Drug Discovery Exam 1 content

Phenotypic drug discovery

ones where you observe that the compound elicits a particular response (e.g. kills cancer cells) before you know the target. compound based

Genotypic drug discovery

ones where you identify a target that you believe will elicit a desired biological response and then find ways of interacting with that target. target based

Drugs can be broken into 2 categories:

1) Stop a process from happening

2) Keep a process going

But ultimately, every drug acts by acting on one (or more) biochemical targets

Main classes of drug targets

Enzymes, Receptors, Other proteins, Nucleic Acids, Others

What are enzymes?

Biomolecules that catalyze chemical reactions, are not consumed, and mainly proteins with possible different forms (isoforms)

What are receptors?

Biomolecules that “change” when they bind to other molecules

Other proteins

Transport proteins (move molecules around), Chaperones (helps messes and de-aggregate in correct way to adopt the correct orientation, allows proteins to return to normal state), Protein-protein interactions

Drugs

chemicals that are recognized (if not approved) by a legitimate regulating agency as having a medicinal effect

Drug Candidates/Development Candidates

chemicals that are under investigation as potential drugs

Xenobiotics

chemicals that are not natural, or are present in abnormally high concentrations, in an organism

Are all drugs xenobiotics?

Yes, all drugs are xenobiotics, but not all xenobiotics are drugs

Ligands

chemicals that bind with a biomolecule

Substrates

chemicals that bind to and are changed by a biomolecule

Products

chemicals that are formed as a result of a reaction

Agonists

chemicals that interact with a biomolecule in a way that entices/induces that biomolecule to perform its normal function

Antagonists

chemicals that interact with a biomolecule in a way that prevents that biomolecule from performing its normal function

What type of environment do enzymes exist in?

Enzymes are proteins (polypeptides) that exist in an aqueous environment

Enzyme composition

They typically have a “hydrophilic” shell that protects a hydrophobic pocket.

The enzymatic activity usually occurs inside of that hydrophobic pocket

The site where the enzymatic activity takes place is called the “active site.”

The enzyme folds to have a hydrophobic pocket and expells water, makes sense Energy wise (hydrophobic effect)

Bonding types

•Covalent bonds - > 200 kJ/mol

•Ionic bonds - ~ 30 kJ/mol

•Hydrogen bonds - ~ 20 kJ/mol (all enzymes exist in water, which has hydrogen bonding)

Interaction types

•Dipolar interactions - ~ 5 kJ/mol

•London/Van der Waals interactions - ~ 3 kJ/mol

Enzyme inhibition

•Competitive Inhibitors: take up space on the active site, so the enzyme can’t bind. Competition depends on concentration

•Noncompetitive Inhibitors (allosteric): change the shape of the active site so the enzyme's active site doesn’t work

•Hybrids (noncompetitive inhibition)

• Reversible inhibitor: inhibitor binds to the enzyme, or to the enzyme and substrates, and disables it

•Irreversible Inhibitors: covalently bound; makes the enzyme useless unless degraded completely, which takes a lot of energy and time

Solubility

Hydrophilic exterior, hydrophobic interior —> site where molecules interact (usually this is the target for drugs)

Enzymes can be activated at allosteric sites

Activators: bind and make active site more accessible/ speed up process

Most drugs are inhibitors

•Competitive/Non-competitive

•Active Site/Allosteric

•Reversible/Irreversible

Isoforms

Multiple enzymes do the same thing. Enhances redundancy

They can be:

•Specific or non-specific

•Reversible or irreversible

•Repair

two types of receptors

•Membrane-bound receptors

•Intracellular receptors

Membrane-bound receptors

•G-protein coupled receptors (GPCRs, 7TM)

•Other enzyme-linked receptors

•Ion channel receptors

•Others

Intracellular receptors

Nuclear receptors

•Secondary receptorsNuclear receptors

•Secondary receptors

Cellular membranes

•Composition of membranes (phospholipid bilayer)

•Hydrophilic surface

•Hydrophobic middle

•Designed to prevent free flow of fluids in/out

Membrane-bound receptors: GPCRs/7TMs

•A great biological target

• Crosses the membrane 7 times

•Signaling cascade

•many membrane-bound receptors are actually not monomers but are often dimers. Meaning that instead of ~1,000 different GPCRs, there are actually
~1,000,000 receptor combinations~

Intracellular receptors: Nuclear receptors

Receptor takes the hormone into the cell, into the nucleus to change or turn on genes

Intracellular receptors: Secondary receptors

enzymes on the E.R. that help with transcription or translation

Agonist

keep it operating normally

Antagonist

Stop operating normally

Full antagonist

death

Partial agonist/antagonist

modulate effects by using both, less activation. the most common

Super agonist

a full agonists, almost a covalent bond. the receptor cannot be turned off normally. the cell cannot survive with the receptor stuck on, so the cell destroys the receptor (converts it back to amino acids) through endocytosis.

a super agonist essentially becomes an antagonist because the receptor is taken out, and cannot do its job anymore

Nucleic acid targets

biological targets. DNA typically targeted by DNA damaging agents

Targeting RNA: new technique, silence RNA by using a complimentary RNA to make dimer, mRNA vaccines is an example

Assay

An examination or determination of characteristics

•For purposes of target discovery, we need to be able to measure whether a process takes place

Concentration

expression of an amount based on volume. For example, molarity (moles per liter)

Dose

expression of an amount, usually (but not always) based on the weight of the organism receiving the dose. For example, milligrams/kilogram (mg/kg)

IC₅₀

The concentration at which 50% inhibition takes place

The lower the IC_50, the more potent the process is

But it is effectively a titration, so there is inherent error

IC₉₀

The concentration at which 90% inhibition occurs

•IC₉₀ is typically determined when evaluating infectious diseases/disease targets

•There is often less error associated with IC₉₀

MIC

•Many infectious disease evaluations also use “minimum inhibitory concentration” (MIC)

•Though useful for early phase studies, it’s not widely used in drug discovery because you generally don’t care about the concentration that inhibits “visible” growth

EC₅₀

The concentration at which 50% of the targeted activity is observed

•Typically used for agonists

•Again, a titration curve with errors

All IC₅₀ are EC₅₀ but not all EC₅₀ are IC₅₀

True

ED₅₀

The dose at which 50% of the targeted activity is observed

LC₅₀

The concentration at which 50% of the targeted population is killed

LD₅₀

The dose at which 50% of the targeted population is killed

Therapeutic Index (TI)

The ratio of the LD₅₀ to the ED₅₀

TI= L/E (TILE)

TI=LD_50/ED₅₀

TI= (lethal 50/ effective dose 50)

no official number that says whether a TI is “good” or “bad.” Rather, it’s a risk/reward analysis

•There is no right or wrong number for a TI. But a larger TI is always preferred to a smaller TI

•In drug discovery, sometimes TI is not the most critical factor, whereas in others it might be…

Assay: Measure formation of a product

•if the end result of a biological process is the formation of a particular protein, we have the ability to detect proteins with a reasonable level of certainty

molecular weight and change of protein

western blot

high res mass spec

Assay: measure formation of a reactant

kinases convert proteins into something else ex: phosphorylation ATP to ADP to measure the ATP or ADP

ex: lightning bugs generate light through proteins and ATP. so measure how much ATP if light is formed and measure light amounts

In order to determine if a potential drug candidate is inhibiting something, we first need to observe what?

a) what it looks like when the process works, and

b) what it looks like when it doesn’t

•The assay evaluates what our potential drug candidate does at different concentrations or doses in order to determine whether it’s inhibiting (or activating) the process

Some common and useful assay detection methods

Colorimetric

•Fluorometric

•Chemiluminescent

•Radiometric

colorimetric

•really a measurement of light absorption

•Using a particular wavelength (color) allows for selectivity (Spectrometer)

problems: other compounds are different colors, making it unclear if inhibition worked or not

fluorometric

assay typically has increased signal-to-noise ratio because incidental interference is decreased

•Twice as “selective” for the wavelength absorbed and emitted

more wavelengths involved (2), more accurate than colors

chemiluminescent

assay accurately measures light given off as a result of a reaction (ex: for measuring amount of ATP by using the reaction of lightning bugs)

radiometric

assay measures the presence of a radioactive element

•Many radioactive elements, including ³H, ¹⁴C, ³²P, ³⁵S

Modern assays: Green Fluorescent Protein (GFP)

•GFP (green fluorescent protein) stems from a relatively small (27 kDa) protein originally isolated from jellyfish

so it means the protein is fluorescent

You can add GFP to the end of any protein and it can be in other colors than green like red or yellow. it helps detemine if a protein is being produced or if its inhibited and not made

Modern assays: ELISA (Enzyme-Linked ImmunoSorbent Assay)

uses a specific antigen/antibody relationship for detection

Direct ELISA: Uses an enzyme-labeled primary antibody that binds directly to the coated antigen on the plate.

Indirect ELISA: Uses an unlabeled primary antibody and a second, enzyme-linked secondary antibody to boost the signal

expensive but can work

•The presence of just about any molecule can be detected and quantified if an antibody is available or can be found

•That antibody can then be modified to utilize one of the aforementioned detection methods

How it works: uses an enzyme attached to an antibody. When you add a special chemical liquid (a substrate) at the end, the enzyme changes it. This turns the liquid a specific color. If the target substance is in your sample, the well changes color. If not, it stays clear. You measure the color with a machine to see how much target substance is present

Modern Assays: FRET (Fluorescence Resonance Energy Transfer)

is a measurement of fluorescence between two different molecules and depends on distance

energy moves from one light-absorbing molecule (the donor) to a nearby second molecule (the acceptor) without emitting a photon.

Think of it like two matching tuning forks: hit one, and the vibration transfers directly to the other through the air if they are close enough

In a FRET assay, scientists use this trick as a microscopic "ruler" to see if two molecules—like two interacting proteins—are touching or within a few nanometers of each other inside a cell

So, it sees if the 2 proteins come together or not, or if they are inhibited and seperates.

Degraded: signal is lost

High concentration of proteins: see flourscence

USEFUL FOR RECEPTORS

IF receptor open: the molecules should be far apart and no signal

Receptor closed: close together and signal

Critical points for assays

Good controls

•You have to be able to show that the process occurs AND that it can be stopped. You need to know what it looks like while working and what it looks like when stopped.

Good reproducibility

•Reagents are reliably available and don’t have batch-to-batch variability

•Not cost prohibitive

How many concentrations to generate an IC₅₀ curve?

•run our assay using varying concentrations of our test compound(s)

•For example, to generate a 10 point IC₅₀ curve, you could use final test compound concentrations of 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 mM, 3 mM, 10 mM and 30 mM

In Log (x) format: 10^-9 M, 3x10^-9 M, 10^-8 M, 3x10^-8 M, 10^-7 M, 3x10^-7 M, 10^-6 M, 3x10^-6 M, 10^-5 M and 3x10^-5 M

Needs to be in beween 5nM (enzyme too low) and 30μM (substrate too high)

Any changes in parameter menas you start over for all data and retest

words of caution for ALL assays

Always run the standards and controls every time

Changing ANY parameter prevents comparison between assays

Be wary of numbers that are very high or very low

•[Enzyme] ~ 5 nM

•[Test compound] in water (& DMSO) < 30 mM

Remember there are error bars associated with assay results – don’t overvalue numbers!

Inadvertent errors - the best results are confirmed multiple times

Anticipate and investigate false positives

•Colored compounds

•Compounds that interfere with detection

•Compounds that act via an undesirable mechanism

False negatives

Operator error

Qualities for good drug candidate

just because a compound is the most active (or selective), that doesn’t mean it’s the best drug candidate.

•Once a potential candidate was optimized for activity, a second optimization campaign was initiated to examine

•Pharmacokinetics

•Pharmacodynamics

Pharmacokinetics

What the body does to the “drug”

ADME

Pharmacodynamics

What the “drug” does to the body

ADME

•Absorption – How the “drug” gets into the bloodstream

•Distribution – How the “drug” gets from the bloodstream to the rest of the body

•Metabolism – How the “drug” is chemically transformed in the body

•Excretion – How the “drug” is eliminated from the body

primary methods of drug administration

•iv – intravenous

•p.o. – per os (by mouth – oral)

•inh - inhalation

•ip – intraperitoneal (only relevant in animal models- directly to stomach)

•How do oral “drugs” get into the bloodstream?

•They don’t pass between the epithelial cells

•They have to go THROUGH the epithelial cells of the small intestine

•Xenobiotics that can pass through the epithelial cells of the small intestine are deposited into the hepatic portal vein

•The portal vein takes these xenobiotics to the liver

•How do inhaled “drugs” get into the bloodstream?

•The alveoli are lined with epithelial cells as well

•Drugs pass THROUGH those cells

•Inhaled “drugs” pass from the alveoli into the pulmonary vein

•Important difference – these xenobiotics do not have to pass through the liver before entering the heart

•Once “drugs” are in the bloodstream, how do they reach their desired destination?

•Helps to consider what exactly is IN the bloodstream

Fenestrated capillaries: leaky, they let small molecules leave the bloodstream without going through cells/tissues

Plasma: proteins like Albumin

Albumin

Binds to random molecules that don’t belong (like drugs), and albumin is a large molecule so it cannot fit through the holes in capillaries

Albumin is more appealing than water for drugs to bind to due to solubility

So you need a drug that is able to bind to albumin but not too tightly where it cannot leave

Problems for drugs to get into the body

Efflux pumps: pump everything out, including drugs

Blood brain barrier (BBB): no fenestrated capillaries, only epithelial cells that are very discimintive

Placenta barrier: also very selective

Oral drugs pass through the liver, which is the site of metabolism

True. Drugs not taken orally have the chance to bypass going to the liver

Liver metabolism

Oxidation:

•Cytochrome P450 (CYP)

•Monoamine oxidase (MAO also exists outside of the liver, and is responsible for the metabolism of many neurotransmitters, which is why MAO inhibition is a viable CNS target)

•Dehydrogenation

Bioconjugation: bioconjugation processes (phase II metabolism), there are two prominent bioconjugation reactions that we should discuss

•Glucuronidation: makes xenobiotic compounds more water soluble and “marks” them for elimination

•Sulfation:

First pass metabolism

process where the concentration of an orally administered drug is greatly reduced by the gut wall and liver before it reaches the rest of the body. ALWAYS happens to drugs given orally

Depot effect

•Albumin binding can actually protect some xenobiotics from metabolism by keeping them in the bloodstream and not releasing them into the liver. So the drug may avoid first pass metabolism by binding to the Albumin

Clearance (CL)

How a drug comes out of the bloodstream

CL= rate of elimination / [drug concentration]

CL= Dose / AUC(0−∞)

CL= vss x t (1/2) / 0.693

Exposure or AUC(0−∞)

how much of the drug is in the bloodstream over an infinite amount of time

AUC(0−∞) = [drug] * t

Units: M*h (molar hours) —> (moles/liter)*hour or mole*hour/liter

Dose

The amount of “drug” that is administered to the organism (mg/kg or mpk)

Find the CL

•CL = Dose/AUC

•CL = [0.5 mg/kg]/269 mM*h

•Dose = 0.5 mg compound/kg animal

•MW = 211.267 g compound/mol compound

•269 mM = 269 mmol/L = 0.269 mmol/L

•0.269 mmol/L * 211.267 mg/mmol = 56.8 mg compound/L (THIS IS AUC value in correct units)

So, CL = Dose/AUC

•CL = [0.5 mg/kg]/56.8 mg*h/L

•CL = 0.0088 L/h*kg

•CL = 8.8 mL/h/kg

C_max

The highest concentration of the “drug”

•For iv dosing, effectively occurs instantly

•Critical to know from an overdose perspective

•C_max = highest [drug]

•Use in tandem with LD₅₀

You can tell by simply looking at the graph

•T_max

Time required to achieve C_max

You can tell by simply looking at the graph

half life (t_1/2)

•Half life is how long it takes the concentration of the “drug” to decrease by half

•t_1/2 = T_{[max drug]/2} - T_max

•Critical for determining how long the [drug] will be effective and dosing frequency

V and V_ss

relates the amount of drug in the body to the blood concentration

•V = (Dose/[drug])*e^-lt

•steady state V

•V_SS = CL*l (where l = exponential decay constant)

•V_SS = CL*t_1/2/ln 2

•V_SS = CL*t_1/2/0.693

Memorize the 0.693

•V_SS is an attempt to quantify how well distributed the drug is throughout the tissues of the body

•The approximate volume of a 150 pound (68 kg) human body is approximately 42 L, or ≈ 0.618 L/kg

V_ss calcuation

•V_SS = CL*t_1/2/0.693

•V_SS = 8.8 mL/h/kg*34.4 h/0.693

•V_SS = 437 mL/kg = 0.437 L/kg

Dosing abbreviations

•qd = once per day (quaque day)

•Routinely used in discovery, but now discouraged by pharmacists (od – omne in die)

•qd# = once every # of days (sometimes qdx#)

•bid = twice per day (bis in die)

•qid = four times per day (quattuor in die)

•qh# = once every # of hours

•The ideal regimen is qd

•In practice, the best regimen is (2 to 3) *t_1/2

Model I

iv

Model II

po

Bioavailability

•is the fraction of the drug that reaches the bloodstream (F)

•By necessity, F_IV = 100

•Bioavailability via other routes is often presented as % F

•%F = AUC (po) / AUC (iv)

Calculate %F (19f)

•8,241 x 10 = 82,410

•49,744/82,410 = 0.6036 ≈ 60.4 %

Over 100% %F?

Due to a slow drip of constant drug that stays in the system for a long time

Assays: partition coefficient P

•P = [compound]_1-octanol/[compound]_water

•logP regularly used

THE TARGET RANGE FOR LogP IS 0 - 5

It is the 1-octanol to water ratio, 0 means equally bound to both and above 5 means too bound to octanol

Assays based on cellular/membrane permeability: PAMPA – parallel artificial membrane permeability assay

Can be run at a variety of pH levels

Done in well plates

If permable: goes from top to bottom

Cons: Only uses the phospholipid bilayer, does not include any receptors that line and outnumber the bilayer

Assays based on cellular/membrane permeability: caco-2 cell permeability

•caco-2 cells are immortalized human colon cancer cells

•Epithelial cells – mimic intestines

•A monolayer of these is examined as in PAMPA

•Allows evaluation of method of transport via the use of an efflux activator (which those are used to get through BBB or placental barrier)

•Better data, much lower throughput

•Assays based on cellular/membrane permeability

•Artificial membranes can be immobilized, and therefore used for rapid evaluation

•By creating HPLC columns of artificial immobilized membranes, the retention times can provide insight into membrane permeability

•Different membrane types (intestine, hepatic, brain blood vessels, etc.) can be rapidly evaluated

•K_IAM

MDCK

•MDCK (Madin-Darby Canine Kidney) cells are another epithelial cell line used to measure absorption

•Typically, an MDCK assay refers to a particular line that has been transfected with the MDR1 gene with overactive Pgp-efflux

•A particularly good predictor of blood brain barrier permeability

Assay for distrubition

•All assays for absorption may be used

•Measurement of protein binding provides a glimpse of how much free drug (vs. protein bound drug) is available in the blood

you dont want the drug to be super binded so it can leave the bloodstream

but you want it binded enough to attach to albuimin throughout the first pass metabolism so the drug can then be distrubted throughout the body

Metabolism Assays

•Liver microsomal oxidation

•Hepatocyte oxidation

•Allows for a full examination of all metabolism, not just CYPs (e.g. bioconjugation, alkylation, etc.)

•The compound is incubated with oxidant for a period of time and the disappearance is measured

ALSO assay to evaluate CYP inhibition (Cocktail assay)