Drug Discovery Exam 1

Scientific Method

Hypothesis, testing hypothesis, and using tests to refine hypothesis. Does not work with confirmation bias

Pharmacokinetics

What the body does to the “drug” (caffeine affecting ur adenosine receptors, for example). Interactions of a drug candidate and the body.

ADME

Pharmacodynamics

What the “drug” does to the body (is ur headache gone?)

ADME

Absorption, Distribution, Metabolism, Excretion

Absorption

How drug gets into the bloodstream. Routes of administration: iv (intravenous), po (by mouth), inh (inhalation), ip (intraperitoneal - only relevant in animal models)

Distribution

How the drug gets from bloodstream to rest of body.

Metabolism

How the drug is chemically transformed in the body

Factors that effect distribution

Albumin: protein in blood that has hydrophobic pockets that other proteins can attach to and then get prevents them from passing through pores in capillaries. Can help avoid first-pass metabolism if drug is highly bound to it. (Depot effect)

Fenestrated Capillaries: molecules not captured by albumin can pass through these capillaries to the cells

Transporters/Efflux pumps: push out xenobiotics from the cells

Drug efflux can cause multidrug resistance to cells (cell starts pushing out all drugs). Cancer cells could just get better at pushing out any treatment drug through efflux pumps.

Blood Brain Barrier (BBB): brain has this instead of fenestrated capillaries.

Placental Barrier: protects fetus from being attacked by mother’s immune system, very impermeable. Must take care to see if drug (or metabolites) for pregnant woman can affect fetus.

Excretion

Metabolized compounds begin passing through kidneys. Blood filtered and metabolites are concentrated for excretion. Can also sweat out metabolites.

FDA

Food and Drug Administration. Evaluates food and medical drugs, approving based on safety and efficacy.

Blood Brain Barrier

Much more selective and less permeable than fenestrated capillaries

Oral Drug enter how?

Through epithelial cells of small intestine. Goes through intestines and hepatic portal vein, meaning it passes through liver first. Goes straight through liver - first pass metabolism.

How are drugs modified chemically by the body

Liver is the site for all metabolism: oxidation and bioconjugation

Liver oxidation mechanisms

Cytochrome P450 (CYP)

Monoamine oxidase

Dehydrogenation

CYP (Cytochrome P450)

Has a heme group. Oxidizes hydrocarbons, providing more polar and more reactive functionalities. Multiple isoforms. Play a big role in metabolizing foods and toxins. Major 4. Activation, inhibition, bioactive metabolities. Plays big role in drug/drug interaction. Many natural CYP inhibitors.

Converts testosterone to hydroxy-testosterone, which is more reactive and actually causes effects

Tamoxifen - used for breast cancer. Oxidized by CYP, which can then get delivered to cells. Metabolized version. is what is active against cancer cells.

Grapefruit juice interferes with CYP3A4 (discovered via LSD use).

Monoamine oxidase

Oxidizes amines in the liver to form Hoffman elimination.

Alcohol dehydrogenase

Metabolic enzyme in liver that converts primary alcohols to aldehydes and secondary alcohols to ketones.

Bioconjugation

Phase II metabolism. Alcohol (or amine) gets added to glucuronic acid, which can happen at any nucleophilic site on a xenobiotic compound. Bunch of OHs added to a drug, making them more water soluble and marks for excretion

Sulfation (typically of alcohol) has same effect

First pass metabolism used how

Can measure the effects by knowing the hepatic blood flow rates. 21 ml/kg in people (typically slower the bigger ur organism is). Depot effect can occur to bypass first pass metabolism.

Microbiome

Can have effect on metabolism of xenobiotics. Can impact effectiveness of drug. Metformin is one of them.

Model I of PK

IV bolus of dose, assume that it is spread through entire body. Top concentration achieved immediately and decreases over time. Not dependent on size of dose. Curve looks same.

Clearance (CL)

Rate of elimination of a drug.

CL = Rate of elimination/[concentration drug]

CL = Dose/AUC_0-infinity

Exposure

Amount of drug in blood stream over time. Measured by AUC or AUC segment (area under the curve).

AUC_0-infinity = [drug]*t

Example: M*h (molar hours) = (Moles/liter)*hour can also be smaller like micromolar hours

Dose

Amount of druge administered to organism. mg/kg or mpk (kilogram of person)

C_max

Highest concentration of drug. For iv dosing, occurs pretty much instantly. Critical to know to prevent overdose

T_max

Time it takes for drug to reach Cmax. Close to 0 for iv dosing. Important for overdose prevention.

Half life: t_1/2

How long it takes to go from Cmax to half of Cmax

Penicillin

Lister sees that penicillium mold is antibacterial on wounds, Fleming later sees that it prevents Staph aureus growth. Concentrate it into penicillin. Florey develops mass production method. Bayer notes thatsulfonamides were antibacterial at the same time, leading to sulfa drugs.

Thalidomide

German pharma company wanted to launch in US as sleep aid and morning sickness reliever for pregnant women, giving samples to physicians to give to patients. However major birth defects. Frances Kelsey blocked it from FDA approval, wom award.

Phenotypic drug discovery

When a compound elicits a specific response before the target is identified. (this drug kills cancer cells, but idk what it’s targeting to do that).

Genotypic drug discovery

Most modern drugs discovered this way. A target that you believe will elicit a desired biological response is identified first, and then you think of ways to interacting with the target to acheive that. (I want to inhibit this transcription factor somehow).

Antagonist

chemicals that interact with a biomolecule in a way that prevents that biomolecule from performing its normal function. most inhibitors are this. Inderal is beta-blocker (beta receptors) that lowers blood pressure, tremors, chest pain.

Agonist

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

Two categories of drugs:

Agonist and antagonist. But ultimately, every drug acts on multiple biochemical targets to carry out its function.

Early stage research and drug discovery:

Target identification, hit generation, lead generation.

Target identification

First step of genotypic drug discovery. Most drugs target membrane proteins or enzymes (especially protein kinases).

Main classes of drug targets

Enzymes, receptors, other proteins, nucleic acids.

Enzymes

Biomolecules that catalyze chemical reactions. Lower free energy of activation. Not consumed in reaction. Primarily proteins but usually have additional co-factors. Can have isoforms, multiple functions/substrates. Exist in an aqueous environment, so typically have a hydrophilic shell protecting a hydrophobic pocket, where active site usually is.

HMG-CoA Reductase

Enzyme that catalyzes conversion of a precursor to cholesterol and other steroids. Atorvastatin acts as a competitive inhibitor for this enzyme to prevent cholesterol production.

ATPase

Enzyme that converts ATP to ADP.

Cyclooxygenase (COX)-2

Used to produce prostaglandins, which are important for inflammatory response and fever. Different from COX-1, which protects stomach lining. Celecoxib drug specifically blocks COX-2 to reduce inflammation/pain/fevr.

Receptors

Biomolecules that change when binding to other molecules. Potention drug target

P2Y₁₂ (GPCR)

ADP receptor on platelet cells. Clopodrigel blocks the receptor, which helps prevent platelet clumping and blood clot formation.

CysLT₁₂ (GPCR)

Leukotriene receptor in lungs. Montelukast sodium binds to it, stops leukotriene-induced inflammatory events, including cell migration, airway edema, capillary permeability, and bronchoconstriction. Useful for asthma, allergies

GABA_A Receptor

Cl- ion channel that decreases neuron activation. Ambien increases the frequency of how often the chloride channels open when GABA binds to the receptor, slowing brain activity. Used as sedative, treating insomnia. Xanax is also agonist of receptor.

Other protein targets

Transport proteins, chaperones, protein-protein interactions.

FKBP-mTOR

Two proteins that elicit immune response when combined with a ligand. Rapamune blocks this to suppress immune system. Protien-protein interaction

DNA Damaging Agent

Drug attacks DNA to kill cells (useful in chemotherapy)

aminoglycosides

another drug target. inhibit protein synthesis via an unproven mechanism

Drugs

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

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

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

Equilibrium in drug discovery

Most chemical reactions have an equilibrium, and this is true in the body too. Diseases often arise when equilibrium is disturbed. Affecting an equilibrium can also impact other equilibria.

Bond types

Covalent, ionic, hydrogen. Strength in that order

Molecule interaction types

Dipolar, London/Van der Waals. Strength in that order.

Competitive inhibitor

inhibitor takes spot of active site, so substrate can’t bond

Noncompetitive inhibitors (allosteric)

Inhibitor bonds to another spot on enzyme, changing active site shape so substrate cannot bind

Hybrid inhibitor

Inhibitor binds to part of the active site, so substrate can’t fit

Protein structure levels

Primary, secondary, tertiary, quaternary

Reversible inhibitors

Inhibitor can detach easily, reaction can go both ways

Irreversible inhibitors

Inhibitor forms strong bond and permanently inactivate enzyme. ex: Imbruvica is a permanent kinase inhibitor that’s used to treat blood cancer.

Enzyme activators

Allosteric activator can bind to allosteric site and activate enzyme so substrate binds or gets released.

Enzyme isoforms

Reversible/irreversible, specific/nonspecific, repair

Membrane-bound receptors, structure?

On the membrane of the cell. G-protein coupled receptors (GCPRs, 7TM). Other enzyme-linked receptors, ion channel receptors, etc. Often dimers, which is important for signal amplification (homodimer), diversification (heterodimer), and preventing accidental activation.

Intracellular receptors

Inside the cell. Nuclear receptors, secondary receptors

7TM/GCPR

Receptor that passes through membrane 7 times. G-protein coupled receptor. Phosphorylation cascade. When signaling molecule attaches on outside. G-protein’s alpha subunit exchanges GDP for GTP, then breaks off and causes other reactions.

Partial agonist/antagonist to receptor

When alone, a partial agonist provides a low-level therapeutic effect (submaximal response). When mixed with a full agonist, it blocks the full agonist from binding, lowering the overall cellular response

Superagonist to receptor

Overactivates receptor, often causing downstream regulation to shut down. Zoladex - Initially overstimulates the pituitary gland to ultimately shut down the body's production of sex hormones

What makes a good drug

Active against the target, able to be given to patients in a manner to derive their effect (need to understand its pharmacokinetics)

CYP isoforms

1A2, 2C9, 2D6, 3A4

Volume of Distribution (V)

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

V = (Dose/[drug])*e^-lambda*t

Helps you figure out how much of drug is spread through body

Steady State V (V_ss)

Attempt to quantify how well distributed the drug is throughout the tissues of the body

V_ss = CL* lambda (lambda is exponential decay constant)

V_ss = CL*t_1/2/0.693

Approx volume of a 150 pound human is about 42 L or 0.618 L/kg

Why half-life and V are important

Half life and V are important since drugs typically administered multiple times. Ex: acetaminophen can be taken 4x a day. Not always the case, some drugs don’t fully clear out and gradually build up over multiple doses (V is important for this!)

Dosing abbreviations and ideal regimen

qd = daily

qd# = every # of days

bid = 2x a day

qid = 4x a day

qh# = every # of hours

Ideal regimen is qd

In practice, best regimen is 2-3*t_1/2 (8-12 hour half life good for qd drugs)

Complications with dosing and measuring

Oral drugs have to enter blood stream before concentration measured

Distribution and non first pass metabolism occur simultaneously with absorption

Bioavailability

Fraction of drug that reaches bloodstream (F)

F_iv = 100

Other routes typically represented as %F.

For example, Chantix po has 87.9% F.

Can compare AUC of oral vs iv (make sure dosage is the same)

F% can be over 10. IV form saturates the proteins quickly, preventing it from getting metabolized, whereas po lets you slowly have greater concentration

Interspecies PK Variability

Mouse, rat, dog, monkey, humans - order of animal testing

Organisms tested on can have very different Cmax, CLmax, tmax, half life, etc. Cause different metabolic enzymes, transport proteins, blood flow rates

How to fix poor PK

Assays. Focusing especially on ADM.

How oral drug solubility tested

Pampa, logP, caco-2 cell permeability, HPLC columns with artificial membranes, MDCK

Can manipulated pH to see how absorbed it is in different parts of intestines

PAMPA

Parallel artificial membrane permeability assay. Wells stacked on phospholipid bilayer. See if compound moves through bilayer.

logP

Oral drugs go through epithelial cells of small intestine (multiple membranes). One way to approximate solubility in a membrane is via partition coefficient P.

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

logP regularly used

0 means equal amounts in water, octanol-1. Target range for logP is 0-5, which indicates drug is reasonably able to permeate membranes and can be absorbed.

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 transport method via use of an efflux activator

Better data, much lower throughput

HPLC columns with artificial membranes

chromatography column using artifical membranes to see how much passes through.

MDCK

Madin-Darby Canine Kidney. epithelial cell line used to measure absorption, good predictor of blood brain barrier permeability

Measuring distribution of drug

Evaluate if drug is stable in different pH and fluid conditions. Use same assays as absoprtion. Can test in plasma.

Measurement of protein binding provides an idea of how much free drug is available in blood. Put drug in plasma with membrane separating other side. See how much passes to see how protein binding(albumin) inhibits or doesn’t inhibit drug,

DMPK

Drug Metabolism and Pharmacokinetics

metabolism assay things to consider

• at what rate is compound metabolized

• does compound inhibit metabolism

• is compound metabolized into an inhibitor of metabolism

Drug metabolism measurement

Liver microsomal oxidation (isolate liver enzymes and test)

Hepatocyte oxidation take liver cells to test drug. Allows for full examination of all metabolism, not just CYPs (bioconjugation, alkylation, etc).

Measuring metabolizing rate

Compound incubated with oxidant for a period of time and disappearnce is measured

Measurign if drug inhibits metabolism

Vast majority of xenobiotics oxidized by few isoforms. Assay to evaluate CYP inhibition.

1A2 substrate and inhibitor

phenacetin (antiarthritic agent)

inhibited by furafylline

2C9 substrate and inhibitor

tolbutamide (antidiabetic agent)

sulfaphenazole

2D6 substrate and inhibitor

dextromethorphan (antitussive agent) mucinex

quinidine

3A4 substrate and inhibitor

has two different oxidizing sites (need to see if drug inhibits either site)

midazolam (antiseizure/anxiolytic agent)

testosterone (steroid hormone)

inhibited by ketoconazole

Why target chaperone proteins?

Since they help other proteins fold properly, inhibiting or enhancing them can affect how well those other proteins fold.

Nucleic Acid Drug Target

DNA-damaging drugs. Targeting RNA is an emerging technique. Latest vaccines are mRNA-based, like the Pzifer COVID vaccine. RNAi (destroys mRNA of a certain protein).

Assay

Examination or determination of characteristics. For target discovery, need to measure whether a process takes place