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A lack of drugs for antibiotic-resistant Gram-negative bacteria

Payne et al. recently reported an excellent overview of a target-based approach to new antibacterial development and the lack of new antibacterial drugs in late-stage development several years ago1. This observation has also been made by the participants in the recent forum2 of anti-infective research and development. Also, the Infectious Diseases Society of America recently identified six top-priority dangerous pathogens — extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, Acinetobacter baumannii, Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus and Aspergillus species — for which there are few or no drugs in late-stage development. This could further limit future safe and effective choices for treating these infections3.

Three of these six pathogens are antibiotic-resistant Gram-negative bacteria. Recently, antibacterial drugs against ESBL-producing Gram-negative bacteria accounted for 15% (2 out of 13) of all antibacterial drugs undergoing development in Phase II trials or later clinical studies3. However, there are no drugs being developed against class C ESBL-producing Gram-negative bacteria. Here, we draw attention to important aspects of urgently needed antibacterial drugs against class C ESBL-producing Gram-negative bacteria, which have been overlooked by these reports. We also suggest that the category of ESBLs should be expanded.

ESBLs are a group of enzymes for which the substrate spectrum has extended to third-generation oxyimino-cephalosporins (for example, cefotaxime and ceftazidime)4. Most of the known ESBLs are class A and D β-lactamases, but recently, several class C ESBLs were reported in Gram-negative bacteria: KL5, HD6, CMY-10 (Ref. 7) and CMY-19 (Ref. 8). The hydrolytic efficiency (kcat/Km) of class C ESBLs for ceftazidime was higher than or similar to that (0.029 μM−1 s−1) of SHV-38 (SHV stands for sulphydryl variable)9, a typical class A ESBL.

Most of the class C β-lactamases have hydrolysing activity against cephamycins (that is, second-generation cephalosporins: cefoxitin and cefotetan), which are not hydrolysed by class A or D ESBLs4,5,6,7,8. Cefepime (a fourth-generation oxyimino-cephalosporin) was also inactivated by KL, HD and CMY-19 ESBLs5,6,8. Rubinstein and Zhanel have noted that physicians are increasingly being forced to use the carbapenems (for example, imipenem or meropenem) and fluoroquinolones (for example, ciprofloxacin or levofloxacin) as first-line therapy for ESBL-producing Gram-negative bacteria; indeed, the situation will become even more severe as ESBL-producing organisms increasingly become concomitantly resistant to the fluoroquinolones2.

However, we recently found that the CMY-10 ESBL had higher imipenem-hydrolysing activity than OXA-23, a class D carbapenemase7,10. Gram-negative bacteria producing such class C ESBLs could present a major therapeutic challenge, and so new antibacterial drugs against class C ESBL-producing Gram-negative bacteria are urgently needed.

To develop these antibacterial drugs, it is necessary to understand the operative mechanism of class C ESBLs to extend their substrate spectrum. Our kinetic data and crystal structure7 of a plasmid-encoded class C ESBL (that is, CMY-10) clarify this mechanism. The region responsible for the extended substrate spectrum is the R2-loop (amino-acid residues 289–307)7. Our sequence alignment of four class C ESBLs shows that the R2-loop includes all regions responsible for the extended substrate spectrum in all class C ESBLs, compared with P99 (a class C non-ESBL) (Fig. 1):

  • three amino-acid deletion (residues 303–305) of CMY-10 (Ref. 7);

  • four amino-acid deletion (residues 293–296) of HD6;

  • the single amino-acid substitution (L296H) of KL5;

  • the single amino-acid substitution (A292S) of CMY-19 (Ref. 8).

These natural mutations in the R2-loop can change the architecture of the active site in class C ESBLs, thereby affecting their hydrolysing activity. Owing to the deletion in CMY-10, for example, the R2-loop in the R2 active site (that is, the region that accommodates the R2 side-chain at C3 of the β-lactam nucleus in oxyimino-cephalosporins) displays noticeable structural alterations. The shortened path of the connection R2-loop between α10 and β11 induces the 2.5 Å shift of α9 and α10 relative to the adjacent helix α11 in CMY-10 compared with both P99 (Ref. 11) and GC1 (Ref. 12) β-lactamases, thereby opening the gap between α9–α10 and α11 (Ref. 7). Therefore, the bulky R2 side-chain of oxyimino-cephalosporins could fit snugly into the significant widening of the R2 active site in this way.

Clinically available β-lactamase inhibitors (for example, clavulanic acid, sulbactam or tazobactam) co-administered with less effective β-lactams are effective against class A beta-lactamases, but have little or no activity against class C β-lactamases. Because Gram-negative bacteria producing class C ESBLs are becoming an increasingly common cause of nosocomial infections5,6,7,8, there is an urgent need to develop an inhibitor of class C ESBLs or to discover new antibacterial drugs for these class C ESBL-producing clinical isolates. Such efforts could be aided considerably by the structural information on class C ESBLs highlighted above13. At present, a few academic research groups (such as our group and Shoichet's laboratory14) and small pharmaceutical companies (for example, Novexel15, which was spun out of Aventis) are seeking such novel β-lactamase inhibitors.

Figure 1: A sequence alignment of amino-acid residues near the H-9 (α9) and H-10 (α10) helix of class C β-lactamases with extended substrate spectrum.
figure 1

Alignment among CMY-10 and P99 β-lactamases for which structures are available is performed based on their superimposed structures. The image above the sequence alignment indicates secondary structure annotation of CMY-10. A partial amino-acid sequence alignment of CMY-10 (Enterobacter aerogenes K9911729; GenBank accession no. AF357598; PDB code, 1ZKJ); CMY-19 (Klebsiella pneumoniae HKY466; GenBank accession no. AB194410); HD (Serratia marcescens HD; GenBank accession no. AY336102), KL (Escherichia coli KL; GenBank accession no. AY533244); and P99 (Enterobacter cloacae P99; GenBank accession no. X07274; PDB code, 2BLT) is shown. The R2-loop of residues 289–307 is shaded. CMY-10, CMY-19, HD and KL are class C extended-spectrum β-lactamases (ESBLs), whereas P99 is a class C non-ESBL.

Accession codes

Accessions

GenBank/EMBL/DDBJ

Protein Data Bank

References

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Correspondence to Sang Hee Lee.

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S.H.L. has received research grants from the National Institute of Health of KCDC in Republic of Korea, the beamline 6B and 6C of PLS supported by MOST and POSCO, the Driving Force Project for the Next Generation of Gyeonggi Provincial Government in Republic of Korea and the Second-Phase of Brain Korea 21 Project. S.S.C. has received a research grant from the 21C Frontier Functional Proteomics Center in Republic of Korea. S.H.J. has received a research grant from the Korea Research Foundation (KRF-2006-331-E00455). J.H.L. declares that he has no conflicts of interest.

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Lee, J., Jeong, S., Cha, SS. et al. A lack of drugs for antibiotic-resistant Gram-negative bacteria. Nat Rev Drug Discov 6, 938 (2007). https://doi.org/10.1038/nrd2201-c1

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