PHMD 213 Quiz 3

Looking at saquinavir, what do enzyme subsites S1, S1’ describe?

• corresponds to substrate/pockets on enzyme positions P1, P1’ (peptides)

• S numbers run toward N-terminus while S’ runs towards C-terminus (scissile bond btw P1 & P1’)

• central OH sits btw two catalytic Asp25/Asp25’ residues

Looking at saquinavir, what is the general drug design strategy?

substrate/peptide → substrate-bade, transition-state-analog inhibitor

• scissile amide bond replaced w/ non-hydrolyzable isostere

• side chains kept/optimized to fit S3, S2, S1, S’ & S2’ (hydrophobic + H-bonding groups)

• made bulkier + rigid + less peptide-like (improves potency, stability, and oral availability)

Looking at saquinavir, why does it work well? What kind of inhibitor is it?

• it mimics tetrahedral transition state of peptide hydrolysis (hydroxyl takes place of water-derived tetrahedral intermediate & H-bonds to catalytic Asp25/Asp25’; enzyme binds transition state more tightly than substrate so it can’t be cleaved)

• competitive inhibitor

How does HIV protease’s active-site symmetry influence the design of other inhibitors?

• HIV protease: C-2 symmetric homodimer (each monomer w/ one catalytic Asp25 → symmetric active site)

• C2-symmetric/pseudo-symmetric inhibitors w/ central hydroxyl/diol that contacts both Asp residues & matching groups on each side (eases synthesis for good fit + acts selectivity; human aspartyl proteases = single-chain + asymmetric)

How does phosphorylation work?

reversible covalent, post-translational modification (kinase transfers gamma-phosphate of ATP to hydroxyl of Ser, Thr, or Tyr)

What is the phosphorylation reaction?

Protein-OH + ATP → Protein-O-PO₃^2- + ADP

What is the dephosphorylation reaction?

Protein-O-PO₃^2- + H₂O → Protein-OH + P_i

What are kinases? What do they do?

• enzymes that transfer phosphate group from ATP → substrate (usually protein)

• acts as molecular switches in signal transduction & regulate enzyme activity, protein-protein interactions, localization + cell cycle

• many = drug targets

What activates protein kinase A (PKA)?

cAMP

• inactive PKA holoenzyme = tetramer (two regulatory + two catalytic subunits); cAMP binds regulatory subunits → releases active catalytic subunits

• inactive form: regulatory subunit has pseudosubstrate sequence in active site (autoinhibition)

Inhibitor or pseudosubstrate has an alanine instead of Serine, why can it bind without being phosphorylated?

it still has recognition site + makes same contacts → binds active site (alanine has no hydroxyl group so it can’t accept phosphate; stays bound + blocks site, acts as competitive inhibitor)

Myoglobin (Mb)

• structure: monomer, 1 heme

• function: O₂ storage in muscle

• substrate: O₂ (heme Fe²⁺)

• binding behavior: non-cooperative

• binding curve: hyperbolic, high affinity

• inhibitor/regulators: CO (competes w/ O₂ for heme Fe) and CN^-

Hemoglobin (Hb)

• structure: tetramer, 4 hemes

• function: O₂ transport from lungs to tissues

• substrate: O₂ (heme Fe²⁺)

• binding behavior: cooperative (binding of one O₂ raises affinity for next)

• binding curve: sigmoidal (S-shaped), lower overall affinity

• inhibitor/regulators: CO + CN^-, allosteric effectors (H⁺, CO₂, 2,3-BPG) lower O₂ affinity

What are the two different states of Hb?

• Tense (T state): low O₂ affinity + deoxygenated form

• Relaxed (R state): high O₂ affinity + oxy form

• O₂ binding: T → R (cooperativity)

What does logP measure? What does higher or lower logP mean?

• measures lipophilicity (log of partition coefficient of neutral compound between octanol and water)

• high logP: more lipophilic, prefers membranes + lipid (less water-soluble), low logP: more hydrophilic, water-soluble (cross membranes poorly)

True or False: logP equals better drug

False

• drugs need balance (very high logP = poor solubility + extensive metabolism + promiscuous binding + toxicity, very low logP = poor membrane permeability + absorption)

What does ADME stand for?

• Absorption: drug moves from site of administration → blood

• Distribution: drug moves from blood → tissues + fluids

• Metabolism: drug chemically modified, mostly in liver (more polar & inactive)

• Excretion: drug & metabolites removed from body (thru kidney/urine & bile/feces)

How do the processes of ADME differ from each other?

movement into blood (A), movement among compartments (D), chemical change (M), and removal from body (E)

What does cytochrome P450 do?

family of heme-containing (Fe) monooxygenases in liver (ER membrane) & gut wall that oxidizes drugs + other xenobiotics thru hydroxylation (handles half of all drugs)

What common fruit inhibits CYP450? Why/how?

• Grapefruit

• furanocoumarins = mechanic-based (irreversible) inihibitors of CYP3A4 (especially intestinal wall); result = less 1st pass metabolism, higher bioavailability, + blood lvl of drugs, risk of toxicity

What is the overall CYP hydroxylation reaction?

R-H + O₂ + NADPH + H⁺ → R-OH + H₂O + NADP⁺

Does CYP catalyze phase 1 metabolism or phase 2 metabolism?

Phase 1

Is the ligand binding specific or broad?

Broad

• CYPs have large, flexible, hydrophobic actives sites + accept many structurally dif substrates (why drug-drug interactions common)

What structural changes can increase metabolic stability?

• block labile (soft spot) site

• reduce lipophilicity/add polar groups

• replace electron-rich aromatic rings w/ electron-poor ones

• add steric hindrance near labile groups/replace easily oxidized groups

• cyclize/rigidfy structure

What are the different types of CYP inhibition?

• reversible: inhibitor binds non-covalently & activity returns when removed

  • competitive: competes w/ substrate for active site (ex: azole antifungals + cimetidine; N lone pair coordinates heme ion)

  • non-competitive/mixed inhibition can also occur

• mechanism-based (irreversible): CYP oxidizes inhibitor → reactive intermediate that covalently modifies/destroyes heme/apoprotein (activity returns when new enzyme synthesized)

Phase 1 metabolism

• functionalization

• introduces/exposes polar function group thru oxidation, reduction/hydrolysis (enzymes include CYPs, ADH, ALDH, MAO + esterases)

Phase 2 metabolism

• conjugation

• attaches large, polar endogenous molecule to drug/phase 1 metabolite (product highly water-soluble + easily excreted; enzymes = transferases)

Does a drug always to undergo Phase 1 before Phase 2 metabolism?

No

• drug w/ suitable handle (polar function group) can be conjugated directly; some drugs excreted unchanged, while others undergo phase 1 only

What are some general transformation strategies?

• phae 1 oxidation: aliphatic + aromatic hydroxylation, epoxidation, N- & O-dealkylation, oxidation deamination, S-oxidation, alcohol + aldehyde oxidation

• phase 1 reduction & hydrolysis

• phase 2: glucuronidation, sulfation, acetylation, methylation, glutathione conjugation, amino acid conjugation

What regions of antibody can recognize molecular targets?

• variable regions of Fab arms (specifically complementarity-determining regions/CDRSs)

• Fc region doesn’t bind target (mediates effector functions + half life)

Can therapeutic antibodies be immunogenic?

Yes

• patient can make anti-drug antibodies (neutralizes drug, speeds clearance/causes reactions)

• risk greatest w/ murine antibodies (-omab), lower w/ chimeric (-ximab) + humanized (-zumab) + fully human (-umab)

What is PEGylation modification and what does it do?

• PEGylation = covalent attachment of polyethylene glycol (PEG) chains to drug

• effects: large hydrodynamic size → less renal filtration + longer half-life, shields drug from proteases + immune system → better stability + lower immunogenicity, higher water solubility + less aggregation, trade-offs: can reduce binding/activity & anti-PEG antibodies possible

Which parts of nucleic acids can be modified?

• phosphate

• sugar (ribose)

• nuclease

What are some of the potential benefits of these modifications?

• nuclease resistance (better stability + longer half-life)

• higher binding affinity to target

• reduced innate immune activation

• better pharmacokinetics

• improved translation (mRNA), cellular uptake + fewer off-target effects

Enzyme + phase for transformation of primary alcohol (RCH₂OH) → aldehyde (RCHO), NAD⁺

• alcohol dehydrogenase (ADH)

• Phase 1

Enzyme + phase for transformation of secondary alcohol (RR’CHOH) → ketone, NAD⁺

• alcohol dehydrogenase (ADH)

• Phase 1

Enzyme + phase for transformation of aldehyde (RCHO) → carboxylic acid (RCO₂H), NAD⁺

• aldehyde dehydrogenase (ALDH)

• Phase 1

Enzyme + phase for transformation of primary amine (RCH₂NH₂) → aldehyde (RCHO)

• monoamine oxidase (MAO, oxidative deamination)

• Phase 1

Enzyme + phase for transformation of R-OH + UDP-glucuronic acid → beta-D-glucuronide conjugate + UDP

• UDP-glucuronosyltransferase (UGT)

• Phase 2

Enzyme + phase for transformation of phenol/alcohol + PAPS → sulfate conjugate

• sulfotransferase (SULT)

• Phase 2

Enzyme + phase for transformation of amine + PAPS → sulfamate conjugate

• sulfotransferase (SULT)

• Phase 2

What is an amphiphile?

molecule w/ both a hydrophilic (head) & hydrophobic (tail) region

How/why do amphiphiles self-assemble in water?

hydrophobic effect

• water forms ordered sheel around exposed nonpolar tails (lowers water’s entropy)

• tails clustered + hide from water → ordered water molecules release + entropy of system increases

• polar heads stay facing water, while van der Waals forces btw tails help stabilize assembly (spontaneous + non-covalent)

Triacylglycerol (TAG)

• structure: glycerol + 3 fatty acids joined by ester bonds (entirely nonpolar, not amphiphilic)

• function: energy storage (not membrane comp bc can’t form bilayer)

Phospholipid

• structure: amphiphile w/ polar phosphate-containing head + 2 hydrophobic tails

• function: main structural lipids of membranes (forms bilayers)

Sterol

• structure: rigid fused 4-ring steroid core, small polar OH head, short hydrocarbon tail

• function: inserts btw phospholipids in animals membranes + modulates fluidity & rigidity (precursor of steroid hormones + bile acids)

What bonds connect glycerol to fatty acids in a triacylglycerol?

ester bonds

Reaction of esterification

carboxylic acid + alcohol → ester + water

Reaction of hydrolysis

ester + water → carboxylic acid + alcohol

Glycerophospholipid

glycerol backbone + 2 faty acids + phosphate w/ alcohol head group

Sphingolipid

sphingosine backbone (amino alcohol) + 1 fatty acid attached by amide bond (making ceramide) + head group

• major comp of myelin

Glycolipid

lipid w/ carbohydrate/sugar head & no phosphate (sit on outer leaflet + involved in cell recognition)

What lipids does a membrane contain?

mainly phospholipids + cholestrol + glycolipids

How do you distinguish saturated, cis-unsaturated, and trans-unsaturated fatty acids?

• saturated: no C=C double bonds, straight + flexible chains that pack tightly

• cis-unsaturated: C=C w/ both chains on same side (rigid 30 degree kink that disrupts packing)

• trans-unsaturated: C=C w/ chains on opposite sides (nearly straight chain that looks + packs like saturated)

How do full and partial hydrogenation change fatty acids?

• full hydrogenation: adds H₂ across all C=bonds (gives saturated fatty acids straight chain, tight packing, higher melting point)

• partial hydrogenation: saturates some double bonds + catalyst isomerizes remaining ones from cis to trans (gives semi-solid fats w/ straight trans chains that raise LDL + unhealthy)

What are gel and liquid-crystalline lipid phases?

• gel phase: lipid tails tightly packed, extended + ordered, little lateral movement (low fluidity, low temp)

• liquid-crystalline phase: tails disordered + flexible, lipids diffuse laterally (high fluidity, high temp)

• melting temperature = transition from gel → liquid-crystalline

Predict melting temperature of cis vs. trans chains

• cis: kinks disrupt packing (lower Tm)

• trans: chains straight + pack better (higher Tm)

Predict melting temperature of long vs short chains

• long: more van der Waals contact + pack more strongly (higher Tm)

• short: lower Tm

What is membrane fluidity?

how easily lipids + proteins move within bilayer (depends on temp, chain length, saturation, cholesterol content)

How does cholesterol affect fluidity?

acts as fluidity buffer

• high temp: rigid ring system restricts motion of neighboring chains (decreases fluidity)

• low temp: prevents tight packing + gel formation (increases fluidity)

• broadens phase transition so membrane (moderately fluid across range of temps)

How does the myelin sheath support rapid signaling?

myelin = lipid-rich multilayer wrapping made by Schwann cells (PNS) + oligodendrocytes (CNS)

• acts as electrical insulator (reduces ion leakage + membrane capacitance so signal travels quickly along axon; signal jumps from one gap to the next/saltatory conduction)

Where are action potentials regenerated?

node of Ranvier (unmyelinated gaps where voltage-gated Na⁺ channels concentrated)

Micelle

• small sphere w/ hydrophobic tails inside + heads facing water (hydrophobic core w/ no water)

• single-tailed amphiphiles w/ conical shape

bilayer sheet

• two leaflets w/ tails facing e/o in middle & heads facing water on both sides (flat sheet w/ edges that expose tails to water)

• two-tailed, cylindrical lipids

liposome

• bilayer sheet that closes on itself into hollow sphere (aqueous interior + no exposed edges)

• phospholipids

What protein structures are likely in integral proteins?

membrane-spanning segments = alpha-helices/beta-barrels (hydrophobic core)

What protein structures are likely in integral proteins?

polar/charged surfaces (globular domains held by electrostatics + H-bonds; sit on surface)

Integral protein

embeeded in membrane & removable only w/ detergents/disrupting bilayer

transmembrane protein

integral protein that spans whole bilayer (domains on both sides)

Peripheral protein

protein not embedded/bound to surface by non-covalent interactions w/ lipid heads/intergral proteins (removable w/ mild treatment)

lipid-anchored protein

protein covalently attached to lipid that inserts into bilayer (protein stays outside)

Ranking easy to difficult for molecules that have different properties to cross bilayer (small nonpolar, small uncharged polar, large uncharged polar, ions/charged)

Easy to difficult

1. small nonpolar

2. small uncharged polar

3. large uncharged polar

4. ions + charged

Simple diffusion

• no protein needed

• no energy needed/passive

• down conc/electrochemical gradient

Facilitated diffusion

• protein used

• no energy needed/passive

• down gradient (saturable + specific)

primary active transport

• protein used

• direct ATP hydrolysis

• against gradient

secondary active transport

• protein used

• indirect: uses gradient established by primary active transport

• one gives down gradient, other moves against gradient

What determines the direction of movement?

• passive transport: concentration gradient (ions: electrochemical gradient/concentration + membrane potential)

• active transport: energy input/ATP overrides gradient (solute moves from low → high conc)

Phagocytosis

cell eating

• engulfs large particles w/ membrane extensions into large vesicle/phagosome (done by specialized cells + receptor-triggered but not constitutive)

Pinocytosis

cell drinking

• nonspecific uptake of extracellular fluid + dissolved solutes in small vesicles (happens continuously in most cells + non-selective)

Receptor-mediated endocytosis

ligands bind receptors that cluster in clathrin-coated pits (pinch off as coated vesicles; highly selective + efficient, concentrating cargo)

Lipid rafts

small, dynamic membrane microdomains enriched in cholesterol + sphingolipids

• tightly packed order phase makes them more rigid/liquid-ordered than surrounding membrane

• thought to organize + concentrate signaling proteins + receptors, aid in vesicle trafficking + serves as platforms for signal transduction