Crystal Structures of KPC-2 and SHV-1 β-Lactamases in Complex with the Boronic Acid Transition State Analog S02030

Abstract

Resistance to expanded-spectrum cephalosporins and carbapenems has rendered certain strains of Klebsiella pneumoniae the most problematic pathogens infecting patients in the hospital and community. This broad-spectrum resistance to ␤-lactamases emerges in part via the expression of KPC-2 and SHV-1 ␤-lactamases and variants thereof. KPC-2 carbapenemase is particularly worrisome, as the genetic determinant encoding this ␤-lactamase is rapidly spread via plasmids. Moreover, KPC-2, a class A enzyme, is difficult to inhibit with mechanism-based inactivators (e.g., clavulanate). In order to develop new ␤-lactamase inhibitors (BLIs) to add to the limited available armamentarium that can inhibit KPC-2, we have structurally probed the boronic acid transition state analog S02030 for its inhibition of KPC-2 and SHV-1. S02030 contains a boronic acid, a thiophene, and a carboxyl triazole moiety. We present here the 1.54-and 1.87-Å resolution crystal structures of S02030 bound to SHV-1 and KPC-2 ␤-lactamases, respectively, as well as a comparative analysis of the S02030 binding modes, including a previously determined S02030 class C ADC-7 ␤-lactamase complex. S02030 is able to inhibit vastly different serine ␤-lactamases by interacting with the conserved features of these active sites, which includes (i) forming the bond with catalytic serine via the boron atom, (ii) positioning one of the boronic acid oxygens in the oxyanion hole, and (iii) utilizing its amide moiety to make conserved interactions across the width of the active site. In addition, S02030 is able to overcome more distantly located structural differences between the ␤-lactamases. This unique feature is achieved by repositioning the more polar carboxyl-triazole moiety, generated by click chemistry, to create polar interactions as well as reorient the more hydrophobic thiophene moiety. The former is aided by the unusual polar nature of the triazole ring, allowing it to potentially form a unique COH. . .O 2.9-Å hydrogen bond with S130 in KPC-2.

Phenylboronic Acid Derivatives as Validated Leads Active in Clinical Strains Overexpressing KPC‐2: A Step against Bacterial Resistance

“…Overall, both derivatives 2 and 4 adopt a deacylation transition‐state analogue conformation with an inverted boron configuration. In such a conformation, previously described for other class A BLs, the positions of the boronic acid oxygen atoms are as follows: one is located in the oxyanion hole and the other one displaces the deacylation water normally positioned between Glu‐166 and Asn‐170 …”

“…In a possible derivatization of phenylboronic acid ( 1 ), we reasoned that the introduction of a carboxylic group mimicking the C3(4)′ carboxylate of β‐lactam antibiotics, a key recognition feature in BLs, might improve KCP‐2 binding through a large interaction network with Arg‐220, Ser‐130, Thr‐237, and Thr‐235. All mentioned residues are located in the β‐lactam carboxylate binding pocket, in which the sulfate group of avibactam, as well as the carboxylic group of other known class A inhibitors, are known to bind …”

“…All mentioned residues are located in the b-lactam carboxylate binding pocket, in which the sulfate group of avibactam, as well as the carboxylic group of otherk nownc lass Ai nhibitors, are knowntob ind. [17,27] Whereast he presence of the boronic acid group guarantees covalentl inking to Ser-70, sinking de facto the inhibitor in the active site, ap roperl inker could correctly orient the lateralc arboxylic chain in the targeted site without preventing other classical interactions between the ligand and the Thr-237, Glu-166, and Asn-170 residues,a ss een in the KPC-2-3-nitrophenylboronic acid complex. [26] With the purpose of mimicking distance, chemistry,a nd interactions of the C3(4)' carboxylate of b-lactam antibiotics, structuralc onsiderations suggested to introduce ac arboxylic moiety,p roperly spaced by at wo atomic vinyl chain,i nt he ortho positiona djacent to the boronic group of compound 1.…”

“…In such ac onformation, previously described for other class AB Ls, the positions of the boronic acid oxygen atoms are as follows:o ne is located in the oxyanion hole and the other one displaces the deacylation water normally positioned between Glu-166 and Asn-170. [27,34] Compound 2 orients its phenylr ing against one hydrophobic region defined by Trp-105 andL eu-167 in proximity of the opening of the binding site. The phenylr ing of 2 is involved in cation-p and p-p interactions with Asn-132 and Trp-105, respectively.F inally,a se xpected, 2 orientsi ts ortho carboxyvinyl lateral chain toward the C3(4')c arboxylate binding pocket, highly conserved in all serine BLs.…”

Boronic Acid Transition State Inhibitors Active against KPC and Other Class A β-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design

“…In the study described in the companion article, the crystal structure of KPC-2 and SHV-1 with 2b (S02030) revealed multiple conformations of 2b in the active site of KPC-2 and SHV-1 (25). The boronic acid preserved the tetrahedral interactions, forming the bond with catalytic serine, positioning one of the boronic acid oxygens in the oxyanion hole (T237:N for KPC-2) and the other toward S130.…”