Diagnósticos sobre el acceso de los indígenas a la educación

As NDM-1 is one of the most recent MBLs to be identified in bacteria from hospital acquired infections, there has been limited work conducted into finding new inhibitors. Currently there are no known clinically relevant inhibitors of the MBLs however there are a few classes of inhibitors in development. These include metal extractors, thiols and transition state mimics all of which are designed to be given in a combination with a current β-lactam antibiotic.

2.5.1 Metal extractors

The metal chelator EDTA (2.16) has been shown to inhibit NDM-1 by binding strongly to the zinc atoms present in the active site44 (Figure 25). However EDTA is non-selective in nature meaning it binds to other metal sites in the body as well as the NDM-1 enzyme which causes many potential issues with its use.

Figure 25: Structures of metal extractors EDTA (2.16) and Aspergillomarasimine A (2.17) Aspergillomarasmine A (AMA, 2.17)66, has recently been identified as an NDM-1 inhibitor. It acts by coordinating to the zinc ions in the active site of the enzyme. This compound, which is similar structure to EDTA, acts as a ‘suicide’ inhibitor by removing the zinc ions from the active site of the enzyme, leaving the enzyme unable to function. Wright et al., who identified AMA, showed it can overcome bacterial resistance in NDM-1 producers towards the carbapenem antibiotic meropenem. Although initial studies show promising results, limited selectivity data is available to show whether the molecule will be selective towards its target or have a detrimental effect by coordinating to other zinc-containing enzymes in the body.

2.5.2 Thiols

Captopril (2.18, Figure 26) is one of the few compounds that effectively inhibit NDM-1. Chen et al. investigated scaling back captopril to simpler derivatives to identify the pharmacophore for NDM-1 inhibition. Captopril is a peptide mimic consisting of a proline residue and a 3-mercapto-2- methylpropanoic acid subunit (2.19). They concluded that the captopril 3- mercapto-2-methylpropanoic acid subunit demonstrated inhibitory activity against the NDM-1 enzyme. This highlighted the potential for thiol containing compounds which could bind strongly to zinc containing systems. Even the smallest of thiols have been seen to give an inhibitory effect such as mercaptoacetate (2.20) which was used to help elucidate the structure of B3 MBL SMB-1.67

Figure 26: Thiol-based inhibitors. a) Captopril (2.18) and active 3-mercapto-2- methylpropanoic acid subunit (2.19). b) mercaptoacetate (2.20)

Work has also been conducted into using cysteine-containing peptides. The 3-mercapto-2-methylpropanoic acid subunit seen in captopril is a peptide mimic of cysteine so activity is also expected to be observed with cysteine. Page et al. showed that micromolar inhibition can be achieved against Bacillus cereus zinc β-lactamase using cysteine-containing peptides.42 This class of compound is discussed further in Chapter 6.

Recently Klingler et al. showed that a number of existing FDA approved thiol-containing drugs can inhibit a range of MBLs.68 The study found that four compounds DL-captopril (2.18), DL-Thiorphan (2.21, Figure 27) , 2,3- Dimercaprol (2.22) and Tiopronin (2.23) showed micromolar levels of affinity against the MBLs. Further studies using thermal shift measurements confirmed that the inhibitors were co-ordinating to the zinc after observing higher melting temperatures than the enzyme with no inhibitor as the enzyme gained increased stability. This is in contrast to EDTA, which gave a

lower melting temperature due to the increased instability of the enzyme once the zincs had been abstracted.

Figure 27: The structures of thiol based inhibitors DL-Thiorphan (2.21), 2,3-Dimecarpol (2.22) and Tiopronin (2.23)

Rhodanines are known to inhibit class A and C β-lactamases and some PBP.69 Although rhodanines have often been identified as pan-assay interference compounds (PAINS),70 a recent report of a broadly active rhodanine based inhibitor claims the inhibitor to be an uncompetitive/non- competitive submicromolar inhibitor of clinically relevant MBLs: VIM-2 (Ki = 183 ± 24 nM) and IMP-1 (Ki = 930 ± 97 nM).71 The inhibitor is also seen to recover the activity of imipenem against clinical isolates of VIM-2, IMP-1 and NDM-1. Brem et al. conducted crystallographic and biological studies to elucidate the mechanism by which the inhibitor works.72 They showed the rhodanine (2.24) hydrolyses to form a thioenolate (2.25) active species which binds through the thiol to the zinc atoms (Figure 28).

Figure 28: Rhodanine inhibitor (2.24) undergoes hydrolysis to give active thioenolate (2.25).

2.5.3 Transition state mimics

A number of ‘suicide’ inhibitors have been designed which mimic the β- lactam antibiotics transition state on hydrolysis. Since β-lactam-containing antibiotics are peptide mimics and since β-lactamases catalyse peptide bond cleavage, researchers hypothesised that a tetrahedral transition state may be formed during the hydrolysis mechanism. It was thought that a chemically stable β-phospholactam transition state mimic may be a good inhibitor of the MBLs (2.26, Figure 29) Examples have seen β-phospholactams used to

inhibit ~50 % of NDM-1’s activity at 100 μM.73

Although inhibition through this type of mimic was seen it was significantly weaker than binding with metal chelators or thiols.

The same principle has been applied with boron containing compounds forming a boronic acid transition state inhibitor (BATSI) (2.27, Figure 29).74 In these compounds, the boronic acid acts as the tetrahedral intermediate.75 These compounds have been shown to inhibit serine-β-lactamases and VIM- 2 in the nanomolar region but have yet to be tested against NDM-1, IMP-1 and SPM-1 (See Chapter 6).

Figure 29: Transition state β-lactamase inhibitors. Phospholactam (2.26) and BATSI (2.27)

2.5.4 Other MBL inhibitors

Recently Worthington et al. have identified a 2-aminoimidazole-derived small molecule (2.28) that is able to suppress resistance of a NDM-1 producing strain of K. pneumoniae76 (Figure 30). This small molecule is able to lower carbapenem MICs by up to 16 fold, while exhibiting little anti-bacterial activity itself. The molecule was derived by examination of previously reported inhibitors of MRSA, an example of drug repurposing.77 The mechanism of action of this compound has yet to be determined.

Figure 30: 2-aminoimidazole scaffold (2.28)

A study was carried out by Weide et al. to identify selective VIM-2 inhibitors41 The most potent compound (Figure 31), a NH-1,2,3-triazole-based inhibitor (2.29), was found to have a Ki of 0.41 ± 0.03 µM activity against VIM-2. The

Minond et al. discussed the click chemistry synthesis of these compounds and studied further derivatives to look for more potent inhibitors.78

Figure 31: Selective VIM-2 inhibitor (2.29) identified by Weide et al.

Toney et al. identified a number of 2,3-(S,S)-di-substituted succinic acid inhibitors (2.30).79 The identified best succinic acid inhibitor, which contained aromatic groups at R1 and R3, gave an IC50 value of 9 nM against IMP-1

(Figure 32) It is expected that the aromatic portions form good π stacking interactions within the enclosed hydrophobic pocket.

Figure 32: Succinic acid inhibitor (2.30) identified by Toney et al.

As well as the described inhibitors above, which would be given in combination with a current β-lactam antibiotic, a number of combinations of non β-lactam containing drugs have been used to treat infections caused by NDM-1 producing bacteria, such as tigecycline and colistin. In 2011 Stone et al. reported a patient with calciphylaxis and co-infection with NDM-1 producing E. coli and K. pneumoniae, both being susceptible to tigecycline and colistin.80 Colistin is an antimicrobial agent composed of a complex mixture of polymyxins which inhibits the cell membrane structure and function. Tigecycline is a glycylcycline with a wide spectrum of activity against bacteria, which inhibits protein biosynthesis. This work highlights that resistance was being observed when treatment was with tigecycline alone and that the colistin was required to fully treat the patient.81

Chapter 3