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Flashcards in Antibiotics blocking protein synthesis Deck (22):
1

PG Assembly

Vancomycin blocks transpeptidation step in addition to penicillins and cephalosporins by binding DalaDala on the end.

2

Mycobacterial Cell Wall

The mycobacterial cell envelope is extremely hydrophobic and forms a strong barrier: Mtb is highly resistant to AB.
The mycobacterial cell wall contains long fatty acids (C60-C90, lipophilic aliphatic carbons), the mycelia acids. Channel forming proteins functionally similar to the well known porins of GN bacteria have been demonstrated, revealing how hydrophilic molecules can pass through their hydrophobic cell wall.
In addition to PG, there is an arabinoglactan.
Encoated in grease; big layer that makes it hard to treat; in part reason it is highly resistant to drugs.

3

Isoniazid

AB exclusively used for mycobacterium TB.
Analog of nicotinamide
Clinical use:
-In combination with other drugs for the treatment of TB.
-As monotherpay for treatment of latent TB/
-In combination regimen for nonTB mycobacterial infections (less frequently).
Mode of action: complex
-Acts as a mimic of nicotinamide.
-Complex, Isoniazid-NAD adduct inhibits INHa
-The best defined mechanism is inhibition of mycelia acid (big greasy barrier on outside) biosynthesis; also different mechanisms; not precisely defined; unique to mTB because of mycolic acids.
Beta lactams that hit cell walls are more active against fast growing.
TB is slow growing
-Bactericidal but less active against fast growing cells.
Side effects:
-Low in general
-Peripheral neuropathy and CNS toxicity.
Other antiTB drugs: Rifampin, Streptomycin, Pyrazinamide, Ethambutol, Ethionamide.
Typical treatment: Rifampin, Isoniazid, Ethambutol, Pyraziamide 6-9 months for non-resistant TB).
Drug sensitive; typically 4 drugs for 3-6 months, then drop to 2 drugs for another 9 months.
Resistant: 2 years of therapy.

4

Inhibitors of protein synthesis

Efflux not as important are not as important then for things that need to be inside the cell to act.
Mechanisms of resistance.
AB need to be inside the cell to act.

5

Inhibitors of Protein Synthesis

Structure of the ribosome; 2 parts, one of the 50S and one is the 30S.
Antibiotics block protein biosynthesis by acting on one or more steps that occur on the 30S and 50S subunits of the bacterial ribosome.
Drugs that act at the 50S:
-Macrolides
-Lincosamides
-Streptogramins
-Everninomycins
-Oxazolidinones
-Lincosamides.
Drugs that act at the 30S subunit:
-Aminoglycosides
-Tetracyclines
Polypeptide exit tunnel
tRNAs
Ribosome has 3 sites:
A site: Aminoacyl
P site: peptidyl
E site: Exit
Crystallized structure finally gave us a view of how these AB work.
RNA in ribosome; drugs bind and interact with RNA, not structural only.

6

Ribosome Diversity

Bacteria
Ribosome Size: 70S
Small subunit:
Size- 30S
Mass (MDa)-0.8
rRNAs-16S
Number of r-proteins-20
Large subunit:
Size-50S
Mass (MDa)-1.6
rRNAs-23S, 5S
Number of r-proteins-34
Higher the number the bigger the size.
Eukaryotes: much larger ribosome: individual subunits are different sizes, rRNAs are different
Comprised of proteins and RNA.
Why we can target protein synthesis in bacteria and not have toxicity in humans; because of huge differences.
54 totally proteins to put together.

7

Basic ribosome structure: mRNA and tRNA

Threading of the mRNA into 30S subunit decoding region.
Placement of the ammoniacal (A), peptide (P), and peptide exit (E) codons of the mRNA in the decoding site.
Architecture of a tRNA highlighting the anti-codon loop that recognizes the codons on mRNA and the CCA talk where the main acid is covalently tethered and activated.
The mRNAs provide the instructional template.
The tRNAs transfer amino acids to the growing peptide chain and provide the anticodons for Watson Crick pairing at the A and P site at the 30S-mRNA interaction site.
tRNA responsible for tethering the right AA.
mRNA provides code for tRNA to bind; which AA will be put into growing protein.
A is where the tRNA comes in and the anticodon loop bp with that region and dictates which AA is next in line for potion synthesis.
P: action happens here; where incoming AA attached to CCA end of tRNA is pushed into, catalyzed to grow the peptide chain.
Signed up by shine dalgarno sequence; interact with 30S and lines up whole initiation complex here.
30S is the decoding subunit.
50S helps us catalyze things.
1. 30S- responsible for reading mRNA codons
2. Initiator tRNA for methionine in green-decoded in the A site.
3. If there is a match, the interfaces come together and translation begins.
See AA end of tRNA harbored in the 50S, where the chemistry happens.
30S decodes mRNA and tRNA; protein synthesis happens in the 50S.

8

Bacterial ribosome: the peptidyltransferase cycle

Step 1: Select the proper amino-acyl tRNA at A site; match with mRNA.
Step 2: Elongate the growing peptide-tRNA (at P site) as it is translocated onto the attacking ammoniacal group (at A site); P site have growing or nearly made peptide chain attached to previous tRNA; AA attached via carboxyl group to OH on the sugar at the CCA end of tRNA; amino group on AA is positioned in ribosome and ribosome catalyzes the nucleophilic attack of N on that mine group onto the attached polypeptide chain in the P site; when this happens, AA from A site to then form the new amide bond with peptide chain at P site and move on...
Step 3: Move the deacylated tRNA to E site (exited) and relocated the peptidyl-tRNA to P site.
Step 4: Select the next ammoniacal-tRNA to A site to start a new cycle of peptide elongation.
Antibiotics could interrupt the timing and specificity of any of these steps, and such disruptions are likely to slow down growth and/or be lethal to the bacteria.
No clear cut rules of whether tidal or just slow down growth.

9

Interact with the 30S Ribosomal Subunit

Tetracycline
Spectinomycin (not approved)
Kanamycin
(aminoglycosides)
Streptomycin; the first early protein synthesis inhibitor.

10

Interact with the 50S Ribosomal Subunit

Erythromycin; early members of this class
Ketolide
Troleandomycin
Azithromycin (zythromax); modified form of erythromycin.
Telithromycin
Cethromycin
Streptogramin A
Streptogramin B
Pristinamycin IIA
Virginiamycin M
Dalfopristin
Pristinamycin IA
Quinipristin
Linezolid

11

30S Subunit of the Ribosome (decoding)

1. Aminoglycosides: Interfere with initiation
2. Tetracyclines and Glycylcyclines: Block the incoming ammoniacyl tRNA.

12

Business end in terms of the peptide transfer center: 50S

3. Macrolides and Ketolides: Bind at the polypeptide exit tunnel; block exit and stars biosynthesis of proteins.
4. Lincosamides: Inhibt transpeptidation/translocation
5. Amphenicols: Peptidyl transfer; chloramphenicols as example.
6. Streptogramins: Inhibit transpeptidation/translocation
7. Oxazolidinones: Initiation Inhibitor
8. Mutilins: Peptidyl transfer

13

Aminoglycoside Antibiotics: Structures

Gentamicin C
Tobramycin
Netilmicin
All have same core in center
PRIMARILY USED FOR GN BACTERIAL INFECTIONS.
Streptomycin discovered in 1944
Aminoglycosides have been widely used in clinical setting due to bactericidal action, and synergy with other antibiotics such as beta lactams.
The hydrophilic sugars with multiple amino groups are protonated at physiological pH to function as polycations to target 16S rRNA; a lot of RNA where things are decoded and where peptide transfer occurs; pka of amino group is around 9, 99% protonated; physiology pH all these N are protonated; highly charged molecule in the gut does not transfer very well across mucosal membranes; not orally available, available via IV.
Not first line of AB therapy-pretty toxic.
Act on hairs in ears and effect hearing
Major side effects: nephrotoxciity and ototoxicity (ear).

14

Aminoglycoside AB: Mode of Action

Aminoglycosides bind the 16S rRNA on the 30S subunit notably at the A site for ammoniacal-tRNA binding.
RNA: big phosphate backbone; molecule with charged amino groups; nice attraction, negative and positive charges drives binding of aminocglycosides to ribosome.
Effects: see the inhibition of initiation of porin synthesis; blocks further translation and elicits premature termination.
Also results in incorporation in incorrect AA.
Cidal AB; complicated mechanism of doing more the stalling synthesis; hangs out, not diving and rapidly growing because cannot make proteins but won't kill; doing this however, sending chunks of protein that aren't fully formed=kill.
A site; where maniacal tRNA is coming in and binding.
Interaction of Gentamicin with minimized A site.
Resistance of AC; interaction of 3 rings of Gentamicin with the bases involved in fundamental A site.
See these positively charged groups interacting with P, idea that these positive charges interacting with specific P on RNA A site drives the inhibition of protein synthesis.
Ionic interactions are strongest; breaking interactions have to do with resistance mechanisms of AC.
Potent inhibitors of protein synthesis.

15

Tetracyclines and Glycylcyclines

Tetracycline
Chlortetracycline
Oxytetracycline
These last 3: 1940s-50s
Doxycycline (1950s)
DMG-MINO (1970s)
Tigecyline (2000s); glycylcycline; glycine on the side.
Structures are the major members of the tetracycline family of ABs
The recent approval of tigecycline, which is resistant to efflux, indicate continued interest in this class of ABs.
Primary mode of resistance to tetracycline is efflux (outside of cell less sensitive to efflux resistance mechanisms).
Major side effects: GI irritation, photosensitivity, discoloration of teeth.

16

Tetracyclines and Glycylcyclines: Mode of Action

Tetracyclines:
Binding site for tetracycline with 16S rRNA on the 30S subunit and; block incoming aminoacyl tRNA; bind in 30S so incoming tRNA cannot bind.
-Tetracyclines work at the 30S subunit to block binding of the incoming ammoniacal-tRNAs to the A site.
-Tetracyclines are bacteriostatic; stall proteins synthesis, bacteria can survive and hang out.

17

Erythromycin Clas of Macrolide ABs

Erythromycin: Interactions with the 23S RNA.
Mode of action of the macrolide ABs:
-binding of macrolides at the 50S polypeptide exit tunnel
Interaction with the 23S RNA base
Bacteriostatic
-The architecture of the macrolactone and the interactions of the sugar are key determinants of binding and specificity for interaction with the 23S RNA.
-Erythromycin binds at the entrance to the peptide export channel, allowing (peptides of 6-8 AA) 6-8 oligopeptidyl-tRNA buildup before elongation is blocked and prematurely terminated.
A2058 is very important in terms of resistance; methylate this and mutants who have different base=complete resistance to erythromycin.

18

Three generations of Macrolides Antibiotics

1. Narrow-spectrum: erythromycin (first one discovered); not very stable in stomach, not great tissue penetration
2. Expanded-spectrum macorlides: clarithromycin and azithromycin: less GI tract irritation, more acid stable, better tissue penetration (in lungs treating resp infection), long half lives, a single dose a day.
Change OH on EM to OCH3 you get clarithromycin.
3. Broad-spectrum ketolides: telithromycin: does not induce rRNA methylation, resistance gene expression.
RNA methylation is one route to resistance for macrolides; induced by the molecules, not telithromycin.
Major side effects: GI irritation, hypersensitivity.

19

Overlap of Macrolides and other protein biosynthesis inhibitors

Overlap with the binding sites for clindamycin, chloramphenicol, erythromycin, and telithromycin.
Structures of antibiotics that are overlapped in A
Macrolides: block polypeptide exit
Lincosamides (such as clindamycin): inhibit peptidyltransferase center.
Chloramphenicol: inhibts peptidyltransferase center.
Cross resistance: MLSb resistance (macrolides, lincosamides, and streptogramin B family members).
Resistance profiles; all overall and close to A2058; resistance one of these, typically have resistance to all of them; resistance to erythromycin, will not pick clindamycin as next drug, will pick something totally different.

20

Synergistic Peptide Combinations: Synercid

Quinupristin and Dalfopristin are the antibiotic components in Synercid.
Few classes of AB where it is combination therapy.
Natural products produced by bacteria.
On their own, not useful.
rRNA on 50S; when dalfoprostin binds; U2585, without D bound; when D binds, cause a rotation in uridine to flip up and see this, now Q has tight binding of that structure.
Idea is that D binds first and provides sight for Q to bind; when both bound, complex is nice strong complex and inhibits protein synthesis.
-Work synergistically to block polypeptide translation by the 50S subunit of bacterial ribosome at sites partially overlapping those targeted by the macrolides; hence resistance profile overlaps.
-Members of group A (or group 1) are cyclic peptides.
-Members of group B (or group 2) are cyclic peptide-polyketide hybrids; biosynthetically stem from acetate biosynthesis.
-The modifications improve water solubility and have allowed clinical approval for the treatment of VRE infections.
Originally not very water soluble; both have amine and form a salt; reasonably pharmaceutical properties, not great but useful.
-Major side effects: mild.

21

Linezolid: Synthetic Oxazolidinone Antibiotic

Linezolid (Zyvox): the only totally synthetic antibiotic in clinical use that blocks protein synthesis.
FDA approval in 2000
The core pharmacophore, the oxazolidinone ring, was the first structurally novel AB to be introduced in 3 decades; carbamate but oxygen carbonyl, nitrogen functional group in 5 membered ring. Drives the name!
Since linezolid: daptomycin (Cubicin-2004), fidaxomycin (Dificid-2011).
Mode of action:
Binds to the P site in the peptidyl transferase center, hence blocking the first peptide bond-forming step in protein synthesis.
-Most active against GP bacteria including VRE and has high oral bioavailability.
-Major side effects: hypersensitivity.
High oral bioavailability.
Without this you need patient in hospital to give drug.

22

Tedizolid: Second Genration Oxazolidinone

Recent approval
Active against linezolid resistant Staphylococci
~10 times more potent than linezolid against MRSA.