Can Precision Electrophile Signaling Make a Meaningful and Lasting Impression in Drug Design?

For several years, drugs bearing reactive electrophilic appendages have been developed. These units typically confer prolonged residence time of the drugs on their protein targets, and may assist targeting shallow binding sites and/or improving drug-protein target spectrum. Studies on natural electrophilic molecules have indicated that in many instances natural electrophiles use similar mechanisms to alter signaling pathways. However, natural reactive species are also endowed with other important mechanisms to hone signaling properties that are uncommon in drug design. These include ability to be active at low occupancy and elevated inhibitor kinetics. Here we discuss how we have begun to harness these properties in inhibitor design.

Effect of Asparagine Substitutions in the YXN Loop of a Class C β-Lactamase of Acinetobacter baumannii on Substrate and Inhibitor Kinetics

ABSTRACTClass C cephalosporinases are a growing threat, and inhibitors of these enzymes are currently unavailable. Studies exploring the YXN loop asparagine in theEscherichia coliAmpC, P99, and CMY-2 enzymes have suggested that interactions between C6′ or C7′ substituents on penicillins or cephalosporins and this Asn are important in determining substrate specificity and enzymatic stability. We sought to characterize the YXN loop asparagine in the clinically important ADC-7 class C β-lactamase ofAcinetobacter baumannii. Mutagenesis at the N148 position in ADC-7 yields functional mutants (N152G, -S, -T, -Q, -A, and -C) that retain cephalosporinase activity. Using standard assays, we show that N148G, -S, and -T variants possess good catalytic activity toward cefoxitin and ceftaroline but that cefepime is a poor substrate. Because N152 variants of CMY-2, another class C β-lactamase, are more readily inhibited by tazobactam due to higher rates of inactivation, we also tested if the N148 substitutions in ADC-7 would affect inactivation by sulfone inhibitors, sulbactam and tazobactam, class A β-lactamase, andA. baumanniipenicillin-binding protein (PBP) inhibitors within vitroactivity against ADC-7. The 50% inhibitory concentrations (IC50s) for tazobactam and sulbactam were improved, with 7-fold and 2-fold reductions, respectively, for the N148S variant. A homology model of the N148S ADC-7 enzyme in a Michaelis-Menten complex with tazobactam showed a loss of interaction between N148 and the sulfone moiety of the inhibitor. We postulate that this may result in more-rapid secondary ring opening of the inhibitor, as the unbound sulfone is an excellent leaving group, leading to more-rapid formation of the stable linearized inhibitor.

Penicillin Sulfone Inhibitors of Class D β-Lactamases

ABSTRACT OXA β-lactamases are largely responsible for β-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the β-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D β-lactamases. To overcome this challenge, we explored the abilities of β-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D β-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low Ki values for the OXA-1 β-lactamase (0.70 ± 0.14 → 1.60 ± 0.30 μM) and demonstrated significant Ki improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low Ki s for the OXA-10, -14, -17, and -24/40 β-lactamases (0.20 ± 0.04 → 17 ± 4 μM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [tn ] = 0) and tn s ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA β-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 β strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type β-lactamases.