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Overview
Metallo-β-lactamase inhibitors: Promise for the future?
Jeffrey H Toney* & Joseph G Moloughney
number of metallo-•-lactamases (MBLs), three subclasses (B1, B2 and B3) have been characterized based on their known Department of Chemistry and Biochemistry sequences [4•]. MBLs expressed in Bacillus cereus (BcII), Richardson Hall Room 352 1 Normal Avenue Bacteroides fragilis (CcrA, also CfiA) and Pseudomonas aeruginosa (IMP-1) have been characterized in greatest detail. The IMP-1 enzyme is of particular interest since it is encoded by both plasmids and integrons [5]. These mobile fragments of DNA could be responsible for the future widespread dissemination of *To whom correspondence should be addressed Current Opinion in Investigational Drugs 2004 5(8):
X-ray crystal structures are now available for several MBLs, The Thomson Corporation ISSN 1472-4472 with and without bound inhibitors, and offer the potential for structure-aided drug design. Examples include BcII [6], IMP-1 Carbapenem resistance continues to erode the effectiveness of [7•,8•], CfiA [9•] and subclass B3 Stenotrophomonas maltophilia antibiotics such as imipenem and meropenem in the clinic. Resistance mechanisms can include interplay between porin loss (membrane (L-1) [10]. The crystal structure of IMP-1 bound to a permeability), mutation of penicillin binding proteins necessary for mercaptocarboxylate revealed three key interactions in the cell division, and expression of class A, B and D β-lactamases. enzyme active site and includes contacts between the inhibitor and a conserved lysine, the zinc atoms and a hydrophobic Bacterial resistance to β-lactams such as penicillin or amoxicillin has pocket. The X-ray crystal structure of subclass B1 been overcome in the clinic using several strategies, including Chryseobacterium meningosepticum (BlaB) MBL bound to an development of antibiotics not susceptible to hydrolysis by β- lactamases, or co-administration of the antibiotic with β-lactamase inhibitors. This overview will focus on progress since 2000 in MBL inhibitors reported prior to the year 2000 include identifying inhibitors of class B, or metallo-β-lactamases with the aim trifluoromethyl alcohols and ketones [12], thioester derivatives of reversing carbapenem resistance. [13-16], thiols [17], biphenyl tetrazoles [18,19], hydroxamates Keywords Antibiotics, antibiotic resistance, β-lactam antibiotics,
(amino-acid derived) [20], phenazines [21] and 1β- metallo-β-lactamases, metallo-β-lactamase inhibitors methylcarbapenems [22•,23•]. This overview presents novel chemical classes of MBL inhibitors reported since 2000. Introduction
β-Lactam antibiotics have proven to be an overwhelming N-arylsulfonyl hydrazones
success in the treatment of bacterial infections. The dynamic
N-arylsulfonyl hydrazones are inhibitors of IMP-1. This nature of bacteria allows for their alarmingly facile adaptation chemical class is exemplified by compound 1 (Figure 1) [24], the to a changing environment. Bacteria have developed several most potent inhibitor of the series. Compound 1 exhibited a K i mechanisms of resistance to antibiotics. The reduced value of 0.7 ± 0.1 µM, characteristic of a competitive inhibitor. permeability of the cell wall [1], alterations in target enzymes Exploration of this chemical class revealed that enzyme (penicillin-binding proteins) [2] and production of various inhibition increased with bulky aromatic substituents that had forms of β-lactamase all contribute to the diminishing little electron withdrawing capacity (eg, compound 1 versus effectiveness of antibiotics. β-Lactamases have been grouped compound 3 (Figure 1)). Activity against the BcII enzyme of this into four molecular classes [3•]; classes A, C and D are serine series was much weaker than that reported for IMP-1. No active site β-lactamases, while class B enzymes are antimicrobial synergy data involving the compounds was metalloproteins that are zinc dependent. Due to the growing Figure 1. N-arylsulfonyl IMP-1 inhibitors.
Succinic acids
against the L-1 enzyme was shown. SB-236049 (Table 1) was Screening of the Merck chemical collection led to the the most potent of the series, with IC values of ≤ 2 µM identification of a series of succinic acids that are potent against the CfiA and BcII enzymes. SB-238569 (Table 1) was inhibitors of IMP-1 [8•]. A 2,3-(S,S)-disubstituted succinic most effective against the CfiA enzyme with a K value of 3.4 acid was found to have an IC value of 0.0027 µM. X-ray µM. The tricyclic compounds showed no metal ion-chelating crystallography and molecular modeling, structure-activity activity when incubated in the presence of 100 µM ZnSO . In relationships studies revealed that potent inhibition addition, the compounds displayed selectivity against MBLs required two hydrophobic groups on the succinic acid by exhibiting minimal or no inhibition against the center in a (2S,3S) configuration. Representative succinic mammalian metalloprotein ACE. The tricyclic inhibitors acids increased sensitivity to imipenem in carbapenem- displayed synergy with meropenem in bacterial strains resistant clinical isolates of P aeruginosa [25]. Thiomandelic acid
Mercaptocarboxylate
Thiols and mercaptoacetic acid thiol esters are reported to be A mercaptocarboxylate compound, 2-[5-(1-tetrazolylmethyl)- broad spectrum MBL inhibitors [26]. Recently, thiolate- thien-3-yl]-N-[2-(mercaptomethyl)-4(phenyl-butyrylglycine)] disulfide exchange has been used as a novel strategy to was found to be a potent inhibitor (IC = 90 nM) of IMP-1. In inhibit the MBL CcrA from B fragilis [27•]. D-captopril, an addition, B fragilis (CfiA) and S maltophilia (L1) enzymes angiotensin-converting enzyme (ACE) inhibitor used to treat were inhibited by the compound with IC values in the hypertension, has been shown to inhibit of the MBL range of 100 to 500 nM. No antimicrobial synergy data on
expressed in C meningosepticum [11] as well as the MBL the compound were available.
expressed in Fluoribacter gormanni, FEZ-1 [28]. The most
potent compounds contained both thiol and carboxylate Cysteinyl peptides
functional groups. The thiol group was necessary for Kinetic studies have shown that cysteinyl peptides are inhibition of the enzyme while the carboxylate group was competitive inhibitors of the B cereus (BcII) enzyme [29]. The not critical. Comparison of compounds 4, 7 and 8 (Figure 2) thiol group is believed to be necessary for inhibition as this reveals that inhibition of the B cereus BcII enzyme is greatest functional group coordinates to the zinc ion located in the when the thiol and carboxylate functional groups are in active site of the enzyme. The thiol inhibitors were non- proximity. Thiomandelic acid and two para-substituted chelators but enzyme inhibition by dithiol derivatives were analogs were the most effective inhibitors (compounds 4, 5 dependent on zinc ion concentration. N-carbobenzoxy-D- and 6; Figure 2) and thiomandelic acid was a competitive cysteinyl-D-phenylalanine was the most potent inhibitor of inhibitor of the BcII enzyme. In addition, thiomandelic acid the BcII enzyme with a K value of 3.0 µM. No antimicrobial was an effective inhibitor (K ≤ 0.80 µM) for eight distinct MBLs included in the study except for the Aeromonas hydrophilia (CphA) enzyme. No antimicrobial synergy data were provided. Thioxocephalosporin and penicillin-derived
inhibitors
Tricyclic natural products
Recently, the hydrolytic product of thioxocephalosporin, a Tricyclic compounds were identified and extracted from the thioacid, was found to be a modest inhibitor of B cereus fungal strain Chaetomium funicola. When tested for inhibition (BcII) [30], and demonstrated competitive inhibition with a against B cereus (BcII), P aeruginosa (IMP-1), B fragilis (CfiA) K value of 96 µM. In addition, a thioamide, cyclic and S maltophilia (L-1) MBLs, these compounds competitivly thioxopiperazinedione, produced by intramolecular inhibited the BcII, IMP-1 and CfiA enzymes. No inhibition aminolysis of thioxocephalexin was shown to competitively Figure 2. Comparison of thiomandelic inhibitors of B cereus (BcII).
Ki values assume competitive inhibition (SD < 20%) Table 1. IC50 values for tricyclic natural product inhibitors.
Compound
IC50 (µM)
B cereus II
SB-236049
SB-236050
SB-238569
inhibit the BcII metallo-enzyme with a K value of 29 µM. A RP: The structure of beta-lactamases. Philos Trans R Soc
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separate report identified a series of (mercaptomethyl)- penicillinates with micromolar to sub-micromolar IC Galleni M, Lamotte-Brasseur J, Rossolini GM, Spencer J, Dideberg O, values for the L1 MBL and low micromolar IC values for Frere JM, the metallo-b-lactamase working group: Standard
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penicillinate sulfones were better inhibitors of L1 and the • A consortium of research groups studying metallo-b-lactamases proposed a (mercaptomethyl)penicillinate sulfides displayed good standard numbering scheme of these enzymes to facilitate comparative analysis of protein structure and catalytic mechanisms. activity against BcII. Several of the inhibitors exhibited synergy with piperacillin against an IMP-1 producing strain Arakawa Y, Murakami M, Suzuki K, Ito H, Wacharotayankun R, Ohsuka of E coli and a single compound had activity against clinical S, Kato N, Ohta M: A novel integron-like element carrying the
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Fabiane SM, Sohi MK, Wan T, Payne DJ, Bateson JH, Mitchell T, Conclusions
Sutton BJ: Crystal structure of the zinc-dependent beta-lactamase
Several novel chemical classes of MBL inhibitors have been from Bacillus cereus at 1.9 A resolution: binuclear active site with
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information on the chemical details of the enzyme-inhibitor interaction. While none of these classes constitute the 'ideal Concha NO, Janson CA, Rowling P, Pearson S, Cheever CA, Clarke
BP, Lewis C, Galleni M, Frere JM, Payne DJ, et al.: Crystal structure
MBL inhibitor', the challenge for the pharmaceutical of the IMP-1 metallo beta-lactamase from Pseudomonas
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presented here for sufficient broad spectrum activity against binding determinants of a potent, broad-spectrum inhibitor.
Biochemistry 2000, 39:4288-4298.
the most important clinical pathogens expressing MBLs [32]. • The SmithKline Beecham group determined the X-ray crystal structure of the Careful monitoring of clinical isolates for the presence of IMP-1 MBL with and without an inhibitor bound, revealing that the MBLs, within the context of other important resistance "hydrophobic flap" extending above the enzyme active site is displaced 2.9 A mechanisms, will provide the ultimate test of whether development of an MBL inhibitor will add value to the 8. Toney JH, Hammond GG, Fitzgerald PM, Sharma N, Balkovec JM, current antibiotic armamentarium. Rouen GP, Olson SH, Hammond ML, Greenlee ML, Gao YD: Succinic
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