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PCL302 > Final Exam > Flashcards

Final Exam Flashcards

1
Q

Type 1 Diabetes

A

loss of insulin production by pancreas

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2
Q

Type 2 Diabetes

A

Resistance to the actions of insulin

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3
Q

5 abnormalities due to insulin deficiency

A
  1. hyperglycemia
  2. increased lipolysis
  3. acidosis
  4. increased LDL; decreased HDL
  5. osmotic diuresis (increased urination)
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4
Q

Insulin replacement therapy

A

insulin associates as hexamer

rate limiting step for capillary absorption is the dissociation of hexamers into dimer and then monomers

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5
Q

5 different types of insulin

A
  1. ultra fast/ short acting: lispro
  2. short acting: regular
  3. intermediate acting: NPH
  4. long acting: lente /ultra lente
  5. ultra long acting: glargine
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6
Q

2 insulin treatment regimes

A
  1. Intensive insulin treatment (frequent monitoring of blood glucose, 3 or more daily injections of insulin)
  2. Continuous subcutaneous insulin infusion (insulin pump with lispro or regular insulin, allows control of dawn phenomenon, patient triggered bolus before meals)
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7
Q

4 adverse effects of insulin therapy

A
  1. hypoglycemia (especially dangerous in Type 1 diabetes)
  2. allergy and resistance to insulin
  3. lipohypertrophy
  4. lipoatrophy (due to impurities: switch to highly purified insulin)
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8
Q

Insulin resistance is due to?

A

decrease insulin receptor number
decrease insulin receptor kinase activity
post receptor defects
decrease GLUT4 translocation from impaired signalling (less glucose can be transported into cell)

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9
Q

Impaired islet function is due to?

A

loss of first phase insulin secretion

increase secretion of proinsulin

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10
Q

Treatment of type 2 diabetes

A
  1. diet and exercise: most effective, increase insulin sensitivity
  2. monotherapy with oral agent
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11
Q

Sulfonylureas (Glyburide)

A

Most effective when B cell function has not been severely compromised
Increased insulin secretion favors lipogenesis

inhibit voltage gated K channel -> activates voltage gated Ca channel -> more intracellular calcium -> cause insulin secretion

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12
Q

Repaglinide and Nateglinide

A

decrease ATP-sensitive K+ conductance
similar to sulfonylureas
maybe more specific for B-cell sulfonylurea receptor (fewer off targets effects)
action maybe glucose dependent
insulin release is rapid and brief
taken with meals for postprandial hyperglycemia

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12
Q

Repaglinide and Nateglinide

A

decrease ATP-sensitive K+ conductance
similar to sulfonylureas
maybe more specific for B-cell sulfonylurea receptor (fewer off targets effects)
action maybe glucose dependent
insulin release is rapid and brief
taken with meals for postprandial hyperglycemia

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13
Q

Biguanides (Metformin)

A

antihyperglycemic
inhibit gluconeogenesis
inhibit glucose-6-phosphatase activity -> glycogen sparing
decrease insulin resistance
mediated by activation of 5’AMP-activated protein kinase (AMPK) in hepatocytes and muscle
DO NOT INCREASE INSULIN SECRETION
NOT HYPOGLYCEMIC EVEN AT HIGH DOSES

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14
Q

Thiazolidinedione (Glitazones)

A

Activates peroxisome proliferator-activated receptor Y
increase adipose tissue -> weight gain

decrease TNFa
decrease leptin
increase lipoprotein lipase
increase GLUT4 and GLUT 1

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15
Q

a glucosidase inhibitors (Acarbose)

A

competitive and reversible inhibitors of a glucosidase
delay carbohydrate digestion and absorption
smaller rise in postprandial glucose

taken with first bit of meal
DO NOT PRODUCE HYPOGLYCEMIA, LACTIC ACIDOSIS or WEIGHT GAIN

effective regardless of age, genetic factor, body weight, duration or severity of the disease

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16
Q

PDGF receptors (platelet-derived growth factor)

A

activation: open c terminal loop, phosphorylation
involved in wound healing
released from activated platelets by degranulation
stimulates fibroblast migration, chemotactic and angiogenic
plays a role in arteriosclerosis (overactivity of PDGF)
blocking antibodies have been developed (problem: too much blockade will end up with bleeding disorder, no healing)

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17
Q

3 drugs that bind covalently to enzymes

A
  1. Acetylsalicylic acid (ASA, aspirin)
  2. Penicillin
  3. Phenelzine
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18
Q

Aspirin

A

irreversibly inactivates COX, inhibiting prostaglandin production
inflammation: due to prostaglandin E and F
ASA acetylates both COX1 and COX2, but COX2 is more important because it is inducible by inflammatory cytokines

antithrombotic: inhibition of COX1 leads to antithrombotic effect (prolonged bleeding)
analgesic: inhibition of prostaglandin synthesis decrease perception of pain
antipyretic: decrease prostaglandin synthesis in hypothalamus induce vasodilation and sweating, leads to decrease body temperature

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19
Q

COX2 selectivity for ASA

A

structural difference between the substrate binding channels in COX1 and 2
VAL434, ARG513, VAL532 side pocket absent in COX1

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20
Q

Penicillin

A

inhibit cross wall formation in bacteria -> abnormally elongated cells -> rupture and cell death

Transpeptidase cleaves terminal D-ala and connects it to m-dap, which is inhibited by penicillin

B-lactamase (decoy) give penicillin resistance to pathogenic bacteria

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21
Q

Phenelzine (Nardil)

A

antidepressant agent
mediates by 5-hydroxytryptamine (5-HT) and catecholamines (norepinephrine and dopamine)

inhibits monoamine oxidase (MAO) 
MAO has 2 isoforms A and B
MAO A: 5-HT and norepinephrine
MAO B: phenylethylamine
MAO-A is more important for antidepressant effects

contains a hydrazine moiety similar to substrates of MAO
inactivates enzyme when covalently binds

non-selective MAO inhibitor
requires several weeks for onset for activity
food restriction for tyramine containing (cheese, red wine, beer) -> leads to hypertension

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22
Q

3 drugs that bind reversibly to enzymes

A
  1. Ramipril
  2. Sulfonamide
  3. Neostigmine
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23
Q

Ramipril

A

inhibit ACE enzyme
blood pressure control and congestive heart failure
reduce formation of angiotensin II, decrease blood pressure

adverse effect inhibit bradykinin metabolism, dry cough, throat irritation, dizziness, itching

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24
Q

Sulfonamide (antibiotic)

A

Interfere microbial folic acid biosynthesis
competes with para-aminobenzoic acid (PABA) substrate for dihydropteroate synthase (DHPS)
use in treatment of bacterial infection
resistance developed by overproduction of PABA or structural change to DHP enzyme

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25
Q

Neostigmine

A

muscle stimulant
inhibit hydrolysis of acetylcholine so acetylcholine cannot be metabolized
will release eventually

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26
Q

3 drug that mimic enzyme substrate (pseudosubstrate)

A

Zidovudine (AZT)
Acycloguanosine
Cytosine Arabinoside

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27
Q

Zidovudine (Retrovir, Azidothymidine AZT)

A

first approved drug for HIV treatment
nucleotide reverse transcriptase inhibitors (NRTIs)
site of action at RNA-directed DNA polymerase (reverse transcriptase) enzyme

inhibits reverse transcriptase in virus
lacks 3’ oxygen, cause DNA chain termination when incorporated into a growing DNA strand

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28
Q

Highly active anti-viral therapy (HAAT)

A

combinations of two or three drugs of different classes
AZT or another nucleoside mimics & non-nucleoside reverse transcriptase inhibitors
entry inhibitors = prevent virion from attaching to host cell membrane
integrate inhibitors = stop DNA incorporation into host DNA

decrease viral load
not curable of HIV but can improve quality of life

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29
Q

Acycloguanosine (acyclovir)

A

topical antiviral drug
treat symptoms of herpes simplex virus infections of the skin, mucous membrane and genitals
mimics deoxy guanosine

viral and host kinase convert acyclovir into Acycloguanosine triphosphate and used by viral DNA polymerase.
no tertiary hydroxyl group prevent binding of next nucleotide

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30
Q

Cytosine Arabinoside (cytarabine, araC)

A

pyrimidine analogue
cytotoxic
incorporation of AraCTP into DNA
mimics deoxycytidine

inhibit DNA polymerase activity, block DNA synthesis
inhibit topoisomerase I results in DNA supercoiling during synthesis
OH group modifies DNA structure inhibition of transcription factor binding to DNA

used in treatment of leukemia

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31
Q

3 types of natriuretic peptide

A

A type: 28 amino acid, secreted by myocyte in atria
B type: 32 amino acid, secreted by myocyte in ventricle
C type: 22 amino acid, secreted by vascular cells

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32
Q

3 effects of increased cGMP

A
  1. increase natriuresis (urination)
  2. decrease peripheral vascular pressure (decrease blood pressure)
  3. suppress renin-angiotensin
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33
Q

Mechanism of natriuresis (ANP)

A

ANP increase glomerular filtration
ANP stimulate phosphorylation of ENac (epithelial sodium channel) blocking Na reuptake
net increase excretion of water and sodium

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34
Q

Mechanism of muscle relaxation by Nitric oxide on soluble guanylyl cyclase

A

NO binds -> cGMP production -> cGMP binds to PKG -> PKG phosphorylates IP3 and L-type Ca channel -> decrease [Ca]
-> decrease MLCK activity -> decrease muscle contraction

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35
Q

Half life of nitroglycerin, isosorbide dinitrate and isosorbide mononitrate

A

Nitroglycerin: acute treatment, 2min
Isosorbide dinitrate: prophylactic (long-lasting) treatment, 1 hour
Isosorbide mononitrate: prophylactic treatment, 5 hours

release of NO (rapid dilation of blood vessels)

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36
Q

PDE inhibitor Methylxanthines

A 
increased cortical arousal, more alert and deferral of fatigue
myocardial contraction
relaxation of smooth muscle
increase gastric secretion
increase digestive enzymes
BRONCHODILATION
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37
Q

Theophylline

A 
treats asthma
bronchodilator
agonist, inhibit PDE from turning cAMP to AMP
decrease Ca
smooth muscle relaxation
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38
Q

Rolipram

A

PDE4 inhibitor (cAMP selective)
treats depression
nausea GI disturbance and emesis

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39
Q

Roflumilast

A

PDE4 inhibitor
used in chronic obstructive pulmonary disease (COPD)
still upsets GI

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40
Q

Sildenafil (Viagra) and Tadalafil (longer halftime)

A

PDE5 inhibitor (cGMP specific)
inhibit breakdown of cGMP to GMP
higher cGMP concentration leads to muscle relaxation
leads to increase blood flow to corpus cavernosum of penis
erection restore

HYPOTENSIVE SHOCK if used with NITROGLYCERINE

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41
Q

Ligand binding domain of nuclear receptors

A

11-13 a helices arrange into a three layer antiparallel a-helical sandwich

long helices 3,7 and 10 form two outer layers

4,5,8,9 create ligand binding pockets

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42
Q

Agonists function of nuclear receptors

A

stabilize nuclear receptor formation and maintaining AF-2 helix 12 in the active conformation

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43
Q

Ligand binding pocket specificity is determined by?

A
  1. shape
  2. size
  3. plasticity
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44
Q

Ligand binding pocket specificity is determined by?

A
  1. shape
  2. size
  3. plasticity
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45
Q

Ligand binding of nuclear receptor triggers ? (6 steps)

A
  1. conformational change in LBD
  2. dismissal of corepressor
  3. recruitment of ubiquitylation leads to corepressor degradation
  4. enhanced affinity for co-activators
  5. recruit co-activator complex
  6. activation of gene transcription
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46
Q

Zinc finger domain in DNA binding domain (DBD)

A

P-box (proximal) domain: DNA binding in major groove
D-box (distal) domain: NR dimerization

nuclear receptors can bind to DNA as monomer, homodimers and heterodimers

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47
Q

Estrogen receptors

A

Type 1
activates gene containing estrogen response element (ERE)
can function as homodimers (a-a, b-b) or heterodimers (a-b)

ERa: breast, endometrial, ovarian and hypothalamus tissue (sex organs)
ERb: brain, bone, endothelial, heart, intestine, kidney, lung and prostate tissue (non-sex organs)
have overlapping and non-overlapping function, can oppose each other

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48
Q

What is the natural ligand of estrogen receptor?

A 
17B- estradiol (estrogen)
agonist
induce puberty
derived from cholesterol
primarily produced in ovaries
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49
Q

Estrogen deficiency

A

menopause
ovarian insufficiency
hyperprolactinemia (elevated prolactin to enhance breast development during pregnancy and lactation)

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50
Q

Elevated estrogen level

A 
weight gain
liver toxicity
breast hypertrophy
thrombosis
increase cancer risk
endometrial cancer
breast cancer
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51
Q

Tamoxifen

A

SERMs (selective estrogen receptor modulators)
derived from diethylstilbestrol
metabolized in liver by CYP2D6 and CYP3A4 into hydroxytamoxifen and N-desmethyl-4-hydroxytamoxifen
both have higher affinity for ER than tamoxifen

52
Q

Tamoxifen is a mixed agonist/antagonist in what tissue?

A

Breast tissue: NCOR or SMRT corepressor highly expressed, antagonistic
Uterine tissue: SRC1 coactivator highly expressed, agonistic

53
Q

Androgen Receptors

A

Steroid or Type 1 Receptors on X chromosome
2 isoforms, ARa and ARb

Natural ligands: 5a dihydrotestosterone and testosterone

54
Q

Androgen receptors DNA binding conformation

A

Head to head homodimer
5’-AGAACAnnnAGAACA-3’
most stable conformation to the common hexameric “half site” DR3

55
Q

Prostate cancer initiation

A

Overexpression of TMPRSS2-ERG (fusion protein) leading to cell cycle progression and overexpression of ETS transcription factor

56
Q

Prostate cancer therapy

A 
expectant management or active surveillance
surgery
radiation
cryosurgery
HORMONE therapy
chemotherapy
vaccine treatment
bone-directed treatment
57
Q

Androgen Deprivation Therapy (ADT)

A 
tumor growth is initially androgen dependent
androgen ablation (block the production of androgen) to cause regression of androgen-dependent tumors

Many men eventually fail this therapy and progress onto AIPC (androgen independent prostate cancer)
also called castration resistant prostate cancer (CRPC)

58
Q

castration resistant prostate cancer (CRPC) treatment

A

development is inevitable
lethal
no longer respond to first-line androgen deprivation therapy
treat with CHEMOTHERAPY and anti-mitotic compound DOCETAXEL

59
Q

4 possible mechanism for CPRC

A
  1. increased sensitivity of androgen receptors to its agonist
  2. AR mutation that render the receptor responsive to alternate non-androgen ligands
  3. ligand-independent AR activation (activation by kinase, phosphorylation)
  4. AR-independent mechanism (direct activation of TMPRESS2-ERG)
60
Q

Retinoic acid receptors

A

Type 2 nuclear receptor
constant partner RXR and RAR
already binds to DNA

61
Q

Acute Myeloid Leukemia (AML)

A

aggressive cancer of bone marrow affecting hematopoiesis
rapid accumulation of immature myeloid progenitors (leukemic blasts)
cytarabine and daunorubicin chemotherapy

62
Q

Acute Promyelocytic Leukemia (APL)

A

Incidence highest in young adults
Presentation
Pancytopenia (Anemia, Neutropenia, Thrombocytopenia)
Coagulopathy

Promyelocytes in peripheral blood

most malignant form of AML
immediately start treatment even with suspicion

APL is now associated with the highest proportion of AML patients who are cured of their disease

63
Q

APL pathogenesis

A

Translocation of retinoic acid receptor alpha

95% PML-RARa -t(15;17)

64
Q

PML/RARa protein

A 
dominant negative mutant
forms homodimer (normally form heterodimer)
repress target genes
less responsive to agonist
binds strongly to corepressors
65
Q

ATRA (all-trans retinoic acid)

A

induce terminal myeloid differentiation
but cannot prevent the occurrence of malignant transformation in myeloid progenitor cells

combine with chemotherapy

Mechanism:

  1. bind ATRA to RAR cause ubiquitylation of PML-RARa protein
  2. CoR is dissociated from repressive complex and CoA is recruited.
  3. differentiation of promyelocytes is restored
66
Q

Arsenic Trioxide (As2O3)

A

cause degradation of PML-RARa, promotes differentiation
APL cells extremely sensitive to arsenic trioxide
increase chance of survival with ATRA admission

67
Q

Why choose DNA as drug targets?

A
  1. to a kill a cell by inducing DNA damage and apoptosis

2. to insert delete or edit aberrant genetic information causing disease

68
Q

DNA-damage response proteins (DDR)

A

p53 activation
cell cycle arrest
apoptosis

selectivity achieved as cancerous cells proliferate faster than healthy cells

69
Q

Cyclophosphamide

A

oral prodrug converted by CYP450 to active mustard
forms covalent purine (G) N7 adducts and N7-N7 crosslinks like cisplatin (not a prodrug delivered in active form)

treat cancers

side effects: teratogenic, immune suppression, alopecia, nausea, bladder and liver toxicity and cancer (induce mutation in DNA)

70
Q

Doxorubicin and Actinomycin D

A

form covalent interactions with bases on opposing strands leading to inhibition of Topoisomerase I and replication stall

treat tumors
side effects : cancer (block repair machinery)

71
Q

Gemcitabine

A

transported via SLC nucleoside transporters and gets phosphorylated (prodrug)
resembles deoxycytidine triphosphate

chain termination
treat leukemia and lung cancer

72
Q

Acyclovir and Molnupiravir

A

block elongation of genetic DNA/RNA of virus, halts replication of host cell

prodrug: base is converted into nucleotide by viral kinase
inhibits HSV1/HSV2 herpes

73
Q

Acyclovir and Molnupiravir

A

block elongation of genetic DNA/RNA of virus, halts replication of host cell

prodrug: base is converted into nucleotide by viral kinase
inhibits HSV1/HSV2 herpes

74
Q

Gene augmentation

A

monogenic
copy of gene without mutation is cloned into a DNA plasmid
insert into cells
production of functional protein

if in somatic cell its called
somatic gene replacement therapy (SCRT)

Cystic fibrosis
Duchenne muscle dystrophy
hemophilia
Huntington's
Rett syndrome
75
Q

Issue with delivering DNA into cells

A

small molecules that are hydrophobic can readily diffuse cell membrane
DNA Is impermeable to cell due to size and charge

76
Q

Electroporation

A

use electrical charge to physically poke holes in cell membrane
no size limitation but organ aren’t accessible (ex vivo, because electrical charge maybe interrupt heart)

77
Q

Gene gun

A

fires micro-projectiles containing DNA conjugated to heavy metal into cells
usually for plants

78
Q

Problem with electroporation and gene gun

A

DNA still have to be transported to the correct compartment
random site integration
tissue must still be accessible

79
Q

Chemical transfection of DNA into cells

A

Cationic lipid: interacts with negative charge on DNA, mixed with neutral lipids to form liposome
no size limitation but rapidly eliminated

Cationic polymers: cationic head interacts with DNA, forms condensed aggregates that stabilize DNA, can be attached to cell permeable peptides
no size limitation

80
Q

Viral transduction of DNA into cells

A

Virus for in vivo modification
tropism: can be targeted to specific tissues
replication defective
replication occur in helper plasmid containing coding trans elements

81
Q

Lentiviral vectors

A 
RNA viruses
Key proteins:
GAG: capsid
POL: protease, reverse transcriptase, integrase
ENV: envelope glycoprotein
TAT: nuclear import, transcription
REV: nuclear export of unspliced RNA

enter via receptor mediated endocytosis
can infect non-dividing cells because TAT can bypass nuclear
most virus can only infect dividing cells because during division there is no nucleus so can access DNA directly

disadvantages: random integration, LIMITS ON SIZE (5kB)

82
Q

AAV vectors

A

Two ORFS:
REP: regulatory proteins
CAP: encodes structural proteins/capsid
gene of interest insert between ITRs

enter via receptor mediated endocytosis
can deliver to nucleus

LIMITS ON SIZE (5kB)

83
Q

ADA-SCID “Boy in the Bubble”

A

Adenosine deaminase deficiency, severe combined immunodeficiency) “Boy in bubble”
ADA catalyze conversion of adenosine to nucleoside inosine
impair lymphocyte development
immune system severely compromised

Treatment: ex vivo viral transduction of ADA gene into isolated bone marrow and lymphocytes that are reinjected

84
Q

Hemophilia A

A

blood clotting disorder
mutations in factor 8 (85%) or factor 9 (15%)
internal or external bleeding
X-linked recessive

treatment: in vivo injection of pseudotyped AAV (target liver cells) containing F9 into blood

85
Q

OTC deficiency

A

drawbacks of gene therapy delivery tools

  1. possible immunogenicity
  2. random insertion into genome

ornithine transcarbamylase deficiency: inability to metabolize ammonia

injected adenovirus carrying OTC gene
died of immune reaction to virus

86
Q

Gene editing

A

advantages: can mimic healthy endogenous gene exactly, permanent cure
disadvantages: irreversibility, off target effects, stimulation of DNA damage (p53) when cutting DNA, immunogenicity

87
Q

Zinc finger domains (ZF)

A

recognize triplets

can be linked linearly to form arrays to target larger sequences ( 3 ZFs target 9 bp)

88
Q

Zinc finger nucleases (ZFNs)

A

two zinc finger arrays linked to subunits of the heterodimeric FOK1 endonucleases

FOK1 only active when heterodimer is formed

ZFNs bind to left and right sequences (Part A and Part B)

89
Q

TALEs

A

transcription activator like effector

each TALE DOMAIN binds 1 bp of DNA can be linked to form arrays that target longer sequence

amino acids at position 12 and 13 of each TALE domain dictates which specific base is bound

amino acids 12 and 13 can be mutated to fit certain DNA sequence

90
Q

TALE nucleases (TALENs)

A

Two TALE arrays linked to subunit of the heterodimeric FOK1 endonucleases

assembly cloning can be difficult

91
Q

CRISPR/Cas9 4 step sequence

A

part of the prokaryotic immune system that defends against bacteriophages

  1. invasion
  2. adaptation
  3. production
  4. targeting
92
Q

CRISPR/Cas9 mechanism

A
  1. Cas9 (endonuclease)
  2. crRNA contains 20 bp used to target
  3. trans-activating crRNA (tracrRNA)

Cas9 displays PAM sequence (‘5-NGG-3’) N=ACG or T
crRNA + tracrRNA = guide RNA

  1. Cas9 binds to PAM
  2. Cas9 uses 20 bp in the crRNA to recognize a match with DNA target
  3. If match, Cas9 activates its two nucleases which cut the top and bottom strands (double strand break)
93
Q

2 DNA repair mechanisms

A
  1. non-homologous end joining (NHEJ): inactivation/knockout

2. homologous recombination (HR)/ homology directed repair (HDR): knock in or gene editing by supplying repair DNA

94
Q

Homology directed repair (HDR) and PRIME editing

A

in addition to Cas9 and gRNA, supply a single stranded oligodeoxynucleotide with homology arms flanking the cut site for repair

PRIME editing: uses nCas9-RT (single strand break) and pegRNA (special type of guide RNA) that contains a template for RT which is used to fill in information repaired using exogenous enzymes

95
Q

Base editing

A

dCas9 (dead Cas9) fused to a base modifying enzyme (cytidine deaminase)
endogenous DNA repair recognize damage base and repair it leading to gene conversion

dCas9 is catalytically inactive but still binds DNA

96
Q

CRISPRi (inactivation) and gene silencing

A

dCas9 attached to transcriptional repressor domain such as KRAB (repressor)

  1. dCas9 targets and effector to a specific gene promoter
  2. blocks transcription or recruit other factors to turn gene off
97
Q

Gene activation with CRISPRa

A

dCas9 attached to transcriptional activation domain such as VP64 (activator)

  1. dCas9 targets gene promoter (TATA box)
  2. recruit and stabilize transcription factor
98
Q

Epigenome editing

A

dCas9 attached to DNA or histone modifying enzyme that induce epigenetic change (HERITABLE)

  1. dCas9 targets gene promoter
  2. cause activation or deactivation through methylation or acetylation

dCas9-p300 (histone acetyltransferase): activates gene
dCas9-Tet1 (DNA demethylase): activates gene
dCas9-Dnmt (DNA methyltransferase): silence gene

99
Q

Ribonucleoprotein complex (RNP)

A

CRISPR/Cas9 can be delivered as RNP using a mixture of Cas9 protein and guide RNA in CATIONIC LIPID

can also be delivered using DNA PLASMID

100
Q

Transthyretin amyloidosis (TTRA)

A

mutation of TTR gene made by liver
in vivo editing using lipid nanoparticle carrying mRNA encoding CRISPR/Cas9 and guide RNA targeting TTR
lipid goes straight to liver

knockout gene in liver decreased serum TTR production by mean of 50%

101
Q

CAR-T cells

A

chimeric antigen receptor T-cell composed of extracellular antibody (scFv) domain artificially attached to intracellular T cell activation domain

editing T cell domain by CRISPR/Cas9

ex vivo

potential gene therapy to target cancer (lymphoma and myelomas)

102
Q

CCR5 germline KO

A

CCR5 (HIV co-receptor) edits at embryonic stage

103
Q

DNA antisense oligonucleotides (ASOs)

A

single stranded DNA that is complementary to a sequence contained within the gene being targeted (RNA)

enter nucleus and binds and targets pre-mRNA

generates a RNA-DNA heteroduplex

recognized by RNase H1 and targeted for degradation

104
Q

ASO steric blockers

A
  1. block translation of mRNA
  2. alter splicing of pre-mRNA
  3. block adenylation (stability)

These ASOs are typically RNA or XNA based
do not activate RNase H

105
Q

ASO drugs

A

enhance stability, delivery and efficacy

Kynmaro (Mipomersen): treat familiar hypercholesteremia, binds to mRNA of ApoB-100 (fat carrier) and initiates destruction of RNase H1

Vyondys (Golodirsen): treat DMD, works by inducing exon skipping, defective form of gene is not produced

106
Q

RNAi (interference) - siRNA pathway

A

siRNA pathway
immune defense against double stranded RNA (virus)

siRNA is composed of guide and passenger strand

binds to RISC complex and directs slicing activity of Argonaute 2 (AGO2) target transcription or block

if match perfect = slice
if match imperfect = translational inhibition

107
Q

RNAi - miRNA pathway

A

Pri-miRNAs cleaved by Drosha (enzyme)
contain hairpin structure
processed by Dicer to form 19-25 nt ds RNAs

binds to RISC/AGO complex

often partially complementary to target, act by binding to 3’UTR and blocking translation of multiple targets

108
Q

Delivery of small RNAs into cells

A

Viral vectors: Lentivirus, adenovirus and AAV

Non-viral vectors: cationic polymer (PEI, cyclodextrins) or cationic lipids (DOTMA)

109
Q

RNAi/miRNA Drugs

A

mi(micro)RNA perfect for multigenic disease such as cancer
si(small interfering)RNA for single gene (hemophilia)

Onpapttro siRNA based drug, treats TTRA

110
Q

CRISPR/Cas 13

A 
class II CRISPR system
uses a single guide RNA and cleaves ssRNA but not dsDNA

contain 2 Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, both RNase domains required for cleavage

Cas13 exhibits collateral RNA cleavage

111
Q

Mechanism for Cas13 RNA cleavage

A
  1. binds to RNA and scans for protospacer flanking sequence (PFS) a non G-base
  2. test for annealing of 30 bp sequence to identify target RNA
  3. If match found, HEPN domains activates and cleaves sequence
  4. domain remains in active conformation and subsequently initiate trans cleavage, only observed in bacteria and in vitro
112
Q

RNA base editing using dCas13

A 
one or both HEPN domains are inactivated through mutation
fusion of dCas13b to deaminase domain of ADAR2 enzyme
purposely mismatch (A-C) to allow for deamination changing A to I which is actually G during translation
113
Q

RNA methylation editing using dCas13

A

m6A catalyzed by METTL3/14 complex transfer of Me from SAM
modification alters RNA splicing, stability, export and translation efficiency
fusion of dCas13 to enzyme variants M3-M314 yield site specific m6A

114
Q

Cas7-11

A

class 1 CRISPR system buy individual proteins are fused together (Cas7-11)

No PFS sequence and no collateral cleavage activity (trans)

No HEPN domains

114
Q

Next generation nucleic acid vaccines

A

goal is to directly target APC cells so that proteins are expressed on their surface

115
Q

SARS-COV-2 vaccines

A

ss(single stranded)RNA virus: use an RNA-dependent RNA-polymerase for replication

Spike glycoprotein binds to ACE-2 receptors on lung epithelial cells

Pfizer/Moderna: uses lipid nanoparticles to package modified RNA(pseudouridine)(less reactive to immune system) encoding
SPIKE PROTEIN

targets dendritic cells, present spike protein to B and T-cells to elicit immune response

116
Q

Difference between Gram + and Gram negative bacteria

A

gram negative have 2 membrane while gram positive only have 1 thick peptidoglycan layer (membrane)

117
Q

Penicillin antibiotic resistance

A

bacteria evolved thicker outer membrane so penicillin harder to penetrate

acquisition of penicillinase gene (breaks B-lactam ring to inactive penicillin)

118
Q

Polymyxins (antibacterials)

A

amphipathic peptides attached to fatty acids- able to insert themselves into membrane

mechanism: directly binds to LPS (lipopolysaccharide) in outer cell membrane which leads to holes in membrane, leakage of cellular contents

BACTERIOCIDAL

119
Q

Vancomycin (antibacterial)

A

Delivered intravenously

treatment for complicated skin infections, blood infections and meningitis

inhibit cell wall synthesis by directly binding to D-ALA-D-ALA precursors

prevent incorporation into growing peptide chain of cell wall, halts elongation and cross linking of peptidoglycan matrix

RESISTANCE: when bacteria change the precursor

120
Q

Nystatin (Antifungals)

A

useful for candida infections such as thrush

not orally bioavailable

useful for topical and GI infections (not absorbed)

mechanism: binds to a fungal specific sterol called ergosterol in fungal cell membrane and cause leakage

121
Q

Docosanol (antivirals)

A

used topically, poor oral bioavailability

mechanism: inhibit fusion of viral envelope with the host cell membrane; directly inserts into human cell membrane to protect from diffusion of virus

prevents viral replication

122
Q

LJ-001 (Antivirals)

A

Blocks viral entry into host cell (after virus binding but before virus cell fusion)

poor stability

Mechanism: directly intercalates into viral membrane in irreversible manner, blocking fusion, only work if envelope is present

ONLY WORK ON ENVELOPED VIRUS such as HIV, influenza, filoviruses

123
Q

Osmotic laxatives

A

treat constipation and digestive conditions

natural inorganic salts: MG-CITRATE which get partially absorbed or synthetic compounds that increase ions but aren’t absorbed (PEG 3350, lactulose)

Mechanism: Delivered into intestine orally, pulls water from surrounding tissue into intestine via OSMOSIS (from high to low), softer stools

124
Q

Bulk-forming laxatives (Methylcellulose, Citrucel, psyllium fiber)

A

treat constipation

involve inert natural (psyllium fiber) or synthetic (polycarbophil) polymers that do not get absorbed but pass through digestive system

Mechanism: delivered into intestine orally, polymers absorb water creating a bulkier, liquid like stool that’s easier to pass

125
Q

pH-modifiers

A

a) induce physiological effects in the stomach
b) re-absorption of drugs and mineral in the urine

Calcium carbonate (TUMS) or Magnesium hydroxide (milk of magnesia), neutralize H+ in stomach and increase pH

K-Phos (potassium acid phosphate) urine acidifier to keep calcium solubilized and prevent kidney stone formation

126
Q

Metal Chelators

A

reduce amount of metal ions in the body

Desirox is an orally bioavailable iron chelator to treat iron overdose (or due to blood transfusions)

127
Q

Drugs that target bone:
Biphosphonates
ALENDRONATE (FOSAMAX) and RISEDRONATE (ACTONEL)

A

Bisphosphonates are pyrophosphate analogues used to prevent and treat osteoporosis

converted into non-productive NTPs (nucleotide triphosphate) in cells

mechanism: attach to hydroxyapatite binding sites on bony surfaces, especially undergoing resorption, and kill osteoclasts (resorption cells)

PCL302 (2 decks)

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