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Improved Antimicrobial Activity of Fully Synthetic Macrolides against Macrolide Resistant Gram-positive Isolates
Sushmita D. Lahiri and Richard A. Alm Macrolide Pharmaceuticals, 480 Arsenal St., Watertown, MA 02472
Background: Rapid emergence of resistance by multiple drug resistant (MDR) pathogens has intensified the search for new antimicrobial agents that are safe and efficacious. Methicillin-resistant Staphylococcus aureus (MRSA) and MDR Streptococcus pneumoniae remain a clinical concern for a variety of community-associated and hospital-acquired infections. Macrolides are a class of known antibiotics that are safe and effective. However, the macrolide class has limited activity against Gram-negative pathogens, as well as against Gram-positive isolates carrying macrolide resistance determinants. In particular, the utility of macrolides against MDR gram-positive pathogens, including MRSA, has been hampered primarily by various changes to the binding pocket, including the methylation of the macrolide binding nucleotide by erm methylases. Methods: Novel macrolides were synthesized via a fully synthetic, chemistry platform. These compounds were tested against characterized clinical isolates following CLSI methods. Results: These new compounds demonstrated improved activity against strains resistant to older generations of macrolides. They do not induce the ermA methylase gene in S. aureus or the ermB methylase gene in S. pneumoniae. They were also tested against MRSA strains that contained a variety of alterations in the leader peptide, resulting in constitutive expression of the ermA or ermC gene. The novel macrolides described here showed a >16-fold improvement in MIC compared to in-class comparators which had MIC values of >256 mg/L. Further, these compounds were not affected by efflux-based resistance mechanisms, such as Mef(A) or Msr(A), found in S. pneumoniae and S. aureus, with MIC values of ≤ 1 mg/L against these isolates. Lastly, S. pneumoniae isolates carrying 23S rRNA and ribosomal protein mutations that result in reduced susceptibility to macrolides remain susceptible to these novel compounds. Conclusion: These data demonstrate that leveraging the flexibility of the synthetic chemistry platform should identify promising candidate molecules that circumvent the common pre-existing resistance mechanisms.
Abstract
Introduction
Results
• This synthetic platform will allow introduction of novel diversity to the macrolide scaffold. • Novel synthetic compounds maintain activity against efflux resistant strains and are
unaffected by target mutations that impact other macrolide antibiotics. • Initial exploration has identified several compounds with measurable activity against MRSA
strains that carry constitutively expressed erm methylase. • Combination experiments with Clindamycin suggest that the observed anti-MRSA activity is
mediated through inhibition of the methylated ribosomes.
Activity against Gram-positive pathogens
• Novel macrolide molecules have excellent potency against Streptococcus spp. and are unaffected by target-based (23S rRNA A2058G or RplD) mutations.
• Represent poor substrates for macrolide-specific efflux pumps (MsrA and Mef). • Do not induce erm methylase similar to other ketolides. • Several analogs display some activity against MRSA isolates with constitutively-
expressed ErmA methylase.
Activity against MRSA isolates carrying erm methylases
• Current macrolides and ketolides do not effectively cover MRSA due to erm-mediated resistance mechanism carried on many SCCmec cassette.
• Several novel macrolide analogs had differentiating activity against MRSA isolates with characterized ermA and ermC leader peptide alterations.
Not drawn to scale. LP=leader peptide; A through D are the repeat sequences that form hairpin structures involved in erm induction.
Since the isolation of Erythromycin in 1949, alterations to the macrolide scaffold to identify analogs with improved properties has been primarily done by semi-synthesis. Recently Seiple et al. (1) published a fully synthetic modular platform that enables the complete synthesis of novel macrolides. This chemistry will support the synthesis of analogs that had previously been unattainable. With new points on diversification now being accessible, the construction of compounds that can address the limitations of current macrolide drugs, while maintaining the beneficial drug-like properties emerges as an attainable goal. Acknowledgments
The authors would like to thank past and present members of the laboratory of Prof. Andy Myers at Harvard University, especially Ian Seiple and Ziyang Zhang, for the development of the fully synthetic macrolide platform and synthesis of the novel compounds.
Reference 1. Seiple, IB. et al. (2016). A platform for the discovery of new macrolide antibiotics. Nature 533: 338-345.
Conclusions
Contact: Dr. Sushmita D. Lahiri Macrolide Pharmaceuticals [email protected] +1 617 453-9091
Poster # SUNDAY
485
Component modification of BB-1 to BB-7 allows for multiplicative expansion of structural diversity Division into components enables greater overall complexity
Combination testing
• Susceptibility testing in a MRSA isolate was performed in combination with increasing concentrations of clindamycin which is a potent inhibitor of non-methylated ribosomes.
• The presence of clindamycin would selectively inhibit non-methylated ribosomes. • Azithromycin and Solithromycin both remained inactive even in the presence of high
concentrations of clindamycin • Novel macrolide scaffolds maintain their anti-MRSA potency in presence of clindamycin:
FSM-100490 FSM-100563 FSM-100573 FSM-100426
alone + CLI 4 + CLI 16 +CLI 64
Azithromycin 1 >1024 >1024 >1024 >1024
Clindamycin 0.125 1024 1024 1024 1024
Solithromycin 0.125 >256 >256 >256 >256
FSM-20707 0.5 >64 >64 >64 >64
FSM-100426 0.125 32 16 16 16
FSM-100490 <0.06 32 32 32 32
FSM-100563 0.125 32 32 32 32
FSM-100573 <0.06 16 16 16 16
DrugMSSA
(MP-12)
MRSA (MP-513 - cErm)mg/L of clindamycin
Results
FSM-20707
Strain Species Genotype
Azi
thro
my
cin
Ery
thro
my
cin
Te
lith
rom
yci
n
So
lith
rom
yci
n
FSM
-10
04
79
FSM
-10
04
90
FSM
-10
06
27
FSM
-10
06
33
FSM
-10
05
73
FSM
-10
05
63
MP-12 S. aureus ATCC29213 1 0.5 0.25 0.125 0.06 0.03 0.06 0.125 0.06 0.06
MP-17 S. aureus BAA-977 (iErmA) >256 >256 0.25 0.125 0.06 0.03 0.06 0.125 0.06 0.06
MP-513 S. aureus MRSA (cErmA) >256 >256 >64 >64 >64 64 32 64 16 16
MP-549 S. aureus USA300 (Msr(A)) 128 64 0.25 0.125 0.125 0.06 0.125 0.125 0.06 0.125
MP-19 S. pyogenes ATCC19615 0.015 <=0.002 0.015 0.008 0.008 0.008 0.008 0.004 0.004 0.008
MP-625 S. pyogenes macrolide-R 2 4 0.25 0.06 0.06 0.06 0.03 0.06 0.03 0.03
MP-21 S. pneumoniae ATCC49619 0.06 0.03 0.008 0.008 0.008 0.004 0.008 0.008 0.004 0.008
MP-586 S. pneumoniae macrolide-R (23S) 64 4 0.008 0.008 0.004 0.004 0.004 0.002 0.004 0.004
MP-589 S. pneumoniae macrolide-R (L4) 64 32 0.03 0.015 0.03 0.008 0.015 0.008 0.004 0.015
MP-590 S. pneumoniae macrolide-R (iErmB) >256 >256 0.015 0.015 0.015 0.008 0.015 0.008 0.008 0.015
MP-592 S. pneumoniae macrolide-R (MefA) 1 0.25 0.03 0.008 0.008 0.004 0.008 0.008 0.004 0.008
MP-599 S. pneumoniae fluoroquinolone-R 1 1 0.015 0.008 0.004 0.004 0.008 0.004 0.004 0.008
MP-602 S. pneumoniae fluoroquinolone-R 1 1 0.06 0.015 0.015 0.008 0.015 0.008 0.008 0.008
MP-632 S. pneumoniae macrolide-R (ErmB/MefA) >256 >256 0.25 0.06 0.03 0.015 0.03 0.03 0.03 0.03
MP-24 E. faecalis ATCC29212 4 1 <=0.03 <=0.03 1 0.03 0.03 0.03 0.03 0.03
MP-624 E. faecalis vancomycin-R (ErmB) >256 >256 16 32 32 2 1 1 1 2
Azi
thro
myc
in
Sol
ithr
omyc
in
FSM
-100
426
FSM
-100
427
FSM
-100
490
FSM
-100
563
FSM
-100
573
FSM
-100
593
1 0.125 0.125 0.25 < 0.06 0.06 < 0.06 0.125
>256 0.125 0.06 0.125 0.06 0.06 0.06 0.125
>256 0.125 0.125 0.25 0.06 0.06 0.06 0.125
>256 >128 32 64 >64 64 32 >64
>256 >128 64 >64 >64 64 64 >64
>256 >128 32 32 >64 32 32 64
>256 >128 32 64 >64 32 32 64
>256 >128 16 64 64 32 16 64
>256 >128 32 32 32 32 16 64
>256 >128 16 32 64 32 32 32
>256 >128 32 64 64 32 32 64
>256 >128 32 64 64 32 32 64
>256 >128 32 64 64 32 32 64
MIC (mg/L)
