To die, to sleep - - PowerPoint PPT Presentation

To die, to sleep… Что отличает бактерицидные антибиотики от бактериостатических

Александр Манькин

(Alexander Mankin) Center for Biomolecular Sciences, University of Illinois at Chicago

Ribosome is the most complex and sophisticated molecular machines in the cell

bacteria grow antibiotic plate

Bactericidal drugs kill bacteria Bactericidal

incubate

Bacteriostatic drugs stop bacteria from growing but do not kill them. Bacterial cells resume growth after removal of the antibiotic

Bacteriostatic Antibiotics could be bacteriostatic or bactericidal

The Macrolide Family

O O HO O OH OH HO O O O O OH N O

Ketolides:Telithromycin (TEL)

O N O O O O O O N O O O N N N

Erythromycin (ERY)

Peptide exit tunnel Catalytic center

Macrolides bind in the peptide exit tunnel

Common wisdom: Macrolides plug the tunnel and block synthesis of all the proteins at early rounds of translation

TEL ERY No antibiotic

Different macrolides inhibit synthesis of different proteins Macrolides act as protein- and context-specific inhibitors of translation

RNA as drug target

Do different macrolides exhibit different killing activity?

Streptococcus pneumoniae

Ery is static, but Tel is highly cidal

0.01 0.10 1.00 10.00 100.00 10 20 30 40 50 60 70 80 90 100

% survival Antibiotic concentration, fold MIC

+ Ery + Tel

Surviving cells after 5 h incubation with the drugs

O HO O OH O O O N OH O O O OH OH O N O O N N N O O O O O O N OH

Ery Tel

Erythromycin Telithromycin Solithromycin

O HO O OH O O O N OH O O O OH OH O N O O N N N NH2 O O F O O O O N OH O N O O N N N O O O O O O N OH

BACTERIOSTATIC BACTERICIDAL

Ery Tel Sol

Static ERY and cidal TEL inhibit synthesis of different proteins

no drug Ery Tel

Hypothesis 1: Residual translation defines cidality

10 100 1000 10000 100000 1000000 2 4 6 8 10 12

CFU/mL Incubation time, hrs

+ Tet

x40 MIC Tet

Does macrolide-induced cidality require protein synthesis?

x40 MIC Tet no drug Tet (static) Tel (cidal)

time

KILL TEST

?

Tetracycline Telithromycin

protein synthesis is required for cidality cidality does not depend of residual translation

Tel remains bactericidal even cells could make no proteins

10 100 1000 10000 100000 1000000 2 4 6 8 10 12

CFU/mL Incubation time, hrs

Tel Tet Tet Tel

Hypothesis 1: Residual translation

Hypothesis 2: Affinity of the drug for the ribosome defines cidality

Ribosome + + + + ++ DEAE magnetic beads

[14C]-Erythromycin

• -
• Ribosome

Equilibrium affinity of bactericidal SOL is comparable to the affinity of bacteriostatic ERY

20 40 60 80 100 100 200 300 400 Concentration of added drug, nM Ribosome-bound [14C]ERY, CPM

100 200 300 20 40 60 Bound, CPM Bound/Free

20 40 60 80 100 100 200 300 400 Concentration of added drug, nM Ribosome-bound [14C]SOL, CPM

100 200 300 20 40 60 Bound, CPM Bound/Free

[14C]-SOL [14C]-ERY KD = 4.9 ± 0.6 nM KD = 5.1 ± 1.1 nM Hypothesis 2: Affinity of the drug for the ribosome defines cidality

Hypothesis 3: Kinetics of the drug dissociation defines cidality

Drugs with similar affinities could have significantly different rates of binding and dissociation Kd = [A][B] [AB]

[AB] [A] + [B] Kd = kd/ka

kd ka

1 2 3 4 5 6 24 20 40 60 80 100 Time, hours Ribosome-bound [14C]SOL, %

[14C]-SOL

1 2 3 4 5 6 20 40 60 80 100 Time, hours Ribosome-bound [14C]ERY, %

[14C]-ERY

Measurement of dissociation rate

Bactericidal drug dissociates very slowly from the ribosome!

Slow-dissociating drugs are bactericidal; fast-dissociating drugs are bacteriostatic

Bound to the ribosome after 30 min after drug removal

The slow off-rate requires an extended side-chain

Drug-free ribosome (%)

U2609

ERY TEL Side chain makes specific contacts with the ribosome

A752

antibiotic plate incubate

Few surviving cells (10-3 - 10-4)

Re-grow

Selection of mutants that can resist killing

U A A U

mutant: A2058U

0.001 0.01 0.1 1 10 100 0 10 20 30 40 50 60 70 80 90 100 Survival cells, % SOL concentration, fold MIC

wild type mutant

Mutant cells are susceptible to solitromycin

Bactericidal Solithromycin stop mutant cells from growing but does not kill them! Most likely the dissociation of SOL from the mutant ribosomes is fast

WHY DO SLOWLY DISSOCIATING ANTIBIOTICS KILL BACTERIA?

Protein synthesis Protein degradation

If the dormant cell runs out of any of the key factors, translation and cell growth cannot be restarted!

synthesis degradation Steady-state level of every protein is defined by the rates of synthesis and degradation Growing cells synthesis degradation Dormant cells

Lack of any key component will prevent translation from restarting (or cell from re-growing and dividing)

drug addition drug removal dissociation from the ribosome live cells During prolonged period of dormancy the cell runs out of one or several critical proteins drug addition drug removal dissociation from the ribosome dead cells

Bacteriostatic antibiotics kill cells upon the long enough exposure

0.01 0.1 1 10 100 6 12 18 24 30 36 Survival cells, % Time, hrs

Erythromycin

x4 x10 x40 x100

Implications: Ø Antibiotics need to be optimized not only for their affinity but also for the proper kinetics. Questions to address: Ø What is the limiting factor? Ø Could we convert bacteriostatic antibiotics into bactericidal if we inhibit the ‘limiting factor’?

Maxim Svetlov