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Ceftazidime / avibactam Michel Arthur Laboratoire de Recherche Molculaire We're gonna geed a bigger boat Spellberg B, Bonomo RA sur les Antibiotiques Centre de Recherches des Cordeliers INSERM UMR S 1138 Equipe 12 A new generation of

  1. Ceftazidime / avibactam Michel Arthur Laboratoire de Recherche Moléculaire “We're gonna geed a bigger boat” Spellberg B, Bonomo RA sur les Antibiotiques Centre de Recherches des Cordeliers INSERM UMR S 1138 Equipe 12

  2. A new generation of β -lactamase inhibitors: Structure 1 st generation : β -lactam 2 nd generation : Diazabicyclooctane Clavulanate Sulbactam Avibactam Tazobactam

  3. Different modes of action E S ES ES* E S* Detoxified β -lactamase + β -lactamase + Substrate Non-covalent Acylenzyme β -lactam complex β -lactam Imipenem HO HO Bla Bla Bla H 2 O E + I EI EI* E I* Non-covalent Acylenzyme + H 2 O β -lactamase + Detoxified complex inhibitor I = Inhibitor Clavulanate Bla Secondary acylenzyme + CO 2 E + I EI* E I* EI Avibactam Acylenzyme + H 2 O β -lactamase + Non-covalent Detoxified complex inhibitor Bla

  4. Inactivation spectrum of avibactam β -lactamase Questions to be addressed:  Are there naturally- occurring “resistant” class A β -lactamases? Class A Active  Are variations in the efficacy of avibactam and clavulanate Class B Inactive positively correlated? negatively correlated? Class C Active independent? Class D Variable  Is acquisition of resistance to avibactam- β -lactam combinations possible following changes in the sequence of the β -lactamases under the selective pressure of the drugs?

  5. Naturally occurring β -lactamases not inactivated by avibactam β -lactamase Inhibitor BlaC M . tuberculosis Bla Mab M . abscessus k 2 / K i = 230,000 M -1 s -1 Rapid inactivation No inactivation, Hydrolysis Clavulanate No deacylation k - 2 = 0 k cat / K m = 210,000 M -1 s -1 Insignificant hydrolysis k 3 = 0.0001 s -1 Rapid inactivation Slow inactivation k 2 / K i = 480,000 M -1 s -1 Avibactam k 2 / K i = 24 M -1 s -1 k -2 = 0.00002 s -1 k 1 k 2 k 3 Association Acylation Hydrolysis Red: Full “ i rreversible” E + I* E + I EI EI* inactivation Dissociation Deacylation Blue : Partial or no k - 1 k - 2 inactivation K i = k -1 / k 1

  6. β β Structural data provided a clue β -lactamase Motif Avibactam Clavulanate Inactive Bla Mab SDN Active BlaC Inactive Active SDG SDN or SDG Catalytic serine (S70) acylated by clavulanate 73 K/K 70 S/S 276 K/E 132 N/G 237 G/T 166 E/E 105 W/I Bla Mab M . abscessus BlaC M . tuberculosis

  7. Impact of SDN ↔ SDG substitutions Efficacy of clavulanate hydrolysis 6 Only Avi Both active Log( k cat / K m ) 5 Bla Mab (N 132 ) 4 3 Only Clav Both active 2 1 BlaC (G 132 ) 0 0 1 2 3 4 5 6 Efficacy of inactivation by avibactam Log( k 2 / K i )

  8. Impact of SDN ↔ SDG substitutions Efficacy of clavulanate hydrolysis 6 Only Avi Both active Log( k cat / K m ) 5 Bla Mab (N 132 ) BlaC G 132 N 4 3 Only Clav Both active 2 Bla Mab N 132 G 1 BlaC (G 132 ) 0 0 1 2 3 4 5 6 Efficacy of inactivation by avibactam Log( k 2 / K i )

  9. Conclusions  BlaC is a naturally- occurring avibactam “resistant” class A β -lactamase  Variations in the efficacy of avibactam and clavulanate are inversely correlated and determined by the SDN versus SDG motif  SDN → SDG acquisition of avibactam resistance (but increased susceptibility to clavulanate) → Do these conclusions apply to β -lactamases from Enterobacteria?

  10. Impact of SDN ↔ SDG substitutions Efficacy of clavulanate hydrolysis 6 Only Avi Both active Bla Mab N 132 Log( k cat / K m ) KPC-2 N 132 5 CTX-M-15 N 132 4 3 Both active 2 N 132 G N 132 G 1 N 132 G Only Clav 0 0 1 2 3 4 5 6 Efficacy of inactivation by avibactam Log( k 2 / K i )

  11. Conclusion  The SDN → SDG substitution has similar impacts on the spectrum of inhibition of distantly related Class A β -lactamases from mycobacteria and enterobacteria → Does this substitution lead to resistance to β -lactam/inhibitor combinations?

  12. MIC of amoxicillin (S) against E. coli producing Bla Mab (E) with or without avibactam (I) β -lactamase MIC (µg/ml) Active (black) and inactive (white) pathways PBP inactivation None 2 S PBP inactivation Bla Mab E + S S ES ES* + H 2 O E + S* >512 PBP inactivation S ES ES* + H 2 O E + S* S + Bla Mab + Avibactam 4 E + I EI EI* PBP inactivation Bla Mab N 132 G >512 E S ES ES* + H 2 O E + S* + S PBP inactivation S ES ES* + H 2 O E + S* S + Bla Mab N 132 G + Avi 64 E + I EI EI*

  13. Emergence of ceftazidime-avibactam resistance in enterobacteria (Resistance: MIC of ceftazidime > 8 µg/ml in the presence of 4 µg/ml of avibactam)  In vivo : Emergence of resistance to ceftazidime-avibactam in 8% (3/37) of the patients infected with carbapenem-resistant Enterobacteriaceae and treated with the ceftazidime-avibactam combination D 179 Y V 240 G D 179 Y + T 243 M Shields RK, Clin Infect Dis 63:1615 – 8 Antimicrob Agents Chemother 61:e02097-16  In vitro : ca . 2 x 10 -9 (ceftazidime 8 µg/ml + avibactam 4 µg/ml) D 179 Y

  14. Impact of D 179 Y in KPC-2 on the : MIC of β -lactams against E. coli Efficacy* of β -lactam hydrolysis Efficacy of inactivation by avibactam k 2 / K i (M -1 s -1 ) k -2 (s -1 ) KPC-2 D 179 Y β -lactamase β -lactam KPC-2 KPC-2 Ceftazidime 3,700 70 290,000 0.001 KPC-2 D 179 Y 0.4 0.00005 Aztreonam 69,000 Not detected Meropenem 67,000 Not detected k 1 k 2 Imipenem 730,000 Not detected Association Acylation E + I EI EI* Ceftriaxone 250,000 3,500 Dissociation Deacylation k - 1 k - 2 Clavulanate 140,000 Not detected K i = k -1 / k 1 * k cat / K m (M -1 s -1 )

  15. Conclusions  D 179 Y is sufficient for resistance to the combination since it enables the combination of: Sufficient residual ceftazidime hydrolase activity (2%) Very low acylation efficacy by avibactam (0,0001%)  D 179 Y: Abolishes resistance to aztreonam, imipenem, and meropenem Increases the efficacy of β -lactamase inactivation by clavulanate → Alternative therapies for isolates producing KPC D 179 Y? → Combine therapies to prevent emergence of D 179 Y? → Is emergence of resistance to β -lactam/inhibitor combinations possible in other Class A β -lactamases?

  16. CTX-M β -lactamases are refractory to gain of ceftazidime-avibactam resistance  Pre-existing polymorphisms: 9 single amino acid variations in the Ω loop of 172 CTX-M sequences  None was associated with ceftazidime-avibactam resistance but Two of them increased the MIC of ceftazidime (in the absence of avibactam) when introduced in CTX-M-15: P 167 S (4 fold) and L 169 Q (16 fold) Selection cefta + Avi S 130 G  L 169 Q (rare) and D 240 G (only in CTX-M-15 and derivatives) are prerequisites for the emergence of ceftazidime-avibactam resistance

  17. Cytoplasmic membrane 60-80 % Transpeptidase β -lactamase 5 paralogues Peptidoglycan Arabinogalactan Mycolic acid Porin Efflux determinants resistance Avibactam Multiciplicity of β -lactamase inhibitor β -lactam

  18. Cytoplasmic membrane 60-80 % Transpeptidase β -lactamase 5 paralogues Peptidoglycan Arabinogalactan Mycolic acid Porin Efflux avibactam Avibactam Dual role of β -lactamase inhibitor β -lactam

  19. Additional target(s) for avibactam and derivatives Inhibition efficacy ( k 2 / K i M -1 s -1 ) MIC of amoxicillin (against Δ bla ) Strain β -lactamase None Avibactam 15a Avibactam 15a M. abscessus Bla MAb >256 (4) 16 (4) 16 (4) 170,000 22,000 M. tuberculosis BlaC 128 (1) 8 (1) 16 (1) 24 < 5 Organic synthesis of avibactam derivatives S-carbamoylation of 100 Slow-binding inhibition catalytic Cys 442 of L , D -transpeptidases 80 Free enzyme (%) 60 40 20 0 0 20 40 60 80 100 120 Pre-incubation time with avibactam (min) Ldt fm (40 μM ) avibactam (1,000 μM )

  20. “We're gonna need a bigger boat” Spellberg B, Bonomo RA  Chemistry L Iannazzo, M Ethève-Quelquejeu, M Fonvielle, F Bochet  Crystallography I Galley, H van Tilbeurgh, M Fonvielle  Microbiology F Compain, JL Mainardi, E Le Run  Enzymology Z Edoo, F Compain, JE Hugonnet  A more potent “irresistible” inhibitor and/or  Diverse inhibitors (pan resistance to all β -lactam-inhibitor MycWall project combinations is at the very least uncommon)  Formulation of inhibitors independently from β -lactams → access to combinations for therapy (and research!) PhD fellowship to ZE PhD fellowship to ELR

  21. MIC of amoxicillin against E. coli strains producing BlaC from M. tuberculosis E = BlaC S = Amoxicillin β -lactamase MIC (µg/ml) I = Clavulanate PBP inactivation None 2 S PBP inactivation E + S S ES ES* + H 2 O E + S* BlaC 512 PBP inactivation S S ES ES* + H 2 O E + S* + BlaC + Clav 8 E + I EI EI* + H 2 O E + I* PBP inactivation BlaC G 132 N 512 E S ES ES* + H 2 O E + S* S + PBP inactivation S S ES ES* + H 2 O E + S* + BlaC G 132 N + Clav 64 E + I EI EI* + H 2 O E + I*

  22. MIC of amoxicillin against E. coli strains producing BlaC from M. tuberculosis E = Bla Mab S = Amoxicillin β -lactamase MIC (µg/ml) I = Clavulanate PBP inactivation None 2 S PBP inactivation E + S S ES ES* + H 2 O E + S* Bla Mab >512 PBP inactivation S S ES ES* + H 2 O E + S* + Bla Mab + Clav >512 E + I EI EI* + H 2 O E + I* PBP inactivation Bla Mab N 132 G >512 E S ES ES* + H 2 O E + S* S + PBP inactivation S S ES ES* + H 2 O E + S* + Bla Mab N 132 G + Clav 32 E + I EI EI* + H 2 O E + I*

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