Brem, J

Lohans, CT

Schofield, CJ

Lohans, Christopher

Brem, Jurgen

Schofield, Christopher

Oxford University Research Archive

	 1	New Delhi metallo-b-lactamase-1 catalyses avibactam and aztreonam hydrolysis 1	 2	Christopher T. Lohans,a Jürgen Brem,a* and Christopher J. Schofielda* 3	 4	aDepartment of Chemistry, University of Oxford, Oxford, UK, OX1 3TA 5	 6	*Address correspondence to co-corresponding authors: 7	Prof. Christopher Schofield, Tel: +44 (0)1865 275625; Fax: +44 (0)1865 285002; Email: 8	christopher.schofield@chem.ox.ac.uk 9	Dr. Jürgen Brem, jurgen.brem@chem.ox.ac.uk  10		 2	Main Text 11	Metallo-b-lactamases (MBLs) threaten the clinical utility of b-lactam antibiotics by 12	hydrolysing penicillins, cephalosporins, and carbapenems. Moreover, they can also hydrolyse all 13	clinically used inhibitors (e.g., clavulanic acid, sulbactam, and tazobactam) that protect b-lactam 14	antibiotics from multidrug resistant bacteria (1). Even the diazabicyclooctane (DBO)-based serine-15	b-lactamase (SBL) inhibitor avibactam, which was recently FDA approved, is hydrolysed slowly 16	by some MBLs (2). 17	The combination of avibactam and the monobactam antibiotic aztreonam has recently 18	passed phase II clinical trials for the treatment of multidrug resistant Gram-negative bacteria 19	producing MBLs (3). While SBL-mediated resistance to aztreonam has long been known via the 20	evolution of SBLs (4), MBLs are not thought to hydrolyse aztreonam (5–7). Due to structural 21	similarities between avibactam and aztreonam (Figure 1A), particularly the sulfonate/sulfate 22	substituent on the b-lactam/urea nitrogen, we were interested in examining the interaction between 23	more recently discovered MBLs and aztreonam, and the potential for new clinically relevant MBLs 24	with monobactam hydrolysing activity. 25	We tested the hydrolysis of aztreonam by recombinant enzymes covering all three 26	subclasses of MBLs (i.e., B1, B2, and B3). Following overnight incubation of a 1:10 ratio of MBL 27	and aztreonam, the extent of hydrolysis was determined by nuclear magnetic resonance (NMR) 28	spectroscopy. While Verona integron-encoded MBL (VIM)-1 (subclass B1), VIM-4 (B1), CphA 29	(B2), and L1 (B3) did not hydrolyse aztreonam (within our limits of detection), the model MBL 30	BcII (B1) showed partial hydrolysis, and the New Delhi MBL (NDM)-1 (B1) fully hydrolysed 31	aztreonam under our assay conditions (Figure 1B). The BcII data is in agreement with the 32	previously observed ‘non-productive’ binding of aztreonam to BcII (8). Interestingly, no 33		 3	interaction between aztreonam and NDM-1 was observed by 19F-NMR (9), suggesting that the 34	binding interaction (e.g., KM) is quite weak. Therefore, more detailed kinetic analyses were 35	performed. 36	The hydrolysis of aztreonam by NDM-1 was monitored over a shorter time scale (Figure 37	1C), yielding a specific activity of 3.7 ± 0.4 nmol min-1 mg-1 using 10 µM NDM-1 and 1 mM 38	aztreonam. The dependence of aztreonam hydrolysis on NDM-1 activity was confirmed by 39	inhibition in the presence of EDTA and D-captopril, both inhibitors of MBLs (Figure 1C). The 40	hydrolysis of avibactam by NDM-1 was also shown by NMR, which indicated that avibactam is 41	hydrolysed more quickly than aztreonam (Figure 1C). 42	The kinetics of avibactam and aztreonam hydrolysis by NDM-1 were further investigated 43	by UV-Vis and NMR spectroscopy (Figure 1D). Due to poor substrate turnover and the limitations 44	associated with these detection methods, a full kinetic characterization was not possible; while the 45	values obtained are expected to be imprecise, they may serve as estimates of substrate affinity and 46	turnover. Although apparent KM and kcat values of ~3 mM and ~0.02 s-1 were obtained for the 47	hydrolysis of avibactam by NDM-1 as monitored by UV-Vis, the limited substrate concentrations 48	prevent accurate nonlinear regression analysis (Figure 1D). Instead, based on the NMR studies 49	which employed a wider range of substrate concentrations, avibactam has an apparent KM of ~24 50	mM and an apparent kcat of ~0.15 s-1 with NDM-1. 51	Aztreonam has an apparent KM of ~9 mM and an apparent kcat of ~0.014 s-1 with NDM-1 52	under the NMR assay conditions. By comparison, BcII has an approximate maximal kcat of ~3 ´ 53	10-4 s-1 with aztreonam, while the other MBLs tested (for which we did not observe aztreonam 54	hydrolysis by NMR) have calculated maximal kcat values of ~6 ´ 10-5 s-1 based on the sensitivity 55	limits of the NMR assay. To provide context with other b-lactamases, the kcat values for avibactam 56		 4	and aztreonam with NDM-1 are comparable to those determined for class D b-lactamases with 57	carbapenems (10) and for class C b-lactamases with cephalosporins (11); these classes of enzymes 58	are thought to contribute to resistance to these antibiotics in vivo. 59	The hydrolysis of avibactam and aztreonam was tested with a panel of twelve b-lactamases, 60	as monitored by NMR (Figure 1E, 1F). The b-lactamases tested belong to classes A (TEM-1, CTX-61	M-15), B (BcII, NDM-1, VIM-1, VIM-4, CphA, L1), C (AmpC), and D (OXA-10, OXA-23, 62	OXA-48). While aztreonam was hydrolysed efficiently by the class A and class D b-lactamases 63	tested, the class C b-lactamase AmpC poorly catalysed aztreonam hydrolysis. As indicated above, 64	while most MBLs tested did not hydrolyse aztreonam, NDM-1 (and BcII to a lesser extent) 65	displayed activity. Although no avibactam hydrolysis was observed for the class A, C, and D b-66	lactamases tested, MBLs belonging to subclasses B1, B2, and B3 all catalysed avibactam 67	hydrolysis (Figure 1F). 68	 These results challenge the widely-held view that MBLs cannot hydrolyse aztreonam. 69	Although the hydrolysis of avibactam and aztreonam by NDM-1 may well not be clinically 70	relevant at the rate we observe, the evolution of MBLs to more efficiently hydrolyse both substrates 71	is likely. This proposal is analogous to what has been observed with the TEM SBLs; while TEM-72	1 does not efficiently hydrolyse aztreonam, TEM mutants with increased aztreonam hydrolysing 73	activity have been identified (12). Furthermore, MBL variants with greater activity may already 74	exist in clinical isolates. Therefore, the potential for MBL-mediated resistance should be 75	considered when evaluating the clinical use of avibactam and aztreonam, individually or in 76	combination, as well as other DBOs and monobactams. 77	 78	Acknowledgements 79		 5	We thank the Medical Research Council for funding our work on MBLs. CTL is grateful to the 80	Canadian Institutes of Health Research. 81	  82		 6	References 83	1. Prosperi-Meys, C, Llabres, G, de Seny, D, Soto, RP, Valladares, MH, Laraki, N, 84	Frere, JM, Galleni, M. 1999. Interaction between class B β-lactamases and suicide 85	substrates of active-site serine β-lactamases. FEBS Lett 443:109–111. 86	2. Abboud, MI, Damblon, C, Brem, J, Smargiasso, N, Mercuri, P, Gilbert, B, Rydzik, 87	AM, Claridge, TD, Schofield, CJ, Frère, JM. 2016. Interaction of avibactam with class B 88	metallo-β-lactamases. Antimicrob Agents Chemother 60:5655–5662. 89	3. 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Potron, A, Rondinaud, E, Poirel, L, Belmonte, O, Boyer, S, Camiade, S, Nordmann, P. 112	2013. Genetic and biochemical characterisation of OXA-232, a carbapenem-hydrolysing 113	class D β-lactamase from Enterobacteriaceae. Int J Antimicrob Agents 41:325–329. 114	11. Rodríguez-Martínez, JM, Poirel, L, Nordmann, P. 2009. Extended-spectrum 115	cephalosporinases in Pseudomonas aeruginosa. Antimicrob Agents Chemother 53:1766–116	1771. 117	12. Cantu, C, Huang, W, Palzkill, T. 1996. Selection and characterization of amino acid 118	substitutions at residues 237-240 of TEM-1 β-lactamase with altered substrate specificity 119	for aztreonam and ceftazidime. J Biol Chem 271:22538–22545. 120	  121		 8	Figure Legends 122	Figure 1. Hydrolysis of avibactam and aztreonam by NDM-1. (A) Structures of avibactam and 123	aztreonam. (B) Extent of aztreonam (100 µM) hydrolysis by VIM-4, BcII, and NDM-1 (all 10 µM) 124	after 16 h. Hydrolysis was monitored by NMR spectroscopy (700 MHz); the peak labeled with an 125	asterisk corresponds to the aztreonam proton indicated with an asterisk in panel A, while the peak 126	labeled with two asterisks indicates the hydrolysed product. (C) Extent of hydrolysis of a mixture 127	of aztreonam (ATM; 1 mM) and avibactam (AVI; 1 mM) by NDM-1 (10 µM) after 50 min, with 128	100 µM ZnCl2 in 50 mM Tris-d11, pH 7.5, 10 % D2O. The addition of D-captopril (500 µM), 129	EDTA (2 mM), or removal of ZnCl2 inhibited aztreonam hydrolysis. (D) Preliminary kinetic 130	characterisation of avibactam and aztreonam hydrolysis by NDM-1 as monitored by NMR and 131	UV-Vis. The NMR studies used 10 µM NDM-1, 100 µM ZnCl2, and the indicated concentration 132	of avibactam or aztreonam in 50 mM Tris-d11, pH 7.5, 10 % D2O. The UV-Vis studies (monitoring 133	at 230 nm) used 10 µM NDM-1, 20 µM ZnCl2, and the indicated concentration of avibactam in 5 134	mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5 (2). Nonlinear 135	regression analyses were performed using Prism 7 (GraphPad). The hydrolysis of (E) aztreonam 136	and (F) avibactam by a panel of b-lactamases covering classes A (black), B (red), C (blue), and D 137	(green). Hydrolysis was measured by NMR after 24 h for samples consisting of enzyme (10 µM) 138	and avibactam (400 µM) or aztreonam (1 mM) in 50 mM Tris-d11, pH 7.5, 10 % D2O. 139	  140		 9	Figure 1 141	 142		143	

New Delhi metallo-beta-lactamase 1 catalyzes avibactam and aztreonam hydrolysis

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