Target: Bacterial ribosome (50S subunit, peptidyl transferase center) [1]
Binding affinity Kd ~ 3 nM for E. coli and S. aureus ribosomes [1]
Inhibition of bacterial coupled transcription/translation IC50 = 0.33 ± 0.02 μM (E. coli S30 lysate) [1]
Inhibition of peptidyl transferase IC50 below tight binding limit (~100 nM) [1]
Partial inhibition of fmet-tRNA binding to P-site IC50 = 17.4 ± 2.1 nM (maximum inhibition 80%) [1]

Retapamulin is a potent inhibitor of protein synthesis with an IC50 of 0.33 μM in lysates prepared from erythromycin-susceptible E. coli cells. Retapamulin (100 μM) is ineffective in inhibiting eukaryotic translation when tested in a rabbit reticulocyte lysate system with the cellular components necessary for mammalian protein synthesis. Retapamulin binds to Erys ribosomes and fully displaces the labeled ligand with an IC50 of 26.1 nM. Retapamulin partially inhibits the ability of charged, N-blocked tRNA to bind to the P-site of E. coli ribosomes, with an IC50 of 17.4 nM (maximum inhibition of 80%). Retapamulin inhibits Staphylococcus aureus and Streptococcus pyogenes with MIC90 of 0.12 μg/mL and ≤0.03 μg/mL, respectively. Retapamulin inhibits S. aureus subset with MIC50/90 values of 0.06/0.12 μg/mL. Retapamulin shows excellent activity against these isolates, with only two requiring a MIC of 0.06 μg/mL. Retapamulin is very active against the S. pyogenes isolates tested with MIC90 of 0.016 μg/mL, and based on MIC90s, is 32- and >1,024-fold more active than mupirocin and fusidic acid, respectively. Retapamulin binds to a unique site on the bacterial ribosome, and by virtue of its novel mode of action. Retapamulin (<2 mg/L) inhibits 37/52 (71%) strains of the B. fragilis group and 85/87 (98%) of the other Gram-negative bacilli. Retapamulin is more active than clindamycin, metronidazole and ceftriaxone against Propionibacterium acnes and anaerobic Gram-positive cocci. Retapamulin inhibits total viable cells (TVC), Protein synthesis and 50S subunit synthesis in both wild-type (wt) Staphylococcus aureus strain RN1786 with IC50 of 12 ng/mL, 5 ng/mL and 27 ng/mL, respectively.

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In Vitro: Retapamulin potently inhibited bacterial protein synthesis in an E. coli coupled transcription/translation assay with IC50 of 0.33 ± 0.02 μM [1].
Retapamulin was ineffective in inhibiting eukaryotic translation in rabbit reticulocyte lysate, achieving less than 20% inhibition at 100 μM [1].
Retapamulin bound to E. coli erythromycin-susceptible ribosomes with high affinity, displacing a fluorescently labeled pleuromutilin derivative with IC50 of 26.1 ± 3.6 nM in a fluorescence polarization competitive binding assay [1].
Retapamulin bound to both E. coli and S. aureus ribosomes with similar potencies (Kd ~ 3 nM), with association and dissociation rates: for E. coli, kon = (3.0 ± 0.4)×10^5 M^-1 s^-1, koff = (8.3 ± 1.0)×10^-4 s^-1; for S. aureus, kon = (6.3 ± 2.9)×10^4 M^-1 s^-1, koff = (2.0 ± 0.2)×10^-4 s^-1 [1].
Retapamulin inhibited ribosomal peptidyl transferase activity in a puromycin-based scintillation proximity assay with IC50 below the tight binding limit (~100 nM) [1].
Retapamulin partially inhibited the binding of [3H]fmet-tRNA to the P-site of E. coli ribosomes with IC50 of 17.4 ± 2.1 nM (maximum inhibition 80%) [1]. Schild analysis indicated that the inhibition was non-competitive and allosteric [1].
Retapamulin enhanced the reactivity of nucleotides A2058, A2059, and A2062 in 23S rRNA to dimethyl sulfate (DMS) modification: A2058: 147% enhancement, A2059: 635% enhancement, A2062: 44% enhancement, as measured by fluorescent chemical rRNA footprinting [1].

Enzyme Assay: For the fluorescence polarization competitive binding assay, bacterial ribosomes were incubated at 37°C for 15 min before dilution in binding buffer (20 mM HEPES pH 7.5, 50 mM NH4Cl, 10 mM MgCl2, 0.05% Tween 20). A BODIPY-labeled pleuromutilin derivative (SB-452466) at 5 nM was preincubated with 30 nM E. coli erythromycin-susceptible ribosomes for 30 min. Test compounds diluted in 10% DMSO were mixed with the ligand-ribosome mixture in a 96-well plate (final volume 40 µL per well) and incubated at room temperature for 2 h. Fluorescence polarization was measured (excitation 485 nm, emission 530 nm). IC50 values were determined by fitting binding data to a four-parameter IC50 equation [1].
For radiolabeled competitive binding kinetics, bacterial ribosomes were incubated at 37°C for 15 min then diluted in binding buffer (for S. aureus: 25 mM MgCl2). A radiolabeled pleuromutilin ([3H]SB-258781) was used. 20 nM unlabeled retapamulin was mixed with ribosomes (5-15 nM) and [3H]SB-258781 (15-19 nM) in 100 µL final volume. Binding was competed at room temperature over time (2 h for E. coli, 5 h for S. aureus). Free and bound ligands were separated by filtration through a filter plate using a cell harvester. Nonspecific binding was determined by displacement with 10 µM SB-268091. Radioactivity was measured by scintillation counting. Binding data were fit to kinetics of competitive binding model [1].
For the puromycin assay (peptidyl transferase inhibition), E. coli tRNAfmet was aminoacylated with [3H]methionine and formylated. MVF mRNA (120 nt) was transcribed. Reaction mixtures containing P buffer (50 mM HEPES pH 7.5, 100 mM NH4Cl, 15 mM Mg(OAc)2), 1 mM DTT, 50 nM 70S ribosomes, 125 nM mRNA, and compound (1% DMSO) were incubated at 37°C for 10 min. [3H]fmet-tRNA (50 nM) was added and incubated 30 min at 37°C. After cooling, 500 nM biotin-puromycin was added and incubated 3 h. Reactions were quenched with streptavidin-SPA beads. The [3H]fmet-puromycin-biotin product was quantified by scintillation proximity counting [1].
For P-site binding assay, [3H]fmet-tRNA, tRNAphe, and retapamulin in 10% DMSO were added to a 96-well plate. E. coli ribosomes (50 nM final) were incubated at 37°C for 15 min, diluted in P buffer, and added to the plate (final: 50 nM ribosomes, 100 nM [3H]fmet-tRNA, 50 nM tRNAphe). Binding reactions were incubated at 37°C for 30 min, then placed on ice. Bound and free ligands were separated by filtration using a filter plate, washed twice with P buffer, dried. Radioactivity was measured by scintillation counting. For Schild analysis, retapamulin was titrated at four different concentrations of [3H]fmet-tRNA (10-200 nM) [1].
For fluorescent chemical rRNA footprinting, E. coli MRE600 ribosomes (200 nM) were activated at 42°C for 5 min and incubated with 200 µM compound at 37°C for 20 min then ambient temperature for 10 min in buffer (40 mM HEPES pH 7.4, 150 mM KCl, 10 mM MgCl2, 6 mM β-mercaptoethanol). Chemical modification of adenine residues was performed by adding DMS (1:12 in ethanol) and incubating at 37°C for 10 min. Reactions were stopped by ethanol precipitation. Methylation pattern was monitored by primer extension using reverse transcriptase with a 5,6-carboxyfluorescein-labeled DNA primer (oLM7: 5'-CCT ACA CAT CAA GGC TC-3') to screen A2058, A2059, and A2062. Samples were separated by capillary gel electrophoresis and analyzed using GeneScan software [1].

Cell Assay: For the bacterial coupled transcription/translation assay, an E. coli S30 lysate was used with a luciferase reporter gene. The 2× TnT premix contained 625 µM each of 20 natural L-amino acids, 700 mM potassium L-glutamate, 5 mM ATP, 1.25 mM each CTP, UTP, GTP, 50 mM phosphoenol pyruvate, 44 mM HEPES, 44 mM Tris-acetate, 125 mM ammonium acetate, 2.5 mM cyclic AMP, 50 µg/mL folic acid, 250 µg/mL E. coli tRNA, 22.5 mM magnesium acetate, 2 mM IPTG, 5 mM DTT, and 87.5 mg/mL polyethylene glycol 8000 (pH 8.0). Final reaction conditions: 1% DMSO, 1× TnT premix, 4 mg/mL S30 lysate, 0.02 mg/mL pDNA-luc, and test compound (0-50 µM). Reaction mixtures (20 µL) containing compound, TnT premix, and S30 lysate were preincubated for 15 min at 37°C. Reactions were started by addition of 5 µL pDNA-luc and incubated at 37°C for 45 min, then ambient temperature for 10 min. Luciferin substrate reagent (25 µL) was added, and luminescence was measured. IC50 values were calculated [1].
For the rabbit reticulocyte assay (eukaryotic translation inhibition), a rabbit reticulocyte lysate system was used with luciferase mRNA as template. Inhibitors were tested from 2 nM to 100 µM with final DMSO concentration of 1%. Full-length luciferase was quantified using a luciferase assay system. Retapamulin showed less than 20% inhibition at 100 µM [1].

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the use of Retapalline during lactation. Because Retapalline is poorly absorbed after topical application, it is unlikely to enter the infant's bloodstream, nor will it cause any adverse effects on the breastfeeding infant if the mother applies the medication to areas other than the breast. Only water-soluble creams or gels should be applied to the breast, as ointments may expose the infant to high concentrations of mineral oil through licking. ◉ Effects on Breastfeeding Infants
As of the revision date, no relevant published information was found. ◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

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Antimicrob Agents Chemother.2006 Nov;50(11):3875-81. Epub 2006 Aug 28.

Retapamulin is a carboxylic acid ester and a cyclic ketone. Retapalline is a truncated pleurotin class of antibacterial drugs. See also: Retapalline (note moved to).

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Additional Info: Retapamulin is a semisynthetic pleuromutilin derivative being developed as a topical antibiotic for treating bacterial infections of the skin. It is potent against susceptible and multidrug-resistant organisms commonly associated with bacterial skin infections [1].
Unlike macrolides (erythromycin, azithromycin, clarithromycin), retapamulin does not show cross-resistance with these classes; macrolides did not displace pleuromutilin ligand from ribosomes [1].
Retapamulin showed a low propensity for development of resistance, indicating low likelihood of resistance via target mutation during therapy [1].
The mode of action of retapamulin is distinct from other classes of antibiotics: it binds to the peptidyl transferase center, inhibits peptide bond formation, and partially inhibits P-site tRNA binding through an allosteric mechanism [1].
X-ray crystallographic data for tiamulin (a related pleuromutilin) show interactions with both A- and P-sites of the ribosome, and retapamulin is expected to have a similar binding mode [1].

C30H47NO4S

517.76

517.322

C, 69.59; H, 9.15; N, 2.71; O, 12.36; S, 6.19

224452-66-8

6918462

White to off-white solid powder.

1.2±0.1 g/cm3

594.9±50.0 °C at 760 mmHg

313.6±30.1 °C

0.0±3.8 mmHg at 25°C

1.571

5.45

1

6

6

36

895

10

S(C([H])([H])C(=O)O[C@]1([H])C([H])([H])[C@](C([H])=C([H])[H])(C([H])([H])[H])[C@]([H])([C@]([H])(C([H])([H])[H])[C@]23C([H])([H])C([H])([H])C([C@@]2([H])[C@@]1(C([H])([H])[H])[C@]([H])(C([H])([H])[H])C([H])([H])C3([H])[H])=O)O[H])C1([H])C([H])([H])[C@]2([H])C([H])([H])C([H])([H])[C@]([H])(C1([H])[H])N2C([H])([H])[H]

STZYTFJPGGDRJD-QPCPVAGTSA-N

InChI=1S/C30H47NO4S/c1-7-28(4)16-24(35-25(33)17-36-22-14-20-8-9-21(15-22)31(20)6)29(5)18(2)10-12-30(19(3)27(28)34)13-11-23(32)26(29)30/h7,18-22,24,26-27,34H,1,8-17H2,2-6H3/t18-,19+,20-,21+,22-,24-,26+,27+,28-,29-,30+/m1/s1

(3aR,4S,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-(((1R,3s,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)thio)acetate

SB-275833; SB 275833; SB275833; Retapamulin, trade names Altabax and Altargo.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 104~110 mg/mL ( 200.86~212.45 mM )
Ethanol : ~104 mg/mL

Solubility in Formulation 1: ≥ 2.75 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.75 mg/mL (5.31 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)