Baker - pushing electrons & drug development Flashcards
What does a reaction mechanism of a catalytic cycle show?
- determines where electrons are
- decides which bonds broken and which made
- describes flow of e-s
In what level of hybridisation are most molecules?
- sp3
What is the arrangement of an sp2 hybridised molecule?
- planar trigonal
How is a σ bond formed in methane?
- 1s orbital of H overlaps w/ sp3 orbital of C
What bonding is there in ethene?
- σ bond formed between C-C (sp2-sp2 overlap)
- σ bond formed between C-H (sp2-s overlap)
- π bond formed between C-C (p-p overlap)
- double bond between C-C (4 e-s shared)
What is a π bond?
- regions of e- density above and below internuclei axis, but none along axis
- so no free rotation between 2 C atoms
What do Lewis structures show?
- represent e-s in a structure
What is the process of working out a Lewis structure?
- write mol skeleton
- assume all bonds covalent
- count available valence e-s
- add σ bonds and give each atom 8e-s (2 for H)
- if no. e-s in structure same as valence e-s then correct
- if larger add π bonds
Is sp hybridisation common?
- no, triple bonds rare
What arrangement does sp hybridisation give?
- linear
What is formal charge?
- charge assigned to atom in molecule, assuming e-s in bonds shared equally
How is formal charge calc?
- count total no. e-s contributed from each element
- what group is that element?
- group no. - total no. e-s
Why do some molecules have more than 1 poss Lewis structure?
- indiv representations of ONE actual structure
- due to e-s being delocalised across no. of atoms
- some structures more likely than others
What is the actual structure when there are multiple poss Lewis structures?
- weighted av of diff resonance structures
What do diff Lewis structures of same molecule have in common?
- same relative positions of all atoms in compound
- same no. paired and unpaired e-s (sum of formal charge identical)
- all important ones have similar energies
What do curly arrows show?
- direction of e- movement
- base at original location of pair of e-s
- head at destination of e-s
- 2 barbs = pair of e-s
- 1 barb = single e-
What are the Bronsted definitions of an acid and base?
- acid = proton donor
- base = proton acceptor
What are the Lewis definitions of an acid and base?
- acid = can coord w/ lone pair of e-s
- base = lone pair of e-s available for sharing
If [A] / [HA] = 1 then what is the ratio of [H] to [HA]?
- they are equal
How can the Henderson-Hasselbach equation be rearranged when pH = pKa?
- log10 ( [A] / [HA] ) = 0
- 10^0 = 1, therefore [A] / [HA] = 1
What are the factors that determine acidity of the organic compound Y-H?
- strength of Y-H bond
- electronegativity of Y
- factors that stabilise Y- compared to YH
- nature of solvent
- for enzymes need to consider –> electronegativity of Y and stability of conjugate base
Which are the stronger acids out of Asp, Ser, Tyr?
- Asp (pKa ≈ 4.5) –> electronegative, 2 resonance structures, -ve charge delocalised in π system, conjugate more stable, therefore a weak acid
- Tyr (pKa ≈ 10) –> O electronegative and -v charge moves around ring
- Ser (pKa ≈ 15) –> O fairly electronegative but no other stabilisation, therefore NOT an acid
What causes the diff in pKa between Asp, Ser and Tyr?
- stability of conjugate base
What 2 routes can drug dev follow?
- compound –> physiological effect –> mol target
- mol target –> compound –> physiological effect
How can drug candidates bind to target molecules?
- DIAG*
- H bonding
- hydrophobic cleft (Ile, Ala, Val)
- stacking of planar aromatic ring w/ others, forming π interactions
Where was aspirin derived from?
- salicylic acid
Why were derivatives of salicylic acid made?
- known to have lots of effects, eg. reducing inflammation
- but acidic, so problems w/ irritation and absorption in stomach
What is the structure of aspirin?
- DIAG*
- acetylsalicylic acid
How does aspirin act on cyclooxygenase component of prostaglandin H2 synthase, and what are the results?
- acylates crucial Ser in cyclooxygenase
- DIAG*
- acylated Ser blocks hydrophobic tunnel, preventing substrate arachidonic acid from reaching active site
- prostaglandin synthesis blocked
- signalling pathways interfered w/ –> affects inflammation, fever, pain, blood clotting
Why do drug candidates often contain chiral centres?
- chirality v important in binding to target
What is the structure of ibuprofen, and what form is this?
- DIAG*
- S (active) form
How is ibuprofen related to aspirin?
- inhibits same enzymes and has similar effects
In what form is ibuprofen sold?
- racemic mixture
- and body converts all to S form
How was chirality a problem in relation to thalidomide, and how is thalidomide now used?
- 1 form had therapeutic effects and other causes problems w/ dev of foetus
- tried to make chirally pure, but body converts it back to both forms
- used now in leukemia patients where no risk of affecting a foetus
What do the majority of drugs target, and why is this a problem?
- at least half target membrane proteins
- problem as determining structure difficult, so hard to design drugs
What are some of the common drug targets?
- enzymes
- G protein-coupled receptor
- VG ion channels
- solute carriers
What is Kd a measure of, and what does a low value mean?
- strength of interaction between drug candidate and target
- lower Kd = stronger interaction
How is Kd calc in relation to ligand binding in drug dev?
- DIAG*
- RL R + L
- Kd = [R] [L] / [RL]
How is the fraction of binding sites calc?
- [RL] / [R] + [RL]
What is true about the Kd equation for ligand binding if half binding sites are occupied?
- [RL] = [R]
What is a biological assay which can be used for drug potency?
- DIAG*
- biological response (%) (=bacteria killed) against [ligand]
- EC50 = conc to elicit 50% response
- EC90 = conc to elicit 90% response
When is IC50 analogous to EC50?
- when target is enzyme
How can enzyme activity (%) be compared to [I]?
- DIAG*
- IC50 = conc of inhibitor to elicit 50% reduction in activity
- IC90 = conc of inhibitor to elicit 90% reduction in activity
How does the conc of substrate affect conc of drug req to inhibit enzyme?
- DIAG*
- if Km small (high affinity) then [S] high and IC50 = Ki x big no.
- if Km large (poor affinity) then [S] low and IC50 = Ki x small no.
What is Ki?
- inhibition constant analogous to Kd –> a specific type of Kd
How is IC50 calc?
- IC50 = Ki (1 + [S] / Km)
Why do we need to consider selectivity of drug molecule?
- foreign molecule, so need to consider if binds to other proteins, DNA, RNA, metabolites, lipids etc. in cell
How is selectivity of a drug molecule calc, and what value is wanted?
- Kd binding of drug to other molecules / Kd binding of drug to target molecules
- want small value
What are effects caused by drug binding to other molecules known as?
- off target effects –> can be good or bad
Why do pharma companies want most drugs to be taken orally?
- IV much more expensive
What is the problem w/ taking drugs orally?
- need to be absorbed from stomach into bloodstream
What is the conc of drug at target site affected by?
ADME
- Absorption (getting into blood)
- Distribution (travelling around body)
- Metabolism (drugs xenobiotic so need to stop metabolism getting rid of them, OR metabolism may turn them into more active form = prodrug
- Excretion
How do drugs reach their targets, and why must they have suitable properties to do this?
DIAG
What is Lipinski’s rule of 5?
- poor absorption likely when…
1) mol weight > 500
2) no. H bond donors > 5
3) no. H bond acceptors > 10
4) partition coefficient, log(P) > 5
What is the partition coefficient?
- tendency for molecule to dissolve in membranes –> want hydrophobicity so can dissolve, allowing diffusion, as active transport would req energy
- correlates w/ ability to dissolve in organic solvents compared to aq solvent
How is the partition coefficient found?
- compare by measuring solubility in water and octanol
- [ligand] (aq) [ligand] (1-octanol)
- log(P) = log10 [ligand (1-octanol)] / [ligand (OH)]
What would a log(P) value of 5 mean?
- 100,000x more soluble in hydrophobic than aq solvent
- would be so hydrophobic that would race through membrane into aq blood and come out of solution
Why are some drugs fluorinated, and when is this done?
- often makes it more bioavailable
- done if they have good effects but poor ADME properties
How is fluorination poss?
- fluorine is good mimic of hydrogen
- VdW radius F = 1.45Å and H = 1.1Å
- C-F bond length = 1.35Å and C-H = 1.1Å
- is slightly bigger than H, but smaller than C=O, so has v little steric effect
What can sub of C-H for C-F in pharmaceuticals achieve?
- alt lipophilicity (usually increase in log(P))
- increase binding affinity of ligand to target (Kd)
- improve metabolic stability (M of ADME)
- increase bioavailability (A of ADME)
What effect does the electronegativity of fluorine have on drug compounds?
- v electronegative so can shift pKa of neighbouring group
- adding F decreases basicity of neighbouring group, so compound more lipophilic, so distributed better
In what cases does fluorination not improve lipophilicity, and why might this be?
- when F adj to O (approx when F<3.2Å from O)
- due to polarisation of O leading to stronger H-bonds to water
- or increase in overall polarity of molecule, leading to gain in solvation energy
How does fluorination modulate binding efficiency and bioavailability in serotonin receptor ligands?
- w/ no Fs –> IC50 and EC50 low, pKa 100:1 and v low bioavailability
- adding 1 F makes binding to target a bit worse, but improves bioavailability, so can now get into bloodstream
- adding 2 Fs decreases pKa, but IC50 too high and now has 2 biological effect
- so adding 1 F is optimal
How is distribution an important property of drugs to consider?
- many compounds (hydrophobic and others) not soluble in bloodstream
- carried round body by human serum albumin (HSA), which has a no. of hydrophobic binding pockets
- want compounds to be in free state, so can be released when reach target (fluoridation can modulate binding of ligand to HSA)
How can distribution of drug be tracked through the body?
- positron emission tomography (PET)
- use radioactive 18F isotope
What is the major problem w/ metabolism for drug candidates?
- xenobiotic compounds (foreign to host) metabolised before excretion
What are the 2 phases of xenobiotic metabolism?
- phase 1 = oxidation
- phase 2 = conjugation
How does phase 1 of xenobiotic metabolism occur in ibuprofen (what half reactions occur)?
- DIAG*
- R-H + H2O R-OH + 2H+ + 2e- (OX)
- NADPH NADP+ + H+ + 2e- (RED)
- O2 + 4H+ + 4e- 2H2O (OX)
What is the purpose of oxidation in xenobiotic metabolism?
- makes molecules more able to undergo addition reactions
What is the role of P450 isozymes, and how many are there?
- cat hydroxylation reactions in liver
- interconversion of FeII / FeIII (OX + RED)
- human genome encodes >50 diff P450 isozymes
What is the role of conjugation in xenobiotic metabolism, and when does it occur?
- adds lots more polar quality so can’t get across membrane anymore
- often occurs after ox has added functional group to drug
What is the effect of fluoridation on metabolism?
- fluoridation can mod metabolism
- C-F bond stronger than C-H
- ox of C-F by cytochrome P450 more difficult
- metabolism reduced, so bioavailability increased
What is the 1st pathway for excretion of drugs?
- drugs absorbed in kidneys and excreted in urine
- blood filtered by glomeruli (capillaries)
- compounds <60kDa pass through
- glucose, nts, water etc. and some drugs reabsorbed
- other compounds excreted
What is the 2nd pathway for excretion of drugs - enterohepatic cycle?
- compounds that avoid filtration in kidneys can be actively transported into bile and then intestine
- further metabolism can occur (excreted in stool) or drug reabsorbed to enter circulation
- decreases rate of excretion of drug
How can excretion kinetics be calc?
- often complex, but sometimes fixed % removed over given period = exponential loss
- [L]0 / 2 = [L] t1/2
- may not be exactly exponential
How can drug toxicity limit drug effectiveness?
- modulate target too effectively –> can stop activity of target completely, good for pathogen, but may not be good for signalling molecule etc.
- off target effects in same family
- off target effects in diff family –> commonly drugs bind to hERG K+ channel, which stops heart if blocked, so all drugs tested for this
- toxic metabolic byproducts
What is the problem w/ taking high doses of paracetamol, and how is it treated?
- DIAG*
- liver glutathione drops dramatically
- left w/ highly toxic N-acetyl-p-benzoquinone imine
- treatment is N-acetyl cysteine –> promotes biosynthesis of glutathione so imine can be converted
What is the lethal dose (LD50), and what is iit comparable to?
- amount req to kill half animals in test
- comparable to EC50 (effective dose)
How is therapeutic index calc, and what value is wanted?
- LD50 / EC50
- want high no. –> indicates big diff between effective and lethal dose
What are diff methods of drug discovery?
- serendipity
- screening
- design
How did penicillin become an available drug, and why does it not have the best ADME properties?
- Fleming observed it killed S. aureus
- beta lactam, inhibits transpeptidase of peptidoglycan
- Pain 2st to use it to treat patients
- Florey and Chain synthesised it to powdered molecule
- only 20% absorbed, 80% excreted in urine, so used to extract it from patients urine when levels low
How is Aspirin an eg. of designing a drug via screening?
- salicylic acid extracted from willow bark
- Hoffman screened for derivatives
- acetylated it –> acetylsalicylic acid = less irritating and more effective
- inhibits prostaglandin synthesis
How do HIV proteases work?
- DIAG*
- break peptide bond, by activating water molecule to attack it
- forming tetrahedral intermediate
How was making HIV protease inhibitors an eg. of structure based design?
- 1 part soluble w/ low activity and other low solubility but high activity
- when joined together they are like transition state analog
- used knowledge of structure of protein and compounds identified by screening
How was mod of HIV protease inhibitor poss?
- structure showed poss at 1 position, as adding groups here doesn’t affect binding of drug to protein as out of way and coming out into solvent –> adding pyrimidine derivative best (IC50 not as good as others tried, but much higher serum conc so EC50 much better, and good solubility halfway between hydrophobic and hydrophilic)
- can aromatise ring to get active form of Crixivan
What are the main steps to designing a new drug?
- identify target
- design/screen for lead compound
- improve bioavailability/efficacy (ADME) and toxixcity
What potential targets are there for drugs?
- KO essential genes in pathogens
- pot targets in human genome
- -> 500 kinase genes
- -> 800 7 TM receptors (350 odorant receptors) = GPCRs
- -> changes in gene expression, protein localisation, PTM from diseased organisms
- -> gene expression in particular tissues or cell types
What is an eg. of how genetic diffs can modulate drug efficacy?
- beta blockers acting on beta-1-adrenergic receptor for hypertension
- get allelic variation of 2 crucial residues, S49 and R389
- -> G49/R389
- -> S49/G389
- DIAG*
- SR/SR = greatest reduction in blood pressure
- SR/GR = 1 mutation, so 1 pair not as good, but still works
- SR/SG = even worse mutation
- GR/SG = doesn’t work at all
Why are most drugs today tested on young males, and why might this be a problem?
- risk young females pregnant or could affect future children –> so less ethical
- but wrong to assume men and women have exactly same metabolism, so drug may be more/less effective
How do you design a lead compound?
- use biochem knowledge for 1st inhibitor
- enzyme target –> substrate or transition state mimic
- receptor target –> natural ligand mimic or monoclonal antibody (eg. prevent dimerisation in signal transduction)
- natural substrate/ligand conc will affect drug potency
What can combinatorial chem produce for screening a lead compound?
- large libraries (millions) of compounds
What is the problem w/ using screens to get large libraries of compounds?
- approx 10^40 poss compounds w/ mw <750
- can’t make all, so limited possibilities investigated
- eg. if add phosphate in 1 position, can’t add anything else there, and carry on w/ that molecule, so missed out on lots of chem
How is a screen for a lead compound carried out to create a large library?
- bind compound to agarose beads (substrate)
- add 1st reactant set –> one to each set of components
- pool
- add 2nd reactant set and repeat
What are the diff steps in designing a new drug and getting it to clinical use?
- preclinical drug discovery (v expensive)
- phase I –> safety, tested on healthy people
- phase II –> safety, efficacy, dosage, on patients
- phase III –> safety, efficacy, dosage, on patients but bigger scale
- phase IV –> low freq side effects, double blind trials
