Med Chem Final Lectures 10-END

PCSK9 Nonsense Mutation=> higher or lower levels of LDL-c?

lower

PCSK9

protease that binds to LDL receptor and blocks LDL-c uptake/digestion

Biophysical Assay

Calorimetry, NMR, X-ray crystallography; low cost

Biochemical assay

enzymatic activity, receptor activity, channel conductance; low cost

Cell-based assay

cytotoxicity, phenotypic

Animal assay

Behavior (learning, memory, pain), organ level, toxicity; high cost

Fluorescence Assay with PLpro

substrate is designed such that it becomes fluorescent when PLpro cleaves the LRGG from coumarin; PLpro active when fluorescent, PLpro inhibited when less fluorescence. absorption of high energy photon and emission of low energy photon

68-95-99.5 Rule

68% f the data is within 1 STDEV of the mean

Z’ = 1

ideal assay

Z’ = 0.5

acceptable assay for screening

Z’

measures the robustness of the assay

Resin beads

porous beads that display functional sites that reactants can access

Fmoc groups

protecting group that reduces the nucleophilicity of the amine; removed with piperidine deprotections

hERG

cardiovascular toxicity assay; hit activity is measured in a probe competition binding assay; hERG active hits will displace probe = bad

Ames Assay

determines whether the compound mutates DNA; salmonella histidine auxotrophs require histidine to grow

PAINs

Aggregators make large inhibitory complexes

metal ligands nonspecifically inhibit metalloenzymes

redox cyclers make reactive oxygen species which are very active

electrophiles

Validation workflow

synthesize hits and confirm structure/activity (IC50) → perform secondary assays → perform toxicity assays/identify PAINs → obtain x-ray/cryo-EM structures

ADME

absorption = uptake of drug into circulation, usually by mouth or lung

distribution = movement of drug around the body, points of accumulation

metabolsim = transformation of drug in the body, enzymatic action, increases of polarity

excretion = removal of drug from the body through urine or feces

bioavailability

fraction of drug making to circulation defines as the drug concentration in circulation divided by the drug concentration administered

Intraveneous injection is 100% bioavailable?

yes

Range of bioavailability for most orally administrated drugs

40-60

Lipinski’s Rule of 5

cLogP <= 5, H bond acceptors (everything) <= 10, H bond donors (just H) <= 5, molecular weight <= 500

Veber Rules

Rotatable bonds <= 10, permeation coefficient > 10^-6 cm/s

Rotatable bonds are defined as…

Sigma bonds between nonterminal atoms (not H, F, Cl, Br I), and not just twirling protons (CH3, OH)

Phase 1 Metabolism

oxidation catalyzed by cytochrome P450 followed by an oxidation cascade by other dehydrogenases, NADPH dependent, generally installs -OH groups on aliphatic carbons and further oxidizes carbonyls

P450

oxidizes the most exposed least sterically hindered; demethylates heteroatoms that leaves formaldehyde; phase 1

FMOs

flavin monooxygenases that oxidize heteroatoms S and N; phase 1

Proteases and Peptidases

hydrolyze proteins and peptides like Pepsin in the stomach and Trypsin in the gut; phase 1

Esterases

hydrolyze esters to carboxylic acids like carboxylesterases (CES); phase 1

Phase 2 Metabolism

glucuronidation catalyzed by UGT that occurs on P450 oxidation products (alcohols, acids, thiols, sulfanimides, hydroxylamines)

Glutathionylation

occurs on electrophilic species, conjugates amino acids to make more polar for secretion; adds large polar groups; phase 2

Drug elimination

follows first order kinetics and steady state concentration is achieved at 7 half lives

Dosing interval

therapeutic level is above the EC50, toxic level defines onset of adverse effects, and therapeutic window is the range between the two

-CH3 isosteres

-NH2, -OH, -SH, -F, -Cl

-Br isosteres

-CHCH3CH3

-I isostere

tertbutyl

-H isostere

-D, -F

Benzene isostere

pyridine, thiophene

Is removing chiral centers good?

yes, you want to remove unecessary chiral centers to ease synthesis/purification, and scale up

Rotatable bonds in context of entropy

more negative entropy of binding (because binding will decrease entropy of the system since the highly rotatable molecule has high entropy) leads to more positive delta g and thus lower binding affinity overall; rigidify the structure via steric blocks and linker modifications

pharmaceutic phase

administration → distribution

pharmacokinetic phase

distribution → target

pharmacodynamic phase

drug acting on the target

Do polar or nonpolar molecules transport through the membrane better?

nonpolar

Order in increasing polarity: pyridine, pyrimidine, benzene, 1,3-difluorobenzene, 1,3-dichlorobenzene, triazine

dichlorobenzene, difluorobenzene, benzene, pyridine, pyrimidine, triazine

Increasing alkyl substitution for an amine does what in terms of basicity, solubility, and nucleophilicity?

decreases basicity nucleophilicity and solubility

Why is morpholine a common group implemented on drugs?

used to increase polarity, decrease basicity of nitrogen to pH 7.4, amine is aliphatic, oxygen is nonnucleophilic

delta g int?

intrinsic protein-ligand binding energy

delta g sf?

solvation energy of the free ligand and protein

delta g sb

the solvation energy of the bound ligand and protein

delta g exp

binding energy measured in solution experimentally

Polarity optimizing ligands

polar modification added to original ligand to aid solubility; designed to be solvent exposed to avoid desolvation penalty

bioisostere

bioactivity equivalent functional group

Bioisostere of carboxylic acid

tetrazole

Bioisostere of amide

pyyrole, triazole, thiazole

Steric shields

bulky substituents around the site discourage enzyme bonding that can cause an undesirable change in structure (hydrolysis/oxidation)

Blocking metabolic soft spots

replace -CH3 and -H with bioisosteres -Cl and -F which makes it resistant to P450 phase 1 metabolism

Methods to prevent amide hydrolysis:

steric shielding, N-methylation, amide replacement with bioisostere, stereochemical inversion

Oligonucleotide vaccination steps

mRNA enters cell and recruits ribosomes to initiate translation

mRNA encodes immunogenic viral protein (the immunogen)

translated immunogen is digested to antigen fragments

antigen presented via the major histocompatibility complex (MHC)

antigen presentation promotes antibody production in the lymph

Why are oligonucleotides not membrane permeable?

have negatively charged phosphate backbone that prevents membrane crossing

How can you mask phosphate backbone in oligonucleotides?