Kinetic Study of Two Novel Enantiomeric Tricyclic β-Lactams Which Efficiently Inactivate Class C β-Lactamases

A detailed kinetic study of the interaction between two ethylidene derivatives of tricyclic carbapenems, Lek 156 and Lek 157, and representative β-lactamases and d-alanyl–d-alanine peptidases (dd-peptidases) is presented. Both compounds are very efficient inactivators of the Enterobacter cloacae 908R β-lactamase, which is usually resistant to inhibition. Preliminary experiments indicate that various extended-spectrum class C β-lactamases (ACT-1, CMY-1, and MIR-1) are also inactivated. With the E. cloacae 908R enzyme, complete inactivation occurs with a second-order rate constant, k(2)/K′, of 2 × 10(4) to 4 × 10(4) M(−1) s(−1), and reactivation is very slow, with a half-life of >1 h. Accordingly, Lek 157 significantly decreases the MIC of ampicillin for E. cloacae P99, a constitutive class C β-lactamase overproducer. With the other serine β-lactamases tested, the covalent adducts exhibit a wide range of stabilities, with half-lives ranging from long (>4 h with the TEM-1 class A enzyme), to medium (10 to 20 min with the OXA-10 class D enzyme), to short (0.2 to 0.4 s with the NmcA class A β-lactamase). By contrast, both carbapenems behave as good substrates of the Bacillus cereus metallo-β-lactamase (class B). The Streptomyces sp. strain R61 and K15 extracellular dd-peptidases exhibit low levels of sensitivity to both compounds.