Aspergillomarasmine A overcomes metallo-β-lactamase antibiotic resistance

Abstract

The emergence and spread of carbapenem-resistant Gram-negative pathogens is a global public health problem. The acquisition of metallo-β-lactamases (MBLs) such as NDM-1 is a principle contributor to the emergence of carbapenem-resistant Gram-negative pathogens that threatens the use of penicillin, cephalosporin and carbapenem antibiotics to treat infections. To date, a clinical inhibitor of MBLs that could reverse resistance and re-sensitize resistant Gram-negative pathogens to carbapenems has not been found. Here we have identified a fungal natural product, aspergillomarasmine A (AMA), that is a rapid and potent inhibitor of the NDM-1 enzyme and another clinically relevant MBL, VIM-2. AMA also fully restored the activity of meropenem against Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. possessing either VIM or NDM-type alleles. In mice infected with NDM-1-expressing Klebsiella pneumoniae, AMA efficiently restored meropenem activity, demonstrating that a combination of AMA and a carbapenem antibiotic has therapeutic potential to address the clinical challenge of MBL-positive carbapenem-resistant Gram-negative pathogens.

Extended Data Figure 1

¹H NMR spectrum of AMA in D₂O

Extended Data Figure 2

¹³C NMR spectrum of AMA in D₂O

Extended Data Figure 3

¹H-¹H COSY NMR spectrum of AMA in D₂O

Extended Data Figure 4

¹H-¹³C HSQC NMR spectrum of AMA in D₂O

Extended Data Figure 5

¹H-¹³C HMBC NMR spectrum of AMA in D₂O

Extended Data Figure 6. IC₅₀ inhibition profiles for select SBLs and ACE

Experiments were done as in Fig. 1b for (a) ACE and (b) CTX-M-15 (●), KPC-2 (○), and TEM-1 (■). Error bars denote standard deviation of at least two replicates.

Extended Data Figure 7. Effects of meropenem dosage on spleen burden

CD-1 mice were infected with K. pneumoniae N11-2218 by i.p. injection. Mice were treated with either PBS (n=6), or various doses of meropenem (n=3 per group) by s.c. injection. Mice were euthanized 48 h after infection, and the bacterial load in the spleen was determined by selective plating. Data are the means with standard error.

Fig. 1. AMA inactivates MBLs

(a) Chemical structure of AMA. (b) AMA inhibits NDM-1 (●) (IC₅₀ 4.0 ± 1.0 μM) and VIM-2 (○) (IC₅₀ of 9.6 ± 2.4 μM). Activity of OXA-48 (■) was unaffected by AMA. (c) Removal of AMA via PD10 column does not restore NDM-1 activity, confirming irreversible inactivation. (d) Addition of excess ZnSO₄ restores activity post5 inactivation. (e) The rate of inactivation of NDM-1 and VIM-2 is saturable with [AMA]. (f) ICP-MS confirms depletion of Zn from NDM-1. For all experiments error bars denote standard deviation of three technical replicates.

Fig. 2. AMA potentiates the activity of meropenem against CRGNP

(a, b) Microdilution checkerboard analysis showing the combined effect of AMA and meropenem selectively against CRE (a, K. pneumoniae N11-2218 MIC meropenem = 32 μg/ml) but not a carbapenem sensitive strain (b, E. coli BW25113 MIC = 0.008–0.016 μg/ml). Heat plots shows the average of two technical replicates. (c) VIM- and NDM-expressing Gram negative pathogens were highly susceptible to meropenem/AMA combination (respectively 2μg/ml and 8 μg/ml) while AIM-, IMP-, and SPM-1-expressing isolates remained resistant.

Fig. 3. AMA rescues meropenem activity in vivo

CD-1 mice were given a sub-lethal dose of K. pneumoniae N11-2218 (meropenem MIC 32 μg/mL) by i.p. injection. (a, b) Groups of mice were treated with a single dose of meropenem (10 mg/kg), a combination of meropenem (10 mg/kg) + AMA (10 mg/kg), or PBS by s.c. injection. Bacterial load in the spleen (a) and liver (b) was determined by selective plating. Data are the means with standard error from two separate experiments (n=7 per group). (c) For survival experiments, CD-1 mice were given a lethal dose of K. pneumoniae N11-2218, and treated with a single dose of meropenem (10 mg/kg), a combination of meropenem (10 mg/kg) + AMA (30 mg/kg), AMA alone (30 mg/kg), or PBS by s.c. injection. Data are the means with standard error from four separate experiments (n=12 per group, except AMA only treatment where n=13). Groups were analyzed using a non-parametric Mann-Whitney t-test. P-values <0.05 were considered statistically significant.