a lower concentration after some time. At every dose it gets back to its highest concentration in your body.

implications
- however, maybe when a drug is at its lowest concnentration it is not effective enough for the targetted reason, and thus the concentration of the initial dose should increase.
- for ex, for antibacterial drugs, if the drug falls too low in the body, maybe some bacteria that isnt yet killed can still penetrate and effect the patient
- thus we would have to raise the inital concentration up, so that the factor it decreases by, is still above the threshold of which the drug is effective.
- this threshold is usually 50 nM

Question 32

what is the problem with plasma proteins and their interaction with drugs in the bloodstream

Answer 32

the bloodstream has plasma proteins which attract drugs and bind to them

this leaves a lower concentration of drugs to bind to the actual target, instead of the plasma proteins

many drugs have a plasma protein binding of >90% leaving only 10% of target binding. so if we want an actual effect we may have to use a much higher concentration of an initial drug to account for plasma binding

Question 33

first challenge for a drug is solubility.

explain the challenge, the types of soluble drugs, its relation to potency, and how solubility is calculated

Answer 33

challenge:
If the drug cannot dissolve, then it will just pass straight through and be excreted from the gut

Polar drugs generally have better solubility. (N+O)
Charged drugs are even better (-NH3+ or –CO2-)
Lipophilic drugs generally have poor solubility (predominantly CH)
- small additions of functional groups can have a huge effect on solubility

solubility and potency:
high potency = need okay solubility

low potency = need much greater solubility of the drug

calculations of solubility
- we can calculate our required solubility
- or just estimate a number
- usually >100 μM of solubility

Question 34

second challenge is permeability

explain the challenge, the types of permeable drugs, how we can measure permeability at the discovery stage where we need to test a large number of compounds, and how good the permeability needs to be

Answer 34

challenge:
If the drug cannot pass through the gut wall to reach the circulation, then it will just pass straight through and be excreted from the gut

• Lipophilic drugs generally have better permeability through a membrane.
• Polar drugs generally have poor permeability.
• Charged drugs have very low permeability but can sometimes flip to a neutral form.
• different connectivity and functional groups can affect permeability

How do we measure permeability in vitro at the discovery stage where we need to test a large number of compounds?

Permeability assays measure the rate at which a compound passes through a layer of cells. Depending on the cell type, it can mimic either passage out of the gut, or passage into the brain

how good does the permeability need to be
estimate: >150 nm/sec

why?:
96% of drugs which act in the brain (the central nervous system – CNS) have permeability of >150 nm/sec.
thus our drug which will also effect the CNS will relativley be at the same permability level

Question 35

not only is it challenging to get the drug in, but to have the drug to stay in is a whole different problem.

when might the efflux of a drug be a problem, what is this issue mediated by, which drugs are susceptible to efflux, how do we measure the efflux, what is the accepted efflux rate, do small changes effect efflux?

Answer 35

when might the efflux of a drug be a problem?
The brain is very good at throwing drugs straight back out – as are bacteria and cancer cells.
* not in the gut because the high drug concentration tends to overwhelm any efflux issues
* but efflux is a problem in the brain because lower concentration of the drug by the time it reaches the brain makes it more of an issue.

what is this issue mediated by?
- Membrane protein
- acts as an efflux pump called P-glycoprotein.
- Also acts as a multi-drug resistant protein-1 (MDR-1)
- one of the main reasons that resistance develops to drugs for cancer.

which drugs are susceptible to efflux
- almost all drugs

how do we measure the efflux
* If drug goes from B to A (out of the system) faster than it goes from A to B (into the system) then it is a “P-gp substrate”.
ie. membrane protein that acts as an efflux pump
* The efflux ratio (ER) is the permeability “out” / permeability “in”
- It is estimated that over 95% of drugs that could be used to treat CNS disorders (depression, schizophrenia etc) do not get into the brain and thus cannot work

what is the accepted efflux rate
ER< 2

do small changes effect efflux?
Yes! connectivity and functional groups can have a huge effect