exam #1 Flashcards
explain Vmax and Km
Vmax (a constant) - the theoretical maximum rate at which a enzyme can turn substrate into produce
Km (a constant) is 1/2 the Vmax
high Km means weak binding, low Km means tight binding
the higher the substrate concentration, the faster the reaction rate (V)
what do enzyme catalysts do?
reduce activation energy required, increasing the rate of reaction
For an enzyme that follows Michaelis-Menton kinetics, Km is equal to which of the following:
a. the [s] at one-half Vmax
b. the Vi at one half Vmax
c. The [s] at one-half Vi
d. The Vi at one-tenth Vmax
e. Two times the Vmax
a. the [s] at one-half Vmax
what is the Lineweaver-Burk Transformation?
inverse of the velocity curve slope of the line = Km/Vmax the y intercept is i/Vmax X intercept is -1/Km 1/V = Km/Vmax(1/S) + 1/Vmax
What are the two categories of enzyme inhibitors?
Reversible inhibitor: interact with enzyme via noncovalent associations
Irreversible inhibitor: interact w/enzyme via covalent associations
What are the classes of reversible inhibition?
competitive inhibitors: bind only to E, not to ES: bind to active site & compete with natural substrate for that site
noncompetitive inhibitors: bind either to E and/or to ES: bind to allosteric sites and alter structure so substrate doesn’t bind as it did naturally
How do the saturation curves for an enzyme catalyzed reaction change with different types of inhibitors?
with competitive inhibitor, Vmax stays the same, but there is a different Km (Km is larger)
noncompetitive inhibitor, Km stays the same, but Vmax is lower
how can you reverse the effect of a competitive inhibitor?
increase substrate concentration
By looking at a lineweaver-Burk Transformation graph (the one with straight lines, not the curve), how do you tell what kind of enzyme has been used?
Competitive inhibitor: the slope of the reaction with the inhibited enzyme is steeper than the uninhibited reaction. The two lines intersect on the Y axis which is the 1/Vmax point (because Vmax doesn’t change with a competitive inhibitor)
Noncompetitive inhibitor: Inhibited slope is still steeper than the uninhibited slope, but the lines intersect on the X axis (-1/Km) because Km stays the same and Vmax is reduced.
Describe mixed inhibition
Mixed inhibition binds at a separate site from the active site to either the free enzyme or the enzyme-substrate complex.
resembles the non-competitive graph, except that the enzyme-inhibitor-substrate complex has residual enzymatic activity, so some product can still be formed even when inhibitor is bound.
Vmax will be reduced
Km may be increased OR decreased
What is uncompetitive inhibition?
inhibitor binds only to the enzyme-substrate complex, at a separate site from the active site and NOT with the free enzyme.
Vmax and Km are reduced by the SAME amount
(on the straight-line graph, the inhibited enzyme line is parallel but to the left of the uninhibited line, and on the curved-line graph, the inhibited starts off more steeply, but then levels out lower than the uninhibited curve)
Serine Protease Inhibitor is an example of what type of enzyme inhibitor?
Irreversible enzyme inhibitor
proteases typically will attack protein amide bonds. can also attack & break ester bonds.
What is clavulanic acid?
Molecular target: beta-lactamase
MOA: irreversible inhibitor
Disease/symptom: anti-bacterial agent used in combination with penicillin overcoming drug resistance
inhibitor of bacterial beta-lactamase:
beta-lactamase is a serine protease & can hydrolyze beta-lactams, such as penicillin antibiotics (responsible for penicillin-resistant bacteria)
Clavulanic acid is a beta lactam and is preferentially taken up by beta-lactamase & hydrolyzed. during the Clavulanic acid molecule’s cleavage, a Michael acceptor is formed which alkylates a nucleophilic residue on the beta lactamase causing IRREVERSIBLE INHIBITION.
so combining beta lactamase inhibitors like clavulanic acid with penicillin helps combat antibiotic resistance
What are the functions of Acetylcholine in the peripheral and central nervous systems?
Peripheral: activates muscles when it binds to ACh receptors on skeletal muscle fibers and causes muscle contraction. In Cardiac muscle fibers, it binds to muscarinic receptors and INHIBITS contraction.
In autonomic nervous system, ACh is released in the following sites: all pre and post ganglionic parasympathetic neurons, all preganglionic sympathetic neurons, preganglionic sympathetic fibers to suprarenal medulla, modified sympathetic ganglion;
when stimulated by ACh, the suprarenal medulla releases epinephrine and norepinephrine … and more stuff that I don’t think we have to know
Central Nervous System: ACh acts as neuromodulator & effects plasticity, arousal and reward. important role in enhancement of sensory perceptions when we wake up & sustaining attention.
Damage to cholinergic (ACh-producint) system in the brain has been associated with memory deficits associated with Alzheimers disease. ACh also shown to be the most important inducer of REM sleep.
What is muscarine?
Molecular target: Muscarinic receptor
MOA: agonist
Disease/symptom: glaucoma
a natural agonist of the parasympathetic nervous system muscarinic receptors. Mimic of ACh-ase
It comes from the amanita muscaria (a mushroom)
used to treat glaucoma, reducing intraocular pressure
if improperly ingested, it is highly toxic
What is atropine?
Molecular target: Muscarinic receptor
MOA: antagonist
Disease/symptom: used to treat organophosphate poisoning and resuscitation
natural antagonist of the parasympathetic system muscarinic receptors: blocks ACh from binding
comes from Atropa belladonna
used to treat organophosphate poisoning from nerve gas and many other therapeutic effects including resuscitation
What is Physostigmine?
a reversible AChE inhibitor from the African Calabar Bean
Used to treat myasthenia gravis, glaucoma, Alzheimer’s disease and delayed gastric emptying
What is the deal with Sarin gas?
an agent that causes organophosphate poisoning
binds to the esteratic site on AChE
then it goes through an aging process where the second phosphate ester is hydrolyzed and makes the phosphate-ester bond irreversible
you need to get treated right away if you are exposed to war gas, otherwise you are hosed.
What is pralidoxime?
Molecular target: Acetylcholinesterase
MOA: antidote used to remove irreversible organophosphate inhibitors from AChE
Disease/Symptom: organophosphate poisoning
Organophosphate poisoning antidote
War gas is an irreversible inhibitor, but pralidoxime can combat it.
It has an OH group bound to a quaternary nitrogen which makes it a strong nucleophile. Strong enough to pull the phosphate group (bound to AChE) off of the AChE
lots of electron density around the oxygen of the pralidoxime, and little electron density around the phosphate (because it is surrounded by 3 oxygens) of the war gas
What is pyridostigmine bromide?
Molecular target: Acetylcholinesterase
MOA: prophylactic to block or prevent organophosphate poisoning
Disease/symptom: used to prevent organophosphate poisoning but has been linked to Gulf War Syndrome
given to gulf war soldiers as a prophylactic
weak inhibitor of AChE, designed so that it doesn’t enter the CNS
BUT the theory is that under stressful conditions, it does leak into the CNS - may cause Gulf War Syndrome - wreaks havoc on AChE receptors
How does a protein kinase phosphorylate a protein?
the protein has a serine, threonine or tyrosine side chain with an OH group.
ATP and protein kinase phosphorylate the protein at that side chain (put the phosphate group onto the O of the OH. That is the on switch
to turn the protein “off”, protein phosphatase takes off the phosphate group. (regulation of the signaling events)
The signal in causes a protein kinase to phosphorylate (and therefore activate) the enzyme.
role of kinases in cancer
kinases have been shown to play a profound role in cancer tumor progression and initiation. Many are oncogenes (RAS) - overexpression or mutation of RAS can lead to tumor initiation & progression
tyrosine Kinases are targets of anticancer drugs: tyrosine kinase inhibitors - targeting those signaling pathways
What is imatinib?
Molecular Target: BCR-ABL
MOA: Competitive BCR-ABL inhibitor, binds to inactive ABL
Disease/Symptom: used to treat various types of cancer but mainly Leukemia, including: ALL, CEL, CML
What is the Philadelphia Chromosome?
first known fusion enzyme: BCR-ABL fusion protein
ABL part from one chromosome and BCR part from another translocate, overlap and then generate an actual protein with both parts of those enzymes
this fusion protein is hyper-activated in chronic myeloid leukemia
the kinase signaling pathway is the on signal that is never shut off
CML proliferation
lead to the discovery that kinases could be viable targets in cancers and other diseases
What is Dasatinib?
Molecular target: BCR-ABL
MOA: binds to active form of ABL
Disease/symptom: used to treat imatinib resistant leukemia that is Philadelphia chromosome positive
(Sprycel)
a derivative of Imatinib
a tyrosine kinase inhibitor
describe the discovery process of Imatinib
- through HTS, identified a protein kinase C inhibitor fragment. Used fragment based drug design to add a fragment that increased cellular uptake in PKC inhibition (increased activity). BUT determined that it was a “dirty” PKC inhibitor - inhibited many kinds of PKC’s and didn’t inhibit BCR-ABL
- Added another fragment for inhibition of BCR-ABL after more drug design. Still inhibited PKC and other kinases, so had to make it more specific
- Added one methyl group paired to amide increased the BCR-ABL specificity and lost connectivity to PKC
- Drug was further enhanced by adding another functional group which increased water solubility and bioavailability - very important for Imatinib and all kinase inhibitors
How do you treat Imatinib-resistant BCR-ABL?
Dasatinib it inhibits imatinib-resistant BCR-ABL the difference between Imatinib and Dasatinib is the orientation of the activation loop Imatinib binds to inactive ABL Dasatinib binds to active ABL
What is Neostigmine?
Molecular target: Acetylcholinesterase
MOA: reversible inhibitor
disease/symptom: used to treat myasthenia gravis, an autoimmune disease
Nerve gas agents
Sarin, tabun, DFP and VX
Molecular target: Acetylcholinerase
MOA: irreversible inhibitors
Disease/symptom: none but are used in war as chemical war agents and by terrorists
What are the key HIV genes?
Structural genes:
Gag (structural proteins): encodes: matrix, capsid, nucleocapside
pol (enzymes): encodes: protease, reverse transcriptase, integrase
env (surface antigens): encodes: gp 160 consisting of go 120 and gp 41
what does HIV1 protease do?
cuts polypeptide chain (inside mature viral particle) into active proteins
what does integrase do?
incorporates viral DNA into human genome
What are the major targets for HIV drug therapy?
HIV1 protease
Reverse transcriptase
What is the structure of Reverse Transcriptase
ribbon structure
shaped like a hand
palm is the business end - where it does the work with RNA
How does reverse transcriptase bind to deoxy Nucleotide Triphosphate (dNTP)?
the polymerase active site is composed of 3 catalytic carboxylates in the palm subdomain that binds two Mg2+ ions that are required for catalysis. Mg positions the oxygens in the right orientation so that it’s in proximity to the phosphate on the dNTP.
Components of Nucleic Acids
Nitrogen bases: DNA only --> thymine DNA & RNA --> adenine, guanine, cytosine RNA only --> uracil Sugars and phosphate DNA only --> 2-deoxyribose RNA only --> ribose Nucleoside = base + sugar Nucleotide = base + sugar + phosphate
What are the pyrimidine and purine bases?
pyrimidine bases:
cytosine (C) and thymine (T)/ Uracil (U)
Purine bases:
Adenine (A) and Guanine (G)
What are 2 examples of Reverse Transcriptase (RT) inhibitors?
AZT: Nucleoside/nucleotide RT inhibitors (NRTI)
Nevirapine: Non-nucleoside/nucleotide RT inhibitors (Non-NRTI)
what are NRTIs
structurally diverse analogs of the natural substrates of DNA synthesis.
all approved NRTIs lack the 3’-OH and act as chain terminators when incorporated in viral DNA by RT. For NRTIs to be effective against HIV, they have to be taken up by the host cell and then phosphorylated by cellular enzymes to convert them to their active form, the NRTI triphosphates.
efficiency of conversion to active metabolite & stability of NRTIs in presence of catabolic enzymes are important considerations in antiviral therapies, because these factors help determine the concentration of the inhibitor in the bloodstream that is required for the NRTI to be effective
What are NNRTIs
non-competitive inhibitors & do not directly interfere with the binding of either the dNTP or the nucleic acid substrates of RT. Pre-steady state kinetic analysis of single nucleotide addition in the presence of NNRTIs has shown that binding of NNRTI interferes with the chemical step of DNA synthesis
What are some examples of NRTIs
zidovudine lamivudine stavudine didanosine zalcitabine abacavir emtricitabine tenofovir Key point to remember is that the structural activity relationship is all around the sugar.
What are examples of NNRTIs?
Nevirapine Delavirdine Efavirenz Dapivirine Etravirine Rilipivirine No bases or sugars here, just aromatic rings that were found to bind to an allosteric site. Very lipophilic, so lots of lipophilic ring systems and nitrile functionalities
What is the MOA of nucleoside RT inhibitors?
suppress HIV replication by attacking reverse transcriptase
NRTIs are similar in structure to the building blocks that make up DNA. They incorporate themselves into the DNA nucleoside chain being produced by reverse transcriptases, they stop attachment of further nucleosides & so prevent ongoing viral DNA synthesis.
What is the MOA of non-nucleoside reverse transcriptase inhibitors?
Attach to the reverse transcriptase and affect the activity of the enzyme by restricting its mobility and making it unable to function
What is the MOA of AZT?
it is a pro-drug
activated via phosphorylation by host cell, mimicking tri-phosphorylated DNA base
then RT recognizes AZT-PPP & tries to incorporate it into the newly sequenced DNA.
AZT does not contain a hydroxyl functionality so DNA chain termination results.
What are two mechanisms of resistance of HIV to NRT inhibitors
- exclusion: enhanced discrimination at the time the NRTI/TP is incorporated. M184V/I mutation is an example of exclusion mechanism. That mutation selectively reduces incorporation of 3TC and FTC by steric hindrance
- Selective removal of NRTI from end of the viral DNA after it has been incorporated by RT. Excision mechanism; example is AZT resistance caused by set of mutations including M41L, D67N, K70R, L210W, T215F/Y, K219E/Q
What are the most frequently observed resistance mutations in patients treated with approved NNRTIs?
K103N (lysine 103) and Y181C
resistance mutations can occur singly or in combinations
resistance to 1st and 2nd generation NNRTIs can evolve relatively quickly
How can you combat resistance of HIV to NNRT inhibitors?
drug cocktails: NNRT, NRT and protease inhibitors
What does an enzyme catalyst do to activation energy?
lowers it - so increases the rate of reaction
enzyme stabilizes the ES transition state more than it stabilizes ES
how does drug development relate to transition states?
if you design a drug that mimics the transition state of the substrate, then that enzyme will prefer to bind to the drug than to the substrate
How does HIV protease work?
it mediates post-translational modification of core proteins into structural proteins.
HIV genome has regions that are genes (like gag and gag-pol genes) that are translated as polyproteins & form immature viral particles. The precursor protein molecules are cleaved by a viral pol-encoded aspartic proteinase to form the structural proteins of the mature viral particle.
HIV protease also activates RT & plays an important role in release of infectious viral particles.
So inhibiting HIV protease & pol gene is a drug target:
What do HIV protease inhibitors do?
act on HIV protease & prevent post translational processing and budding of immature viral particles from the infected cells.
major breakthrough in treatment of HIV when used in combination with RT inhibitors.
What was the first HIV-1 protease inhibitor?
Saquinavir
What are scissile bonds?
areas along the virus polypeptide where the enzyme recognizes it needs to cut to make active proteins
What is the HIV protease enzymatic mechanism?
Active site triad on the protease (Asp25-Thr26-Gly27) is located in a loop whose structure is stabilized by a network of hydrogen bonds. Carboxylate groups of Asp25 from both chains are nearly coplanar & show close contacts. It is a rigid network due to the “fireman’s grip” interaction in which each Thr26 OG1 accepts a hydrogen bond from the THR26 main-chain NH of the opposing loop.
Drug design of saquinavir
started with a lead structure and then designed off of that using the grow scaffold method (start with fragment & then grow off functional groups)
1. Started with peptidomimetic of a natural substrate of HIV-1 protease. Cbz was added to fill the S2 pocket (Cbz is a standard protecting group) Used “aspartyl warhead” to mimic transition state of peptide substrate. it binds with much greater affinity than the natural substrate would, so it’s a strong competitive inhibitor. Aspartyl warhead is stable & can’t be hydrolyzed like the natural substrate could. Added a t-butyl ester group to the existing proline to strengthen the binding interactiosn to pocket S1’
2. carbon spacer between Cbz group is added with a side chain containing a primary amide group filling pocket S2 - allows the Cbz group to interact with pocket S3. creates significant increase in enzyme inhibition from 6500 nM to 140 nM
3. Cbz group exchanged for a quinoline ring –> increases lipophilic interactions & introduces stronger pi-stacking interaction due to more electron deficient nitrogen ring. - enzyme inhibition goes from 140nM to 23 nM.
4. proline residue was exchanged for a decahydroisoquinoline, occuies the S1’ pocket w/hydrophobic interactions & extends t-butyl ester group into the S2’ pocket. Now enzyme inhibition is 0.4 nM or 400 pM
extremely specific for HIV-1 protease & reduces off target effects
What are some FDA approved HIV-1 protease inhibitors (transition state analogs)?
Amprenavir Saquinavir Indinavir Lopinavir Ritonavir Nelfinavir all of these are peptidomimetics & used along with reverse transcriptase inhibitors
What is Darunavir?
second generation HIV protease inhibitor.
designed the drug better (than saquinovir), increase interactions with the inhibitor and overcome drug resistance (mutations that led to minimal structural changes in the active site of HPI).
Has many characteristics of saquinovir with isosteric replacement
What is the basic timeline of bacteria/antibiotic discovery?
1670’s bacteria discovered by Leeuwenhoek
1859 - Pasteur demonstrated that microorganisms grow rather than spontaneously generate (germ theory of disease)
1905 - Koch - proved germ theory
1940’s - 1st mass produced antibiotic - penicillin- used during WWII
What kind of products are antibiotics?
natural
Fleming discovered penicillin when he noticed clear circles around mold on a petri dish (the mold was penecillium)
Explain selective toxicity
common processes mediated by different proteins: because of evolutionary distance between bacterial cells and eukaryotic cells, so a compound that inhibits a biosynthetic bacterial enzyme has little or no effect on eukaryotic counterpart
cell wall vs. plasma membrane: bacterial cells have some structures that are unique, so we can target those things - like components of bacterial cell wall
How does bacterial resistance arise?
bacterial mutations accumulate rapidly
antibiotics place strong selective pressure on bacterial populations driving evolution of resistant strains
-exposure to low antibiotic blood concentration gives bacterial population time to accumulate sufficient mutations to develop resistance
How do bacteria exchange genetic information?
Transformation: fragments of DNA from dead bacteria are picked up by recipient
Transduction: viruses (bacteriophage) pick up a piece of genome accidentally and insert it into recipient upon infection
Conjugation: physical bridge formed between cytoplasms. F plasmid (fertility factor) is expressed by donor, it is copied & copy is transferred to recipient. THIS is the most common form of exchange
What are the mechanisms of resistance
- Bacterium may have altered the target such that antibiotic binding affinity is vastly reduced
- bacterium may have eliminated the target altogether
- bacterium may either restrict access or actively pump the antibiotic out of the cell
- bacterium may express an enzyme that inactivates the antibiotic
- bacterium may express a biofilm that restricts access
- bacterium may have entered a state of quiescence.
What are the beta-lactam ring antibiotics?
Inhibit cell wall
Penicillins, cephalosporins, carbapenems, monobactams
all share Beta-lactam ring structure which is essential to the function of these compounds: beta lactam ring is highly reactive due to the geometry of 4 atoms in the ring (looks like a square)
Describe the structure of gram positive bacterial cell wall
simpler structure of the two (gram positive & gram negative) outermost layer is carbohydrates and proteins, below that is cross-linked peptidoglycans (sugar chains with amino acids that are in D conformation (not L)) arranged in layers that are cross-linked laterally and above and below (this makes the rigid cell wall). Under that is lipid bilayer containing proteins. Some of those proteins are responsible for constructing the cell wall - they are called PBPs (penicillin binding proteins) b/c they serve as targets for this class of antibiotic. There are 7 different PBPs & different penicillins have different patterns of binding to subsets of these proteins
Describe the structure of a gram negative bacterial cell wall
outer lipid membrane that is distinct & different from inner lipid membrane. It contains porins which function to exclude certain molecules while allowing others to pass. Porins make entry of antibiotics more difficult. Other proteins induce septic shock in the host & may supply antigenic determinants which can be used by the immune system.
below the lipid membrane is a peptidoglycan layer (thinner than in gram positive). This region is called the periplasmic space.
Below this is the inner plasma membrane that contains PBPs along with other proteins that are involved in the transport of nutrients and waste
What do penicillin binding proteins do?
build and repair the bacterial cell wall
7 different proteins - each is an enzyme that constructs some aspect of the peptidoglycan layer
because there are multiple PBPs, different penicillins are going to bind differently & inhibit different proteins; they are going to work differently in different bacteria, because each bacteria has a different set of PBPs.
What does PBP-1 do?
it is a transamidase that crosslinks peptidoglycan strands
accepts a terminal D-ala-D-ala peptide into its active site (that is what is going to cross link)
certain penicillins take the place of D-ala-D-ala.
When PBP-1 tries to convert it, the Beta-lactam ring breaks & attaches permanently to the enzyme, inactivating it. (covalent bond)
Why are there so many beta-lactam antibiotics?
each beta lactam antibiotic has a different (but overlapping) spectrum of activity because it has different abilities to inactivate different PBPs.
amino acid sequences are different in the various PBPs, so any one specific antibiotic will bind with different affinities
What is the major form of bacterial resistance to beta-lactam antibiotics?
ability of the bacteria to express an enzyme that cleaves the beta-lactam ring (beta-lactamases)
many different beta-lactamases are produced by different bacteria, and different penicillins have various sensitivities to these inactivating enzymes.
–> Different species of bacteria are differently resistant to certain penicillins
what is the basic structure of penicillin?
Pentagonal ring attached to a beta-lactam ring.
Carbon 6 is on the beta-lactam ring, the side chain on carbon 6 determines the specific type of penicillin
What were the first penicillins?
fermentation derived penicillins
these were secreted by the penicillium fungus. The side chain was derived from substances in the media, so it was possible to alter the media to create Penicillin G (Benzylpenicillin) or Penicillin V (Phenoxymethylpenicillin)
They work against most non-resistant gram positive bacteria, cheap, relatively non-toxic (except for allergies)
Penicillin V is more acid stable than G, so it survives the stomach better - better for oral administration
What is an example of a parenteral penicillin?
Methicillin
Semi-synthetic penicillinase-resistant parenteral penicillin
could remove the side chain completely by leaving out that constituent of the media where penicillin was grown. Parent molecule was harvested, and synthetic side chains were added at C6.
Methicillin is not used today due to drug resistance
What are examples of semi-synthetic penicillinase-resistance oral penicillins?
Oxacillin (Bactocil)
Cloxacillin (Cloxapen)
Dicloxacillin (Dycill, Pathcil)
What are examples of penicillinase-sensitive, broad-spectrum oral penicillins?
Amoxicillin
Ampicillin
these are effective against some gram negative as well as gram positive bacteria
What are examples of semi-synthetic penicillinase-sensitive, broad spectrum, parenteral penicillins?
Carbenicillin (Geocillin)
Ticarcillin (Ticar)
Mezlocillin (Mezlin)
Piperacillin (Pipracil)
What is Augmentin?
Amoxicillin (broad spectrum orally active penicillin which is sensitive to beta-lactamases) and Clavulanic acid (beta lactam molecule which is weak by itself as an antibiotic, but great inhibitor of beta-lactamases.
successful strategy: combine an antibiotic with an inhibitor of one or more resistance genes.
Why so many penicillin allergies?
6-8% of US population is allergic to penicillins
one of the major reasons is that penicillins can form haptens - a small molecule covalently attached to a protein which creates a potent epitope for an immune response.
the beta-lactam ring can be hydrolyzed in the liver leading to protein conjugation.
What are Cephalosporins?
Beta-lactam molecules
original (natural one) was not very potent, but quite resistant to beta-lactamases.
synthetic chemistry has generated 1000’s of different molecules - there are several places we can add groups (unlike penicillins where it’s just the C6) - allow us to maintain activity & alter function
can be broad spectrum and are being used against some very dangerous pathogens - hospital acquired infections are one example
Give examples of 1st through 3rd generation cephalosporins
1st generation: Cephalexin
3rd generation: Cefotaxime
4th generation: Cefepime (he didn’t give an example of 2nd)
What are classes of synthetic antimicrobials?
And what are their MOAs
Sulfonamides Trimethoprim Quinolones Nitroheteroaromatic compounds Inhibit various aspects of nucleic acid (DNA and RNA) biosynthesis and function.
Sulfonamides
Examples: Sulfamethoxazole, Sulfadiazine
Inhibit dihydropteroate synthase (an intermediate in DNA synthesis)
These used to be broad spectrum, but resistance has vastly decreased their use
resistance involves mutations in the dihydropteroate synthase enzyme
subsequent mutations allow folate to be harvested through other pathways.
Examples of Quinolones, how they work, and mechanisms of resistance
Examples: Ciprofloxacin, levofloxacin
Inhibit bacterial DNA gyrase and topoisomerase IV. This inhibits gene transcription (rather than inhibiting DNA synthesis, we are inhibiting DNA function)
Resistance mechanisms:
-mutations in DNA gyrase
-inhibition of drug entry (gram negative bacteria)
-increased drug efflux (gram negative bacteria)
Examples, MOA and resistance mechanisms of nitroheteroaromatic compounds
Examples: Nitrofurantoin, Metronidazole
MOA: thought to work by inhibiting nucleic acid (DNA and RNA) synthesis
These compounds need to be activated to function
Activation step occurs through the action of an enzyme unique to anaerobic organisms.
Resistance: mutation of the activating enzyme
What are the classes and broad mechanism of action of protein synthesis inhibitors?
aminoglycosides, macrolides, lincosamides, tetracyclines
Examples: Gentamicin, erythromicin
MOA: bind to bacterial ribosomal subunits
-block translation (if the ribosome has not initiated)
-Incorporate incorrect amino acids (if the ribosome is actively reading mRNA)
What are resistance mechanisms for aminoglycosides?
and when are they used?
- bacteria have evolved enzymes which add modifications to these compounds
- destroys their ability to bind to ribosomes (reminiscent of our p450 system in the liver)
These are broad spectrum antibiotics which are reserved for serious infections. They have equally serious potential side effects including destruction of cells in the ear required for balance & necrosis of kidney tubules.
Bacterial resistance mechanisms and therapeutic uses for Macrolides
Resistance:
- bacteria sometimes modify their own ribosomes - results in an organism which has a much harder time synthesizing proteins, but also has a vastly reduced affinity for macrolide antibiotics
- other bacteria can efflux the drug (costs the bacteria energy)
Therapeutic use:
- considered some of the safest of the antibiotics in common use
- often used to treat respiratory tract gram positive infections
What are bacterial cyclic peptides?
bacterial cyclic peptides are constructed from amino acids by enzyme complexes & contain both normal and modified amino acids
-NOT made by ribosomes and not coded as genes
function as weapons against other bacteria by inhibiting cell wall biosynthesis
Examples: vancomycin, daptomycin
they wrap around the cell wall precursor so enzyme can’t access it
MOA and resistance
Vancomycin
Effective against sensitive gram positive bacteria
binds to D-ala-D-ala terminals of peptidoglycan. This blocks the PBP from crosslinking the peptidoglycan strands and the cell wall falls apart
Resistance:
bacteria have substituted D-ala-D-ala with D-ala-D-lactate
Penicillin and cyclic peptide: similarities and differences
both penicillins and cyclic peptides target the peptidoglycan crosslinking step
penicillin inhibits the enzyme
vancomycin masks the substrate
Conventional chemotherapy (pre-2000) vs current
In the past, cancer tumors were thought to be all cancer cells. Chemo (developed in the last 100 yrs) goal was to damage tumor DNA ( all chemotherapy drugs before Glevac). Since then, due to genetic information, we know tumors have many different types of cells (immune, cancer, vascular, tumor initiating cells), so drugs are being developed to target tumor initiating cell phenotype.
Hallmarks of Cancer
Sustaining proliferative signaling Evading growth suppressors Activating invasion and metastasis enabling replicative immortality Inducing angiogenesis Resisting cell death
What is the last frontier of cancer research?
Activating invasion and metastasis: most animals have the capacity for cells to differentiate into different phenotypes, but tumor cells hijack the mechanisms and are no longer regulated by growth factors. Become irreversible & promote invasion and metastasis.
how much does it cost to get a drug developed?
1.5 billion
ADMET
absorption, distribution, metabolism, excretion and toxicity
aromaticity and bonding
cyclic ring systems
shared electrons are conjugated by resonance
these compounds are unusually stable
these compounds are flat and planar
Pi electrons are delocalized around the ring above & below the plane
how to determine formal charge
Count covalent bonding electrons around each atom as 1.
Count the loan pair electrons (each pair) as 2 (1 each).
If number of electrons = valence #, neutral charge
if number of electrons > valence #, negatively charged
if number of electrons < valence, positively charged
what is the difference between a nucleophile and an electrophile
a nucleophile is electron-rich, negatively polarized (base)
an electrophile is electron-poor, positively polarized (acid)
how many points of attachment of a drug to a receptor site is essential for the desired effect?
3 points minimum
What kinds of forces are involved in drug-target interactions?
covalent
ionic
ion-dipole/dipole-dipole
hydrophilic (h-bonds)
charge transfer (pi stacking/base stacking)
hydrophobic (Van der Waals)
Most useful drugs bind through the use of multiple weak bonds (ionic & weaker)
What is an example of a covalent interaction in drug design?
CC-1065 - anti-tumor antibiotic
alkylates the N3 position of adenine in the minor groove of DNA –> suicide structure - irreversible alkylation & kills the DNA
What is a bioisoster?
substituents or groups that have chemical or physical similarities which produce broadly similar biological properties
What is an example of a successful isometric replacement?
5-FU: chemotherapeutic agent where H in 5 position is replaced with F –> drug kills thymidylate synthase, it’s a suicide substrate which leads to inhibition of DNA synthesis & cell death
What does HSP90 do?
helps fold other proteins (molecular chaperone): Geldanamycin is a drug that fits in the pocket of HSP90 - preventing HSP90 from folding other proteins –> cancer target
Most naturally occurring medicinal agents exist as single isomer or racemic mixtures?
single isomer forms (optically active forms).
more than half the number of drugs in use are chiral & usually only one enantiomer has the beneficial effect
two types of stereoisomers
Enantiomer: 3D arrangements that are non-superimposable mirror images
Diastereomers: Not mirror images
Can be S and R
E and Z
Which form of epinephrine is active?
R-isomer- interacts with 3 distance points on the adrenergic receptor. S epinephrine only interacts with 2 points
Which form of ibuprofen is active?
S (left handed)
Which form of aspartame is sweet?
S
What is the difference between Quinine and Quinidine?
They are stereoisomers of each other.
Quinine is antimalerial drug
Quinidine is anti arrhythmic drug
display different biological activity because of different structures.
Stereochemistry of Albuterol
sold as racemic mixture, but only the R form is the active bronchodilator
S albuterol indirectly antagonizes the activity of R-albuterol, but it’s cheaper to cell the racemic mixture
What is the stereochemistry of ritalin?
sold as a racemic mixture
R Ritalin is 35 times more active than (s), and pure (R) form’s effects last longer than racemic mixture
Stereochemistry of Cannabinoids
Natural THC: R Synthetic THC (S) - much less active: (S) Dexanibinol - no psychoactive properties but potent neuro-protective agent in clinical trials for traumatic brain injury/stroke
What is an example where stereochemistry is harmful?
Thalidomide: sedative & used as chemotherapy agent for multiple myeloma. Used to be prescribed for morning sickness. R-isomer is effective drug, S-isomer is teratogenic
Triprolidine
active stereoisomer, molecular target, disease/symptom
(E)-Triprolidine is active ((Z) is inactive)
Histamine H1 antagonist
Anti-histamine
Ibuprofen
(S)-Ibuprofen is active, (R) is inactive
COX inhibitor
NSAID Anti-inflammatory agent
Quinine
(3R, 4S, 8S, 9R) Quinine is active
Off target effect of inactive stereoisomer is an anti arrhythmic to regulate heart beat
Unknown what the molecular target is
anti-malarial
Albuterol
(R)-Albuterol is active form
inactive stereoisomer antagonizes activity of R-form
Beta2-adrenergic receptor agonist
Asthma drug bronchodilator
Ritalin
(R)-Ritalin is active
(S) form causes loss of activity and long term drug effect
Norepinephrine-dopamine reuptake inhibitor
treats Epilepsy in Europe
5-Fluorouricil (5FU)
H is replaced with F (isometric replacement
Thymidylate Syntase inhibitor
Anti-cancer drug
Dexanabinol
(S) Dexanabinol is active form
R form has similar effects to natural THC (psychoactive)
NMDA receptor antagonist
Neuro protective agent to treat traumatic brain injury/stroke
Epinephrine
(R)-epinephrine is active
acts on adrenergic receptors
treats glaucoma
What is molecular modeling?
Molecular Graphics: 3D visualization of interactions between compounds & proteins, which help scientists understand the mechanism of action of the drug and to improve the design of a drug
Computational Chemistry: Docking compounds into proteins computationally (size/shape, polarity, orientation, flexibility)
What is the Flow chart for docking using discovery studio software?
PDB ID (protein file)
Ligand file
Imported to the software
Preparation:
Prepare ligand
Prepare protein
Binding site identification
Docking:
dock ligand to the binding site
analyze poses
Calculated binding energy
How do you know when to use SBDD, LBDD, Receptor-based drug design, De novo design & Homology modeling?
Known protein structure, known ligand = structure-based drug design (SBDD) (Molecular docking: protein-ligand interaction/ Drug-Receptor interaction)
Known protein structure, no known ligand = Receptor-based drug design, De novo design (build or find the key that fits the lock)
Unknown protein structure, known ligand(s): Ligand-based drug design (LBDD) (1 or more ligands: similarity searching, several ligands: pharmacophore searching, many ligands (20+): quantitative Structure-Activity Relationships (QSAR)
Unknown protein structure, no known ligand: Homology modeling (HTS, Combinatorial Chemistry, Virtual Screening (Build the lock, then find the key))
Structure Based Drug Design
Given a protein structure and or its binding site, find a new molecule that changes the protein’s activity
Ligand-Based drug design
given an active ligand, find a new molecule that changes the protein’s activity
Grow Scaffold
Used in SBDD
Scaffold in active site/ define search site sphere –> define link points, define fragment libraries –> calculate interaction sites –> search fragment libraries/enumerate library
De Novo Evolution uses polar and hydrophobic interaction sites inferred from the protein to identify linking fragments
Ligand optionally positioned in a protein active site, select fragment to be replaced, replace fragment: start with core & grow outward
Fragment-based drug design
Fragments in active site/ define search site sphere –> calculate interaction sites –> search library for link hits/score and save hits
De Novo Link can be used to identify single linking fragments to grow a scaffold, like Grow Scaffold, but is uniquely and best able to identify bridging fragments
start out and go in
SBDD summary
Probe the binding site (PLACE) Adding fragments (GROW) Modifying Scaffolds (REPLACE)
LBDD
Structure of targeted protein unknown:
looking for chemical similarity to known ligands then test experimentally
Resources for CADD
Binding activity data bases
2D Substructure Matching
Chemical Fingerprints
3D Substructure Matching (Pharmacophore model)
Pharmacophore Model
3D Substructure matching:
Set points in space defining the binding of ligands with target
Key factors in developing such a model are the determination of functional groups essential for binding, their correspondence from one ligand to another, and the common spatial arrangement of these groups when bound to the receptor
Examples of Block Buster Drugs developed using CADD
Saquinavir (HIV-1 Protease inhibitor)
Resuvastatin (HMG-CoA Reductase Inhibitor)
Gleevec (BCR-ABL Protein Kinase Inhibitor
Sorbinil (Aldose Reductase Inhibitors - diabetic complications)
Protein Data Bank
Archive of experimentally determined 3D structures of biological macromolecules
Direct Design
SBDD: followed when spatial structure of the target is known: Grow, replace, De novo (fragment)
Indirect design
LBDD
Followed when the structure of the target is unknown
what is HTS
High-throughput screening: a technique for empirically discovering chemical modifiers of biological action.
1st done by Pfizer - screened 100K soil samples to discover new antibiotics & discovered terramycin
What is the advantage of HTS?
decrease cost by developing ways to remove human element, rapidly screen compounds
requires HIGH investment up front, but it costs 1.5 billion & 20 yrs on average to get a new drug to market
Biochemical assays
Little functional data collected. Kinases and other enzymes. Simple ligand-receptor interactions. Readily available to HTS.
Cell-based assays
Functional data (agonist, antagonist, intra-cellular pathways) GPCRs, ion channels, and other membrane receptors tested in this manner. Harder to adapt to miniaturized HTS format gives you a lot more information than biochemical assay
Homogeneous assays
All assay components exist in solution phase at the time of detection (e.g. none of the components are in beads or cells). Technically no component scatters light. No wash steps. Mix and read
Heterogeneous assays
one or more assay components are present in solid phase at time of detection (e.g. ELISA, SPA, cells). May involve wash steps.
Hit
an activity discovered by primary assay high-throughput screening
Lead
an activity confirmed in a secondary assay that is typically a more functional end point
Lead Candidates
show activity in animals with promising pharmacology (ADMET/toxicology)
Drug
Lead candidates that have effective activity in humans
What are considered druggable targets in human disease?
Protein kinsases GPCRs Ion channels Serine proteases Protein phosphatases
Differences in laboratory bench top & HTS assays
Protocol: BT can be complex, HTS simple (only 5-10 steps)
Assay volume: much smaller in HTS
Reagents: BT more flexible - can have unstable, limited and variable, HTS has to have single batch, stable over long period of time
Variables:BT can handle more than HTS
Assay container: BT can be varied, HTS: micro titer plate
Time of measurement: BT: milliseconds to months, HTS: minutes to hours
Output formats: more variable in BT, HTS: plate reader- mostly fluorescence, luminescence & absorbance
Reporting format: BT: statistical analysis of manually entered data, HTS: automated analysis of all data using statistical criteria
Optimization process for HTS
Time: requires robotics and automation -time/well -wells/day -screens/yr -project time
Costs: minimizing requires assay miniaturization -reagents -consumables -instrumentation -personnel
Quality:
- few false positives
- few false negatives
- S/N, H/L, Z’-factor
- validated binders
What are the types of assays utilized in HTS drug discovery?
In Vitro Enzyme assays
Cell based assays (give more significant information)
What are the main assay formats?
Biochemical assays (isolated proteins or enzymes)
- Fluorescence/Forster Resonance Energy Transfer (FRET)
- Scintillation proximity (SPA) “radioactive”
Cell-based Assays (Recominant or primary cell lines)
-Luciferase reporter-genes (luminsecent)
Other Assays
-whole animal (sea elegins)
there are many more
SPA
Scintillation Proximity Assay
example of heterogeneous assay
Kinase acceptor is attached to a scintillation bead - becomes phosphorylated by another kinase
kinase donor is labeled with p33 (radio label)
once p33 is bound to kinase acceptor, it will emit a beta particle which will be absorbed by scintillation bead & then bead will emit luminescence.
If you see loss of luminescence, that means you’ve inhibited that enzyme
Some drugs that have been discovered using scintillation proximity assay:
Gleevac
Roscovitine
Iressa
FRET
Fluorescence resonance energy transfer: distance-dependent interaction between electronic excited states of 2 dye molecules in which excitation is transferred from donor molecule to acceptor molecule without emission of a photo. Efficiency of FRET is dependent on intermolecular separation.
We are looking to inhibit yellow fluorescence - much safer to work with than SPA.
Cell based reporter assays
You have gene of interest - gene promotor is recombinantly attached to luciferase (firefly luminescence). When luciferase is expressed in cell, it glows when treated with luciferin.
Vimentin-Promoter-Firefly Luciferase Cloning
Luciferase is a surrogate reporter for vimentin gene expression. If you inhibit vimentin expression, you inhibit luciferase activity, and so you don’t see the glow
How do you know your assay is high quality?
Z’ factor
if your Z’ factor is 0.5 or greater, you have a good assay
How do you make a Uniform Single MDA-MG-231 Breast Carcinoma Spheroid for HTS?
have to have ECM. Without it, you won’t get any spheroid formation.
What are some examples of modern nature product drugs used today?
morphine
Vincristine (anti-cancer - from Vinca rose)
Artemisinin (from Artemisia annua - antimalarial)
Taxol1 (from Taxus brevifolia - anticancer)
antibiotic penicillins from penecillium sap
Red Tides
caused by dinoflagellates: produce powerful toxins. As fish swim through a bloom of dinoflagellates, they get disrupted or killed & release neurotoxins in the water
When humans eat affected fish or shellfish, they can be affected: ciguatera (from eating affected fish) and paralytic shellfish poisoning (PSP) from eating affected shellfish
What is the dinoflagellate toxin?
Maitotoxin: 164 C’s + a bunch of ether rings with sulfonate esther functionalities sticking out.
induces opening of ion channels & causes influx into cells, causing cell death
What percentage of drugs that have been developed come from natural products?
75%
What are strategies for isolating and identifying natural products?
Older:
- focus on chemistry of compound but not on activity
- isolation & identification of compounds followed by biological activity testing (mainly in vivo)
Modern:
- bioassay-guided isolation & identification of active “lead” compounds from natural sources
- production of natural products libraries
- production of active compounds in cell or tissue culture, genetic manipulation, natural combinatorial chemistry etc.
- more focused on bioactivity
What are the steps of bioassay guided natural product isolation?
take crude material, do some processing on the bench [fractionate] (HP20: a solid-phase adsorbed, HPLC: high performance liquid chromatography, partition etc) to get pure compound. Test fractions for purity and activity.
determine structure of pure compound (NMR, MS, IR (infrared spectroscopy), UV etc)
Several cycles of fractionation are needed to obtain a pure compound
explain extraction and fractionation
Step 1:
natural source is lyophilized (dried) and ground to a powder
step 2:
powder is extracted with organic (non-polar) and aqueous (polar) solvents.
The remaining powder is filtered off & the liquid extract (containing natural products) is concentrated.
Step 3:
extract may be pre-fractionated to remove unwanted products using aqueous organic solutions
pre-fractiosn are tested in biological assay models of human disease to identify active fractions
Active fractions are then further purified using HPLC (high performance liquid chromatography)
Explain high-Throughput bioassay guided purification
- natural product extracts are purified using HPLC using HTS formatted deep well plates (2mL volumes) or using standard test tubes of various sizes.
Step 2:
HPLC fractions are concentrated & formatted into HTS plates & sent out for biological testing
Active wells or fractions are then further purified using same method but by adjusting HPLC method to obtain optimized separation/purification
Describe the natural product drug discovery pipeline
Initial bioactive sample –> processing experiments to get bioactive concentrate (from that, identify compounds/known compounds) –> bioassay-guided fractionation to get pure bioactive compound (1-10 micrograms) –> structure determination to get novel bioactive compound (1-10 mg)–> evaluation of natural SAR Shotgun synthesis of derivatives scale-up purification to get potential lead (1-10 g)–> medicinal chemistry to get development track candidate (100g)
have to keep increasing the amount of material as you proceed through this process
Prialt
(Ziconotide)
MOA: selective blocker of voltage gated Ca channels. used for pain. Discovered in 1960s by prof Olivera at University of Utah
from Cone snail
Yondelis
(Trabectedin)
MOA: DNA backbone cleavage. Used for cancer. from Ecteinascidia turbinate
Vira-A
(Vidarabine, Ara-A)
MOA: Viral DNA polymerase is the target. Blocks viral DNA synthesis
Antiviral agent, notably Herpes -s1 & Herpes-s2,
Cytosar-U
(Cytarabine, Ara-C)
MOA: DNA damage
cancer
from Tethya sp.
Soblidotin
anti-cancer agent
peptido-mimetic
from Symploca sp.
Eribulin
(Halaven)
Cancer
from Halichondria okadai
blocks microtubule formation
Bryostatin 1
MOA: inhibitor of protein kinase C modulates PKC activity cancer from Bubula neritina failed clinical trials
E7974
anti cancer
from Hemiasterella minor
Salinosporamide A
Marizomib
from Salinispora tropics
anti cancer
MOA: irreversible proteasome inhibitor
Taxol
Pacletaxel
from Taxus brevifolia (Pacific Yew Tree bark)
took a long time to elucidate the complete structure from 1965 (time of purification) took almost 10 yrs to identify structure
1992 Taxol is FDA approved for cancer Tx
inhibits microtubule dynamics - stabilizes them so they can’t add or subtract
binds to taxane site
Vinblastine
Antimitotic drug
Inhibits microtubule dynamics
binds to the vinca domain
Cochicine
antimitotic
inhibits microtubule formation
used to treat gout
binds to cochicine domain
Microtubule structure
have alph and beta subunits
have negative & positive end
add subunits to the positive end
remove units on the negative end
depolymerize & polymerize
important in mitosis - pull apart daughter cells
good for cancer drug b/c you inhibit cell growth
Docetaxel
(Taxotere)
synthesized derivative of Taxol
used to treat breast cancer, gastric cancer, lung cancer, prostate cancer, squamous cell carcinoma
Therapeutic targeting of the Hallmarks of Cancer
EGFR inhibitors –> sustaining profliferative signaling
Cyclin-dependent kinase inhibitors –> evading growth suppressors
Immune activating anti-CTLA4 mAb –> avoiding immune destruction
Telomerase inhibitors –> enabling replicative immortality
Selective anti-inflammatory drugs –> tumor-promoting inflammation
Inhibitors of HGF/c-Met –> Activating invasion & metastasis
Inhibitors of VEGF signaling –> inducing angiogenesis
PARP inhibitors –> Genome instability & mutation
Proapoptotic BH3 mimetics –> resisting cell death
Aerobic glycolysis inhibitors –> deregulating cellular energetics
Classes of DNA-interactive agents
DNA-DNA crosslinker Intercalator Double-stranded break Code-reading Protein-DNA complex Secondary DNA structures
lots of adverse effects, but still work because majority of cells in our body are in a quiescent state (somewhat protected against these compounds)
alkylating agents began as a result of WWI chemical warfare
How to DNA alkylators work?
2 regions on DNA act as nucleophiles, N3 of adenine and N7 of guanine (lone pair on N)
Minor groove of DNA is adenine rich - so lots of N3 alkylations occur there
alkylators are strongly electrophilic - form covalent bonds with nucleophilic groups on DNA –> irreversible inhibitors of transcription and translation
simple nucleophiles like methane sulfonates and ethyleneimines tend to react via SN2 mechanism.
Nitrogen mustards can form aziridinium ions that react via SN1 mechanism
Mephalan
nitrogen mustard derivative
MOA: interstrand DNA crosslinker
multiple myeloma
Mitomycin C
Interstrand DNA crosslinker
Stomach and GI tract cancer
made by a bug
Doxorubicin
MOA: stabilizes topoisomerase II - DNA cleavable complex
used for rhabomyosarcoma, breast cancer, adult acute leukemia, endometrial cancer, stomach cancer, cervical cancer, non-Hodgkins lymphoma
Etoposide
MOA: stabilizes Topoisomerase-II-DNA cleavable complex
Testicular cancer, small-cell lung cancer
What is the structure of Mustard DNA alkylating agents?
N with an R group & two, 2 carbon + Cl chains
Cl groups are leaving groups, lone pair N attacks the C-on the Cl and forms an electrophilic aziridinium ion (3 ring system)
DNA nucleophilic attack (N7 on guanine or N3 on adenine) bonds DNA to one side, then it happens again to bond a 2nd DNA strand to the second side = cross linkage ville.
What is the mechanism of mitomycin C DNA alkylation
Step 1: bioreduction (have to reduce the carbonyl groups*** so electrons on phenol can delocalize) followed by loss of methoxy generating quinone methide (looks like an iziridinium ion)
Step 2: Quinone methide is attached by the N7/N3 of guanine
Step 3: Loss of carbonate generating a second quinone methide.
Step 4: second quinone methide is attached by another N7/N3 guanine leading to cross linked DNA
What do platinum drugs do?
irreversibly alkylate DNA
attack by nucleophilic DNA base (like mustard drugs) and the reaction sequence can occur a second time to cross-link DNA
What are some examples of platinum drugs?
Cisplatin, Carboplatin, oxaliplatin, Pyriplatin
How do topoisomerase II catalyzed reactions work?
modulates DNA topology - can knot it up or catonate it (link it like a chain) or relax or supercoil it
What is the topoisomerase II DNA interaction mechanism
ATP binds to enzyme in ATP binding sites, then enzyme will interact with DNA via DNA binding domain - uses ATP to make conformational changes
Enzyme makes double stranded cleavage points on it. (makes 2 cuts). In step 3 it cuts the DNA, then bottom piece of DNA moves to the top in step 4 where it is a stable cleavage complex. Then enzyme will re-ligate DNA back together - closed clamp complex.
What is the main area of drug development with topoisomerase II
drugs function by stabilizing topoisomerase DNA complex at the cleavage complex. Drugs interact with the protein but also intercalate DNA - form a stable complex that accumulates in the nucleus; you get DNA damage & cell death
Etoposide
stabilizes the cleaved complex
binds at the C-terminal DNA-Cleavage/Religation domain on Topoisomerase IIA
ICRF-193
stabilizes the closed clamp
binds to ATP binding sites at the N-terminal ATPase domain
Topoisomerase IIA drug
What are the side effects of Topoisomerase II Drugs
genotoxicity resulting from topolla
slow growth rates - quiescence - mitotic failure (decreasing concentration)
Recombination Mutagenesis translocations –> apoptosis (cell death), DNA translocation - cancer (increasing concentration. - secondary malignancies
they keep you alive, but they give you more cancer
Anthracyclin Antitumor Antibiotics
6 different circled structural differences
Doxorubicin: Ch2-OH (only one with CHOH)
Daunorubicin: only one with C=O in the circle
Idarubicin: H
4’-Iodo-doxorubicin: I
Epirubicin: OH
Pirarubicin: chair conformation
Daunorubicin
Original anthracycline first developed in Europe
Doxorubicin
Developed from mutated strain of streptomyces.
What is the problem with anthracyclins?
Early Cardiotoxicity
happens during treatment or first year after its completion
Late Cardiotoxicity: at least 1 yr after completion of treatment, cumulative, dose-related & can result in congestive heart failure & left ventricular dysfunction
generates ROS
Strategies to reduce cardiotoxicity
Dexaroxane (blocks anthracycline-iron complex & redox cycle of semiquinone)
Antioxidants (block reactive oxygen species)
ACE inhibitors/Beta blockers (block early & late cardiac toxicity)
Mustard agents
target DNA
Alkylate and can cross link DNA
for Multiple myeloma
Mytomycin C
Target DNA
Alkylate and can cross link DNA
Stomach & intestinal cancer
Platinum drugs
target DNA
Alkylate & cross link DNA or can alkylate a protein forming protein-DNA cross link
Conventional chemotherapy
Anthracyclins (e.g. doxorubicin)
target Topoisomerase IIa
stabilize a transient complex with DNA known as the cleavage complex
for Breast, endometrial, acute leukemia, cervical, stomach & lymphoma cancer
Etoposide
target Topoisomerase IIa
Stabilize a transient complex with DNA known as the cleavage complex
Lung and testicular cancer
ICRF-193
Target topoisomerase IIa poison but also can chelate cellular iron preventing ROS
stabilizes the closed clamp complex with DNA but is more effective at chelating iron
Used to circumvent anthracyclin induced ROS and cardiotoxicity.
