PHCL 4001 Exam 1 Review Flashcards
What second messenger acts as a low glucose sensor in the regulation of the lac operon?
A. cAMP
B. Ca2+
C. Phospholipids
D. cGMP
A. cAMP (Correct)
You are working as part of a research group to develop a new generation of cyclooxygenase-targeting small molecules for the treatment of inflammatory pain. You’ve just received the results of a saturation radioligand binding study. Compound UMN567 has a KD of 500 nM. Compound UMN784 has a KD of 10 nM. You want to test the compound with the highest affinity for the target in your animal model. Which compound should you choose?
A. UMN567
B. UMN784
B. UMN784 (Correct)
Pharmacology
The study of the effect of chemical substances on the function of living systems
Drugs
A chemical substance, typically of known structure, which, when administered to a living organism, produces a biological effect
Pharmacodynamics
what the drug does to the body
the study of the biochemical, physiological, and molecular effects of drugs on the body
(mechanisms of action)
Pharmacokinetics
What the body does to the drug
The study of the movement of drugs in and out of the body and specific tissues
(absorption, distribution, metabolism, excretion)
Mid-19th century
pharmacology as a scientific discipline was born
- morphine to opium
- cell theory
- structural formulas, chemistry
- bacteria as cause of disease by Louis Pasteur
20th century
synthetic chemistry began to revolutionize the pharmaceutical industry
new synthetic drugs were made
new area of antimicrobial chemotherapy:
- treat syphillis
- first antibacterial drugs
- florey and chain develop penicillin
Following events in chronological order from earliest to most recent:
- The earth is formed
- First written accounts of herbal medicine came from China and Egypt
- Cell theory is popularized by Rudolf Virchow
- Louis Pasteur links bacteria with disease
- Florey and Chain develop penicillin as an antibiotic
- End of World War II
How is the cell cycle regulated
There are 3 cell cycle checkpoints. The major checkpoint in mammalian cells is at the G1-S boundary.
The machinery that controls this transition (G1-S checkpoint) are
cyclin-dependent kinases (Cdks)
Cyclin-dependent kinases (Cdks) are regulated by
1) cyclins,
2) activating phosphates,
3) inhibitory phosphates,
4) phosphatases that remove inhibitory phosphates, and
5) CKIs
cell cycle
G1 phase
S phase
G2 phase
M phase
M phase
Prophase
prometaphase
metaphase
anaphase
telophase
cytokinesis
List what needs to ‘go wrong’ for a cancer to develop.
1) Inappropriate expression of proteins that drive cell cycle progression (oncogenes)
2) A loss of function of proteins that inhibit cell cycle progression (tumor suppressors)
Identify types of proteins that could potentially be targeted in new cancer therapies.
Lots! Use your imagination. p53 (gene therapy to deliver functional copies?), Cdks (hard to get a specificity but there are isoforms), Cdk regulatory proteins
Oncogene
(derived from a proto-oncogene)
a gene that has been inappropriately overexpressed or mutated and promotes progression through the cell cycle
Proto-oncogenes
are genes that cause normal cells to become cancerous when they are mutated. Proto-oncogenes are normal cellular genes that regulate cell growth and differentiation
Tumor suppressor
a gene whose expression inhibits cell cycle progression
Wee1 Kinase (p-o/ts)
(Cdk/cyclin) Active —-> Inactive
tumor suppressor
Cdc25 (p-o/ts)
Inactive —-> Active (Cdk/cyclin)
proto-oncogene
Cdk Inhibitor proteins or CKIs (p-o/ts)
tumor suppressors
p53 (p-o/ts)
tumor suppressor
p53 is
a transcription factor that will lead to the expression of CKI proteins and inhibit Cdks
increases the expression of cyclin-dependent kinase inhibitors (CKIs)
is a tumor suppressor
the protein E2F
- initiates transcription of genes needed to transition to S-phase
- is a transcription factor
- is sequestered by the retinoblastoma protein (Rb)
Put the stages of mitosis in order
prophase
prometaphase
metaphase
anaphase
telophase
Cdc25
is a phosphatase that removes an inhibitory phosphate group from a Cdk-cyclin complex
All mechanisms by which a Cdk can be regulated
- inhibitory phosphates
- activating phosphates
- phosphates that remove inhibitory phosphates
- Cdk inhibitors (CKIs)
- cyclins
The retinoblastoma protein (Rb) family are:
tumor suppressors
Cell cycle in order
G1 Phase
G1-S checkpoint
S phase
G2 phase
G2-M checkpoint
M phase
Mitosis won’t progress until the spindles have attached to the sister chromatids and these chromatids are aligned at the equatorial plate. This checkpoint is called
the metaphase to anaphase transition
Cdk inhibitor proteins (CKIs) are targeted for degradation by:
ubiquitin ligases
The nucleus of a cell is divided into two identical daughter cells during the process of
mitosis
the cytoplasm of a cell is divided into daughter cells during the process of
cytokinesis
The primary cellular machinery that controls the G1-S transition are
cyclin dependent kinases (Cdks)
Explain how gene regulation is related to Pharmacology.
Many disease states, including cancer, are the result of over or under expressed genes. Changing gene expression can be achieved pharmacologically and lead to therapeutic benefits.
Explain how the lactose operon in E. coli is regulated
The lactose (lac) operon is regulated via both positive and negative regulation. E. coli’s preferred energy source is glucose.
When glucose is present (e. coli)
the bacteria don’t transcribe genes involved in the breakdown of less-preferred energy sources, such as lactose.
When glucose is low (e. coli)
When glucose is low, and lactose is present, the bacteria will turn on a set of genes (called an operon) that encode for proteins involved in lactose catabolism.
The lac operon is turn on when (e. coli)
1) glucose is low via an upregulation of cAMP, which binds the CAP protein, initiating an allosteric change that allows it to associate with DNA, bind RNA polymerase, and promote transcription initiation, and
2) when lactose is present via the binding of lactose metabolite to a transcriptional repressor. This binding causes an allosteric change in the repressor that promotes repressor dissociation from the DNA.
Transcription is changed by
regulated recruitment of transcription factors
Turn-on a gene
turning on a transcription factor, recruiting an active polymerase to the promoter to increase transcription
Turn-off a gene
preventing an active polymerase from binding the promoter to initiate transcription
List examples of how turning-on or turning-off genes could be exploited pharmacologically to treat a disease like cancer.
Use your imagination! Turning off a gene that is causing inappropriate cell cycle progression (i.e., oncogene). Turning on a gene that could halt cell cycle progress (i.e., tumor suppressor).
Note that this could be done by targeting transcription factors directly or by targeting them indirectly via the signaling pathways that regulate them.
E. coli’s preferred carbon source is
glucose
The lac operon is turned on when
glucose is low
lactose is present
cAMP is high
adenylyl cyclase is active
What does the lac operon encode?
genes involved in breaking down lactose
Transcription factors are exclusively found in the nucleus
false
most base pairs in the human genome code for proteins
false
regulators of transcription in eukaryotes need to bind within a couple of base pairs of the promoter to effectively change transcription efficiency or rate
false
a shared characteristic of transcription factors (as a class) is that they have
a DNA binding motif
All of the following are DNA binding motifs
Helix-turn-helix (HTH)
leucine zipper
zinc finger
A repressor protein prevents transcription of gene X unless a specific environmental condition is met. this type of regulation is
negative regulation of transcription
How is specificity of gene expression achieved?
regulated recruitment of transcription factors
Name the simplified components of a signaling pathway.
- Signal molecule (first messenger)
- Receptor protein
- Intracellular signaling proteins (second messengers or kinases)
- Target proteins
Endocrine signaling
depends on endocrine cells, which secrete hormones into the bloodstream for distribution throughout the body.
Paracrine signaling
depends on local mediators that are released into the extracellular space and act on neighboring cells.
Autocrine signaling
occurs when a cell secretes a signaling molecule that binds back to its own receptors.
Contact-dependent signaling
requires cells to be in direct membrane–membrane contact.
Synaptic signaling
is performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses
Describe the characteristics of second messengers.
- Small molecules
- Rapidly generated (short half life)
- Generated from readily available sources (e.g., ion gradients, plasma membrane phospholipids)
- Readily reversible (i.e., can be turned off quickly by phosphodiesterases or ion transporters
Select all the second messenger molecules
calcium
cAMP
DAG
IP3
Explain how second messengers are generated (i.e., from what sources, by what mechanism) and inactivated, including examples.
They are generated from readily available sources, including bembrane sources (IP3, DAG), nucleotide sources (cAMP, cGMP), ion gradients (Ca+2, Na+, K+),
They are inactivated by phosphodiesterases (cAMP, cGMP), ion transporters (Ca+2, Na+, K+), enzymatic inactivation (prostaglandins, IP3, diacylglycerol)
A characteristic of second messengers is that they have a long half-life
false
a characteristic of second messengers is that they are rapidly formed from readily available precursors allowing for a quick response
true
Types of signaling
autocrine
contact-dependent
synaptic transmission
endocrine
paracrine
Which second messenger molecule is increased under conditions of low glucose and binds the CAP protein to promote RNA polymerase binding to the lac operon promoter?
cAMP
Which of the following is a mechanism by which second messengers are rapidly inactivated
phosphodiesterases
enzymatic inactivation
ion transporters
c-Myc (a protein in the Myc family) is a
proto-oncogene
in a signaling pathway, there can only by one second messenger
false
Select all of the second messengers that are membrane-derived:
IP3
DAG
List types of receptors and state their location in the cell (e.g., plasma membrane, cytosol).
cell surface receptors
intracellular receptors
nuclear receptor signaling molecules
enzyme linked receptors
Describe how (i.e., via what transducer, involved enzymes, etc.) activation of a G protein-coupled receptor can lead to increases in intracellular calcium.
Activation of GPCR: When a ligand (such as a neurotransmitter, hormone, or other signaling molecule) binds to the extracellular domain of a GPCR, it induces a conformational change in the receptor.
Differentiate allosteric compounds from orthosteric compounds in terms of receptor binding site.
Allosteric compounds and orthosteric compounds interact with receptors via different binding sites and mechanisms:
orthosteric compounds bind to the primary binding site of the receptor and directly compete with the endogenous ligand, while allosteric compounds bind to a different site on the receptor and modulate the receptor’s activity without directly interfering with the orthosteric ligand binding.
hallmarks of cancer
Growth signals
apoptosis
anti-growth signals
limitless replication
angiogenesis
tissue invasion and metastasis
List the key steps in the drug development process.
1) Preclinical
1. invitro and invivo testing
basic research
early discovery
pre clinical
2) clinical
2. Human testing
clinical development
phase 1, 2, 3
- Data review
FDA review - Surveillance
Post market marketing
Name 5 sources of new drugs
natural products
synthetic chemicals
biotechnology
repurposing/repositioning
structure-based in silico docking or screening
natural products
~50%
Ex. Aspirin - derived from salicylic acid found in the bark and leaves of the willow and poplar trees
many are originally derived from microbial sources such as antibiotics
synthetic chemicals
synthetic dyes
coal tar (Ex. acetaminophen)
large chemical libraries
biotechnology
Ex. Human insulin for diabetes
one of the first uses of recombinant DNA technology
Repurposing/repositioning
identify new indications for already approved or already-clinically tested drugs
plays a crucial role in drug development
lowers overall developmental costs by ~$300 million
Ex. Sulfonamides for many indications, viagra for erectile dysfunction
structure-based in silico docking or screening
instead of screening 1 million compounds in a pharmacological assay, in silico screening allows for the assessments of 1 billion compounds, virtually
Ex. of de novo in silico screen to an FDA approval
none
still relatively new
Phase I Clinical trials
20-100 healthy volunteers
purpose: mainly SAFETY (several months)
also involves dosage optimization, measurement of distribution, metabolism and elimination
Phase 2 clinical trials
several hundred volunteer patients, with diagnosis according to target indication
purpose: SAFETY, EFFICACY, dosing (several months)
Phase 3 clinical trials
several hundred to greater than 5,000 patients, with diagnosis according to targeted indication
purpose: SAFETY and EFFICACY (1-4 years)
Describe the drug development process in terms of length, expense, and likelihood of success.
Overall failure rate in drug development is >96%, including a 90% failure rate furing clinical development
mean cost of developing a new drug is between $314 million to 2.8 billion
Which of the follow is a source of new drugs
plants
microbes
repurposing
in silico screening
clothing dyes
coal tar
The process of identifying new indications for already approved or already clinically tested drugs is called
repurposing or repositioning
Which value most closely is approximately the median cost of developing a single anti-cancer agent in the US in 2018.
$800,000,000 (eight hundred million dollars)
Repurposing a new drug for a new indication is usually more expensive than starting a de novo drug discovery and development effort
false
The clinical phase at which the highest percentage of new drugs fail is:
phase II
Based on the percent of new drugs progressing between Phase I, PHase II, and Phase III trials, most new drugs fail because they are not
Effective
The likelihood a new compound starting Phase I trials will eventually gain FDA approval (average across all indications) is:
10%
The average time from target identification to FDA approved therapeutic is:
10-15 years
Once a drug is approved by a regulatory body (ex. FDA), it will remain approved and available indefinitely, regardless of how safe it is
false
Post-marketing surveillance refers to the process in which regulatory bodies monitor the safety of drugs once they reach the market after the successful completion of clinicals. This phase is also known as:
Phase IV
Agonist
preferentially binds (i.e., has a higher affinity for) and stabilizes the active receptor confirmation
Antagonist
binds with equal affinity to the active and inactive receptor
Inverse agonist
preferentially binds (i.e., has a higher affinity for) and stabilizes the inactive receptor conformation
List intermolecular interactions that may contribute to drug binding.
ionic
covalent (some irreversible, like aspirin)
hydrogen bonds
hydrophobic interactions
Explain how radioligand binding can be used to determine a drug’s affinity for a target and be able to look at a specific binding curve and ballpark binding affinity (KD).
Radioligand binding assays are widely used in pharmacology to determine the affinity of a drug for its target receptor
To ballpark the binding affinity (KD) from a specific binding curve, one would typically fit the data to a binding equation (such as the Hill equation for sigmoidal curves or the Michaelis-Menten equation for hyperbolic curves) using nonlinear regression analysis. The KD value can then be estimated from the curve fit parameters.
Additionally, graphical methods such as the Scatchard plot can be used to estimate KD from binding data.
How Radioligand binding assays work
Radioligand Preparation: Synthesize a radiolabeled ligand.
Tissue/Cell Membrane Preparation: Isolate membranes containing the target receptor.
Binding Assay: Incubate radioligand with membranes and varying concentrations of unlabeled compound.
Separation: Separate bound and free ligand.
Radioactivity Measurement: Measure bound radioactivity.
Data Analysis: Plot percentage of specific binding vs. unlabeled compound concentration.
KD Estimation: Determine KD from the binding curve.
Describe what structure-activity relationship studies are and why they are performed.
to predict biological activity from molecular structure. This powerful technology is used in drug discovery to guide the acquisition or synthesis of desirable new compounds, as well as to further characterize existing molecules.
ligand
any molecule that binds a receptor
a drug’s specific binding is determined by adding together (summing) its total and nonspecific binding
false
You can determine whether there is a correlation between derivatives of a drug that bind to the presumptive receptor and a pharmacological effect by performing a
Structure-activity Relationship or SAR study
When performing a saturation radioligand binding assay to measure nonspecific binding, the concentration of non radiolabeled (i.e., ‘cold’) compounds needs to be ______ than the concentration of radiolabeled compound
higher
