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 . 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 , IMP-1
Carbapenem resistance continues to erode the effectiveness of [7•,8•], CfiA [9•] and subclass B3 Stenotrophomonas maltophiliaantibiotics such as imipenem and meropenem in the clinic. Resistance mechanisms can include interplay between porin loss (membrane (L-1) . 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 , thioester derivatives
of reversing carbapenem resistance.
[13-16], thiols , biphenyl tetrazoles [18,19], hydroxamates
Keywords Antibiotics, antibiotic resistance, β-lactam antibiotics,
(amino-acid derived) , phenazines  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) , 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 , alterations in target enzymes
Exploration of this chemical class revealed that enzyme
(penicillin-binding proteins)  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 .
Thiomandelic acid Mercaptocarboxylate
Thiols and mercaptoacetic acid thiol esters are reported to be
A mercaptocarboxylate compound, 2-[5-(1-tetrazolylmethyl)-
broad spectrum MBL inhibitors . Recently, thiolate-
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  as well as the MBL the compound were available. expressed in Fluoribacter gormanni, FEZ-1 . 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 . 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 Aeromonashydrophilia (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) , 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 Lond B Biol Sci. 1980, 289:321-31.
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 numbering scheme for class B b-lactamases. Antimicrob Agents
the BcII MBL . In general, the (mercaptomethyl)-
Chemother 2001, 45:660-663.
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 metallo-beta-lactamase gene blaIMP. Antimicrob Agents Chemother
isolates of P aeruginosa producing either VIM-1 or SPM-1.
Fabiane SM, Sohi MK, Wan T, Payne DJ, Bateson JH, Mitchell T,
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
reported recently and there is now a rich source of structural
features of a mononuclear enzyme. Biochemistry 1998, 37:12404- 12411.
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
industry will be to continue to explore the chemical classes
aeruginosa and its complex with a mercaptocarboxylate inhibitor:
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 .
• 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 acids as potent inhibitors of plasmid-borne IMP-1 metallo-beta- lactamase. J Biol Chem 2001, 276:31913-31918. References
• The Merck group identified succinic acids as potent IMP-1 inhibitors via high throughput screening. The X-ray crystal structure of the enzyme bound to two different succinic acids was determined. Interplay of impermeability and chromosomal beta- lactamase activity in imipenem-resistant Pseudomonas 9.
Payne DJ, Hueso-Rodriguez JA, Boyd H, Concha NO, Janson CA,
aeruginosa. Antimicrob Agents Chemother 1992, 36:2046-2048.
Gilpin M, Bateson JH, Cheever C, Niconovich NL, Pearson S, et al.:
Identification of a series of tricyclic natural products as potent
Massova I, Mobashery S: Kinship and diversification of bacterial broad-spectrum inhibitors of metallo-beta-lactamases. Antimicrob penicillin-binding proteins and beta-lactamases. Antimicrob Agents Agents Chemother 2002, 46:1880-1886. Chemother 1998, 42:1-17.
• The GlaxoSmithKline group identified tricyclic inhibitors using natural
• Extensive review on sequences alignments amongst penicillin-binding product screening and determined the X-ray structure of the CfiA MBL bound proteins and beta lactamases.
10. Ullah JH, Walsh TR, Taylor IA, Emery DC, Verma CS, Gamblin SJ,
22. Nagano R, Adachi Y, Imamura H, Yamada K, Hashizume T, Morishima
Spencer J: The crystal structure of the L1 metallo-beta-lactamase
H: Carbapenem derivatives as potential inhibitors of various beta- from Stenotrophomonas maltophilia at 1.7 A resolution. J Mol Biol lactamases, including class B metallo-beta-lactamases. Antimicrob
1998, 284:125-136. Agents Chemother 1999, 43:2497-2503.
• The Banyu Tsukuba group showed that 1beta-methylcarbapenems can
11. Garcia-Saez I, Hopkins J, Papamicael C, Franceschini N, Amicosante
sensitize resistant strains of IMP-1 producing Serratia marcescens when co-
G, Rossolini GM, Galleni M, Frere JM, Dideberg O: The 1.5-A structure of Chryseobacterium meningosepticum zinc beta-lactamase in complex with the inhibitor, D-captopril. J Biol Chem 2003,
23. Nagano R, Adachi Y, Hashizume T, Morishima H: In vitro antibacterial 278:23868-23873. activity and mechanism of action of J-111,225, a novel 1beta- methylcarbapenem, against transferable IMP-1 metallo-beta-
12. Walter MW, Felici A, Galleni M, Paul-Soto R, Adlington RM, Baldwin JE,
lactamase producers. J Antimicrob Chemother 2000, 45:271-276.
Frere JM, Gololobov M, Schofield CJ: Trifluoromethyl alcohol and
• The Banyu Tsukuba group showed that 1beta-methylcarbapenems can ketone inhibitors of metallo-b-lactamases. Bioorg Med Chem Lett sensitize resistant strains of IMP-1 producing Serratia marcescens as well as
1996, 6:2455-2458. Pseudomonas aeruginosa in the presence of imipenem.
13. Greenlee ML, Laub JB, Balkovec JM, Hammond ML, Hammond GG,
24. Siemann S, Brewer D, Clarke AJ, Dmitrienko GI, Lajoie G, Viswanatha
Pompliano DL, Epstein-Toney JH: Synthesis and SAR of thioester IMP-1 metallo-beta-lactamase: effect of chelators and and thiol inhibitors of IMP-1 metallo-beta-lactamase. Bioorg Med assessment of metal requirement by electrospray mass Chem Lett 1999, 9:2549-2554. spectrometry. Biochim Biophys Acta 2002, 1571:190-200.
14. Hammond GG, Huber JL, Greenlee ML, Laub JB, Young K, Silver LL,
25. Huber J, Young K, Painter RE, Rosen H, Silver LL: Inhibition of IMP-1
Balkovec JM, Pryor KD, Wu JK, Leiting B, et al.: Inhibition of IMP-1 metallo-beta-lactamase in clinical isolates by two succinic acid metallo-beta-lactamase and sensitization of IMP-1-producing derivatives. 40th Interscience Conference on Antimicrobial Agents and bacteria by thioester derivatives. FEMS Microbiol Lett 1999, 179:289-
26. Mollard C, Moali C, Papamicael C, Damblon C, Vessilier S, Amicosante
15. Payne DJ, Bateson JH, Gasson BC, Khushi T, Proctor D, Pearson SC,
G, Schofield CJ, Galleni M, Frere JM, Roberts GC: Thiomandelic acid,
Reid R: Inhibition of metallo-beta-lactamases by a series of thiol a broad spectrum inhibitor of zinc beta-lactamases: kinetic and ester derivatives of mercaptophenylacetic acid. FEMS Microbiol Lett spectroscopic studies. J Biol Chem 2001, 276:45015-45023.
27. Boerzel H, Koeckert M, Bu W, Spingler B, Lippard SJ: Zinc-Bound
16. Payne DJ, Bateson JH, Gasson BC, Proctor D, Khushi T, Farmer TH,
Thiolate-Disulfide Exchange: A Strategy for Inhibiting Metallo-beta-
Tolson DA, Bell D, Skett PW, Marshall AC, et al.: Inhibition of metallo- lactamases. Inorg Chem 2003, 42:1604-1615. beta-lactamases by a series of mercaptoacetic acid thiol ester
• This study proposes a novel mechanism for inactivation of metallo-beta-derivatives. Antimicrob Agents Chemother 1997, 41:135-140. lactamases, targeting the Zn-bound Cys for thiolate-disulfide exchange.
17. Goto M, Takahashi T, Yamashita F, Koreeda A, Mori H, Ohta M,
28. Garcia-Saez I, Mercuri PS, Papamicael C, Kahn R, Frere JM, Galleni M,
Arakawa Y: Inhibition of the metallo-beta-lactamase produced from
Rossolini GM, Dideberg O: Three-dimensional structure of FEZ-1, a Serratia marcescens by thiol compounds. Biol Pharm Bull 1997, monomeric subclass B3 metallo-beta-lactamase from Fluoribacter 20:1136-1140. gormanii, in native form and in complex with D-captopril. J Mol Biol
18. Toney JH, Fitzgerald PM, Grover-Sharma N, Olson SH, May WJ,
Sundelof JG, Vanderwall DE, Cleary KA, Grant SK, Wu JK, et al.:
29. Bounaga S, Galleni M, Laws AP, Page MI: Cysteinyl peptide Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacillus cereus zinc beta-lactamase. Bioorg Med Chem inhibitors of Bacteroides fragilis metallo-beta-lactamase. Chem Biol
1998, 5:185-196. Metallo-beta-lactamase inhibitors: could they give old
19. Toney JH, Cleary KA, Hammond GG, Yuan X, May WJ, Hutchins SM,
antibacterials new life?Curr Opin Investig Drugs 2003, 4:115-116.
Ashton WT, Vanderwall DE: Structure-activity relationships of biphenyl tetrazoles as metallo-beta-lactamase inhibitors. Bioorg
31. Tsang WY, Dhanda A, Schofield CJ, Frere JM, Galleni M, Page MI: The Med Chem Lett 1999, 9:2741-2746. inhibition of metallo-beta-lactamase by thioxo-cephalosporin derivatives. Bioorg Med Chem Lett 2004, 14:1737-9.
20. Walter MW, Hernandez Valladares M, Adlington RM, Amicosante G,
Baldwin JE, Frere JM, Galleni M, Rossolini GM, Schofield CJ:
32. Buynak JD, Chen H, Vogeti L, Gadhachanda VR, Buchanan CA, Palzkill
Hydroxymate inhibitors of aeromonas hydrophilia AE036 metallo-b-
T, Shaw RW, Spencer J, Walsh TR: Penicillin-derived inhibitors that lactamases. 1999. simultaneously target both metallo- and serine-beta-lactamases. Bioorg Med Chem Lett 2004, 14:1299-304.
21. Gilpin ML, Fulston M, Payne D, Cramp R, Hood I: Isolation and structure determination of two novel phenazines from a Streptomyces with inhibitory activity against metallo-enzymes, including metallo-beta-lactamase. J Antibiot (Tokyo) 1995, 48:1081- 1085.
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