Inhibition of Class D -Lactamases by Acyl Phosphates and Phosphonates

The resistance of bacteria to -lactam antibiotics is to a large extent due to -lactamases (14). There is a wide range of -lactamases, although from a structural standpoint, they can be divided into four classes, A, B, C, and D (11). Each class contains many variants of widely differing substrate specificities and thus clinical importance. Of the canonical four classes, it is perhaps class D that at present is least well studied at the molecular level. The class D -lactamases, also generally known as oxacillinases because of their general specificity for oxacillin and its derivatives, represent a diverse class of enzymes (1) that hydrolyze a broad spectrum of substrates (2). Like class A and C -lactamases, the class D enzymes are serine hydrolases, catalyzing substrate hydrolysis by way of a covalent acyl-enzyme (acyl-serine) intermediate and thus by a double-displacement mechanism. Structural studies indicate that the class D -lactamases are more closely related to class A than to class C, although there are distinct differences in active-site structures and thus, presumably, in chemical mechanisms (20). Although class D -lactamases are clinically significant, there are no specific inhibitors known for them other than certain inhibitory -lactams (8, 12) and a series of anthraquinone dyes (15). The classical mechanism-based inhibitors of class A -lactamases are not generally effective against class D (16). A variety of phosph(on)ates have been found to be covalent inhibitors of class A and class C -lactamases (4, 5, 9, 17–19). This paper describes the screening of a panel of these compounds, 1 to 9, against the class D OXA-1 -lactamase. This enzyme is representative of one subclass of the D enzymes (1) and is clinically important in its own right; a crystal structure is also available (20). The more effective compounds of 1 to 9 (Fig. 1) were also tested against the OXA-10 enzyme, a representative of another major class D subgroup (1). The OXA-1 and OXA-10 -lactamases were prepared and purified as described previously (8, 20). The various phosph(on)ates were available from previous studies (4, 5, 9, 17–19). The phosphates 2 and 4 to 8 and the phosphonates 1, 3, and 9 were all irreversible or slowly reversible inhibitors of the OXA -lactamases, and the inactivation step could be described simply by scheme 1 as follows: