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New Approaches to Metallo-β-Lactamase Inhibitors

Periodic Reporting for period 1 - MBLIs (New Approaches to Metallo-β-Lactamase Inhibitors)

Reporting period: 2015-07-01 to 2017-06-30

The increasing problem of antibiotic resistance is a major global public health concern in Europe. The β-lactam antibiotics (BLAs) remain the most important antibiotics representing >60% of small molecules used for antibacterial therapy in clinical use. β-Lactam antibiotics contain the β-lactam ring which is critical for their mechanism of action which involves inhibition of penicillin-binding proteins (PBPs) that are essential for bacterial cell wall biosynthesis. BLA efficacy is declining due to resistance mechanisms including by the widespread occurrence of β-lactamases, which catalyse the hydrolysis of the β-lactam ring to give inert β-amino acids. There are two classes of β-lactamases (based on their mechanism of action): (a) the serine β-lactamases (SBLs; subdivided into class A-penicillinases, class C-cephalosporinases, and class D-oxacillinases) which employ a nucleophilic serine residue in catalysis, and (b) the metallo-β-lactamases (MBLs; class B) which require one or two zinc ions to activate a “hydrolytic” water molecule. The MBLs were long-considered as of little clinical relevance; however, they now present a serious global threat to the use of almost all BLAs (including carbapenems; “last resort” antibiotics) - with the only exception being Aztreonam – rendering the development of MBL inhibitors (MBLIs) important. Due to the variations in MBL structures, a major challenge in MBL inhibition is the development of compounds with the breadth of selectivity necessary for clinical use. This work aimed at the synthesis of broad-spectrum MBL inhibitors active against a panel of clinically representative MBLs, whilst maintaining selectivity against human MBL-fold enzymes which have related active sites, and some are crucially involved in DNA-repair (SNM1A and SNM1B), and other human metallo-enzymes. To date there are no clinically useful MBL inhibitors, in part due to selectivity issues. The work employed organic synthesis and was supported by dynamic combinatorial chemistry (DCC), MBL assay screening, biological NMR, structural biology, and medicinal chemistry. To address to problem of MBL resistance mechanism, novel synthetic methods were developed and led to the synthesis of a wide range of organic families including rhodanine derived enethiols, α-sulfanyl phosphonic acids, α-amino- phosphonic and -boronic acids as ‘transition state analogue’ based inhibitors, and indole carboxylates. The combined results revealed that low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs. An important outcome of the work is the dual inhibitory activity of some organic families for both SBLs and MBLs, as well as the inhibition of PBPs, thus representing a promising line not only for the protection of β-lactam antibiotics from both MBLs and/or SBLs, but also for direct inhibition of PBPs.
"The structural diversity and differences in metal utilisation by metallo-β-lactamases (MBLs) makes the discovery of a broad spectrum MBL inhibitors challenging. Optimised protein production and purification procedures (for all subclasses B1, B2 and B3 of MBLs) have been performed and their crystal structures have been obtained. Initially phosphonic acids were chosen for inhibitor discovery, however, in all cases the compounds were near inactive supporting the importance of both thiol and carboxylate groups for binding and inhibition. Additional MBL screens revealed the potential of using indole carboxylates for DC screens. Synthetic work on this family of compounds, yielded promising results with respect to applying DC chemistry (boronate ester exchange) on the 7- position of indole carboxylate, couple to analysis by x-ray crystallography. Rhodanine derived enethiol inhibitors of Metallo β-Lactamases: In this work, efficient methods were developed for the synthesis of analogies of the rhodanine ML302 and the ene-thiol ML302F, derived by hydrolysis of the former. The overall results revel that rhodanine derived species have potential as broad spectrum MBL inhibitors, and when combined with other results, also as serine β-Lactamase and penicillin binding protein inhibitors. Cyclic boronic and phosphonic acids: Recent work from the host laboratory has found that cyclic boronates are potent inhibitors, not only on MBLs, but also on SBLs, some of which also show weak activity against penicillin binding proteins. The syntheses of boronic acids was investigated via two independent routes involving: (a) Matteson’s homologation wherein boronic esters containing the chiral pinanediol auxiliary can undergo α-chloromethylene homologation to give α-chloroboronic esters which are further transformed to α-amino boronic esters, and (b) catalytic asymmetric hydroboration of aldimines. Pyridine, Pyrrole and Indole carboxylates: Synthetic methods were developed for the preparation of pyridine, pyrrole and indole carboxylates some of which showed broad spectrum activity with IC50 values <5nM (e.g. for VIM-2 at 200pM). Work in the Schofield group has led to one publication in Future Medicinal Chemistry [""The road to avibactam - the first clinically useful non-β-lactam working like a β-lactam"", 2016, 8, 1063-1084]. Work on rhodanine derived enethiols, pyridine, pyrrole and indole carboxylates, phosphonic and -boronic acids as ‘transition state analogue’ based inhibitors, as well as results from cross screening on different biological targets are currently in preparation and will be disseminated in the near future."
The increasing problem of antibiotic resistance is a major global public health concern. In the EU 25,000 patients die each year due to infections caused by multi-resistant bacterial pathogens. The EU spends at least 1.5 billion euro per year on healthcare costs and productivity losses due to resistance. Society is now in an alarming situation with respect to β-lactam antibiotic resistance. The successive development and applications of different β-lactam antibiotic generations (e.g. penicillins, cephalosporins, penems) has promoted resistance via the evolution of serine- and metallo- β-lactamases which are a major public health issue. Thus there is a clear need for the development of MBL inhibitor:β-lactam-based combination therapies. Although the project was mainly addressed at basic science questions, the results together with the track record of the host laboratory suggests that translational applications are feasible. My work formed part of a larger programme involving both industrial and academic collaborations aimed at developing new antibiotics. Improved antibiotic therapies will result in quicker recovery and greater number of successful treatments. Thus EU member countries will benefit by strengthening productivity, and reducing social costs. Commercial exploitation of the results will help to give Europe a competitive advantage in new antibiotics. Thus, the project was a small building block in the construction of the 'European Innovative Union' leading to economic growth, improved European employment, and enhanced quality of life. This work has led to the most potent MBL inhibitors yet identified.
Inhibition of PBPs by b-lactams and Resistance mechanisms.