We focus our project on hitting the Achilles' Heel of metallo-beta-lactamases (MBLs), a vital class of beta-lactamases without available clinical inhibitors, produced by drug-resistant pathogens. We want to obtain the broad-spectrum inhibitor of MBLs and lay the foundation for clinical trials in the future.

Firstly we used synthetic biology methods to express a series of MBLs in E. coli. Besides, a virtual screening model has been built to discover the therapeutically effective compounds, in order to predict potential inhibitors. Next, effective compounds were screened via high-throughput screening with fluorescent probe (CDC-1) from FDA approved drug libraries and traditional Chinese medicine libraries. Also we assessed their inhibitory ability in living bacterial cells by UV-vis spectroscopy. Finally we have obtained a series of effective inhibitors of MBLs, including an excellent broad-spectrum inhibitor, which can prevent living resistant bacteria from hydrolyzing beta-lactam antibiotics. We hope these inhibitors can be ideal candidates for therapeutics for diseases caused by drug-resistant pathogens.

Metallo-beta-lactamases result in beta-lactam resistance

The introduction of antibiotics represented one of the most important medical interventions in the history of global health, resulting in a dramatic reduction in human morbidity and mortality caused by bacterial infections^[1]. However, the overuse of antibiotics has made the production of drug-resistant bacteria a global burden. The World Health Organization has named antibiotic resistance as one of the three most important public health threats of the 21st century^[2].

The beta-lactams are the most successful class of antibiotic drugs but they are vulnerable to inactivation by a growing cadre of beta-lactamases that now number more than a thousand variants^[3]. These enzymes cleave the amide bond of the beta-lactam ring thus inactivating the antibiotic.

The Ambler system divides beta-lactamases into four classes, termed A, B, C and D. This year, we focus on class B that called metallo-beta-lactamases, or MBLs. Much different from other beta-lactamases, MBLs require zinc for activity and catalysis does not proceed via a covalent intermediate but rather through direct attack of a hydroxide ion that is stabilized by the Zinc ions in the active site^[4]. MBLs confer resistance to most beta-lactam antibiotics including carbapenems(often seen as last line drugs)^[3], making it a global threat to public health.

At present, due to the huge cost and low return of developing new antibiotics, the trend of developing new antibiotics has gradually declined in recent years. Alongside improvements to antibiotics themselves, combinations of existing beta-lactams with MBLs inhibitors represent the more feasible and common strategy to extend the usefulness of these existing beta-lactams.

For other beta-lactamases, a number of these beta-lactam-beta-lactamases inhibitor combinations are approved for clinical use previously (for example, Amoxicillin-clavulanate is effective in the treatment of community-acquired respiratory infections^[5] )，and many others are in clinical trials.

Unfortunately, the discovery of a clinically useful, specific inhibitor of metallo-beta-lactamases is made difficult by the fact that this compound must remain inactive towards the human proteins which are members of the metallo-beta-lactamase superfamily, or other metallo-enzymes such as the angiotensin converting enzyme. Another difficulty is to find a compound active on all subclasses of metallo-beta-lactamases and even on all the enzymes within a same subclass^[6]. Indeed, despite extensive academic effort, no MBL inhibitor is yet close to the clinic^[7].The dilemma of MBLs clinical therapy has inspired us to seek for the Achilles' Heel of MBLs.

Build gene circuits

We use synthetic biology methods to design and construct gene circuits. Moreover, using the prokaryotic cell (E. coli) as the chassis cell. We built multiple genetic circuits that ultimately ensured that each one we used matched perfectly with the chassis, resulting in our target protein.

Special fluorescent probe CDC-1

In our project we used a special fluorescent probe CDC-1, which contains a beta-lactam ring and a fluorescent group. When the beta-lactam ring is hydrolyzed by metallo-beta-lactamases, the probe emits an easily recognizable fluorescence. It is a property that will be exploited many times in our subsequent steps.

Determination of Enzyme Activity

We used fluorescent probe CDC-1 as the substrate to determine the kinetic parameters of related enzymes and the optimal buffer system, and established four enzyme activity systems for the screening of inhibitors in the next step.

Fluorescent High-throughput Screening

At the molecular level,referring to the established virtual screening model, we set up high-throughput screening system based on the fluorescent probe CDC-1, and selected effective inhibitors, in which there is a broad spectrum inhibitor, whose inhibition efficiency is more than 90% from over 4000 drugs in FDA approved libraries and traditional Chinese drug libraries. We also measured IC₅₀value of the screened inhibitors to assess the inhibition ability.

Living bacterial cells detection based on UV-visible Spectroscopy

At the cellular level, we combined some of the typical beta-lactams with selected inhibitors to act on E.coli which can express target MBL, and evaluated the effects of inhibitors on living cells by measuring the absorbance curve of antibiotics.

We want to address the problem that there are no clinically available inhibitors of beta-lactamase. We hope that we can contribute to solving issue, especially the mechanism about MBLs in the treatment of disease clinical problem. For this, we screened out broad spectrum inhibitors that are expected to be used clinically to regenerate antibiotics, which can possibly solve the current medical dilemma of drug-resistant bacteria. Besides, we tested therapeutic effect in live bacteria by combining beta-lactams with inhibitors, and proved that the inhibitors we screened were effective in preventing MBLs-carrying bacteria from hydrolyzing antibiotics. We have successfully hit the Achilles' Heel of MBLs!

For the further research, we will use clinically collected resistant strains to test the effectiveness of our inhibitors.

For therapeutics, we consider putting the screened inhibitors into applied medicine. We also hope that with mature results, the combination of beta-lactam/beta-lactamase inhibitors therapy can establish novel medicine and save lives clinically, which will be the progress in the clinical treatment of bacterial infection.

[1] Paulo Durão, Roberto Balbontín, Isabel Gordo. Evolutionary Mechanisms Shaping the Maintenance of Antibiotic Resistance. Trends in Microbiology, Volume 26, Issue 8, 2018, pages 677-691. ISSN 0966-842X, https://doi.org/10.1016/j.tim.2018.01.005.
[2] Jose M. Munitaand Cesar A. Arias. Mechanisms of Antibiotic Resistance. Microbiol Spectr. 2016 Apr; 4(2): 10.1128/microbiolspec.VMBF-0016-2015.
[3] Caitlyn M Rotondo and Gerard D Wright. Inhibitors of metallo-b-lactamases. Microbiology 2017,39:96–105.
[4] Palzkill T. Metallo-beta-lactamase structure and function. Ann N Y Acad Sci. 2013;1277:91–104. doi:10.1111/j.1749-6632.2012.06796.x.
[5] Anthony R. White, Clive Kaye, James Poupard, Rienk Pypstra, Gary Woodnutt, Brian Wynne, Augmentin ® (amoxicillin/clavulanate) in the treatment of community-acquired respiratory tract infection: a review of the continuing development of an innovative antimicrobial agent , Journal of Antimicrobial Chemotherapy, Volume 53, Issue suppl_1, February 2004, Pages i3–i20, https://doi.org/10.1093/jac/dkh050.
[6] Carine Bebrone. Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochemical Pharmacology, Volume 74, Issue 12, 2007, Pages 1686-1701, ISSN 0006-2952, https://doi.org/10.1016/j.bcp.2007.05.021.
[7] Tooke CL, Hinchliffe P, Bragginton EC, et al. beta-Lactamases and beta-Lactamase Inhibitors in the 21st Century. J Mol Biol. 2019;431(18):3472–3500. doi:10.1016/j.jmb.2019.04.002.
[8] Fan Gao. Combination of two lead compounds for the treatment of TB based on beta-lactamase as a target. 2018.
[9] Ying Ge, Ya-Jun Zhou, Ke-Wu Yang, Yi-Lin Zhang, Yang Xiang and Yue-Juan Zhang. Real-time activity assays of b-lactamases in living bacterial cells: application to the inhibition of antibiotic-resistant E. coli strains. Royal society of Chemistry. 2017.
[10] Ke-Wu Yang, Yajun Zhou, Ying Ge and Yuejuan Zhang. Real-time activity monitoring of New Delhi metallo-b-lactamase-1 in living bacterial cells by UV-Vis spectroscopy. Royal society of Chemistry,2017.