2.2. Development of antibacterial drugs for diabetic infections

Based on the β-lactam ring-opening mechanism, β-lactamases can be subdivided into two major groups. Serine β-lactamases (SBLs) use an active-site serine residue to covalently attack the β-lactam ring resulting in an inactive ring-opened product. Fortunately, several SBL inhibitors (such as clavulanic acid, sulbactam, tazobactam and avibactam) are clinically available as codrugs that are coadministered with β-lactam antibiotics to overcome SBL-related resistance. Metallo-β-lactamases (MBLs), typified by recently emerged New Delhi metallo-β-lactamase-1 (NDM-1), employ an active-site zinc-stabilized OH anion that acts as a nucleophile in β-lactam ring opening. MBL-producing pathogens are resistant to virtually all clinically used β-lactam antibiotics, including the last-resort antibiotics carbapenems. In fact, the first NDM-1-producing pathogen has been found in a diabetic patient. Despite the rapid spread of MBL-producing bacteria, which is a major threat to public health, no inhibitors of NDM-1 or other MBLs are clinically approved so far. Thus, there is a great need to develop selective MBL inhibitors as clinically relevant codrugs to restore the activity of β-lactam antibiotics.

The fungal natural product aspergillomarasmine A (AMA) has recently been identified as a selective and potent NDM-1 inhibitor and a promising codrug candidate both in vitro and in vivo (King et al., 2014, Nature 510:503-506). We have recently developed a simple biocatalytic method to prepare AMA and related aminopolycarboxylic acids (Poelarends et al., manuscript under review in Nature Catalysis). In this project, we will synthesize various aminopolycarboxylic acids, including novel analogues of AMA, as well as smart pro-drug versions of these compounds, and explore their bioactivity, target selectivity and therapeutic potential. As such, the proposed project will contribute to the development of new chemotherapeutic strategies to fight antibiotic-resistant pathogens, which are more prevalent in individuals with diabetes.

In this project, the following methods are used:

1. Chemical and chemoenzymatic synthesis of new aminopolycarboxylic acids, including analogues of the natural products Aspergillomarasmine A & B and ethylenediamine-N,N-discuccinic acid, as well as smart and potentially selective pro-drug versions of these compounds;
2. Characterization (structural identity and stereochemistry) of the newly synthesized compounds by mass spectrometry, NMR spectroscopy, chiral HPLC, and optical rotation measurements;
3. Evaluation of the inhibitory properties of the newly synthesized aminopolycarboxylic acids and corresponding pro-drugs against NDM-1, VIM-2 and other metallo-β-lactamases, as well as possible off-target metallo-enzymes, using in vitro dose-dependent enzyme inhibition assays and inactivation kinetics;
4. The most potent and selective aminopolycarboxylic acids and pro-drugs will be studied for their ability to rescue the activity of existing β-lactam antibiotics in NDM-1- or VIM-2-positive clinical isolates;
5. Selected compounds will be tested for their ability to restore the activity of β-lactam antibiotics (e.g. meropenem) in worms, insect larvea and/or mice infected with a NDM-1 or VIM-2 expressing bacterial strain;
6. During the project we will continuously assess opportunities for patent applications as well as for collaborations with academic and industrial partners to further develop the products.

Please note that Prof. Poelarends is also participating in the Centre for Sustainable Antimicrobials (CeSAM), which comprises advanced facilities for fundamental research to develop novel antibiotics and therapeutic concepts to fight resistant bacterial strains as well as high-throughput (animal) testing and adequate patient-screening facilities. This existing network of collaborators will further support the proposed project.

1. Recent advances in the synthesis of the fungal natural products Aspergillomarasmine A and B and related aminopolycarboxylic acids;
2. Chemoenzymatic synthesis and evaluation of novel aminopolycarboxylic acids as clinically relevant metallo-β-lactamase inhibitors;
3. Chemical and enzymatic synthesis of aminopolycarboxylic acid-based metallo-β-lactamase inhibitors: Exploration of structure-activity relationships;
4. Development of aminopolycarboxylic acid-based pro-drugs: Exploration of activity, target selectivity and therapeutic potential;
5. Recent progress in the development of β-lactamase inhibitors.

The immunocompromised state and diabetic complications lead to high risk for infections in diabetic patients. Indeed, infectious diseases are more prevelant and often more serious in diabetic patients, which potentially increases their morbimortality. Furthermore, higher antibiotic resistance rates in diabetic patients compared to those without diabetes have been reported, further complicating the treatment of the infectious disease. Due to common and recurrent infections, diabetic patients need more antibiotic treatments, which may further select for the presence and increase in antibiotic-resistant pathogens. New chemotherapeutic strategies to fight antibiotic-resistant pathogens, particularly in patients with diabetes, are therefore urgently needed. The proposed project contributes to precision medicine because it aims to develop a clinical inhibitor of metallo-β-lactamases, which could be used as a codrug to rescue or potentiate β-lactam antibiotics in combination therapies. In this way resistance can be overcome, allowing more efficient treatment of infectious diseases in diabetic as well as other patients.

The proposed project fits in the Drug Development domain and will contribute to the development of new chemotherapeutic strategies to fight antibiotic-resistant pathogens, which are more prevalent in individuals with diabetes. As such, it may also become linked to drug application.