Structures of FOX-4 Cephamycinase in Complex with Transition-State Analog Inhibitors

Abstract

Boronic acid transition-state analog inhibitors (BATSIs) are partners with β-lactam antibiotics for the treatment of complex bacterial infections. Herein, microbiological, biochemical, and structural findings on four BATSIs with the FOX-4 cephamycinase, a class C β-lactamase that rapidly hydrolyzes cefoxitin, are revealed. FOX-4 is an extended-spectrum class C cephalosporinase that demonstrates conformational flexibility when complexed with certain ligands. Like other β-lactamases of this class, studies on FOX-4 reveal important insights into structure–activity relationships. We show that SM23, a BATSI, shows both remarkable flexibility and affinity, binding similarly to other β-lactamases, yet retaining an IC50 value < 0.1 μM. Our analyses open up new opportunities for the design of novel transition-state analogs of class C enzymes.

Class C β-Lactamases: Molecular Characteristics

“…The first crystallographic structure of an AmpC was reported in 1994, for the covalent complex between a phosphonate transition-state analog and the E. hormaechei P99 (formerly known as E. cloacae P99) cephalosporinase, which belongs to the ACT family (141). Many structures from different families of class C β-lactamases have since been published, including ACC (142,143), ACT (141,(144)(145)(146)(147)(148)(149)(150)(151)(152)(153)(154)(155)(156), ADC (103,(157)(158)(159)(160)(161)(162), CMH (163), CMY (154,(164)(165)(166)(167)(168), EC (165,, FOX (207)(208)(209), MOX (210,211), PDC Figure 2 here…”

Structural Insights into Inhibition of the Acinetobacter-Derived Cephalosporinase ADC-7 by Ceftazidime and Its Boronic Acid Transition State Analog

“…Despite the high mobility of the oxyimino moiety, the dimethyl-carboxylate portion of this group has been implicated in improved inhibition in class C -lactamases. For example, a cefotaximelike BATSI that lacks the terminal dimethyl-carboxylate binds 15-fold and 2-fold worse in E. coli AmpC 13 and FOX-4 32 , respectively. Since boronic acids are reversible inhibitors, these Kis reflect true binding affinities and suggest this group makes specific interactions with the enzyme.…”

“…MICs were performed as previously described [28][29][30][31][32] and according to Clinical and Laboratory Standards Institute (CLSI) guidelines, using a 6 x 104 cfu/mL inoculum.…”

“…Utilizing nitrocefin (NCF) as a colorimetric substrate of ADC-7, the inhibition constant (Ki) of LP06 BATSI with ADC-7 was determined using competition kinetics as previously described. [28][29][30][31][32] The measurements of the initial velocities were performed with the addition of 100 µM NCF after a 5 min pre-incubation of the enzyme (…”

β-Lactamases: Sequence, Structure, Function, and Inhibition

“…More evidence for the utility of boronates as inhibitors of class C enzymes is provided by Lefurgy et al [16]. They report on four compounds designed as boronic acid transition-state analogs (BATSIs) that were tested against the extended spectrum class C cephalosporinase FOX-4 (named for its substrate preference for the cephamycin cefoxitin).…”