Biomedical Drug Development - Exam 2

GPCR

7 transmembrane receptor that is coupled to a G protein.

heterotrimeric G proteins

also known as guanine nucleotide-binding protein. consist of alpha, beta, and gamma subunits. subunits act as intracellular molecular switches

nucleotide exchange

GPCR regulates when GDP is exchanged for GTP, activating G proteins

GRK

inhibits activated GPCRs by phosphorylating them. once phosphorylated, beta-arrestin can bind to the receptor and block G proteins from binding there and can lead to the internalization of the receptor

• all GRKs have a conserved AGC region, important for their kinase activity

RGS

promote the association of the alpha, beta, and gamma subunits. terminate signaling

ligand

any substance that binds specifically and reversibly to a biomacromolecule to form a larger complex that alters its activity or function

light-sensitive compounds

photosensitive compounds can undergo chemical reactions when exposed to light

endocrine

hormones that are released into the bloodstream

paracrine

hormones that act on nearby cells

autocrine

cells that are affected by the hormones they released

intracrine

signaling molecules that acts intracellularly and is not secreted

hormone

a class of signaling molecules that are sent to distant tissues or organs to regulate physiology and behavior

first-in-class drug

a prototype drug that uses a new mechanism of action to treat a disease

B_max

represents the total number of receptors in the assay that can bind to the radioligand

EC₅₀

Refers to the concentration of any bioactive compound that induce a response halfway between the baseline and maximum after a specific exposure time

K_d

equilibrium dissociation constant of the radioligand

K_i

equilibrium dissociation constant for nonradioactive ligand

K_on

association rate constant, the rate that R and L will form RL

K_off

dissociation rate constant, the rate that RL will separate for form R and L

K_a

equilibrium affinity constant (K_on/K_off)

stoke’s shift

the difference between the maximum excitation and maximum emission spectra of a fluorophore  

DELFIA

ligand-based fluorescence that uses lanthanide chelates. this allows for a large stokes shift value which makes it easier to differentiate between the maximum emission and maximum excitation. large stoke shift increases sensitivity of assay format

CADD

computation-assisted drug discovery

• virtual ligand screening (VLS)

• QSAR - there is a relationship between how a molecule acts in vivo and its chemical structure 

• synthon-based hierarchical screening (V-SYNTHES) use docking to predict the affinity between fragments and target proteins. the fragments with the highest affinity had synthons added to create more complete molecules. docking and synthon-adding can be repeated and ultimately lead to in vitro testing

LRI-based screening approaches

1. radioligand binding assay

2. ligand-based fluorescence

3. SPR

4. ligand-based NMR

5. downstream-based ligand measurements 

GRAFS

Glutamate - family C

Rhodopsin - family A

Adhesion - family B2

Frizzled/Taste - family F

Secretin - family B1

structure of glutamate GPCR family

has a venus fly trap extracellular domain

structure of rhodopsin GPCR family

basic GPCR structure

structure of adhesion GPCR family

has an autoproteolysis-inducing domain

structure of frizzled/taste GPCR family

cystine-rich domain

structure of secretin GPCR family

large extracellular domain

5 categories of GPCR ligands

1. light

2. odor

3. hormones

4. proteins

5. neurotransmitters

functions of beta arrestin

1. terminate GPCR signaling

2. can mediate downstream responses, regardless of g protein activation

3. plays a role in GPCR internalization and trafficking

4. acts a scaffold for MAPK, JNK, AKT

difference between hormone and neurotransmitter

hormones are secreted by endocrine glands and travel throughout bloodstream. neurotransmitters are released by presynaptic nerve terminal into the synapse

general steps of drug development process prior to IND (investigational new drug) filing

1. hit finding and validation: use bioinformatic analyses and high through screening to identify potential drug targets

2. hit to lead: aka lead generation. where small molecules from the previous step are evaluated and optimized to identify promising compounds

3. lead optimization: synthesize lead compounds, new analogs that are more potent, less off-target activities, and reasonable in vivo pharmacokinetics

4. preclinical regulatory: pharmacodynamics (what drug does to body), pharmacokinetics (what body does to drug), [absorption, distribution, metabolism, excretion] (ADME), toxicology

4 peptide modifications used in drug discovery

1. N-methyl amino acids (arginine and lysine)

2. acetylation

3. glycosylation

4. adding PEG groups 

features of SPR method in evaluating ligands

1. rapid method for characterizing membrane protein and ligand interactions

2. label-free detection of biomolecular interactions in real time

3. can be used on many membrane systems

4. potential for medium-throughput screening of ligands for drug discovery

working mechanism of SPR

1. a polarized light strikes an electrically conducting surface at the interface between two media (glass slide and gold film)

2. this causes plasmons, electron charge density waves

3. plasmons cause the light intensity to decrease, resulting in a resonance angle that is proportional to the mass on the sensor’s surface

DEL library

used in drug discovery with GPCR

1. molecules are bound to a DNA tag that acts as a barcode

2. an immobilized protein, or in the case GPCR in a live cell, is introduced to the molecules

3. the molecules that can bind to the protein stay attached, while the others are washed away

4. the molecules that could bind are identified, using the “barcode” PCR and DNA sequencing

5. these molecules can be used in further testing 

3 GRK domains

1. RGS - regulator of g protein signaling domain

2. PH - pleckstrin homology domain (only GRK2/GRK3)

3. AGC - AGC sequence important for kinase activity

why do peptide drugs have to be delivered intravenously

they risk degradation by peptidases and renal elimination

key features of chemokines

they attract immune cells (macrophages, t cells, mast cells, granulocytes) to site of infection

• 8-10kDA

• conserved region has 4 cystine residues

• 4 families: C, CC, CXC, CX3C

G_αq calcium production signaling pathway

1. agonist binds to gpcr

2. αq activate PLC

3. PLC allows PIP2 to be converted into IP3 and DAG

4. IP3 will bind to IP3 receptor on ER/SR

5. this allows Ca2+ to leave the ER/SR and enter cytoplasm

6. Ca2+ will work with DAG to activate protein kinase C

G_αs calcium production signaling pathway

1. agonist binds to gpcr

2. αs activates adenylyl cyclase so that ATP → cAMP

3. cAMP increases production PKA

4. PKA phosphorylates RyR, L-type Ca2+ channels, and mysoin binding protein C on ER

5. allows calcium to leave ER and enter cytoplasm

G_αi/o calcium production signaling pathway

1. agonist binds to gpcr

2. G_αi/o inhibits adenylyl cyclase and voltage-gated calcium channels

3. β and γ activate PLC

4. PLC allows PIP2 to be converted into IP3 and DAG

5. IP3 will bind to IP3 receptor on ER/SR

6. this allows Ca2+ to leave the ER/SR and enter cytoplasm

7. Ca2+ will work with DAG to activate protein kinase C

list 3 signaling indicators

1. measure cAMP levels: use radio-immunoassays, immunodetection assays, lyse-cell based cAMP measurement

2. track nucleotide exchange using BODIPY-FL dye

3. track GTP hydrolysis using GTPase-Glo assay. the more active the GTPase, the less light will be produced

tools to measure beta arrestin bias

1. presto-tango-based

2. nanobit-based

3. path hunter-based

tools to measure g protein bias

1. BRET

explain PRESTO tango assay

• uses HEK239 cells. these have a luciferase reporter gene

1. a ligand will bind to gpcr

2. this allows beta-arrestin-TEV protease complex to cleave the transcription factor (tTA) from the V2 tail of the receptor

3. now the free tTA can travel to the nucleus and activate transcription of luciferase reporter gene

explain FRET

1. the donor and acceptor have to be in close proximity to each other

2. excitation of photons from the ground to the excited state of the donor

3. energy transfer from excited donor to acceptor through the dipolar coupling between donor emission and acceptor excitation dipole moments

explain BRET

• similar to FRET, but does not require an external light source to excite the donor protein

1. when the donor and acceptor proteins are far away, only the donor’s Rluc emission will be detected (480 nm)

2. when the donor and acceptor proteins are close to each other, the energy transfer can take place and the acceptor’s EFYP emission will be detected (530 nm)

explain what BRET has to do with G protein activation

• alpha and beta/gamma subunits close together = inactive G protein = high BRET

• alpha and beta/gamma subunits far away = active G protein = low BRET

general tools for structural biology

1. x-ray crystallography

2. nuclear magnetic resonance 

3. cryogenic electron microscope

explain vapor diffusion technique

a mixture of precipitate (e.g., ammonium sulfate, isopropanol) and sample is placed in a sealed chamber above a large volume of more precipitate

• used for crystallizing proteins

x-ray diffraction

changes in direction of x-ray beam due to interactions with electrons around atom

when was the first structure of GPCR resolved

2000

when was the first human gpcr structure resolved

2007

when was the first GPCR-G protein complex structure resolved

2011

when was the first cryo-EM GPCR-G protein complex structure resolved

2017

when was the first GPCR-b-arrestin structure resolved

2015

when was the first GPCR-GRK structure resolved

2022

The conserved structural feature of GPCR DRY-E motif between TM3 and TM6

ionic lock, important for g protein coupling and receptor activation

advantage of cryo-em

1. can image large/dynamic molecular complexes

2. preserve native-like functional state

3. need less protein

4. no crystallization

5. captures different conformational states

disadvantage of cryo-em

1. resolution limitation

2. computational demands

3. sample homogeneity

4. small protein inefficiency

5. specilaized equipment

advantage of smFRET

1. high resolution

2. direct observation of dynamics

3. quantitative measurements

4. only need small amount of sample

5. can preform in-cell measurement 

disadvantage smFRET (single molecule fluorescence resonance energy transfer)

1. limited dynamic range

2. distance innacuracy from moving fluorophores

3. big fluorophores cause function issue in protein

4. photobleaching and blinking, can be caused by nearby F and Y residues

5. data analysis can be biased

explain FUAA

• used to explore intermediate states

1. orthogonal aminoacyl tRNA synthetase adds the FUAA onto orthogonal tRNA

2. the amber codon (TAG) allows FUAA to be incorporated into the protein

• amber codon is used since TAG is least used stop codon, less likely to cause widespread negative issues with translation

5 challenges in current gpcr study and drug discovery

1. correlation between conformational states and downstream signaling partners/pathways to design conformation-specific drugs. maybe use FUAA or use BRET and PRESTO as solution

2. some methods used in drug discovery cant distinguish between the different beta and gamma subunits. maybe can be solved using biased signaling

3. for them to be studied, membrane proteins like gpcr need micelles to mimic natural enviornment or else they will unravel or denature

4. dynamic proteins make it difficult to resolve all amino acids. can use NMR to do this

5. issue with latest version of BRET 2012: light scattering in tissue. issue could be fixed using near-infrared probes

The conserved structural feature of GPCR npxxy motif in tm7

important for receptor stability and activation

transducer

molecules that convert the extracellular signal to an intracellular signal allowing an intracellular signaling cascade to occur

binder

a ligand that binds to its receptor

full agonist

Ligands that elicit maximal signaling

Partial agonist

Ligands that elicit submaximal signaling

Antagonist

Ligands that bind the receptor but do not affect constitutive receptor activity

Inverse agonist

Ligands that inhibit constitutive receptor signaling

IC50

the concentration of any bioactive compound that inhibits a response by 50%

Ca2+ Mobility

Activation of GPCRs is known to trigger a cascade of events that lead to the modulation of cytosolic Ca²⁺ dynamic.

biased signaling

is a ligand-based signaling preference observed when multiple signal pathway co-exit in a signaling process.

Biased ligand

compounds that promote selective activation of specific signaling pathways in GPCR

EFRET

varies as the inverse sixth power of the distance between the two molecules

fret efficiency

phage and yeast display

involves displaying peptides or proteins on the surface of bacteriophages to study the stability and affinity of proteins

smFRET

Is a biophysical technique that uses two fluorescent dyes to measure the distance and conformational changes between molecules or within a single molecule.

DEER

(double electron-electron resonance) is a pulse derived from EPR technique that measure distances between two electron spins in the nanometer range, often in spin-label biomolecules.

orthosteric site

or the binding pocket

used to measure gpcr conformational changes

BRET and NMR