N -Arylsulfonyl Hydrazones as Inhibitors of IMP-1 Metallo- (cid:2) -Lactamase

Members of a family of N -arylsulfonyl hydrazones have been identiﬁed as novel inhibitors of IMP-1, a metallo- (cid:1) -lactamase of increasing prevalence. Structure-activity relationship studies have indicated a requirement for bulky aromatic substituents on each side of the sulfonyl hydrazone backbone for these compounds to serve as efﬁcient inhibitors of IMP-1. Molecular modeling has provided insight into the structural basis for the anti-metallo- (cid:1) -lactamase activity exhibited by this class of compounds. The ever-increasing incidence of microbial resistance to (cid:2) -lactam antibiotics has been traced, in a large number of instances, to the ability of the organisms to produce (cid:2) -lacta-mases, enzymes capable of deactivating these antibiotics by hydrolysis (16, 22, 28). Consequently, there is a considerable interest in the development of (cid:2) -lactamase inhibitors that would allow for the continued use of (cid:2) -lactam antibiotics in clinical therapy. To date, four distinct classes, designated A, B, C, and D, of (cid:2) -lactamases Metallo- (cid:1) -lactamase assays. A spectrophotometric method based on the use of a chromophoric substrate, nitroceﬁn (27), was used for the measurement of (cid:2) -lactamase activity with the aid of a Cary5 spectrophotometer (Varian, Missis-sauga, Ontario, Canada). All performed HEPES buffer (50 mM, pH 7.3) at The procedure involved the preparation of following solutions: (i) nitroceﬁn (2 mM) in HEPES buffer (50 mM, pH 7.3) containing dimethyl sulfoxide (DMSO) (5% [vol/vol]) and (ii) metallo- (cid:2) -lactamase (400 nM) in HEPES buffer containing (100 (cid:6) M) and (BSA) stock (cid:2) -lactamase denaturation at protein for a reliable and reproducible assessment of the initial rate of enzyme-catalyzed hydrolysis of its substrate, observations in agreement with At the low concentration (1 (cid:6) g/ml) in the assay, BSA no detectable interaction with the product of nitroceﬁn hydrolysis. the degree of inhibition was not signiﬁcantly inﬂuenced by the presence of BSA up to a concentration, in the assay, of at least 20 (cid:6) g/ml. In a typical assay (ﬁnal volume, 1 ml), metallo- (cid:2) -lactamase (4 nM) in HEPES allowed to equilibrate at 30°C for 1 min. The reaction was initiated by the introduction of the substrate (100 (cid:6) M for IMP-1 and 200 (cid:6) M for BcII), and the increase in the absorbance at 482 nm was recorded. For determination of the concentration required to effect 50% inhibition of enzyme activity (IC 50 ), the enzyme was preincubated with the desired compound for 1 min at 30°C prior to the initiation of the assay by the addition of the substrate. The compounds to be tested were dissolved in DMSO prior to incu- bation with the enzyme, with the ﬁnal concentration of DMSO in the assay not exceeding 1% (vol/vol). IC 50 s were deduced from a plot of percent loss of activity versus inhibitor concentration. The precision of these determinations was within the range of (cid:4) 10%. of steady-state kinetic constants. Estimates of steady-state kinetic parameters for the hydrolysis of nitroceﬁn ( K m and k cat ) were achieved by ﬁtting the initial velocity data to the Michaelis-Menten equation software